key: cord-0953551-xyzp6z2h
authors: Fotopoulou, Eirini; Ronconi, Luca
title: Application of Heteronuclear NMR Spectroscopy to Bioinorganic and Medicinal Chemistry☆
date: 2018-12-31
journal: Reference Module in Chemistry, Molecular Sciences and Chemical Engineering
DOI: 10.1016/b978-0-12-409547-2.10947-3
sha: 3a1bc2e8f2a19555b5874e22a20347af04c30a6b
doc_id: 953551
cord_uid: xyzp6z2h

Abstract The aim of this article is to provide a comprehensive overview on the use of heteronuclear (in particular, metal) NMR spectroscopy to investigate the behavior of metal ions in biological systems, focusing on some key results, and successful applications of NMR spectroscopy involving direct detection of metals. In this regard, although there is growing interest in the applications of solid-state NMR to biological systems, this subject lies outside the scope of this work, which will be therefore limited to NMR studies carried out in solution. Starting from some review papers published in the last few years, we are here covering relevant results in the field, including the most recent findings reported in the literature to date.

Although the list may not be complete, about 25 elements are currently believed to be essential for mammalian life, among which 14 are metals or metalloids. 1 Inorganic elements are essential components in many aspects of the biochemistry of living organisms. For instance, they can act as catalytic cores at the active sites of enzymes or as second messengers for a variety of small molecules and receptors, or they can stabilize the tertiary structure of proteins and nucleic acids. 2 In particular, metals in biological systems usually exist as electron-deficient cations (Lewis acids) that may interact with electron-rich biomolecules that are "natural ligands" (Lewis bases) capable of binding metal ions to perform important biological functions. Additionally, a significant number of clinical trials involve nowadays both essential and nonessential metal-based compounds to be used in either therapy or diagnosis, including anticancer, antibacterial, antiviral, antiparasitic, anti-inflammatory, antineurodegenerative, and magnetic resonance imaging (MRI) contrast agents. 1 The growing interest in understanding the coordination chemistry of biometals and the development of structure-based drug design of inorganic pharmaceuticals require suitable and reliable analytical tools to study the speciation of metal ions in biological systems. In this context, nuclear magnetic resonance (NMR) spectroscopy is a powerful and versatile technique that can provide sitespecific information about chemical bonding, structure and dynamics in molecular systems, and a description at atomic level of the intermolecular receptor-metal ion interactions responsible for molecular recognition. 3 Advances in the field have been providing detailed structural information on proteins, nucleic acids, and carbohydrates, including investigations of cells and perfused organs under physiological conditions, thus allowing quantification of the dynamic properties of metabolites and kinetics of enzymatic reactions at steady state or through real-time monitoring. 4 To date, most successful applications of NMR to biological systems have been typically carried out in aqueous solutions by exploiting nuclei with nuclear spin quantum number I ¼ 1/2, such as 1 H, 13 C, 15 N, and 31 P (Table 1) , whereas it proved less powerful when applied to quadrupolar nuclei (i.e., I > 1/2), that is, those having nonspherical charge distribution and a nuclear electric quadrupole moment (Q). Unfortunately, for a large number of biologically relevant elements, the only NMR-active isotopes are quadrupolar, thus giving rise to broad lines together with low detection sensitivity. 5 When dealing with the NMR spectroscopy involving 'exotic nuclei' (i.e., other than 1 H, 13 C, 15 N, and 31 P), it is worth pointing out that:

• quadrupolar nuclides account for nearly 75% of the stable NMR-active isotopes in the periodic table;

• the inherent sensitivities of I ¼ 1/2 and some quadrupolar nuclei are not similar (e.g., 45 Sc, 59 Co, 51 V (I ¼ 7/2), and 93 Nb (I ¼ 9/2) are much less sensitive than 1 H although being all over 99.5% naturally abundant);

• although many useful empirical correlations exist between structure and NMR parameters of I ¼ 1/2 nuclei, such correlations are not so widely explored for the quadrupolar counterparts;

• from both the chemical and biological point of view, there is nothing inherently more interesting about I ¼ 1/2 nuclei than there is about quadrupolar nuclei (if they were of equal practical difficulty, 17 O NMR would be, at least, as widely used as 1 H or 13 C NMR);

• if the problem of line broadening of resonances from quadrupolar nuclei could be overcome, then there would be enormously increased research activity in this area.

The aim of this article is to provide a comprehensive overview on the use of heteronuclear (in particular, metal) NMR spectroscopy to investigate the behavior of metal ions in biological systems focusing on some key results and successful applications of NMR spectroscopy involving direct detection of metals. In this regard, although there is growing interest in the applications of solid-state NMR to biological systems, this subject lies outside the scope of this work, which will be therefore limited to NMR studies carried out in solution.

Starting from some review papers published in the last few years, 6 we will cover relevant results in the field, including the most recent findings reported in the literature to date. The present article is organized as follows:

À the first section provides an overview of metal NMR spectroscopy emphasizing the practical limitations related to quadrupolar nuclei; À the second section describes illustrative NMR data for biologically relevant metallic and semimetallic elements belonging to s-, p-, d-, and f-block; À the last section deals with some significant biological data obtained by exploiting nonmetallic NMR-active nuclei other than 1 H, 13 C, 15 N, and 31 P, whose detection to probe, indirectly, the coordination chemistry of biometals has been extensively reviewed. 7

Nuclides reported in Table 2 are, at least in principle, observable by direct NMR spectroscopy in solution but, to the best of our knowledge, there are no relevant reports on the exploitation of their NMR properties in bioinorganic and medicinal chemistry; therefore, they will not be discussed. For both the experimental details and the practical setup of the NMR experiments, the reader is referred to the appropriate literature cited throughout the text.

The development of metal (in particular transition metal) NMR spectroscopy has been very uneven because of the very small number of nuclei with favorable nuclear properties for high-resolution NMR. The majority of metal nuclei have I > 1/2; therefore, contrary to I ¼ 1/2 counterparts (having spherical nuclear charge distribution), they show a nonspherical distribution of the nuclear electric charge (i.e., oblate or prolate spheroid). Consequently, in addition to their magnetic moment, they also possess a nuclear Table 1 NMR properties of the most common I ¼ 1/2 nuclei Isotope A (%) g (Â10 7 rad T À1 s À1 ) Relative receptivity to 13 electric quadrupole moment (Q) but, whereas the orientation of the magnetic (spin) dipole is quantized relative to the external magnetic field, the orientation of Q is quantized relative to the electric field gradient (EFG) at the observed nucleus, arising from the local electronic environment (i.e., electrons and other surrounding nuclei). EFGs exert a torque on the quadrupolar nuclei and the tumbling of the molecule can then trigger transitions among the various nuclear spin states. The quadrupolar coupling constant is defined as w ¼ e 2 Q(EFG)/h where h is Planck constant (6.626070 Â 10 À34 J s).

The main drawback of NMR spectroscopy of quadrupolar nuclei is that the spin-lattice relaxation time (T 1 ) can be very short and broad lines (or even none) may be recorded. The nuclear energy levels depend on both the EFG and the applied magnetic field. In the liquid phase, rapid and isotropic molecular tumbling averages both dipolar and quadrupolar interactions. On the other hand, relaxation of Q upon fluctuations of the EFG (e.g., due to molecular collisions) relaxes the nuclear spin as well, and the fast relaxation often results in short-lived nuclear states with broad resonance lines (quadrupolar broadening). 8 The quadrupolar relaxation rate is defined as

where is the asymmetry parameter of the EFG and t c is the rotational correlation time for isotropic tumbling. Anyway, it is worth reminding that this equation is valid when the molecular motion is characterized by an isotropic tumbling correlation time, and 1/ T 1Q ¼ 1/T 2Q ; t c ¼ 4p 0 r 3 /3k B T, where 0 is the viscosity of the medium, k B is Boltzmann constant (1.3806488 Â 10 À23 J K À1 ), T is the temperature, and r is the molecular radius. The linewidth depends on the linewidth factor

Accordingly, favorable properties to metal NMR spectroscopy are the following.

• For quadrupolar nuclei, larger values of I minimize Q, thus decreasing line broadening and increasing receptivity (see succeeding text).

