key: cord-023225-5quigar4
authors: nan
title: Posters
date: 2012-08-21
journal: J Pept Sci
DOI: 10.1002/psc.2449
sha: 
doc_id: 23225
cord_uid: 5quigar4

No abstract is available for this article.

Laboratory of Molecular Biology and Immunology, Department of Pharmacy, University of Patras, Patras, Greece Antimicrobial peptides (AMPs) are an important component of innate immune system of most living organisms. They have recently gained much attention as new anti-infective drugs with new modes of actions and few or no side effects. Their antimicrobial spectrum covers gram-positive and -negative bacteria as well as fungi and certain viruses 1 . Fish have proven to be a rich source of antimicrobial peptides. Three chrysophsin peptides (chrysophsin-1, -2, -3) have been identified in the gills of the red sea bream, Chrysophrys major, which are all bactericidal to pathogenic bacteria at low concentrations 2 . They are cationic α-helical peptides, rich in histidine residues and all end in an unusual RRRH motif. However, in addition to its high antimicrobial potency, chrysophsins have considerable hemolytic activity. The development of new analogues which would preserve high antimicrobial potency, but would lack the undesired hemolytic activity, could be a useful tool with possible commercial and clinical applications. In the present study, we synthesized a series of analogues of chrysophsin-1 with different ratios of Lys and Leu residues, utilizing the Fmoc/But solid phase methodology 3 . The synthesized analogues were purified and isolated by RP-HPLC. The antimicrobial properties of the above peptide analogues are currently testing in 3 Gram positive (S. aureus, S. epidermidis, E. faecium) and 2 Gram negative (E. coli, P. aeruginosa) bacteria. The goal is to identify the minimum bacteriostatic and bactericidal concentrations of the analogues, under conditions that simulate the best possible that of the human organism. Hemolytic or cytotoxic activity of the peptides will also be determined. 1 The rise of antibiotic resistance demands the development of new antimicrobial agents. These should exhibit a novel mechanism of action so as to overcome the resistance and be invulnerable to 'not yet acquired resistance mechanisms'. Such criteria are difficult to meet. However, cationic host defence peptides (HDPs) have emerged as promising candidates.

HDPs target and disrupt bacterial membranes. In order to evade such a threat a bacterium would need to make substantial changes to its membrane composition disfavouring the development of resistance (1) . However, exact role and mechanism of HPDs in the regulation and monitoring of microbial invasions remain to be established. Herein we will present new potential mechanisms of antimicrobial regulation by helical HDPs using de novo (2) and native systems (3) . Biophysical and microbiology aspects of the experimental designs will be discussed. The low number of the newly discovered antibiotics, emergence of multiple-drug resistance, and the alarming death rate due to the infection disease led to development the alternative means to combat the infections. The researchers accumulate information about antimicrobial drugs that could be result of the innate immunity mechanisms. Armed only with the innate immunity, the insect has developed into the most widespread class in living kingdom. They produce several antimicrobial peptides with complementary and rapid mode of action. So far there are hundreds of antimicrobial peptides isolated from insect and lot of them are waiting to be discovered. The fleshly Neobellieria bullata was chosen for isolation of these active compounds. Its larvae in the third instar were squeezed to collect the haemolymph, which was gradually centrifuged and precipitated by acidified methanol. Supernatant was subsequently separated by chromatographic methods (SPE column, RP-HPLC) to obtain fractions of short peptides. Identification and characterization of these fractions were performed by tricine electrophoresis, mass spectrometry MALDI-TOF analysis and N-terminal sequencing. Several fractions showed antimicrobial activity against Institute of Chemical Kinetics and Combustion, 630090 Novosibirsk, Russian Federation In this work, we extracted 3D-structural information on newly synthesized, medium-length, double spin-labeled peptaibiotics using PELDOR spectroscopy. We investigated the magnetic dipole-dipole interactions between spin labels and the orientation selectivity effects.

In particular, the medium-length peptaibiotics tylopeptin B 1,2 and heptaibin 3 , double spin-labeled with the nitroxyl probe TOAC (4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-4-carboxylic acid), were studied by means of X-Band PELDOR spectroscopy. This study was conducted on tylopeptin labeled at positions 3 and 13 (T313) and heptaibin labeled at positions 2 and 14 (H214) in frozen glassy methanol solutions at 77 Κ. PELDOR data analysis was carried out using the theory developed for short interspin distances. The distance distribution functions between spin labels for Τ313 (maximum at 1.78 nm, halfwidth of 0.08 nm) and Η214 (maximum at 2.30 nm, half-width of 0.05 nm) were determined. The intramolecular distances observed between the labels allowed us to assign an essentially α-helical conformation to Τ313 and a largely prevailing 310-helical structure to Η214 under the aforementioned experimental conditions. are amidated at the C-terminus, as a result of a posttranslational enzymatic reaction. Temporins are particularly active against Gram-positive bacteria and are not toxic to eukaryotic cells. In this study we designed a series of analogues of TB with the aim to improve the peptide antimicrobial activity against both Gram negative and Gram positive strains and then to structurally elucidate the mechanism of interaction of active peptides with LPS. The peptides have been synthesized substituting one or two amino acids with an alanine and lengthening the sequence with positively charged amino acids. Among the 16 designed peptides, one of the analogues, TB_KKG6A, showed highly increased activity against Gram negative bacteria and also a slightly increased activity against Gram positive bacteria with a total lack of hemolytic activity. To develop LL-37-derived short AMPs with prokaryotic selectivity and lipolysaccharide (LPS)neutralizing activity, a series of amino acid-substituted analogs based on IG-19 (residues 13-31 of LL-37) were synthesized. Analog a4 showed the highest prokaryotic selectivity, but much lower LPS-neutralizing activity compared to LL-37. The analogs, a5, a6, a7 and a8 with higher hydrophobicity displayed LPS-neutralizing activity comparable to that of LL-37, but much lesser prokaryotic selectivity. These results indicated that the proper hydrophobicity of the peptides is crucial to exert the amalgamated property of LPS-neutralizing activity and prokaryotic selectivity. To increase LPS-neutralizing activity of the analog a4, we synthesized Trp-substituted analogs (a4-W1 and a4-W2), in which Phe 5 or Phe 15 of a4 is replaced by Trp. Despite their same prokaryotic selectivity, a4-W2 displayed much higher LPS-neutralizing activity compared to a4-W1. This result suggested that the effective site for Trp-substitution when designing novel AMPs with higher LPS-neutralizing activity, without a remarkable reduction in prokaryotic selectivity, is the amphipathic interface between the end of the hydrophilic side and the start of the hydrophobic side rather than the central position of the hydrophobic side in their α-helical wheel projection. Furthermore, D-enantiomeric peptides (a4-W1-E and a4-W2-E) of a4-W1 and a4-W2 possessed not only more improved prokaryotic selectivity and retained LPSneutralizing activity compared to a4-W2 but also protease stability. Taken together, a4-W1-E and a4-W2-E can serve as promising templates for the development of therapeutic agents for the treatment of endotoxic shock and bacterial infection. Department of Zoology, Faculty of Science, Charles University, Prague, Czech Republic Antimicrobial peptides (AMPs) are among the most promising lead compounds for developing medicines in the fight against resistant pathogenic bacteria. We have already shown that the venom of wild bee is a rich source of pharmacologically interesting antimicrobial peptides [1] [2] [3] [4] . From the venom of solitary bee Macropis fulvipes, we isolated and characterized the novel antimicrobial peptide named macropin (MAC-1). By Edman degradation and mass spectrometry, its primary sequence was established as GFGMALKLLKKVL-NH2. MAC-1 possesses potent antimicrobial activity against both Gram-positive andnegative bacteria and moderate hemolytic activity against human red blood cells. CD spectra confirmed that MAC-1 can form an amphipathic α-helical secondary structure in the presence of membrane-mimicking substances as sodium dodecyl sulfate or organic solvents like trifluoroethanol. We prepared a series of MAC-1 analogs to study the effect of incorporating D-amino acid residues into the sequence in various positions on antimicrobial and hemolytic activity, α-helicity and serum stability. The substitution of L-amino acid residues at N-terminal part of sequence by D-amino acid residues led to the improving hemolytic activity with maintaining or increasing antimicrobial activity. These modifications increased peptide stability in human serum. Effect of the incorporation of D-amino acid residues into the MAC-1 sequence on its α-helical structure will be discussed. The neutralization of endotoxins (lipopolysaccharide, LPS) by suitable compounds has been shown to be a key step in the treatment of infectious diseases, in particular in the case of Gram-negative bacteria. The active endotoxic center of LPS is lipid A, its lipophilic part. An effective antimicrobial peptide against Gram-negative bacteria is magainin 2, which was originally found in the skin of an African frog. Here, we studied the interaction of hexa-acyl bisphosphoryl lipid A 1 prepared from Erwinia carotovora LPS with magainin 2 with some minor substitutions in the amino acid pattern. By using Fourier-transform infrared spectroscopy, the gel to liquid crystalline phase transition of the acyl chains of lipid A, the conformation of their phosphate groups due to peptide binding, and the profile of the secondary structure of the peptides was investigated. The Zeta potential of lipid A aggregates in the presence of the peptides was determined by measuring the electrophoretic mobility. Small-angle X-ray scattering was performed for the elucidation of the aggregate structures in the absence and presence of the peptides, and isothermal titration calorimetry was applied for evaluating the thermodynamics of binding between peptides and lipid A. The data show that Asp-or Glusubstituted peptides improved the binding activity to lipid A correlated with characteristic changes in the physical parameters, which were stronger expressed for the Aspsubstituted peptide. The new hydrogen bond connection between Glu and Asp by carboxylic acids apparently leads to a more pronounced -structure of the peptide. The conformation change of the peptide enhanced the activity of incorporation into the lipid A aggregates, along with changes in biochemical and biophysical parameters. Royal College of Surgeons, Dublin, Ireland Cationic antimicrobial peptides (CAPs) have been reported to exhibit anticancer activity 1 . One such peptide, P18, has been shown to inhibit the growth of several cancer cell lines, with inhibiting concentration (IC50) in the range of 10 to 20 μM 2 . However the concentration at which P18 and other CAPs act is too high to be clinically relevant. The enhancement of their activity can be achieved through the modification of their amino acid composition or the addition of other molecules. Conjugation of naturally produced hydroxylated fatty acids to P18 showed a 10-fold improvement in its anticancer activity on a variety of human-derived cell lines.

In addition to the enhancement of activity we wished to understand the mechanism of action of the peptide and conjugates. We investigated the uptake of conjugated and unconjugated peptides into Hela (cervical) and MiaPaCa (pancreatic) human cancer cells and the localisation of the peptide in the cell once taken up. We investigated the effect of altering the carbon number of the hydroxylated fatty acids ranging from hydroxyhexanoic acid (R6) to hydroxydodecanoic acid (R12) conjugated to P18 peptide and tested on Hela and MiaPaCa cell lines. Circular dichroism studies were performed to investigate the effect on α-helical content due to amino acid composition alteration and hydroxyalkanoic acid conjugation. The effect of the position of the hydroxyl moiety on enhancement of activity was also investigated. In the current study P18 and its derivatives also lacked haemolytic activity with concentrations up to 66 fold higher than IC50 values needed to observe any haemolysis. When current antibiotics become less efficient, there is a promise that some antibiotics can be replaced by other nature's substances, e.g. peptides. Halictines are novel antimicrobial peptides isolated from the venom of the eusocial bee Halictus sexcinctus. We obtained four analogues of the native peptide HAL 1 from IOCB AV CR. They already characterized structural properties of these peptides and their antimicrobial activity against selected bacteria 1 . The analogues were prepared by point mutations of native peptide, which could increase antimicrobial activity and decrease undesirable hemolytic activity.

Our aim was to characterized membrane permeation activity of Halictines through the use of a basic model of biological cells -large unilamellar phospholipid vesicles LUVs. We prepared two basic types of leakage assays based on LUVs with free dyes entrapped inside and one assay with Laurdan content. We used classical Steady State Fluorescence Spectroscopy 2 and advanced fluorescence methods 3 for study of dyes escape from LUVs and we also used Laurdan Generalized Polarization technique GP 4 for better understanding peptide insertion into membrane. In this way we received complementary information and we can conclude that the most active peptides are the native HAL 1 and analogue HAL 1/6. However HAL 1/6 requires presence of negatively charged phospholipids in membrane which may explain its higher selectivity against bacteria. Furthermore, FCS results have shown that the leakage happens via pore formation. Results from GP revealed that peptide insertion in the membrane do not lead directly to formation of pores.

against a wide range of microorganisms, mainly by perturbing the permeability of bacterial membranes through the formation of pores. However, AMPs effects on membrane properties probably extend beyond poreformation. We performed a systematic spectroscopic analysis of the effects on membrane structure and dynamics of two very different AMPs: the cationic PMAP-23, which creates pores according to the "carpet" model 1 , and alamethicin, which forms "barrel-stave" channels 2 . By using fluorescence anisotropy measurements on liposomes comprising probes localized at different depths in the bilayer, we measured peptide effects on membrane fluidity and order. Laurdan spectral shifts provided indications about water penetration in the bilayer. In the case of PMAP-23, it was possible to focus specifically on the lipids surrounding the peptide by following the membrane-probe fluorescence due to FRET from the peptide Trp residues. Finally, peptide-induced perturbation of lateral mobility and domain formation were determined by several methods. All experiments were compared with liposome-leakage measurements: while for PMAP-23 all membrane-perturbing effects are correlated with the vesicle leakage process, alamethicin does not significantly influence membrane dynamics at the concentrations in which it forms pores. Surprisingly, in all cases the most significant peptide-induced effect is a reduction in membrane fluidity. We have reinvestigated 20-residue peptaibols named metanicins from an ascomycetous fungus originally described as Metarhizium anisopliae strain CBS 597.80 (CBS = Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands). However, due to unusually shaped conidia and based on RNA-sequencing of its internal transcribed spacer (ITS) region, the identification of CBS 597.80 as Metarhizium has been withdrawn and this particular strain is currently under taxonomic reinvestigation 2 . Sequencing of four isolated peptides by FAB-MS, ESI-MS and Edman degradation of partial hydrolysates revealed structural relationship to 20-residue peptaibol antibiotics paracelsins from Trichoderma reesei (=Hypocrea jecorina). Sequences determined are: Ac-U-A-U-A-U-A(U)-Q-U-V-U-G-L-U-P-V-U-U(J)-Q-Q-Fol (exchange positions in parenthesis; Ac, acetyl; U, Aib, α-aminoisobutyric acid; J, D-isovaline; Fol, L-UPMC Univ Paris 06 Laboratoire des BioMolécules; CNRS UMR 7203; ENS LBM; address: Laboratoire des BioMolécules, ENS Dpt de Chimie, 24, Rue Lhomond F-75005, Paris, France Current data suggest that the cellular uptake of cellpenetrating peptides (CPPs) occur by two processes: direct translocation across the plasma membrane and endocytosis 1 . The large diversity of CPP sequences described in the literature (derived either from fragments of proteins, structurally constrained synthetic peptides, peptide libraries 2 or dendrimers) has hampered the identification of general rules for their efficacy of internalisation. We have used a reductionist approach, restricting the CPP functional groups (amide and guanidinium) and tailoring CPP amphiphilic properties. Two families of CPPs have been designed: 1) primary amphiphilic CPPs corresponding to tetra-arginines functionalised with fatty acid chains of different lengths and 2) secondary amphiphilic CPPs containing arginine and alanine or tryptophan residues 3 . These CPPs were linked by a disulfide bridge to a peptide inhibitor of protein kinase C (PKCi). The efficiencies of internalisation of the conjugates were quantified by a method based on MALDI-TOF mass spectrometry previously developed in our group 4 . The mechanism of internalisation was studied by comparing the amounts of cell-surface bound and internalized PKCi cargo on CHO-K1 cells and glycosaminoglycan-deficient CHO cells at 4 o C and 37 o C. Conjugates were found to enter by both direct translocation and glycosaminoglycandependent endocytosis. In addition, the primary amphipathic CPPs were found to be more efficient than the secondary amphipathic ones.

Furthermore, structural or mechanistic novelty does not guarantee immunity from resistance, with strains resistant to linezolid identified prior to FDA approval. Therefore, modifying existing antibiotics to overcome resistance mechanisms presents an opportunity to rationally develop effective new drugs more rapidly than screening for new structures. Vancomycin is a glycopeptide commonly used as a front line treatment for infections caused by methicillinresistant Staphylococcus aureus (MRSA). The emergence of vancomycin-resistant Enterococci (VRE), vancomycinintermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) has prompted the development of semisynthetic glycopeptides 3 . We have generated a variety of glycopeptide derivatives that show superior antibacterial activity against MRSA and VRE compared to vancomycin and second generation lipoglycopeptides. This was undertaken by employing a combination of solid phase and solution phase chemistry to attach a membraneassociative element that selectively binds to bacterial membranes in preference to eukaryotic membranes, thus increasing the local concentration at the Lipid II D-Ala-D-Ala peptidoglycan cell wall precursor target site. Three novel antimicrobial peptides, named panurgines (PNG), were isolated from the venom of wild bee Panurgus calcaratus. One of them is dodecapeptide with sequence LNWGAILKHIIK-NH2 (PNG-1). The next two peptides are almost identical. These are cyclic peptides containing 25 amino acid residues and two intramolecular disulfide bridges LDVKKIICVACKIXPNPACKKICPK-OH (X=K PNG-K and X=R PNG-R). All peptides exhibited antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria, antifungal activity and low haemolytic activity against human erythrocytes. We prepared 11 analogues of α-helical amphipathic PNG-1 with the aim to improve its biological properties and a linear analogue of PNG-R to elucidate the importance of disulfide bridges for its activity.

In the second part of the study, we followed the effect of panurgines on the degree of membrane disruption by observing the leakage of fluorescence dye (calcein) entrapped in artificial phospholipids vesicles [1] . Specifically, we investigated membrane interactions of PNGs with the vesicles made from negatively charged 1:2 DOPC/DPPG and 1:2 DOPC/DOPG vesicles as a general model of bacteria membrane and 15:80:5 DOPC/DOPG/CL as a possible model for a membrane of Bacillus subtilis. The membrane interaction of PNGs was also investigated on uncharged DOPC vesicles as potential model membrane for erythrocytes. PNGs exhibited weak dye-leakage activity for neutral vesicles, while they effectively induced dye leakage in the presence of negatively charged vesicles.

These results indicate that PNGs have stronger potency to disrupt bacteria-mimicking anionic membranes than those which mimic eukaryotic cell membrane. Department of Biochemistry and Toxicology, University "Lucian Blaga", 550012 Sibiu, Romania A common tool to bias the conformation of linear peptides is the insertion of side-chain modified amino acids or sidechain/main-chain conformationally restricted building blocks. An alternative approach is a simple backbone modification. In this connection, backbone amide replacements with (almost) isosteric surrogates were extensively used. These modifications may impart resistance to enzymatic degradation and better bioavailability to the peptides, but also influence the secondary structure. A thioamide (ψ[CS-NH]) is perhaps the closest structural mimic of an amide. However, it possesses different and attractive features: (i) Its NH group forms stronger hydrogen bonds, being more acidic than that of the amide. (ii) Its C-N bond undergoes cis/trans isomerization by irradiation at 260 nm (π→π* transition). (iii) It may act as a "minimalist" fluorescence quencher. For all these reasons, we started a programme aimed at exploring how the endothioamide bond affects peptide folding and bioactivity. In this communication, we describe the synthesis and conformational results of the three analogs of the membrane-active peptaibiotic trichogin GA IV listed below: n-octanoyl-Aib-Gly-ψ[CS-NH]-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-OMe (2/3) n-octanoyl-Aib-Gly-Leu-Aib-Gly-ψ[CS-NH]-Gly-Leu-Aib-Gly-Ile-Leu-OMe (5/6) n-octanoyl-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-ψ[CS-NH]-Ile-Leu-OMe (9/10) The syntheses of the three peptides were accomplished in solution according to a fragment condensation approach. Appropriate thioamide-containing tri-or tetrapeptides were prepared by treating the corresponding all-amide precursors with the Lawesson reagent. FT-IR absorption, 2D-NMR and CD conformational investigations on the three analogs were conducted in comparison with the naturally occurring peptaibiotic. All three analogs maintain the capability to interact with the DOPE/DOPG model phospholipid membranes and exhibit a comparable bioactivity against S. aureus.

Peptide-peptide interaction of Lactococcin G class IIb two peptide bacteriocin H. Etayash, W.Soliman and K. Kaur* Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, T6G 2E1

Lactococcin G, a class IIb two-peptide bacteriocin, consists of two complementary peptides LcnG-α and LcnG-β that act as one functional unit with optimal antimicrobial activity achieved by the presence of both peptides in approximately equal amounts. In this study we have investigated the mechanism of pairing of the two complementary peptides as well as explored any specific interaction that could take place between the peptides. Molecular dynamics (MD) simulation was employed to study the interactions at the atomistic level. Four different MD simulations with the peptides in a lipid bilayer system were conducted. MD results from these simulations confirmed and pointed out that (i) the two putative GxxxG motif, G7xxxG11 in LcnG-α and G18xxxG22 in LcnG-β, were attracted and came closer to each other, showing the role of these motifs in attracting the two peptides to each other. Closer views, however, showed no clear interactions between these two motifs. Most likely, nonspecific interactions play a role in bringing the two peptides together; (ii) Variations and loss in the secondary structure in both the peptide fragments were confirmed among the four simulations. On the contrary, stability of helical regions was identified between residues W3-G9 and D26-Q29 in LcnG-α and V9-E14 in LcnG-β; and (iii) Role of tryptophan at the N-terminal regions in positioning and setting the peptide orientations were confirmed which matched the previous reported results. Faculty of Pharmaceutical Sciences, Unesp -Univ. Estadual Paulista, Araraquara, Sa͂ o Paulo, Brazil Antibiotic resistant bacterial strains represent a global health problem. Antimicrobial peptides (AMPs) are promising novel antibiotics because they have displayed little or no resistance effects. It is well known that the charge, amphipathicity, hydrophobicity and helicity of the peptide are fundamental for the biological activity. In addition, covalent dimerization appears as a new parameter to be studied. In this way, several bioactive sequences were dimerized obtaining pharmacotechnics advantages like enhanced antimicrobial activity, solubility and proteases resistant. However, the effect of this modification is unclear since dimeric versions of some AMPs are toxic 1 . To evaluate the effects of dimerization on the structure and biological activity of the AMP Aurein 1.2, the monomeric version (AU) and the C-and N-terminal dimers, (AU)2K and E(AU)2, respectively, were synthesized. Circular dichroism results indicated that dimeric versions showed more defined structures in aqueous solution. E(AU)2 showed "coiled coil" structure while (AU)2K an αhelix structure. In contrast, AU displayed typical spectra for disordered structures. In TFE and LPC, all the peptides acquired a high amount of α-helix structure. The antimicrobial activity against bacteria and yeast decreased with dimerization. However, dimeric peptides promoted the aggregation of C. albicans. Hemolytic and vesicle permeabilization assays showed that AU has a concentration dependence activity, an effect that can be assigned to a "carpet" like mechanism peptide, whereas this effect was less pronounced for dimeric versions, suggesting that dimerization may change the mechanism of action. In conclusion, our studies showed that the effects of AMP dimerization are complex and still unclear. 1 , the first antimicrobial peptide generated in vivo and isolated from the gut contents of the cattle tick Boophilus microplus 2 . We have shown that these peptides are equally lethal to Candida albicans MDM8 and practically not active on human erythrocytes 1 . To examine the properties and mode of action of Hb40-61a, we synthesized it and its fluorescently labeled analogue (FAM-Hb40-61a) by the solid-phase method at 60 o C, purified them by RP-HPLC and characterized their purified forms by LC-ESIMS. At low salt concentration, both peptides were found to inhibit the growth of Candida albicans ATCC 90028, Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258, but Hb40-61a was two-fold more active (MICs of 12.5; 12.5 and 50.0 μM, respectively). At those concentrations, both peptides also kill the fungi. Assays with human erythrocytes showed that, likewise Hb40-61a, FAM-Hb40-61a present activity lower than 25% at 100 μM. Apparently, Hb40-61a targets the membrane cell because confocal microscopy analysis revealed that, at the half of MIC value, FAM-Hb40-61 accumulates on the fungal cell membrane. In contrast, Fluorescence Activated Cell Sorting (FACS) analysis revealed that, at the MIC, more than 90% of the FAM-Hb40-61a penetrates into the cell. Membrane permeability assay using Hb40-61a, C. albicans ATCC 90028 and the Kit Live/Dead Funga Light confirmed progressive membrane damage associated with an increase in peptide concentrations. The use DiBAC4 (5) and FACS analysis showed that Hb40-61a alters the plasma membrane potential, leading to cell death. Supported by FAPESP, CNPq and CAPES. Lasso peptides form a growing class of 16 to 21 residue ribosomally-synthesized and post-translationally modified peptides produced by bacteria. They share a rigid and compact interlocked structure consisting of a macrolactam ring at the N-terminus and a C-terminal tail that is looped back and threaded through the ring, forming a typical [1] rotaxane 1, 2 . The macrolactam is formed by condensation of an Asp8 or Glu9 side-chain with the free amino group of a Gly1 or Cys1. The lasso fold is stabilized either by steric hindrance assumed by bulky amino acid side-chains and/or by disulfide bonds between cysteines from the tail and the ring. Given this structure, lasso peptides display a high stability against proteolytic and chemical degradation. They are biologically active on various enzymatic targets, which confer them in some cases an interesting antimicrobial activity. Given its characteristics, the lasso scaffold thus represents a promising tool for biotechnological application in the development of bioactive peptides. Until now, nine peptides had been structurally characterized as lasso peptides. Based on a genomics-based approach, we identified a novel lasso peptide from Streptomyces sviceus that we termed sviceucin. It was produced in high yield by heterologous expression in S. coelicolor and submitted to structural analysis by mass spectrometry and NMR. Sviceucin is 20residue long and stabilized by two disulphide bonds. Their connectivities were identified mainly from the typical NOEs between the beta-protons of the cysteines. The lasso structure of sviceucin was obtained by NMR-based molecular modelling. Sviceucin was shown to exhibit antibacterial activity directed against Gram positive bacteria, while Gram-negative bacteria and fungi showed resistant. The Penicillium chrysogerum antifungal protein (PAF) is a cysteine-rich, cationic protein that inhibits the growth of a variety of filamentous fungi without toxic effect on mammalian cells 1 . Although PAF is used to be produced in P. chrysogerum or a similar microorganism, preparation of analogues of the protein for structural and functional investigations requires an efficient chemical method.

The unsuccessful continuous synthesis of the 55-mer small protein prompted us to use native chemical ligation 2 . The syntheses of the fragments were performed by solid-phase method applying tBoc chemistry. Using the acid-labile tBoc protecting group, the thioester end of the N-terminal fragment remains intact during the course of the synthesis.

The first attempt was the synthesis of peptides with pmethylbenzyl groups on the side chains of all of the six cysteine residues. Under no circumstances oxidative folding provided the natural disulphide bridge pattern. The failed attempts led us to orthogonal protection of the sulphydryl groups. Different sets of protecting groups were tried and evaluated. Our experiments showed that basic treatment triggered rearrangement of the previously formed disulphide pattern. Thus, base-labile protecting groups (such as 9-fluorenylmethyl, Fm) have to be avoided in the synthesis of PAF. The alarming increase and spread of antibiotic resistance among bacterial pathogens has stimulated the development of new antibacterial agents with innovative mode of action. Antimicrobial peptides with broad spectrum activity are widely distributed in nature and play an important role in innate immunity in several species, including humans. Tigerinins are a unique family of 11-to 12-residue antimicrobial peptides found in skin secretion of the Indian frog Rana tigerina 1, 2 . Characterized by a disulfide-bridged loop composed of nine amino acids, tigerinins do not show primary structural homology to any known antimicrobial peptides from amphibians. Tigerinins could provide novel lead compounds for the design of effective antimicrobial peptides with a new mode of action. The peptide MurDp1 has been identified after the screening of phage display libraries against Pseudomonas aeruginosa cell wall biosynthesis MurD amide ligase enzyme 3 . MurDp1 is a low micromolar range inhibitor of MurD enzyme and showed good antimicrobial activity. Composed of nine amino acids, it is also characterized by a nine residues disulfide-bridged loop containing two prolines. This great similarity with tigerinins, led us to investigate if MurD enzyme could be a potential target for these peptides. In silico analyses using modelling, molecular dynamics and docking with P. aeruginosa MurD showed that MurDp1 and tigerinin-1 and -2 make similar interactions in the binding site. These results suggest that MurD may be an intracellular target for tigerinin-1 and tigerinin-2. Synthesis, MurD enzymatic inhibition assay, antibacterial activity evaluation and structure-activity relationships of MurDp1 and tigerinins analogs will be presented. 

H. Etayash, L. Norman, T. Thundat*, K. Kaur* Faculty of Pharmacy and Pharmaceutical Sciences, Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta T6G 2E1, Canada Listeria monocytogenes is a gram positive bacterium that accounts for about 28% of the deaths resulting from food borne illnesses in North America. Moreover, L. monocytogenes is considered one of the most difficult bacteria to detect in contaminated food products. While standard microbiological and biochemical assays currently used are accurate and sensitive, they are time consuming and often require specialized instruments operated by a trained user making on-site testing difficult. To this end, we propose the development of an antimicrobial peptide (AMP) or peptide fragment sensor for the on-site detection of L. monocytogenes. Leucocin A, which is a naturally occurring AMP consisting of a 37 amino acid sequence, is known to exhibit specific activity against L. monocytogenes at pico to nanomolar concentrations. For this reason, we have synthesized a shorter peptide fragment of Leucocin A consisting of 24 amino acids using solid phase peptide synthesis. The peptide was purified by reversed phase HPLC and MALDI-TOF mass spectrometry indicates the desired biological entity was achieved. By including an N-terminal cysteine group, the tailored AMP was readily immobilized at a gold interface. The resulting thickness and molecular orientation, determined by ellipsometry and grazing angle infrared spectroscopy, respectively, indicate that the helical peptides were adsorbed on the interface with a preferred orientation parallel to the surface. The bacterial specificity of the anchored Leucocin A fragment was tested against three gram positive bacteria and results reveal that the adsorbed AMP exhibits a limit of detection of approximately one bacterium/μL which is a clinically useful detection range. Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic During the last few years we have identified three novel defensins from arthropods. Two of them, lucifensin 1 and lucifensin II were purified from various tissues of Lucilia sericata and L. cuprina larvae, respectively. Larvae of these flies are routinely used in the hospitals around the world for the treatment of non-healing infected wounds in the procedure known as maggot therapy. These 40 amino acid residues and three disulfide bridges peptides differ from each other only in one amino acid residue in position 11 (Val-Ile). Linear precursor of lucifensin was prepared by Fmoc-SPPS chemistry which was then subjected to the oxidative folding yielding a peptide with a pattern of disulfide bridges identical to that of native lucifensin and other insect defensins. This was examined by the identification of the fragments resulting from the thermolysin digestion of lucifensin by means of mass spectrometry. However, this cyclization reaction proceeded via an intermediate having incorrect pairing of disulfide bridges. From the hemolymph of blood sucking tick Dermacentor marginatus (D.m.) we purified defensin containing 38 amino acids and three disulfide bridges. Its sequence determined by Edman degradation and mass spectrometry was identical to that previously determined by molecular biology methods 2 . Sequence of D.m.defensin shows no homology to insect defensins. By SPPS prepared linear precursor of D.m.-defensin was subjected to oxidative folding under the open air. The linear peptide was straightforwardly folded into cyclic one which was identical to the native peptide. In antimicrobial assay using a set of different bacteria all three studied defensins show activity preferentially against Gram-positive bacteria including Staphylococcus aureus but are inactive against Gram-negative ones. The importance of disulfide bridges on tertiary structure of defensins and their antimicrobial activity will be presented. Recently, the chemical structure and conformation of pseudodesmin A has been determined through X-Ray diffraction and NMR spectroscopic analysis 1 . In this way pseudodesmin A was identified as a new member of the viscosin group of antimicrobial peptides (AMPs). In addition, it was demonstrated that individual molecules self-assembly in apolar environment into a supramolecular pore-like structure, providing structural support for its biological activity 2, 3 .

To further explore the structure-function relationship, a viable synthesis strategy for pseudodesmin A analogues was developed, based on side-chain attachment of the first amino acid to the solid support, followed by stepwise Fmoc solid-phase synthesis of the linear peptide precursor and on-resin head-to-tail cyclization. NMR study confirmed the molecular structure and thus the development of an efficient and successful synthesis of this type of AMP's. These results and the synthesis route will be presented. Trichogin GA IV, isolated from the fungus Trichoderma longibrachiatum 1 , is the prototype of lipopeptaibols, a subclass of short-length peptaibiotics exhibiting membranemodifying properties. Its primary structure is as follows: n-Oct-Aib 1 -Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile 10 -Lol, where n-Oct is n-octanoyl, Aib is α-aminoisobutyric acid, and Lol is the 1,2-amino alcohol leucinol. This peptaibol is predominantly folded in a mixed 310-/αhelical conformation with a clear, albeit modest, amphiphilic character 2 . In this work, we synthesized by solution and solid-phase methodologies a set of trichogin GA IV analogs in which the four Gly residues, lying on the poorly hydrophilic face of the helical structure, are substituted by one (or more) strongly hydrophilic Lys residues. Moreover, we synthesized another set of analogs where one (or more) Aib residues are replaced by Leu. The conformational preferences of these analogs were assessed by X-ray diffraction, CD, and 2D-NMR techniques 3 . We tested the role played by the substitutions on the peptide bioactivity, e.g. protease resistance, cytotoxicity, and hemolysis. Cytotoxicity was tested using three in vitro cell-based assays: (i) human red-blood cells lysis; (ii) cell mortality in total human blood leukocytes and in separate subpopulations; (iii) cell mortality in three tumor-derived stable cell lines (HeLa, A541, and A431). Our data show that some of our trichogin analogs are active against tumor cells, leaving the leukocytes unaffected. 

A convenient post-screening ring opening approach for the decoding of one-bead one-compound cyclic peptide libraries A. Girard, E. Biron* Faculty of Pharmacy, Université Laval and CHUQ Research Center, Quebec, Canada Combinatorial chemistry has been widely used as an effective method for the generation and screening of synthetic peptide libraries. Amongst the different combinatorial methodologies to discover new bioactive peptide-based compounds, we were particularly interested in the one-bead one-compound (OBOC) approach 1 . This powerful approach fully exploit the great molecular diversity accessible with peptides and has been used to identify a great number of ligands and modulators for a wide variety of biological targets. However, its use with cyclic peptides is limited by difficulties in sequencing hit compounds by Edman degradation or tandem mass spectroscopy due to the lack of free N-terminal amine and complicated fragmentation patterns, respectively. This problem has been overcome by Pei and coworkers by using a bead segregation strategy in which the outer layer exposes the cyclic peptides and the inner layer the linear counterpart for sequencing 2 . More recently, Lim et al.

reported an elegant method to prepare and sequence OBOC cyclic peptoid libraries without encoding by using a ring opening approach with triazine-based cyclic derivatives 3 . Unfortunately this method is incompatible with amino acids bearing some functionalized side chains. Based on this strategy, we have developed an efficient method to prepare OBOC cyclic peptide libraries that does not require encoding by using a simultaneous ring opening/cleavage approach. The procedure is compatible with commonly used amino acids and allows rapid and efficient sequencing of selected hits after on-bead screening. The synthesis of an OBOC cyclic peptide library, ring opening methodology and sequencing by mass spectrometry will be presented. Cyclotides are a very abundant class of plant peptides that display immense sequence variability around a conserved cystine knot motif and a head-to-tail cyclized backbone conferring them with remarkable stability 1 . Their intrinsic bioactivities combined with tools of peptide engineering make cyclotides an interesting template for the design of novel agrochemicals and pharmaceuticals 2 . However, laborious isolation and purification prior de novo sequencing limits their discovery and hence their use as scaffolds for peptide-based drug development 3 . Here we extend the knowledge about their sequence diversity by analyzing the cyclotide content of a violet species native to Western Asia and the Caucasus region 4 . Using an experimental approach, which we named 'sequence fragment assembly' by MALDI-TOF/TOF-based peptidomics, we were able to characterize novel cyclotides from Viola ignobilis. Amino acid sequencing of various enzymatic digests of cyclotides allowed the accurate assembly and alignment of smaller fragments to elucidate their primary structure, even when analyzing mixtures containing multiple peptides. Using in-source decay and high energy collision induced dissociation of digested cyclotides allowed to distinguish isobaric residues Ile and Leu. Overall this work underlines the immense structural diversity and plasticity of the unique cyclotide framework. The presented approach for the sequence analysis of peptide mixtures facilitates and accelerates the discovery of novel plant cyclotides. Glycation is a nonenzymatic reaction occurring between reducing sugars and reactive amino groups of biomolecules. The reaction leads to a formation of a heterogeneous mixture of compounds which are classified as early, intermediate or advanced glycation end products (AGE). These compounds, especially advanced glycation end products, are involved in many pathological processes, mainly diabetic complications, and could be markers of certain diseases. Detection of early products of glycation (Amadori products) is a relatively easy task and can be performed by various methods including e.g. MS/MS techniques, isotopic labeling and affinity chromatography on immobilized boronic acid 1,2 . However, the diverse structures of AGEs make detection of these compounds more challenging. The aim of the study was testing a new method of AGEs identification based on isotopic 13 C labeling. A model protein (hen egg lysozyme) was modified with an equimolar mixture of [ 12 C 6 ]Glc and [ 13 C 6 ]Glc. Then the glycated protein was subjected to reduction of the disulfide bridges followed by enzymatic hydrolysis. The obtained digest was analyzed by LC-MS methods. The glycation products were identified on the basis of characteristic isotopic patterns resulting from the use of isotopically labeled glucose. This method allowed for identification of 41 early Maillard reaction products and 8 different structures of the glycation end products. Isotopic labeling technique combined with LC-MS is a new and very sensitive method for identification of the advanced glycation end products even if their structures are unknown. This method could be also used as an alternative method of detection of Amadori products. In the course of a project aimed to assess the significance of antibiotics for the producing organism(s) in the natural habitat, we screened a specimen of the fungicolous fungus Hypocrea phellinicola growing on its natural host Phellinus ferruginosus 1 . Using a peptaibiomics approach 2,3 , we detected 19-and 20-residue peptide sequences by (U)HPLC/HR-ESI-QqTOF-MS. Structures of peptaibiotics found were independently confirmed by analyzing the peptaibiome of an agar plate culture of H. phellinicola CBS 119283 (ex-type) grown under laboratory conditions. Notably, H. phellinicola could be identified as a potent producer of 18-, 19-, (culture) and 20-residue (specimen) peptaibiotics of the suzukacillin-type4. Minor components of the 19-residue peptaibols, herein named suzukacillins C, are assumed to carry a C-terminal residue tentatively assigned as tyrosinol (Tyrol). In addition, the previously isolated suzukacillin B 4 was sequenced and shown to be a microheterogeneous mixture of 11-residue peptaibols. In order to further investigate the significance of antibiotics for the producing organism(s) in the natural habitat, we screened specimens of the fungicolous fungus Hypocrea pulvinata growing on its natural hosts Piptoporus betulinus and Fomitopsis pinicola 1 . Using a peptaibiomics approach2, we detected 17-, 18-, 19-(major sequences), and 20-residue peptide sequences in the five specimens analyzed by (U)HPLC/HR-ESI-QqTOF-MS. Structures of peptaibiotics found were independently confirmed by analyzing the peptaibiome of pure agar cultures obtained by single-ascospore isolation from the specimens 1 . Major, 19-residue peptaibols were assigned as deletion sequences of the trichosporins B3 lacking the Ala/Aib residue in position 3 . Our results corroborate that: i) peptaibiotics are, indeed, biosynthesized in the natural habitat, thus, ii) their membrane-perturbing formation of ion channels may support the parasitic life style of a fungicolous fungus. Based on methodology that we have developed in our lab 2 , we identified specific and selective substrates for these serine proteases. We used a 10,000 membered PNAencoded peptide library to screen 10,000 possible peptide substrates in a single experiment. The library was incubated with the protease of interest and then hybridized on a custom designed DNA microarray. Microarray scanning and data analysis allowed the measurement of the changes in FAM/TAMRA ratios resulting from the protease activity and the determination of the protease specificity. To verify the predicted activity and specificity, FRET peptides were synthesized, incubated with the enzymes and the hydrolysis reaction was followed by monitoring fluorescence emission. Specificity constants Kcat/Km were calculated and the cleavage sites of the peptides were identified. DUBs were, moreover, found to be associated with several diseases and as such are emerging as potential therapeutic targets 2 . Several directions have been pursued in the search for lead anti-DUB compounds. However, none of these strategies have delivered inhibitors reaching advanced clinical stages due to several challenges in the discovery process, such as the absence of a highly sensitive and practically available high-throughput screening assay 3 . In this study, we report on the design and preparation of a FRET-based assay for DUBs based on the application of our recent chemical method for the synthesis of Ub bioconjugates 4 . In the assay, the ubiquitinated peptide was specifically labeled with a pair of FRET labels and used to screen a library comprising 1000 compounds against UCH-L3. Such analysis identified a novel and potent inhibitor able to inhibit this DUB in time-dependent manner with kinact = 0.065 μM and Ki = 0.8 μM. Our assay, which was also found suitable for the UCH-L1 enzyme, should assist in the ongoing efforts targeting the various components of the ubiquitin system and studying the role of DUBs in health and disease. 3 . More recent work based on RNA interference experiments on a mouse model suggested that isoform-specific inhibitors against NMT1 might be effective anti-cancer agents as a knockdown of NMT1 inhibits the tumour growth, whereas knockdown of NMT2 has no effect 4 . If residual NMT2 activity can compensate for loss of NMT function in healthy cells, potential toxicity may also be minimised.

We developed a method to identify peptide or protein substrates of NMT1 and/or NMT2. Peptides/ proteins are exposed to NMT1 and/or NMT2 and an alkyne-tagged analogue of myristoyl CoA. Subsequent azide-alkyne "click" cycloaddition allows visualisation of the myristoylated substrates in fluorescence or chemiluminescence, using a fluorescent or a biotin moiety on the capture reagent. This labelling technology was applied to peptide libraries prepared on microarrays to investigate NMT1/2 isozyme substrate specificity using recombinant NMT1 and NMT2. Peptides made of the first 8 or 15 amino acids at the Nterminus of known myristoylated proteins were functionalised with a biotin moiety at the C-terminus and immobilised on an avidin-functionalised glass plate before being screened for activity. Selective peptide substrates will be developed as isozyme-specific inhibitors and applied in cancer cell lines. Using chemical proteomics and the labelling technology, a selective NMT1 or NMT2 inhibitor could also be used to identify protein substrates of one isozyme. For this purpose computer programs are created which can generate fragments of one compared structure and to reveal homology by their scanning along the amino acid sequence of another. Our analysis was performed by comparing the primary structures of all possible protein fragments with the amino acid sequences of all presently known natural regulatory oligopeptides. The oligopeptides were extracted from the EROP-Moscow database1 which at the time of analysis contained data on the structures and functions of more than 10,000 natural oligopeptide regulators. The structure-function analysis was performed using a specialized software package. The input data were the complete amino acid sequences of the proteins used as a source of fragments with a specified length. Then the initial sequence was fragmented in a stepwise manner. For example, in the case of dipeptide fragments, this procedure produced fragments with the following numbers of amino acids from the N-terminus -1-2, 2-3, and so on until the fragment that started at the second residue from the C-terminus. The cases when the amino acid sequence of a fragment coincided with part of the primary structure of a natural oligopeptide were recorded in the Total protein chemical synthesis requires a case by case design and optimization which is governed by factors such as the solubility of the individual peptide segments, their primary sequence and in particular the presence of "difficult" amino acid residues at ligation junctions such as proline or the location of cysteines. Usually, a subset of chemical tools are selected among a vast array of methodologies to match the specificities of the target protein.

In this context, methods enabling the assembly of three peptide segments in the N-to-C and C-to-N direction play a central role and must be considered as complementary as they can be selected for building subdomains of the target protein.

To date, most of the proteins were assembled in the C-to-N direction.

Only few methods are available for the N-to-C sequential assembly of proteins, whose design is highly challenging.

We have recently reported that SEA ligation, that is the reaction of a bis(2-sulfanylethyl)amido group (called SEA) with a cysteinyl peptide, allows the formation of a native peptide bond in water and at neutral pH 1 .

In this communication we will show that Native Chemical Ligation and the unique chemical properties of SEA group 2,3 can be combined in order to design a highly efficient one-pot three segments protein assembly procedure, working in the N-to-C direction 4 Amylin is one of the most amyloidogenic peptides, its fibrils are responsible for causing type II diabetes. Amyloid formation mechanism is investigated both to find amyloid inhibitors as potential medical drugs, and to use amyloids as potential self-assembling biomaterials [1] . Amyloid formation of amylin 10-29, its reverse and designed analogue beta-sheets and beta-sheet stacks was studied by molecular dynamics (MD), Amber 9.0, f99 force field. MD revealed that for amylin 10-29 and its reverse analogue both the parallel and antiparallel beta-sheet and beta-sheet stack structures are stable suggesting that this could explain the high tendency of amylin to form amyloid fibrils. Parallel amylin 10-29 beta-sheet stacks are kept together by two hydrophobic cores, while for the antiparallel system the dominating is the backbone hydrogen bonding between neighbor strands. Also the bent form of the amylin 10-29 beta-sheet is stable. This is in concordance with transmission electron microscopy (TEM) experiments stating that all three peptides, amylin 10-29, its reverse and designed analogues, exhibited significant fibrillar polymorphism [2] University of Gdansk, Poland Molecular dynamics (MD) of two peptides DLSFMKGE (MK) and DLSFKKGE (KK) not related to any known disease was run to investigate the mechanism of the amyloid formation. The parallel and antiparallel [1] betasheets of MK and KK peptides were simulated by molecular dynamics (MD), Amber 9.0, f99 force field, NTP protocol. It was found that antiparallel beta-sheets both of MK-and KK-peptides show much higher stability than the corresponding parallel beta-sheets. This MD result was supported by ATR-FTIR spectroscopy [2] . The betasheet stacks built from six ten stranded antiparallel beta-sheets of MK-and KK-peptides: 10x6xMK and 10x6xKK, were subjected to MD. It was found that the MK-system, 10x6xMK, is strongly kept together due to hydrophobic core built from two metionines, two phenylalanines and two leucines, but the KK-system, 10x6xKK, which differs only by one mutation M5K dissolves already at 20 ns of MD run, because the separate beta-sheets don't hold togather in the betasheet stack due to lost hydrophobic core. The hydrophobic core of the MK-system consists of hydrophobic units centered on the two phenylalaninetwo metionine hydrophobic interactions, and two leucines from the both sides stabilize the unit. This mechanism could be used in amyloid based biomaterials. Urokinase plasminogen activator (uPA) is a serine protease involved in the metastasis of several tumor types. uPA is therefore an interesting target in cancer therapy. Upain-2 is a new analogue of a highly specific peptidic inhibitor (upain-1) of uPA. The peptide contains twelve amino acids and is cyclized through the cysteines at its termini (S 1 -S 12cyclo-Ac-CSWRGLENHAAC-NH2). Upain-2 inhibits uPA with a Ki of approximately 40 μM. 1 One method to improve binding affinity is multivalent exposure of the inhibitor, where the local concentration at the binding site is increased. Fusion of upain-1 to the trimeric tetranectin showed improved binding affinity compared to the single peptide. 2 Here, we report efforts towards novel chemically linked upain-2 peptides to allow multivalent display. The Ki value of an upain-2 dimer, linked by a short PEG chain through the N-termini, was almost halved compared to that of the single peptide (23 μM). This motivated us to explore the role of the site (N-or Cterminal) and the size of the linking segment on the binding affinity. Additionally, the influence of the number of upain-2 peptides in the molecule (two vs. four) was investigated by synthesizing a carboprotein that displayed four upain-2 peptides. We present two novel NMR spectroscopic approaches to study reversible self-assemblies in solution. Both methods were applied on the self-assembling pseudodesmin A, a Pseudomonas produced cyclic lipodepsipeptide that has the capacity to form pores in cellular membranes. 1, 2 The first method is based on the dependence of the 13 C α relaxation rate constants on the anisotropy of the assembly. 3 When the monomer conformation is known and the multiple CH bonds in the monomer sufficiently sample all orientations, the rotational diffusion coefficients can be assessed, revealing assembly shape information. In addition, the orientation of the monomer within the assembly is obtained. The second method is based on fitting translational diffusion coefficient data as a function of concentration in a model-free way, i.e. without assuming an oligomer shape beforehand. Here, it is assumed that the diffusion coefficient's dependence on the oligomer size behaves as a power law, which dramatically simplifies the expression for the average diffusion coefficient (measured by PFG-NMR) as a function of concentration. The fitted value of the exponent of the power law fully embeds all shape information of the assembly, and may be related to the socalled fractal dimension of the oligomer. Moreover, this approach reveals mechanistic information concerning the assembly formation.