• Large natural abundance (A) of the NMR-active nucleus (which may be accomplished by isotopic enrichment) and large magnetogyric ratio (g, effectively the ratio of the magnetic moment to the nuclear spin quantum number) increase the intrinsic NMR receptivity (i.e., the intensity of the signal) given by | g 3 |AI(I þ 1). I ¼ 1/2 nuclei with low-g values have a further problem of too slow relaxation since the rate depends on g, so long accumulation times and/or sensitivity enhancement techniques may be required.

In general, quadrupolar nuclei display broad resonances in NMR spectra, unless they are in highly symmetrical electrical environments, which reduce the magnitude of the EFGs at the nuclei (extreme narrowing conditions). Biological macromolecules that introduce high EFGs, in conjunction with nonaveraging motional characteristics, can make quadrupolar interaction effective. However, the binding of quadrupolar metal ions can be studied by NMR spectroscopy, potentially even in the cells. This is possible because the binding of metal ions to a biomolecule can cause a substantial increase of the effective t c , thus leading to a favorable change in relaxation behavior of the ions. Another severe hindrance to the development of transition metal NMR spectroscopy is caused by the fact that some nuclei in specific oxidation states, such as high-spin Fe(II), Fe(III), Co(II), Ni(II), Cu(II), and Ru(III), are paramagnetic. Paramagnetic compounds contain unpaired electrons whose density has a drastic effect on both the chemical shift and the linewidth of signals in the NMR spectra of metallobiomolecules containing one or more paramagnetic transition metals.

Line broadening of NMR signals corresponding to the paramagnetic metal itself and to the nuclei in the neighborhood of the paramagnetic center is a severe limitation for high-resolution NMR spectroscopy. Relaxation of the unpaired electron(s) gives rise to the major source of line broadening of resonances for paramagnetic compounds in solution spectra, as the relative electron relaxation is sensed by the resonating nucleus through dipolar coupling. In addition, the delocalization of the unpaired electron(s) throughout the molecule is another factor that causes extreme line broadening. 9 In some cases, extreme line broadening prevents the detection of any NMR signal.

The hyperfine shift is another factor to be taken into account when NMR studies are carried out on paramagnetic systems. It is defined as the difference in chemical shift between that of a paramagnetic molecule and that of an analogous diamagnetic system. Both contact (through-bond) and pseudo-contact (through-space) shifts are important contributors. In particular, the hyperfine shifts of nuclei in paramagnetic molecules can be well outside the window of signals for diamagnetic counterparts (even hundreds of ppm). The wide range of chemical shift values observed for paramagnetic compounds (large high-frequency and low-frequency shifts) is often attributed to the different resulting spin delocalization mechanisms. 9 On the other hand, in some circumstances, the effects induced by a paramagnetic metal ion can be used to probe the active sites of metallobiomolecules and analyzed to give additional constraints for structural calculations on paramagnetic proteins, leading to NMR structures with greater precision. In fact, many NMR structures of a variety of paramagnetic metalloproteins have been reported to date. 9 To summarize, the development of NMR techniques for the direct detection of metal ions in biological systems has been hampered by the quadrupolar and paramagnetic properties of some of the biometals of interest. Nevertheless, several successful studies have been carried out in the field, which are reported in the following sections. (Note to chemical shift referencing: Given a specific NMR signal recorded for the nucleus X, its chemical shift (d) is defined as the difference between its resonance frequency (n X, sample in Hz) and the resonance frequency of the reference substance (n X,ref in Hz), divided by the operating frequency of the spectrometer (n X,spec in MHz): d ¼ (n X,sample n X,ref )/n X,spec . Owing to the different units of numerator (Hz) and denominator (MHz), chemical shifts result in Â10 À6 unitless figures. Therefore, for greater convenience, the factor of 10 6 difference is discarded and d is appropriately represented by the unit ppm (parts per million). NMR referencing is a rather delicate topic, and this issue is even more pronounced in the case of metal NMR spectroscopy. In fact, different reference substances and sign conventions have been used throughout the past decades, thus making the comparison of data reported in the literature difficult and somewhat misleading. Moreover, since salts are generally used as references, the actual chemical shifts may be strongly influenced by the nature of the counterion and by the concentration of the salt itself. In this article, the reported NMR parameters, including the reference samples and conditions, are those recommended by the International Union of Pure and Applied Chemistry (IUPAC Recommendations 2001, revised and updated in 2008). 10 Nevertheless, the reader should be aware of the existence of a number of alternative references and conventions and should refer to the individual papers cited throughout the text for a correct comparison.) Lithium is generally present only at trace levels within the human body, and neither nutritional nor biological roles have been undoubtedly recognized so far. On the other hand, there are several medical uses of Li(I) derivatives, in particular, for the treatment of bipolar disorders. 11 Naturally occurring lithium consists of the 6 Li and 7 Li isotopes, which are weakly quadrupolar (compared to other alkali metals) owing to their small quadrupole moments reflecting the small size and simple electronic structure. Both isotopes yield narrow spectral lines and long T 1 (typically 2-8 s), but the higher natural isotopic abundance and receptivity and the more favorable relaxation properties make 7 Li the isotope of choice for most studies (although 6 Li NMR has been also employed). 12 Owing to the relatively simple aqueous chemistry of the Li þ ion, the chemical shift range is rather small and spectra usually show a single (averaged) resonance from all environments, thus complicating the interpretation of the NMR signals of the usually heterogeneous biological systems. Therefore, it is often advantageous to resolve signals from the different compartments by either exploiting differences in relaxation properties or adding a shift reagent since each local Li(I) environment may potentially have a characteristic concentration and spin relaxation behavior. 7 Li NMR is particularly suitable for studying transmembrane transport and competitive countertransport of Li þ and other alkali metals. 13 Nevertheless, a major application relates to the development of noninvasive in vivo analytical tools to measure brain lithium concentration and speciation in humans to elucidate the mechanism(s) of therapeutic action and toxicity of lithium-based drugs in clinical practice. 14 Remarkably, NMR investigations of the human brain at high magnetic field (3 T) allowed the determination of the relationship between the amount of lithium in the brain and in serum in lithium-treated bipolar patients. 14c The beneficial effect of Li in mania and depression results from its action on the brain and, hence, the evaluation of its amount is an important parameter with regard to patient response. As brain lithium cannot be monitored readily at present, its concentration is measured in the serum. Moreover, due to the sedative and possible neurotoxic side effects, Li þ concentration needs to be monitored and maintained within the safe therapeutic window by measuring it in serum or plasma. Although plasma lithium values are used to monitor the therapy, the lithium in red blood cells (RBCs) may provide a better estimate of levels in brain. 15 For example, rats were treated with different amounts of Li 2 CO 3 and, 24 h after administration, blood was collected, added with a Tm (III)-based shift reagent, and analyzed by 7 Li NMR, providing a clear discrimination between plasma and RBC lithium signals. 16 The 7 Li spectrum ( Fig. 1) shows two peaks about 13 ppm apart corresponding to plasma (extracellular) and RBC (intracellular) lithium.

By comparing peak intensities with standards prepared using blood from untreated rats, it was possible to quantify the amount of lithium and results were in good agreement with other commonly employed techniques.

Hyperpolarized 6 Li NMR was recently shown to provide insights into hemoglobin oxygenation levels. Hyperpolarization by dissolution dynamic nuclear polarization (DNP) is a relatively new technique that enhances the NMR signal intensity of insensitive long-T 1 nuclei such as 6 Li. 17 6 Li is a I ¼ 1 nucleus with an exceptionally small quadrupole moment that can be hyperpolarized by dissolution DNP and, subsequently, detected even in vivo. 18 Accordingly, Mishkovsky and coworkers exploited hyperpolarized 6 Li to evaluate blood oxygenation in human and rat blood and plasma. 19 Based on the assumption that 6 Li longitudinal relaxation of Li þ ions in aqueous solution is strongly dependent on the concentration of paramagnetic species, 20 the measurement of the longitudinal relaxation time of 6 Li þ in presence of oxygenated hemoglobin (diamagnetic) and deoxyhemoglobin (paramagnetic) allowed the reproducible mapping of blood oxygenation in a number of samples, and results were fully consistent with those obtained by means of the usual oxygen gas analysis.