Both methods thus allow structural information of the assembly to be obtained, even when there is little or no prior knowledge available on the mechanism of the selfassembly. Nucleotides and α-amino acids are crucial building blocks for living organisms. These chiral molecules are the biosynthetically precursors of two of the most important classes of biopolymers, DNA and proteins, respectively. The 3D-structures of biomolecules are currently studied using a variety of techniques, while helical handedness is routinely detected by means of light pulses of opposite circular polarization. The difference in the UV absorption of these two circularly polarized pulses is called electronic circular dichroism (ECD). In Nature, biomolecules explore a wide range of conformations with intrinsically strong ECD signals in the 200-300 nm region, but these signals are essentially absent in the visible. Nanomaterials such as metallic nanoparticles (depending on their sizes) display absorptions in the visible region but are achiral. As a result, when biomolecules are co-assembled with nanomaterials their chirality is transferred to create a plasmon-induced ECD signal in the visible region. In this work, we present our results which underscore the occurrence of moderately strong ECD bands in the range 300-550 nm resulting from a series of appropriately thiolfunctionalized peptide oligomers (based on alternating L-Ala and Aib residues) covalently anchored to 2-2.5 nm sized gold nanoparticles. We related the (positive or negative) signs of the ECD plasmonic signal with the oligopeptide length, that in turn is strictly associated with their secondary structure. This latter property was simultaneously monitored via ECD in the 200-250 nm range. We believe that in our systems a peptide-tometallic surface chirality transfer would take place. Light can be controlled with high temporal and spatial precision. If a specific molecule is made light-sensitive, then a precise spatiotemporal control of some of its properties can be achieved. Azobenzene is the most widely used photochromic group due to its propensity to pass reversibly from the cis to the trans state under irradiation with light of the appropriate wavelength. The cisand transazobenzene isomers exhibit different spatial arrangement of the aromatic moieties that give rise to significantly distinct physical and chemical properties. The design of novel azobenzene-based molecules with precisely placed photochromic groups able to induce photomodulation of macroscopic properties is currently attracting much interest.

In this work, we explored the behaviour of the conjugate formed by linking each of the four hydroxyl groups of pentaerythritol to the carboxylic function of bis[p-(phenylazo)benzyl]glycine. This C α -tetrasubstituted α-amino acid bears two side-chain azobenzene groups.

The resulting system exhibits tetragonal symmetry, with a total of eight azobenzene moieties, and can be viewed as a central core surrounded by a shell of azobenzene groups at the periphery. Up to eleven (out of the possible fifteen) discrete states produced by sequential trans-to-cis isomerization of the individual azobenzene units have been observed depending on the time of exposure to UV-light. This process is fully reversible (cis-to-trans) under Vis-light irradiation for several cycles. In addition, this compound has been shown to exhibit photomodulated physical properties, such as polarity and hydrodynamic volume. Moreover, it shows a high propensity to self-assemble in aqueous solution, giving rise to supramolecular vesicles. Light-scattering and electron microscopy experiments confirmed that a conformational reorganization of the vesicles can be triggered under exposure to UV or Vis light. The total chemical synthesis of native or modified proteins is gaining increase importance in the study of protein function, but also in the development of protein therapeutics. It is usually achieved by assembling in water unprotected peptide blocks using so-called native peptide ligation methods. Recently, our group has developed a novel native peptide ligation method based on a peptide featuring a bis(2sulfanylethyl)amido (SEA) 1 group on its C-terminus in reaction with a cysteinyl peptide in water at pH 7. We will discuss in this communication the scope and limitations of SEA native peptide ligation. For this, model SEA peptides featuring all the possible proteinogenic amino acids were synthesized. Their rate of SEA native peptide ligation with a model Cys peptide were determined in the absence of presence of guanidinium hydrochloride or other additives frequently used in NCL. We will present also experiments intended to clarify the mechanism of SEA ligation such as the effect of pH on the rate of ligation, or the ability of the transient thioester SEA form produced by in situ N,S-acyl shift to participate in thiol-thioester exchange 2,3 .

Overall, the data show that SEA ligation is an interesting method for native peptide ligation at various X-Cys junctions, and thus an interesting alternative to NCL. PLGA copolymers were used as the support for inducing controlled biomarkers releasing system. Visualization of the penetration in the hippocampus of mice with confocal microscope was carried out by testing both peptide-free and peptide-bearing nanoparticles, previously labeled with the phthalocyanine fluorescent probe. The encapsulation degree of the larger (12-24) segment was less effective than the others thus stressing the importance of the peptide length to this internalization process. The results showed that all peptide-containing nanoparticles were able to cross the blood-brain-barrier thus indicating improved bioavailability and uptake for peptide delivery into the brain. In regard to the radiolabeling approach, the 99m Tc radioisotope was used to label the peptide sequences at His residues, as previously described 3 . Stable metal-peptide complexes were obtained in 10 -5 -10 -6 M peptide concentration range. Noteworthy, higher metal labeling yield was achieved with peptide segments bearing His residues at peptide C-terminal position, thus pointing to a positiondependent effect for the 99m Tc coupling reaction. In conclusion, the findings indicate potentials for the proposed encapsulation and radiolabeling strategies applicable for in vitro and in vivo diagnostic assays with these peptides for the study of amyloid plaques. We have used bifunctional short peptides (Ac-CG n C-NH 2 , n=2, 4, 6) to selectively link gold nanorods in an end-to-end manner. Additionally, we have manipulated the gap distance between the rods by changing the length of the peptide linker. The presence of the peptide in the gaps was shown by incorporating a propargylglycine residue in the sequence, which was detected with surface-enhanced Raman spectroscopy (SERS). The acetylene moiety will allow further chemical modification of the linker in the gaps, opening a wealth of interesting molecular systems to be placed and studies inside self-assembled nanogaps. 1 In this work, the fragmentation pathways of alitame, neotame and andvantame in comparison to those of aspartame and aspartame-d3, were studied by negative ion electrospray ionization (ESI) high resolution mass spectrometry (Thermo Orbitrap mass analyzer). Accurate mass spectra of the dipeptides allowed proposing specific fragment ions. Neotame and advantame, which are the N-(3,3dimethylbutyl) and N-[3-(3-hydroxy-4-methoxyphenyl)propyl] derivatives of aspartame, presented similar fragmentation to that of aspartame.

For neotame and advantame, the "diketopiperazine'' pathway seemed to be the major one, while a pathway resulting to the formation of a pyrrolidine-2,5-dione derivative, through the involvement of the side chain carboxyl group of aspartate, was also observed. For alitame, the "pyrrolidine-2,5-dione" pathway was recorded. Similarities in the fragmentation using either orbitrap or triple-quadrupole mass spectrometry have been observed. Elucidation of the fragmentation is very useful for the trace-level determination of the artificial dipeptide sweeteners in complex matrices. 

Generation of Silver Nanoparticles in the Presence of Oligoproline Derivatives P. Feinäugle, H. Wennemers* ETH Zürich, Switzerland In the last years, the generation of silver nanoparticles (AgNPs) attracts, due to its unusual physical and chemical properties, more and more attention. AgNPs offer great opportunities for applications in molecular electronics, catalysis, imaging and for antimicrobial coatings. 1 The characteristics depend on their shape and size. 1 Many efforts have been made to optimise the generation process by, for example, varying the reducing agents, which usually are used for the synthesis or using manifold additives which should guide the nucleation and also stabilize the resulting particles. Nevertheless, the generation of AgNPs in defined sizes and shapes still remains a challenge. We address this goal by utilizing functionalized oligoprolines that form a conformationally well-defined and rigid helical secondary structure (PPII) 2 as additives.

Recently, we showed that by decorating this template with aldehydes which allow for in situ reduction of the silver, they act as scaffolds in the generation process and allow the formation of defined nanoparticles. 3 We will report the results of the generation of AgNPs with various oligoprolines as additives, which differ in the attached functional groups as well as in the length of the peptides. Laser desorption/ionization mass spectrometry (LDI-MS) using specific inert surfaces to promote ion formation has been widely investigated the last decade [1] . In addition to porous silicon through the original DIOS technique, different materials were tested as potent LDI-promoting agents. We explored a variety of inert silicon-based UVabsorbing materials that were presenting different physico-chemical properties for the analysis of peptides [2] [3] [4] . Both material architecture (amorphous powders, structured particles, structured surfaces) and material hydrophilic/hydrophobic character tuned by specific chemical derivatization (oxidation, silanization) were probed as crucial parameters for achieving efficient and robust detection of an home-made array of model peptides covering a wide structural and mass diversity. Through this set of experiments, we were able to compare the performances of all investigated silicon-based supports, especially taking into account peptide detection sensitivity (down to femtomolar concentrations) and reproducibility/repeatability (intra-spot/inter-spot signal variations) as well as the method robustness using conventional MALDI-TOF/TOF instrument. Having illustrated the capability to achieve both peptide detection and sequencing on these ionizing surfaces in the same run, high-throughput identification of protein tryptic digests by a rapid MS profiling and subsequent MS/MS analyses was achieved. Comparison of the MS and MS/MS data with those obtained with sample conditioned in organic matrix [5, 6] showed a great behavior for low mass responses demonstrating the capability of LDI on nanostructured silicon supports to be a complementary method to MALDI in proteomic workflow. The dipeptides aspartame, alitame, neotame and advantame are low caloric artificial sweeteners. Advantame 1 , which is the N-[3-(3-hydroxy-4-methoxyphenyl)propyl] derivative of aspartame, is the most recent among them. An application for its approval has been applied in USA, Australia and New Zealand. Such sweeteners are used in food products and beverages and they can help in managing body weight and disorders like obesity and diabetes. 2 In this work, the simultaneous determination of aspartame, alitame, neotame and advantame by negative and positive electrospray ionization (ESI), under Hydrophilic Interaction Chromatography (HILIC), is presented. Advantame, neotame and intermediates were synthesized in our laboratories for the present application. The key-step for the synthesis of advantame and neotame was the reductive amination of H-Asp(OBu t )-Phe-OMe with 3-(3-hydroxy-4methoxyphenyl)propanal and 3,3-dimethylbutanal, respectively. The chromatographic behavior of the artificial sweetener dipeptides was studied on two HILIC columns: Kinetex HILIC (a fused core silica column) and ZIC-HILIC column (a sulfoalkylbetaine column). The separation of dipeptides was achieved on Kinetex HILIC using 5 mM ammonium formate buffer pH 3.5 / methanol / acetonitrile (15/10/75), with a flow rate of 100 μL/min at 50 o C column oven temperature. At this pH, silica is neutral and the dipeptides are in positively charged form. The retention mechanism of all analytes seems to be partition to the water layer as well as hydrogen bonding. 3 Département de Pharmacologie, Université de Sherbrooke, Sherbrooke, QC, Canada Plasma and in vivo stability are essential requirements for the successful development of potential drug candidates or diagnostic imaging probes. Rapid degradation of compounds in plasma may result in insufficient concentration to produce the desired pharmacological activity or to be used as a diagnostic agent. There are several strategies to improve plasma half life of peptides including PEGylation, modification of Nand Cterminal fragments of peptide, replacement of labile amino acids, and cyclization 1 .

We have previously reported on probes which specifically detect matrix metalloproteinase-2 (MMP-2) activity with magnetic resonance and optical imaging 2,3 . MMPs are zincdependent endopeptidases degrading the extracellular matrix (ECM) and involved in cancer progression. The main goal of this work was to find more stable probes without sacrificing enzyme specificity. We have selected specific MMP-2 substrates 4 and their stability was evaluated in three different conditions: in plasma, in plasma with a MMP inhibitor and in a MMP-2 solution. The samples were analyzed by HPLC to detect the degradation pattern of our compounds and by LC-MS to determine the molecular mass of peptide fragments. Based on these studies, the most stable peptide was selected and incorporated in a solubility switchable probe with radiolabelled (68)Ga-DOTA. Its in vivo stability was estimated up to 30 minutes, making it a suitable candidate for further investigations. Cancer of thyroid gland is the most common malignancy of the endocrine system. The treatment improvement could be achieved by early diagnosis. The aim of the study was to identify cancer specific markers using the libraries of artificial receptors immobilized on the cellulose. An array of supramolecular structures formed from N-lipidated peptides attached to cellulose via aminophenylamino-1,3,5-triazine was prone to formation of monolayer of "holes" and "pockets" in dynamic equilibrium. This selforganized structures were found capable of binding small guest molecules very efficiently recognizing the shape, size, and polarity of ligands, thus resembling arti cial receptors 1 . Recognition and binding properties of guest molecules by artificial receptors depends mainly on the character of peptidic pockets and structure of the fatty acid. Proper construction of the binding pocket allows selective binding components of mixtures of compounds from a living organism 2 .

The preliminary data indicates that it is possible to construct an array of artificial receptors with diversified structures of peptidic pockets which are able to distinguish between components of homogenates from tumor and normal tissue. century. Most of opioid alkaloids and their derivatives have μ-opioid affinity, while endogenous enkephalins are rather δ-than μ-selective. Morphine is still the drug of choice for treating severe pain caused by cancer or surgical operation, but its side effects are the reason for the searching and development of new, selective MOR agonists. The aim of our study is to choose within recently published crystallographic structures templates for homology modeling of the human μ-opioid receptor. We generated several models using different templates and all of them were evaluated by docking procedure (Gold 5.1) Ligands used in this investigation were synthesized and evaluated for their biological activity in our previous studies. They are enekphalin analogues with substitutions in second position. The best model of the human mu-opioid receptor was chosen according to data obtained from docking and in vitro biological activity of analogues and endogenous enkephalins. Acknowledgments: This work was supported by NFSR of Bulgaria project DVU 01/197 and COST Action CM0801 project DO 02-135/31.07.2009. pneumoniae, H. pylori, Proteus sp. are considered as important factor contributory to development of rheumatoid arthritis (RA). The aim of this study was to investigate the level and specificity of antibodies binding to the synthetic peptides corresponding to the bacterial ureases "flap" region sequences in the rheumatoid arthritis patient's sera. For these investigations, peptides with amino acid sequences derived from "flap" regions of different ureases were synthesized using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate 1 (DMT/NMM/BF4) as coupling reagent. Peptides were immobilized on a cellulose membrane. The level of antibody binding as well as specificity of them was analyzed by quantitative dot blot method using sera 40 sera from rheumatoid arthritis patients (RAP) and sera from 38 volunteer blood donors (VBD). The results of studies suggest that "flap" region may be involved in arising antibodies participating in autoimmunological processes but not to fight infection. This effect indicates that the peptides analyzed by us could be useful for investigation of RA pathogenesis. This suggestion was confirmed by the antibodies absorption experiment which indicates that specificity of antibodies present in RAP serum is slightly lower in comparison with VBD serum. It has been found that antibodies present in RAP serum recognize not only a specific peptide but also peptides containing fragments with different amino acid sequences. It means that immune system of RAP is unstable and may produce a wide spectrum of antibodies recognizing not only a specific epitope but also a set of similar structures. Autoantigen-specific T-cells also play a crucial role in the initiation and perpetuation of Dsg3/Dsg1-specific T-cell responses. T-cells recognize epitopes from Dsg3 protein and produce different cytokines, e.g. interferon-γ (IFNγ).

Functional T-cell epitopes of Dsg3 protein have outstanding importance in immunopathological research, development and the design of novel diagnostic tools.

Our previous studies have shown that certain T-cell epitope peptides are able to stimulate the peripheral blood monomorphonuclear cells (PBMC) of PV patients more effectively than those of healthy donors. Our aim was to select a set of T-cell epitope peptides as potential synthetic antigens which are reliably able to distinguish between donors based on the in vitro T-cell stimulating activity. We have prepared synthetic Dsg3 oligopeptides by Fmoc/tBu solid phase methodology. After cleaving from the resin with TFA the peptides were purified by RP-HPLC, and they were characterized by RP-HPLC, mass spectrometry and amino acid analysis. PBMC of PV patients and healthy donors were isolated; and the cultures were stimulated by Dsg3 peptides in a concentration of 0.025 mM for 20 hours, and the rate of IFNγ production was determined from the supernatants in sandwich ELISA. Synthetic Dsg3 oligopeptides induced different in vitro IFNγ production rate on PBMC obtained from PV patients and healthy controls determined by ELISA. Our approach identified a synthetic antigen set as a promising biomarker for pemphigus vulgaris. [1] . In particular, CAP2b (pELYAFPRVamide) has been shown to elicit antidiuretic activity in the Green Stink Bug Acrosternum hilare [2] , an important pest of cotton and soybean in the southern United States. Analogs of CAP2b containing either an (E)-alkene, cisPro or a transPro isosteric component [3] were synthesized and evaluated in an in vitro stink bug diuretic assay, which involved measurement of fluid secretions of Malpighian tubules isolated from A. hilare [2] . At a concentration of 1 μM, the conformationally constrained transPro analog demonstrated significant antidiuretic activity, whereas the cisPro analog failed to elicit any activity. The results provide strong evidence for adoption of a trans orientation for the Pro in CAP2b neuropeptides during interaction with the receptor associated with the antidiuretic process in the stink bug. The work further identifies a scaffold with which to design biostable mimetic CAP2b analogs as potential leads in the development of environmentally favorable pest management agents capable of disrupting CAP2bregulated diuretic systems. The enkephalins are pentapeptides (Tyr-Gly-Gly-Phe-Met/Leu) with a proven antinociceptive action. It is believed that the interaction between them and the lipids composing the membranes is important for converting the peptides into a "bioactive" conformation 1,2 . Using Langmuir's monolayer technique the interaction of a synthetic Methionine-enkephalin (Met-enk) and its amidated derivative (Met-enk-NH2) with mixed lipid monolayers composed of palmitoleoylphosphatidylcholine (POPC), sphingomyelin and cholesterol was studied. The surface pressure-area (π-A) isotherms with regard to πmin, π max and the hysteresis curve shape of the pure lipid monolayers and after the addition of the respective enkephalins were detected. In addition, by using Brewster angle microscopy (BAM), the surface morphology of the mixed lipids-enkephalins monolayers were determined. Our results suggest that there is a strong penetration effect of the enkephalins studied into the mixed monolayers. Moreover, our results demonstrate the potential of lipid monolayers formed in Langmuir's through in combination with BAM to be successfully used as an elegant and simple membrane models to study lipid-peptide interactions at the air/water interface. Acknowledgments: This work was supported by Bulgarian Ministry of Education, Youth and Science, projects N DO02-107/08, DRG 02/5 and MY-FS-13/07. Dept Pharmacolgy, Temple Univ, Philadelpha, PA 19140, USA Bioinformatic algorithms has predicted the existence of several potential hormone-like peptides transcribed from the Ecrg4 gene 1 . Previous publications indicated a highly level of gene expression of Ecrg4 products has been found in the pancreas 1 , choroids plexus, epithelial cells, leukocytes, and macrophages 2 . However, the presence in the hypothalamus and the major form of derived peptides in each tissue haves not been clearly identified.

Knowingledge of the precise peptide generatesd within a given tissue is essential to understanding its functions. We have generated the peptide specific antibodies to against Ecrg4-derived Pprepro-augurin(71-147) and developed a specific RIA kit for the quantification of the such peptide in question. A method for the purification of endogenous Ecrg4-derived peptides from bovine hypothalamus also has been established. Using RIA to monitor the immunoreactive fractions and MALDI-TOF to identify the endogenous peptides, we foundhave detected the presence of Ecrg4 derived molecular of bovine preproaugurin(71-147) from the homogenates of bovine hypothalamus. Immunohistochemistrycal staining by antibody aalso confirmed the presence of thee peptide in some of the hypothalamic cells. of hypothalamus. The amount of prepro-augurin(71-147) in the hypothalamus although not soas high as pancreas, but is one third of the augurin level of the pituitary. Conclusions: The native peptide derived from augurin preproteinEcrg4 has been discovered.identified. We have confirmed the property of purified peptide,s prepro-augurin(71-147), along with the synthetic peptide standards. The present study provides the necessary procedures such as the elution from (1) C18 column, (2) P6 sizing column, and (3) a further purification conditions for HPLC in order to enhance the immunoreactivity from tissue fractions and yield enough amount for identification. This DSIP-related peptide (KN-DSIP or KND) differs from DSIP by only 2 amino acid residues in positions 2 and 5. We do not consider the homology between DSIP and KND as accidental, bearing in mind functional significance of histone demethylases of the JmjC-group. Methylation-demethylation of histones is known as an important mechanism of posttranslational modification playing a prominent role in epigenetic regulation of chromatin structure and gene transcription. DSIP is also known as an effective "normalizer" and protector from homeostatic disorders induced by stress related disturbances. We suggest that histone demethylase of the JmjC-group containing DSIP-related region can be considered as a possible protein precursor of endogenous peptides with DSIP-like activity.

In order to test our hypothesis we synthesized KND and studied its biological effects. In a preliminary assay cited below [1] KND showed similar and probably more pronounced effects than DSIP as an agent that stimulates endurance and stress-resistance of animals in the forced swimming test. Also KND provided a more active detoxifying action after administration of a semi-lethal dose of the cytostatic agent. In the present work we assessed neuroprotective and antioxidative potency of both peptides in vivo and confirmed the higher efficiency of KND. This study is supported by the Moscow government. is a tridecapeptide (pGlu 1 -Leu 2 -Tyr 3 -Glu 4 -Asn 5 -Lys 6 -Pro 7 -Arg 8 -Arg 9 -Pro 10 -Tyr 11 -Ile 12 -Leu 13 ) highly expressed in the central nervous system. This peptide elicits an analgesic response following peripheral or central administration. Importantly, NT exerts a more potent analgesia than morphine at an equimolar dose, without having the associated side effects of opioid drugs. 1 Structure-activity studies have identified the C-terminal fragment NT(8-13) as the biologically active minimal sequence. However, nor the full or truncated peptides cross the blood-brain barrier (BBB), thus hampering its clinical development.

The substitution of Pro 10 by an unnatural amino acid silaproline 2 (Sip) increased bioavailability and plasma stability. Structural properties conferred by the Pro 10 were also retained as determined by NMR and IR. 3 Aiming at delineating the mode of action of CL, three new CL derivatives bearing suitable labeling moieties, i.e the fluorescent molecule FITC, the streptavidin-counterpart biotinyl-group and the 99m Tc-radiometal chelating unit dimethylGly-Ser-Cys, were designed, synthesized, purified, and characterized to be applied in in vitro and in vivo evaluation studies. The structure of the CL derivatives in aqueous solutions was studied with NMR, in parallel and in comparison with the parent molecule CL, in order to examine whether the presence of the labeling moieties has induced changes to the structure of the biologically active part of CL. Cell survival assays with CL and the CL derivative bearing the FITC moiety were conducted in the PC12 cell line in order to explore their rescue effect. In parallel, the CL derivative bearing the dimethylGly-Ser-Cys moiety was successfully radiolabeled with 99m Tc and its stability was assessed over time in its synthesis reaction mixture and in plasma. This 99m Tc-radiolabeled derivative was subsequently administered to Swiss albino mice in order to determine the biodistribution of CL in the living organism and its route of excretion, a study that has not been carried out so far for any peptide of the Humanin family. Furthermore, the potential interaction of CL with β-amyloid peptide, the hallmark of AD pathogenesis, was explored with circular dischroism. The results of this multifaceted approach to the biological action of CL will be presented. Institute of Biochemistry and Biotechnology, Martin-Luter University, Halle-Wittenberg, Germany Kinins, such as the nonapeptide bradykinin, are important mediators of various physiological and pathophysiological responses including inflammatory disease, asthma, rhinitis, cell division, pain, vascular permeability, allergic reactions, pathogenesis of septic and endotoxic shock. There are two types of receptors for kinins, known as B1 and B 2 . B 2 receptors are constitutively expressed in wide variety of cells and required entire BK sequence for recognition, while B1 receptors have normally very limited expression and respond to [desArg 9 ]BK. B 1 receptors gene is turned on following either tissue damage or inflammation. Accumulated evidence indicates that most of the clinically relevant effects of BK are functions of B2 receptors this being the reason why research on their antagonists is a topic of great interest. In our previous study we described the synthesis and some pharmacological properties of four new analogues of bradykinin (BK), designed by substitution of position 7 or 8 of the known [D-Arg 0 ,Hyp 3 ,Thi 5,8 ,D-Phe 7 ]BK antagonist with L-pipecolic acid (L-Pip) (both analogues were also prepared in N-acylated form with 1-adamantaneacetic acid (Aaa)). Our results showed that presence of L-Pip in position 7 slightly increased antagonistic potency in the blood pressure test, but it turned the analogue into an agonist in the rat uterus test. Replacement of Thi by L-Pip in position 8 also enhanced antagonism in the rat pressure test but preserved the antagonism in the rat uterus test. In the present study we continue our previous investigations to find structural requirements which in the case of BK analogues result in high B2 antagonistic activity. Several new bradykinin analogues modified in their Cterminus with D-pipecolic acid were synthesized using SPPS method. The biological properties of the analogues were assessed by their ability to inhibit vasodepressor response of exogenous BK in conscious rats and by their ability to inhibit the contractions of isolated rat uterus evoked by BK. Acknowledgements This work was supported by the University of Gdansk (DS/8453-4-0169-2).

Peptides with beta-turn structure in peptide/MHC complexes A. Stavrakoudis Department of Economics, University of Ioannina, Greece Major histocombatibility complex (MHC) molecules interact with small peptides and form complexes. In most of the cases, peptide's structure in these complexes is found in extended conformation. However, notable exceptions exist where the peptide forms a beta-turn structure. This happens mainly in the central part of the peptide in class I complexes [1] , or at the C-terminal of class II complexes [2] . Several peptide/MHC complexes were, derived with xray studies, were extensively subjected to molecular dynamics simulations [3] in order to investigate the stability of this turn-like structural feature and to explore the factors that possibly contribute to this stability. It was found that both intra-peptide and peptide/MHC interactions might be responsible for peptide's conformation. The peptides were found to undergo several structural transitions indicating conformational plasticity and not a completely rigid structure inside the MHC groove. The results might be of special importance in designing defective peptide vaccines and beta-turn pharmaceuticals. The heptapeptide Met-enkephalin-Arg6-Phe7 (MERF) with the sequence of YGGFMRF is a potent endogenous opioid located at the C-terminus of proenkephalin-A (PENK), the common polypeptide precursor of Met-and Leuenkephalin. Our systematic bioinformatic survey revealed considerable sequence polymorphism at the heptapeptide region of different PENK prepropeptides among 56 vertebrate animals. Four orthologous heptapeptides with single or double amino acid replacements were identi ed among 15 animals, such as YGGFMGY (zebra sh), YGGFMRY (newt), YGGFMKF (hedgehog tenrek) and YGGFMRI (mudpuppy). Each novel hepta-peptide, together with the mammalian consensus MERF and Metenkephalin, were chemically synthesized and subjected to functionality studies, using radioligand binding competition and G-protein activation assays in rat brain membranes [1] . Equilibrium binding af nities changed from good to modest as measured by receptor type selective [ 3 H]opioid radioligands. The relative af nities of the heptapeptides reveal slight mu-receptor (MOP) preference over the delta-receptors (DOP). [ 35 S]GTPγS assay, which measures the agonist-mediated G-protein activation, has demonstrated that all the novel heptapeptides were also potent in stimulating the regulatory G-proteins. All peptides were effective in promoting the agonist induced internalization of the green uorescence protein-tagged human mu-opioid receptor (hMOP-EGFP) stably expressed in HEK293 cells. Thus, the C-terminally processed PENK heptapeptide orthologs exhibited satisfactory bioactivities, moreover they represent further members of the so-called "natural combinatorial neuropeptide library" emerged by evolution. Corticotropin releasing factor (CRF) exerts most of its physiological and pathophysiological actions by interacting with its type 1 receptor (CRF1) and activating different intracellular signalling pathways. The CRF1 is a plasmamembrane protein, which belongs to the family B of G-protein coupled receptors (GPCRs) and like the other GPCRs consists of an amino-terminal extracellular region, a carboxyl-terminal intracellular tail and seven, mostly hydrophobic, membrane-spanning segments (TM1-TM7), connected by alternating intracellular (IL) and extracellular loops (EL). Binding of CRF and its related peptides, such as sauvagine, to the extracellular regions of CRF1 is associated with receptor activation and subsequent activation of different G-proteins and regulation of diverse signalling pathways. Using a mutagenesis approach in combination with a radioligand binding study we found that Trp259 and Phe260 in the second extracellular loop of CRF1 interacted with the amino-terminal portion of CRF and sauvagine. Interestingly only the interaction of sauvagine with Trp259 and Phe260 is important for CRF1-mediated stimulation of cAMP accumulation. In marked contrast the interaction between CRF and the residues Trp259 and Phe260 was unimportant for the activation of adenylate cyclase. Thus it is possible for Trp259 and Phe260 of CRF1 to regulate distinct signalling pathways, or different sets of them, after their interaction with different peptides. We are now performing experiments to fully elucidate the signalling pathways that are regulated by the interaction of CRF and sauvagine with Trp259 and Phe260. These studies will advance the development of CRF1-selective selective signalling-specific peptides that would be extremely useful for the elucidation of the role of CRF1 in many physiological and pathophysiological situations, and possibly for the treatment of several CRF1-related diseases. Thymus humoral factor gamma-2 (THF-γ2), an octapeptide, purified from crude THF, retains essentially all the biological properties of THF [1] [2] . It regulates clonal expansion, differentiation and maturation of T-cell precursors, stimulates the production of lymphokine, maitains the normalization of impaired ratios between helper(CD4+) and suppressor / cytotoxic (CD8+) subsets and augments IL-2 production in spleen cells. THF-γ2 has a calculated molecular weight of 918 and has the following amino acid sequence: Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu. Its poor stability towards protein enzyme limits its extensive application. With the inte ntion to promote its bioavailability, bioactivity and develop ideal immunoregulatory drug candidat, four series of derivatives of THF were designed and synthesized: 1. N-and Cterminal acylation. 2.Restitution the flexible segment Gly-Pro by unnatural amino acids 6-aminohexanoic acid (Aca) in order to shorten the synthetic steps and simultaneity improve the bioavailability and biostability of peptide; 3. Reserve protected group of some amino acid residus as spot mutation. 4. Mannich-based cyclization was carried out on resin [3] , Phe was replaced by Tyr serving as the active hydrogen component, a proline was introduced at the N terminal as the amine component and formaldehyde was used as the only component in solution.

The bioactivity of synthesized products were detected. The leukocytopenia model in mice was induced by cyclophosphamide intraperitioneal injection. White blood cell count, thymus index and spleen index were detected to evaluate the immune function of compounds in mice.

The results show that those compounds play a significant role in improving immune function in mice. The activity of compound LHL21 and LHL22 are also better than authentic compound TP-5 and Tα1. Marine organisms have been recognized as a promising source for the development of new pharmaceuticals. In the course of screening for antitumor substances from marine organisms, we found cyclic peptides containing many nonribosomal amino acids such as hydroxyasparagine, hydroxyleucine, or other supporting a hydrophobic side chain that were shown to be a key element for their biological activity. The laxaphycine B, a cyclic lipopeptide isolated from marine cyanobacteria Anabaena torulosa harvested in French Polynesia 1 constitutes an example of this peptide class. This compound has attracted our attention because of its micromolar cytotoxic activities on different cancer cell lines as well as its antiangiogenic properties which seems to be due to an interaction with the VEGF receptor-1 2-3 . The synthesis of the non-natural amino acids 4-5 and of laxaphycine B analogues will be presented along with their preliminary biological activities. Immune response suppressors are used in the medical praxis to prevent graft rejection after organ transplantation and in the therapy of some autoimmune diseases including dermatology. Cyclolinopeptide A (CLA) c(Pro 1 -Pro 2 -Phe 3 -Phe 4 -Leu 5 -Ile 6 -Ile 7 -Leu 8 -Val 9 -), a cyclic, hydrophobic nonapeptide isolated from linseed, possesses strong immunosuppressive and antimalarial activity. 1 It has been suggested that both the Pro-Pro cis-amide bond 2 and an 'edge-to-face' interaction between the two aromatic rings of adjacent Phe residues 3 in tetrapeptide unit are important for biological activity. This edge-to-face interaction can be influenced when phenyl rings are replaced by naphtyl substituent.

In this communicate new analogues of CLA modified by 2naphtylalanine (2-Nal) in positions 3 or 4 or both 3 and 4 (1-3 linear analogues, 4-6 cyclic analogues) will be presented.

The synthetic strategy and biological activity as well as conformational analysis will be evaluated. The onset of type II diabetes mellitus (T2DM) coincides with the deposition of fibrillar material in the islet of Langerhans in the pancreas that is a clinical hallmark of more than 95% of patients suffering this disease. 1 The main component of the pancreatic amyloid deposits is a 37-residues polypeptide hormone called Islet Amyloid Polypeptide (IAPP) or amylin. 2 In this work we have examined, by means of CD spectroscopy and ThT-fluorescence, the conformational polymorphism of both full-length 1-37 hIAPP, and the related fragment hIAPP17-29, and compared the results with the respective rat counterparts. Moreover, the cytotoxic activity was determined toward different pancreatic β-cells lines in the attempt to correlate IAPP's fibrillogenic properties with the ability to mediate cell death. Together the results suggest that β-sheet conformational transition, that generally preludes to fibril formation, is not a prerequisite for eliciting toxicity toward β-cells cultures. Interestingly, confocal microscopy indicated that both hIAPP1-37 and hIAPP17-29 can enter the cell and might exert their toxic action at intracellular level. Acknowledgments: This work was supported by MIUR, FIRB-MERIT project RBNE08HWLZ. Due to its physiological functions, 26S proteasome is considered the target molecule in overcoming several diseases [1] . Its core particle 20S has three types of active sites: chymotrypsin-, trypsin-and caspase-like. Many natural and synthetic compounds were tested for their ability to inhibit proteasome. A recent report describing the inhibition of 20S by the serine proteases inhibitor -bovine pancreatic trypsin inhibitor -was considered by us with great attention [2] . Our scientific interest is focused on peptide inhibitors and their interaction with serine proteases. Sunflower trypsin inhibitor (SFTI-1) is the smallest and the most potent peptide inhibitor in the Bowman-Birk family. Owing to its size and the rigid structure (disulfide bridge and "head to tail" cyclisation) SFTI-1 is willingly chosen as the lead structure in the search for new inhibitors [3] . Its sequence is shown below (Lys 5the P1 residue responsible for specificity): & 1 Gly-Arg-Cys(& 2 )-Thr-Lys 5 -Ser-Ile 7 -Pro 8 -Pro-Ile-Cys(& 2 )-Phe-Pro-Asp& 1 Since native SFTI-1 is not able to inhibit 20S [2] , we have designed its monocyclic analogues (with disulfide bridge only) with Lys or Arg in position 5 (P 1) and at least one basic amino acid (Lys or Arg) in positions 7 (P 2') and/or 8 (P3'). All analogues inhibit chymotrypsin -(IC50 at the range of 1÷3 μM) and caspase-like (IC 50 at the range of 0.7÷7μM) activities in vitro, whereas their activity towards trypsin-like specificity is much weaker. in several rat tissues, our view on RAS has changed. Metabolism of the Ang-(1-12) may represent alternative pathway of Ang II formation, importantly, independent on renin and ACE activity 1,2 . Ahmad 2 et al. have described metabolism of Ang-(1-12) by human atrial tissues and showed that Ang II is formed mainly by chymase. This renin-inependent Ang II production could explain the "resistance" regarding use of ACE inhibitors in patients with hypertension or diabetic nephropathy. Noteworthy, the role of Ang-(1-12) in circulation is still unclear and there are no information about possible pharmacological modulation of its metabolism. In our study, we compared the ex vivo metabolism of angiotensinogen (fragment 1-14) in hypertensive (SHR) and normotensive (WKY) rats in organ bath of aorta and heart using LC-MS method 3 . Surprisingly, we identified Ang-(1-12) formed via reninindependent pathway to be a main product of angiotensinogen metabolism in rat aortic tissue and heart. In this setting, Ang-(1-12) appeared to be not only prevalent metabolite of angiotensinogen, but also served as a substrate for generation of Ang I and Ang II. As compared to WKY rats, formation of Ang II, from Ang-(1-14), was much higher in SHR aortas but not in the heart. The functional consequences of these findings require further investigation. This study was supported by the grant N N401 293939 Polish Ministry of Science and Higher Education. The lysosomal cysteine protease cathepsin C (Cat C), also known as dipeptidyl peptidase I (DPPI), activates a number of granule-associated serine proteases with proinflammatory and immune functions by removal of their inhibitory N-terminal dipeptides. Activity of this protease is associated with several pathologies in human body [1] . In this work the characterization of Cat C specificity using combinatorial chemistry methods will be described. The main goal of this work was to determine of substrate specificity of the prime region of this enzyme. The chemical synthesis and deconvolution of two libraries will be described. The hemostatic mechanism has the crucial role to prevent loss of blood from injured blood-vessels. This loss is prevented by the integrity of the vessel walls, by platelets aggregation or by blood coagulation, which in normal conditions is limited onto the local trauma of the vessel wall. In generally, the blood coagulation mechanism is important for maintaining vascular integrity and thus for the precaution of an organism from bleeding, which may also occur by blood coagulation caused by thrombin production. The diversion rate of this production leads to an expansion of thrombin to the general blood circulation. Thus, when thrombin generation is not controlled by the mechanisms of inhibition, a widespread undesirable intravascular thrombosis is occurred. The whole process of platelets adhesion requires the presence of clotting factor VIII (FVIII), a necessary for the blood coagulation cascade glycoprotein, which takes part in the intrinsic pathway and acts as a coenzyme for the activation of factor IX, a serine protease depended on the thrombin production.

The target of the present research is the synthesis of biologically active cyclic, head to tail, peptides, analogs of the sequence 558-565 of A2 subunit of FVIII, which are potentially capable to block FVIIIa-FIXa complex, reducing the thrombin production and thus the blood coagulation. The synthesized peptides are investigated for their inhibitory activity and tested for clotting deficiency by measuring the chronic delay in the activated partial thromboplastin time (APTT) and the reduction of the % value of the FVIIIa, which they generate in samples containing recombinant FVIIIa, in vitro. The blood coagulation is part of an important host defence mechanism, which under pathological conditions results in inappropriate intravascular coagulation when thrombin is produced. Clotting sequence is the result of a cascade of two biochemical pathways, intrinsic pathway, so called because all components are present in blood, and extrinsic pathway, in which tissue factor is required in addition to circulating components. The activated form of factor VIII (FVIIIa) is a key component of the fluid phase of the blood coagulation and plays an important role formatting a trimolecular complex with factor IXa, Ca 2+ and negatively charged phospholipids of the cells membrane. This complex is called tenase and participates in activation of prothrombin, which acts on fibrinogen to generate fibrin monomer, polymerized rapidly to form fibrin clot. The FVIII is comprised of a heavy (A1-A2-B) and a light (A3-C1-C2) peptide chain, both cleaved by proteases at three sites, resulting in alteration of its covalent structure and conformation. Its deficiency is known as haemophilia A.

Our research effort is focused on the synthesis, identification and biological evaluation of peptide analogs, expected to inhibit selectively the increasing of thrombin production. Their sequence is based on the regions in which the FVIII interacts with FIX, specifically on the sequence 558-565 of the A2 subunit. The synthesized peptides are examined for their activity and tested for clotting deficiency by measuring the chronic delay in the activated partial thromboplastin time (APTT) and the reduction of the % value of the FVIIIa, which they generate in samples containing recombinant FVIIIa, in vitro. inhibitor with the following structure: Ac-LLLLRVKR-NH2, which has potent effects on the proliferation of prostate cancer cells. The potency and stability of this compound was subsequently enhanced by substitution of Arg residue in position P1 with its conformationally restricted mimetic -4-amidinobenzylamine (AMBA). Nevertheless, the specificity toward PACE4 was significantly reduced by this modification. Thus, in order to improve its selectivity without sacrificing inhibitory potency we decided to use positional scanning approach. In this study we present synthesis of two series of peptide libraries, which were designed by substitution of Leu in the P5, P6 position of our control peptide (Ac-LLLLRVKR-AMBA) with each of nineteen amino acid residues in order to verifying its influence on activity and selectivity of the resulting analogues. All peptides were synthesized by a combination of solid phase peptide synthesis and solution synthesis and tested for their inhibitory potency against furin and PACE4. The P2-P8 fragments were synthesized by Fmoc/tBu SPPS strategy on hydrazinobenzoyl or acid labile 2chlorotrityl chloride resin. Then coupling of the 4-amidinobenzylamine · 2 HCl was performed. The best modifications were combined to give as several multipoint substituted inhibitors. We believed that our work, will provide new important information about structure-activity relationship of these class of analogs in order to obtain potent and highly specific PACE4 inhibitor. Institute for Research in Biomedicine, Parc Cientific de Barcelona, Barcelona -Spain A bacterial toxin-antitoxin (TA) system is composed of two genes organized in an operon encoding a toxin and an antitoxin that regulate the growth and death bacterial cell under various stress conditions. The operon parDE encode a TA system formed by ParE toxin and its antitoxin ParD. ParE is a 12 kDa protein that inhibits DNA gyrase activity and thereby blocks DNA replication. However the ParE-gyrase interactions and the gyrase activity inhibition mechanism have not been explored. As an approach for understanding of this mechanism and to elucidate the ParE region responsible for protein-protein interactions we have designed and synthesized a series of linear analogues of ParE and investigated the ability of peptides to inhibit DNA topoisomerases activity. So, based on structural data inferred from ParE three-dimensional model 1 , 12 peptides were synthesized by solid-phase method. Four peptides (ParELC3, ParELC8, ParELC10 and ParELC12), showed complete inhibition of DNA gyrase supercoiling activity, by gel electrophoresis assay 2 , with an IC100 of 20 to 50 μmol.L -1 . In addition, intrinsic fluorescence and fluorescence anisotropy assays showed that inhibition process must occur by interaction with the GyrA subunit. Differently of wild type ParE, the peptide analogues were able to inhibit the DNA relaxation of topoisomerase IV with lower IC100 values. Interesting was that only ParELC12 displayed inhibition of the relaxation activity of human topoisomerase II. Our results suggest a new class of molecules with simultaneous inhibitory activity in DNA gyrase and topoisomerase IV. Furthermore, we have obtained the first example of a synthetic peptide from a bacterial toxin with inhibitory activity on human topoisomerase II. Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovakia The renin-angiotensin system (RAS) has long been recognized as an important regulator of systemic blood pressure and electrolyte homeostasis. Our understanding of RAS has experienced remarkable change over the past two decades. Besides, angiotensin II, the new biologically active peptides [e.g. Ang-(1-7), Ang-(1-12), Ang IV, Ang-(2-10)] and pathways [e.g. angiotensin converting enzyme 2 -ACE2] have been described 1 ; some of them, like Ang-(1-7) may oppose many actions of Ang II. Importantly, despite all components of classical RAS are found in adipose tissue 2 , the data about fat formation of various angiotensins remain scarce. In our study, we compared the ex vivo metabolism of angiotensinogen, Ang-(1-12) and Ang I in hypertensive (SHR) and normotensive (WKY) rats in organ bath of retroperitoneal and periaortic fat tissue using LC-ESI-MS method. Additionally, qPCR measurements of mRNA expression of main enzymes involved in Ang I metabolism were performed. Both in the periaortic and epidydymal fat, the formation of Ang-(1-7) was higher than production of Ang II. Fat tissue formation of two main Ang I conversion products, Ang II and Ang-(1-7), differed significantly between SHR and WKY rats. Compared to WKY rats, the formation of Ang-(1-7) in periaortic fat tissue was decreased in SHR. In opposite, in epidydymal fat tissue formation of Ang-(1-7) and Ang II was higher in SHR. Interestingly, there were no differences in aorta formation of Ang II and Ang-(1-7) between SHR and WKY rats. Our results suggest that in hypertension visceral fat production of angiotensin peptides is increased, while generation of "beneficial" Ang-(1-7) in periaortic fat is decreased. However, the functional importance of such finding require further investigation. Department of Chemistry and Biochemistry, University of Washington, USA Phospholipases A2 (PLA2) are a superfamily of enzymes involved in various inflammatory diseases. 1 In particular, human secreted GIIA sPLA2 is an attractive target for the development of novel medicines. 1 We have shown that 2oxoamides based on γ-or δ-amino acids are potent inhibitors of cytosolic GIVA PLA2. Very recently, we have demonstrated that a long chain 2-oxoamide based on (S)leucine displays inhibition of human and mouse GIIA sPLA2s (IC50 300 nM and 180 nM, respectively). 2 A combined experimental/computational study was undertaken to further understand the role of the α-amino acid of 2-oxoamides for the inhibitor-enzyme binding. The crystal structure of GIIA sPLA2s reveals a highly conserved Ca 2+ -binding loop and a catalytic dyad consisting of His47/Asp91. 2-Oxoamides based on hydrophobic αamino acids showed better binding score prediction compared to polar α-amino acid derivatives. A number of new 2-oxoamides based on α-amino acids were synthesised and tested for their inhibitory activity against GIIA, GV and GX PLA2. The 2-oxoamide based on (S)-valine displayed potent inhibition of GIIA sPLA2 (IC 50 218 nM) in accordance with the predicted docking score. Docking results reveal that (S)-valine-based inhibitor forms key interactions with the active site of the enzyme. The carboxylic group participates in a hydrogen bonding with Gly31 and Lys62, and 2-carbonyl group with Gly29. Furthermore, both carbonyl groups are in the proximity with Ca 2+ . The side chain of (S)-valine adopts a suitable orientation to interact with Tyr51 and Lys62. The long aliphatic 2-oxoacyl chain is accommodated in the hydrophobic region of the active site and creates proximal contacts with Leu2, Ile9, His6 and Phe5. The search for novel classes of pharmaceutical molecules with enhanced therapeutic power has been the subject of numerous research groups all over the world. Moreover, systems of immobilization and controlled release which are adapted to these new classes of molecules, has proven to be an area of extreme importance to provide the same therapeutic efficacy. Using solid-phase chemistry a series of CcdB toxin analogous peptides were synthesized and were synthesized and tested against the capacity of inhibition of bacterial enzymes DNA gyrase and topoisomerase IV (topo IV). Subsequently those peptides were detained in drug delivery systems (DDS) to be tested against the inhibition of growth of different bacterial species. In this data we could observed that the analogue CcdbSG2 could inhibit only DNA gyrase and not the topoisomerase IV. In the other hand the analogue CcdBSG1 presents a hard inhibition potential against topo IV specially because of their structural difference. Is possible conclude that topoisomerase IV presents the tertiary structure very similar to DNA gyrase, but those mechanisms of action must be clearly distinct 1 . In the in vitro studies, as expected, results revealed that the drug delivery systems are the key to the power efficiency of peptide analogues against the bacterial growth inhibition which cannot be observed when the peptides are free in solution. Some of the different lipid compositions of the DDS are demonstrating to be more efficient in the membrane cell transverse and this data previously assumes that it is possible to apply different types of DDS to promote the peptide molecules transport across the cellular membranes according to several specific therapies. With this studies we have obtained more knowledge about the interaction system of enzyme-toxin and hopes which helps in future studies to development a new antimicrobial molecules class. It is urgent to develop less toxic and more efficient treatments for Leishmaniases and Trypanosomiases. We propose to target an ancestral form of the proteasome, the HslVU protease, which is present in the parasite's single mitochondrion, essential for the growth of these organisms and has no analogue in the human host. Originally discovered in eubacteria, this complex is constituted by two central hexameric HslV protease rings sandwiched between two hexameric HslU ATP-ase rings. As HslV shares a similar enzymatic mechanism with the host proteasome, we propose to inhibit the assembly of the complex in order to be selective. According to studies on bacterial HslVU, 1,2 the C-terminal segment of HslU is essential in HslV activation and in complex assembly, therefore representing a privileged target. We produced recombinant HslV, which is inactive alone, and showed that a synthetic C-terminal HslU peptide was able to induce the digestion by HslV of a fluorogenic substrate that we developed. With this enzymatic test in hands, we started the characterization of the interaction of the C-terminal portion of HslU with HslV. We will present the results obtained with various series of analogues of the original C-terminal HslU peptide, including truncated forms, Ala scan, constrained analogues and multivalent constructions. Helped by molecular modelling studies, the aim is to establish structural requirements, which could lead to high affinity and stable ligands able to inhibit the interaction between the HslU and HslV rings, obligatory for the degradation of proteins by the HslVU complex. Finally, we checked that HslV was inhibited by classical active-site directed proteasome inhibitors like bortezomib. 

F. Babos a,d , E. Szarka b , Gy. Nagy c , Z. Majer d , G. Saŕmay b , A. Magyar a , F. Hudecz a,d citrullinated filaggrin peptide (CCP) were detected in RA sera and anti-CCP positivity is widely used for diagnostic purposes. Identification of new epitopes of filaggrin 1 would be useful in the diagnosis of anti-CCP3 seronegative patients. In order to achieve optimal immune recognition of biotinylated epitope peptides it is important to analyse the effect of the labelling moiety on antibody binding. For these studies 5-as well as 19-mer peptides with Nor C-terminal biotin were synthesised manually by SPPS, using Fmoc/ t Bu strategy. Biotinylation was performed by using biotin, biotinyl-6-aminohexanoic acid or 4,7,10-trioxa-1,13-tridecanediamino succinic acid linker 2 modified biotin. Labelled peptides were used in an indirect ELISA, on NeutrAvidin pre-coated plates and the binding was detected by anti IgG-HRP. To examine the role of the presence/position of biotin in the secondary structure of the peptides, electronic circular dichroism (CD) method was used. We found that the CCP3+ serum samples specifically recognized the C-terminally biotinylated 5-mer filaggrin peptides, while showed no binding with the N-terminally biotinylated compounds. In case of the 19-mer epitope peptides there was no difference between the recognition of Nand C-terminal biotinylated analogues. Data presented suggest that the position of the biotin in case of the short filaggrin epitope peptides markedly influence the serum antibody binding. Upon activation process, they are released from the granules and then involved in immunoresponse of the organism. When out of the cell those enzymes remain in free form or become associate with the cell membrane.

The physiological role of this proteases is manifestated in several processes such cytokine and chemokine processing, platelet activation, and degradation of extracellular matrix's proteins [1] .

In this work results of the specificity of two members of NSPs PR3 and HNE evaluated using the combinatorial chemistry methods will be presented . Both enzymes share primary specificity and to obtain the selective substrate that will be recognized only by one enzyme, the prime sites should be investigated. The general formula of the designed library is as follows:

where in positions X1', X2' and X3', the set of 19 proteinogenic amino acids (except Cys) was introduced. ABZ is 2-amino benzoic acid served as donor of fluorescence and 3-nitro-L-tyrosine as acceptor. Eukaryotic proteasome is a highly organized protease complex comprising a catalytic 20S core particle (CP) and two 19S regulatory particles (RP), which together form the 26S structure. The main function of this large intracellular protease is to degraded ubiquitine labeled proteins. The catalytic particle of the proteasome displays three distinct enzymatic activities: trypsin-like, chymotrypsin-like and glutamyl-like. The increase activity of the proteasome is associated with several disease including cancer [1] . The main aim of this work is to synthesized the cell permeable FRET displaying peptides that will selective cleaved by single proteasome activity. Additionally each peptide when independently cleaved by the proteasome subunit, should emit the fluorescence energy in a different spectral region. Our intention was designing substrates which would allow to monitor simultaneously (in a single experiment) and independently of three proteasome activities In this report, we will describe the chemical synthesis of several peptides modified at on Cand N-termini by synthetic fluorescent amino acids The general formula of these peptides is as follows:

where X is a non proteinogenic amino acid that serve as a donor of fluorescence, Y amino acid that is a acceptor of fluorescence.

The obtained fluorescent peptides were examined for their ability to cross the cell membrane. Also kinetic parameters (k cat, KM, kcat/KM) with proteasome will be presented. Approximately 100 DUBs are encoded in the human genome and are involved in a variety of regulatory processes, such as cell-cycle progression, tissue development, and differentiation. Recently, several groups have introduced various methods for linking ubiquitin to different substrates via nonhydrolyzable isopeptide bonds, which resist the action of DUBs. Using these methods, one could explore the function and the mechanism of DUBs and apply them in activity based profiling. Here we present a new and convenient strategy for preparing nonhydrolyzable ubiquitinated peptides and proteins by Nmethylating the isopeptide bond. Using this method we prepared several nonhydrolyzable ubiquitinated peptides with different lengths derived from ubiquitinated H2B and examined their affinity to different DUBs.

F.I. Nollmann, C. Dauth, D. Reimer, H.B. Bode* Goethe Universität, Frankfurt, Germany Bacteria of the genus Xenorhabdus and Photorhabdus are gram negative gamma proteobacteria that live in symbiosis with nematodes of the genus Steinernema. Undergoing their partly entomopathogenic life cycle these bacteria not only produce antibiotics 1,2 and insecticides 3 but also several different small molecular compounds and peptides. 4 For the most part the biological benefits of these secondary metabolites have not fully been understood yet. 5 With the help of inverse feeding experiments, HR-MS and NMR as well as molecular engineering we were able to characterize and/or isolate some of these peptides. Since they are mostly produced in trace amounts, we synthesized them in order to make them accessible to continuative testing. Given that not only linear but also highly methylated or cyclic peptides are produced, the synthesis was quite challenging. Nevertheless, we were able to establish in our laboratory a general synthesis route for cyclic peptides 6 and depsipeptides 7 , as well as highly methylated hydrophobic linear sequences. Testing several of these peptides has revealed activity against insect cells and against the causative organisms of neglected tropical diseases. Cyclotides are a large class of plant peptides defined by a head-to-tail cyclized backbone and three conserved disulfide bonds in a knotted arrangement. These unique structural features confer them with remarkable stability and due to a range of bioactivities they are extensively investigated as templates in drug discovery 1 .

Based on the use of Oldenlandia affinis in traditional African medicine for its uterotonic principle we investigated crude plant extracts and semi-pure cyclotide fractions for the ability to induce uterine contractions using a collagen-gel contractility model2. Pharmacological analysis of the effects led to the identification of the oxytocin receptor, a representative of the G-protein coupled receptor (GPCR) family, as a molecular target for cyclotides. Mass spectrometry-based sequence analysis of 'active' fractions revealed cyclotides with high similarity to the human oxytocin (h-OT) peptide that exhibited weak binding to the human oxytocin receptor. We further analyzed synthetic cyclotide-derived small OT-like peptides and grafted the h-OT sequence into the stable cyclotide frame. These peptides showed increased binding and activation as compared to native cyclotides.

These findings may open new avenues for the discovery of GPCR ligands from natural peptide sources. GPCRs are promising drug targets and ~5 0% of currently used drugs act via binding to these receptors. Natural combinatorial peptide libraries are likely to play an important role in identifying novel GPCR ligands 3 . Particularly plant cyclotides cover a large chemical space based on their high sequence diversity. Together with their range of bioactivities and unique stable structure suggests that cyclotides are of current and future interest for drug discovery and development. Acknowledgements: This work is funded by the Austrian Science Fund FWF (P22889). Drosha and Dicer are two key endonucleases for biogenesis of microRNAs (miRNAs) that regulate target mRNA. Drosha converts pri-miRNA to ~7 0 nucleotide (nt) pre-miRNA in nucleus and Dicer converts pre-miRNA to linear ~2 2 nt single-stranded miRNAs in cytosol. Even though Dicer is potentially important to control availability of mature trans-acting RNAs in cytosol, the enzyme itself does not seem to be the suitable target controlling miRNA processing due to the lack of its substrate specificity. Nature, however, might be intelligent enough to differentiate a variety of pre-miRNA, so that a certain specific pre-miRNA is converted to mature miRNA in case it needs. Therefore, other component(s) in the enzyme complex could be involved in recognition of auxiliary proteins from out sources to give extra specificity.

We have synthesized Trp-containing amphiphilic peptides against several pre-miRNA. Peptide 2b showed a picomolar binding affinity and a large specificity against pre-let7a-1. In vitro miRNA processing, Dicer activity was also selectively enhanced in the presence of this peptide.

On treatment with this peptide on hct116 colon cancer and P19EC cell lines, let7a-1 miRNA was more processed than reference miRNAs. The toxicity of furan is known to rely on its selective oxidation in the liver by Cyt P540 enzymes transforming it into the very reactive butenedial, which quickly reacts with proximate nucleophiles. 1 This principle was used in our laboratory to develop a high yielding DNA interstrand crosslinking methodology. 2 In view of the demonstrated site-selectivity, the method further holds promise for sitespecific crosslinking DNA to its binding proteins, which is highly relevant in the study of transient protein-DNA interactions. Furthermore irreversible DNA binding can be achieved through such a covalent linkage, which is potentially useful for new generation therapeutics. 3 The reactive furan moiety can in principle be incorporated either in the DNA or in the protein. In the former case, a furan modified nucleotide was built into an oligonucleotide positioning the furan moiety at the periphery of the DNA, to avoid interstrand crosslinking. For the latter approach, we initially chose to synthetically access a furan modified DNA binding protein mimic. Next to a previously described non-covalent GCN4 mimicking dimer, 4 we have also investigated a new type of steroid-based dipodal DNA binders. 5 Synthesis of the latter constructs has proven challenging in view of the immobilization of two peptide chains with helix forming tendency at close distance on the template. Results, showing the power of microwave assistance will be discussed. In an alternative approach, a full length protein was modified with furan by amber suppression based on the structural similarity between a furan modified amino acid and pyrrolysine. 6 Pharmaceutical Institute, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany Human matriptase-2 is a 80 kDa protein with trypsin like specificity. This protein exhibits a domain organization similar to family of membrane-bound serine proteinases known as type II transmembrane serine proteinases. Among many ascribed function in human body, this enzyme is a potent negative regulator of hepcidin, the peptide involved in iron homeostasis [1] . Matriptase-2 has a similar fold as other TMSP members, however their detailed specificity still remain unclear. The aim of this study was to determine the substrate specificity of this physiological important enzyme using combinatorial chemistry approach. In order to characterize the matriptase-2 specificity, the tetrapeptide library with C-terminal amide of aminocoumarin (ACC-NH2) that serve as a fluorophore, was synthesized. Its general formula is given below: X4-X3-X2-X1-ACC-NH2, where in position X4-X2 the set of proteinogenic amino acid residues are present, whereas in position X1 Lys or Arg was introduced. Deconvolution of such library was performed using iterative approach in solution. The results obtained indicate that matriptase-2 display diverse P4-P2 specificity as compare to matriptase-1. The most efficient hydrolyzed amino acid residue in position P4 appear to be Ile, that is followed by Arg in P3 and Ser in P 2 . The Arg in position P1 is 30% faster hydrolyzed then Lys. For selected substrates, the kinetic parameters (kcat, K M ) were determined. Amyotrophic lateral sclerosis (ALS) is a chronic progressive disease. It is characterized by degeneration of upper or lower motor neurons, but its pathogenesis is still unknown and no effective treatment currently exists. It is known that antibodies to gangliosides have been found in some ALS patients, and these antibodies are also well known to be present in the patients affected by a variety of autoimmune diseases including multiple sclerosis. Up to now anti-gangliosides antibodies are detected in clinical immunology laboratories using isolated non consistent antigen mixtures. Therefore, we are interested in developing reliable and univocally characterized synthetic antigens for efficient antibody detection. CSF114(Glc) is a family of structure-based designed glycopeptides that we previously developed as Multiple Sclerosis (MS) synthetic probes. These N-glucosylated peptides are able to detect specific autoantibodies in the sera of an antibody-mediated form of MS. 1 Autoantibody recognition was favored because of the exposition of the sugar amino acid on the tip of type 1' β turn structures. Aim of this study is the introduction, in the type 1' β turn peptide structure, of the sugar moiety specific for anti-gangliosides antibody recognition by synthesizing specific building blocks. These building blocks are amino acids carrying glycans mimicking the biological activity of complex oligosaccharides. We selected Sialic acids (in particular the N-acetylneuraminic acid -Neu5Ac) 2 because they are involved in a significant number of biological events. Neuraminic acid and its derivates are widely distributed in animal tissues and in bacteria, especially in glycoproteins and gangliosides. Therefore, we synthesized Fmoc-L-Asn(Neu5Ac)-OH and Fmoc-L-Ser(Neu5Ac)-OH. These building blocks will be introduced in the type 1' β turn structure for the detection of anti-gangliosides antibodies in ALS. As a distinct pattern of MS could involve an antibodymediated demyelination, identification of autoantibodies as specific biomarkers is a relevant target. Even if interesting data focused on the diagnostic and prognostic role of the detection of antibodies to myelin oligodendrocyte glycoprotein (MOG) in adults' serum, its value remains dubious due to many other contrasting results. Our research group identified CSF114(Glc), an Nglucosylated peptide, able to detect disease-specific autoantibodies in the sera of a statistically significant number of MS patients. 1, 2 Since this synthetic antigen may be considered as a mimic of aberrant post-translational modification (i.e. N-glucosylation) of myelin protein(s) triggering autoimmunity in MS, our goal is to obtain the extracellular domain of MOG properly glucosylated thanks to a simplified native chemical ligation approach. 3 For this purpose, the N-glucosylation will be introduced in a synthetic peptide fragment following the building-block approach by SPPS. The other protein fragment bearing an N-terminal cysteine will be expressed in E. coli after introduction of a selective point mutation into MOG. Finally, our aim is to test the semi-synthetic protein by SP-ELISA to study the ability to detect autoantibodies in MS patients' sera and to find a potential cross-reactivity with CSF114(Glc). This peptide is an endogenous ligand of the opioid receptorlike 1 (ORL1), previously referred to as "orphan" receptor, structurally and functionally related to the classical opioid receptors. Also the hexapeptide Ac-RYYRWK-NH2 is shown to be a selective ligand for the NOP receptor with marked analgesic effect. With a view to developing ligands for the NOP receptor with more potent analgesic activity, new series of the Ac-RFMWMK-NH2 and Ac-RYYRWK-NH 2 , modified at position 4 and 5 respectively with newly synthesized β 2tryptophan analogues were synthesized 1 . The aim of the present study was to examine the effects of naloxone (Nal) and JTC-801 (NOP receptor antagonist) in the analgesic activity of newly synthesized hexapeptide analogues. All peptides (10 μg/kg), Nal (1 mg/kg) and JTC-801 (0,5 mg/kg) were injected intraperitoneally (i.p.) in male Wistar rats. Antinociceptive effects were evaluated by two nociceptive tests -paw-pressure (PP) and hot-plate (HP) and statistically accessed by ANOVA. The results will be discussed compared to the referent compound in both tests used and mechano-and thermo-receptors are involved. [1] . SOCS1 and SOCS3 have many similarities as well as some intriguing differences. Both can block signalling by direct inhibition of JAK enzymatic activity yet apparently require different anchoring points within the receptor complex. While the primary SOCS1 interaction is with a critical pY residue within the JAK catalytic loop [2] it interacts also with pY residues in the IFNαR1 and IFN R1 subunits in a JAK1-independent manner; the SOCS3-SH2 domain also interact with Y1007 in JAK2, albeit with slightly lower affinity, but subsequent studies demonstrated a high affinity interaction with pY residues located within receptor subunits [3] . Mutagenesis studies identified small regions at the N-termini of the SOCS1 and SOCS3-SH2 domains, and at the C-terminus of the SOCS3-SH2 domain, which were critical for phosphotyrosine binding. In order to gain insights in molecular discriminants for the interaction of both SOCS1 and SOCS3 toward JAK2 and TYK2 we designed and synthesized peptides encompassing regions involved in proteins recognition. We set up a SPR assay to evaluate the affinities of complexes formation. Then through an Alascanning approach we have designed new peptide sequences containing un-natural amino acids that are able to better recognize wild sequences and whole proteins. Cellular experiments on STAT1 activation signaling suggest their potential application as modulators of disorders involving SOCSs overexpression. Targeting proapoptotic death receptors (DRs) to trigger apoptosis in cancer cells is a promising anticancer therapeutic approach. TRAIL (TNF-Related Apoptosis Inducing Ligand) is a transmembrane homotrimeric protein belonging to the TNF family that triggers selective tumour cell apoptosis upon binding to its cognate receptors DR4 and DR5. Several strategies are being developed to exploit the unique cancer selectivity of the TRAIL-DR pathway in therapy, including the use of recombinant TRAIL targeting DR4 or DR5. [1] Recently, a disulfide-bridged macrocyclic 16-mer peptide (derived from phage display) that binds selectively to DR5 has been identified. [2] Oligomeric versions of this macrocyclic peptide display increased binding avidity to the receptor and exhibit the capacity to activate the TRAIL apoptotic pathway both in vitro and in vivo. [3] However, disulfide bonds are susceptible to reduction and scrambling in vivo potentially resulting in the loss of the desired biological activity. Among alternative linkages with increased redox stabilities, lanthionine thioethers, in which one of the sulfur atoms of the disulfide bond is removed have previously been introduced into biologically active peptides with some success. [4] Disulfide bridges can undergo a -elimination in alkaline conditions, followed by a Michael addition to give a thioether bridge. Optimization of this reaction led to the desulfurized analogue of the DR5-binding peptide. The native DR5-binding peptide and its desulfurized analog have been compared for their structural (NMR conformational analysis) and biological properties (affinity to DR5 and signaling pathways). The apelin/APJ complex has been detected in many tissues and is emerging as a promising target for a number of pathophysiological conditions. In the central nervous system, apelin/APJ was detected in brain regions involved in spinal and supraspinal control of pain, such as the amygdala, hypothalamus, dorsal raphe nucleus and spinal cord. We propose the hypothesis that apelinergic agonists represent a potential new approach to pain modulation and that the synthesis of stable analogues would lead to compounds with antinociceptive properties. There is currently little information on the structure/activity relationship (SAR) of the apelin hormone. In an effort to better delineate SAR, we synthesized analogs of apelin-13 modified at selected positions with unnatural amino acids, with a particular emphasis on the C-terminal portion. Analogs were then tested in binding and functional assays by evaluating Gi/o mediated reduction in cAMP levels and by assessing β-arrestin2 recruitment to the receptor. The plasma stability of new analogs was also assessed. Several were found to possess increased binding and higher stability compared to the parent peptide. There is compelling evidence that the neuropeptide 26RFa and its cognate receptor GPR103, are involved in the control of food intake and bone mineralization. 1 Among the GPCRs whose structures have been solved, GPR103 exhibits the highest sequence homology with the beta2adrenergic receptor. The aim of this work was to experimentally characterize predicted ligand-receptor interactions by site-directed mutagenesis of GPR103 and design of point-substituted 26RFa analogs. Starting from the X-ray structure of the beta2-adrenergic receptor, a 3D molecular model of GPR103 has been built. The bioactive C-terminal octapeptide 26RFa(19-26), KGGFSFRF-NH 2 , 2 was subsequently docked in this GPR103 model and the ligandreceptor complex was submitted to energy minimization.

In the most stable complex, the Phe-Arg-Phe-NH2 part was oriented inside the receptor cavity whereas the N-terminal lysine remained outside. A strong intermolecular interaction was predicted between the Arg 25 residue of 26RFa and the Gln 125 residue located in the third transmembrane helix of GPR103. In order to study this interaction, we have investigated the ability of 26RFa and Arg-modified 26RFa analogs to activate the wild-type (wt) and the Q125Amutant receptors transiently expressed in CHO cells. The platelet receptor αIIbβ3 plays a critical role in the process of platelet aggregation and thrombus formation. Upon platelet activation its conformation changes leading to an increased affinity for fibrinogen. The αIIbβ3 activation is regulated by "outside-in" and "inside-out" signaling. Among the protein-protein interactions, which contribute to «inside-out» signaling, the most important is that of talin with the β3 cytoplasmic tail. It has been recently suggested that talin-mediated αIIbβ3 activation relies on the cooperative interaction of the membrane proximal (MP) and the membrane distal (MD) β 3 regions with talin F3 domain and that the -N 744 PLY 747 -motif of β3, which can be phosphorylated at Y 747 , plays a critical role in this process. 1 To evaluate the interaction of talin with the β3 tail of integrin we designed and synthesized three peptides corresponding to the MD and MP parts of β3 in their carboxyfluoresceinlabeled form (MD: CF-R 736 AKWDTANNPLYKE 749 -NH 2 , CF-N 743 NPLYKEA 750 -NH 2 and MP: CF-K 716 LLITIHDRKE 726 -NH2). Emission and anisotropy fluorescence spectroscopy was used to quantitatively assess the affinity of these peptides for talin. Furthermore, to challenge the role of the Y 747 phosphorylation in talin-α IIb β 3 interaction we also studied the binding of talin to the modified analogues of MD, CF-R 736 AKWDTANNPL(pTyr)KE 749 -NH 2 and CF-N 743 NPL(pTyr)KEA 750 -NH 2 . Our experiments revealed that the MD and MP parts of β3 bind tightly to talin and that Y 747 phosphorylation has an inhibitory effect on this binding. 

Functionalized oligoprolines as multivalent scaffolds in tumor targeting P. Wilhelm, H. Wennemers* ETH Zurich, Zurich, Switzerland Oligoprolines are known to be structurally well-defined molecular scaffolds. In aqueous media, even short chain lengths of six proline residues adopt a polyproline II helix (PPII). This secondary structure is a highly symmetric helix where every third residue is on top of each other in a distance of about 9.5 Å. [1] The incorporation of Azidoproline (Azp) allows facile and versatile functionalization either via copper-catalysed azide-alkyne cycloaddition (CuAAC) or an acylation that followed a Staudinger reduction. [2] Based on the structural integrity of the oligoproline scaffold, targeting vectors can be conjugated via coppercatalysed azide-alkyne cycloaddition in defined distances. Recent studies on radiolabeled oligoproline-bombesin conjugates, to target the gastrin-releasing peptide receptor (GRP-R), showed in vitro and in vivo superior internalization in prostate cancer cells compared to the established monovalent ligands. [3] A facile route to synthesize alkynylated ligands has been developed successfully. We are currently expanding this concept to the integrinligand c(RGDyK) as well as to [Tyr 3 ]-Octreotide, which binds to somatostatin-receptors. The monovalent analogue of the latter, DOTA-[Tyr 3 ]-Octreotide (DOTATOC), is well established for diagnosis [4] and therapy [5] of somatostatinpositive tumors such as neuroendocrine tumors. The Cu I -catalyzed azide-alkyne addition 1 (CuAAA), the useful variant of "click chemistry," has emerged as a powerful technique for specific addition. That chemistry is also commonly used for conjugation, and cyclization of peptides. It is known that cyclization can increase the metabolic stability of peptides, as well as enhance potency or selectivity. Another useful application of the CuAAA, which we are reporting, is the N-terminal crosslink of two synergic peptides to gain their potency. CuAAA reaction is performed on solid phase (Merrifield resin) where one of the peptide components with azido group on the linker (6azido-hexanoic acid) is "clicked" with second peptide component in solution, made by Fmoc strategy in partially protected form containing at N-terminal side alkyne group (Fmoc-L-propargylglicine). CuAAA coupling is performed in DMF/t-BuOH/H2O with presence of CuI and sodium ascorbate when reacting mixture was degassed. Linked peptides are cleaved finally from resin and purified. As an application example we picked two endothelin active peptide analogues: BQ123 derivative 2 (a highly potent and selective ETA antagonist) and IRL-1620 derivative 3 Angiogenesis is a key step in the transition of tumors from a dormant state to a malignant state. The vascular endothelial growth factor (VEGF) is a major contributor to tumor angiogenesis. Its pro-angiogenic activity is mainly mediated through binding to two tyrosine kinase receptors located predominantly on the surface of endothelial cells: VEGFR-1 and VEGFR-2. VEGF binding to these receptors triggers the activation of different signal transduction pathways responsible for the proliferation, survival and migration of endothelial cells 1 . VEGF/VEGFR system constitutes a target to stop tumour growth. An attractive approach is the development of peptides, or small-molecules, with a high affinity for the extracellular domain of the receptors to prevent VEGF binding. Based on the X-ray structure of VEGF and the D2 domain of VEGFR-1 2 , cyclic peptides had been developed in our group 3 . Such peptides, mimicking simultaneously the 3-4 loop and helix ·1 of VEGF, can bind to D2 domain of VEGFR-1 and inhibit receptors phosphorylation and thus MAP kinase pathway 4 . We describe here our strategies to optimize peptidic antagonists of VEGFR-1. Chemical modifications are made in order to better mimic peptide conformations and to increase their receptor binding affinities. We introduce a hydrophobic functional group at the C-terminal of the original cyclic peptide 4 , some of such modified peptides reveal improved VEGFR-1 binding affinity. Otherwise, as the helix ·1 presents most of the important residues in VEGFR1 binding according to alanine-scan in the literature5, we try to stabilize the helical conformation by insertion of Aib residues or by peptide cyclisation. The peptides affinities are evaluated by an ELISA test developed previously 3 . Institute of Chemistry and Biochemistry, Freie Universität Berlin, Thielallee 63, D-14 195 Berlin, Germany New polypeptide was isolated from the Azemiops feae viper venom by combination of gel filtration and reversephase HPLC and called azemiopsin. Its amino-acid sequence (DNWWPKPPHQGPRPPRPRPKP) was determined by means of Edman degradation and mass spectrometry. It consists of 21 residues and does not contain cysteine residues. According to circular dichroism measurements, this peptide adopts a β-structure. Peptide synthesis was used to verify the accuracy of the determined sequence and to prepare sufficient peptide amount for biological activity studies. Azemiopsin efficiently competed with α-bungarotoxin for binding to Torpedo nicotinic acetylcholine receptor (nAChR) (IC50 0.18±0.03 M) and with lower efficiency to human α7 nAChR (IC50 22±2 μM). Ala scanning showed that amino-acid residues at positions 3-6, 8-11 and 13-14 are essential for binding to Torpedo nAChR. In biological activity azemiopsin resembles waglerin, a specific blocker of muscle-type nAChR from Tropidechis wagleri venom. However the sequences of these peptides are markedly different, and azemiopsin is the first natural toxin to block nAChRs that does not possess disulfide bridges. Laboratory of Peptide Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan While neutrophils infiltrate into damaged sites immediately after tissue injury, endogenous factors which induce their acute transmigration and activation have not been thoroughly elucidated. For the candidates, we recently identified two novel neutrophil-activating cryptides, mitocryptide-1 (MCT-1) and -2 (MCT-2), which were hidden in mitochondrial cytochrome c oxidase and cytochrome b, respectively [1] [2] [3] . In addition, the presence of many neutrophil-activating peptides other than MCT-1 and -2 was observed during their purification. These findings suggest that neutrophils are regulated by many unidentified peptides. Here, we purified a novel neutrophil-activating octadecapeptide whose primary structure was identical to mitochondrial cytochrome c (68-85) from porcine hearts. We named this functional peptide as mitocryptide-CYC (MCT-CYC). The structure-activity relationships of cytochrome c on β-hexosaminidase release from neutrophilic differentiated HL-60 cells demonstrated that cytochrome c (70-85) was the most potent cryptide among cytochrome c-derived peptides. Since cytochrome c is known to be involved in the apoptotic process, our present results suggest that cryptides produced from cytochrome c play an important role in scavenging toxic debris from apoptotic cells by neutrophils. anthracis spores are very resistant and can remain dormant in soil for decades. Therefore, an effective detection system for B. anthracis is urgently needed. Recently, it was found that one of the components of the B. anthracis exosporuim is a collagen like protein whose carbohydrate portion is composed of the tetrasaccharide with the highly specific monosaccharide upstream terminal, named anthrose. 1 Since anthrose was not found on other bacterial spores, including those closely related to B. anthracis, this monosaccharide is an attractive target for the development of new B. anthracis detection and identification methods. Peptide cyclization represents particularly interesting approach for the design of artificial receptors for anthrose, because cyclic peptides provide the possibility of having a spherical lipophilic binding site of appropriate size and shape for a particular carbohydrate substrate. 2 The presence of hydrogen donor/acceptor groups within a three-dimensional structure permits carbohydrate substrates to be encapsulated, thereby allowing their binding in water. In order to determine whether the cyclic peptide receptor can selectively detect the anthrose, we have successfully prepared cyclic peptide combinatorial library (total 6859 peptides) by the process of divide, couple and recombine ("tea-bag" technology) using standard Fmoc solid-phase peptide synthesis. 3 Prepared combinatorial library is screened for anthrose binding in fluorescence-based assay, and individual cyclic peptides with enhanced affinity toward anthrose are identified by the positional scanning deconvolution process. 3 Cyclization of linear sequences is a well-known approach used to restrict the flexibility of peptides. Cyclization often increases selectivity of peptides towards one specific receptor type, increases metabolic stability and generally increases lipophilicity, which often improves the bloodbrain barrier permeability of peptides. In our previous study [1] we have reported on the synthesis of a cyclic endomorphin-2 (EM-2) analog, Tyr-c(D-Lys-Phe-Phe-Asp)-NH2, which elicited analgesia after peripheral administration. Encouraged by the fact that this analog was able to cross the blood-brain barrier we designed and Aliskiren is the first orally active, direct renin inhibitor to be approved for the treatment of hypertension. Its structure and conformational analysis were explored using molecular dynamics (MD) simulations. For the first time, MD calculations have also been performed for aliskiren at the receptor site, in order to reveal its molecular basis of action. It is suggested that aliskiren binds in an extended conformation and is involved in several stabilizing hydrogen bonding interactions with binding cavity (Asp32/255, Gly34) and other binding-cavity (Arg74, Ser76, Tyr14) residues. Of paramount importance is the finding of a loop consisting of residues around Ser76 that determines the entrapping of aliskiren into the active site of renin. The details of this mechanism will be the subject of a subsequent study. Additionally molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) free energy calculations for the aliskiren-renin complex provided insight into the binding mode of aliskiren by identifying van der Waals and nonpolar contribution to solvation as the main components of favorable binding interactions. adamantyltripeptides and phospholipids in liposomal bilayers 1 . Now, we were primarily interested to study incorporation profile of mannosylated adamantyltripeptides. We have demonstrated that the adamantyl moiety, due to its liphophilic properties, penetrates into the lipid core of the bilayer while the hydrophilic part with the mannosyl moiety is exposed on the liposome surface. After concanavalin A (Con A), a lectin, which specifically binds α-D-mannosyl residues, was added to the liposome preparation, increase in liposome size and appearance of aggregates has been observed. The enlargement of liposomes was ascribed to the specific binding of the Con A to the mannose present on the surface of the prepared vesicles. The AFM analysis revealed that the adamantyltripeptide molecules grouped into small domains that raise above the bilayer surface. The molecule size and molecular geometry, as well as the hydrophilic and hydrophobic surfaces in the structure of mannosylated adamantyltripeptides, are responsible for arrangement of molecules in the lipid bilayer. This approach might be a useful model for investigation of specific protein interactions with membrane receptors. Also, the adamantyl moiety may be considered as a potential membrane anchor for different carbohydrate or other molecules of interest, which could be bound on it and thus exposed on liposome surfaces and as such used in targeted drug delivery. The assay is carried out in a 96 well format P122 and images are captured throughout the course of the assay, thus we can not only determine a ligand's propensity to induce internalization, but also its efficacy and internalization rate. Addition of test compound, followed by the standard agonist at a later interval, enables differentiation between agonist or antagonist activities. In the positional scanning format [1] , while the possibility of agonists and antagonists working against each other within a mixture exists, the effects are minimized in screening the whole library as there are as many arrangements of the sub-libraries as there are defined positions. Therefore while an agonist and antagonist might be present in a particular mixture in one sub-library they will be in different mixtures in all other sub-libraries. We have used this assay format to simultaneously screen for novel agonists and antagonists against the Orexin 2 receptor. Assay development and library screening will be presented. [1] . Since the Pro residue in position 2 of EM2 is very important in the proper conformational alignment of the two aromatic residues Tyr 1 and Phe 3 in EM2 molecule at the receptor site, it is possible that structural modification around the Pro 2 residue yields compounds with unique biological properties and improved metabolic stability. In the present study, we synthesized seven EM2 analogues containing isoPro or constrained residues with oxopyrrolidine or oxopiperadine ring, instead of Pro residue in position 2. All peptide analogues were synthesized solid phase method. Incorporation of oxopyrrolidine and oxopiperadine rings were carried out on a solid support by the methods of Gellerman, et al. [2] and Mohamed, et al. [3] , respectively. Opioid receptor binding activity for μ and δ-receptors using The development of resistance to mainstay cancer therapies has become a major limitation for the treatment of many cancers. There is an urgent need to develop new antineoplasic agents with innovative anticancer approaches. To overcome resistance to cancer therapies, our attention has turned to proteins that regulate multiple signalling pathways essential for tumour survival. Among the few known nodal proteins upregulated in cancer cells and involved in many hallmarks of cancer, we are interested in survivin. An essential regulator of cell proliferation and apoptosis, survivin is sharply overexpressed in cancer cells and plays a major role in resistance. 1 Being a small protein, its bioactivity is relies mainly on protein-protein interactions (PPI) with different partners. A critical point for its multiple functions in cancer is its association with Hsp90, which is required for its stability. A nonapeptide from survivin called shepherdin has been shown to modulate the interaction of survivin with Hsp90 by binding to Hsp90 and to induce death of tumour cells. 2 Unfortunately, shepherdin is not cell permeable, has low proteolytic stability and shows poor bioavailability, limiting its use as anticancer therapeutic agent. To improve pharmacological properties of shepherdin, cyclic and peptidomimetic analogs of shepherdin have been synthesized followed by structure-activity relationship studies. In Hsp90 binding studies, some cyclic hexa-and heptapeptidic analogs showed increased affinity compared to shepherdin. The synthesis of cyclic and peptidomimetic analogs and the results from the binding assays and the conformational analyses will be presented. The hexapeptides with formula Ac-RYYR/KW/IR/K-NH2 have been identified as shortest peptide sequence with high NOP receptor affinity, selectivity and marked analgesic effect. It was found that the following peptides act as partial or full agonists or antagonists of NOP receptor in different in vivo and in vitro systems. These hexapeptides were used as chemical templates in SAR studies 1,2 . The aim of the present study was the synthesis and the biological screening of new analogs of Ac-RFMWMK-NH2 and Ac-RYYRWK-NH 2 , modified at position 4 and 5 respectively with newly synthesized β 2 -tryptophan analogues 3 . These non natural amino acids were prepared using reaction of asymmetric Friedel-Crafts alkylation of various indoles with a chiral nitroacrylate to provide optically active β-tryptophan derivatives. The four newly synthesized ligands for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) have been prepared by solidphase peptide synthesis-Fmoc-strategy. These compounds will be tested for agonistic activity in vitro on electrically stimulated smooth-muscle preparations isolated from vas deferens of Wistar rats. Bacterial infections are a common problem associated with dermal wounds. These infections can prolong or impair wound healing. Hydrogel materials that display inherent activity against bacteria can be used to directly treat accessible wounds to prevent or kill existing infection. In this work, we describe the design and utilization of injectable gels prepared from self-assembling β-hairpin peptides having a high content of arginine. These gels were found to be extremely effective at killing both gram-positive and gramnegative bacteria, including multi-drug resistant P. aeruginosa. Importantly, no added antibacterial agents are necessary since the nanostructure of the gel, itself, is the active agent. Using self-assembling peptides for material construction allows facile structure-activity relationships to be determined since changes in peptide sequence at the monomer level are directly transposed to the bulk material's antibacterial properties. Structure-activity relationships studies show that arginine content largely influences the hydrogel's antibacterial activity, and influences their bulk rheological properties. These studies culminated in an optimized gel, composed of the peptide PEP6R. PEP6R gels prepared at 1.5 wt % or higher concentration, demonstrate high potency against bacteria, but are cytocompatible towards mammalian mesenchymal stem cells. The general mechanism by which PEP6R exerts its action was explored and it is suggested that involves membrane disruption that occurs when cells come in contact with the gel's surface. Atomic Force Microscopy (AFM) was used to study the effect of the gel on the cell envelope morphology of E. coli.

Rheological studies indicate that the gel is moderately stiff and displays shear-thin recovery behavior, allowing its delivery via simple syringe. 1 They are intimately involved in the molecular process leading to the delicate nano-patterned silica shells of diatoms.

Deciphering the mechanisms of silica-biogenesis in diatoms will inspire the development of novel routes for the biomimetic synthesis of silicon-based materials under mild conditions and expand the scope of biotechnological applications, e.g. for immobilization of enzymes in silica matrices. We synthesized silaffin peptides derived from C. fusiformis that carry posttranslational modifications such as phosphorylation or polyamines linked to lysine side chains. A distinct alteration of silica precipitation activity depending on the particular modifications of the silaffins emerged. 2 These modified silaffin peptides were covalently linked to recombinant proteins by expressed protein ligation leading to stable protein-silaffin conjugates. 3 Using eGFP as model protein, we could show that eGFP-silaffin conjugates can induce biomineralization of silica and ensure an efficient and homogeneous immobilization of eGFP into silica particles, superior to simple co-biomineralization approaches. Moreover, a significant stabilization of immobilized eGFP against denaturing agents was observed. We established a method for controlled immobilization of biomolecules based on covalent attachment of silaffin peptides with well-defined silica precipitation properties. Currently this method is applied to the immobilization of biotechnological relevant enzymes in order to test their activity and the stabilization effect. Herein, we present the covalent functionalization of multiwalled CNTs (MWCNTs) with organocatalysts based on proline or proline derivatives carrying either a dipeptide or a sulfonamide moiety. Two different approaches were followed, namely, covalent grafting of the organocatalysts either at the tips or at the sidewalls of the CNTs. For the former approach, MWCNTs were oxidized in order to introduce carboxylic units at their tips and make them easily dispersed in aqueous solutions. Then, oxidized MWCNTs readily reacted with proline-based derivatives carrying a free amino unit yielding the corresponding hybrid materials. For the latter approach, the functionalization methodology based on in-situ generated aryl diazonium salts was followed. In this context, MWCNTs were modified with aryl units carrying free amino terminal groups, which were subsequently conjugated with proline-based derivatives carrying a free carboxylic unit. All newly formed hybrid materials were fully characterized with complementary spectroscopic (ATR-IR, Raman), thermal (TGA) and microscopy (TEM) techniques. The catalytic evaluation of the activity of the CNT-based organocatalysts in aldol reactions is in progress. Financial support from GSRT/ΕΣΠΑ 2007-2013 ΣΥΝΕΡΓΑΣΙΑ through 09ΣΥΝ-42-691-ΝΑΝΟΚΑΤΑΛΥΣΗ project is acknowledged.

Novel organogels based on self assembly of rationally designed pseudopeptides C. Pappas, N. Sayyad, A.G. Tzakos, I. Plakatouras Section of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina, Ioannina, GR-45110, Greece Self-assembly is becoming a rather intriguing way to build an array of nano-and micro-structured materials. Low molecular weight organogelators can self-assemble into various architectural types in organic solvents through weak intermolecular interactions. Such organogelators have potential applications in the generation of novel materials for nanobiotechnology1. Herein, we report the synthesis of rationally designed pseudopeptides and the conditions to form organogels. The obtained gels are responsive to temperature, and the sol-gel process is thermoreversible. The architecture of the constructed organogels was characterized via TEM and spectroscopic techniques. Diffusion Ordered NMR spectroscopy (DOSY) was further utilized to determine differences in the molecular shape of the different pseudopeptides. Applications of the resulted compounds in nanotechnology will be reported. Since 2000, organocatalysis has met such a great rate of expansion that is nowadays considered the third major branch of modern asymmetric catalysis along with the transition metal catalysis and biocatalysis. After the seminal work of List, Lerner and Barbas on the enantioselective aldol reaction between acetone and 4-nitrobenzaldehyde catalyzed by proline, it became clear that amino acids and peptides could serve as an abundant pool full of potential to develop novel organocatalytic motives. Following our recent report that the combination of a prolinamide with a thiourea group having as a spacer a chiral diphenylethylenediamine leads to an efficient organocatalyst for the aldol reaction, 1 we recently considered the possibility to couple the prolinamide unit with an urea moiety. One of our main interests was the substitution of the diphenylethylenediamine spacer by a gem diamine derived from an α-amino acid. The gem diamine is easily synthesized via a Curtius rearrengement of the corresponding acyl azide. After synthesis and evaluation of a number of potential catalysts, the prolinamide derivative bearing a gem diamine derived from (S)-phenylalanine and an aryl urea moiety proved to provide the best results in the reaction between cyclic ketones and aldehydes. Utilizing 10 mol% of our organocatalyst, the aldol products were obtained in high to quantitative yields (up to 98%), high to excellent diastereoselectivities(up to >98:2) and high to excellent enantioselectivities (up to 99% ee). Peptide self-assembled monolayers are of current interest to study physicochemical properties of modified metal (e.g. Au) surfaces. Rigid peptide scaffolds could enhance the interaction between gold surfaces and labels by reducing and precisely monitoring the distance between the supported monolayers and gold. The C α -tetrasubstituted αamino acid 4-amino-1,2-dithiolane-4-carboxylic acid (Adt) 1 , which contains a cyclic disulfide system, is interesting in this respect because it may allow the parallel binding of the peptide helical chain to the metal surface. Adt occurs in Nature 2 and has been utilized in medicinal chemistry 3 and in a model compound of [FeFe] hydrogenase. 4 We synthesised a series of constrained helical peptides, based on the Ala-Ala or the Ala-Aib sequence, containing one or two Adt residues. These peptides were functionalised with spectroscopic or opto-electronic labels. Among the large number of reactions involving the formation of carbon-carbon bond, the addition of ketones to nitroolefins is a powerful tool for the synthesis of γ-nitrocarbonyl compounds, useful intermediates for pharmaceutical industry. Our recently reported primary amine-thioureas based on tert-butyl esters of natural amino acids exhibit excellent performance for the Michael reaction of ketones with nitroolefins providing the products quantitatively and almost stereospecifically (>99% ee). 1, 2 Using this methodology, enantiopure baclofen and phenibut (analogs of GABA) have been synthesized. 2 Polymersupported organocatalysts constitute a great challenge for the Michael reaction. In the current study, we report the immobilization of amine-thiourea catalysts containing (1S,2S)or (1R,2R)-diphenylethylenediamine and tert-butyl aspartate, on various polymer supports, either directly or through spacer units. The solid-supported catalysts evaluated in the reaction between acetone and βnitrostyrene and highlighted the importance of the choice of the polymer as well as the presence of the spacer or not. The direct attachment of the primary amine-thioureaaspartate to a crosslinked polystyrene-divinyl benzene resin containing a uniform distribution of aminomethyl groups provides a supported catalyst that affords the product of the reaction between acetone and β-nitrostyrene quantitatively and in high enantioselectivity (91% ee 

A. Theodorou, G.N. Papadopoulos, C.G. Kokotos* Laboratory of Organic Chemistry, Department of Chemistry, University of Athens, Athens, Greece After the pioneering report that proline can catalyze efficiently the intermolecular aldol reaction between acetone and a variety of aromatic aldehydes, 1 it became evident that amino acids and peptides 2 can afford a plethora of different structural scaffolds for novel catalysts. Along the first decade of its life, Organocatalysis has grown to such an extend that now it is considered the third major branch of asymmetric catalysis. Recently, researchers have paid special attention to other amino acids rather than proline. Some primary amino acids have already been applied to a number of transformations with success. 3 Usually improved catalytic properties are observed when derivatives of primary amino acids are utilised. We have undertaken a study on the application of simple and cheap primary amino acids and amino acid derivatives, either commercially available or easily obtained, as organocatalysts for the asymmetric α-amination of aldehydes. In the present work, we report that the use of simple derivatives of primary amino acids like phenylalanine and aspartic acid can efficiently catalyze this transformation leading to products in high to quantitative yields and enantioselectivities up to 94% ee. The majority of the organocatalysts developed up to now for asymmetric organic transformations employ more than one functionalities in the catalytic mechanism that act through either covalent or non-covalent interactions. For example, proline employs the pyrrolidine nitrogen and the carboxylic acid group, while chiral thioureas combine the thiourea functionality with a tertiary or a primary amino group. We have recently shown that an amide of proline with a diamine carrying a thiourea group is a very good catalyst for the enantioselective aldol reaction. 1 Trying to improve the activity, we have found that a tripeptide-like thiourea having as building blocks (S)-proline, (1S,2S)diphenylethylenediamine and (S)-di-tert-butyl aspartate provides the products of the reaction between ketones and aromatic aldehydes in high to quantitative yields and high stereoselectivities (up to 99:1 dr and 99% ee). A number of structural modifications of the catalyst were undertaken in order to understand the role of the hydrogen bond donors of the catalyst, i.e. the prolinamide hydrogen and the two hydrogen atoms of the thiourea group. We have come to the conclusion that the importance of the hydrogen bond donors of the catalyst follows the order: thiourea hydrogen originated from aspartate › amide hydrogen › thiourea hydrogen originated from diphenylethylenediamine. g Eldrug S.A., Patras 26504, Greece A convenient and facile synthesis and in vitro biological evaluation of N-substituted 5-butylimidazole derivatives as potent Angiotensin II (ANG II) receptor type 1 (AT1) antagonists have been reported in the present study. A series of imidazole based compounds bearing the biphenyl moiety at the N-1 position, a halogen atom at the C-4 and polar substituents such as hydroxymethyl at the C-2 position were synthesized. 1,2 These compounds were evaluated for binding to human AT1 receptor and for ANG II antagonism in vitro on isolated rat uterus. In particular, 5butyl-1-[[2΄-(2H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole derivatives complexed with the AT1 receptor and showed high binding affinity. These analogues were also found to be active in the rat uterotonic test. Importantly, their binding affinities and potencies were comparable to those of losartan. These results indicate that the hydroxymethyl at the C-2 position of the imidazole ring is favorable for high affinity binding and antihypertensive activity and in line with the activities of the losartan counterparts. Experimental findings are in good agreement with docking studies, which were undertaken in order to investigate ligand/AT1 receptor interactions. Z-Leu-Glu-His-Asp-aLuc, Suc-Leu-Leu-Val-Tyr-aLuc) are good substrates for bioluminescence assays, for example in the detection of caspase activity during apoptosis 1 . These substrates generally offer significant advantages, such as increased sensitivity, ease of use, and high throughput screening capacity. Luciferase-based assays are typically 10-to 100-fold more sensitive than the comparable fluorescent assays (rhodamine 110, 7-amino-4-methylcoumarin (AMC) and 7-amino-4trifluoromethylcoumarin (AFC)). The synthesis of different type peptide-amino-luciferin conjugates and their precursors have been published 2 and some of them are commercially available. However, because of their high price the in vivo application of these conjugates is limited. To solve this problem we successfully worked out a new, easier and more convenient and economical method for the preparing these derivatives starting from 2-chloro-benzothiazole. Moreover this products have excellent purity (>99%) and adequate yield (82-93%). Major health problems arising from bacterial resistance towards existing antibiotics make discovery of antibacterial drugs with new mechanisms of action pertinent. Although proof of concept for a novel antimicrobial approach using peptide nucleic acid (PNA) antisense targeting of essential bacterial genes was obtained a decade ago, this technology is still limited by the lack of carriers that facilitate effective bacterial delivery and confer optimal pharmacokinetic properties to the prospective drugs. [1, 2] In the past two decades, parallel efforts of exploiting naturally occurring antimicrobial peptides (AMPs) as drugs have been made. The cationic AMP subclass appears to be directly involved in the innate immune response towards microbial infections. [3] So far only few cell-penetrating peptides, with activity on mammalian cells, and other membrane-active peptides, have been investigated as potential vehicles for bacterial delivery. For instance, cationic AMPs with an internal target appear not to have been investigated for bacterial delivery of antibiotics. The aim of this project is to develop highly potent genetic antibiotics by exploiting naturally occurring antimicrobial peptides as potential delivery vehicles for antisense peptide nucleic acid oligomers. The AMPs are chosen from AMPs reported to act via intracellular targets, and thus must possess an inherent ability to permeate bacterial cell membranes without direct killing of the bacteria. Faculty of Chemistry University of Gdansk, Gdansk, Poland AZT (3'-azido-2'3'-dideoksythymidine), a modified nucleoside used in antiretroviral therapy and peptide plant hormone -systemin were used as substrates of 1,3-dipolar cycloaddition (click chemistry). Systemin is 18-aa peptide defense hormone released in response to plant (tomato, tobacco) damage or pathogen attack. We examinated whether systemin's fast movement through plant tissues could be used for cargo (AZT) transport.

The Huisgen cycloaddition also known as 1,3-dipolar cycloaddition is a chemical reaction belonging to the larger class of cycloadditions. Reaction between organic azide and alkyne appended substrates allows the synthesis of the desired conjugate in high purity and yields irrespective of the sequences and functional groups on either of the two substrates [1, 2] . Conjugate of AZT-systemin has been synthesized by click chemistry, using systemin modified at N-terminus with propiolic group and AZT. The conjugation was catalyzed by Cu(I). The reaction was fast, efficient and regioselective. Its progress was easily monitored by capillary electrophoresis (CE). CE was also applied for characterization of systemin and AZT-systemin stability and movement throughout tomato leaf and stem. Despite the fact that systemin moves rapidly through tomato tissues, our calorimetric (ITC) studies showed that the peptide does not interact with liposomes-cell membrane model. Universitätsklinik für Nuklearmedizin, Inselspital, Bern, Switzerland Regulatory peptides (e.g., somatostatin, bombesin) have been shown to be suitable vectors for the specific delivery of radioactivity to tumors for diagnostic and therapeutic applications in nuclear oncology. 1 A potential drawback of such vectors is their inherent instability in vivo. Thus, new strategies are needed for the stabilisation of radiopeptides in order to improve their bioavailability and, consequently, increase their accumulation in the targeted tissue. It has been suggested that 1,2,3-triazoles, readily obtained by CuAAC, are suitable amide bond surrogates which are resistant to proteases. 2 In the present study, we report the synthesis and pharmacological evaluation of radiolabelled, triazole-containing analogues of the gastrin releasing peptide receptor (GRPR) targeting peptide bombesin (BBN).