Relative receptivity to 13 Sodium ions have a number of functions, including the cotransport of solutes and the maintenance of both concentration gradients across cell membranes and the pH (Na þ /H þ transport). Moreover, Na þ is involved in several pathological processes, such as cell death, edema formation, tumor growth, electrophysiological processes, and ion transport, all showing alterations in either intra-or extracellular sodium levels. 21 Among the NMR-active nuclei in biological samples, 23 Na is favored because of its high natural abundance, relatively high sensitivity to detection, and high concentrations in tissues (5-150 mM) . Notwithstanding its quadrupolar properties, lineshape analysis of 23 Na NMR resonances can provide information on Na þ binding, and the study of relaxation processes can describe the dynamics of sodium ions in living systems and correlate such dynamics with the pathological or normal state of the system itself.

In most living cells, the transmembrane gradients of sodium ions are essential for proper cell function. Therefore, changes in ion concentrations at each side of the membrane may result in severe functional disorders of the cell. In this regard, 23 Na NMR relaxation techniques are able to discriminate between different adjacent sodium ion compartments, whether or not they are physically separated, as long as they show different molecular motions and, thus, induce different relaxation characteristics. The use of multiple echoes to provide relaxation information has greatly increased the usefulness of 23 Na NMR spectroscopy, and addition of paramagnetic shift reagents or contrast reagents allows the differentiation of intra-and extracellular sodium pools. The first widely used aqueous shift reagent for cations was reported by Gupta and coworkers in the attempt at finding suitable compounds impermeable to cell membrane and capable to split the NMR resonances. They succeeded with [Dy(PPP) 2 ] 7À (PPP ¼ tripolyphosphate) in experiments with sequential frequency-selective radiofrequency pulses to excite selectively the wellresolved resonances of shifted and nonshifted sodium and produce separate images of the different sodium pools. 22 Their discovery was followed by many other lanthanide-based shift reagents used for many different applications, including the study of ion transport and metabolism, the measurement of temperature and pH, the enantioselectivity ratio of a substance, and the encapsulation efficiency of liposomes. 23a-f For example, [Tm(DOTP)] 5À (DOTP ¼ dioctyl terephthalate) was successfully used to study Na þ transport in human erythrocytes and to quantify the movement of ions across the membrane. 23g Other recent examples encompass the use of 23 Na NMR in the evaluation of the intra-and extracellular diffusion of sodium ions in rat skeletal muscle (and its effect on ischemia), 23h the study of sodium ion gradients in microorganisms, 23i the investigation of complex I driven sodium transport, 23j and the determination of the stoichiometric relationship between Na þ ions transported and glucose consumed in human erythrocytes. 23k Multiple quantum and multiple-quantum filtered (MQF) pulse sequences were proved useful to define the interaction of quadrupolar nuclei like 23 Na with biologically relevant macromolecules, including in vivo NMR and MRI applications. 24 In particular, the relaxation profiles of specific magnetization or coherence of a quadrupolar ion can be used to determine sodium binding to biomolecules. For example, Torres and coworkers have used the relaxation properties of selected 23 Na magnetization coherences to determine the apparent binding constants for Na þ and a protein (bovine serum albumin, BSA), a nucleic acid (yeast RNA), and a self-associating macroassembly (the detergent sodium dodecyl sulfate, SDS). 25 These three macromolecular systems were chosen as models of different classes of biomolecules that are likely to have diverse Na þ -binding environments, and the results confirmed the occurrence of a strong binding to RNA and weak binding to BSA, whereas both strong and weak binding sites were identified in SDS.

Geraldes and coworkers used 23 Na MQF NMR spectroscopy to characterize the isotropic and anisotropic binding and dynamics of intra-and extracellular Na þ ions in different cellular systems in the absence and presence of Li þ . 26 This study provided a detailed evaluation of extra-and intracellular molecular sites of Na þ and Li þ binding, enabling the differentiation between isotropic and anisotropic Na þ binding sites and the determination of the extent of Li þ competition for those sites. Remarkably, this represents a solid proof-of-concept to the exploitation of 23 Na MQF NMR to decipher the pharmacokinetics and binding sites of lithium-based drugs in vivo. 11 Guanine quadruplexes (G-quadruplexes) are noncanonical nucleic acid structures present in guanine-rich nucleic acid sequences, in which four guanine bases are interconnected via Hoogsteen hydrogen bonds to form planar G-quartets that stack to each other. In the center of the cycle formed by the four guanines, completely dehydrated alkali ions (usually K þ or Na þ ) are coordinated by the buried carbonyl oxygens of the nucleotides. Stacked G-quartets form an ion channel, and the presence of the cations is crucial for the formation, stability, and function of G-quadruplexes. The presence of G-quadruplexes in different regions of the genome suggests a biological relevance for these systems. 27 Detection of alkali cations in G-quadruplexes has usually relied on either direct solid-state techniques, such as X-ray crystallography and solid-state NMR, or indirect methods using surrogate I ¼ 1/2 probes, such as 15 NH 4 þ and 205 Tl þ , in solution NMR experiments. 28 On the basis of these studies, two types of alkali metal-binding sites were generally found in G-quadruplex structures, one type being loosely coordinated to phosphate groups and the other residing inside the G-quadruplex channel. For several years, direct detection by solution NMR of alkali ions was considered unfeasible due to low signal intensity and unfavorable quadrupolar properties. Nevertheless, Won and coworkers reported the first direct solution NMR detection of the Na þ residing inside G-quadruplex channel structures formed by 5 0guanosinemonophosphate (5 0 -GMP) and the DNA oligomer, d(TG 4 T). 29 Fig. 2A shows 29 Na NMR spectra for 0.8 M Na 2 (5 0 -GMP) in an aqueous solution at pH 8. The signal centered at 0 ppm exhibits a bi-Lorentzian lineshape arising from the slow exchange of Na þ cations between phosphate-bound and free states. The small peak at À17 ppm is due to the Na þ cations residing inside the 5 0 -GMP channel. The signal intensity for the channel Na þ cations decreases as the sample temperature increases from 278 to 308 K, an indication of "melting" of the 5 0 -GMP aggregates. Analogously (Fig. 2B ), 23 Na NMR spectra of d(TG 4 T) show the channel Na þ signal at À17 ppm but, in this case, the total integrated area for this signal remains approximately unchanged between 278 and 293 K, indicating that the G-quadruplex structure of d(TG 4 T) does not melt at 293 K. These findings opened up many new possibilities in the study of cation binding and transport dynamics in G-quadruplexes including the measurement of cation binding affinity for the channel site in a direct and site-specific way. 30

Relative receptivity to 13 Together with sodium, potassium represents the most abundant monovalent cation in the cells. In particular, it is the major ion in intracellular fluids (typically $140 mM), whereas Na þ is found at higher concentration outside the cell (145 vs. 5 mM). K þ is involved in the control of transmembrane potentials and regulates the equilibrium of cellular electrolytes and osmotic pressure. It acts primarily as counterion for negatively charged solutes and is involved in counterbalancing the high negative charge density associated with the nucleic acid phosphate backbone. The interaction with most biological ligands is weak and, as such, its direct binding is unlikely to be involved in the triggering of biological activity, although it seems to be required for the activation of a number of enzymes. 21 Conventional analytical methods for the detection of intracellular ions (e.g., flame photometry and radioisotope tracer techniques) rely on time-consuming destructive methods to achieve separation of intra-and extracellular compartments. Furthermore, there could be uncertainty associated with the nonspecific binding of ions to the cell membrane and with ion fluxes during the separation procedure. Conversely, the possibility to develop nondestructive and noninvasive methods such as NMR spectroscopy is highly desirable. Naturally occurring potassium consists of the quadrupolar 39 K, 40 K, and 41 K nuclides, the former being the isotope of choice for most NMR studies because of its higher natural abundance. Unfortunately, low sensitivity (compared to 7 Li and 23 Na) was proved a major drawback for the development of such analytical technique, as confirmed by the relatively few data reported in the literature. Nevertheless, as technology evolves, the detectability and quantification in different tissues by means of NMR spectroscopy have been improving over time. For instance, the recent development of a triple resonant ( 39 K/ 23 Na/ 1 H) radiofrequency coil setup allowed the unprecedented acquisition of tissue images of a rat head in vivo by 39 K MRI, 31 and, more recently, a detailed investigation of the 39 K magnetic resonance imaging of human muscle tissue in vivo has been reported. 32 Most intracellular K þ is bound to ribosomes as it stabilizes the negatively charged ribose-phosphate backbone and specific structural motifs. In general, acting as charge screen, together with divalent metal ions, it is crucial for the establishment of the correct structure of RNAs and is also associated with DNAs. The first direct solution NMR detection of K þ residing inside G-quadruplex channel structures formed by 5 0 -GMP and the DNA oligomer d(TG 4 T) was reported by Wong and coworkers in 2005. 29 As shown in Fig. 3 , two 39 K resonances are clearly observed at 278 K. The peak at 0 ppm is due to surface/free K þ cations, whereas the signal at about 18 ppm was assigned to the channel K þ cations, whose intensity decreases as the sample temperature is increased, thus indicating the "melting" of the 5 0 -GMP aggregates. It was also found that potassium ions move through the G-quadruplex channel at a much slower rate than sodium counterparts. 39 K NMR studies were also carried out to investigate the interaction of K þ with ribosomes 33 and quadruplex DNA, 34 and attempts have been made to quantify intracellular K þ in vitro. 35 K þ transport in human erythrocytes was successfully studied by employing a Dy(III)-based paramagnetic shift reagent, 36 which allowed intra-and extracellular 39 K NMR signals to be discriminated (Fig. 4) , thus establishing the suitability of NMR spectroscopy for measuring K þ fluxes and ion concentrations in human erythrocytes.

A (%) I g (Â10 7 rad T À1 s À1 ) Q (fm 2 ) Relative receptivity to 13 Rubidium has two quadrupolar NMR-active isotopes and, despite the higher natural abundance of 85 Rb, 87 Rb is the isotope of choice for NMR studies because of the more favorable spectroscopic properties.

Rubidium has no acknowledged natural biological role but Rb þ is an established probe for K þ . Rb þ uptake data can be used to approximate K þ influx provided that there are no significant differences in ion selectivity of the different K þ -transporting systems. 37 This implies similar ion selectivity of the systems transferring these ions inside and outside cells, thus supporting the role of Rb þ as a mimic for K þ . The rates of influx and efflux of Rb þ in living tissues and isolated cells have been measured by 87 Rb NMR spectroscopy. 38 The relatively high natural abundance, low biological abundance, and high NMR sensitivity of 87 Rb (compared to 39 K) make it a good tracer for K þ influx and efflux studies by NMR in cells and perfused organs. 37 With reference to Fig. 5 , Rb þ uptake rates determined by 87 Rb NMR spectroscopy are similar to those measured by atomic emission spectroscopy, thus allowing estimates of K þ influx rates. The method does not require shift reagents because of the 8.5 times faster kinetics of Rb þ equilibration in the extracellular space (compared with the intracellular space) and the much higher concentration of Rb þ in the latter.

Cardiac sarcolemmal K ATP channels are crucial in adaptation to stress caused by metabolic inhibition and moderate exercise, which requires not only downregulation of energy spending but also upregulation of mitochondrial ATP synthesis. In order to investigate sarcolemmal and mitochondrial effects of a Kir6.2 (K þ ion-selective subunit of the channel) knockout, 87 Rb NMR has been used, showing that Kir6.2 knockout results in a lack of stimulation of the unidirectional potassium efflux from the heart that creates a primary defect leading to the development of non-insulin-dependent (type 2) diabetes. 39 During the last decade, imaging the distribution of 87 Rb, which mimics K þ as a substrate for the Na þ /K þ -ATPase pump in myocardial cells, by MRI was shown promising in distinguishing between necrotic and reversibly damaged tissue. 40 Subsequently, 87 Rb MRI has been moving to in vivo applications to monitor, upon replacement of K þ with Rb þ , brain potassium in animal models and to study K þ dynamics in live rats with focal ischemic stroke. 41 

A (%) I g (Â10 7 rad T À1 s À1 ) Q (fm 2 ) Relative receptivity to 13 Interest in the biological roles of cesium ions arises from three areas: (i) applications related to alkali metal ion transport and enzyme activation, (ii) toxicological considerations related to the uptake and passage through food chains of radioactive 137 Cs produced in fission reactions, and (iii) applications of cesium derivatives in the treatment of behavioral depression. 2 The development of 133 Cs NMR spectroscopy for Cs þ analysis has triggered the interest in the biochemistry and physiology of Cs þ in biological systems. It accumulates in the intracellular space, primarily through the action of Na þ /K þ -ATPase, thus making it a valuable tool for noninvasively probing of its congener K þ . A major advantage of 133 Cs NMR is that the chemical shift range is much larger than the other alkali metals, and 133 Cs signals are extremely sensitive to changes in chemical environment, solvents, temperature, and counterions present. 42 133 Cs has a small quadrupole moment, resulting in narrow NMR lines and a relaxation rate approximately 200 times smaller than the other NMRactive alkali metal nuclei. Cs þ is 100% visible by NMR spectroscopy and its biomedical applications have been extensively reviewed. 43 Intra-and extracellular 133 Cs NMR resonances in Cs þ -loaded cell suspensions can be distinguished without the addition of shift reagents (required to resolve resonances of other alkali metal ions such as 7 Li, 23 Na, and 39 K). In this regard, intra-and extracellular resonances were readily resolved for suspended human erythrocytes treated with CsCl, the resulting spectra exhibiting two resonances separated by 1.0-1.4 ppm (Fig. 6) . 44 In addition, it is possible to resolve the NMR signals of 133 Cs in different tissue compartments on the basis of chemical shift or relaxation properties. This compartmental resolution applies not only to the intra-and extracellular spaces but also to the subcellular compartments. For example, Cs þ binding to human RBC was evaluated by means of relaxation measurements, showing that it binds more strongly to 2,3-bisphosphoglycerate and RBC membranes than to any other intracellular component in RBC at physiological concentrations. 45 133 Cs NMR spectroscopy was also proved useful to evaluate the ion transport across membranes and the kinetic/chemical environment of the intracellular space in systems ranging from RBC to rat brain. 43 Together with K þ , Mg 2þ is one of the major intracellular ions (typically 30 mM) and is 90% bound to the ribosomes. It is regarded as a natural RNA cofactor and, besides acting as charge screener to allow proper folding and correct establishment of RNA tertiary interactions, it was shown to be also directly involved in ribozyme catalysis. 47 Moreover, magnesium is an essential cofactor for many RNA-and DNA-processing enzymes and for enzymes using ATP, ADP, or AMP as substrates. 2 As metallotherapeutic drugs, magnesium salts are frequently prescribed to treat diseases such as gestational hypertension, preeclampsia and eclampsia, asthma, strokes, acute myocardial infarction, and arrhythmias. 48 Mg 2þ readily forms complexes with biological substrates to either define peculiar conformations or catalytically activate specific chemical functionalities, and its role is critically dependent on the coordination mode adopted in a defined biological context.