To study the effect of backbone modifications in the minimal GRP-binding sequence, we synthesized a series of analogues of [Nle 14 ]BBN (7) (8) (9) (10) (11) (12) (13) (14) , in which each amide bond is individually replaced by a 1,4-disubstituted 1,2,3triazole. After radiolabelling of the peptidomimetics, their binding affinity and internalization kinetics were determined using PC-3 cells. Metabolic stability was evaluated in blood serum. A number of the novel tumor-targeting peptide analogues presented exhibit both a retained high affinity (nM) towards the GRPR and an improved serum stability. First preclinical data on the in vivo evaluation of the most promising candidate will be presented. To the best of our knowledge, this is the first report of the systematic replacement of amide bonds with 1,2,3triazoles within the binding sequence of linear, high affinity peptides. The methodology can be applied to a variety of peptide vectors and thus, holds great potential for the development of novel, stabilized peptide-based radiopharmaceuticals. DNA is the molecular target for many of the drugs that are used in cancer therapeutics, and is viewed as a nonspecific target of cytotoxic agents. Although this is true for chemotherapeutics, other agents that were discovered more recently have shown enhanced specificity. 1 The development of new site-specific DNA binders, which are associated with the recognition of the DNA major groove, are based on the design of transcription factor mimics that bind the DNA as a dimer 2 , and prevent specific genes from being transcribed. These could ultimately result in interesting biomedical applications as designed genome interfering agents or diagnostics. 3 In order to approach this biological constructs, we choose the bZip Leucine zipper transcription factor as a model to mimic. As the entire structure cannot be synthesized without expensive, complicated and time-consuming biotechnological methods, the substitution of the dimerization domain by a less complex scaffold is the first step in the design. Thus, we consider a steroid based scaffold as a candidate. The specific choice of the steroid scaffold as substituent is inspired by its known ability to enhance proteolytic stability of attached peptides, by its conformational properties ensuring correct positioning of the two appended chains and by its potential to increase bioavailability. This transcription factor binds specific DNA sequences by dimerization and inserting short α-helices into the DNA major groove. In order to attach the peptides to the scaffold, different strategies were studied. Firstly, applying the well-known click chemistry, functionalizing the scaffold with an alkyne moiety, the peptide with an azide and viceversa. Secondly, via the unknown resin to resin transfer reaction (RRTR), which has not been applied on peptide chemistry so far. This unprecedent methodology consists on the reaction of a peptide, which is attached on a safety-catch resin, with a second resin bearing a nucleophilic amino terminus resulting in amide bond formation. During the process, the peptide on solid support undergoes cleavage. An hexapeptide was synthesized on a preloaded safetycatch resin. Deoxycholic acid derived scaffold with orthogonally protected amines was attached to tentagel resin that acts as acceptor resin. RRTR experiments were performed at both C12 and C3 positions of the deoxycholic acid derivative.

In addition, this convergent strategy can be applied to other different peptide conjugated systems. We recently described a new kind of cyclized peptide in which the cyclization is performed between the side-chains of two diaminoacyl residues via a diversely substituted guanidine bridge. 1 We showed that the degree of bridge substitution could impact on the orientation of the bridge inside the cycle and therefore the peptide conformation. We prepared two series (22 and 15 atoms cycle size) of cyclic enkephalin analogues to assess the potential effect of this kind of bridge on the biological activity. The compounds were synthesized on the solid support via the formation of a thiourea bridge and with the variable substituent being introduced at the last step before cleavage. It is noteworthy that the synthesis afforded at least two stable and separable conformers for each analogue of the shortest cycle series. Generally, one major and one minor species were recovered. But in the case of di-substituted compounds with a cyclic moiety (pyrrolidine or piperidine substituents), three significant species were obtained. Analogues were submitted to various biological assays (binding to μ and δ opioid receptors and functional assays). We observed a significant variation in affinity and selectivity for the receptors as a function of the degree of bridge substitution. A structural analysis by 2D NMR has been undertaken and correlated the variation in activity with a variation in conformation. The origin of the multiple conformers observed for the analogue with a pyrrolidine susbtituent was also investigated. This kind of cyclization could represent a useful tool to easily modulate the conformation and biological activity of a unique peptide sequence. The T-cell response is triggered by the formation of the trimolecular complex between the major histocompatibility complex (MHC), the immunodominant myelin protein epitopes and the T cell receptor (TCR). Herein, we report the design and synthesis of non-peptide analogues with the ability to mimic the immunodominant epitope 83-99 of MBP 1,2 . The mimetics were designed to block the formation of the trimolecular complex and therefore the T-cell activation 3, 4 . More specifically, indole analogues were synthesized with substitution at positions 2 and 4 or 6. These molecules contain a carboxyl or an ethyl ester group in position 2 and a benzylamino or phenylamino group in position 4 or 6. The synthesis of the indole ring was achieved by Fischer reaction followed by catalytic hydrogenation, reductive amination or arylation and ester hydrolysis. The synthesized molecules were purified using liquid chromatography, and they were identified by mass spectrometry and 1H-NMR. Laboratory of Peptide Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Japan Amyloid β peptide (Aβ), the main component of senile plaques in the brain of Alzheimer's disease (AD) patients, is formed by proteolysis of amyloid precursor protein (APP). As β-secretase (BACE1: β-site APP cleaving enzyme 1) triggers Aβ formation by cleavage at the Aβ domain Nterminus, it is a molecular target for AD therapeutic intervention. 1 Previously, we reported potent pentapeptidic 2 and non-peptidic 3 BACE1 inhibitors containing a substrate transition-state mimic. Although these inhibitors exhibited potent inhibitory activities, their molecular-sizes appeared a little too big (MW>600) for developing practical drugs. In this study, we designed a series of small molecular peptides, with BACE1 inhibitory activity, lacking the P1-P1' region on the basis of the conformational structure bound in BACE1. 4 Design and synthesis of new 3'-peptidyl-tRNA analogues, in particular "hydrolysable" analogues, which represent covalent conjugates of peptide-nucleic acid (PNA) with "stop-peptides," were carried out. Such compounds are of interest as tools to study the ribosome functioning and as inhibitors of protein biosynthesis. (2 Aminoethyl)glycine PNA models 3'-end tRNA sequence CCA in designed structures. Computer simulations showed the formation of Watson-Crick pairing of the PNA cytosine residues with 23S rRNA nucleotides G2251 and G2252 involved in interactions with peptidyl-tRNA during its specific binding in P site of the ribosomal peptidyl transferase center (PTC). Short "stop-peptides" were planned for conjugation with PNA. These peptides form stable complexes with the ribosomal tunnel (RT) that leads to ribosome stalling and translational arrest. Structures of "hydrolysable" 3'peptidyl-tRNA analogues that could form peptide bond with amino acid residue of aminoacyl-tRNA in A-site of PTC included 2'-deoxyriboadenosine instead of the PNA adenine containing residue. Such conjugates would permit to identify the chemical nature of specific sites localized in RT and responsible for interactions with amino acid residues of the nascent polypeptide chain. PNA and "stop-peptide" as well as PNA-"stop-peptide" conjugates were prepared by solid phase synthesis on SASRIN polymer using Fmoc/Bhoc(Boc) strategy. Synthesis of "hydrolysable" conjugates included modification of the 3'hydroxyl of 5'-protected 2'-deoxyadenosine by N-blocked "stop-peptide", deprotection of the 5'-hydroxyl, its conjugation with N-protected PNA and removal of protecting groups from the resulted conjugate.

The binding of the new 3'-peptidyl-tRNA analogues with ribosome will be tested by chemical probing and in the cell free translation system. This study was supported by the Russian Foundation for Basic Researches (grant 10-04-01187-a). A close structural similarity of endomorphin-2 and another atypical opioid peptide, morphiceptin, which both have a Phe residue in the third position, encouraged us to study antinociceptive activity of these two peptides and their analogues.

In order to improve the affinity and chemical stability of these opioid peptides, we have designed, synthesized, and analyzed 9 novel analogues. The first modification included endomorphin-2 and morphiceptin analogues, where halogenated phenylalanines in position 3 or 4 were incorporated as surrogates of the native phenylalanine. Another important modifying element is non-protein amino acid canavanine (Cav) and its analogue (sArg). It is well documented that Cav and sArg exhibit strong analgesic activity. Two new morphiceptin analogues were synthesized by introducing Cav and sArg in position 3. We further characterized their antinociceptive activities by the paw pressure (PP) test. The experiments were carried out on male Wistar rats (180-200 g), treated with i.p. doses of 1 mg/kg. e Eldrug S.A., Patras 26504, Greece The Renin Angiotensin System (RAS) has been a prime target for the therapy of cardiovascular diseases. Angiotensin II type 1 (AT1) receptor mediates vast majority of biologically detrimental actions. Non-peptide AT1 receptor blockers are presently the most specific means to block the RAS enzymatic cascade. The DuPont group was the first to develop Losartan (DuP 753), an orally effective Angiotensin II receptor blocker, which is metabolized in vivo to the more potent antagonist EXP 3174. Herein, we report on the preparation of E-urocanic1 acid based analogs, focusing our attention on the introduction and structural modifications of the substituents on the imidazole ring as well as the modifications on the acrylic side chain. In particular, we have designed and synthesized a series of urocanic acid analogs bearing the biphenylmethyl tetrazole moiety at the N-1 of the imidazole ring.2 Additionally, the rigid acrylic chain was lengthened by esterification resulting in the ethyl ester and on the other hand the latter was readily converted to the corresponding acrylic alcohol or aldehyde which may proved to be effective structural elements for enhancing biological activity. Finally, a lipophilic alkyl chain such as the n-butyl group was introduced at the 2-position of the ring which may possibly enhance the antihypertensive activity. Docking studies and biological evaluation of the synthesized analogs are being undertaken. 

University of Athens, Department of Chemistry, Laboratory of Organic Chemistry, 15701, Panepistimiopolis Zografou, Athens, Greece The backbone modification of bioactive peptides with replacement of a scissile peptide bond in enzymatic hydrolysis is a well-established strategy for developing protease inhibitors. 1 In particular, for zinc metalloproteases, which contain a zinc atom in their active site, several successful modifications have been reported over the past years. 2 Phosphinic pseudopeptides are among the best candidates when addressing the challenge to potent and selectively inhibit zinc proteases. A thorough search in the literature3 revealed the absence of any reference regarding thiophosphinic pseudopeptides. We thought that this class of compounds would add a valuable tool in the field of zincbinding groups.

In the present study, we describe in detail the first synthesis of a new class of phosphorous compounds, thiophosphinyl dipeptide isosters (TDIs). We prepared several fully protected thiophosphinate pseudodipeptides of the general formula PG-Phe-Ψ[P(S)(OX)CH2]-Gly-PG' starting from the corresponding phosphinate pseudodipeptide using Lawesson's reagent. Selective deprotection of these compounds was also studied and the results are disclosed. These compounds can be used as building blocks for the synthesis of longer thiophosphinic pseudopeptides after suitable deprotection and elongation as well as transition transition state-mimicking inhibitors for several zinc metalloproteases. In the last decade, trypsin inhibitor SFTI-1 isolated from sunflower seeds [1] has become one of the most studied peptidic inhibitors of serine proteases. Owing to its small size and a strong trypsin inhibitory activity (Ka = 1.1×10 10 M -1 ), SFTI-1 is considered to be a very attractive template for designing proteinase inhibitors with the potential use as pharmacological agents [2] . It could also serve as an affinity probe for the isolation of trypsin like (SFTI-1) or chymotrypsin like ([Phe5]SFTI-1) proteinases. Following this idea, we decided to synthesize a set of cell-permeable monocyclic SFTI-1 analogues with a fluorophore moiety attached at their N-termini. The presence of the fluorophore in the molecule enabled us to show that the analogues can cross the cell membrane. The cell penetration assay was performed using multiple cell lines (HeLa cells and human fibroblasts cell line (46BR.1N) was obtained from European Collection of Cell Cultures (ECACC)). For all the obtained peptidomimetics, we determined the association constants with cognate proteinases. Selected peptides were also used as a probes for the detection of inhibitor -proteinase complex, which was achieved by the means of gel filtration chromatography equipped with fluorescence detector and acrylamide native gel electrophoresis. The functional reconstruction of folded protein surfaces with peptide-based mimics is an enormous scientific challenge. The majority of proteins show activity through a small area of their folded surface: "the binding site". However, linear peptides are too flexible and seldomly adopt the correct 3D-structure of the binding site spontaneously. Therefore, they show limited or no activity at all 1 . Crucial for activity is to control the secondary (αhelix, β-sheet and/or β-turn) and tertiary structure (relative orientation of subdomain structures). We present the development of a new type of watersoluble scaffolds that have the potential to control both secondary and tertiary structure of discontinuous (i.e. double-loop) protein mimics. The new scaffolds contain a first pair of reactive functionalities to constrain the linear peptide conformation via a 'CLIPS' reaction2, stabilizing the secondary structure. Next to this, a second functionality allows for ligation of two dissimilar constrained peptides to form a discontinuous binding site mimic via oxime-ligation or CLICK-reaction. These ligations offer the ability to position different peptide loops in 3D, thus mimicking the tertiary structure of the native protein. Most unique to our approach is the fact that all chemical conversions are performed in aqueous media, using side-chain unprotected peptides 3 . Growth hormone-releasing peptide 6 (GHRP-6) is a synthetic hexapeptide (His-D-Trp-Ala-Trp-D-Phe-Lys-NH2), which interacts with two kinds of receptors: growth hormone secretagogue receptor 1a (GHS-R1a) and cluster of differentiation 36 (CD36). The latter is a membrane glycoprotein member of the class B scavenger family, and decreases the internalization of oxidized lipids into macrophages, as well as causes inhibitory effects on angiogenesis associated with binding to thrombospondin.

To increase activity and selectivity for the CD36 receptor, different analogues of GHRP-6 were synthesized. In particular, substitution of Trp 4 in GHRP-6 by aza-amino acids has given selective analogs, due likely to induction of a β-turn secondary structure. For aza-peptide synthesis, a submonomer solid-phase approach has proven effective to introduce side chains onto the semicarbazide residue. Studying influences of benzylidene, benzhydrylidene and fluorenylidene residues during the alkylation of the semicarbazide, superior conversion was observed with fluorenone derivative, and mild alkylation conditions employing Et4NOH as base have improved yields and minimized racemisation. Our presentation will focus on the improved submonomer synthesis method for optimization of selective and potent CD-36 ligands with antiatherosclorotic and anti-angiogenic effects. For instance, the integrin αvβ3, vitronectin receptor, is expressed in a number of cell types and has been shown to mediate adhesion of osteoclasts to bone matrix, vascular smooth muscle cell migration, and angiogenesis. Integrin αvβ3 also play a significant role in tumor growth, invasion and metastasis, and is a receptor for the extracellular matrix proteins with the exposed arginine-glycine-aspartic (RGD) tripeptide sequence. RGD has been shown to be potent antagonist of the integrin αvβ3, and has excellent anti-angiogenic properties including its suppression of tumor growth in animal models. In this context, drug design based on the RGD structure may provide new treatments for diseases such as thrombosis, osteoporosis, and cancer. We designed and synthesized series of short RGDmimetics containing the sequence Xaa-GD, where Xaa is Arg-mimetic. As promising candidates we have chosen canavanine (Cav) and canaline (Can) instead of the basic residue Arg. In order to improve antitumor activity of the parent molecule, C-terminal modifications were also applied.

Their cellular uptake was determined on human breast (MCF7) cancer cell lines. Furthermore, the in vitro cytostatic effect was evaluated by MTT assay on human liver hepatocellular carcinoma (HepG2) and human breast (MCF7) cancer cell lines after 24, 48 and 72 hours of treatment. In the case with the human tumor cell lines (HepG2, MCF7) and C-modified analogues, statistically reliable results were achieved for the most of concentrations used. Acknowledgements: This work was supported by Bulgarian Ministry of Education and Science, project MY-FS-13/07.

Microwave assisted solid phase synthesis of urea and urea/amide based foldamers K. Pulka, C. Douat-Casassus, G. Guichard* European Institute of Chemistry and Biology, University of Bordeaux 1 -CNRS UMR 5248, Pessac, France Foldamers 1 are fully arti cial molecules that structurally and functionally mimic variety of biopolymers. Among them, aliphatic N,N'-linked oligoureas with proteinaceous side chains can adopt extremely robust helical folds stabilized by intramolecular three-centred H-bonds. 2 Owing to their resistance to enzymatic degradation, diversity of side chains and structural predictability urea-based foldamers represent unique scaffolds to elaborate functional mimetics of α-polypeptides. Of note, heterogenous oligo(urea/γamides) backbones obtained by substituting NH groups by CH2 display very similar folding propensities. In our laboratory we are investigating the solid phase synthesis of urea and urea/γ-amide oligomers. Urea bonds are incorporated into the growing chain by reaction of active succinimidyl carbamates. Previously we have applied two different strategies involving Fmoc-or Bocchemistry, but both methodologies suffer some limitations. 3 Therefore a new strategy (compatible with the use of TFA sensitive linkers and side chain protecting groups) featuring azide as a masked amine group has been developed. The synthesis of 15 new azido protected succinimidyl carbamate building blocks is reported. They were obtained in 6 steps from α-amino acids (12-45% overall yield). The Staudinger reduction with PMe3 was successfully applied to restore the amine group after urea formation on solid support. In addition, microwave irradiation has been found to dramatically accelerate the synthesis. Overall, this azide-strategy combined with microwave irradiation was found to be very effective for the solid phase synthesis of oligoureas and related hybrids, surpassing previously developed approach utilizing Fmoc chemistry. These antibiotics should have a mechanism different from currently used antibiotics to circumvent existing resistance mechanisms 1 . Previous results have shown that "genetic" antibiotics operating by gene silencing in bacteria via RNA interference may be successful new candidates. Efficient silencing requires efficient crossing of cell membrane. This step can be alleviated using cell penetrating peptides (CPP) as carrier of drug candidates, such as peptide nucleic acids (PNAs) which inherently have poor internalization properties 2 . The aim of this study is to elucidate mechanisms of uptake in bacteria using PNA-CPP conjugates, which previously have shown promising antibacterial effects 2 .

The fate of the PNA and CPP parts of the conjugates, once inside the cell, is investigated regarding localization and possible degradation within the cell. Furthermore, a method for toxicity testing of PNA-CPPs is being developed using histamine release in RBL-2H3 cells as a quantitative measure of allergenicity of PNA-CPPs. The prospect of this information is to define boundaries within which CPPs can be found, thereby rationally designing novel efficient antibacterial biomolecular drug delivery systems. Oxytocin and its fragments have the potential to influence behavioral and cognitive functions, including their disturbances in some brain disorders. Therefore, there is an interest to synthesize new peptide-steroids chimeras for potential therapeutic use. Oxytocin analogue was synthesized in solution by coupling azido-phenylalanyl residue or p-azidopegylated handle to the N-terminal end of oxytocin molecule. Its C-terminal fragment Pro-Leu-Gly-NH2 (MIF-1) was elongated at proline residue by the same type of azido handles as well. Both peptides were marked for fluorescent detection of their possible binding on brain slices. Peptide chimeras with the suitable steroids were prepared via azide click to the triple bond on the modified steroid counterpart like (17α)-17-hydroxypregn-4-en-20-yn-3-one, 24-norchol-5-en-22-yn-3β-ol. Steroidyl-peptides were then used in the trials using rat-brain slices. The sites of the peptide-steroids chimeras bound to the brain tissue were identified with the aid of fluorescent microscopy. The suitable chimeras will be tested for their penetration through blood brain barrier for the pharmacological effects. indicating that the orientation of the n-butyl group is of primary importance. Docking studies revealed that the highly active analog affords an additional hydrophobic binding feature compared to losartan which fits to an extra hydrophobic cavity. These results may contribute to the discovery of new biologically active molecules by a convenient and cost effective synthetic strategy.

the context of pain research, the co-administration of opioid agonists and NK1 antagonists previously led to an enhanced antinociceptive potency, 1 and recently Largent-Milnes and co-workers have shown that a hybrid opioid-NK1 octapeptide was able to attenuate tolerance development, related to sustained opioid treatment. 2 Our group has prepared a compact opioid agonist-NK1 antagonist peptidomimetic chimera Dmt-D-Arg-Aba-Gly-NMe-3',5'-Bn(CF3)2 1 that served as a lead structure. 3 We report a solid phase method for the synthesis of the 4amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Aba) structure, which is used as a central unit in the investigated dual ligands. This method allowed the rapid assembly of new bifunctional ligands containing the Aba structure. Variations of the D-Arg 2 , Gly 4 and N-benzyl substituents were made. The introduction of D-Cit 2 , a Gly 4 → β-Ala4 substitution and the removal of the trifluoromethyl substituents in 1 caused considerable shifts in receptor binding. The obtained structure-activity relationships will be presented. Hence, a promising approach for the treatment of DMD is the use of drugs to force PTC readthrough. (+)-Negamycin 1 is a dipeptidic antibiotic containing a hydrazide structure. Although (+)-1 was not clinically developed due to some toxicity, it was recently reported that (+)-1 restore dystrophin expression in the muscles of mdx mice, an animal model of DMD. 1 Therefore, (+)-1 is a promising therapeutic candidate for diseases caused by nonsense mutations. Based on our own efficient total synthetic method of (+)-1, 2 structure-activity relationship (SAR) study was perfromed to discover derivatives with a potent readthrough-promoting activity. We found a derivative, (5R)-5-hydroxy-6-aminohexanoyl-glycine exhibited not antimicrobial activity but a similar readthrough activity to (+)-1, suggesting that the PTC readthrough mechanism can be distinguished from the antimicrobial mechanism. 3 Moreover, we synthesized 5-epi-negamycin and found that this analog exhibited a similar activity to (+)-1 in in vitro readthrough assay. This result hence prompted us to synthesize a 5-dehydro-derivative, e.g., 5-dehydro-3-epinegamycin 2, which is a natural product with little antimicrobial activity. Surprisingly, we found that 2 showed a higher in vitro readthrough-promoting activity than (+)-1. This result suggests that Mother Nature independently evolved readthrough-promoting products like suppressor tRNA, in distinction from aminoglycosides, which show both antimicrobial and readthrough-promoting activities. Agricultural University of Athens, Athens, Greece High interest has been paid to synthetic structural motifs that promote specific conformations because of their importance for the development of new therapeutic peptidomimetics. 1 In addition, such motifs may show catalytic activity for asymmetric organic transformations. During the last two decades, various synthetic structural motifs that promote reverse turns have been studied. Following our interest on chiral prolinamide-thioureas that present interesting organocatalytic activity, 2 we have undertaken a combined experimental/computational study to understand the structural features that may stabilize a reverse turn in short-length peptidomimetics containing a thiourea functionality. Compounds with the sequence R-Pro-diphenylethylenediamine-thiourea-Asp(OBut)-OBut (R: Boc or Fmoc, or Boc-Ala), were synthesized and studied by NMR spectroscopy (TOCSY, 1H-13 C HSQC, NOESY, ROESY spectra) for the sequential assignment and the exploration of the dipolar connectivities. Sampling of the conformational space was driven by the NOE intensities while molecular dynamics simulations were further applied to the consistent with the experimental data conformers in order to monitor the stability of the formed hydrogen bonding interactions in the course of time. Energy refined produced conformers were subsequently modified by applying all combinations of D-and L-amino acids at each site in a stepwise manner. The modelled structures were studied in silico aiming to explore the combinations of heterochiral residues which would promote a folded structure and would favour the potential of β and γ turn motif. The most promising combinations were chosen for synthesis and subsequent NMR characterization. 3 In this research project we will deal with chemical strategies to produce suitable surface modifications in order to induce multidirectional cellular migration along gold surfaces. To achieve this objective we want to use and characterize self-assembled monolayers (SAMs) of thiolated DNA chains (DNA-SH) adsorbed on gold surfaces through the hybridization with complementary modified single-stranded PNAs. PNA is a structural DNA mimic obtained by polymerization of N-(2aminoethyl) glycine monomers that replace the ribose-phosphate backbone characteristic of natural nucleic acids. It is an achiral, uncharged, and relatively rigid biopolymer of high biological and chemical stability, 4 and it can bind complementary DNA strands with higher affinity than the corresponding DNA sequences.For all these reasons we have chosen PNA as a key molecule to promote and assist the movement of cells. By producing a chemical gradient of DNA-SH along a gold surface in the presence of a chemotactic molecule it will be possible to obtain and control a directed cellular migration. The Norwegian Structural Biology Centre and The Centre for Theoretical and Computational Chemistry, Department of Chemistry, University of Tromso, 9037 Troms , Norway Renin is a highly selective aspartic protease which catalyzes the hydrolysis angiotensinogen, a protein secreted from the liver, to the decapeptide angiotensin-I. 1 Angiotensin-I is further processed by the relatively nonspecific angiotensin converting enzyme (ACE) to give the octapeptide angiotensin-II, a potent vasoconstrictor and the dominant peptide produced by the reninangiotensin system. Renin catalyses the rate determining step in the formation of angiotensin-II, and has for several decades been an established therapeutic target for drug development in relation to hypertension. 1 In the search for renin inhibitors, substituted piperidine derivatives have been identified as promising, 2-4 and piperidines have proven to be efficient scaffolds for the development of novel non-peptide aspartic protease inhibitors, particularly towards renin. [5] [6] [7] We herein describe a series of 4-triazolyl substituted piperidine derivatives that have been synthesized from N-Boc protected trans-4-ethynyl-3-hydroxy piperidine and tested as novel renin inhibitors. Piperidine derivatives containing a 1-substituted 1,2,3-triazol-5-yl substituent were found to be most active and molecular docking experiments provides a rank order that is in very good agreement with experimental data. The CXCR4/SDF-1 axis is involved in many biological processes such as hematopoiesis, immune cell migration, as well as in cancer metastasis. CXCR4 also mediates the infection of T-cells with X4-tropic HIV functioning as a coreceptor for the viral envelope protein gp120. CXCR4, as a pharmaceutical target, is of utmost importance but the lack of synthetic agonists has seriously slowed down drug development. It has been recently described by our research group 1 , that grafting the SDF-1 N-terminus onto a side-chain of the inverse agonist T140 2 . generated high affinity synthetic agonists as well as partial agonists for the chemokine receptor CXCR4. To remain stable towards proteases and act as useful pharmaceutical tools, the PK-ADME properties need to be improved with a gradual transition to peptidomimetic structures. Medicinal chemistry witnessed major advances with the discovery of small synthetic molecules that mimic the natural peptidic substrates. These small molecules do not undergo proteolytic degradation, an advantage they hold over natural counterparts. In order to improve stability against proteases, part of the SDF-1 chain was replaced with variable lengths of Polyethylene glycol and unnatural amino acids at differents positions. Here, we have produced a series of compounds, most of which showing nanomolar affinities for CXCR4 and some are displaying partial agonistic properties. TLRs are the innate immunity receptors that recognize the epitopes found on surfaces of various cells and therefore they initiate and sustain the atherogenic inflammatory response [1, 2] . We assume that the use of small STAT1 mRNA−binding PNA−inhibitors to manipulate the activity and expression of STAT1 could prove an attractive therapeutic strategy in treatment of atherosclerosis. To that end we synthesized a specific STAT1 mRNA−binding PNA inhibitor as well as a non-specific PNA to compare their inhibition of gene expression. In our work we developed effective method of synthesis of PNA−peptides conjugates by means of "click chemistry". Determination of optimal conditions for conjugation (connection of PNA with the peptide) will allow for the design of compounds useful in gene therapy. The specificity of PNA hybridization to complementary DNA fragment was verified by capillary electrophoresis (CE). As an artificially synthesized somatostatin analogue, Tyr 3octreotate (TOCA) can specifically bind to somatostatin receptor (SSTR), which are usually over-expressed on many tumor cells. Carbohydration of N-terminus of TOCA has resulted in improved pharmacokinetics and tumor targeting (1) . 18 F is an ideal nuclide for positron emission tomography (PET) imaging; there may be significant uses of 18 F labeled Glucitol-TOCA and its analogues as tumor probes for the diagnosis of SSTR-positive tumors. In order to explore a novel PET probe for diagnosis of SSTRpositive tumors, we designed a synthetic route to synthesize n-Gluc-Lys(NOTA)-TOCA, which uses 1,4,7-Triazacyclononane-1,4,7-triacetic acid (NOTA) as the chelating reagent. n-Gluc-Lys([Al 18 F]NOTA)-TOCA is radiosynthesized quickly and efficiently using the chelation reaction of Al 18 F complex and n-Gluc-Lys(NOTA)-TOCA. The aim of this study is to develop an efficient method for the synthesis of monomers of triazolic nucleic acid (TNA), a new class of artificial nucleic acids. But-2-yne-1,4-diol and nucleobases derivatives will be substrates of the monomers synthesis. TNA oligomers could be used as specific inhibitor of TAR RNA HIV-1, the regulatory RNA structure crucial for HIV replication. "Click chemistry" based on 1,3-dipolar cycloaddition will be used to conjugate an alkyne and azide derivatives of monomers subunits. A Ru (II) complex will be used as a catalyst of internal alkyne (but-2-yn-based) cycloaddition. The reaction gives exclusively of 1,4,5-trisubstituted derivative of triazole ring 1 . The monomers will be characterized using RP HPLC, capillary electrophoresis (CE) and 1 H and 13 C NMR. The resulting monomers containing Fmoc-protected amino group and a free carboxyl group will be used for the classical SPPS method to synthesize TNA oligomers. TNA sequences will be designed against TAR's bulge and an external loop. 1 through the recognition that the repertoire of polypeptide conformations can be greatly expanded by the creation of structures incorporating β-amino acids. 2 Moreover, the numerous advantages of hybrid (mixed α-and β-) backbone peptidomimetics with respect to homogeneous ones were quite recently outlined. 3 We describe here various β-amino acid-based β-hPhe-β-hPhe dipeptide derivatives, also conformationally constrained, and their application to the synthesis and biological evaluation of hybrid analogues of the opioid endogenous peptide endomorphin-2 (EM-2). The opioid system mediates a wide variety of pharmacological and physiological processes, including pain perception and modulation. The amidated tetrapeptide EM-2 has been shown to be μ-opioid receptor (MOR) agonist exhibiting a very high μ-receptor affinity and selectivity, and it is an important model in the search towards new analgesics. 4 Structural investigation of EM-2 reveals the high conformational freedom of the Phe side chains and also the inherent flexibility of the peptide backbone, indicating many probable bioactive conformations, ranging from βturns to extended conformations. With the aim of better clarify the relevant role of the proper spatial orientation of the aromatic rings and in particular of the benzyl side chains at position 3 and 4, 1 H NMR studies, molecular modelling, and molecular docking to a homology MOR model of our hybrid analogues are currently under way. The lantibiotics represent a class of antimicrobial peptides, in which the unusual amino acids dehydroalanine and dehydrobutyrine and the intramolecular thioether bridges (lanthionines) are important structural features for bioactivity.The lipid II -nisin complex is responsible for pore-formation since the C-terminal part of nisin is inserted into the bacterial cell membrane which ultimately results in cell leakage and collapse of vital ion gradients. In order to increase the metabolic stability of nisin, the oxidationsensitive thioether bridges can be replaced by metabolically stable dicarba moieties, as successfully demonstrated by the synthesis of nisin AB(C) analogs containing alkane/alkene bridges [1] . To obtain more insight into the importance of the cross-bridged DE-Ring structure (i→i+3, i+2→i+5 connectivity) on nisin's bioactivity, we synthesized a series of all four diastereomers of the crossed alkene-bridged DE-ring mimic, using ring-closing metathesis. All four diastereoisomers were obtained by HPLC and structurally characterized by NMR spectroscopy. An orthogonal protection scheme was used, to enable the independent N-or C-terminal modification of the bicyclic hexapeptides with azide/alkyne functionalities. Via Cu(I)-catalyzed cycloaddition chemistries, alkyne-functionalized natural ABC-fragments of nisin, which were obtained by tryptic digestion of full length nisin followed by HPLC purification, have been conjugated to synthetic DE-ring mimics to obtain novel nisin derivatives and their affinity toward lipid II and pore-forming capacity have been studied. Herein, we report on the details of the synthesis and characterization of the geometric isomers of the synthetic DE-ring mimics, and their use as synthons in Cu(I)-catalyzed click chemistry to obtain newly designed nisin hybrids as potential novel peptide antibiotics. Università di Ferrara, Dipartimento di Biochimica e Biologia Molecolare, Ferrara, Italy miRNAs play an important role in regulation of gene expression, being involved in numerous processes such as cell proliferation, cell differentiation, apoptosis and also in the progress of diseases as cancer and cardiovascular disorders. miRNAs associated to diseases recently become targets for the development of new drugs based on antisense oligonucleotides or analogues complementary to the chosen miRNA, in order inhibit the binding of the miRNA to its mRNA target. Therapeutic silencing of miRNA has been also observed in several animal disease model. 1 In this work we propose a new approach to interfere in the miRNA function, based on Peptide Nucleic Acid (PNA) oligomers designed to be complementary to selected regions of the miRNA precursor (pre-miRNA). As the pre-miRNA bases belonging to the stem are not perfectly complementary, we hypothesized that the mismatched duplex of the pre-miRNA could be opened by PNAs inhibiting of its maturation into miRNA. Two PNA sequences, targeting respectively the "sense region" and the " 5' end region" of the pre-miR210 were designed. PNAs were conjugated to different carrier peptides, HIV-TAT, R8, K4 and two Nuclear Localization Signal (NLS and biNLS), in order to increase their cellular uptake. To verify the ability of the designed PNAs to give strand invasion on the pre-miRNA, we conjugated also PNAs to the thiazole orange, a probe which lights-up upon hybridization 2 The development of privileged molecular scaffolds efficiently mimicking reverse turn motifs has attracted remarkable interest when structural constraints are exploited to increase both binding and selectivity of model peptides. One of the successful approaches to restrict peptide conformation is the disubstitution in the α position of an α-amino acid, leading to a conformational constraint and a stereochemically stable quaternary carbon center. In particular, spirocyclic scaffolds are able to provide, upon the attachment of appropriate functional groups, useful high-affinity ligands, relevant to the field of drug discovery. 1 At present, we are interested to spirocyclic tryptophan (Trp) analogues, in order to develop new reverse turn nucleating moieties able to be inserted into pharmacologically relevant peptidomimetic compounds. Among peptides sharing a tryptophan-containing β-turn motif of which the Trp residue is critical for binding, we looked at the hormone peptide somatostatin, 2 acting in various organ systems as a neuromodulator and a neurotransmitter, as well as a potent inhibitor of various secretory processes and cell proliferation. 3 Somatostatin and its analogue octreotide (Sandostatin® drug, clinically used for the treatment of endocrine tumors and acromegaly) are thought to interact with the sst1-5 receptors mainly by inserting a β-turn substructure, carrying a lysine (Lys) and a Trp side chain into a pocket of the G protein-coupled somatostatin receptor.

We report here the preparation and structural characterization of a new 1,2,3,4-tetrahydro-β-carboline (THBC)-based spirocyclic lactam as type-II β-turn model compound and the application of its core structure to the synthesis of a somatostatin mimetic, whose biological evaluation is under way. 

The analogues of SFTI-1 modified in the P 1 position by, βand γ-amino acids and N-substituted β-alanines R. Lukajtis, a M. Filipowicz, a A. Legowska, a D. Debowski, a A. Lesner, a K. Rolka a a Faculty of Chemistry, University of Gdansk, 80-952 Gdansk, Poland Serine proteinases play very important roles in many physiological processes in humans, such as: food digestion, fertilization of the ovum, blood clotting and dissolution of blood clots, immune response. However, their uncontrolled activity can evoke serious pathological conditions. Therefore, serine proteinase inhibitors are considered to be a promising class of therapeutic agents. Trypsin inhibitor SFTI-1, on which we focused our attention in the last decade, is an attractive template for the design of such compounds. Its primary structure is shown below: & 1 Gly-Arg-Cys(& 2 )-Thr-Lys 5 -Ser 6 -Ile-Pro-Pro-Ile-Cys(& 2 )-Phe-Pro-Asp& 1 The inherent feature of natural peptides and proteins is their low stability towards proteases, which seriously reduces their bioavailability. There is a growing need for the development of artificial biopolymers with diverse side chains, capable of mimicing peptide function. β-and γpeptides are an interesting class of peptidomimetics with significant chemical and biological properties. The present communication describes the chemical synthesis and inhibitory activity of a series of trypsin inhibitor SFTI-1 monocyclic analogues (with disulfide bridge only) modified in P1 position by βand γamino acids and N-substituted β-alanine (β-peptoid units). The following mimetics of proteinogenic Lys or Phe were used: β 3 hLys, β 3 hPhe, γ 4 hhLys, γ 4 hhPhe, βhNlys, βhNphe. All compounds were synthesized manually on solid support. β-Peptoid monomers were introduced into the peptide structure by two steps method [1] . Newly obtained SFTI-1 analogues modified in P1 position by β-derivatives of Lys and Phe were able to inhibit bovine β-trypsin and bovine αchymotrypsin, respectively, whereas the remaining ones (except for [βhNphe5]SFTI-1) appeared to be inactive. The notion that early soluble Aß intermediates are endowed with cytotoxic effects suggests that a major effort should be directed toward the inhibition of amyloid aggregation at very early stages. Inhibiting Aß self-oligomerization could, therefore, provide a useful approach to treating and controlling the pathogenic pathways underlying Alzheimer's disease (AD). Likely, agents that target the basic molecular recognition process preceding the formation of early intermediates are the most valuable candidates. We have conjugated a trehalose moiety to the known ß-sheet breakers pentapeptides LPFFD. 1 Trehalose has received a special interest because it has been found to be effective in the treatment of neurodegenerative diseases associated with peptide or protein aggregation. 2 The glycosidic moiety was covalently linked to different regions of the peptides' primary sequence, including the N-terminus or C-terminus or the aminoacid side chain. This new class of peptides showed an increased resistance to proteases. 1 In this work, the inherent ability of these peptides to recognize and bind the monomeric form of recently reported a D-amino acid-containing HIV protease inhibitor with a sulfonyl group showed an activity enhancement against drug resistant viruses. X-ray crystallographic study of the derivative revealed existence of four bridging water molecules. We suggest that the additional indirect interactions through water molecules induced the inhibitor's flexibility in binding conformation, keeping the affinity with the mutated proteases. Oxalyamide, so-called oxamide, has two carbonyl oxygen atoms as hydrogen bonding acceptor similar to sulfonyl group, which is promising to interact with water molecules. To increase the numbers of bridging water molecules, we built-in two oxamide structures to both terminals of pseudo-symmetric compounds with hydroxymethylcarbonyl-hydrazide isostere. The derivatives were tested for inhibitory activity using wildtype HIV protease and a highly mutated protease with lopinavir resistance. We found that the loss of potency against the mutated protease was relatively small in the oxamide derivatives. The molecular dynamic simulations suggested the ability of bridging water formation of the two oxamide groups.

Optimization of the PNA-synthesis using different bases for Fmoc-deprotection S. Rawer 1 , K. Braun 2 , R. Pipkorn 2 1 Life Technologies, Darmstadt, Germany 2 DKFZ, Heidelberg,Germany PNA (peptide nucleic acids) are considered as highly sensitive and specific tools for antisense strategies especially conjugated with cell penetrating peptides. Individual designed shuttle systems can be applied in cancer diagnostics and possible therapy (1) . It is, however, undisputed that proper PNAs' syntheses prove to be a challenge for coupling and Fmoc-deprotection. Due to the structure-formation the success of the synthesis depends strongly from parameters, like activator's quality and deproctection kinetics correlating to the length of the PNA polymer SPPS product. Using the example of the SPPS PNA synthesis' results of the coding sequence of c-myc human Exon II, different bases, acting as Fmoc-deprotection reagents, are compared and analyzed aiming at optimizing the PNA synthesis strategy (2) Peptidoglycans are central structural components of the cell wall of bacteria. Several plant receptors are known to recognize peptidoglycan fragments. It is believed that these receptors form part of the defense mechanism against bacterial infections in several plant species. Peptidoglycans consist of long chains of alternating β(1-4)linked GlcNAc and MurNAc moieties that are crosslinked by short, non-ribosomal peptides. These peptides consist of several D-amino acids and the symmetrical (R,S)diaminopimelic acid (meso-DAP). In particular, the latter complicates the synthesis of peptidoglycan fragments due to the requirement for individually addressing the two pairs of functional groups. Some chemical syntheses of peptidoglycan fragments have been reported [1] [2] [3] [4] , hhich involved multi-step formation of an orthogonally protected DAP moiety, and elaborate oligosaccharide synthesis.

Here we present a new and simple approach to peptidoglycan synthesis which is based on the use of commercially available building blocks for the DAP and oligosaccharide components. This allows easy access to a range of peptidoglycan fragments for structure-activity studies. The introduction of solid-phase peptide synthesis (SPPS) and the subsequent refinement of resins, linkers, coupling reagents and amino acid protecting groups allowed access to a wide range of peptides. Therapeutic peptides, in particular, have benefitted from the maturation of SPPS, as complex peptides can be synthesized more efficiently in comparison to conventional solution phase synthesis. However, peptides containing multiple disulfide bonds often still remain difficult to make due to a lack of orthogonal cysteine protecting groups that can be used in routine SPPS. The cysteine protecting group S-tertbutyl mercapto (S-tBu) is commercial and orthogonal to other cysteine protecting groups. Removal of the protecting group is facilitated by reducing agents (e.g. thiols or phosphines) and is stable to TFA and piperidine, hence compatible with Fmoc/O-tBu peptide synthesis. However, the protecting group cannot be used in routine SPPS due to long deprotection times (4-24h) . In certain cases it has been shown to be impossible to remove due to proximity of bulky protecting groups and sensitivity to certain sequences. Additionally, reports of desulfurization of S-tBu protected cysteine to dehydroalanine, by the use of prolonged exposure to reducing agents, show the limitations of this protecting group. The concept of cysteine protecting groups labile to reducing agents is promising due to orthogonality to other cysteine protecting groups and the limitations of S-tBu initiated an investigation into novel reductive cysteine protecting groups. Herein, we introduce S-TMP as a novel cysteine protecting group that is very labile to reducing agents. The increased lability, in comparison to S-tBu, allows utilization of reducing agent labile protecting groups in routine peptide synthesis of disulfide containing peptides. As modern automated SPPS protocols allow the assembly of larger and increasingly complex peptides, a precise control of the coupling reactions is a crucial prerequisite in peptide synthesis. Monitoring the progress of synthesis allows the detection of undesirable products caused by side reactions, incomplete couplings or deprotections. 1 Although different methods have been developed for monitoring SPPS, we observed that the use of colorimetric test or continuous-flow UV absorbance of the reaction column effluent was not informative enough to identify difficult steps in the synthesis.

In this study we demonstrate the usefulness of the combination of a MW-assisted mini-cleavage protocol and the UPLC-ESI-MS analysis for monitoring the quality of the reaction steps. As a proof of concept, based on this strategy, we monitored the synthesis of PTHrP(1-34)NH 2 (synthesised by Fmoc/tBu RT-SPPS, Liberty™, CEM), characterised by a cluster of arginine residues in the 19-21 region. 2 By the use of MW irradiation during the mini-cleavage protocol, we optimized time for mini-cleavages particularly in case of multi-arginine containing peptides, protected by Pbf group. The results obtained by UPLC-ESI-MS showed that the complete removal of the Pbf groups from the arginine sidechain residues required 1h at RT. On the other hand, the MW-assisted mini-cleavage monitoring let us to obtain final results just in 15 min, confirming that the use of microwave irradiation in mini-cleavages is an efficient strategy to monitor also difficult peptide couplings, such as multiarginine peptides. Identification of some deletion sequences was helpful to recognise critical couplings in order to adopt more efficient coupling strategies and therefore to optimise the final yield and purity of the crude peptide. Development of green sustainable chemistry is currently regarded as a challenge in science and technology to reduce the use of organic solvents and utilize less toxic solvents instead. Water and aqueous-based solvent systems represent an increasingly significant choice for replacing traditional solvents in synthetic chemistry. Until recently, peptide synthesis in aqueous solution has remained largely unexplored. This is because the most common building blocks are sparingly soluble in water and are considered inappropriate for in-water peptide synthesis. We have developed a method for solid-phase peptide synthesis in water, which utilizes water-insoluble Fmoc-amino acids that are converted to water-dispersible nanoparticles. In this way, the solubility problem is overcome. Our technology, which uses suspended nanoparticle reactants for the coupling reaction, offers many advantages in terms of reaction efficiency over inwater synthesis using water-soluble or non-disperse reactants. However, there are two main problems with this nanoparticle approach; (i) slow reaction rates compared to general peptide synthesis in ordinary organic solvents (ii) poor yields for the synthesis of long peptides because the protected peptide chains on the resin have a tendency to aggregate in water. MW assisted SPPS is particularly attractive because of the widespread availability of the new technology, including automated peptide synthesizers. A trial of MW assisted inwater solid-phase synthesis using non-disperse Boc-amino acids has been reported by Albericio previously. Currently, Fmoc-amino acids are routinely used as building blocks for solid-phase peptide synthesis. With this in mind, we have developed a MW irradiation procedure aimed at reducing reaction time and increasing reaction yield for in-water solidphase synthesis using water-dispersible Fmoc-amino acid nanoparticles. And we demonstrated in-water solid-phase synthesis of difficult sequence peptide with MW irradiation. 

M. Lebl, Z. Flegelova Spyder Institute Praha, Czech Republic Cotton was shown as a convenient solid phase support earlier 1-3 , but did not find wide acceptance by the peptide community. We decided to try its application as (i) a support of choice for the synthesis driven by combination of capillary forces and gravity, (ii) support for synthesis utilizing in situ neutralization Boc based protocol, (iii) support for combinatorial synthesis based on easy labeling and physical separability of cotton substrate, and (iv) support for multisupport synthesis.

-We have built a simple synthesizer in which the cotton carrier (functionalized thread) is placed inside the capillary tubing and the appropriate reagents are introduced by connecting the inlet with appropriate reagents. The speed of "pumping" the reagents is driven by the difference between the elevation of the inlet and outlet of the capillary tubing.

-We have shown that Boc solid phase synthesis utilizing in situ neutralization is compatible with cotton substrate and provides high quality products. Combining with the fact that cotton by itself 4 acts as the self-association breaking agent, makes cotton a suitable carrier for synthesis of "difficult" sequences.

-Labeling of individual solid support particles can be easily based on the length of the cotton thread pieces, number and positions of knots, or their attachment to a secondary carrier. In addition, it is possible to synthesize peptides differing by the partial structure (alternative linkers, terminal modifications, etc.) in a mixture of classical resin with labeled cotton carriers, which are easily separable at the end of the synthesis. . Use of microwave irradiation provides peptides in a fraction of time compared to conventional methods, and the peptides are also often generated in higher yield and purity. While microwave technology is particularly suited for the synthesis of "difficult" to synthesize peptides, this tool can routinely be used for the synthesis of a wide variety of peptides without the need for extensive method optimization. The focus of this study is to demonstrate how a peptide can be synthesized on a small scale (for example 25 μmol) up through larger scales (>1 mmol) with ease. As a biologically relevant model peptide the last 13 residues of the human platelet factor 4 protein (HPF4 57-10) was selected due to its significant antimicrobial activity. 1 However, the problem of developing a robust Fmoc thioester method is that the deprotection of the Fmoc group with base at each cycle is not compatible with an active ester at the C-terminus. Many ingenious approaches have been developed to generate the required thioester peptide. 2, 3, 4 The most popular has been to use an Nacylsulfonamide as a base and acid stable (safety-catch) linker for peptide synthesis. Alkylation of the sulfonamide after peptide assembly makes the linker labile to cleavage with nucleophiles. 5 Whilst popular, it has been plagued by notoriously low yields which originate from the incomplete acylation of the resin-bound sulfonamide with the C-terminal residue, incomplete alkylation of the sulfonamide and the incomplete thiolysis of the resin-bound protected peptide. In this poster we describe the development of a novel dual linker strategy 6 , involving anchoring of the sulfonamide linker to a standard acid-labile resin. This variation overcomes many of the current limitations of the sulfamylbutyryl linker approach and provides a simpler and scalable method for peptide ligation via Fmoc SPPS.

M. Ziovas, D. Tataraki, P. Manousou, N. Parveri, F. Satoglou, D. Gatos and K. Barlos Department of Chemistry, University of Patras, 26500 Patras, Greece Solid phase peptide synthesis is traditionally performed by the attachment of the C-terminal amino acid through its α-carboxyl function on a suitable solid support and elongating peptide chain towards the amino terminal of the peptide by adding sequentially the amino acid residues in the gradually growing peptide chain. Several thousands of publications and patents describe this methodology and its application for the production of peptide pharmaceuticals. In contrary to the attachment of the C-terminal carboxyl function, attachment of amino acids and peptides through an amino acid side-chain on suitable resins and their application in SPPS is described in a small number of publications and patents. Most of these publications describe the attachment of the amino acids through a side-chain carboxyl group of Asp and Glu. In the present work peptides were synthesized very efficiently in high yield and purity by anchoring of side-chain hydroxyl, amino or thiol groups of amino acids, amino acid amides, amino alcohols or small peptides on resins of the trityl or benzhydryl type. Several peptides of pharmaceutical interest, such as exenatide, octreotide, pramlintide, calcitonin, bivalirudin, insulin B-chain and others were produced as examples of this technology, either by the step-by-step procedure or by fragment condensation in solution and on solid phase.

Step Given that some of these diseases are caused by a mutation and/or malfunction of an essential protein, a better understanding of the structure and function of such proteins will allow us to prevent, slow down or even cure these diseases. To increase our knowledge, the synthesis of the target protein, a fragment involved in its activity or interacting peptides that modulate the protein activity is often required. In some cases the preparation of a protein analogue that improves its efficacy is envisage. However, conventional solid-phase peptide synthesis methods have some limitations when attempting to achieve these complex sequences of considerable length.