The interaction of 25 Mg quadrupole moment with EFGs provides a very effective relaxation mechanism. 49 The observed lineshapes are sensitive to the motion and exchange dynamics of the Mg 2þ ions, thus providing insights into the interaction with various biomolecules. Owing to the low natural abundance and low receptivity, only the use of isotopically enriched 25 Mg 2þ allowed a total lineshape analysis of 25 Mg NMR spectra to determine the association constant (K a ), the free energy of activation (DG Ã ), outer-sphere association (K os ), and off-rate (k off ) and on-rate (k on ) constants for magnesium binding to several biologically relevant macromolecules, as summarized in Table 3 . 50 Indirect methods have also been used to study RNA-Mg(II) interactions. Normally, such investigations rely on changes of 1 H chemical shifts upon titration with Mg 2þ , allowing Mg(II)-RNA affinity constants to be determined. Moreover, the different binding modes of Mg 2þ to RNA (i.e., inner-and outersphere coordination) can be mimicked by employing different probes like Mn 2þ , [Co(NH 3 ) 6 ] 3þ , and Cd 2þ . 51 25 Mg NMR was also proved useful to investigate the interaction between Mg 2þ and calmodulin (a calcium-binding protein that can bind to and regulate a multitude of different protein targets), thereby affecting a number of cellular functions. 52 Tsai and coworkers demonstrated that Mg 2þ shows opposite site preference relative to Ca 2þ and binds to sites I and II of calmodulin with a binding constant of $2000 M À1 , whereas it binds weakly to sites III and IV. Since the intracellular concentration of Mg 2þ is higher than that of Ca 2þ , they hypothesized that sites I and II are constantly occupied by Mg 2þ at the resting state. Analogously, the interactions between Mg 2þ and other biomolecules, such as adenylate kinase (a phosphotransferase enzyme requiring Mg 2þ to catalyze the production of ATP from ADP) 53 and several biological polyelectrolytes, 54 have been studied by 25 Mg NMR spectroscopy. In both cases, it was demonstrated that magnesium ions bind loosely to the investigated actin filaments and, thus, show a behavior typical of counterions.

Relative receptivity to 13 Calcium is an important element in the human body and is located principally in bones and teeth as apatite, a calcium phosphate mineral. It is distributed throughout all tissues where it plays special roles in controlling blood pressure, nerve impulse transmission, muscle action, blood clotting, and cell permeability. 21 Together with Na þ , Ca 2þ is a major extracellular ion (typically 4 mM). Its fundamental role in initiating biological reactions results from rapid exchange kinetics and strong ligand binding capability. Many processes of signal transduction involve the release of Ca 2þ as part of an interconnected set of pathways. Increased intracellular levels are sensed by a family of calcium-binding proteins, including calmodulin, which use changes in the intracellular calcium concentration to activate a variety of enzymes, such as protein kinases, NAD kinase, and phosphodiesterases, and some Ca 2þ -ATPases. 2 Calcium supplements (usually carbonate, citrate, gluconate, or lactate salts) are administered to protect against osteopenia, osteoporosis, and hypertension, and calcium-based drugs may be used to prevent from high blood cholesterol, diabetes, and major pregnancy complications. CaCO 3 is also marketed as antacid to relief the pain and discomfort of indigestion, heartburn, and other symptoms related to excess stomach acidity. 55 43 Ca is the only NMR-active calcium isotope and its NMR properties are rather unfavorable due to the low resonance frequency, low natural abundance, and strong quadrupolar relaxation, thus explaining the paucity of data available. In this regard, the most recent biologically related application of 43 Ca NMR in solution appears to date back to 2008 when Kwan and coworkers reported the first direct NMR evidence for Ca 2þ ion binding to G-quartets by combining natural abundance 43 Ca NMR spectroscopy with extensive quantum chemical calculations. 56 In the past three decades, such technique had provided a few insights into the structural and motional characteristics of calciumbinding sites in a number of calcium-binding proteins. 57 For example, the 43 Ca NMR signals from Ca 2þ ions bound to the Ca-binding proteins parvalbumin, troponin C, and calmodulin have been detected 58 by using isotopically enriched 43-calcium. The signals of 43 Ca 2þ bound to all three proteins were recorded at similar chemical shifts, and all showed similar magnitude of the Table 3 Determination of kinetic and thermodynamic parameters for Mg 2þ binding to phosphate-containing ligands by 25 Mg NMR spectroscopy quadrupole coupling constant. This observation supported the hypothesis that the Ca-binding sites have the same arrangements of oxygen donors coordinated to the Ca 2þ ion. An analogous study has been carried out to evaluate the exchange rates and the binding constants of Ca 2þ ions to the high-affinity and low-affinity binding sites on calmodulin. 59 The calcium-binding properties of equine and pigeon lysozyme and those of bovine and human a-lactalbumin have been also investigated. 60 An example is shown in Fig. 7 . Upon addition of isotopically enriched 60 Ca 2þ to equine lysozyme, a broad peak (Dv ½ ¼ 253 Hz) appears at À5.3 ppm, and the signal intensity increases linearly up to 1 equiv. of metal ion. This resonance corresponds to calcium bound to the single high-affinity calcium site. In the presence of excess metal ion, a sharp signal (Dv ½ ¼ 10 Hz) is also observed at l ppm, in the proximity of that of free calcium. All proteins were found to contain one highaffinity calcium-binding site, and a second weak calcium-binding site was observed for bovine a-lactalbumin only. The chemical shifts, linewidths, relaxation times, and quadrupolar coupling constants for the respective 43 Ca NMR signals associated with highaffinity binding sites were quite similar, indicative of a high degree of homology between those sites in the four proteins. On the other hand, both the chemical shifts and the quadrupolar coupling constants are quite distinct from those observed for typical EF-hand calcium-binding proteins, thus suggesting a different geometry for the calcium-binding loops.

p-Block: Group 13 (B, Al, Ga, In, and Tl)

Relative receptivity to 13 Boron is regarded as a nonessential element for mammalian life, although it may turn out to be a necessary "ultratrace" element. The main medicinal application of boron is the tumor-targeted delivery of boron derivatives for boron neutron capture therapy (BNCT). 61 The therapy is based on the nuclear reaction that occurs when the stable isotope 10 B is irradiated with neutrons at appropriate energy to produce 11 B in an unstable form, which then undergoes instantaneous nuclear fission to produce high-energy alpha particles and recoiling 7 Li nuclei ( 10 B þ n th ! [ 11 B] ! a þ 7 Li). These heavy charged particles have pathlengths of approximately one cell diameter (10-14 mm) and deposit most of their energy within the boron-containing cells. Provided that boronbased therapeutics are delivered and accumulate preferentially in tumors and enough low energy thermal neutrons (n th ) reach the target site, cancer cells undergo necrosis as a result of the 10 B(n,a) 7 Li capture reaction. BNCT has been used clinically to treat patients with brain cancers, such as glioblastoma multiforme, with high-grade gliomas, and a much smaller number with primary and metastatic melanoma. Delivery vehicles synthesized for this application include sodium borocaptate (BSH, Na 2 B 12 H 11 SH), borano phosphonates and borano bis(phosphonates), borano phosphate oligodeoxynucleotides, in which BH 3 is linked to the phosphate backbone of antisense oligodeoxynucleotides, 61b and carboranes. 61d Boron NMR is a suitable technique to study the pharmacokinetics of boron-based therapeutics and to evaluate their binding to biologically relevant molecules. Both 10 B and 11 B are NMR-active, but 11 B has higher sensitivity and natural abundance. Therefore, although 10 B is the nucleus used in BNCT treatment, the more sensitive 11 B is more appropriate for NMR studies, since the isotopic difference does not alter the structure, binding, or the pharmacokinetic effects of the BNCT agents. Both isotopes are quadrupolar and their relaxation times are rather short. NMR research efforts have been primarily moved toward two directions: first, to investigate the metabolism and pharmacokinetics of BNCT agents in vivo and, second, to use localized NMR spectroscopy and/ or MRI for noninvasive mapping of the administered molecules. While the first goal can be accomplished by 11 B NMR for natural abundance samples, molecules used in BNCT are generally enriched (>95%) in the less favorable (in terms of NMR properties) 10 B nucleus. Anyway, the use of 10 B MRI was proved successful to evaluate the distribution in vivo of these agents 62 and, in this regard, much attention has been recently given to the exploitation of boron nitride nanotubes containing Fe paramagnetic impurities 63 and to dual gadolinium/boron compounds as contrast agents. 64 The first attempts to investigate the interaction between boron derivatives and biomolecules by 11 B NMR spectroscopy date back to 1990s. Peptide boronic acids are exceptionally potent inhibitors of serine proteases, which are well known to play crucial roles in biological systems. Therefore, the high affinity and specificity of boronic acid-based inhibitors make them considerably interesting as both research tools and potential therapeutic agents. 11 B chemical shifts are very sensitive to changes in the coordination geometry of the boron atom, and resonances belonging to trigonal species appear downfield compared to tetracoordinated ones. Accordingly, for a number of such boron-containing inhibitors, 11 B signals were recorded at about 17 ppm, consistent with the formation of boron-histidine and boron-serine adducts with a-lytic protease in a tetrahedral geometry. 65 11 B chemical shift and relaxation rate changes can be also used to monitor the interaction of BNCT agents with proteins. An example of such application of 11 B NMR spectroscopy is the study of the reaction between BSH (Fig. 8 ) and serum albumin, the latter likely being involved in its pharmacokinetics. 66 Similarly, the binding of borate ions to cytochrome c surface has been investigated by 11 B NMR spectroscopy. Cytochrome c is a globular heme protein acting as electron carrier in the cell mitochondria. It has a positively charged surface that is believed to bind mitochondrial enzymes, small anions, and metabolites. Such surface interaction influences the efficiency of the proton transfer and the mobility of the protein itself. Borate ions were shown by 11 B NMR to bind specifically to cytochrome c surface, and this interaction could be used as a model for anion-protein surface interaction. 67 Fig. 9A shows the 11 B spectrum at 11.4 T of a 100 mM borate solution in presence of 4 mM ferricytochrome c at pH 9.7 at 5 C. The large peak at 7.1 ppm (peak z) represents the weighted average resonance arising from 70% B OH ð Þ 4 À exchanging with 30% B(OH) 3 . The peaks at 2.1 ppm (peak y) and 1.8 ppm (peak x)