Using novel technologies, such as a microwave-assisted solid phase synthesis, commonly found in many peptide synthesis labs, here we performed the step-wise solidphase synthesis of a protein holding more than 100 residues (D-VEGF). This synthetic achievement indicates the suitability of this approach for synthesis of long proteins or their analogues. The detailed synthesis of the enatiomeric version of VEGF and the selenomethionine substituted analogues of HuPrP (106-140),1 proteins involved in angiogenesis and prion protein amyloidoses respectively, are described as study cases, where the use of microwaves allow us to obtain them in a fast and efficient manner. Therefore, the development of novel peptide analogues with enhanced in vivo stability could potentially provide therapeutic alternatives. The pharmacological evaluation of a bioactive peptide [Des-Gly 10 ,Tyr 5 (OMe),D-Leu 6 ,Aze-NHEt 9 ]GnRH, analogue 1, is presented herein. In vitro (kidney mouse membranes) and in vivo (clinically relevant pharmacokinetic mouse model) bioassays were coupled to liquid chromatographytandem mass spectrometry. Analogue 1, an agonist of the GnRH receptor with a binding affinity in the nanomolar range, caused testosterone release in mice that was acutely dose-dependent, an effect blocked by cetrorelix.

Repeated dosing studies in mice demonstrated that analogue 1 was well tolerated and had potency similar to that of leuprolide, based on plasma and testis testosterone reduction and histopathological findings. Analogue 1 also shared with leuprolide similar significant antiproliferative activity on androgen-dependent prostate cancer (LNCaP) cells.

On the basis of pharmacokinetic advantages, we expect that analogue 1 or analogues based on this new design will be therapeutically advantageous for the treatment of cancer and endocrine disorders. Cortexin is a polypeptide drug isolated from cattle and porcine brain cortex. Cortexin is effective in monotherapy and in combination with traditional methods of treatment.

Cortexin produces tissue-specific, regulatory, and reparative effects on the brain cortex and contains active low-molecular-weight neuropeptides (<10 kDa) penetrating through the blood-brain barrier. Cortagen is a tetrapeptide H-Ala-Glu-Asp-Pro-OH (Geropharm) produces nootropic and neuroprotective effects. Oleylcortagen is a lipophylic analog of cortagen C 17 H 32 O-Ala-Glu-Asp-Pro-OH was created for increased proteolytic stability and increased penetrating through the blood-brain barrier. The main aim of our investigation is the analysis of psychopharmacological profile of 3 peptide preparations in comparison with piracetam. Have been shown oleylcortagen (1 mg/kg) and piracetam (200 mg/kg) possess activating effect on motor and research components of behavior in «open field» test. Two of peptides (oleylcortagen, cortexin) decreased period of immobilisation and demonstrated antidepressant effects on rat behavior in the Porsolt's test, on other hand cortagen demonstrated depressant action. Therefore, the significant psychoactivating properties are typical for oleyl-cortagen, cortexin. The mechanism of the action of these peptides can be explained from the viewpoint of the regulatory cascade. They produce a direct information impact on cell structures of the brain, and then promote release of the regulatory peptides, which in turn, induce the release of the next group of peptides. Neurology, Queen Square, London WC1N 3BG, UK One of the hypotheses of Alzheimer's disease neuropathology involves beta-amyloid (βA) binding with proteins on neuronal cell surface which leads to cell lysis and amyloid plaque formation. According to the latest data α7-type of the nicotinic acetylcholine receptor (AChR) and the prion protein can be the target for betaamyloid toxicity [1, 2] . Aggregated βA causes many pathological changes in cultures of mixed neurons and astrocytes such as sporadic cytoplasmic intracellular Ca 2+ -signal, activation of reactive oxygen species (ROS) production and cell death. In the present work we demonstrated the ability of affinity purified antibodies to synthetic fragment 173-193 of α7-subunit of the AChR (AchRabs) or to peptide 95-123 of the prion protein (PrPabs) to protect cells from βA induced cell death. We also showed that both antibodies did not block βA induced Ca 2+ -signal in astrocytes. However, preincubation of cortical co-culture of neurons and astrocytes with AChRabs or PrPabs significantly reduced the rate of caspase 3 activation and the rate of βAinduced ROS production via modulating NADPH-oxidase. More detailed research of involvement of α7-type AChR revealed that α-bungarotoxin was also very effective in the inhibition of caspase 3 activation and superoxide production. The observed positive effect of antibodies to α7-type AchR or to the prion protein gives an additional explanation regarding the involvement of these proteins in AD pathology and provides new approach into an anti-AD vaccine design. Capturing and macrophage-aided clearance of amyloid beta by surface modified proteineous particles M. Richman, S. Rahimipour Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel Imbalanced homeostasis and oligomerization of Amyloidβ (Aβ) peptide in the brain are hallmarks of Alzheimer's disease (AD). Microglia and macrophages play a critical role in AD progression by clearing Aβ from the brain or inducing inflammation. Recent evidence suggests that the phagocytic pathway of Aβ may be defective in AD microglia/macrophages that contributes to the build-up concentration of Aβ in the brain. 1,2 Therefore, efforts have been directed toward developing treatments that trigger these cells to clear Aβ through alternative mechanisms. We have recently demonstrated that protein microspheres modified at their surface with multiple copies of an Aβrecognition motif can strongly bind Aβ, inhibit its aggregation and directly reduce its toxicity by sequestering it from the medium. 3 Here, we describe how the Aβ-bound microspheres can stimulate microglial cells and be phagocytosed through a mechanism that is distinct from that of Aβ. The phagocytosis was mostly effective with microspheres having diameter size of about 0.7-1 mm and introduction of polyethylene glycol to the surface of the microspheres changed the kinetics of the phagocytosis. Moreover, while aggregated Aβ induced a significant inflammatory response that was manifested by the release of TNF-α from the microglial cells, the Aβ-bound microspheres dramatically reduced the amount of the released cytokine. Our data suggest that surface-modified microspheres could be utilized to detoxify other pathogenic or misfolded proteins that their accumulation may lead to genesis of other diseases. Vasoactive intestinal peptide (VIP) and its derivatives have been thought to be promising drug candidates for airway inflammatory diseases. However, the therapeutic potential of VIPs is highly limited because of rapid metabolic degradation and systemic side effects following systemic administration. Previously, to overcome these drawbacks, our group developed a novel VIP derivative, [Arg 15, 20, 21 , Leu 17 ]-VIP-GRR (IK312532), with improved metabolic stability (1), and respirable powder (RP) formulation of IK312532 (IK312532-RP) for pulmonary administration (2) . These attempts successfully led to enhanced pharmacological effects of IK312532 in the airway system and reduced systemic exposure; however, further chemical modification of IK312532 with a focus on metabolic stability might provide better clinical outcome. The present study aimed to design a PEGylated VIP derivative with improved metabolic stability and to develop its respirable powder (RP) formulation for inhalation therapy. IK312532 was chemically conjugated with PEG (5 kDa, P5K), the physicochemical and biochemical properties of which were characterized by CD spectral analysis, binding assay, and metabolic stability. The RP formulation of PEGylated IK312532 (IK312532/P5K) was prepared with a jet mill, and in vitro inhalation performance and in vivo pharmacological effects in antigen-sensitized rats were also evaluated. The CD spectral analysis demonstrated that PEG conjugation had no impact on the solution structure of IK312532. Although receptor-binding activity of the IK312532/P5K (IC50: 82 nM) was estimated at ca. 30-fold less than that of IK312532 (IC50: 2.8 nM), metabolic stability for the IK312532/P5K was highly improved. According to the laser diffraction and cascade impactor analyses, IK312532/P5K-RP had fine in vitro inhalation performance. Insufflation of IK312532/P5K-RP (150 μg of IK312532/P5K) in antigen-sensitized rats resulted in marked attenuation of inflammatory events, as evidenced by significant decrease of inflammatory biomarkers and granulocyte recruitment in pulmonary tissue at 24 h after antigen challenge. From these findings, PEGylation of VIP derivative, as well as its strategic application to the RP formulation, might be a viable approach to improve its therapeutic potential for treatment of airway inflammatory diseases. The previous studies have shown that trkb (tropomyocin receptor kinase) acts as an oncogenic agent and its binding to BDNF (brain derived neurotrophic factor) activates signaling angiogenesis of tumor proliferation [1] . For finding the most stable and potentially effective peptides against the trkb, we applied the following protocol. At the first step of this protocol we designed a peptide library by using sequence tolerance method in Rosetta 3.3 package, then peptide energy optimization performed by backrub protocol for finding the most stable peptides. The five best peptides in energy optimization selected based on backrup scores by using R package [2] . 3D-structure prediction of the selected peptides was performed by using molecular dynamic in Hyperchem 7 software. Docking of peptides with trkb receptor was carried out in HADDOCK software. We used cyclotraxin, a selective TrkB inhibitor as positive control for this protocol. Cyclotraxin and the peptides were compared by ANOVA or T-test. The peptides are going to be tested against the trkb in an in vitro model. Dirucotide (MBP 82-98 ) is a synthetic peptide analog of , that consists of 17 amino acids and tested in a phase trial were failed to reach his tolerance level on previous phase II in RPMS patients. One of the major disadvantages of peptide therapy is the activation of proteolytic enzymes, leading to peptide degradation. To address this problem cyclic peptide analogues have been synthesized. Thus we synthesise a linear and cyclic analogues of dirucotide. The two analogues were synthesized by changing the amino acid residue at position 91 from to The synthesis of the linear peptide, as well as of the cyclic one, was carried out by the Fmoc/tBu methodology, utilizing the 2-chlorotrityl chloride resin (CLTR-Cl). The purification was achieved using semi-preparative RP-HPLC and the identification was assessed by analytical RP-HPLC and by mass spectrometry (ESI-MS). The linear and cyclic peptide analogues will be used in human T-cell cultures to test their immunogenicity in patients versus healthy controls. 1 In the first approach a reporter moiety was introduced to diagnose and treat CXCR4 related diseases. Therefore, an anchor point to attach additional molecules to the ligand was elucidated. Using SAR studies to optimize the linker from the ligand to the detectable moiety the excellent receptor affinity could be retained. In a final step the reporter moiety was introduced to give a ligand for diagnosis via PET imaging and for possible endoradiotherapeutic applications. 2 Originating from the dimeric motif present in many active CXCR4 ligands several dimers were prepared using a monomeric ligand identified in the prior study. Comparison of monomers and dimers yielded a possible subsite binding mode explaining why the dimers exhibit enhanced affinity using a model derived from the origins of the monomer. 3 Several peptidomimetic modifications were introduced to the ligand to reduce the peptidic structure. In a conformationally guided approach introduction of a peptoid motif could enforce a single active conformer that was enhanced through subsequent modifications. This yielded a compound 100 times better than the original CXCR4 antagonist which is the most affine CXCR4 ligand reported so far. Our previous studies have demonstrated that PACE4 represents a potential therapeutic target for the treatment of prostate cancer 1 . Moreover, we have developed potent and selective PACE4 inhibitor, (20-fold specificity over furin), known as Multi-Leu or ML peptide, which has a significant inhibitory effect on the proliferation of prostate cancer cell lines. Peptide-based drug candidates can be limited by their poor metabolic stability and low bioavailability. Thus, we performed structure-activity relationship studies to improve the pharmacokinetic properties of our ML inhibitor. We have designed and synthesized new ML peptide analogs having various chemical modifications. First, based on our previous results, we combined the most effective modifications of position P8 (D-amino acids) and P1 the arginine mimetic 4amidinobenzylamine (AMBA) to improve overall properties of our leading compound. Second, the N-terminus of the resulting analogs was modified with a fatty acid, in order to enhance their cell permeability properties. Third, we modified the inhibitors with a PEG moiety to increase their stability and bioavailability. We tested the inhibitory activity, stability in plasma, cellular uptake, and cytotoxicity of each inhibitor. The results of this study demonstrate that the presence of the N-terminal extension (C8 or PEG8) does not affect activity of our inhibitors. On the other hand, we show that introduction of the PEG moiety does not increase cytotoxicity of ML analogs. It is interesting to note that the PEGylated analog Ac-PEG8-D-Leu-LLLRVKR-AMBA has better cell-permeability activity than its counterpart without PEG unit. This combination of pharmacological properties makes our new ML analogs promising candidates for the development of potential anti-prostate cancer agents. [1] Peripheral coadministration of RF9 with opioid analgesics led to confirm the involvement of NPFF receptors as a part of the antiopioid system. Indeed, RF9 was able to reverse the opioid induced hyperalgesia in rat (Randall Selitto test). Then, a complete structure-activity relationships analysis was performed with RF9, assessing the involvement of each moiety for affinity and selectivity towards both NPFF receptors. A first exploration of the N-terminus part of RF9 (>80 synthesized derivatives) led to replace the hydrophobic adamantane moiety by a hindered aromatic group, providing a subnanomolar NPFF1 ligand with more than two log-units selectivity against NPFF2. Then, the removal of the Cterminal amide function led to reduce the dipeptide Arg-Phe-NH2 into an arginine derivative. In spite of an initial loss of affinity, optimization of the phenethyl moiety at the Cterminus part of arginine led to non-selective nanomolar ligands for both NPFF1 & 2 receptors. Next, we applied efficient methodologies in order to synthesize non-natural analogs of arginine, leading to various compounds exhibiting selectivity for either NPFF1 or NPFF2 receptors. In particular, compound RF313 was identified as a selective NPFF1 antagonist (NPFF1, Ki = 64 nM; NPFF2: Ki > 13 μM). Lacking of analgesia properties, oral administration of RF313 (1 mg/Kg per os) to rats was able to fully reverse fentanylinduced hyperalgesia. RF313 is the first orally available NPFF1 antagonist capable of reversing opioid induced hyperalgesia at low dose. Moreover, this result allows identifying for the first time NPFF1 receptor as a key-partner of the anti-opioid system. Administration of multiple antiplatelet agents has become the mainstay in the treatment of acute coronary syndromes in everyday clinical practice. We have previously reported significant antiplatelet effects of novel synthetic peptides' single administration on experimental carotid artery thrombosis in rabbits 1 . In the present study we sought to investigate the peptides' effects when administered in marginally effective doses (significantly lower than those utilized in the past), in animals that had previously received low doses of aspirin. The peptides when co-administered with aspirin preserved the carotid artery's blood flow, in contrast to the total artery occlusion observed in animals receiving aspirin and placebo. Blood flow at 90 min after electrical stimulation was reduced to 56.7±7.9% and 33.2±0.3% in the YMESRADR and PSRCDCR-NH2 groups respectively (p<0.001 vs aspirin and control). Thrombus weight was significantly reduced in animals receiving YMESRADR and PSRCDCR-NH2 versus aspirin and control (3.9±0.3 mg and 3.1±0.4 mg, vs 7.8±2.2 mg and 5.7±0.8 mg respectively, p<0.05). Platelet aggregation was significantly inhibited in the YMESRADR and PSRCDCR-NH2 groups by 36.0±14.1% and 45.0±12.0% for ADP (p<0.05 vs aspirin and control), and 35.5±6.3% and 54.2±5.6% for AA (p<0.05 vs aspirin and control), respectively. Blood loss did not significantly differ among the various groups. Administration of novel synthetic peptides, even at marginally effective doses, in animals previously treated with low doses of aspirin results in enhanced antiplatelet effects in an experimental model of arterial thrombosis. The study of peptide metabolism is particularly important when examining anticancer peptides; it can provide pivotal clues for the evaluation and improvement of stability of a peptide drug leading to enhanced pharmacokinetics and efficacy. GnRH analogues are used for the treatment of prostate cancer. As with other peptides a drawback is their short half-life due to their metabolic degradation. In order to examine the stability of these analogues we have developed several in vitro peptide stability and metabolism assays using specific tissues, isolated membranes, cancer cells that are analyzed subsequently using LC-MS/MS based approaches. Such in vitro studies are followed up with pharmacokinetic studies in mice in order to establish the correlation between in vitro and in vivo approaches. The kidney is the main metabolic organ for peptide metabolism and for that reason we employed a peptide stability and metabolism assay previously described by our group using isolated mouse kidney membranes for the evaluation and comparison of different GnRH analogues. We tested GnRH analogues in other tissues such as mouse plasma, which is the "distributing" tissue for these drugs and mouse brain homogenate, a tissue known for its abundance in peptidases and relevance for centrally mediated effects. Stability studies were also performed in cancer cells. In all cases LC-MS/MS based assays were developed for measurement of peptide drug and the resulting metabolites. For triptorelin, and a series of GnRH analogues, degradation and metabolite formation was studied by our mouse kidney membranes assay. Studies of coadministering the peptide of interest with inhibitors that are presumed to block the metabolism in mice are ongoing. The vulnerability of GnRH analogues was verified after incubation with plasma and brain homogenate and by metabolite identification we obtained clues about the key cleavage sites. The described in vitro/in vivo protocols provide valuable information that could lead to the synthesis of more stable anticancer peptides with improved anticancer efficacy. In this work, we explored the use of a high-throughput synthesis and screening approach with peptide arrays to identify and structurally optimize shortened HIV-1 fusion inhibitors. The peptide array technology involves miniaturized synthesis of immobilized peptides, followed by affinity testing with a five-helix bundle, as a mimetic of the fusogenic gp41 protein. This exercise resulted in the identification of a class of truncated peptides which demonstrates a surprisingly high antiviral potency, despite the absence of the pocket-and the lipid-binding domain. The propensity of these peptides to adopt the bioactive α-helical conformation in solution as determined by circular dichroism, could be the key factor for this unexpected potency. These peptides are promising leads for the treatment and prevention of HIV. The pathological role of platelets in cardiovascular disease (CVD) is well established. Platelets secrete ADP to recruit additional platelets to a thrombotic site. We have previously identified novel cell-permeable peptide modulators of platelet function by using a bioinformatic screen based on patterns of evolutionary conservation in transmembrane proteins 1 . In this study we further explored peptides derived from cadherin cell adhesion molecules. We explored a range of 14 overlapping peptides derived from different cadherins varying from 6-15 amino acids long.

Peptides are synthesized and analyzed in a high-throughput platelet ADP secretion assay. Peptides (0.05-50μM) were assessed in the presence and absence of thrombin receptor activating peptide (TRAP;4μM). We identified CADH-5 and 6 proteins, but not CADH 1 or 2 in human platelets using western blotting and mass spectrometric analysis. Peptides derived from paralogous juxta-membrane, cytoplasmic regions of these cadherins are potent modulators of platelet secretion. By systematically deleting amino acids from C or N-terminus of active peptides, we established that the minimal functional sequence for biological activity was a short six-residue motif, which corresponds to the known catenin-binding region of cadherin tails (KEPLLP) 2 . Peptides alone have no biological activity. However, they potentiate the response induced by the platelet agonist TRAP at low doses. Thus we have identified a cadherin-derived peptide that can modulate platelet secretion events. This highlights a previously unknown role of cadherins in the regulation of platelet function. Agents that interfere with cadherin signaling in platelets might have a therapeutic role in CVD. Ageing of the brain leads to impairments in cognitive and motor skills, and is the main risk factor for several common neurological disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD). Altered protein handling (proteolysis, repair system, chaperones) forms a basis for a large number of protein conformational disorders. Extra-and intracellular, as well as intranuclear accumulation of abnormal proteins, in the form of protein inclusions and aggregates, and dysfunction of the quality control mechanisms are common in all these disorders. Alterations in protein homeostasis occur with age, causing molecular changes such as protein misfolding and aggregation. Many biologically active proteins lack stable secondary and tertiary structure, these are called intrinsically disordered proteins (IDPs), some of them (e.g. β-amyloid, α-synuclein) are coupled to neurodegenerative disorders. IDPs exist as assemblies of rapidly fluctuating structures undergoing coupled folding and aggregation process. Protein aggregation is characterized by polymorphism, where a mixture of soluble oligomers, amyloid fibrils and amorphous aggregates is the final product. Soluble oligomers are inevitable formed during the self-association process and might initiate the neurodegenerative cascades of AD, PD and similar diseases. The emerging consensus that protein misfolding (leading to IDPs) is the cause of several neurodegenerative disorders now offers the opportunity to develop a general therapy. Soluble oligomers with ID regions are potential drug targets. Recently short peptide fragments and small peptidomimetic molecules have been found also in our laboratory; these molecules bind to the ID regions of β-amyloid and are putative drug candidates. Precise control of bleeding is ensured by anticoagulant mechanisms, which under normal conditions prevail over coagulants mechanisms. Disrupting the balance between procoagulant and anticoagulant systems due to congenital or acquired defects leading to thrombotic disorders. Anticoagulants are substances that inhibit blood clot formation. Their action consists in inhibiting the synthesis of prothrombin, the substances forming thrombus as well as some coagulation factors. Many peptides and proteins with different molecular weight, such as antistasin (ATS), ghilantens, hirudin, etc. showing high anticoagulant activity are isolated from salivary glands of ticks and leeches. They inhibit the action of serine proteinases from blood coagulation cascade. This creates an opportunity for targeted synthesis of low molecular weight analogues of some of these proteins, which can be used in the prevention and treatment of thrombotic disorders. ATS is competitive, slow-binding inhibitor of factor Xa. It is well known that blood coagulation could be blocked at different stages of the coagulation cascade through inhibition of various enzymes. Therefore, it is interesting to determine the place of action of newly synthesized antithrombotic agents in the blood coagulation cascade. This can be done by determining the inhibitory constants of newly synthesized peptides on different enzymes of this cascade.

Herein we report on our kinetic investigation of newly synthesized peptide amides, analogues of isoform 2 of ATS 1 . Ki, Km, Vmax and type of inhibition on the factor Xa, thrombin and plasmin were determinate. Some interesting differences between type of inhibition of ATS, free acids and amide analogues of ATS were revealed. To evaluate the relative anti-platelet aggregation activities of each peptide, the lebetins were chemically synthesized and fully characterized.

Here we described the synthesis, the solution structure of lebetin G1-G38 from the venom of Vepera lebetina by 1 H bidimensional NMR and the relation structure-activity. This peptide has been demonstrated to be associated with a potent anti-platelet aggregating activity. The G1-G38 three dimensional structure consists in a compact β-bulged hairpain core from which emerges one loop and the Cterminus and the N-terminus. We report on an approach whereby ligands are designed to bind and stabilize the 13-26 region of Aβ in an α-helical conformation. These ligands reduce Aβ toxicity to cells in culture and to hippocampal slice preparations. In addition, when these inhibitors are administered to Drosophila melanogaster expressing human Aβ (1-42) in the central nervous system, a prolonged lifespan, increased locomotor activity, and reduced neurodegeneration is observed 1 . Stabilization of the central Aβ α-helix appears to counteract polymerization into toxic assemblies and indicates that this approach holds promise for the development of orally available compounds against Alzheimer's disease. Encouraged by the above results we are currently developing a second generation of designed ligands. This involves synthesis of different new peptoids and unnatural amino acids. Additional support for the concept comes from recent molecular dynamics simulations that also uncover details of the mechanism of unfolding of the Aβ central helix 2 as well as retardation of the folding in presence of ligands designed to interact with the native helical conformation 3 . Synthesis and methodology for new ligands, which includes synthesis of novel amino acids, will also be presented. Triostin A is a well-known natural product with antibiotic, antiviral, and antitumor activities. It inhibits RNA synthesis by bifunctional intercalation into DNA base pairs through its two quinoxaline units showing CpG selectivity. Triostin A must adopt an altered conformation upon DNA bisintercalation that is substantially different than its preferred native X-ray or solution conformation. This fact suggests that the destabilizing conformational change in the cyclic octadepsipeptide counteracts much of the gains derived from a second intercalation. Nonetheless, the wide range of pharmacological activities exhibited by this compound prompted interests in identifying novel and additionally potent lead compounds with improved pharmacokinetic properties for clinical applications.

Herein, a library of twelve simplified Triostin A analogues has been synthesized by solid-phase peptide synthesis.

The introduction of the key quinoxaline units was carried out in solution.

The analogues' conformation corresponds to the staple form that bisintercalator cyclic (depsi)peptides adopt when binding to DNA and, in addition, some of the synthesized compounds showed improved solubility. Our library was evaluated for its antiproliferative activity against four human cancer cell lines and one analogue showed greater cytotoxicity than Triostin A and even comparable activity to doxorubicin, a very commonly used drug in cancer chemotherapy nowadays. Surprisingly, little is known about its mechanism of degradation in solution or the degradation products. A recent study identified monomeric polysulfides and dimeric degradation products, postulated to derive from β-elimination followed by deamidation and dimerization. 1 We recently reported that degradation of oxytocin and its analogues in aqueous solution at pH 7.4 produced monomeric polysulfides with up to 6 sulfur atoms as well as dimeric products. 2 Unexpectedly, incubation of OT or of various analogues modified in position 1 resulted in identical dimeric degradants. We concluded that β-elimination via breakage of the C-S bond of Cys1 must be a key step of the process, and that the resulting ΔAla 1 residue would have to undergo further modifications to yield the same dimeric products independently of the substitution of the N-terminal nitrogen.

Here we further clarify the degradation mechanism and propose a structure for the dimers. We postulate that hydrolysis of the ΔAla 1 residue yields an N-pyruvoyl linear peptide, which loses one sulfur atom and subsequently forms dimers, which we found are linked by one disulfide bridge and one non-reducible bond. The putative linear N-pyruvoyl oxytocin intermediate was synthesized and found to degrade to the same dimers as the ones in the incubations of OT. A [U-13 C]Cys 1 OT analogue gave degradation products with 13 C-NMR spectra consistent with a non-stereospecific aldol-type condensation. Detailed experimental procedures, structures of the degradants and the postulated mechanism of OT degradation in near neutral solutions will be presented. INSERM U765 Paris, France αIIbβ3 is the main platelet integrin and is responsible for platelet aggregation. A lipid-modified peptide corresponding to αIIb intracellular sequence 1000-1008 (palmitoyl-K-L 1000 EEDDEEGE 1008 , pal-K-1000-1008), is platelet permeable and inhibits human platelet aggregation induced by thrombin 1 . YMESRADR, a peptide corresponding to the extra-cellular sequence 313-320 of αIIb, is a platelet activation and aggregation inhibitor 2 . The aim of the present study was to investigate the cooperativeness of the intracellular and extra-cellular peptides on platelet aggregation and their effect on the phosphorylation of FAK and ERK. Pal-K-1000-1008 together with the extracellular YMESRADR peptide, at concentrations lower than their IC50 values, showed cooperative inhibition of platelet aggregation. The peptide combination inhibited also fibrinogen and PAC-1 binding to activated platelets. FAK phosphorylation is a postaggregation event related to outside-in activation of the receptor. The combination of peptides inhibited FAK phosphorylation. ERK phosphorylation is independent from platelet aggregation, and is enhanced by RGD-peptide inhibitors. The combined peptides inhibited ERK2 phosphorylation. Ovarian cancer (OC) is considered a rare disease and represents the fifth most common cause of death from cancer in women. The standard first-line treatment consists of a combination of paclitaxel and carboplatin (DDP) or carboplatin alone. In the case of progressive disease or drug resistance to platinum-based agents, either alone or in combination, investigational compounds should be used (1) . The mechanisms behind acquired resistance to DDP and its derivatives are not clear yet, although it is evident that the process is multifactorial, including enhanced DNA repair processes. Some peptides designed from the interface subunit of the human Thymidylate synthase (TS) have been identified recently (2), as effective anticancer agents against sensitive and resistant OC cells. One of them was also able to recover the cellular sensitivity towards cisplatin in resistant OC cells in the μM range. To improve its potency and selectivity structural studies have been performed in combination with cellular assays aimed at understanding the mechanism of action. A label-free quantitative proteomic approach has been undertaken to study the effects of the peptide on the proteins involved in the modulated metabolic pathways, in particular those involved in the folate metabolism. Structure-activity relationships (SAR) have been performed to improve the lead peptide pharmacodynamics. All the compounds have been assayed and a protein profile set was studied to mark and validate their behavior as inhibitor of OC cell growth. Hepatitis C is a liver disease provoked by a virus known as HCV. The disease is insidious. HCV causes anorexia, nausea, vomiting, fever, fatigue and jaundice. In about 40% of sufferers the disease is short, but others become chronic. In the chronic form in about 20% of cases the final result is cirrhosis of the liver and in the remaining 20% it leads to liver cancer. HCV is a very serious problem today. About 3% of people infected with HCV worldwide, i.e. about 4 million are residents of Europe. 170 million people carry the disease as a chronic illness with the potential to develop into cancer in their liver. All these people represent a "reservoir" for storage and distribution of HCV. Ribavirin, the nucleoside analog 1-β-D-ribofuranosyl-1,2,4triazole-3-carboxamide, known by the trade name Virazole (also known as Rebetron in combination with interferon-α), exhibits antiviral activity against a variety of RNA viruses (paramyxoviruses, flaviviruses, etc.) as well as some DNA viruses. In humans ribavirin is currently used in combination with interferon-α to treat HCV infections. This lack of strict specificity and a broad spectrum of activity are due to its multifunctional mechanism of action against viruses. These characteristics have made ribavirin a drug of substantial research interest. Unfortunately, ribavirin shows a significant toxicity, causing bleeding in accumulation [1] . Herein, we report a total synthesis of modified in position 5' of ribose residue, ribavirin in order to be further linked to cell penetrating peptides (CPPs). In addition the synthesis of some CPPs as well as bonding between two parts of final hybrid molecules will be reported. In our case the design of new hybrid molecules is done in order to: (a) vectorize ribavirin into liver cells; (b) transport ribavirin molecule trough cell membrane and (c) decrease toxicity of ribavirin. To obtain oligomeric Aβ peptide, our laboratory uses a precursor depsipeptide of Aβ. This precursor, termed as "iso-Aβ" has an ester bond between the side chain of Ser 26 and Gly 25 . At physiological pH this ester bond becomes an amide bond via an O→N acyl shift. Binding partners by which the oligomeric Aβ mediates its toxic effect has not been yet investigated in the proteome or subproteome level. We used protein array technology to study the interaction of oligomeric Aβ with 8163 recombinant human proteins, immobilized on a protein chip. Aβ binding proteins were identified with the aid of a monoclonal Aβ antibody. Altogether 324 proteins were found to interact with our Aβ-oligomers. These proteins were grouped according to their function. One of the major groups contained 24 proteins participating in translation. These proteins were found in ribosomes. To prove our proteomic results ribosomes from rat hippocampus were isolated. ELISA experiment revealed that Aβ binds to ribosomes in a dose-dependent manner.

Using the sequence of the 324 Aβ-binding proteins a homology search was performed to find oligopeptides, that possibly bind to Aβ. Based on these sequences a peptide chip containing hexapeptides was prepared. Aβ interacting peptides were identified with a monoclonal antibody.

Several peptides were synthesized and tested on MTT assay. Two out of four compounds inhibited the toxicity of Aβ on rat hippocampal slices. Summarizing our results Aβ binding proteins and peptides were identified. Knowledge about Aβ binding proteins can help to understand the pathogenesis of AD, such us the possible involvement of ribosomes. Oligopeptides can be lead compounds of future drug development. Huge proteolytic complex named proteasome catalyzes protein degradation in every eukaryotic cell. It consists of 31 subunits forming four stacked rings and one or two regulatory caps. Two inner rings of the proteolytic part contain three catalytic β-subunits that possess different substrate specificity. Higher vertebrates can express γinterferon-inducible immuno-β-subunits. Proteasome plays an essential role in continual turnover of intracellular proteins and in antigen processing. Autoimmune diseases such as multiple sclerosis and its murine model EAE are believed to rise from breakdown of tolerance of the immune system. It assumed that immunoproteasome could play an important role in autoimmune diseases. Several classes of chemicals proved to be inhibitors of proteasome and the most active are boronate peptide derivatives. These inhibitors totally inactivate proteasome and result in full stop of intracellular protein turnover and cell death via apoptosis. Another class of inhibitors, epoxy ketones, was shown to be more selective for immunoproteasome and could be used not for full stop of proteasome function, but for fine tuning of altered proteasome functioning. We examined properties of several inhibitors of four different classes, namely peptide boronate bortezomib, peptide aldehyde MG132, lactam lactacystin, and peptide epoxyketones epoxomicin, MG132ek, UK101 and PR-957. For inhibition experiments we used proteasome isolated from eukaryotic cell lines CHO, NSO and HEK, treated and non-treated with γ-interferon, as a model cells contatinig constitutive and immunoproteasome. The upregulation of proteasome immunosubunits was revealed in CHO and NSO cells treated with γ-interferon.

The IC50 values for all studied inhibitors were obtained, and Ki in some cases were calculated. The epoxyketones were shown to selectively inhibit in submicromolar concentrations the proteasome sample which contain high amount of immunosubunits. In order to find an effective antimalarial, this study refers to some Angiotensin II (AII) analogues which were considered the important physicochemical characteristics described by Silva et al. 1 to verify the biological activity against Plasmodium gallinaceum and to understand the hydrophobic cluster influence, explained by Tzakos et al. 2 These analogues were synthesized and characterized as described by Silva 1 , as well as the biological assays and comprises, to verify: the hydrophobic cluster activity -A) DRVYHIPF; B) DRVYPR; C) RYHIPF and D) FPHIYVRD; the importance of these residues in AII molecule -E) RYPF; the importance of aromatic residues -F) YHPF and the action of these hydrophobic residues, when interacting with the parasite membrane -G) VIPF. It was observed that in A (94% of bioactivity), the phenol group of Tyr is close to imidazole group of His that could promote a hydrogen bond formation. Besides that, could occur van der Waals interactions between Ile and Phe residues due its proximity and non-polar characteristic. These interactions could not be effective in native AII (88%) 3 , because Ile residue promote a steric influence on the organization of His and Tyr residues 4 that not exist in B (57%). In C (74%) and E (72%) analogues, the influence of the Arg residue could promote a cation-π interaction with Tyr residue 5 and the cluster may have suffered slight destabilization and its antiplasmodial activity was compromised subtly. In D (12%), the electrostatic change, obtained with the total inversion can have disordered its interaction with parasite membrane, since it is not related to membrane receptors, because D-AII presented 91% of biological activity. Moreover, hydrophobic and aromatic residues importance was confirmed through the results obtained 93% and 89% of activity, with G and F, respectively. We conclude that hydrophobic cluster modifications and interactions of amino acid side-chain influences in the biological activity. Closed Joint-Stock Company "Vertex", St-Petersburg, Russia Creatine (Cr), a small molecule synthesized in the kidney, liver and pancreas plays important role in ATP synthesis, replenishing its store even in the absence of oxygen. Cr is able to protect brain cells against ischemic damage; however it has poor ability to penetrate the blood-brain barrier without specific carrier protein. Thus, synthesis of stable hydrophobic derivatives capable of crossing the BBB by alternative pathway is of great importance for the treatment of different neurological diseases including stroke, traumatic brain injury and hereditary CRT deficiency.

Here we describe the synthesis and biological activity of new hybrid compounds -creatinyl amino acids. Originally the title compounds were synthesized by guanidinylation of sarcosyl peptides. However, for large scale synthesis better results can be obtained using direct Cr conjugation with amino acid or peptide derivatives by isobutyl chloroformate method. Addition of equivalent amount of ptoluenesulfonic acid as lipophilic counterion ensures efficient Cr dissolution in DMF along with its simultaneous protection towards intramolecular cyclization. It excludes the application of expensive guanidinylating reagents and permits to simplify the synthetic procedure. Purification of final product and its conversion into appropriate salt form can be achieved by IEC followed by crystallization from organic solvents. Synthesized creatinyl amino acids and peptides exhibited significant biological activity in different assays including platelet aggregation test, ischemic stroke and NaNO2-induced hypoxia model. One of the most effective compounds -creatinyl-glycine ethyl ester increases life span of experimental animals more than two times in hypoxia model and has neuroprotective action in brain stroke model when applied both before and after ischemia. These data evidenced that creatinyl amino acids can represent promising candidates for the development of new drugs useful in stroke treatment. The efficient recognition and destruction of tumor cells via specific cellular markers is a major goal in cancer therapy. Various growth factor receptors such as EGFR, HGFR, VGFR and their downstream signaling networks have been proven to be effective molecular targets, as they are frequently involved in cancer proliferation and metastasis. Downregulation of these receptors and/or blocking their signaling pathways have clear anti-tumoral effects. 1 Drugs based on monoclonal antibodies (mAb) targeting such cell surface receptors have attracted a lot of attention as a new generation of therapeutics. However, their production is costly and identifying new, variable routes to modified molecules with similar properties is currently a major focus. 2, 3 Here we present an approach to chemically synthesize a molecule that combines the mode of action of antibodies with the advantages of smaller, chemically accessible molecules. These "synthetic antibody" (SAB) molecules contain a chemoattractant that activates the innate immune response and resembles the Fc domain of a typical antibody. Specificity is imparted by two binder peptides that assume the function of the variable antibody domains and bind to a cell surface target. The Fc and Fab domains of the SAB molecules are connected via polyethylene glycol linkers. SAB molecules are prepared by solid phase synthesis, a flexible technique that allows fast production, full control of their properties and targeting two different cell surface receptors (bispecific tumor targeting). They are currently tested in vitro and in vivo for their effect on the innate immune system, general toxicity and selective binding to cancer cells. The key enzyme in the processing of polyproteins translated by viral RNA genome of SARS-CoV is a 33kDa protease called 3C-like protease (3CL protease). SARS 3CL protease is a cysteine protease containing a Cys-His catalytic dyad, and cleaves precursor poly proteins at as many as 11 conserved site involved a conserved Gln at the P1 position and a small amino acid (Ser, Ala, or Gly) at the P'1 position. Due to its functional importance in the viral life cycle, SARS 3CL protease is considered to be an attractive target for drug design against SARS. Recently, we found tetrapeptide aldehyde, Ac-Thr-Val-Cha-His-H, showed high inhibitory activity with IC50 value of 98 nM toward 3CL-R188I mutant protease 1,2 . To compare the inhibitory activity of small compounds with those containing active functional groups, we synthesized serine-derivatives within the essential functional groups and evaluated its inhibitory activity. The synthetic scheme was started from Fmoc-Ser(tBu)-OH, following modification of C-terminal carboxyl group with P2, N-terminal amine with P4 and side chain alcohol with P1 functionalities. 5 steps overall reaction led to obtain 44 novel serine derivatives for the small molecular inhibitors of SARS 3CL protease. The assay with 3CL R188I mutant protease was examined to evaluate the inhibitory activity of the synthetic serine derivatives. Then, molecular docking study of complex of 3CL protease with the ligand was carried out. Docking simulation experiment with R188I (PDB ID: 3AW0) and the inhibitor, which has the best activity in the serine derivatives, indicated that P1 fitting S1' pocket. At the result of assay, P1, P2 and P4 positions of the inhibitor should be modified by benzoyl group, cyclohexyl group and cinnamoyl group, respectively. Their bioactivities are underpinned by their distinctive structure with exceptional stability, thus making cyclotides exciting, not only for agricultural and pharmaceutical purposes, but also as a template in drug design. In all of the reported activities, cell membranes seem to be the primary target for cyclotide activity. To unravel the importance of lipid membranes on the reported activities of cyclotides, a set of cyclotides belonging to Möbius and bracelet subfamilies were compared in their mode of action. The lipid selectivity and membrane affinity were compared with their efficiency against different target cells (e.g. red blood cells, bacteria, HIV particles). We have found that the bioactivity of cyclotides is dependent on the lipid composition of the target cell membrane and independent of a protein chiral receptor. In particular, all the native cyclotides tested target the cell membrane through specific binding to phospholipids containing phosphatidylethanolamine (PE)headgroups, but the membrane binding affinity is further modulated thorough non-specific peptide-lipid hydrophobic interactions, which are dependent on the specific cyclotide.

In addition, the bioefficiency of cyclotides broadly correlate with their ability to target and disrupt the cell membrane.

Overall, we have shown that even with a common specificity for membranes containing PE-phospholipids, a fine selection was found across the family. In particular, each cyclotide inserts and disturbs the membrane in a distinct way, which explains the diversity of this family but also their distinct activities.

The observation that all the tested cyclotides have a preference for a specific lipid makes this family truly intriguing and brings insights to optimize the use of the cyclotide template in drug design. Malaria is a disease that affects around 500 million people causing 0.5-1 million of deaths annually. Based on our previous studies, Angiotensin II (AII) presented antiplasmodial activity against Plasmodium gallinaceum, but due to pressure activity, it cannot be used as an antimalarial drug. In an attempt to increase antiplasmodial activity and reduce hypertensive activity, we synthesized by solid phase method, cyclic analogues of AII with i-(i+2) and i-(i+3) lactam bridge scaffold 1 using Asp and Lys residues. The bridge was more effective when inserted next to N-terminal extremity 1 , probably this insertion, on another portion of the peptide, provides a change in the conformation of the molecule and its hydrophobic cluster formed by Tyr, Ile and His 2 , which may have influence in the peptide-membrane interaction. Thus, we have focused in the N-terminal extremity, testing new analogues, using Glu/Asp/Orn/Lys residues as bridgeheads components in i-(i+4) lactam bridge scaffolds, which showed that antiplasmodial activity is increased using Glu residue and that larger lactam rings are better to biologically active. Therefore, new restrict peptides by i-(i+2) and i-(i+3) lactam bridge were designed, using Glu residue as bridgehead element, but the same effect was not verified, getting a maximum of 65% of bioactivity. On the other hand, we promoted an increase in the hydrophobic character of the molecule, replacing the Asp residue of AII sequence by Fmoc-Glu and Asp(OFm), in order to improve the interaction of these compounds in the sporozoite membrane. The replacement by Fmoc-Glu provided a decrease of activity, while that Asp(OFm) kept the AII activity, because there are changes of charge in the peptide, which may have modified the conformation in physiological medium. This kind of approach may offer the basis for development of new drugs and chemotherapy against malaria. Animal venoms are complex chemical cocktails, comprising a wide range of biologically active reticulated peptides that target with high selectivity and efficacy a variety of membrane receptors such as ion channels or G-Protein Coupled Receptors. Venoms can therefore be seen as large natural libraries of biologically active molecules that are continuously selected and highly refined by the evolution process. The vision associated with the VENOMICS project is to investigate in depth the enormous structural and pharmacological diversity of venom peptides through the development, integration and implementation of a novel research paradigm combining cutting-edge "omics" technologies in a high-throughput workflow. This new paradigm enclosed in VENOMICS aims at replicating in vitro the diversity of venoms to generate original peptide banks to be used in drug discovery programs. Herein, we show the different strategies we adopted for efficient solid phase synthesis and folding with an easy purification of peptides rich in cysteine and containing posttranslational modifications (PTM). Angiogenesis depends on the adhesive interactions of vascular cells. The adhesion receptor integrin av b3 was identified as a marker of angiogenic vascular tissue. The αν β3 integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis, mainly by interacting with matrix proteins through recognition of an Arg-Gly-Asp (RGD) motif. Inhibition of the αν β3 integrins with a cyclic RGD peptide impairs angiogenesis, growth and metastasis of solid tumours in vivo. The aim of this study was to investigate the effects of replacement of a-amino acids by aza-β 3 -amino acid analogs in cyclic RGD-peptides as αν β3 -integrin antagonist on angiogenesis, microcirculation, growth and metastasis formation of a solid tumour in vivo. The selectivity profile of these antiadhesive cyclopeptide is rationalized by a special presentation of the pharmacophoric groups. We synthesized cyclic RGD peptidomimetics that include aza-β 3 -amino acid residues.

Modifications were added to the RGD skeleton in order to optimize the peptide activity. Then, we investigated the pharmacokinetics activity of these pseudopeptides in HEK (Human Embryonic Kidney 293) and endothelails cells HUVEC (human umbilical vein endothelial cells) cell by analyzing cell viability and protein involved in the angiogneisis processes. Since tenascin C is a factor expressed highly in the tumorassociated matrix, targeting it would be a desirable first step for targeting the tumor-specific microenvironment In fact, a high level of tenascin C expression has been reported in most solid tumors, including lung cancer, colon cancer and glioblastoma. Therefore, the targeted binding of tenascin C in tumor stroma would inhibit tumor metastasis by modulating cancer cell growth and migration. We isolated a peptide that bound to tenascin C by phage display peptide library selection, and the selected peptide specifically recognized tenascin C protein in xenograft mouse tissue. We also observed exclusive staining of tenascin C by the selected peptide in tumor patient tissues. Moreover, the peptide reduced tenascin C-induced cell rounding and migration. We propose that the tenascin C targeting peptide may be useful as a specific anti-cancer diagnostic and therapeutic tool for most human solid tumors. Radiolabeled pansomatostatins are expected to enhance hsst1-5 tumor-uptake and to broaden clinical indications as compared to currently established sst 2-prefering radioligands. Previous experience has revealed [111In-DOTA 0 ,DTrp 8 ]SS-14 ([ 111 In]AT2S) as a true pansomatostatin analog, exhibiting however poor in vivo stability. In order to enhance metabolic stability, we introduced a second disulfide bridge to the AT2S motif by formation of extra 6/12-amino acid (AA) or 8/12-AA ring generating AT5S and AT6S, respectively. The orthogonally protected sequences were assembled on the solid support, deprotected and cleaved from the resin with TFA. The first Cys 6 -Cys 11 (AT5S) or Cys 5 -Cys 12 (AT6S) cyclization was performed in DMSO, while the second was completed with iodine oxidation after in situ deprotection of Cys 3 (Acm) and Cys 14 (Acm). During hsst 1-5+-autoradiography, AT5S showed unexpected total loss of sst 1-5 affinity, whereas AT6S showed high affinity (IC 50 in nM) to all hsst1-5 (hsst1= 11.5±3.3; hsst2= 6.3±0.6; hsst3= 9.7±3.6; hsst4= 5.4±0.8; and hsst5= 25.7±7.0). Consistent with this finding, only AT6S stimulated sst2 internalization during immunofluorescence-based internalization assays, showing agonistic properties for sst2. Furthermore, [111In]AT6S internalized rapidly and specifically in sst2+ AR4-2J and HEK293-hsst3+-cells. HPLC analysis of 5 min ex-vivo mouse blood samples revealed that >98% [111In]AT6S remained intact. After injection in SCID mice bearing AR4-2J and HEK293-hsst3+ tumors [111In]AT6S specifically localized in the rsst2A+ (1.9±0.2%ID/g vs. 0.8±0.05%ID/g + 100 nmol Tate at 4 h postinjection (pi)) and in hsst3+ implants (3.7±0.4%ID/g vs. 0.3±0.05%ID/g + 80 nmol KE108 at 4 h pi). This study has shown that introduction of an extra disulphide bridge in AT2S confers high metabolic stability. However, in a 6/12member ring combination it leads to total loss of affinity. The reasons for this effect are currently investigated by NMR conformational studies. Transporter compounds are useful tools to solubilise and increase the delivery of therapeutic molecules in the human body. One system to improve the cellular uptake of such therapeutic molecules are cell-penetrating peptides (CPPs). These short peptide chains are either polycationic (containing several Arg and Lys) or show a more amphiphatic structure. 1 It is known that the multivalency effect -the presentation of several copies of a CPP motif on a single molecule -can increase the cellular uptake. 2 Peptide dendrimers represent a group of tree-like, multivalent macromolecules, which are synthesized for different chemical and biological applications in our group. 3 We now combine linear CPPs with peptide dendrimers to get a well defined branched molecule made up of only natural amino acids. In our systematic study of peptide dendrimers decorated with different CPPs we found that the potency of the single CPP as a transporter for small molecules can be increased and that these peptides show usually low cytotoxicity. Additionally we designed new dendritic cell penetrating peptides with similar activities like linear CPPs. All compounds are covalently linked to fluorescein for visualization with flow cytometry and confocal analysis. The results show that the peptides can transport efficiently a hydrophobic cargo into the cells. Chemical stability of esters of acyclovir with amino acid and cholic acids K. Chuchkov, R. Chayov, I. G. Stankova* South-West University "Neofit Rilski", Blagoevgrad, Bulgaria Amino acid esters of antiviral drugs are a very good solution for improving oral bioavailability of the actual medicine. One of the most effective and tolerant prodrugs is valine ester of acyclovir -valaciclovir. Taken orally exhibits three to four times higher bioavailability of acyclovir. The chemical stability of amino acids (4-Fphenylalanine) (R,S) and bile acids (deoxycholic acid and chenodeoxycholic acid) esters of acyclovir was studied in experimental conditions simulating some relevant biological medias (pH 1.0 and 7.4, 37°C).The chemical stability experiments revealed that the examined amino acid ester of acyclovir were relatively unstable in acidic pH, but bile acid ester is stable in the same pH. The examined amino acid and bile acid esters of acyclovir in neutral pH are relatively stable. In pH 7,4 all of tested compounds are more stable than valacyclovir (t1/2 = 13 h) -the first effective prodrug of acyclovir. In acidic pH acyclovirdeoxycholat and acyclovirchenodeoxycholat are more stable than valacyclovir. Acyclovirchenodeoxycholat is the most promising anti-EBV prodrug candidate with high activity and satisfying chemical stability. Cell-penetrating peptides (CPP) have become efficient tools for the cellular internalization of bioactive molecules due to their ability to cross the plasma membrane of diverse cells and cell lines. [1] We recently reported that the CPP sC18, which consists of the residues 106-121 of the C-terminal region of the cationic antimicrobial peptide cathelicidin (CAP18), is an effective carrier peptide for small organic molecules like fluorophors and toxic peptide sequences into various cell lines [2] . However, in general linear peptides are more susceptible to proteolytic degradation than their cyclic analogs [3] . Therefore, we investigated the cyclization of CPP derived from sC18 by means of CuI-mediated azidealkyne cycloaddition (CuAAC) [4] . Furthermore, we examined their conformation and proteolytic stability as well as their internalization efficiency and toxicity against various cell lines, in comparison to their linear equivalent and to other CPP. Looking for the proper prodrug: A peptidomimetic approach to identify and inactivate bacterial mono-ADP-ribosyltransferase toxins M. Beich-Frandsen, R. Jørgensen Division of Microbiology and Diagnostics, Statens Serum Institute, Copenhagen, Denmark Mono-ADP-ribosylation is an endogenous posttranslational modification in eukaryotic cells, simultaneously utilized as virulence strategy by deadly secreted bacterial toxins. Many bacterial toxins have been found to act as mono-ADP-ribosylating enzymes, targeting anything from G-proteins to the actin skeleton. The diphtheria toxin from C. diphtheriae and exotoxin A from P. aeruginosa, both target the diphthamide-group of a unique modified histidine in eEF2, inhibiting protein synthesis by ribosome mimicry 1,2 . We aim to inactivate these NAD+-utilizing toxin enzymes by NAD-conjugated peptidomimetics, in a target-specific prodrug-approach. The ADP-ribosylation reaction follows a random third-order S N 1 mechanism. In the proposed model for the transition state of the reaction, the cleavage of the N C1-NN1 bond of NAD + releases strain and generates a oxacarbenium ion intermediate with a positively charged nicotinamide (N)ribose, subject to a nucleophilic attack from the substrate 3,4,5 . ADP-ribosylating toxins are commonly characterized by a ARTT-motif involved in substrate recognition 2 . Studies suggests conformational rearrangement of the residues surrounding the substrate binding site to be required for optimal geometry 5 of the initial glycosidic NC1-NN1 bond cleavage within NAD + . Subtype specific NAD-conjugated peptides, designed based on previous structural analysis of the ADPribosylation reaction, act as substrate for the enzymatic ADP-ribosyl-transfer, and hereby attach covalently to and inactivate the NAD + -utilizing toxin.