were assigned to B OH ð Þ 4 À specifically bound to two conformations of ferricytochrome c coexisting at pH 9.7, whereas the shoulder at around 6 ppm (peak w) is still unassigned. When MQF NMR experiments were carried out (Fig. 9B) , two narrow signals related to the specific binding sites of ferricytochrome c and a broad peak owing to the borate/boric acid were detected (at $2 and 7.1 ppm, respectively), suggesting two different types of binding sites designated as I (for slowly exchanging borate ion) and II (for fast exchanging borate ion and boric acid). Finally, a recent paper reports on the use of 11 B NMR spectroscopy to study boron-induced enzyme inhibition. 11 B chemical shift analysis was used to prove the formation of ternary complexes between boronic acids, sugars, and a-chymotrypsin as model enzyme. 68 Aluminum

Relative receptivity to 13 Aluminum is the third most abundant metal in the Earth's crust, representing approximately 8% of total weight, and the natural element consists entirely of the 27 Al isotope. The concentration in blood plasma is about 0.005 mg L À1 , corresponding to about 1% of the aluminum body burden, 80% of which is bound to proteins. It accumulates mainly in the liver, bones, and spleen of humans and animals. It is a nonessential element nowadays recognized as potential toxic element linked to neurological problems, especially dialysis encephalopathy and Alzheimer's disease, and also to some bone disorders and problems in the hematopoietic system, muscles, and joints. 69 For a long time, owing to the low solubility under physiological conditions, aluminum was regarded as a nontoxic element, so its biological effects have not been investigated until recently. In particular, due to the increasing acidity of the environment and the concomitant increased dissolution of aluminum minerals, the concentration of this element in freshwaters has become a considerable issue. On the other hand, aluminum-based drugs are sold as antacids (Al(OH) 3 and Al 2 (CO 3 ) 3 ) and for the treatment of malaria (Al(OH) 3 ). Three aluminum salts (alum, Al(OH) 3 , and Al(PO 4 )) have also been added to many vaccines, including widely used formulations for diphtheria, hepatitis B, and tetanus, as adjuvant ('vaccine boosters'). 70a Moreover, the magnetic resonance characteristics of this nuclide also make it potentially suitable for medical applications ( 27 Al-based MRI is feasible). 70b 27 Al is amenable to NMR studies owing to the relatively high receptivity, the existence of only one isotope, and the relatively small quadrupole moment associated with its high nuclear spin. The latter two favorable factors result in a much higher relative peak height (by one order of magnitude or higher compared to the other elements belonging to group 13), thus allowing to achieve sufficient signal-to-noise ratios even for dilute ($0.01 M) solutions of aluminum compounds. 27 Al linewidths may vary from 3 to several kHz, and the signals may even completely vanish into the baseline noise in some instances. This negative aspect of quadrupolar relaxation is counterbalanced by the fact that the magnitude of the quadrupolar line broadenings provides information about the coordination geometry of the aluminum center. The major metal transport protein in blood plasma is the bilobal glycoprotein transferrin, and 27 Al NMR can be used to investigate directly the metal in the specific binding sites and to reveal subtle intersite differences. 71 Human serum transferrin (Fig.  10) is a member of a small group of monomeric nonheme proteins (MW $76-81 kDa), which includes lactoferrin, ovotransferrin, and melanotransferrin. It has two binding sites for Fe 3þ ions in a six-coordinate, distorted octahedral coordination geometry identified as C-and N-terminal sites. Two tyrosines, one histidine, and one aspartate constitute four protein ligands for the metal ion, which requires a synergistic anion for the formation of stable transferrin complexes. The bidentate CO 3 2À serves this purpose by coordinating directly to the metal in the fifth and sixth coordination positions in vivo. Since serum transferrin is normally only about 30% saturated with iron, it retains a relatively high capability for binding to other metal ions. Vogel and Aramini demonstrated the feasibility of 27 Al NMR to probe the binding of Al 3þ to ovotransferrin and its halfmolecules in the presence of carbonate or oxalate as synergistic anions. 72 The ovotransferrin-bound 27 Al NMR signals have some rather unusual properties related to quadrupolar nuclei bound in slow exchange to large macromolecules far from extreme narrowing conditions. First, the maximum intensity of the protein-bound 27 Al signal is substantially lower than that observed for equimolar solutions of the free metal ion. Secondly, the signals exhibit a unique pulse angle dependence where a maximum in peak intensity is attained at pulse lengths that are less than half the 90 degrees pulse for aqueous Al 3þ solutions. Third, 27 Al signals for Al 3þ bound to the half-molecules of ovotransferrin are much broader than those for the intact protein, thus reflecting the importance of molecular motion on the detectability of quadrupolar nuclei. Finally, the increase of the external magnetic field strength causes line narrowing and a 2-4 ppm downfield dynamic frequency shift (Fig. 11 ). 73 The chemical shifts of the 27 Al signals from Al 2 -ovotransferrin were found from þ40 to À46 ppm, which is in accordance with a six-coordinate (octahedral) Al(III) complex. From these assignments and titration experiments, it was found that, in the presence of carbonate, the N-terminal site of ovotransferrin binds Al 3þ with a higher affinity than the C-terminal site. Interestingly, changing the synergistic anion to oxalate alters the specificity (Fig. 12 ). 72a Aluminum neurotoxicity involves also the inhibition of certain enzymes, such as ATPase. It is believed that the Al 3þ ion, once bound to ATP, interferes with Mg 2þ so that any consequent reactions requiring Mg 2þ -ATP complex participation are inhibited. 21 This has prompted the study of the binding of Al 3þ to ATP by 27 Al NMR. The linewidths of these complexes typically fall within the range 150-500 Hz. Detellier and coworkers 74 studied these interactions at pH 7.4 using multinuclear NMR spectroscopy. Such 27 Al NMR investigations allowed them to identify two complexes coexisting in equilibrium: 2:1 {Al(ATP) 2 } and 1:1 {Al(ATP)}species.

More recently, the involvement of glutathione (GSH) in aluminum toxicity was evaluated by detailed spectroscopic studies, including 27 Al NMR. 75 GSH turned out to form various mono-and dinuclear Al-containing species by coordinating the metal center through the Gly and Glu carboxylate groups, although the Glu amino group, the peptide imino, and carbonyl moieties also appeared to be involved in the bidentate and tridendate derivatives. 27 Al NMR spectroscopy has been also employed to study aluminum-containing species present in natural waters. For example, Casey and coworkers studied the rates of solvent exchange in aqueous Al 3þ -maltolate complexes. 76 Maltolate is a natural product that can be isolated from larch trees but is now widely used as a food additive. It is soluble in water, and the corresponding trisderivatives of Al 3þ are toxic and may cause brain disease. A variable temperature 27 Al NMR investigation showed that maltolate can replace the inner-sphere water molecules bound to Al 3þ and labilize the remaining coordinated water molecules. In particular, coordination of a single maltolate ligand into the inner-coordination sphere of [Al(H 2 O) 6 ] 3þ increases the exchange rate of the other bound water molecules with bulk solution by a factor of about 10 2 . Remarkably, the addition of a second ligand, to form the bis-derivative [Al(maltolate) 2 (H 2 O) 2 ] þ , increases the rate by an additional factor of 6-7.