Relying on previous structural studies, and established ligand-binding and kinetic data, an initial peptide library, designed by bioinformatics and evaluated for specificity of common targets in-silico identifies initial leads. Lead-scaffolds are implemented in rational peptide-design, based on high-resolution structural-and biophysical studies of multiple peptide-enzyme complexes, to identify possible prodrug-strategies for enzyme inactivation. Nanoparticles play a crucial role in medicine for their potential application as in vivo carriers of active principles [1] . Liposome display unique pharmacokinetic properties slowly releasing drugs loaded in the inner aqueous cavity. In the last years we have developed supramolecular aggregates labeled by bioactive peptides able to recognize overexpressed receptors on tumour cells membrane delivering doxorubicin chemiotherapeutic drug [2] . Neurotensin(NT), a 13 amino acid peptide, has dual functions of neurotransmitter or neuromodulator. The Cterminus short fragment 8-13 preserve the activity but the half life of wild type form in vivo is very short. NT receptor type 1 (NTS1) is overexpressed in severe malignancies such as small cell lung cancer and colon, pancreatic, and prostate carcinomas. We have designed new amphiphilic molecules containing in the hydrophobic moiety two aliphatic chains and in the hydrophilic moiety a the bioactive portion able to aggregates with phospholipid molecules achieving liposome. We have synthesized neurotensin wild type sequence, the truncated form and the tetra-branched neurotensin(NT1-13) or a truncated form(NT8-13) tetrabranched peptides(NT4) adopting an opportune synthetic strategy on solid phase. All liposome were formulated adding the neurotensin amphiphilic monomer in ratio 5:95 with DOPC in order to evaluate the capability to recognize selectively receptors overexpress on cell membrane surface. The liposomes size was determined by Dynamic Light Scattering measurements, values for the hydrodynamic radius(RH). The selective internalization and cytotoxicity of fully doxorubicin loaded liposomes as compared to pure DOPC liposomes, was tested in HT29 human colon adenocarcinoma and TE671 human rhabdomyosarcoma cells. Recently, small interfering RNA (siRNA), one kind of RNA interference (RNAi) technology represent the most common and, to date, the most effective method to inhibit target gene expression in human cells. It is also a common recognition that non-toxic delivery of siRNA is an urgent problem for the therapeutic application of siRNA. For the efficient gene silencing in vivo, prolonged circulation of siRNA with take efficient and non-toxic cellular uptake and resistance against enzymatic degradation are indispensably required. 1 ) Telomerase activity has been regarded as a critical step in cellular immortalization and carcinogenesis and because of this, regulation of telomerase represents an attractive target for anti-tumor specific therapeutics.

In this paper, we present the efficient and non-toxic cellular uptake of siRNA using novel amphiphilic peptides and the application to silencing of hTERT in human cancer cell lines.

In the present study, we investigated the intracellular delivery of siRNA using some amphiphilic peptides and the silencing effect of siRNA targeting hTERT mRNA in 3 human cancer cell lines, Jurkat, HeLa and K562. The complex of siRNA and a specific amphiphilic peptide or its hybrid with an intracellular transport signal peptide could be effectively taken up into cells. The complex also showed a high silencing effect against hTERT mRNA. Moreover, the combination of siRNA-NES conjugates and the amphiphilic peptides improved silencing effects up to 95.2 %. The amphiphilic peptides and their hybrids showed almost no cyto-toxicity and protected siRNA against intracellular nuclease digestion in 10% FBS (half life time was over 48h). Tumor targeting with the decapeptide gonadotropinreleasing hormone (GnRH) or its analogues is based on the discovery that GnRH receptors are overexpressed in many tumor cells, compared with their expression in normal tissues. Using these peptides as carriers/targeting moieties in a conjugate with therapeutic agents can increase the selectivity and the stability of the conjugates, or eliminate the toxic side effects of the drug. GnRH-III (<EHWSHDWKPG-NH 2) is especially favoured as a targeting moiety because of its antiproliferative effect and weak endocrine activity in mammals. The aim of our work was to synthesize novel short-chain fatty acid (SCFA) acylated daunorubicin-GnRH-III bioconjugates with enhanced enzymatic stability, cellular uptake, in vitro and in vivo antitumor activity. Ser in position 4 of GnRH-III was replaced by Lys and its ε-amino group was acylated with various fatty acids. GnRH-III( 4 Lys(X), 8 In conclusion, Glu 6-10 /DGlu 6-10 substitution in GI-derived radiopeptides enhanced stability and reduced renal values by ~2 .5 fold. On the other hand, removal of the (Glu)5sequence in MG11 suppressed renal uptake but undermined stability. Both modifications, however, compromised tumor targeting, demonstrating that more efficacious structural changes in the GI motif are warranted to improve the in vivo profile of resulting radioligands.

Synthesis and characterization of a 99mTc-labeled rhodamine-conjugated angiotensin peptide as a potential cardiac function imaging agent S.M. Okarvi King Faisal Specialist Hospital & Research Centre, Cyclotron and Radiopharmaceuticals Department, Riyadh 11211, Saudi Arabia Angiotensin II (Ang II) is an 8-amino acid peptide that has been known to play a vital role in cardiovascular system. The actions of Ang II have been implicated in many cardiovascular conditions, such as coronary heart disease and heart failure. In an effort to develop a cardiac imaging agent with enhanced targeting capacity, we attached Ang II to rhodamine (Rh), a lipophilic cation that specifically accumulates in the myocardium. The Rh conjugated Ang II was evaluated for its potential as a heart imaging agent. Rh-Lys-Gly-Cys-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-CONH2 was prepared by solid-phase peptide synthesis according to Fmoc/HBTU methodology. NHS-Rh was attached to the Ang II peptide via the free amino group of Lys residue by manual synthesis. Rh-Ang II conjugate was labeled with Tc-99m by the stannous-tartrate exchange method. In vitro stability was determined in human plasma and in excess of cysteine. In vivo biodistribution and biokinetics were performed in Balb/c mice and Sprague Dawley rats. The Rh-Ang II conjugate radiolabeled efficiently with Tc-99m (>75% labeling efficiency) as determined by HPLC analysis. Tc-99m-Rh-Ang II exhibited good chemical stability against cysteine transchelation and sufficient metabolic stability in human plasma. In mice, the bioconjugate displayed efficient clearance from the blood and excreted mainly through the renal route with some excretion by the hepatobiliary pathway. The uptake in the heart was 1.8±0.5% ID/g as early as 30 min post-injection; whereas, the uptake in the lungs, liver, stomach and kidneys varied between 1-10% ID/g. In rats, the bioconjugate displayed relatively better pharmacokinetic characteristics, with low uptake in the major organs (<4% ID/g). The uptake in the heart (1.7±0.4% ID/g) was found to be higher than the uptake in the blood and muscle, resulting in good heart-to-blood and heart-to-muscle uptake ratios. This initial study towards the development of an effective cardiac imaging agent advocates that the use of hybrid conjugates appears to hold a great promise as a new and attractive approach for rapid and efficient imaging of heart. In humans two isoforms of GnRH are exist, GnRH-I (<EHWSYGLRPG-NH 2, where <E is pyroglutamic acid) and GnRH-II (<EHWSHGWYPG-NH2). In contrast to GnRH-I, exact function of GnRH-II is not well identified. However, it was indicated that GnRH-II derivatives have more potent antiproliferative activity on human endometrial and ovarian cancer cells in vitro than GnRH-I analogs. Some kinds of tumor cells (e.g. breast, prostate, colon) produce GnRH-I and GnRH-II, and express their receptors. However, the presence of functionally active GnRH-II receptors on cancer cells is still not clear 1-2 . On the other hand, GnRH-II analogs, both agonists and antagonists, induce apoptosis in many different cell types. In contrast to GnRH-I agonists, GnRH-II agonist [D-Lys 6 ]GnRH-II does not activate NFκB, therefore does not protect the cancer cells from apoptosis [3] [4] [5] . In the regulation of apoptosis, not only the pro-and antiapoptotic proteins play a role 6 , but other molecules also attend (e.g. CK2, casein kinase II enzyme) 7 . Our aim was to synthesize new proapoptotic peptide or enzyme inhibitor containing [D-Lys 6 ]GnRH-II based derivatives and investigate their in vitro cytotoxic, cytostatic and apoptotic effects. The sythesis of [D-Lys 6 ]GnRH-II, which is one of the most potent GnRH-II agonist, was carried out on solid phase using Fmoc/tBu strategy. Different proapoptotic peptides or enzyme inhibitors were attached directly to the GnRH-II derivative. In vitro cytostatic and cytotoxic effects of GnRH-II conjugates were investigated by MTT-assay using different human breast (MCF-7 and T47-D) and colon carcinoma (HT-29) cell cultures. Early apoptotic effect of GnRH-II conjugates was studied by flow cytometry on MCF-7, T-47D and HT-29 cell lines. According to our results, these conjugates might be potential candidates for cancer treatment, because the combination of a targeting moiety (GnRH derivative) with proapoptotic peptides or enzyme inhibitors and drug molecules might have synergistic activity resulting in highly potent multifunctional therapeutic agents. Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padua, Italy Melanoma is the most deadly skin cancer with an increasing incidence. Its therapy continues to be a challenge since, regardless of the treatment used, longterm survival is quite uncommon. Melanoma cells are characterized by high levels of glutathione S-transferase P1-1 (GSTP1-1) isozyme. This enzyme is also highly expressed in solid tumours and drugresistant cells, where a role for this protein in the regulation of cell proliferation has been described. 1 In the past, Lyttle et al. synthesised glutathione (GSH)linked anticancer prodrugs activated by physiological concentration of GSTs. 2 Structure and mechanistic studies showed that in the GST-GSH complex, the GST Tyr residue placed near the S atom of the Cys in GSH, is in the phenoxide form to facilitate the deprotonation of the sulfhydryl group in GSH, making it able to react with electrophilic species. 3 By taking advantage of this active-site geometry, we synthesised two tyrosinase activated melanoma prodrugs (4-methoxyphenol and N-acetyl-4-S-cysteaminylphenol) linked to GSH. In these compounds an ethoxycarbonyl group was placed between the GSH sulphur, oxidate to sulfone, and the tyrosinase-activated prodrugs. In this way the Tyr phenoxide (Tyr7 in GSTP and Tyr6 in GSTM isozyme) would be able to abstract one of the acidic methylene protons linked to the sulfone moiety. Such a deprotonation should trigger a beta-elimination, resulting in the release of melanoma prodrugs. The stability of the obtained putative prodrugs has been evaluated in different pH buffers and in rat liver and kidney cytosolic fractions. Synthetic peptides represent first choice biomolecules, with respect to proteins and mAb, for the monitoring of population variability and receptor functionality associated to a tumor pathology due to their favorable pharmacokinetic profile. It has been demonstrated that the surface molecule integrin alfa-V beta-3 could be an ideal target for the interaction with melanoma directed radiolabeled peptides. This integrin is indeed overexpressed both in tumor cells and in tumor vessels endothelium, while it is not expressed in the majority of normal tissues. Cyclic peptides with the sequence Arg-Gly-Asp (RGD), are known to bind the integrin alpha-V beta-3 with high affinity and selectivity and could conveniently target a radiotherapeutic to melanoma cells. 1 We previously demonstrated that the cyclic RGDfK peptide interact with high affinity to A375 human melanoma cells. 2 The aim of this work was to conjugate the 99mTc to the peptide by using the [ 99m Tc(N)(PNP)] 2+ system (PNP=aminodiphosphines) to obtain a radiolabeled peptide useful in melanoma imaging. 3, 4 spectrum. A NH(i)→βCH(i-1) cross peak more intense than the NH(i)→βCH(i) cross peak is observed when the peptide adopts the 2.05-helixl conformation. By contrast, an opposite trend of intensities of the same NOE cross peaks indicates the occurrence of a 310-helical conformation. By using this method, we were also able to determine that polar solvents, such as MeCN or MeOH, may induce a 310helical structure in Deg homo-peptides which otherwise adopt the 2.05-helix conformation in CDCl3. This finding allows us to rapidly switch these peptide series from "short" (310-helix) to "long" (2.05-helix) by changing the solvent only, thus making them extremely attractive tools. School of Biological Sciences, University of Auckland, Auckland 1142, New Zealand Naturally occurring anti-freeze proteins (AFPs) enable organisms like polar fish to survive the freezing temperatures of their natural habitat. 1 As well as being cryoprotective, AFPs exhibit properties of ice recrystallization inhibition (RI). 2 The ability of AFPs to influence the size, morphology and aggregation of ice crystals can be used in food technology, where the growth of ice crystals in frozen foods is of primary concern. 3 Chemical synthesis of AFPs enables access to pure material for structural and functional studies. Our new research programme in the 'Food and Health' area, prompted us to develop tailor made anti-freeze peptides for potential applications in the frozen food industry. 'Multiple salt-bridge analogues' of the naturally occurring winter flounder AFP were synthesized and evaluated for their antifreeze activity in comparison to the natural sequence. Details on the molecular modelling, structural characterization and anti-freeze activity analysis on the synthetic AFPs will be presented. Several disorders are now believed to be conformational diseases caused by misfolding in the structure of specific proteins leading to the gradual aggregation and deposition of these abnormal proteins. Attempts to develop effective therapies for these proteopathies (such as Alzheimer's, Parkinson's and Huntington's disease) have been directed toward reducing the production of the proteins, inhibiting their aggregation, or promoting their removal. Conformation-dependent antibodies that specifically target misfolded proteins are proving to be useful tools to study the pathogenesis and as possible treatments of these diseases.

We have previously shown that active immunization with tetrapalmitoylated Aβ1-15 peptide embedded in liposomes (ACI-24) was capable of eliciting a conformation-specific immune response in mice. 1 The antigenic peptide was shown to mimic the aggregated β-sheet conformation of the pathological A β42 peptide on the surface of the liposomes by CD, ssNMR and ThT assay. We have also reported the modulation of the conformation of Aβ 1-15 derived sequences by means of different lipidation patterns, lipid chain lengths and liposome surface charge.

Here we present the characterization of this panel of liposomal peptide constructs by ATR-IR and correlate the results with previous studies by CD.

In summary, ATR-IR secondary structure analysis of a battery of amyloid-derived lipopeptides allowed to study the key parameters responsible for the adopted conformation on the lipid bilayer. The presented strategy to control the conformation of liposomal peptide immunogens could be applied to the rational design of vaccines against a range of protein misfolding diseases, such as Alzheimer's disease. Cyclic lipodepsipeptides (CLPs) are secondary metabolites produced by various bacteria, most prominently Bacillus and Pseudomonas. These often display potential antimicrobial properties, frequently ascribed to the ability to disrupt cellular membranes or form ion-pores. CLPs are a structurally diverse group of molecules always consisting of an oligopeptide chain cyclized by a lacton bond and a fatty acid moiety. They are divided into several groups based on sequence similarity. One such group is the viscosin group, which contains CLPs produced by Pseudomonas that all feature nine amino acids and a conserved sequence pattern of hydrophobic and hydrophilic residues. 1 Recently, we have extensively described the self-assembly properties of one viscosin group member, pseudodesmin A. 2, 3 We have shown that in non-polar organic solvents this CLP forms anisotropic supramolecular structures of unrestricted size, reminiscent of the ability to form ionpores in the non-polar cellular membrane environment.

Here, we report on how the most prominent structural variations within the viscosin group affect the conformation and self-assembly properties. Since the well-defined monomer conformation of pseudodesmin A is key to this self-assembly, the most striking variation within the viscosin group is the variability of the Leu5 stereochemistry. For the first time, a conformational study will be reported for CLPs featuring an L-Leu5 residue, such as viscosin and viscosinamide. The results of this study contribute to a deeper understanding of the structure-function relationship of CLPs in general. Effective design of peptide mimicking protein helical segments is a great challenge. An intriguing approach to improve the helical content in a peptide sequence is achieved through the "peptide stapling" strategy using a hydrocarbon cross-linker (staple) 1 .

In this study we have investigated the effect of the introduction and location of staples on secondary structure of sequences derived from a reported peptide, mimicking Cullin3 binding region to KCTD11 2,3 , to gain an insight into the designing of new molecular entities able to modulate the KCTD11 oncosuppressor activity. Indeed, KCTD11 plays a crucial role in the ubiquitination of HDAC1 by acting, in complex with Cullin3, as an E3 ubiquitin ligase, thereby affecting Hedgehog activity and medulloblastoma growth.

In the designed peptides the stapling has been achieved by ring-closing olefin metathesis between two S-2-(4'pentenyl) alanine residues incorporated at i and i+4 positions and into different sites within the peptide chains.

The stapling position has been chosen on the basis of Cullin3-KCTD11 complex model. Circular dichroism studies have highlighted that the stapled peptides exhibit remarkable and different changes in secondary structure contents in comparison with the linear precursors and the unmodified sequence. A NMR conformational analysis will allow the determination of the conformational preferences of the stapled peptides at atomic resolution with also relevant implications for the KCTD-Cullin3 recognition. Based on our findings from protease-mediated ligation of cleavage-sensitive peptides and proteins using ionic liquids (ILs) as additives 1 and from chemical acylation reactions of peptides in ILs, 2 we hypothesized direct and unique interactions between biomolecules and ILs as the molecular reason for IL-mediated structural effects. To verify this, we performed systematic structural investigations of Suc-Ala-Xaa-Pro-Phe-pNA and Ala-Xaa-Pro-Phe model peptides dissolved in aqueous imidazolium-based ILs using high resolution magic angle spinning (HR-MAS) NMR spectroscopy. Highly resolved NMR spectra are obtained that allow for complete 1 H resonance assignments of the peptides as solutes and the ILs as solvents. The changes in the proton chemical shifts Δδ( 1 Η) of a peptide on dissolution in ILs arise from direct interactions between both, including IL-induced structural/conformational changes of the peptide molecules. 3, 4 The strength, position, and type of these interactions are influenced by the nature of the amino acid residues in the peptide sequence as well as the nature and type of the IL anions. Downfield shifts indicate strong interactions between the ILs and the peptide backbone, whereas upfield shifts point to hydrophobic interactions and stacking effects with the IL imidazolium ring. The pattern of Δδ( 1 Η) along the peptide chain reveals the sites and the type of interactions. The extent of Δδ( 1 Η) implies a significant effect on the structure of the peptides. Different Δδ( 1 Η) were obtained for cis-and trans-configured peptide isomers and point to a conformer-specific IL/peptide interaction. Calcitonin is a 32-amino acid polypeptide hormone, which takes part in calcium metabolism in bones. It belongs to a protein family whose members may form amyloid fibrils. These fibrils (or pre-fibrillar aggregates) are related to serious diseases known as amyloidoses, whereas amyloid-like fibrils may have natural functional roles in many organisms. The amyloid form of human calcitonin is related to medullary thyroid carcinoma. Using our online consensus prediction tool for 'amyloidogenic propensity' of protein molecules "AMYLPRED", a novel hexapeptide of human calcitonin was predicted as a novel, possible amyloidogenic peptide. We investigated experimentally its ability to form amyloid fibrils, utilizing TEM, X-ray fibre diffraction, ATR FT-IR spectroscopy and polarized light microscopy. Analysis of the results shows that this peptide self-assembles into amyloid-like fibrils and that amyloid fibrillogenesis is mediated via nuclei of liquid crystalline nature, known as spherulites. We thank the University of Athens for the financial support. School of pharmacy, University of Norwich NR4 7TJ ; 3 Inserm U982, Université de Rouen, 76821 Mont Saint Aignan 26RFa, a RFamide neuropeptide, is the ligand of GPR103 and the system 26RFa/GPR103 is involved in feeding behaviour [1] . A structural study by NMR showed that 26RFa, in a membrane mimetic medium, possesses a helix P4-R17 -linker K18-G21 -turn F22-R25 motif. SAR studies on truncated fragments of 26RFa indicate that, in addition to the turn motif, additional residues are required to maintain a correct biological activity (26RFa EC50=10,4nm; 26RFa10-26 EC50=37,5nm; 26RFa13-26 EC50=95,3nm; 26RFa16-26 EC50=237nm) [2] . Our aim is to synthesize new GPR103 ligands using an iterative process combining rational conception, peptide and modified amino acid syntheses, biological and structural analyses. Structural and biological results will allow us to find new modifications and conceive more potent analogues. Using NMR, we demonstrated that 26RFa11-26 encompasses a nascent helix and a turn while 26RFa13-26 possesses only the C-terminal turn structuration. The destabilization of the helical structure on fragments with a shorter N-terminal region could explain the decrease of the biological activity of 26RFa fragments. We decided to focus on the importance of a stabilized helix on the biological activity. We chose the Hydrogen Bond Surrogate method which stabilizes a helix by replacing the main hydrogen bond with a covalent link without modifying the side-chains [3] . Syntheses, NMR characterization and molecular modelling of analogues will be discussed. Acknowledgments: This work is supported by ERDF funding through the IS:CE-Chem project and Interreg IV A France-(Channel)-programme. Firstly, it induces a constraint by restricting the ı o-dihedral angle to values around -60°. Secondly, the Xaa-Pro peptide bond is subject to cis-trans isomerization characterized by an increased cis population and an activation energy that is low when compared to the other amino acids. Besides, the five-membered ring of the Pro residue can adopt two main distinct conformations (up-puckered or downpuckered) that are almost equally abundant in peptides and proteins. A variety of mimics and analogs have been designed in order to control the conformation of the peptide backbone and/or to alter the cis/trans ratios and the rotational barriers for cis-trans isomerization. In this presentation, NMR studies 2 and theoretical calculations 3 have been performed on model peptides Ac-Ser(ΨPro)-NHMe, (S,S)-Ac-Ser(ΨH, CF3 Pro)-NHMe, and (R,S)-Ac-Ser (Ψ CF3 ,HPro)-NHMe. 4 Their thermodynamic and kinetic features have been analyzed in chloroform, DMSO, and water, allowing a precise description of their conformational properties. We found that trifluoromethyl C δ -substitutions of oxazolidine-based pseudoprolines can strongly influence the cis-trans rotational barriers with only moderate effects on the cis/trans population ratio. In CHCl3, the configuration of the CF3-C δ entirely controls the ψ-dihedral angle, allowing the stabilization of γ-turn like or PPI/PPII-like backbone conformations. Moreover, in water and DMSO, this C δ -configuration can be used to efficiently constrain the ring puckering without affecting the cis/trans population ratio. Theoretical calculations have ascertained the electronic and geometric properties induced by the trifluoromethyl substituent and provided a rational understanding of the NMR observations. In contrast to formation of the native state, aggregation of misfolded protein molecules into so-called amyloid fibrils is a strongly irreversible, kinetically-controlled process which may lead to structural variants of fibrils composed of chemically-identical protein chains. This poorly understood aspect of polymorphism of amyloid fibrils has important implications in vivo in the course of amyloidassociated neurological disorders -e.g. in the form of prion strains in Creutzfeldt-Jakob disease. In this paper, we examine cross-seeding behavior of a model amyloidogenic polypeptide -bovine insulin (bov-ins) and recombinant Lys B31 -Arg B32 insulin (KR-ins). Fibrils of bov-ins and KR-ins exhibit characteristic "fingerprint" features in the amide I' infrared region. These spectral traits are transferred to daughter generations of fibrils upon cross-seeding of native bov-ins with KR-ins fibrils and vice versa. This study highlights an interesting aspect of a non-anfinsenian behavior of aggregating insulin: the memory effect. Our results indicate that fine infrared features in the amide I region of fibrils from closely related proteins may not reflect different amino acid composition but rather the fact that a particular amino acid sequence favors an amyloidogenic pathway that determines a certain and spectrally-distinct stacking mode of β-strands. An encounter with an alien seed on the amyloidgenic pathway would hijack the yetnative monomers and force them to follow the assembly pattern (and resulting IR characteristics) of the preformed fibril. Phosphorylation and OGlcNAcylation are dynamic intracellular protein post-translational modifications that often are alternatively observed on the same protein serine and threonine residues. Phosphorylation and OGlcNAcylation commonly occur on residues in natively disordered regions of proteins, and often have opposing functional effects. In the microtubule-associated protein tau, hyperphosphorylation is associated with protein misfolding and aggregation as the neurofibrillary tangles of Alzheimer's disease, whereas OGlcNAcylation is stabilizing. A series of peptides derived from the prolinerich domain (residues 174-239) of tau was synthesized, with the free Ser/Thr hydroxyls, with phosphorylated Ser/Thr, with OGlcNAcylated Ser/Thr, and with the dietylphosphorylated Ser/Thr. Phosphorylation and OGlcNAcylation were found by CD and NMR to have opposing structural effects, with phosphorylation favoring polyproline helix (PPII) formation, with OGlcNAcylation opposing PPII, and with the free hydroxyls intermediate. Phosphorylation of serine and threonine residues in prolinerich sequences was found to induce PPII in other proline-rich sequences, with significant conformational restriction at phosphoserine and phosphothreonine residues observed by NMR. Notably, serine and threonine residues are common in proline-rich domains of proteins, and PPII is a common conformation observed for phosphorylated ligands in protein-protein interactions. The Ser/Thr diethylphosphate was found to be a practical mimic of OGlcNAcylation, as an easily synthesized neutral amino acid with similar sterics to OGlcNAc and strongly opposing PPII. Dept. of Biology, University of Padova, Padova 35121, Italy We recently established that designed hairpin peptides interfere with the amyloidogenesis of both human pancreatic amylin and α-synuclein ( α-syn), two 'unstructured' polypeptides associated with human diseases. 1 In the case of α-syn, the most potent 'amyloid inhibitors' act by diverting α-syn to non-amyloid aggregates. The binding sites for these agents and nonhairpin peptides of similar sequence have been determined by 2D-NMR-monitored titration studies using universally 15 N-labeled α-syn. In the case of the hairpins that produce non-amyloid aggregates from α-syn, initial binding occurs in the non-amyloidogenic C-terminal segment of α-syn and the hairpin peptide is incorporated in the resulting aggregates. NMR studies also serve to define binding interactions and conformational changes that may be essential early steps in aggregation and pre-amyloid state processes. MT-II is a long acting, non-selective super-agonist of melanocortin GPCR receptors, 1 characterized by lactam bridge between residues i and i+5 stabilizing a type-II β turn structure. 2 The recently introduced CuI-catalyzed azide-alkyne 1,3-dipolar cycloaddition, presents a promising opportunity to develop a new paradigm for an orthogonal bio-organic and intramolecular side chain-toside chain cyclization. We introduced this [1,2,3]triazolyl-mediated cyclization in a PTHrP model peptide, and demonstrated that i-to-i+4 side chain-to-side chain cyclization offers a powerful approach for generating stable helix mimetic structures. 3, 4, 5 In the current study we explored the potential of the i-to-i+5 side chain-to-side chain 1,4-disubstituted [1, 2, 3] triazolyl-bridge to stabilize -turn conformations. We designed a series of [1,2,3]-triazolyl containing cyclo-heptapeptides, derived from the model peptide lactam MT-II, differing in the size of the bridge, the location and orientation of the [1,2,3]-triazolyl moiety. Conformational and biological studies, performed on this series demonstrate that [1, 2, 3] triazolyl-mediated side chain-to-side chain cyclization results in the conformational stabilization of type-I β turn generating heterodetic cyclopeptides with enhanced in vitro potency and improved receptor subtype selectivity. This study confirms the versatile nature of the [1, 2, 3] triazolyl-mediated side chainto-side chain cyclization. Moreover the chemical accessibility, functional compatibility, and metabolic stability suggest that the [1, 2, 3] International Associated Laboratory Samuel de Champlain, 5 Regional Platform for Cell Imaging of Haute-Normandie (PRIMACEN), Institute for Medical Research and Innovation (IRIB), University of Rouen, 76821 Mont-Saint-Aignan, France Scorpion toxins are polypeptidic molecules capable of selectivity binding to ion channels with the highest affinities and represent natural peptide blockers that are good tools to characterize new or particular types of ion channels. Astroglial cells appear to express all ion channels species even voltage-gated ion channels previously suspected to be present only in excitable cells; but these channels are poorly studied and their function are not still completely elucidated.Thus, the purpose of the present study was to investigate for the first time, the global effect and the potential protective effect of one scorpion toxin acting on Kv channels, the Pi4,on H2O2-induced oxidative stress and cell death in rat astrocytes. Pi4 is a 38-residue toxin crosslinked by 4 disulfide bridges, isolated from the venom of Pandinus imperator1 and is one of the most potent and selective inhibitor of Shaker B and Kv1.2 channels2. In our study, Pi4 was obtained by Solid Phase Peptide chemical Synthesis(SPPS) and named sPi4. The identity and homogeneity of sPi4 were also verified by HPLC, mass spectrometry analysis(MALDI-TOF) and an enzyme-based cleavage to determine the assignment of sPi4 half-cystine pairings.The synthetic sPi4 was found to be indistinguishable from the natural toxin. Incubation of cultured astrocytes with graded concentrations of H2O2 for 1h provoked a dose-dependent reduction of the number of living cells as evaluated by flow cytometric analysis and lactate dehydrogenase assay. Interestingly, pre-treatment of astrocytes with very low concentrations of sPi4(10-8 to10-12 M),dose-dependently prevented cell death induced by H2O2, and the effect of sPi4 was accompanied by a marked attenuation of ROS accumulation. Also, sPi4 stimulated SOD and catalase antioxidant activities in a concentrationdependent manner,and blocked H2O2-evoked inhibition of SOD and catalase activities. In addition, at low concentration (10-8 to10-14 M) the toxin exhibits no toxic effect on astrocytes.Taken together these data indicate that the sPi4 acts as a protective agent on astrocytes from oxidative assault and then might have therapeutic value in the potential treatment of a number of neurodegenerative diseases. [4] . In order to select small active peptides, the library was kept at the minimum size and was built using only 12 selected D-amino acids to avoid structural redundancies. To further increase the chemical diversity [5] a set of different carboxylic acids were coupled to the N-terminus. The iterative screening led to the identification of an N-terminally modified tetrapeptide with a very high radical-scavenging activity. By analyzing several peptide variants, we found that antioxidant properties were strongly sequence-and structure dependent, because scrambled or proline-scan variantswhose secondary structures are deeply altered -are much less effective. Data on peptide structure-activity relationship will be presented. An investigation of the in vitro effects produced in cellular models of oxidative stress are also in progress.

Structural analysis of peptide-analogues of ZP proteins with amyloidogenic properties: Insights into mammalian zona pellucida formation. N.N. Louros, V.A. Iconomidou, S.J. Hamodrakas* Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Athens 15701, Greece A filamentous structure surrounding mammalian oocytes, called zona pellucida, is responsible for species-specific sperm binding. This extracellular porous structure is composed of 3-4 glycoproteins, termed ZP1-4, which assemble into filamentous polymers that are cross-linked by disulfide bonds. All ZP proteins present a unit with high sequence homology at their C-terminal end, designated as the ZP domain. This structural module is found in a family of extracellular proteins with various functions and from a wide variety of organisms (ZP domain proteins). It is a binary structure of approximately 260 amino acids, divided into two domains, the N-terminal region named ZP-N and the C-terminal region known as the ZP-C domain. The ZP-C block is associated with protein function, whereas the ZP-N domain is responsible for protein polymerization. Structural analysis revealed a possible polymerization model based on backbone interactions between specific parts of the ZP-N units. Peptide-analogues of the aforementioned segments were synthesized, since the ''AMYLPRED'' application, an 'amyloidogenic determinant' prediction algorithm developed in our lab, predicted them as being potentially 'aggregation prone'. Our experimental data clearly indicate that these peptides do actually fold and self-assemble into amyloid-like fibrils. Therefore, all the derived data suggest that mammalian zona pellucida is a novel functional amyloid, similar to its biological equivalent, silkmoth chorion that has been established to be a natural, protective amyloid. We thank the University of Athens for the financial support. Biostrucutres and Bioimmaging Institute of National Research Council, Naples, Italy The definition of the molecular basis of human diseases is one of the most important goals of structural biology. Nucleophosmin (NMP1) is an ubiquitously expressed protein is a nucleolar chaperon and has a key role in several biological processes from ribosome biogenesis, to cell-cycle regulation and is a frequently target of oncogenic mutations in acute myeloid leukaemia (AML) [1] . These mutations occur predominantly at the C-terminal domain of NPM1 compromising its stability and causing the aberrant translocation of NPM1 to the cytosol. This domain is structurally characterized by the presence of three alpha helices, and the folding of Cter-NPM1 proceeds via an extended nucleus and the denatured state retains significant malleable structure at the interface between the second and third helices [2] . Here, we have further investigated the structural determinants in the folding process of the C-term NPM1 domain and on the basis of available structural and composition data, several peptides covering the three helices were designed and synthesized. Their conformational properties were investigated by CD and NMR spectroscopy: these studies demonstrated that once removed from the protein architecture helix1 and helix2 substantially miss fully ordered conformations while helix3 retain a high helical content also in mutated variants. Our observations may help structure-based drug-design INSERM U829 University of Evry-Val d'Essonne, Bâtiment Maupertuis, 91025 Evry, France SL21 (STOP-like protein with mass of 21 kDa) is a neuronal protein with a calmodulin-binding and microtubulestabilizing activity. Microtubule binding is inhibited by calmodulin 1,2 . The lack of the STOPs gives rise to defects in synaptic plasticity, as observed in schizophrenia 3 . To study the SL21 protein's structure and its interaction with calmodulin, we have used a 44 residue peptide derived from SL21, containing the microtubule binding motif Mn. The structure of the peptide has been analyzed using circular dichroism (CD) spectropolarimetry. The CD spectra showed that the peptide's secondary structure consists in a predominantly β-sheets, while the tertiary structure consists in rather folded 3D-structure. The temperature dependence of CD indicated that the secondary structure of the peptide remained very stable between 5 °C and 95 °C, while the tertiary structure changed at about 35 °C. The interactions between the SL21 peptide and calmodulin have been studied using the isothermal titration calorimetry (ITC). The results showed that calmodulin binds the peptide with the affinity of 104 M-1, in a Ca 2+ -independent manner, the reaction being driven by entropic contributions. Our results might contribute to understanding the microtubule stabilizing activity of the SL21 protein.

peptides 4, 5 . Based on the structure in polar environment a transition from 310-helical to an α-helical conformation at the water/lipid interface in bilayer membranes is hypothesized 3 The efficient method development for the analysis of proteins, peptides, and synthetic oligonucleotides with a novel hybrid reversed phase column F. Becker *1 , N. Shoji *2 , A. Matsui *2 , C. Yokoyama *2 , T. Sato *2 , T. Takai *2 , and N. Kuriyama *2 *1 YMC Europe GmbH, *2 YMC Co., Ltd.

The recent development of bio-pharmaceutical industry has been remarkable and an effective analytical method with higher sensitivity, superior selectivity, and increased speed has been required in the characterization of peptides, proteins and oligonucleotides by highperformance liquid chromatography (HPLC). The method development of reversed phase HPLC requires optimization of several conditions, such as bonded-phase, column efficiency, solvent type, pH and temperature. Especially pH and buffer type is a most important parameter to control retention of biomolecules which have multiple ionic functional groups. Furthermore, temperature often becomes a key tool to achieve better peak shapes and resolution for larger molecular-weight compounds.

Although silica based reversed phase columns have been widely used for separation of biomolecules, they have low stability under alkaline conditions and a limited usable pH range. To improve the chemical stability at an expanded pH and temperature range , we have developed a new type of organic/inorganic hybrid reversed phase column, YMC-Triart C18 and YMC-Triart C8. The novel technologies of manufacturing particles and surface modification provide outstanding chemical stability and excellent peak shape for many types of compounds under a variety of mobile phase conditions. In this poster, we will show characteristics of this new hybrid column, and some example cases of efficient method development in separation of the biomolecules such as peptides, proteins and synthetic oligonucleotides. The fully-extended peptide conformation (2.05-helix) is the longest possible for a single α-amino acid as it is characterized by an axial translation per residue of about 3.85 Å. Therefore, it is extremely attractive for its application as a molecular bridge. However, it is known that the stability of this type of helical structure (relative to the competing, much shorter 3 10-helix) appears to be dramatically governed by the choice of the C-terminal functionality (only esters seem to be appropriate).

In this work, we examined the series PyrAc-(Deg)n-O-(pNO2)Bzl [where PyrAc is the fluorophore 1-pyrenylacetyl, Deg is Cα,α-diethylglycine, n=1-5, and O-(pNO2)Bzl is para-nitrobenzoxy] with the Deg homo-peptides as fully-extended bridges in a static fluorescence study. To perform a very stringent comparison with our FT-IR absorption and NMR results, the solvent used was CHCl3. The trend observed in the quenching efficiencies for the shortest members of the series, the (Deg)1-3 peptides, steadily decreases from 0.97 (n=1) to 0.89 (n=2), and 0.64 (n=3). This finding is compatible with the conclusion that these three peptides are all essentially fully-extended in CHCl3 solution. However, the quenching efficiencies for the longest Deg homo-peptides. 0.68 for n=4 and 0.65 for n=5, are not further reduced. The inevitable conclusion is that the tetra-and pentapeptides populate at least two different conformations (most probably, the 2.0 5-helix and the 310helix), in which the probe dyads are located at different distances. Notably, these results fit well with those extracted from our FT-IR absorption and NMR studies in the same solvent of low polarity. We conclude that a combination of terminally aromatic groups, as in the Deg homo-peptide series investigated in this work, is not eligible for the fabrication of a stable fully-extended conformation. The surfactant protein C (SP-C) is a 35-residue highly hydrophobic peptide with two covalently linked fatty acyl chains that adopts a mainly helix structure while associated with the membrane. However, it misfolds into a β-rich structure under certain environmental conditions, which suggests that SP-C is in a metastable state 1 . The slow α→β transitions are likely to be caused by the membranedissociation, deacylation1 and exposure to the neutral pH 2 of the alveolar subphase, leading to the formation of amyloid aggregates associated with Pulmonary Alveolar Proteinosis.

We have been studying the pH-dependent conformational behaviour of both the acylated and deacylated isoforms of SP-C in a membrane mimetics chloroform/methanol/water mixture using a constant-pH Molecular Dynamics method [3] [4] [5] . Our simulations identify the increase of pH as a promoter of peptide-peptide interactions, which may contribute to the α→β transitions observed at neutral and alkaline pH conditions in the experimental results 2 . We also observe that the loss of structure is more prone to begin at the C-terminal region and that the β motifs start to form between the N and Cterminal disordered regions of the SP-C. In addition, the presence of the acyl groups results in a slightly higher structural stabilization of the SP-C helix and in a decrease of the N-terminal plasticity at acidic and neutral pH. Since a comprehensive conformational analysis and the elucidation of the mode of association between SP-C and the membrane are essential to understand the interactions involved in structure stabilization and in some specific functions of the peptide, we are now studying both SP-C isoforms in a membrane environment. These studies will not only be the most relevant for the in vivo situation but will also answer to some questions left open by the previous work.

were rather small. VP plays a very important role in controlling resorption of water by the distal tubules of the kidney and in regulating the osmotic pressure of blood in mammals. VT, while exhibiting both oxytocin and vasopressin activities, has not changed over the last 300 million years until appearance of the mammals. Currently, it occurs naturally in the non-mammal vertebrates [1] . Here we report on studies of arginine-vasopressin (CYFQNCPRG-NH2, AVP) and arginine-vasotocin ([Ile3]AVP, AVT) in a mixed dodecylphosphocholine (DPC) and sodium dodecylsulfate (SDS) micelles. DPC micelles are considered good models of eukaryotic cell membrane [2] , while the SDS micelle improves peptide/micelle solubility [3] . For these studies we use 2D nuclear magnetic resonance (NMR) supported with molecular modelling methods. Conformation-activity considerations relationships and the role of the peptide-micelle interactions on the structure of the analyzed peptides will be described and discussed. Vibrational and chiroptical spectroscopic investigation on diamide peptide models K. Knapp, a M. Hollósi, a E. Vass, a Zs. Majer a a Eötvös Lor nd University, Institute of Chemistry, Laboratory for Chiroptical Structure Analysis, Budapest, Hungary Understanding of peptide folding has great importance in many fields of chemistry. Since amino acid residues are the simplest building blocks of peptides, the investigation of their conformational properties could help us in understanding the structures of peptides or in designing peptides of specific 3D structure. Describing the conformational building blocks of even the simplest peptide models (e.g., amino acid diamides) can serve as a good starting point to decipher higher-level structural properties of their polymers. Among the simplest compounds which include the essential structural elements of polypeptide backbone are the N-methylamides of N'acetylamino acids. These compounds have been the subject of extensive spectroscopic analyses (FTIR, VCD, ECD, NMR) [1, 2] . The conformational landscape of these model compounds were analyzed by solution phase VCD and ECD spectroscopy and density functional theory (DFT) computations. This work reports systematic VCD and ECD spectroscopic studies in different solvents on synthesized diamide models (Ac-α/βhomo-Xxx-NHCH3, Xxx = Ala, Pro, Phe). VCD and ECD data, combined with quantum chemical calculations performed at the B3LYP/6-31++G** and 6-311++G** level of theory was particularly useful for identifying the preferred Dipartimento delle Scienze Biologiche, Università di Napoli "Federico II", Napoli, Italy c Dipartimento di Scienze Ambientali, Seconda Università di Napoli, Caserta, Italy β-hairpin is an important secondary structural element used by many proteins for biomolecular recognition, and thus is an attractive tool for mimetic design 1 . Aβ -hairpin motif consists of two antiparallel strands linked by a turn region. In this work we aimed to stabilize a β-hairpin conformation by an interstrand covalent bridge, made through a click chemistry reaction, which yields 1,4-disubstituted 1,2,3, triazole, starting from alkyne and azide reactive groups. In particular, we explored the conformational stability of a series of β-hairpin peptides containing a 1,4-disubstituted 1,2,3 triazole bridge with variable length. We employed the well structurally characterized trpzip2 peptide as a convenient b-hairpin model system. 2 As alkyne amino acids were introduced propargylglycine (Pra), homopropargylglycine (Hpg) or bishomopropargylglycine (Bpg), while the following azide amino acids were inserted: Lβ-azidoalanine (Dap (N3)), L-γ-azidohomoalanine (Dab (N3)), and L-δ-azidoornitine (Orn (N3)). All peptides were synthesized and purified. Successively, the linear unprotected peptides were cyclized in solution by the Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction and purified prior to the spectroscopic characterization. Linear and cyclic peptides were analyzed by a combination of CD and NMR techniques. 3 have been investigated. 1 Their behaviour has been compared with that of proline in homochiral and heterochiral dipeptide sequences including L-or Dphenylalanine. NMR and IR studies as well as X-ray diffraction analysis provide evidence that the additional β-phenyl substituent does not disrupt the tendency of proline to occupy the i+1 position of a β-turn. Moreover, the β-turn conformation is stabilised, with reference to L-Pro, when L-cis(βPh)Pro or L-trans(βPh)Pro are combined with D-Phe, that is, in the heterochiral sequences. The pyrrolidine conformation is significantly affected by the presence of the β-phenyl group. The puckering modes that alleviate most the steric hindrance introduced by the bulky phenyl substituent are preferred. The L-cis(βPh)Pro residue shows a marked propensity for the Cγ-endo/Cβexo half-chair arrangement whereas the trans derivative exhibits a higher flexibility, with different pyrrolidine shapes being accessible. Interactions between the aromatic ring of (βPh)Pro and that of the contiguous L-or D-Phe residue are observed in all the dipeptides crystallised. In solution, they seem to occur only for the heterochiral sequences. This intramolecular aromatic-aromatic interaction may be responsible for the higher β-turn ratio observed for such sequences and also seems to affect the conformational preferences of the pyrrolidine ring. In Fmoc chemistry the Trt and Acm groups are widely accepted as a protecting group for Cys. Upon incorporation of these Cys derivatives onto the growing peptide chain, however, considerable base-catalyzed racemization of Cys is known to always occur at the activating and coupling steps with phosphonium or uronium reagents such as PyBOP or HBTU, respectively. In addition to this, racemization of the C-terminal Cys esterified to resins also occurs during the repetitive N α -Fmoc deprotection using piperidine. In order to find an S-protected Cys derivative applicable for Fmoc chemistry that can efficiently suppress racemization of Cys both when its incorporation is conducted by phosphonium or uronium reagents and when it is linked to a resin via ester linkage, we introduced the 4methoxybenzyloxymethyl (MBom) group into the thiol function on Cys. The MBom group was found to substantially supress racemization of Cys (0.4%) during its incorporation mediated by phosphonium or uronium reagents in comparison with the Trt and Acm groups (8.0% and 4.8%, respectively). Even after a 6-h treatment of the peptide resin, in which its C-terminal Cys was linked to a Trt-type resin, with 20% piperidine in DMF, the MBom group caused a significant reduction in the level of racemization with the C-terminal Cys (6.4%) while Trt and Acm groups led to a considerable extent (30.2% and 27.9%, respectively). Next, we demonstrated the usefulness of the MBom group on Cys by applying it to NCL chemistry involving the coupling of a peptide thioester and a Cys-peptide. In the synthesis of the latter peptide, however, a diastereoisomer with racemization of the Nterminal Cys tends to be difficult to separate from the intact peptide as its chain length increases. Thus, this measure using Fmoc-Cys(MBom) can facilitate the avoidance of ambiguities in the quality of the synthesized peptides. Labeled peptides are important tools in chemical biology to obtain information on their biological function in, for example, cellular communication processes as well as to study the molecular basis of diseases. The functionalization of bioactive peptides with biophysical reporter groups like strongly absorbing chromophores, is most often performed via post-synthetic chemoselective biocojugation reactions, involving amino, thiol, or azide specific reactions. An alternative approach for the installation intense chromophoric organic molecules, is the coupling of nonchromophoric precursor molecules to obtain chromophoric properties in the final product. Recently, we have shown that the [2+2] cycloaddition-cycloreversion reaction between peptide-functionalized electron-rich alkynes and electrondeficient cyanovinyl derivatives results in the formation of a new class of π-conjugated peptidic donor-acceptor chromophores. 1, 2 The chromophoric properties of the final product can be tuned by varying the p-electron conjugation pattern. Herein, we describe the synthesis of the versatile building blocks, N-a-(4-ethynylphenyl)-N-a-(methyl)-glycine and N-a-(4-(1,2,2-tricyanovinyl)benzoyl)-glycine, their subsequent application in SPPS to functionalize peptides with D-p-A chromophore precursor molecules, and their UVvis absorption characteristics. As a typical example, alkyne-functionalized Aβ peptides, Val-Ala-Ile and Lys-Leu-Val-Phe-Phe-Ala-Glu, have been reacted with a series of cyanoolefins to obtain far-red intense imaging chromophores for amyloid-rich peptide aggregates. 

A novel method for regioselective disulfide formation in mini-proteins by kinetic oxidation control T. Lindner, U. Haberkorn, W. Mier Department of Nuclear Medicine, University Hospital Heidelberg, Im Neuenheimer Feld 400, Heidelberg, Germany Rigid frameworks are essential for the development of peptide-based alternatives to immunoglobulins. [1] Disulfideconstriction covalently stabilizes tertiary structures, which allows further miniaturization of protein-derived drugs by chemical synthesis. While the amino acid assembly steps in solid phase peptide synthesis are highly efficient, regioselective formation of multiple disulfide bonds is still a challenging and time demanding task. Even after applying laboriously optimized folding conditions and extensive purification, a successful synthesis of the correctly folded isomer is not guaranteed. [2] To circumvent these problems, a procedure that takes advantage of the different reactivity of free vs. Acmprotected thiols during iodine oxidation was developed. This protocol to sequentially form two disulfide bridges in a one-pot reaction was applied to different peptides, such as (a) Min23, a mini-protein scaffold for phage-display assisted drug design; [2] (b) α-conotoxin ImI, a neurotoxic peptide isolated from the venom of marine cone snails; [3] and (c) endothelin-1, a peptide involved in blood pressure regulation. [4] The presented technique tolerates precarious residues within the to-be-folded-peptide, such as fluorescent dyes or metal chelators as used for bioactivityassays. Compared to the individually optimized literature procedures, this novel approach offers significant improvements: Overall yields >30% are obtained in first attempts and stepwise formation of the disulfide bridges is performed within a few hours instead of days. In recent thirty years, C-terminal modified peptides have been proved to have greater potential as APIs (Active Pharmaceutical Ingredients) due to their increased chemical and enzymatic stability and improved pharmacodynamic properties 2-4 . A prominent example, Octreotide 5-6 , an octapeptidoalcohol, has witnessed as a potent anti-cancer agent targeted for gastro-entero carcinomas.

In view of synthetic methodology, peptidoalcohol can not be directly prepared by standard SPPS protocol becouse of the C-terminal structure released from resin are not alcohol but always peptidoacid or peptidoamide.

To overcome this problem, a novel protocol of shortened n-1 coupling cycles on Merrifield resin and then the ammonolysis of peptedyl resin by an aminoalcohol as the C-terminal residue getting peptido-alcohol as targetting product has been devoloped in our lab. Because of the cleavage treatment of peptidyl Merrifield resin is not under acidic condition, such as HF or TFMSA, but ammonolysis, some side-chain producting groups(SPG) related to Boc chemistry like Bzl, Clz, Tos…; must be avoided in sequence assembly. Therefore a Hybrid Orthogonal Protection (HOP) of Boc/fmoc protocol was adopted for the sake of producing naked peptidyl (without any SPG) resin before ammonolysis. Fifteen peptidoalcohols with different terminal alcohols were conveniently prepared, most of them released form resin with very good yields. Due to its cyclic structure, proline is the coded amino acid with a more restricted conformational flexibility. The incorporation of additional groups into the pyrrolidine ring is a useful means to produce new amino acids that combine the conformational properties of proline with sidechain functionality. This is the case of β-phenylproline, (βPh)Pro, that can be regarded as a proline-phenylalanine hybrid in which the orientation of the aromatic substituent is dictated by the conformation of the five-membered ring and the cis or trans configuration of the phenyl group relative to the carbonyl moiety. Accordingly, cis(βPh)Pro and trans(βPh)Pro combine the conformational properties of proline with an aromatic side-chain functionality that is rigidly oriented with respect to the peptide backbone, and this may be useful in the design of biologically active peptides and other applications relying on specificallyoriented side-chain moieties.

We have developed synthetic procedures for the preparation of the cis(βPh)Pro and trans(βPh)Pro stereoisomers in enantiomerically pure form. The methodology is based on the preparation of racemic precursors of each amino acid and their subsequent HPLC resolution on chiral columns. Multigram quantities of the target amino acids have been isolated in optically pure form and suitably protected for use in peptide synthesis. The importance of peptide cyclization for studying peptide conformation, creating new structures, or for developing peptide therapeutics is well established. In particular, sidechain lactam bridges linking two amino acid residues that are several residues apart in the linear sequence or headto-tail backbone peptide cyclization enable rigidification of the structure and improvement of in vivo stability. Native Chemical Ligation (NCL) is now an established method for producing backbone-cyclized peptides or proteins. The application of NCL to the synthesis of sidechain cyclized peptides is less frequent. Head-to-side-chain cyclization by ligating a C-terminal thioester with a Cys residue located on a lysine side-chain was used by few authors. The alternative tail-to-side-chain cyclization mode is rare, probably due to the difficulty of installing a thioester group on amino acid side-chains such as aspartic or glutamic acids The reaction of a bis(2-sulfanylethyl)amido (SEA on ) group with an N-terminal cysteine residue in water and at neutral pH results in the formation of a native peptide bond. [1] Oxidation of SEA on results in a cyclic disulfide called SEA off having a 1,2,5-dithiazepan-5-carbonyl structure. [2] SEA off is a self-protected form of SEA on . We show here that bis(2-sulfanylethyl)amido side-chain acid(DAB), ornithine and Lysine were selected as building block; 1a and N,N'-Cbz-1-amidinopyrazole (1b) were selected as guanidinylating reagents for specific situation. For synthesis of N-terminus local cyclo-guanidine peptide, designated peptides were assembled on acid labile solid support such as Rink amide resin by Fmoc strategy. Then either Fmoc-Dab(Boc)-OH, Fmoc-Orn(Boc)-OH or Fmoc-Lys(Boc)-OH was incorporated respectively at N-terminus. Fmoc was removed followed by guanidinylating by 1b and then peptide was cleaved by acid. By neutralizing with NMM in acetonitrile solution, side chain amino group and a-guanidine would form 6, 7 or 8 membered local cycloguanidine. The remaining Cbz could be removed by hydrogenation.

For synthesis of backbone side chain cyclic peptide, bis-Fmoc-DAA was introduced in the peptide previously on resin followed by removal of Fmoc. Selective guanidinylate side chain aminogroup by 1a followed by peptide assembling with an insertion of orthogonal protected DAA at 1-4 AA apart from first DAA. For synthesis of a-NH2sidechain cyclic peptide, first DAA should be introduced with orthogonal protected form. 1b was used to guanidinylate a-NH2. After cleavage and neutralization of those two kinds of intermediates, guanidine-bridged marco-cyclic peptide was formed. The resin is also a multipurpose tool for the synthesis of carboxylic acids, esters and thioesters. When the synthesis is completed, the fully protected peptide hydrazide resin is oxidized with either N-bromosuccinimide (NBS) or copper(II) acetate in pyridine. The resulting acyl diazene resin is then cleaved by peptide displacement at the C-terminus with amine. The fully deprotected peptide amide is finally obtained by treatment with trifluoroacetic acid (TFA). In our approach, we used a 4-Fmoc-hydrazinobenzoyl AM NovaGel resin to synthesize a peptide-substituted amide in the C-terminus. First, the oxidative cleavage was carried out with NBS in pyridine and a nucleophile [a protected 4 (aminomethyl) benzimidamide (Amba)]. However, the yield of the reaction was very poor. In the next step, we applied copper(II) acetate in the presence of pyridine and Amba. Following optimization, the efficiency of the process was significantly improved.

Herein we discuss the conditions needed to obtain a reasonably high efficiency of the oxidative cleavage in the synthesis of our C-terminal modified peptides using the aryl hydrazine resin linker.

blood vessels on tumor tissues, similarly to integrin receptors. This observation suggests CD13 as a selective target for targeted delivery of drugs and nanoparticles to tumor neovasculature using NGR peptides as homing motif. In our work, new cyclic-NGR peptides containing a thioether linkage were prepared. The influence of their structure on the speed of succinimide ring formation and deamidation was evaluated and compared with the previously published data on cyclic-NGR derivatives containing amide bond or disulfide bridge in the cycle (c[KNGRE]-NH2 and c[CNGRC]-NH 2 ). To avoid the deamidation under the conditions used for cyclization, the synthetic routes were optimized. The influence of the pH, ionic strength and temperature of the solution on their chemical stability was investigated. The structure of the cyclic peptides was investigated by circular dichroismand NMR-spectroscopy. Receptor binding ability and the influence of the cyclic peptides on the cell adhesion and motility were also evaluated. This work was supported by grants from the Hungarian National Science Fund (OTKA NK 77485 and K 81596) and the National Innovation Office (BIO_SURF, OM-00146/2008).

Clickable Peptides and Their Attachment to Oligonucleotides M. Wenska, M. Alvira, R. Strömberg Department of Biosciences and Nutrition, Karolinska Institutet, Novum, SE-141 83 Huddinge Methodology for the ready conversion of peptides into "clickable" azido-peptides with the possibility of selecting either N-terminus or C-terminus connection is presented. 1 Synthesis of peptide-oligonucleotide conjugates (POC's) include conjugates of oligonucleotides with peptides known to be membrane penetrating and nuclear localization signals. A general procedure, based on a new activated alkyne linker, for the preparation of POC's has been developed. 2 With this linker, conjugation is effective at room temperature in mM concentration and submicromolar amounts. This is made possible since the use of a readily attachable activated triple bond linker speeds up the Cu(I) catalyzed 1,3-dipolar cycloaddition ("click" reaction). The main scheme for conjugate preparation involves sequential conjugation to oligonucleotides on solid support of i) an H-phosphonate based aminolinker ii) the triple bond donor p-(Npropynoylamino)toluic acid (PATA) and iii) azido-functionalized peptides. The method gives excellent conversion of oligonucleotide to the POC on solid support, and only involves a single purification step after complete assembly. The procedure which makes use of a low concentration of copper ions leads to a product with very little copper left (similar or less than in drinking water). The synthesis is flexible and can be carried out in non-specialist laboratories without the need for specific automated synthesizers since it has been designed to utilize commercially available oligonucleotide and peptide derivatives on solid support or in solution. Comparison of alternative deprotection reagents to piperidine for the synthesis of a poly-alanine peptide on the Tribute® peptide synthesizer M.A. Onaiyekan,* J.P. Cain, C.A. Chantell, M. Menakuru Protein Technologies, Inc. Tucson, AZ, USA In peptide synthesis, piperidine is a common agent for Fmoc removal. However, piperidine is a controlled substance which requires special handling and cannot be used in some countries. Therefore, it would be useful to identify alternative deprotection reagents to piperidine for Fmoc removal. It is well known that poly-alanine sequences have a high propensity to aggregate after the fifth residue. In this application, (A)10K-OH was synthesized using the Tribute®'s Intellisynth UVmonitoring and Feedback System to compare the efficiency of Fmoc removal by piperidine vs. three alternative bases (pyrrolidine, cyclohexylamine, and tertbutylamine) in the last 5 cycles of the synthesis. It was found that pyrrolidine produced a higher purity product with fewer deprotection repeats and shorter deprotection times per cycle than piperidine, proving it to be a highly efficient, viable alternative to piperidine for Fmoc removal. The endogenous tripeptide GPE also nammed "Glypromate" is made up by the three N-terminal residues (glycine-proline-glutamate) of the insulin-like growth factor 1 (IGF1). This tripeptide is a partial glutamate antagonist and showed good results in different neuroprotective in vitro and in vivo experiments. 1,2 GPE also binds to glial cells regulating neurotransmitter levels in the brain. 3, 4 However, GPE suffers from poor lipophilicity and a short half-life in vivo. That's why there is a need for more lipophilic and protease resistant analogues of GPE. In this poster we present the synthesis of trifluoromethylated analogues of GPE based on the 2 or 5-CF3-pseudoproline residues. Introduction of fluorine atoms on bioactive compounds is known to deeply modify their physico and biochemical properties increasing lipophilicity and resistance to protease. 5 Thus, developing a trifluoromethylated analogues, we intend to increase the bioavailability of GPE, keeping the benefit of its neuroprotective properties. Our research team is strongly involved in the synthesis of trifluoromethylated alpha-amino acids. Recently we published the synthesis of 2-trifluoromethyl-1,3oxazolidines derived from fluoral and (L)-serine and we demonstrated that these five membered ring 5-CF3pseudoprolines are hydrolytically stable and can be considered as proline analogues. 6 That's the reasons why we are interested to replace the proline residue of GPE by those trifluoromethylated compounds. The development of original coupling conditions and the detailed synthesis of two pseudoprolines analogues of GPE will be presented in this poster. modifications. In combination with automated SPPS, unprecedented access to large peptides and small proteins for biological research has been achieved. We demonstrate the application of this methodology to the synthesis of a variety of peptides on the Prelude® peptide synthesizer.

Exploring the space of fluorine-labeled α-amino acids for solid state 19 F-NMR structure analysis of peptides: rational design, synthesis and applications P. Solid state 19 F-NMR is a powerful method to study membrane-active peptides, as it can reveal their conformation, orientation and dynamics when embedded in biomembranes. 1 For this purpose the native peptide has to be selectively labeled with a suitable 19 F-containing amino acid at several different positions. The resulting battery of singly 19 F-labeled analogues is then analyzed by solid state 19 F-NMR. The main limitation to this approach currently lies in the poor arsenal of available 19 F-labels. We have therefore rationally designed and synthesized several specific amino acids bearing a CF3-reporter group, which fulfil all strict criteria to a "proper" 19 F-label. 2, 3 To allow a geometry-based structure calculation, the CF3group has to be rigidly attached to the peptide backbone. We thus rigidified the side chain using either a [1.1.1]bicyclopentane moiety, a cyclobutane ring, or the intrinsic proline framework. This way, suitable CF3-labeled analogues were created as substitutes for bulky hydrophobic amino acids (Leu/Ile/Val/Met), for aromatic residues (Phe), for polar side chains (Ser/Thr), and for proline (Pro). By now we have applied the developed 19 F-labels for a comprehensive structure analysis of more than ten different membrane-active peptides (Gramicidin S, PGLa, Mag 2, KIGAKI, SAP, Temporin A, BP100, etc). Recently, several new activators have been introduced into the market, and they were evaluated along with some older activators for their ability to synthesize a range of peptides with shorter and longer reaction times on the Symphony® peptide synthesizer. It was found that HDMC, PyClock, COMU, HCTU, and HATU worked well at shorter reaction times (2x1 min), but PyOxim and TFFH only worked well at longer reaction times. The performance of PyBOP at shorter reaction times was poor only for more difficult sequences. These results are important for selecting an appropriate activator for fast SPPS applications. The plant cyclotides form the largest family of cyclic peptides 1 . They contain a signature motif referred to as the cyclic cystine knot, which is derived from the cyclic backbone and three inter-knotted disulfide bonds. Intriguingly, cyclotides can be boiled, treated with chemicals or enzymes without disrupting their overall fold. Thus, they are sometimes labeled as ultra-stable proteins.

In addition, cyclotides are tolerant to mutations, and as a scaffold they can successfully accommodate foreign bioactive epitopes of variable sequences 2 . Cyclotides share many of these properties with another disulfide containing cyclic plant peptide, the sunflower trypsin inhibitor 1 (SFTI-1) 3 . Emerging evidence indicates that cyclotides and SFTI-1 are valuable not only as peptide stabilizing scaffolds; in combination with their cell penetrating properties, these disulfide rich cyclic peptides have significance as intracellular drug carriers. Although both peptides are genetically encoded, studies to ascertain the exact mechanisms of their biosynthesis are currently on going. Thus, the synthesis of cyclotides and SFTI-1 are currently restricted to chemical means. We have recently adapted a Fmoc-SPPS method for cyclic peptide synthesis, via N-acylurea intermediates with the assistance of microwave irradiation. This method is a safe and convenient alternative to Boc-SPPS and has the ability to be automated conveniently. Using this method, parent scaffolds as well as several cyclotide and SFTI-1 analogues with potential antimicrobial and matrix metalloprotease activities were synthesized. With the rising interest in the cyclization concept as a tool to impart stability on unstable peptides, the cyclic peptide synthesis method adapted herein is anticipated to have numerous applications.

fixed configuration. The nonnatural oligomers have an extended conformational space and are supposed to adopt non-canonical secondary structures 2 . In addition, the backbone modification makes these molecules more stable towards proteolytic degradation. The majority of proteins in nature are post-translationally modified, and the most abundant modification is the protein glycolysation, which introduces wide structural variety to proteins. Glycoproteins have an important role in the biological recognition process, such as immunodifferentiation, cell adhesion, cell differenciation and regulation cell growth 3 . New aza-β 3 -amino acids bearing either an azide instead of amine on Lys and Orn chain or an alkyne group will be described and used in solid phase synthesis to finally performed a click chemistry to cyclize pseudopeptides or to introduced a glycosylated function 4 .

true for a series of peptides that display strong corticotropin releasing factor (CRF) antagonistic activity. Seminal studies by Rivier et al. have shown that the incorporation of a lactam bridge in the CRF-sequence resulted in an enormous increase in activity and potency, due to stabilization of the bioactive a-helical conformation of the peptide; and the newly designed peptide was called astressin. 1 Based on the astressin sequence, we started a truncation and deletion study to arrive at astressin analogs with a reduced size but still remain active as CRF antagonists. This study resulted in the smallest active CRF antagonist, astressin(30-41). 2 This sequence was further optimized by the introduction of novel covalent constraints, other than the well-known lactam bridge. As a first approach, the alkene/alkane bridge, which can be introduced via ring-closing metathesis via alkenesubstituted amino acid side chains, and as a second approach, the triazole bridge ('click' macrocyclization), via either a Cu(I)-or a Ru(II)-catalyzed cycloaddition reaction between azide-and alkyne-derivatized amino acid residues were explored. Herein, we will present the details of the synthesis of the alkene-, azide-, and alkyne-functionalized amino acids, their use in SPPS, and the optimized approaches for macrocyclization. Furthermore, the peptides have been characterized by HPLC, NMR, LCMS, and studied by circular dichroism spectroscopy to obtain insight into the helical propensity of the peptides in relation to the cyclic constraint. The synthesis of a nitronyl nitroxide, C α -tetrasubstituted αamino acid (a class of sterically restricted amino acids that promote the formation of peptide β-turns and helical structures) was achieved by derivatisation of racemic 2amino-5-cyano-indan-2 carboxylic acid [Aic(CN)]. Racemic Boc-Aic(NN)-OH was prepared by bis(alkylation) of ethyl isocyanoacetate under phase transfer conditions with 3,4-(bis)bromomethyl benzonitrile as alkylating agent, followed by acidic hydrolysis, N α -Boc protection, and saponification of the ester function. Resolution was achieved through formation of the diastereomeric amides of (S)-phenylglycinol with chromatographic separation and mild acidic hydrolysis. Reduction of the nitrile group to an aldehyde was carried out with Raney nickel in the presence of sodium hypophosphite. Condensation with 2,3-diamino-2,3-dimethylbutane gave the corresponding tetramethylimidazolidine, which was oxidised with 3chloroperbenzoic acid to the desired nitronyl nitroxide. The UV-Vis absorption and EPR spectra of the amino acid were recorded and its magnetic properties were examined. In order to develop the synthesis of this peptide using the Fmoc solid-phase peptide synthetic methodology, orthogonally protected β-hydroxyaspartic acid was needed. More precisely we wish to dispose of (2R,3R)-N -Fmoc-3-TBDM-Silyloxy-aspartic acid α -allyl ester instead of the recently reported DMAB ester 2-3 indeed, in preliminary assay using this protective group we experienced difficulties during the final cyclisation step 4 . The synthesis was developed starting from inexpensive L(+) dimethyltartrate and extended to the others stereoisomers of the β-hydroxyaspartic acid.

structure. For that, we chose to replace proline by silaproline to afford polysilaproline. This study shows the comparison of two polyamino acids: polyproline and polysilaproline polymers. Homopolypeptides were synthesized by polymerization of corresponding amino acid N-carboxyanhydride 3 . Multicomponent reactions (MCRs) represent a chemical process involving at least three reactants for the formation of several covalent bonds in one operation 1 . By definition MCRs are chemo-and regioselective, convergent stepefficient procedures and take place with high atom economy. The copper(I)-catalyzed 1,3-dipolar cycloaddition of organic azides and terminal alkynes (CuAAC) reported by Meldal 2 and Sharpless3 has been involved in various fields of chemistry and biochemistry research. However only few reports describe the implementation of CuAAC and MCRs. 4, 5 Recently our research focused on a novel threecomponent reaction based on a Cu(II)-triggered aminolysis of peptide hydrazide resin and an azide-alkyne cycloaddition sequence. 6 Copper(II)-induced oxidative aminolysis of hydrazides generates Cu(I), catalyst of the azide-alkyne cycloaddition. This feature was exploited to design a solid phase detaching three-component reaction.

The MCR process requires a peptide hydrazide resin, an amino azide linker and an alkyne, resulting in the formation of peptide modified at the C-terminus through an amino 1,2,3-triazole linker. This method can potentially be applied to the synthesis of a large variety of peptide derivatives starting from Fmoc-SPPS assembled peptidyl resins.

Furthermore, it is not practical to compare HPLC spectra from different resin samples (e.g., before and after reaction) directly. A comparison by analyzing the same (mg) amount of resin would involve tedious sample preparation that is extremely error-prone and would be impractical because factors resulting from the increase or decrease of the molecular weight of the resin-bound compounds may have a significant influence on the results. The use of internal reference compounds allows rapid assessment of reactions performed on solid supports. The internal reference compound is bound to the resin together with the substrate and cleaved with the products after completion of the reaction. Commercially available compounds can be used for this purpose, or likewise, the reference compound can be generated from the substrate by partial capping of a functionality. The peak integration of the reference compound in the HPLC-UV spectra can be correlated directly to those of the rest of the compounds present in the reaction mixture and therefore a quantitative interpretation of the spectra with respect to conversion and yield is possible. Here we demonstrate the proof of principle as well as the accuracy of this method. Modifier proteins such as ubiquitin are conjugated to protein substrates in cells and thereby mediate various biological processes. 1 Of high interest, is the ubiquitin fold modifer 1 (UFM1, 83 residues) which has structural similarity to ubiquitin but has no sequence similarity. Unlike ubiquitin, UFM1 has not been extensively studied and little is known about its biological role. To understand UFM1's biological functions, access to pure, homogeneous natural and modified UFM1 protein is essential. Chemoselective ligation techniques are suitable for providing such proteins.

Recently, a variation of the α-ketoacid-hydroxylamine (KAHA) ligation 2 was developed, which utilizes the chemoselective reaction between a C-terminal peptide αketoacid and a N-terminal 5-oxaproline. 3 This modified form of the KAHA ligation furnishes a native peptide bond and a homoserine residue. This ligation is useful for the synthesis of proteins from two unprotected protein segments in aqueous buffers. For the synthesis of larger proteins, a sequential ligation strategy is necessary. Using UFM1 as the model system we have developed a sequential ligation procedure using KAHA ligation with 5-oxaproline. Applying the new sequential ligation strategy we have prepared UFM1 by total chemical synthesis. We have also prepared a Cterminal thioester surrogate of UFM1 protein, which is suitable for conjugation to proteins of interest. The syntheses required the development of a bifunctional peptide segment bearing an α-ketoacid and an orthogonally protected 5-oxaproline. The preparation of the protein segments, their intermediates, the deprotection, and sequential KAHA ligations towards the syntheses of UFM1 protein and C-terminal modified thioester UFM1 protein will be discussed.

affinity and biological activity, we have designed and synthesized new analogues by multiple N-Methylation of hUT-II(4-11) backbone amide bonds. All the peptides were performed by a novel synthetic approach, in which the introduction of N-methyl groups occur during regular solidphase peptide synthesis. On these new ligands we evaluated the binding affinity and biological activity at the UT receptor and performed preliminary NMR conformational studies. Since that time a number of different machines have been used to automate peptide synthesis. Modern machines are following two general setups; the so called "single approach" and the "parallel approach". In the single approach, the machine is developed to synthesize one or few peptides simultaneously. The user is able to optimize the synthesis conditions on each single peptide and each single coupling step. The maximum product quality regarding purity and yield is the major task of this approach. In the parallel approach, the machine is developed to synthesize a huge number of different peptides in the same single setup and time-frame. The user always has to find a synthesis protocol appropriate for the needs of each peptide to reach the maximum quality, knowing that there will be always a number of failed peptides. As a result you will find both types of peptide synthesizers in laboratories all over the world: The Single Machine, for the complicated peptides, and the Parallel Machine, allowing generation of multiple peptides with standardized protocols for each. The Tetras is the first instrument combining the advantages of both machine types and allows the user to synthesize up to 106 different peptides in parallel. Each peptide can have its own individual synthesis protocols, separate of all others. The user can combine different synthesis scales, peptide lengths, and activator reagents in one run. Finished peptides can be removed and new peptides can be started while the Tetras is still running. The Tetras allows the user to establish an uninterrupted production shop using one instrument only.

Siemion, I. Z.; Peptide Res

The Peptides: Analysis, Synthesis And Biology

Monitoring peptide folding in membrane-active peptides: a time-resolved spectroscopic study E. Gatto a

Cordopatis P. 31st European Peptide Symposium

SAR studies of triazolyl-containing cyclopeptides: A defined -turn structure increases potency and selectivity to melanocortin receptor subtypes C. Testa, a,b

Proc. Natl. Acad. Sci

Proc. Natl. Acad. Sci USA

Multicomponent Reactions

Microglobulin: a "difficult" protein S. Abel, M. Beyermann 1

The protein ß2-microglobulin constitutes the noncovalently bound light chain of the major histocompatibility complex class I (MHC) and plays an essential role in the dialysisrelated amyloidosis. [1, 2] To examine the amyloid fibrils of the ß2-microglobulin (ß2-M) via infrared spectroscopy we intended to synthesize 13-C-labeled ß2-M. [3] Due to the two cysteine residues in positions 25 and 80 we used the native chemical ligation (NCL) strategy for assembling the 99-mer protein. This necessitates the synthesis of three segments which was accomplished on solid phase using the Fmoc/t-Bu chemistry. The preparation of the segments had to be optimized with respect to aggregation, aspartimide and piperidide formation, trifluoracetylation, and S-tert-butylsulfonium formation. Additionally, NCL steps had to be optimized, because of "internal" thioester formation, dimerization and the formation of side-products of the activated N-terminal segment

Peptaderm Inc., Krakowskie Przedmie cie Str. 13, Warsaw, Poland Immunosuppressors, such as Cyclosporine A (CsA) and Tacrolimus®, are routinely used in prevention of graft rejection after organ transplantation and in therapy of some autoimmune diseases, including skin inflammation. A naturally occurring in linseed oil Cyclolinopeptide A (CLA, c(-Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val) 1 possesses a strong immuno-suppressive activity, comparable at low doses with that of CsA 2 , but is much less toxic. We synthesized new CLA analogs, containing instead of one proline residue its six-membered mimics, pipecolic acid (Pip): c(-Pip-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val) (1) and c(-Pro-Pip-Phe-Phe-Leu-Ile-Ile-Leu-Val) (2). The incorporation of pipecolic acid residue led to different conformational behavior of the nonapeptide cycle. NMR experiments in CDCl 3 solution showed that CLA analogue 1 with the pipecolic acid residue in position 1 was much more flexible than cyclopeptide 2. The new peptides were devoid of toxicity up to 100μg/mL with regard to human peripheral blood mononuclear cells (PBMC), did not inhibit tumor necrosis factor alpha production in blood cell culture, but exhibited dosedependent, anti-proliferative actions for phytohemagglutinin A-activated PBMC. Since peptide 1 was more potent it was tested for growth inhibition of L-1210 lymphatic leukemia. The peptide was found to strongly inhibit the cell growth even at low concentration (63% inhibition at 5μg/mL). 

HIV-1 has emerged as the largest and the most devastating public pandemic in our days, affecting approximately 70 million people worldwide 1 . Development of an effective, safe and preventive HIV vaccine remains an urgently needed priority. Epitopes for HIV-specific antibodies in elite controllers, a subgroup of long term non progressors, encompassing segments of MPER of gp41 and for the V3 loop of gp120 were identified using the phage display technology 2 . Immunization experiments with epitopes conjugated to an artificial sequential oligopeptide carrier (SOC4), formed by four repeats of the tripeptide Lys-Aib-Gly in tandem, or to the palmitoyl group are currently in progress. All syntheses were performed on a Rink amide resin following the Fmoc technology. Conjugation of epitopes to the SOC4 carrier was realized via a chemoselective ligation approach, which generates an oxime bond between the H2N-O-groups of the modified lysine residues and the aldehyde group of each epitope 3 Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 53-114 Wrocław, Poland 4 Peptaderm Inc., Krakowskie Przedmieście 13, 00-071 Warszawa, Poland Nonproteinogenic amino acids have been a tool to modify the structures of natural peptides since a long time 1 . Bioactive peptides involved in a physiological and biochemical processes cannot be applied in the therapy because of their instability in physiological conditions. That's why the synthesis of their stable active analogues is a challenge for medicinal chemistry nowadays. 4-trans-Hydroxyproline (Hyp) is an important building block of natural collagen. It is responsible for the stabilization of collagen super helix, forcing the trans amide bonds configuration with preceding amino acids 2 . At the same time the impact of trans-4-hydroxyproline on the conformation other than the collagen peptide chains of biologically important compounds is little known. It is known that immunosuppressive activity of CLA is comparable with cyclosporine A and is associated with the presence of the tetrapeptide fragment Pro-Pro-Phe-Phe containing Pro-Pro cis amide bond. 3 Now we present synthesis, conformation and biological activity of new analogues of cyclolinopeptide A (CLA), containing 4-transhydroxyproline instead of proline residues in position 6 or 7. We expected that the introduction of the hydroxyl group in the pyrrolidine ring might influence the biological activity and conformation of the native peptide due to its hydrophilic character and hydrogen bonding ability. The linear precursors of modified CLA analogues were prepared manually by standard solid-phase procedure "step by step" on Wang resin using Fmoc/tBu strategy and TBTU as coupling reagent. The cyclizations of linear peptides have been made under high dilution conditions by means of EDC/HOBt coupling reagents. The biological activity of newly synthesized compounds as well as the conformational study will be evaluated. Dip. di Scienze Ambientali, Seconda Università di Napoli, Caserta, Italy NMR spectroscopy is a powerful method to perform structural studies on peptides. To completely fulfill the potential of NMR, peptides labeled with stable isotopes ( 15 N, 13 C, 2 H) are essential. 1 Peptides are easily prepared on solid-phase but chemical synthesis becomes prohibitively expensive when applied to the incorporation of isotopes. An alternative cost-effective strategy is the recombinant expression of peptides in E. coli as fusion constructs with carrier proteins. 2 The main problem of this approach is the need of chemical reagents or proteases to cleave the target peptide from its fusion partner after purification. Proteases may determine the heritage of extrasequence amino acids at the peptide N-or C-terminus, while chemical reagents require harsh reaction condition that may modify target peptides. An interesting solution is represented by the use of inteins as fusion partner. Inteins are protein elements that can catalyze their self-excision from a flanking sequences in mild conditions, by adding nucleophilic agents such as thiols or simply by shift of pH and temperature, bypassing the use of proteases or chemical reagents. 3 We used the self-cleaving MxeGyrA mini-intein as fusion partner for the preparation by recombinant means of two isotope labeled peptides, HPLW and QK. 4, 5 The two peptides target Vascular Endothelial Growth Factor Receptor (VEGFR) and have been described to modulate VEGF-dependent angiogenesis. Our expression and purification scheme allows to obtain homogeneously isotope labeled peptides. The availability of isotope labeled HPLW and QK opens the way to NMR studies aimed to characterize the folding dynamics of the two peptides and their structures in complex with VEGFR. An NMR method to discriminate between the fullyextended and different helical conformations in a spacer peptide C. Peggion*, M. Crisma, F. Formaggio, C. Toniolo ICB, Padova Unit, CNR, Department of Chemistry, University of Padova, 35131 Padova, Italy The ideal fully-extended, α-peptide conformation, also known as 2.05-helix, is characterized by φ = ψ = ω = 180°t orsion angles. The repeating motif of this foldamer is a pentagonal (pseudo)cyclic structure (called C5), stabilized by an intraresidue H-bond. The N-H and C=O groups in the 2.05-helix are not involved in intermolecular H-bonds. Multiple C5 conformations were observed in homopeptides made up of C α,α -dialkylated glycines with both side chains longer than a methyl. This is the case for C α,αdiethylglycine (Deg), the residue studied in this work. It is known that Deg homo-peptides can adopt the 2.05-helix 1 or the 310-helix depending on environmental factors and Nand/or C-terminal moieties. 2, 3 In this communication, we introduce an NMR method to discriminate between the 2.05-helix and the 310-helix based on the observation of cross-peak intensities in the NOESY Human serum amyloid A (SAA) is a highly conserved apolipoprotein produced by the liver under inflammatory conditions accompanying e.g. atherosclerosis, cancer and amyloidosis [1] . It is also known that SAA1α isoform has the amyloidogenic properties [2] . Till now it is little known about structure of human SAA, as it hampers structural studies due to its facile aggregation. The analysis of protein sequence and CD data together with theoretical studies revealed a typical globular structure of the protein [3] . The C-terminal sequence of SAA contains three proline residues, which probably are responsible for the unordered structure. Recent in vitro studies involving SAA and human cystatin C (hCC) revealed direct interactions between the (86-104) fragment of SAA and the (93-120) sequence of hCC. The results of ELISA test for the (86-104) SAA fragment have shown that it binds to hCC very well. The NMR studies for the wild (86-104) sequence found an unordered structure in phosphate buffer. Based on these data we decided to check how the point mutations Pro→Ala in (86-104) SAA fragment could influence the peptide's structures. We synthesized four peptides with Pro→Ala point mutations and we performed CD experiments at different conditions. The results show that two of them contain disordered structure and two α-helical structures. In this project we analyze the solution structures of these peptides at the atomic resolution using 2D NMR supported with molecular dynamics. Design and conformational analysis of stapled peptides mimicking Cullin3 binding region to KCTD11. I. de Paola, a L. Pirone, a E. Pedone, a S. Di Gaetano, a L. Vitagliano, a R. Fattorusso, b G. Malgieri, b L. Zaccaro*, a Acknowledgement: This study was supported by EU within the European Regional Development Fund (POIG. 01.01.02-00-007/08-04).

Model of Angiotensin II bound to the AT1 receptor in the lipid bilayer environment M.T. Matsoukas, T. Tselios* Department of Chemistry, University of Patras, GR-26504, Patras, Greece The Renin-Angiotensin System () plays a major role in blood pressure regulation. A sequence of enzyme reactions leads to the release of Angiotensin II which interacts principally with the type-1 Angiotensin II receptor (AT1), a 359-residue, which belongs to the G protein-coupled receptor family. In the present study, the human AT1 3D model was constructed using Modeler for the sequence alignment and loop refinement tools. On this basis, the crystal structure of bovine rhodopsin, (pdb code 1U19), was used as a 3D template. The GROMACS software and Amber99SB forcefield were utilized for molecular dynamics calculations [1] in order to evaluate the binding mode of Angiotensin II. The role of the critical amino acids of the binding site V108, N111, L112, A163, K199, S252, H256, N294 and Y292 is being studied. Moreover, newest information on the role of the 2 nd extracellular loop by Unal et. al. [2] have been implemented on the model, therefore we propose the contribution mechanism of the residues F170-Q187 for binding of Angiotensin II to the AT1 receptor for activation and signaling. 