Apart from investigating the biological role of aluminum, 27 Al NMR has been also exploited to assess the binding environment and ligands coordination modes in Al(III)-containing potential biologically active scaffolds, such as bionanomaterials. 77

Gallium, indium, and thallium

Relative receptivity to 13 Although none of the group 13 elements is considered essential to life, their trivalent ions are of great biological interest. Gallium is present in human tissues at a level of only 10 À4 -10 À3 ppm. 78 It has two naturally occurring isotopes and 13 radioactive nuclides. 67 Ga (g, t 1/2 ¼ 3.25 days) and 68 Ga (b þ , t 1/2 ¼ 68 min) are two radioisotopes with appropriate energies and half-lives for g-scintigraphy and positron emission tomography (PET), respectively. Together with imaging applications, the Auger electrons emitted by 67 Ga possess potent cytotoxicity pointing toward potential therapeutic applications of the radionuclide, whereas the positrons emitted by 68 Ga may also have therapeutic applications in the prevention of restenosis by intracoronary radiation therapy. 79 Although most reports on gallium pharmaceutical chemistry relate to applications of its radioisotopes, the tumorseeking and antineoplastic properties of nonradioactive Ga 3þ salts were already recognized in the 1970s when, for example, safety and activity of intravenous gallium(III) nitrate were extensively studied in clinical trials. 80 The development of gallium-based anticancer agents has been pursued as a strategy to circumvent the limitations faced with simple gallium salts. In particular, efforts to improve bioavailability via the oral route have recently led to the selection of the complexes tris(8-quinolinolato)gallium(III) (KP46) and tris(3-hydroxy-2-methyl-4H-pyran-4-onato)gallium(III) (gallium maltolate) for clinical studies. 81 Despite the nonideal imaging characteristics of its gamma emissions, 111 In (g, t 1/2 ¼ 2.82 days) is also a popular radiolabel for targeting biomolecules and is widely employed in nuclear medicine for g-imaging, probably because of the simplicity of its bioconjugate chemistry. Its radioisotopes are administered to the patients in the form of stable chelates. 79 69/71 Ga and 113/115 In are quadrupolar NMR-active isotopes. They are characterized by high sensitivity to detection by NMR and large chemical shift ranges, two factors that make their study relatively easy. 71 Ga has higher receptivity and narrower linewidths than 69 Ga, which makes it usually the more favorable isotope for direct NMR observations despite the lower natural abundance. 113 In and 115 In have large quadrupole moments so their linewidths are very sensitive to the environmental symmetry around the indium nuclei. The low receptivity of 113 In accounts for the lack of NMR studies based on this nuclide. Gallium-and indium-based radiopharmaceuticals are generally chelated with suitable ligands, such as triazamacrocyclic ligands with different types of pendant arms, that form kinetically and thermodynamically stable complexes in vivo. The thermodynamic stability of gallium and indium complexes with potential applications in imaging and radioimmunotherapy has been widely investigated in vitro and in vivo by NMR (some examples are summarized in Table 4 ), and recent advances have been focusing on the development of new generation chelators, including the tripodal 3-hydroxy-4-pyridinone and the quinazoline-derivative DOTA-like ligands. 82 Given the potential imaging applications of Ga(III), recent studies have focused on the exploitation of 71 Ga NMR spectroscopy to gain insights into the structure, internal dynamics and stability in aqueous solution of the corresponding "cold" (i.e., nonradioactive) gallium derivatives. These include, for example, chelates of mixed phosphonates-carboxylate triazamacrocyclic ligands relevant to nuclear medicine 83 and some heterobimetallic Ga(III)-Ru(II) complexes containing histidyl-alanyl-valinyl (HAV) sequences for tumor targeting of potential theranostic agents (combining the anticancer activity of the Ru(II) unit with the imaging properties of 67 Ga(III) labeling). 83b The redox chemistry of thallium is considerably different from the other elements of the group. In fact, under physiological conditions, the most stable oxidation state is þ1, although Tl 3þ may also exist. 69 Thallium salts are poisonous due to the capability of the thallous ion to mimic alkali metal ions, especially K þ . 84 Interestingly, both 203 Tl and 205 Tl nuclides are NMR-active and nonquadrupolar. They have high receptivity, 203 Tl being only slightly less receptive than 31 P, whereas 205 Tl is the third most receptive I ¼ 1/2 nuclide. Because of its similarity to the alkali metal ions, Tl þ has potential as probe for Na þ and K þ in biological systems. 205 Tl NMR can be used to investigate directly the specific binding sites of transferrins. Due to its high receptivity, 205 Tl NMR signals of protein-bound Tl 3þ ions can be observed even at mM concentrations. The first 205 Tl NMR study of human serum transferrin was reported over 40 years ago by Bertini and coworkers. 85 They showed that 205 Tl NMR is a suitable probe to monitor the occupancy of the two available transferrin binding sites, thus characterizing both the dithallium-and the monothallium-transferrin derivatives with carbonate as synergistic anion. The high affinity of the protein for trivalent metal ions was thought to be responsible for the stabilization of the þ3 oxidation state of the metal. Two distinct 205 Tl NMR signals (at þ2075 and þ2055 ppm downfield from aqueous Tl þ ) of similar shape were found for the Tl(III) 2 -transferrin derivative at physiological pH. The two signals are relatively broad (Dv 1/2 $ 100 Hz) and show a different pH dependence (the signal at þ2055 ppm proved more resistant to acidification). At physiological pH, the Tl 3þ ion was shown to bind sequentially to the two sites; the signal at þ2055 ppm appeared first and was assigned to Tl 3þ bound to the acid-resistant C-terminal site. The signal at þ2075 ppm was assigned to Tl 3þ bound to the N-terminal site. By using 13 C NMR spectroscopy to study the 13 CO 3 -205 Tl-transferrin derivative, it was shown that the 13 C nucleus of the synergistic anion is strongly magnetically coupled to the 205 Tl nucleus so that its 13 C signal is split into a doublet. Analogously, in the 13 C NMR spectrum of ( 13 CO 3 -205 Tl) 2 transferrin, two superimposed doublets are recorded, the extent of the coupling constants (290 and 265 Hz) being typical of a 2 J( 205 Tl-13 C) coupling. This result provided evidence of carbonate coordination to the metal. 86 Similar experiments were carried out by Aramini and coworkers to investigate the binding of 205 Tl to chicken ovotransferrin in the presence of carbonate as the synergistic anion. 87 Two 205 Tl NMR signals due to the bound metal ion in the two high-affinity iron-binding sites of the protein were detected and, from titration studies, it was demonstrated that Tl 3þ shows no site preference in ovotransferrin. Again, when 13 C-labeled carbonate was used, two closely spaced doublets in the carbonyl region of the 13 C NMR spectrum of ovotransferrin were recorded due to the coupling between the bound metal ion and carbonate ( 2 J( 205 Tl-13 C)) ranging from 270 to 290 Hz).