A. Stavrakoudis Department of Economics, University of Ioannina, Greece One key step in the immune response against infected or tumor cells is the recognition of the T-cell receptor (TCR) by class I major histocompatibility complexes. It has been found [1, 2] that such peptide/MHC complexes can interact with antibodies as well. This happens mainly in the central part of the peptide in class I complexes [1] , or at the Cterminal of class II complexes [2] . In some cases, the same peptide/MHC complex has been found to interact with both TCR and antibodies [3] . In these study a series of supermolecular complexes have been studied with stateof-art molecular dynamics simulations [4] The dipeptide kyotorphin (Tyr-Arg, Kyo) plays a role in pain modulation in the mammalian central nervous system (CNS), and is one of the most investigated neuropeptides. The Tyr-Arg motif exists widely throughout the brain not only as kyotorphin, but also as the N-terminal part of several endogenous analgesic peptides 1,2 . Also, this peptide is very rapidly degraded by aminopeptidases 3 . One of the successful strategies in the design of neuropeptides with enhanced stability and improved delivery to the CNS is that with the use of non-protein amino acids, like canavanive (Cav), a structural analogue and antimetabolite of arginine (Arg Trichogin GA IV (nOct-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Lol, in which nOct is n-octanoyl and Lol is leucinol) is an antimicrobial lipopeptaibol, a unique group of membraneactive compounds of fungal origin, characterized by a high content of the nonproteinogenic Ca,a-disubstituted glycine Aib (a-aminoisobutyric acid). Owing to the gem-dimethyl substitution on the C a atom, Aib exhibits a strong propensity to induce β-turns and 310/α-helical conformations in peptides. We have previously reported on a fluorescent analog of trichogin GA IV, the primary structure (and acronym) of which are: Fmoc-Aib-Gly-Leu-Aib-Gly-Gly-Leu-TOAC-Gly-Ile-Leu-OMe (F0T8) where Fmoc is fluorenyl-9-methyloxycarbonyl, TOAC is 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid, and OMe is methoxy. The double substitution of an energy donor (Fmoc) at the N-terminus and an acceptor (TOAC) in the trichogin sequence enabled us to make use of time-resolved optical spectroscopies, spanning from the nanosecond to the microsecond time regime, to investigate the conformational propensity and the dynamical features of F0T8. Experimental and computational results indicated that the 3D-structural and dynamical properties of F0T8 are characterized by a transition from an elongated helix to a more compact conformation mimicking a helix-turn-helix motif. To further investigate the role of the flexible Gly5-Gly6 central motif we synthesized a new trichogin analog having the Gly6 residue substituted by Aib: Fmoc-Aib-Gly-Leu-Aib-Gly-Aib-Leu-TOAC-Gly-Ile-Leu-OMe (F0A6T8) Experimental and computational results indicated that also the F0A6T8 peptide populate two conformations, the dynamics of which were studied at different temperatures using time-resolved spectroscopic measurements. This replacement was demonstrated to stiffen the peptide backbone by reducing the flexibility around the crucial -Gly5-Gly6-dipeptide unit. The antigen α4β1, a member of the integrin family, is involved in the migration of lymphocytes through endothelium to the site of inflammation. 1 Thus, α4β1 antagonists may be useful tools for the treatment of various inflammation disorders such as asthma and inflammatory arthritis. In addition, recent studies indicate that α4β1 integrin promotes angiogenesis by allowing the invasion of myeloid cells into tumors, while α4β1 antagonists prevent monocyte-induced angiogenesis, macrophage colonization of tumors and tumor angiogenesis. 2 Aiming to the discovery of novel α4β1 antagonists, a series of new peptide analogues cyclized through cysteine disulphide bonds were synthesized and tested in vivo against angiogenesis in chicken embryo chorioallantoic membrane (CAM model) 3 . SAR results indicated that: YR-c(CDPC)-CONH2 promoted angiogenesis at the higher studied concentration and showed slight inhibition at the lower one, Sal-R-c(CDPC)-OH, Sal=salicylic acid, showed important inhibition of angiogenesis at dose-dependent manner, YR-c(CDPC)-OH and Sal-YR-c(CDPC)-OH both showed no activity on angiogenesis. NMR spectroscopy was applied for the sequential assignment as well as for the elucidation of specific conformational features. Experimental NOE data were further imposed as distance constraints to a thorough conformational search by applying Molecular Dynamics simulations. Energy refined produced conformers were used as template for the generation of the pharmacophore model associated with the antagonistic activity. Such studies are intended to drive a rationalized design and development of this class of inhibitors. Hynes R. O. Cell, 1992, 69, 11-25. Scaffold Discovery by Phylomers: a novel CD40L specific scaffold derived from Glycyl tRNA Synthetase S.R. Stone [1] , K. Hoffmann [1, 2] , N. Milech [1, 2] , P. T. Cunningham [1] , M. Kerfoot [1] , S. Winslow [1] , Y-F, Tan [1] , M. Anastasas [1] , C. Hall [1] , M. Scobie [1] , P.Watt [2] , and R. Hopkins [1, 2] [1] Drug Discovery Technology Unit, Telethon Institute for Child Health Research, 100 Roberts Road, Subicao, 6008, Western Australia [2] Phylogica Pty Ltd, 100 Roberts Road, Subiaco, 6008, Western Australia Biopanning of Phylomer 1 Phage Display libraries against human CD40L yielded a cluster of highly specific overlapping peptide fragments, from three bacterial genomes, corresponding to the highly conserved catalytic domain from the tetrameric Gα2β2 class of Glycyl tRNA synthetases. Structural analysis of the overlapping peptide fragments described a scaffold consisting of a central βsheet, comprising 4 anti-parallel β-strands, flanked by Nand C-terminal α-helices. Further structural analysis revealed that these key structural features, which also encompass the crucial ATP-binding motifs of the catalytic domain, are conformationally conserved across both tetrameric Gα2β2 and dimeric Gα2 Glycyl tRNA synthetases, yet importantly, there is only limited sequence conservation across these classes. Given the identical function of the described domain and it's structural conservation, we postulated that members of the dimeric Gα2 class would display similar CD40L specific binding as the tetrameric Gα2β2 class, despite the sequence dissimilarity. To test this hypothesis, structurally equivalent peptide fragments of representative bacterial, archaeal and eukaryotic genomes comprising the dimeric Gα2 class were tested for CD40L binding in a process we termed ortholog scanning. The results showed that both archaeal (P. horikoshii) and eukaryotic (H. sapiens) structurally equivalent peptides bound to CD40L with reasonable specificity and inhibited the CD40:CD40L interaction with comparable IC50's to the primary Gα2β2 class sequences. Similar results were also observed for the representative bacterial Gα2 class peptides. That the sequentially diverse orthologous peptides display CD40L specific binding has important implications to the affinity enhancement strategies to develop the scaffold as a therapeutic agent, and in improving its "drug-like" properties. We have initiated 1 an investigation related to the effect of radical species upon structures of some peptide segments. In the proposed experimental protocol, aqueous peptide solution was submitted to gamma ray irradiation in controlled 1-15 kGy doses. The generation of peptide analogues, possibly induced by reactive oxygen species were examined by electrospray triplequadrupole tandem mass spectrometry (collision induced dissociation approach) and amino acid analysis of crude and/or purified by-products. Noteworthy, the gamma irradiation process induced, regardless of the peptide sequence, a non-linear and progressive degradation of all peptides assayed. Furthermore, these peptides could be classified in some different classes according to their halflife dose. For instance, the vasoactives angiotensin II (AII), Ang (1-7), bradykinin (BK) and some related peptides were more stable than the melanocyte-stimulating hormone α-MSH, Substance P or the BK 's (305-325) B2 receptor fragment (LVYVIVGKRFRKKSREVYQAI). Usually, the most prominent derivatives generated from this experimental protocol revealed that they are likely induced by oxidation process, yielding a variation of +16 Da in their molecular weight. The main source of peptide modifications seems to lie either on the Phe (hydroxyl group insertion at o-, m-or p-positions of its aromatic side chain) or Met oxydation. In the former case, only Phe 8 and not Phe 5 is oxidized in the BK structure whereas Substance P generates an analogue bearing Metsulfoxide without modifying its Phe 7,8 residues. Thus, collectively, these findings clearly stress the complexity of factors involved in peptide structural modifications induced by gamma ray-type strong electromagnetic irradiation experiment. An additional target of this approach lies indeed, in the production of unusual peptides for further structure-function investigations. University of Bern, Bern, Switzerland Linear peptides are typically poor drug candidates due to their low bioavailability and rapid proteolysis. These limitations can be overcome by rigidifying their structure through head-to-tail or side chain-involving cyclizations. Cyclic constraints may also increase biological activity by stabilizing secondary structures and by reducing the entropic penalty of binding to a protein target. The use of multiple branching amino acids in a peptide sequence, like diamino acids (as used in peptide dendrimers 1 ) or amino diacids, allows to design peptides resembling polycyclic alkanes, a type of topology only rarely found in nature (e.g. amatoxins and lantibiotics). Bicyclic homodetic peptides such as "norbornapeptides" (bicyclo[2.2.1]heptapeptides) were prepared using an orthogonal protection scheme: the first cyclization is performed on resin after selective deprotection of a glutamic acid residue, whereas the second ring closure is achieved by amide bond formation at high dilutions. These peptides are structurally well-defined and cover an almost pristine area of peptide topological space. 2 Their conformational rigidity was investigated by means of 2D-NMR and X-ray crystallography and may offer a platform to design drugs tackling protein-protein interactions. The interaction of peptide ligands with protein receptors face peculiar challenge in recognizing binding surfaces due to availability of a multitude of conformations. Therefore it is essential to constrain the peptide conformations for the recognition of receptors and thus finding the bioactive conformation. The cell surface receptor protein family Integrins recognize "RGD" sequence which is present in different proteins. To determine the bioactive conformation required to bind with receptor αIIbβ3, the peptide sequence "RIPRGDMP" from Kistrin was inserted into CDR 1 loop region of REI protein (REI-RGD34). It helps out in finding the possible bioactive conformation of peptide by restricting the sampling space. The activity of REI-RGD34 was studied and found that as the temperature increased REI-RGD34 showed a higher affinity towards the receptor αIIbβ3. The proposed mechanisms for the increased activity of REI-RGD34 at higher temperature were justified in either of two ways. The modified complex forces the restricted peptide to adopt a bioactive conformation or it unfolds the peptide in a way that opens its binding surface with high affinity for receptor. In this study we model the conformational preferences of "RGD" sequence in octapeptide "RIPRGDMP" at two different temperatures (250 o C and 420 o C) using multiple MD simulations. We found that at higher temperature "RGD" sequence from "RIPRGDMP" adopt turn conformation, while a bend conformation was observed at low temperature. The analysis of various pharmacophoric parameters hint that the turn conformation of "RGD" sequences adopted at higher temperature could be the potential bio-active conformation, and helps out in designing of antagonists for cell surface receptor αIIbβ3. The 14-residue peptaibol antibiotic trichovirin I-4A (TV) of the linear, covalent structure Ac-Aib-Asn-Leu-Aib-Pro-Ala-Val-Aib-Pro-Aib-Leu-Aib-Pro-Leuol (Ac, acetyl; Aib, α-aminoisobutyric acid; Leuol, L-leucinol) has been synthesized1 and very thin (~25 μm) hair-like crystals were obtained from a methanolacetonitrile-water mixture. Diffraction data were collected at 100 K at the Diamond Light Source England, using the microfocus beamline 2 I24 and a X-ray beam focused to a size of 10 μm full-width-half-maximum. Two independent molecules (A) and (B) were located in the crystal's asymmetric unit 3 . Both chains assume 4 complete turns of a curved 310 right-handed helical conformation stabilized by intramolecular hydrogen bonds. Up to now TV represents the longest right-handed 310-helix of a natural peptaibol sequence complementing those of synthetic, protected homooligo-Aib- Insulin is a protein hormone that plays a key role in regulation of blood glucose levels and, thus, has widespread impact on lipid and protein metabolism.Insulin is known to act through binding to the insulin receptor (IR); however, the structure of the insulin-IR complex is not known. The crystal and NMR structures of insulin represent only inactive storage forms. It is widely acknowledged that insulin must undergo structural changes in the C-terminus of the B-chain upon binding to the IR. In addition, the N-terminus of the Bchain may adopt two different conformations in hexamer, known as T-and R-states. The R-state of the N-terminus of the B-chain creates a long B1-B19 central a-helix. The T-state of N-terminus is in an extended conformation. However, the biological relevance of the T/R forms remains elusive 1 . In this study, we have focused on the synthesis of new insulin analogues modified at the Nterminus of the B-chain and subsequently correlated their biological activities with their 3D-structures. The invariant residue GlyB8 seems to be critical for the T/R transition. Glycine can adopt wide range of dihedral angles (φ/ψ) and it occupies significantly diverse dihedral angles in T-and R-states. a-Aminoisobutyric acid (Aib) is an amino acid with a high helical propensity, which often folds into right-or left-handed α-helix. We have introduced Aib at position B3, B5 and B8 with the aim to induce the R-state of the hormone. In contrast, as D-Pro and NMeAla are not able to adopt the φ/ψ angles of the right-handed α-helix we have introduced these amino acids at position B8 to obtain the T-state of insulin. Peptide dendrimers are tree-like molecules formed by alternating functional amino acids with branching diamino acids such as lysine. 1 Unfortunately these molecules have not yielded to structural characterization and little is known about their molecular-level structure. Computational methods seem to be an adequate tool to address these issues.Herein we present a comprehensive structural characterization of peptide dendrimers using molecular simulation methods. 2 Multiple long molecular dynamics (MD) simulations were used to extensively sample the conformational preferences of several third-generation peptide dendrimers, including some known to bind aquacobalamine. We used several conformational analysis procedures (clustering, energy landscapes and multivariate analysis) to analyze conformational changes that can be correlated with particular structural trends.The results point to a high conformational flexibility of these molecules, with no clear "folded state", although two markedly distinct behaviours were identified. Some dendrimers favour mainly loose conformations, while others prefer more compact configurations. Through a series of computational mutations we investigated the influence of the presence and placement of charged residues in dendrimer topology, finding that electrostatic interactions among charged residues are a major determinant in structure acquisition by peptide dendrimers. These conclusions bring new insight into the conformational behaviour of these systems and may provide better routes for their functional design. Acid-mediated prevention of aspartimide formation in solid phase peptide synthesis T. Michels, a R. Dölling, b U. Haberkorn a , W. Mier*, a a Department of Nuclear Medicine, University Hospital Heidelberg, 69120 Heidelberg, Germany; b BIOSYNTAN GmbH, Robert-Rössle-Straße 10, 13125 Berlin, Germany Aspartimide formation is one of the major obstacles that impede the solid phase synthesis of large peptides and proteins. The main reason for aspartimide formation is the piperidine-catalyzed Fmoc cleavage of peptides containing aspartic acid. Several side chain protecting groups have been developed 1 but the complete prevention of aspartimide formation can only be achieved using N-(2hydroxy-4-methoxybenzyl) (Hmb) as backbone protecting group. 2 However, Hmb-protected building blocks are difficult to synthesize and only the dipeptide containing glycine (Fmoc-Asp(tBu)-(Hmb)Gly) is commercially available. Until now, no cost effective strategy to suppress this side reaction has been developed. Formally, aspartimide is the result of an attack of an amidate species at the carbonyl carbon of the OtBu protected side chain carboxylate of aspartic acid, which might be prevented by protonation of the amidates with piperidinium ions. In this work the suppression of aspartimide formation by adding small amounts of organic acids to the deprotection agent piperidine was studied. This procedure was shown to efficiently prevent the formation of aspartimide side products in several peptides, i.e. PreS9-33-y, a 26-mer peptide derived from the HBV surface antigen and a peptide parathyroid hormone (PTH) fragment. The testing of a series of 18 different acids covering a broad range of pKa values showed that this effect is virtually independent of the acid strength. Since aspartic acid is found in most oligopeptides, the authors recommend to generally add 5% (v/v) formic acid to piperidine based Fmoc cleavage mixtures. Decomposition of the resin linkers during TFA cleavage of peptides in Fmoc-strategy leads to alkylation of sensitive amino acids 1 . This side product formation is a crucial drawback, especially during the synthesis of biologically important Cys-containing peptides on Wang support. Through a battery of approaches (1H-NMR, UV and LC/ESI-MS) we detected an unexpected alkylation of the sulfhydryl group of cysteine side-chain residues by the phydroxyl benzyl group from the Wang resin linker. Herein, we present the feasibility for S-alkylation of Cys-containing peptides from Wang linker decomposition. This sidereaction occurs during the final TFA cleavage of the peptide from the solid support, while the position of the cysteine residue within the peptide sequence as well as the resin's substitution influence the extent of Cys-alkylation. The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria Influenza viruses cause epidemics and pandemics all over the world. Therefore, the development of virus resistance to drugs, leads to search for novel derivatives and approaches to chemotherapy for human influenza infection. Antioxidant therapy is known to be one potential approach. The application of combination therapy of antioxidants with antiviral drugs could reduce the complications and lethal effects, caused by an influenza virus 1 . In our study, amino group of neuraminidase inhibitor -oseltamivir, which belong to second generation anti-flu drugs, was covalent conjugated with known antioxidantscysteine, histidine. tryptophan and etc. The study of the role of the modified by antioxidants oseltamivir on proliferation of influenza virus is in progress. Recently we reported a short synthesis of 5 or 6-membered cyclic guanidine via intramolecular reaction of alkyl diamine with N,N'-Cbz-methylisothiourea(1a). Here we report a further application of synthesis two types of cyclo-peptide guanidine-bridged cyclopeptides utilizing this mechanism -N-terminus local cyclo-guanidine peptide and backbone guanidine-bridged marco-cyclic peptide. Three N,N'protected diaminoacids (DAA) including 2,4-diaminobutyric P334. Antifreeze glycoproteins (AFGPs) are found in the deep sea teleost fish in Arctic and Antarctic oceans. These biomolecules are able to inhibit the growth of ice crystals and depress the freezing temperature of the blood serum in fish enough to keep them from freezing in their sub-zero environments while the melting temperature remains unchanged 1 . Despite AFGPs have been consider as a potent cryopreservation, obstacles to develop AFGPs as medicinal and industrial application are mainly due to the lack of access to pure form from natural sources and the problem of understanding how AFGPs inhibit ice crystal growth. As a result, a considerable progress toward the design and synthesis of AFGP analogues has been made several groups 2 . In the course of the studies on the structure-activity relationships of AFGPs, we are interested in peptoids as mimics of α-peptides and synthesized monoglycosylated peptoid analogues by substituting the glyco-thr residue as AFGPs mimics. In this presentation, we will show our studies on how the insertion of peptoid residue into AFGP backbone affects the AFGP activity by measuring both thermal hysteresis (TH) and ice recrystalliztion inhibition (IRI). [1] , both for diagnosis and endoradiotherapy. For this application the peptides can be attached with chelating agents that bind radioactive metals such as 67 Ga, 68 Ga or 111 In for imaging or therapeutic radiometals such as 90 Y and 177 Lu. The chelating agent most frequently applied is the macrocyclic ligand 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetate (DOTA), it is commonly introduced as the tris(tBu ester). The cleavage of the tBu protecting groups on DOTA is known to be sluggish [2] . Several attempts have been made to synthesize DOTA with protecting groups that can be removed under mild conditions. However, these derivatives have not yet found widespread application. Our new approach was to prepare a protecting group for DOTA-based prochelators that is convergently cleaved under the cleavage conditions of the amino acid protecting groups of the peptide. O-phenylisopropyl (OPp) esters are more sensitive towards acid than tBu esters. Deprotection occurs with 2% trifluoroacetic acid in dichloromethane [3] . Therefore, a synthesis of the prochelator DOTA-tris(OPp ester) was developed. The copper-catalyzed azide-alkyne cyclization (CuAAC), the most commonly recognized variant of "click chemistry," has emerged as a powerful technique for ligation, conjugation, and cyclization reactions of peptides. It is known that cyclization can increase the metabolic stability of peptides, as well as enhancing potency or selectivity by stabilizing an active conformation. One application of the CuAAC that has generated interest is the use of this reaction to replace a disulfide bridge with the product triazole, which among other complementary properties may prevent in vivo redox chemistry. In this poster, we synthesize a new analogue of the cyclic cancertargeting peptide CNGRC where we replace the disulfide bond with a triazole linkage using click chemistry and a fully automated, on-resin method using the Single-Shot delivery feature on the Prelude® peptide synthesizer. Unnatural amino acids including D-amino acids are manufactured mainly by the enzymatic process. However, one enzyme can produce only one amino acid due to its high specificity and it takes a long time and a lot of expenses to develop the appropriate enzyme itself. ARCA (Alanine Racemase Chiral Analogue) is an organic catalyst1 which can overcome these drawbacks and can produce almost all kinds of amino acids efficiently. The amine functionality of L-threonine is freely reacted with the aldehyde group of ARCA to form the corresponding imine, which is easily epimerized in the presence of organic base due to the acidity of the alpha proton of imine. The difference in the stability between the imines of the optical epimers rendered them to be shifted to D-allo-threonine derivative dominantly. Once the epimerization reaction reached equilibrium, the reaction mixture was hydrolyzed under acidic condition to give D-allo-threonine and ARCA, which could be recycled repeatedly without significant loss in yield or purity to produce more D-allo-threonine from Lthreonine in excellent yields. Optimization of the reaction conditions with various bases and solvents is discussed and mass production of optically active D-allo-threonine including optical purification is described. The manufacuring process for the preparation of ARCA will be shared as well. Our group is interested in the development of efficient synthetic routes for the preparation of enantiopure atrifluoromethylated amino acids (a-Tfm-AAs) starting from chiral CF3-oxazolidines or imines 1 and their incorporation into a peptide chain. 2 These non-natural amino acids are very attractive compounds for the design of biologically active molecules, particularly peptides, due to the unique physical, chemical and biological properties impart by the CF3 group. 3 As conformationally constrained cyclic amino acids have recently gained considerable interest, we are particularly focused on the preparation of pyrrolidine-type a-Tfm AAs. 1, 4 Incorporation of proline derivatives is known to restrict the amino acyl-proline cis/trans isomerization, to limit the protein folding and consequently to modulate the biological activity of peptides. Based on these observations, Mutter's group introduced pseudoproline building blocks (ψPro) into a peptide sequence as reversible protecting groups for Ser, Thr and Cys. 5 The ψPro residues proved to be versatile tools for overcoming the aggregation caused by hydrophobic interactions encountered during solid-phase peptide synthesis (SPPS). They also turned out to be inducers of βturns containing predominantly cis-amide bond and useful tools in peptide cyclization. Here, we report the results obtained for the preparation of various hydrolytically stable trifluoromethylated pseudoprolines (CF3-ψPro) as well as the methodological studies developed to optimize the synthesis of various C-and N-terminal CF3-ψPro containing dipeptides. Rennes, France Protein strructure and function rely on a still not fully understood interplay of energetic and entropic constraints defined by the permutation of the twenty genetically encoded amino acids. Many attempts have been undertaken to design peptide-peptide interaction pairs and synthetic receptors de novo by using special building blocks. 1 A rational approach starting from hydrazine to create new building blocks based on a tailored metalchelating amino acid analogues was envisaged. To create chemical recognition units, which bind oligohistidine tags with high affinity and stability, several supramolecular entities containing one to three nitrilotriacetic acid analogue (YNTA) moieties were synthesized. These new building blocks additionally contained an amino group or an acido group, which can be flexibly introduced into peptide in N or C-termini or into the peptidic chain by solid phase chemistry in Fmoc/t-Bu strategy. These multivalent chelators were characterized and the corresponding metalchelating peptides could act as metal sensors and synthetic receptors for histidine-tagged proteins. The potential of peptides as drug candidates is often limited by their pharmacokinetic properties. Structural modification of the peptide backbone via N-methylation is a powerful medicinal chemistry tool that confers oral bioavailability to these molecules. N-methylation exerts a strong effect on the backbone conformation and, as a result, many N-methylated peptides show enhanced biological activity and higher receptor selectivity. Another approach to increase the solubility of peptides is by conjugation of PEG to a derivatizable functionality. By combining these two approaches we have developed N-OEGylation. This novel form of peptide modification consists of the attachment of oligoethylene glycol (OEG) chains to the amide bonds. Many bioactive peptides comprise one or more N-Me amino acids which are essential for their activity. Thus, we consider that replacement of a backbone N-Me group by an OEG chain may imply a minimal structural perturbation and may lead to N-OEGylated peptides with preserved biological activity. Furthermore, our strategy is a promising way to improve the bioavailability of cyclic peptides that do not have any site where a PEG could be attached. As a proof of principle, several N-OEG analogs of two bioactive cyclic peptides were synthesized in SPPS. First, we performed a full N-OEG scan of the Sansalvamide A peptide. Next, several analogs of Cilengitide were prepared by replacing the N-Me of valine by OEG chains of different length. Depending on its size, the OEG residue was incorporated by using an N-OEG derivative as building block or, alternatively, using an N-substituted amino acid bearing an attachment site where a PEG was conjugated post-synthetically.The biological activity of all the N-OEGylated peptides was evaluated. Some of the Sansalvamide A peptide analogs exhibited cytotoxicity within the same range as the original peptide, which suggests that backbone amide groups may be useful OEGylation sites in bioactive cyclic peptides. The modification of peptides is an important step in pharmacology to vary the affinity and the stability of peptidic drugs. Whereas a wide range of strategies exists for the functionalization of the N-terminus and the side chains, facile variation of the C-terminus remains an important challenge. We consider peptidyl-phosphoranes as a promising platform to enable orthogonal and mild introduction of a great variety of chemical functionalities at the C-terminus. A convenient method for the synthesis of soluble peptidyl-phosphoranes has been presented by our group recently. 1 In this, 2-bromo-acetyl bromide was coupled to a Wang-resin followed by alkylation of triaryl-or trialkyl phosphine moiety. Deprotonation to the phosphorus ylide and subsequent acylation with an Fmocamino acid created the basis for assembly of the peptide by SPPS. Final acidic cleavage produced a decarboxylated and unprotected, soluble peptidyl-phosphorane. From this point, a variety of orthogonal modification reactions at the peptides C-terminus is possible, e.g. click reaction with azides allow for the incorporation of triazoles as peptide bond mimetics. The Wittig reaction opens up another interesting portal for C-terminal modification, as vinyl ketones are formed by reaction with aldehydes. The described chemistry was applied to modify caspase-3 inhibitors. In order to address the S1 site of caspase-3, the commonly known DEVD inhibitor was varied at the C-terminus by introduction of different residues. The DEVD motif was synthesized as peptidyl-phosphorane and modified in Wittig reactions. The resulting C-terminal vinyl ketones were obtained by the reaction of aliphatic and aromatic aldehydes. A small library was generated, and 12 novel compounds were tested for their potential to inhibit caspase-3. Semmelweis University, Department of Biophysics and Radiation Biology, Budapest, Hungary Considering the impact of UV irradiation on the structure and function of proteins 1 , it is a matter of utmost importance to resolve the conditions of photolysis more deeply. We think that a protein, as a complex unit, gives multiple responses to all impacts therefore the analysis of these responses is a rather complex problem. The main goal of our research is the deeper understanding the tryptophan-mediated photolysis of disulphide bridges in bio-active proteins upon near-UV irradiation using cyclic peptide models, as small protein units, to define the caused functional damage. Cation -pi interaction is increasingly recognised as an important noncovalent binding interaction which plays a role in establishing the final structures of proteins. Within a protein, cation -pi interactions can occur between the cationic side-chains of either Lys or Arg and the aromatic side-chains of Phe, Tyr or Trp 2 . Our earlier results with GLA indicate that new covalent bonds are also formed between Cys and Lys during illumination, which is also a reason why the Lys residue is planned to be included in the sequence of the models. Our aim is to study whether the cation -pi interaction can have an influence on the SS-bridge splitting in small cyclic pentapeptide models. Here we report about the conformational analysis, synthesis and spectroscopic investigation of Lys-and Arg-containing model peptides. The azide functionality is very popular mainly due to azidealkyne click chemistry 1 used in many peptide ligation strategies. Azido-peptides are usually prepared by incorporation of azide containing residues or azide functionalization of aldehyde resins affording C-terminal azido-peptides. 2 Alternatively, the N-terminus can be converted into an azide via a Cu(II)-catalyzed diazotransfer reaction using triflyl azide. 3 Recently, a number of safer, shelf-stable and easily prepared diazotransfer reagents has been developed, of which imidazole-1-sulfonyl azide has been used to introduce azide moieties in proteins under copper-free conditions. Typically, it has not been reported to be used on the solid phase. 4 We provide a very easy, fast and efficient method for conversion of amines into azides on a solid phase support which in our opinion has major benefits over earlier reported methods making using of less stable reagents that require a metal ion catalyst. We demonstrate how the diazotransfer reaction can be performed on a solid phase support using the imidazole-1sulfonyl azide reagent without the need for a metal ion catalyst. Using a model peptide we studied the effect of stoichiometry, added base and solvent. In addition we examined the effect of the nature of the N-terminal residue on the efficiency the diazotransfer reaction. Finally, we found that the optimal conditions to perform the reaction also depend on the nature of the solid phase support the reaction is performed on. The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark Site-selective strategies for post-translational modification of peptides and proteins are essential tools for many areas of research in the life sciences, yet remain a chemical challenge due to the multiplicity of functional groups present. There are powerful chemoselective reactions, however, they aim at introducing only one functionality at each reaction site. Here we present a one-pot, threecomponent dual-functionalization of peptides or proteins based on a 1,3-dipolar cycloaddition between a functionalized malemide, an N-hydroxylamine and a peptide or protein with an N-terminal serine residue at the N-terminus, which is selectively oxidized to a 2-oxoaldehyde. Most common moieties for labeling, e.g. fluorophors and PEG-chains, are commercially available as maleimides. Nitrones were easily obtained by condensation of peptide-aldehydes and primary Nhydroxylamines under aqueous conditions. The chemoselective 1,3-dipolar cycloaddition reaction between the peptide-nitrone, and a functionalized maleimide proceeded in aqueous solution at room temperature or with gentle heating, which provided the stable isoxazolidine product. We envision that this 'one site -two functions' method can be used widely to introduce two separate moieties. The method was used to introduce two separate ligands in a range of other peptides. For example, new multimodal molecular imaging techniques depend on facile chemical methods for site-selective dual-functionalization. We used our new methodology to synthesize a cyclic RGD-peptide for combined PET and optical molecular imaging. Finally, the small protein IPB3 was successfully N-terminally modified, including with a PEG-chain, using this new, general method. Multiple Sclerosis (MS) is the most known chronic, inflammatory, demyelinating disease of the central nervous system (CNS), characterized by a progressive neurodegeneration, caused by an autoimmune response to self-antigens in genetically susceptible individuals. It is nowadays known that post-translational modifications may affect the immunogenicity of self-protein antigens, triggering an autoimmune response and creating neoantigens; in particular aberrant glycosylations affect various parts of the immune response and have profound effects on immune tolerance. In previous studies we demonstrated the value of the glycopeptide CSF114(Glc) which, by virtue of the particular type I' β-turn structure, optimally exposes the minimal epitope Asn(Glc) to autoantibody recognizing in ELISA in multiple sclerosis patients' sera 1 . ELISA assays allowed to conclude that the ability in detecting autoantibodies in multiple sclerosis sera was stricktly linked to saccharidic moieties and to conformation around minimal epitope of the antigenic glycopeptide. Herein, taking advantage of such considerations, we focused our attention on the synthesis of a little library of lysine branched multiple antigen peptides (MAPs), containing the minimal epitope Asn(Glc), in an attempt to increase the antigenicity of linear peptide sequences 2 . With this aim, we performed the SPPS of glucosylated MAPs via the building block approach, studying the role of different long spacers on the dendrimeric core, and the role of different peptide sequences around the sugar moiety, in order to optimize the synthetic process and to evaluate the influence on the affinity and specificity in SP-ELISA. Environmentally induced co-or post-translational modifications of autoantigens are hypothesized to break immune tolerance leading to self reactivity in PBC. 1 It has been previously reported that the use of synthetic post-translationally modified peptides, introducing Fmoc-L-Lys(Nε-(±)-α-Lipoic acid)-OH, as peptidomimetics of natural neoantigens allowed to detect autoantibodies in the sera of patients affected by PBC, and they might be useful diagnostic tools that can be used in earlier stage patients and possibly to monitor disease activity. 2 Only the R-(+)-enantiomer of α-lipoic acid exists in nature and is an essential cofactor of four mitochondrial enzyme complexes. 3 But it remains unclear if the tridimensional structure of the lipoic acid is of any importance in the interactions antibody-peptide during the indirect ELISA tests. Therefore, it is necessary to synthesize each peptide separately with one absolute configuration of the lipoic acid. Herein, we describe the synthesis of the two diastereoisomers Fmoc-L-Lys(Nε -(R)-α -Lipoic acid)-OH and Fmoc-L-Lys(Nε -(S)-α-Lipoic acid)-OH that have to be used in Fmoc/tBu SPPS as building blocks for the synthesis of post-translationally modified peptides. 2 Recently it has been reported the introduction of a new generation of CD diagnostics based on a unique antigen approach, consisting on human tTG cross-linked with gliadin peptides coated on the ELISA plates 3 . On the basis of experimental data obtained by mass spectrometry and indicating which are the fragments of these two proteins that are supposed to be involved in the antibody recognition, we were able to select the most representative tTG and gliadin fragments 4, 5 to design and synthesize by Fmoc-SPPS nine cross-linked eoepitopes. Aim of our study was the characterization of autoantigenic epitopes by testing, in celiac patients' sera, the reactivity of these nine synthetic peptides. These neoepitopes were tested in ELISA to evaluate the IgA and IgG response against tTG-gliadin adducts in celiac patients' sera in order to develop a new ELISA test based on peptides as an even more powerful diagnostic tool in terms of specificity and sensitivity. 1 More than 80 analogs have already been described, wherein the hydroxy acid and the amino acid constituents were replaced by D-amino acids and/or N-methyl amino acids with preserved or altered side chains. For certain types of cancer cells, several of these analogs were found to be more active than the natural product itself. 2 However, it does appear that many of these compounds have limited solubility in water. 2b Here we report the synthesis of 5 novel analogs of the Sansalvamide A peptide bearing an N-oligoethyleneglycyl (OEG) chain attached to the different backbone positions. Attachment of this chain is aimed to enhance the hydrophilicity of the original peptide. Our synthetic strategy to modify the backbone with the N-OEG group relies on the use of N-OEG amino acids, which were synthesized in solution and then used as building blocks in SPPS. As expected, couplings to the N-OEG residues were found to require special conditions. Methods for the coupling to Nmethyl amino acids were applied and this enabled to obtain the different linear pentapeptides, which were cyclized in solution.

Both the synthetic strategies of these demanding peptides as well as the preliminar evaluation of their biological activity will be deeply discussed. Glycoconjugates such as glycoproteins and glycolipids have important roles in cell functions, for example, intercellular recognition, cell proliferation control, and information transmission. In order to study the structurefunction relationship, synthesis of these glycoconjugates is essential. Glycoproteins and glycopeptides are classified into two categories: N-and O-glycosylated derivatives. The N-acetyl-α-D-galactopyranosylated Ser or Thr derivatives [Ser/Thr(α-D-GalNAc)] are important intermediates for O-glycopeptide synthesis. However, the synthesis of Ser/Thr(α-D-GalNAc) derivatives by chemical glycosylation is difficult because of the decreased nucleophilicity of hydroxy function in the glycosyl acceptor due to an unfavorable hydrogen-bonding pattern between the OH and α-NH groups 1 . Several approaches to overcome this problem have been reported 1, 2 . In addition, the O-glycosidic bond is cleaved easily in acidic conditions. In this study, we assumed that the formation of a cyclic structure containing an α-NH group would increase the reactivity of OH function. Thus, we focused on the N, N'isopropylidene derivatives of Ser/Thr containing dipeptides 3 . We found the reaction of mannopyranosyl trichloroacetimidate and the N, N'-isopropylidene dipeptide in the presence of TMSOTf in dichloromethane produced the desired glycosylated dipeptide in good yield. However the selective intermolecular disulfide bond formation is a very difficult and complicated synthetic problem. In this work we report on synthetic approaches for the formation of conjugates with intermolecular thioether or disulfide bonds. For the disulfide bond formation, we use two activation approaches: i) activation of the four Cys residues of the carrier testing two activating reagents, in both solid and liquid phase respectively and ii) activation of the Cys containing bioactive molecule. As bioactive molecule we selected the R 997 PPLEED 1003 sequence derived from the intracellular part of the αIIbplatelet integrin receptor. This region is critically involved in platelet aggregation and is a target of intervention for developing antithrombotic agents 2 . The Ac-[Lys-Aib-Cys(CH2CO-αIIb997-1003)]4-NH2 and Ac-[Lys-Aib-Cys(Cys-αIIb997-1003)]4-NH2 conjugates were synthesized and examined for their ability to inhibit platelet aggregation. The biological assays indicated that the synthesized conjugates penetrate the platelet membrane and inhibit human platelet aggregation, in contrast to the corresponding free peptide analogues. The molecules were reported to exhibit broad-spectrum cytotoxicity against the tumor cell lines. Although these peptides contained the novel β-methoxytyrosine, Lipton et al. reported the synthesis and cytotoxicity of desmethoxycallipeltin B, in which substitution of D-tyrosine for β-methoxytyrosine did not substantially affect the cytotoxicity of callipeltin B 3, 4 . However, a structure-activity relationship study of the molecules has not been shown to date in detail. In the course of our recent research regarding the synthetic study of cyclic depsipeptides, we conducted studies on the synthesis of callipeltins supposed to be efficient structures for CCR5 inhibitors as anti-HIV drugs or anti-cancer agents. In the present study, we report the synthesis of cyclic depsipeptides of callipeltin B analogues consisting of L-, D-amino acids and/or N-methyl amino acids, for a structure-activity relationship study of linear-and cyclic depsi-peptides against HeLa cells5. In the assay of synthetic peptides, all of the synthetic callipeltin B analogues exhibited no cytotoxicity. We supposed that dimethylpyroglutamic acid of callipeltin B was essential structure to show the cytotoxicity against HeLa cells. Monash University, Melbourne, Australia Protein-protein interactions represent a significant portion (15-40%) of all interactions within the cell; 1 as such these interactions are ideal targets for drug discovery. While difficult to target using small molecules, these interactions can be disrupted using a small section of the protein's binding partner. These short peptides must retain the defined secondary structure associated with the protein binding interface in order to inhibit their protein targets. As the secondary structure adopted by the parent protein is not always exhibited by its derived peptides, constraints are introduced as necessary to help define the structure of the peptide. Inducing secondary structure reduces the energy required for organisation, decreasing the energy of binding and has the potential to increase stability with respect to degradation by proteases. 2 Solid phase peptide synthesis was used to make several small peptides corresponding to the structured sections of the binding partners for three protein-protein interactions. These peptides were designed to target heart disease, prostate cancer, and liver cancer respectively. Secondary structure was introduced using lactam bridge constraints. For the αhelical peptides, a side chain constraint approach was used to nucleate helix formation. As hydrogen bonding between the C=O of the i th residue and the NH of the (i+4) th residue stabilises native α-helices, constraints were introduced linking the side chains such that the residues were held in close proximity. For β-pleated peptides, an antiparallel β-sheet arrangement was achieved by introducing turn regions into the peptide in such a way that the β-strands were aligned. Constraints were again introduced using lactam bridges between Lys and Arg or Glu side chains. These peptides were characterised by NMR and CD spectroscopy to verify the correct secondary structure had been induced. Göttingen, Germany Different properties can be combined in a single molecule by using a scaffold arranging functional groups in a predefined topology. The TASP (template-assembled synthetic proteins) concept describes templates to reinforce and direct the folding of designed molecules into a predetermined topology. [1] Due to their resistance to proteolytic degradation and their rigid basic structure, cyclic β -tripeptides are suitable carrier molecules for bioactive compounds; they are further known to form tubelike structures by stacking of the peptide rings leading to higher organization of functionalized peptides. [2] With this scaffold different inhibitory systems were synthesized that feature cell penetrating and fluorescent properties. The signal transducer and activator of transcription 3 (STAT3) protein, which has been described as an oncogenic protein, was selected as the first target. [3] A peptide sequence which targets the SH2 domain of STAT3 was used in two different approaches. It was either directly attached to the cyclic β-tripeptide via a Huisgen [2+3]cycloaddition or the peptide was incorporated into the inhibitor loop of the cystine knot microprotein oMCoTI-II, which was also attached to the cyclic-β -tripeptide. [4] Further, sodium channels are addressed usingconotoxines. First, an alkyne functionalized conotoxin SIIIA was synthesized applying different folding methods. The alkyne linker will be used to attach a fluorophore or to functionalize a cyclic-β-tripeptide. Using single molecule imaging the spatial distribution, local concentration and organization of the ion channels in neurons will be imaged. Further, the cyclic-β -tripeptide templating effect will be used functionalizing with μ-conotoxines. Those proteins are folding helper proteins. Together with chaperones, they form receptor complexes. They catalyze the isomerization of prolyl bonds in various folding states of target proteins. Indeed, their role has been implicated in refolding of denatured proteins, de novo protein synthesis and the biologically active conformation of proteins 1 . Among them, the FKBP subclass comprises the small PPI calstabin 1 and 2. It is of interest to try to understand the way those proteins act, in order to help the overexpression of various types of membrane proteins, aiming at the renaturation, purification and crystallization attempts of receptors. We chose to work on calstabin 2 because this short (107 aa) protein has been described as a sub-family comprising 4 isoforms (from ~3 0 to ~1 00 aminoacids), some of them not being fully described to date. The relative shortness of those proteins together with the fact that the two higher molecular weight ones are catalytically active as prolyl isomerases, facilitate the characterization of the synthetic proteins.In order to obtain the full length calstabin 2, a native chemical ligation (NCL) approach was chosen 2 . An optimized stepwise elongation allowed the obtention of the C-terminal segment up to the Cys 22. Moreover, several methods were compared for the synthesis of peptide 1-21 opportunely functionalized at its C-terminus for the NCL.The ligation at Thr site between peptide 1-21 featuring a bis(2-sulfanylethyl)amino 3 The chemical diagnostics of paintings is a relevant topic in the field of chemical sciences applied to the conservation and safeguard of cultural heritage. Chromatography is a highly sensitive and suitable technique for accurate methods of analysis of the limited amount of sample material typically available from works of art. Paint media deriving from proteins traditionally include egg, milk, animal and fish collagen glue. Egg yolk (egg tempera), egg albumin (glair) and casein (a blend of related phosphoproteins commonly found in milk) are traditionally used as pigments binders. We propose the UPLC-based amino acid analysis as diagnostics technique on non pre-treated or submitted to extraction processes model samples, showing that good results can be achieved with very scarce sample manipulation and great advantage. We applied the amino acids analysis carried out by the AccQ•Tag™ Ultra Performance Liquid Chromatography to the standard and model samples. In particular, after protein hydrolysis (24h, 114 °C, 6M HCl) of the samples, the amino acid derivatization by 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate allowed a reproducible amino acids analysis characteristic of the protein type. The results obtained confirmed the reliability of the data achieved and demonstrated that the AccQ•Tag™ Ultra UPLC method could be a powerful technique to be applied to the relevant field of protein binders diagnostics for paintings conservation. Moreover a Multivariate Analysis that offers a wide variety of tools and methods mainly concerned with mathematical models for the representation of multidimensional data has been proposed and the high model efficiency has been established for sample containing mixture of proteins. Reactions performed on solid supports, such as resin, are commonly monitored by HPLC-UV after cleaving the products from the support. However, UV-absorption coefficients may differ between compounds, and therefore the relation of the area percentage values of the peaks may not directly reflect the molar concentrations of the corresponding compounds. It is for this reason that, for example, in solid-phase peptide synthesis it is difficult to calculate the yield of the coupling of a Fmoc-amino acid or the removal of the Fmoc-group because of its high absorbance. Recently, we reported the identification of minimal phosphopeptides that specifically interact with the PBD of human Plk1, but not those of the closely related Plk2 and Plk3 1 . Comparative analyses of the crystal structures of the Plk1 PBD in complex with the minimal phosphopeptides revealed that the C-terminal SpT dipeptide functions as a high-affinity to the interaction. In an attempt to obtain the adequate cellular permeability and stability in vivo, we have accomplished the peptide-peptoid hybrid or peptomers cyclization using various methods like formation of amide, thioether and triazole and screened the Plk1 inhibition activity on the first cyclic peptomers liibrary using PBD-binding assay. Based on our first screening results, we also carried out the detailed investigation to further increase the activity and also to understand the significance of peptoid mimics as Plk1 inhibitors. The mode of interaction between the cyclic peptomers and PBD might provide a template for designing therapeutic agents that target Plk1.

A synthetic 83 amino acid long peptide corresponding to the minimal metacaspase catalytic domain induces cell death in Leishmania major C. Servis, H. Zalila*, I. Gonzalez, L. Lozano, N. Fasel Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Despite a lot of controversy during the last decade, there is increasing experimental evidence that cell death (CD) is genetically programmed in lower eukaryotes.In the CD proteolytic cascade of plants and protozoa, caspases are likely replaced by metacaspases that are cysteine peptidases recognizing arginines or lysines in P1position. Metacaspases have been found to control cell death in plants. The human protozoan parasite Leishmania major expresses a single metacaspase (LmjMCA) harboring a central domain with the catalytic dyad histidine and cysteine as found in caspases. Metacaspase could therefore be one of the executioners of the death pathway in Leishmania.In this work we showed that, in stress conditions, LmjMCA precursor forms were extensively processed into soluble forms containing the catalytic domain and this domain was sufficient to enhance sensitivity of parasites to hydrogen peroxide by impairing the mitochondrion function. We tested different lengths of the LmjMCA catalytic domain and found that the overexpression of the polypeptide corresponding to amino acids 136-218 was sufficient to sensitize L. major mitochondria to oxidative stress.We synthetized an 83aa long peptide corresponding to the minimal metacaspase catalytic domain (aa136-218) and showed that it has specific metacaspase activity in vitro.We are currently investigating its activity on possible target proteins, which have been identified in a yeast two-hybrid screen. Identifying proteins involved in the metacaspase signaling pathway will shed light on the understanding of CD in Leishmania and open new perspectives in drug target investigation to fight leishmaniasis and other major infectious diseases.

S. Alasibi, G. Ashkenasy Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel Various factors can affect the conformations and folding states of protein molecules and as a consequence their activity. These factors include amino acid mutations, interactions with other macromolecules, binding to regulatory molecules, and also external changes such as pH jump or shining light. In order to control the folding states and to modulate the functions of peptides and proteins by light, photocleavable groups are usually incorporated into specific residues to mask critical interactions. For example, introducing caging groups into coiled-coil proteins recognition interface affects complex formation and template-assisted ligation reactions, in which the coiled-coils serve as templates to catalyze the condensation reactions between two short peptide fragments 1 . Our research group has been studying peptides replication networks, which were made of coiledcoil peptides and analyzed the response of such networks to light as external trigger 1 . It was shown that even replicating networks made up of a small number of molecules can possess complex behavior, considering the wealth of catalytic pathways and transformations. Hence, Boolean logic operations can provide valuable means to analyze and interpret their behavior 2 . Herein, we describe the use of chemical inputs and UV light to manipulate peptides folding and functionality within new synthetic networks. These networks perform complex behavior and, as a result, selective product formation is used to implement Boolean operations that have not been achieved before. Institute of Bioorganic Chemistry of RAS, Moscow, Russia Earlier, we have shown that N-acylated amino acid nitriles and amides react with ethylene derivatives forming the 3amino-and 3-hydroxypyridines and pyrroles [1] . A possible reaction mechanism is the geterodienic condensation of 5aminooxazole derivatives to dienophiles. The higher yields were observed when used the dicarboxylic acids as dienophiles and 5-amino or 5alkoxyoxazole as geterodienes. While the same reactions with the fullerene derivatives, as dienophiles, gave low yields. The nitrile groups of specified pyridines possess ability to react with amino groups of peptides and proteins even at room temperature. In view of high activity of nitrile groups such pyridines can form tetrapyridotetrazoporphyrins and self-condensation products giving appropriate dendrimers (possible due to mobile hydrogen in the 6th position). High molecular weight dendrimers were identified by massspectrometry, gel electrophoresis and dynamic light-scattering. Catalytic oxidizing properties of tetrazaporphyrin derivatives and phtalocyanin were used in synthesis of cyclic peptides and for the S-S bonds formation. The transformation of peptides into heterocycles via an intramolecular reaction of nitrile groups was used to determine the sequence of some peptides, which favored the resistance of transformed compounds to hydrolysis and to the electron impact at mass spectrometry. Diazotization of peptides and their derivatives facilitates identification of amino acid sequence by mass spectrometry due to the peculiarities of their fragmentation. In addition, an amino acid analysis of the diazotized peptide makes it easy to determine the N-terminal amino acid. We present here a multi-disciplinary approach combining X-ray crystallography, computational analyses, and immunological tests to identify epitopes of the oligopeptide-binding protein A (OppA Bp) from the Gramnegative pathogen Burkholderia pseudomallei, the etiological agent of melioidosis. 3 Computational analysis on OppA crystal structure was used to design potential consensus epitopes, that once synthesized as free peptides (COMP 1-3) were found to be immunoreactive against sera from melioidosis patients. Notably, one of the predicted peptides allowed to distinguish between seropositive, seronegative and recovered groups, underlining its potential for diagnostic purposes. Parallel experimental epitope mapping, based on proteolysis and mass spectrometry, allowed us to identify linear peptide epitopes (EXP 4-6) localized in similar protein regions as COMP1-3. Moreover, the match between theoretical and experimental mapping of epitopes was improved by expanding our computational approach, i.e. including an energy based decomposition procedure to divide OppA Bp into separate fragments. Overall, our results illustrate the successful development of a novel integrated structurebased approach for the discovery, design and preparation of epitopes. Nonetheless, given antigen crystal structures, our method is expected to be broadly applicable in the design and generation of new epitope candidates, as being confirmed by on going experiments on different antigens. The application of peptide thioacids as reactive intermediates and building blocks has received considerable attention recently. The chemical ligation reaction between thioacids and azides has been reported for the synthesis of small to larger peptides as well as for the modification of proteins. 1 Fmoc based methods for the preparation of peptide thioacids have to our knowledge not been extensively researched and a facile approach to their synthesis is desirable.We have recently shown that t-butyl thioesters are robuster than previously reported, and can be used for the Fmoc based solid-phase preparation of peptide thioesters being also easily cleavable with thiolates. 2 Peptides attached via a 4-mercapto 4-methylpentanol (MMP) resin can be cleaved using 3-mercaptopropionitrile to obtain protected thioacid peptides with a ß-elminable cyanoethyl group.The thioacid peptides could then be obtained in situ after treatment with DBU (6.5 % in DMF) and further reacted with sulfonyl azides in the presence of 2,6-lutidine in a one pot reaction. By treating the cyanoethyl peptide thioesters with (NH4)2S in a sodium phosphate buffer (pH = 9), various model penta-peptide thioacids could be obtained cleanly at room temperature in up to 75 % overall yield based on initial coupling. These peptides were then further ligated with electron deficient sulfonyl azide functionalized peptides.Larger peptide thioacids could also be obtained using this protocol. A 16mer derivative of penetratin-1, a cell-penetrating peptide from the third helix of the homeodomain of the antennapedia protein, was prepared as a peptide thioacid in a 51 % yield (based on coupling of the first amino acid). In this report, a sensitive, selective and rapid UPLC-MS method was developed for the determination of the [Lys-Gly]5-MOG35-55 peptide in order to control the conjugation of mannan with the [Lys-Gly]5-MOG35-55 peptide. The separation was performed on an Acquity UPLC system with a BEH C18 column packed with 1.7 μm particles. The total run time was 3 min. Calibration curve based on peak area ratio was linear at the concentration range of 20 -90 μg/ml, with a detection limit of 5 μg/ml. The method showed satisfactory reproducibility and confirmed the entire conjugation between oxidized mannan and peptide sequence. The development of simple, low-cost and fast methods for protein purification is of increasing importance both for academic and industrial applications. A very promising approach is inverse transition cycling (ITC) that exploits the temperature dependent aggregation properties of ELPs. 1 Elastin-like polypeptides (ELP) are artificial polypeptides composed of pentameric repeats (Val-Pro-Gly-Xaa-Gly) derived from mammalian elastin. 2 ELPs are characterized by a specific transition temperature (T t ) that depends on the amino acid composition of the pentarepeat; they are water-soluble below and aggregate reversibly above this narrow temperature range (T t ). 3 These properties are transferred to target proteins by N-or Cterminal fusion with ELPs. 4 During ITC these fusion proteins precipitate, while other components remain in solution. Repeated cycles of heating and cooling allow simple recovery of the target protein.We synthesized various ELPs consisting of 5 to 10 pentameric repeats and including different guest residues. The transition temperature of all synthetic ELPs was determined using photometric assays and measuring turbidity. 3 In order to test if ELP properties can be efficiently transferred, we fused ELP to a small recombinant protein (Ras-binding domain, RBD) by expressed protein ligation. 5 This approach will allow the incorporation of ELPs with unnatural amino acids and other chemical modifications into target proteins. Currently we are focusing on biotechnologically relevant enzymes that constitute a major cost factor in industrial processes. The authors thank Süd-Chemie/Clariant for their financial support. Department of Pharmacy, University of Patras, Rio, Greece Peptides penetrating the cell membrane, known as Cell Penetrating Peptides (CPPs), as well as their mimics, used as delivery agents to cells have been reported 1,2 . CPPs can be natural sequences or artificial constructs designed to capture the features of natural formations. CPPs are particularly important in the delivery of peptides, proteins, nucleic acids, small molecule drugs or imaging agents. Incorporation of a heterocyclic motif into a peptide or peptide-like backbone introduces conformational constraints and/or latent reactivity related to the heterocycle's structural profile. Heterocycle-based CPP mimics are, thus, promising candidates for therapeutics 3 Protected synthetic non-ionic peptides, which are for example synthetic intermediates for the production of API's, are often very hydrophobic and not soluble in most common solvents. They are thus difficult to purify by preparative RP-HPLC, classically used for industrial production. It is then challenging to develop alternative purification chromatographic processes using suitable solvents and providing good yields, high purity and sufficient productivity. The technique of support free liquid-liquid chromatography 1 , including both its hydrostatic (Centrifugal Partition Chromatography or CPC) and its hydrodynamic (Counter-Current Chromatography or CCC) declensions, are mainly involved in phytochemical studies 2 but has also been applied to peptide purification 3 . The previously developed biphasic solvent systems are not adapted to the purification of highly hydrophobic protected peptides. To overcome this problem, two new scales of biphasic solvents systems and a ternary biphasic solvent system were developed to overcome solubility problems often encountered with those peptides. The new systems composed of heptane/THF/CH3N/DMSO/water, heptane/Me-THF/NMP/water, and CMPE/DMF/water were efficiently used for the CPC purification of a 39mer protected exenatide and a 8mer protected peptide intermediate of bivalirudin synthesis. The developed scales show a wide range of polarity and should be useful for general use in CPC for the separation of hydrophobic synthetic free or protected peptides. The progressive aggregation of β-amyloid peptide (β-AP) into insoluble amyloid fibrils ultimately leading to formation of toxic amyloid plaques is widely considered to be the central pathogenic cause of Alzheimer's disease. In the last decade accumulating evidence suggests that soluble oligomeric non-fibrillar forms of β-AP are neurotoxic as well. Consequently, inhibiting the aggregation of β-AP is one of the therapeutic strategies against Alzheimer's disease and a number of small molecules have been identified as inhibitors of β-AP aggregation and neurotoxicity. Among these, curcumin, the phenolic yellow pigment and active ingredient of the turmeric herb, is receiving special attention because of its rich pharmacology that includes in vitro and in vivo inhibitory action against Alzheimer's disease insults. In the current work the interaction of β-AP(1-40) with curcumin is investigated with fluorescence, CD, and NMR spectroscopies in water and water-methanol mixtures and at various β-AP(1-40):curcumin ratios. In NMR studies in 100% methanol curcumin behaves like a macromolecular species with a change in the sign of its nOe signal providing direct indication of its association with β-AP(1-40). In 50% methanol the presence of β-AP(1-40) results in great broadening of the 1 H peaks of curcumin, indicative of a complete change in its solution state. Additionally, the fluorescence of curcumin in 50% methanol shows a blue shift with enhanced intensity, observations consistent with a hydrophobic modification of curcumin environment upon interaction with β-AP. Finally, in water the induced circular dichroism spectrum of curcumin in the near UV region provides clear evidence for the loss of symmetry of curcumin molecule due to changes in its microenvironment generated by interaction with β-AP(1-40). Our experimental findings support the direct interaction of β-AP(1-40) with curcumin and establish its importance as a potential aggregation inhibitor of β-AP. [1] . Based on its sequence, we synthesized H-Tyr-D-Trp-NH-1-Ada (1-Adamantane) (YO-14) and H-Tyr-D-Trp-NH-2-Ada (2-Adamantane) (YO-13) and reported they had potent antiproliferative activity on cancer cells (A-430 and SW480), which were comparable to TT-232 and cycloheximide. A structure-activity relationship analysis revealed that lipophylicity of YO-14 and -13 could be responsible for their antiproliferative activity. Now, we described the substitution of Tyr of YO-14 and -13 by Tyr(Bzl), Phe, 1-Nal(1-Naphthylalanine), 2-Nal (2-Naphthyalanine) and the anticancer and DNA polymerase inhibitory activities in order to explore the effect of hydrophobic substituent. Among the compounds, YO-113 and -114 had the highest lipophilicity judging from their retention time and lipophilicity index (YO-113: 45.5 min, 59.9; YO-114: 44.6 min, 55.9). YO-113 and -114 exhibited strong DNA polymerase inhibitory activity as well as antifroliferative activity on HCT116 cells at 100 M. These activities were greater than those of YO-14 and -13. Antiproliferative activity of the compounds containing 1-Ada such as YO-14, -89, -109, -111 and -113, was comparable to that of the compounds containing 2-Ada such as YO-13, -90, -110, -112 and -114. These findings suggest that the lipophilicity well correlates with DNA polymerase inhibitory activity and antiproliferative activity on HCT116 cells. Further structureactivity relationship study is progressing in our group. Multiple Sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) 1,2 . Our aim was to immunologically control the attack of the myelin sheath in MS patients without the total suppression of the immune system. Anthraquinones (Mitoxantrone, Ametantrone) are widely used in cancer therapy as immunosuppressants.Mitoxantrone is also used to treat several forms of advancing MS, including secondary progressive MS, progressive relapsing MS, and advanced relapsingremitting MS 3 . More specifically, Mitoxantrone is an inhibitor of the type II topoisomerase, which disrupts DNA synthesis and DNA repair in both healthy cells and cancer cells. Herein, we report the synthesis of an anthraquinone type compound conjugated to the immunodominant 35-55 myelin oligodendrocyte glycoprotein (MOG35-55) for the selective immunosuppression of the encephalitogenic T cells in MS patients. The anthraquinone was synthesized by a Friedel-Crafts acylation of hydroquinone from phthalic anhydride, followed by reduction of the resulted quinizarine to its leuco form, addition of the appropriate diamine and air oxidation 4 . The synthesized molecules were purified using liquid chromatography, and they were identified by mass spectrometry and 1 H-NMR. The synthesis of the MOG35-55 was performed under microwave irradiation 4 and its conjugation with the anthraquinone was performed in solution. The final analogue was purified by RP-HPLC and identified by ESI-MS. Benzopyrans, diketopiperazines and 1,5benzodiazepin-2,4-diones are well-known and widely investigated scaffolds, e.g. the latter showing anxiolytic and antiarrhythmic effects. Now, we propose a new potential "privileged structure" containing a triazole moiety mimicking the cis-amide bond within the 1,5-benzodiazepin-2,4-dione motif 2 .Molecules based on this [1,2,3]-triazolo [1,5-d] benzo-1,4diazepin-2-one scaffold are synthesized and decorated via a modular approach on Wang resin using α-amino acids, 2-ethynylaniline building blocks and N-alkylating agents resulting in five points of diversity. The methodology involves the attachment of α-amino acids onto a solid support, subsequent removal of the Fmoc group followed by an optimized diazotransfer reaction of the resulting amine yielding a resin-bound azide. Conversion of the latter into a 1,5-disubstituted 1,2,3-triazole moiety is achieved quantitatively by addition of a range of 2-ethynylaniline building blocks using a Ru(II)-catalyst. The desired scaffold can be obtained in high crude purities (>95%) in solution via an acid catalyzed one-step cyclisation-release strategy. Solution-phase N-alkylation finally affords the fully diversified scaffold. Interestingly, N-alkylation induces atropisomeric effects which can be studied via 1 H NMR spectroscopy.Taking into account future screening results of the synthesized libraries, a well-thought decoration of this scaffold leading to discovery of new lead molecules is within reach. Peptide Symposium in the wonderful small seaside town of Porto Carras. Maurice Manning and Lajos Balaśpiri were nominated as captains of the two teams, the Rest of the World and Europe. Similar matches were organized at subsequent European Peptide Symposia. Now, in Greece, the two captains would like to hand over their roles to younger scientists [Professors Gabor Mezö(Hungary) and LaśzlóÖtvös (USA)] to continue this tradition at the coming European and possibly American Peptide Symposia. It seems best to play in the free time (in the evenings after the excursions). Necessary conditions: good weather; a nice large soccer field; a soccer ball, preferably new; and jerseys and shorts (different colours), organized as always by the Organizer Commettee. The captain of the winning team will receive a trophy at the end of the 32 nd European Peptide Symposium. The teams will remember two earlier excellent referees: Professor Lajos Kisfaludy in Porto Carras [1986] and the Soccer Professor + Ferenc Puskaś (Hungary) in Budapest (1998). In the Poster Session, the results from the past 25 years will be presented in about 15-20 pictures. These pictures may possibly be bought free, 0% can be saved at the Poster Session. All Participants are welcome at the new party in Athens. Conclusion will be presented by the players and fans in Athens. The human Lactoferrin-derived peptide, hLF1-11, was proven to be highly active against antibiotic-resistant bacteria 1 . However, the clinical use of this antimicrobial peptide (AMPs) is hampered by the peptide low stability due to fast degradation or to peptide aggregation, as the use of higher peptide concentrations results on higher toxicity levels. AMP immobilization onto a biomaterial surface could be the pathway to overcome these difficulties 2 . The aim of this work is the development of an antimicrobial surface by covalent immobilization of hLF1-11 onto the surface of chitosan thin films. Chitosan ultrathin films were prepared through the spincoating of a 0.4% chitosan solution in gold substrates. hLF1-11 immobilization was performed through an SS bound between hLF1-11 terminal cysteine and an N-acetyl cysteine previously coupled at chitosan films. Surfaces were characterized using ellipsometry (thickness), Infrared reflection absorption spectroscopy (IRRAS) and X-ray photoelectron spectroscopy (XPS). Bacterial adhesion studies were performed using methicillin-resistant S. aureus (ATCC33591). Chitosan films were incubated with this bacterial suspension at 37ºC for 6h and 24h. The viability of the attached bacteria was evaluated using LIVE/DEAD® Bacterial Viability Kit (Baclight TM ) and Fluorescence Microscopy. hLF1-11 peptide was successfully covalently immobilized onto chitosan thin films. Both soluble and attached peptide presented a higher antimicrobial activity than the control chitosan. identified as a potent vasoconstrictor that binds with high affinity to UT receptor. 1 The cysteine-linked cyclic region, hUT-II(4-11), is responsible for the biological activity and has been widely used to elucidate the Structure-Activity Relationship of hUT-II. 2 With the aim to investigate the role of hydrogen bond and the effects of a peptide backbone constraint on binding