The interaction of monovalent thallium with yeast pyruvate kinase was investigated by 205 Tl NMR. 88 Pyruvate kinase from almost all sources requires mono-and divalent metal ions. Potassium is the physiologically relevant monovalent cation, but several other þ1 cations, including Tl þ , can activate this enzyme. Compared to KCl, TlNO 3 was shown to activate pyruvate kinase to 80%-90% activity, in combination with Mn(NO 3 ) 2 as the activating divalent cation. At higher Tl þ concentrations, enzyme inhibition occurs, and the extent of such inhibition is also dependent on the nature and concentration of the divalent cation. In this regard, the effect of Mn 2þ on the relaxation properties of 205 Tl þ was determined by 205 Tl NMR spectroscopy. The distance between Tl þ and Mn 2þ at the active site of yeast pyruvate kinase was calculated from the paramagnetic contribution of Mn 2þ to the longitudinal relaxation rates of the bound Tl þ . Similar measurements were performed to monitor structural alterations introduced at the active site of yeast pyruvate kinase by mutation of Thr298 89 and showed that the mutation influences the interaction between the mono-and the divalent cations, both interacting with the phosphoryl group of the substrate at the active site of the enzyme.

p-Block: Group 14 (Si, Ge, Sn, and Pb)

Silicon and germanium

Relative receptivity to 13 Silicon is a highly abundant element in minerals and soils, but its role in living systems, if any, is poorly understood and it is currently debatable whether it is essential for human life. Silicon is not particularly toxic but finely divided silicates or silica can cause major damage to the lungs. 2 29 Si is the only NMR-active isotope of silicon, and 29 Si NMR can be employed to study the biological and medicinal chemistry of silicon, although low natural abundance and low receptivity have hindered the development of this technique so far. Accordingly, to the best of our knowledge, only a very few studies were recently reported on the use of 29 Si NMR in solution to characterize organosilicon derivatives of biological interest, that is, showing antifungal, antibacterial or cytotoxic activity. 90 On the other hand, solid-state 29 Si NMR studies applied to biocompatible materials have been reported, including the development of siliconcontaining monocomposites for the controlled release of pharmaceuticals 91 and of bioactive silicon-based hybrids for bone regeneration. 91d,92 Additionally, 29 Si MRI has potential for imaging silicone prostheses in humans. 93 29 Si relaxation times of silicone gels average T 1 ¼ 21.2 AE 1.5 s and T 2 ¼ 207 AE 40 ms, with no significant difference between virgin and explanted gels. Three volunteers with silicone gel-filled breast implants and one subject with an intraocular silicone oil injection were thus examined with a total acquisition time of 10-15 min per image. In all 29 Si images, the shape of the silicone object is well depicted and, although conventional proton images are superior in resolution and signal-to-noise ratio, 29 Si imaging has the advantage of optimal specificity, since only the silicone itself is visible. Germanium has no biological role but it is believed to stimulate the metabolism. Nevertheless, organogermanium compounds may exert some biological activity. 94 For example, spirogermanium, a germanium-containing azaspirane derivative, is reported to have in vitro and in vivo cytotoxicity toward a number of preclinical tumor models and to exhibit antiarthritic and immunoregulatory activities. 95 It underwent phase I and II clinical trials but turned out to be poorly active for further clinical use.

The natural abundance of 73 Ge is slightly higher than 29 Si, but recording of 73 Ge resonances is very difficult because of the low value of g, along with its nuclear spin of 9/2 and large quadrupole moment. EFGs around 73 Ge lead to excessive broadening of the signals. Symmetrical germanium complexes give sharp signals in the 73 Ge NMR, whereas the signal broadens as the symmetry lowers. For instance, the halfwidth of the 73 Ge peak for tetramethylgermanium, a compound with high geometric symmetry, is only 1.4 Hz, whereas the corresponding values of germacyclohexane, 1-methylgermacyclohexane, and 1,1-dimethylgermacyclohexane are 15.4, 22.3, and 15.6 Hz, respectively. When either halogen or oxygen atoms are asymmetrically substituted, as in 1-bromo-1-methylgermacyclohexane, excessive broadening takes place to such an extent that observation of signals is impossible. 96 To the best of our knowledge, no 73 Ge NMR studies on biologically relevant systems have been reported to date.

Relative receptivity to 13 Tin may be an essential element for human beings. The chemistry of organotin compounds has attracted much attention during the last 60 years, owing to potential biological and industrial applications. Tin is regarded as the third most important pollutant in the ecosystem, which has raised the concern that it may enter the human food chain and be accumulated in the environment and in other biological systems. 97 Organotin(IV) derivatives have interest as potential metallopharmaceuticals since some exhibit in vitro antitumor activity against a number of human tumor cell lines and antimicrobial, anti-inflammatory, cardiovascular, trypanocidal, antiherpes, and antituberculosis agents. 79c, 98 The merits of tin NMR in assessing structural characteristics of organotin compounds (e.g., substitution pattern on tin, coordination number, influence of solvent, and speciation under physiologically relevant conditions) were recognized in the early 1970s, owing to the reasonably favorable magnetic properties of tin nuclei. Tin has ten natural isotopes, of which three have I ¼ 1/2. Among them, 115 Sn is the least NMR favorable isotope, because of its extremely low natural abundance. As a result, it has been only rarely studied, for example, to overcome difficulties in unraveling strongly coupled homonuclear n J( 119 Sn-119 Sn) scalar coupling satellites in di-tin compounds. 99 The "twins" 117 Sn and 119 Sn nuclei have rather similar properties, but 119 Sn NMR spectroscopy is, by far, the most widely used in tin chemistry because of both its slightly higher magnetic moment and natural abundance. Thus, not surprisingly, the use of NMR in tin chemistry has been widely reviewed. 100 In particular, the use of 117/119 Sn NMR spectroscopy to investigate the interaction between organotin complexes and biomolecules such as amino acids, peptides, carbohydrates, and nucleic acids has been extensively reviewed, including equilibrium, structural, and biological studies. 101 Nowadays, 119 Sn NMR is mainly used as a routine analytical tool to characterize tin complexes, in particular Schiff bases derivatives, as potential antimicrobial and anticancer agents. 102 Lead

Relative receptivity to 13 Lead is probably nonessential for man but its toxicology is of great interest since it is a ubiquitous environmental contaminant. Although the use of lead (as PbEt 4 ) in gasoline and paint has now been banned in most developed countries, lead is still among the ten most common contaminants. 103 On the other hand, lead has been investigated for its possible applications in medicine. The potentially therapeutic radioisotope 212 Pb has a number of interesting features including a-emission by short-lived daughter radionuclides, and its use is encouraged by the development of state-of-the-art generator systems 104 and by the observation that when 212 Pb-conjugated antibodies are internalized, radioactivity is retained inside the cells. 105 207 Pb is the only NMR-active (I ¼ 1/2) lead isotope and has excellent receptivity, relatively high natural abundance, and large chemical shift range. Although 207 Pb NMR spectroscopy has been used extensively to characterize Pb(IV)-alkyl derivatives, relatively few spectroscopic studies have been carried out on the aqueous coordination chemistry of soluble Pb(II) complexes. 106 The toxicological implications of lead derivatives have spurred a number of studies aimed at investigating their interaction with various proteins. 107 For example, Pb 2þ can bind very tightly to, and even displace Ca 2þ from, calmodulin, calbindin, and troponin C. Moreover, it can replace calcium in the activation of several enzymes, including protein kinase C, phosphodiesterase, and myosin light chain kinase, the latter two in a calmodulin-dependent way. Therefore, model systems are needed for lead bound to Ca-binding sites in proteins, as these interactions are thought to account for lead's toxicity. For example, the high-affinity Ca 2þbinding sites of carp (pI 4.25) and pike (pI 5.0) parvalbumins, as well as those of mammalian calmodulin and its C-terminal tryptic half-molecule (TR 2 C), have been investigated by 207 Pb NMR spectroscopy. 108 For the parvalbumins, two 207 Pb signals are observed, with chemical shifts ranging from þ750 to þ1260 ppm downfield of aqueous Pb(NO 3 ) 2 , corresponding to 207 Pb 2þ bound to the two high-affinity helix-loop-helix Ca 2þ -binding sites. Four 207 Pb signals, recorded in the same chemical shift window, can be discerned for calmodulin (Fig. 13 ). Experiments on TR 2 C allowed the assignment of each signal due to 207 Pb 2þ occupying a helix-loop-helix site in either the N-or the C-lobe of the intact protein. 207 Pb and 1 H NMR titration studies on calmodulin have provided evidence that Pb 2þ binding to all four sites occurs simultaneously, in contrast to the behavior of the same protein in the presence of Ca 2þ . The large chemical shift dispersion observed for the 207 Pb signals of the three investigated proteins illustrates the remarkable sensitivity of this parameter to subtle differences in the chemical environment of the protein-bound 207 Pb nucleus.

Pb 2þ can replace calcium and sometimes zinc in both the "hard" oxygen-and nitrogen-rich protein active sites and in the "soft" ones, such as all-sulfur-containing zinc ion coordination sites. Among the sulfur-rich targets for Pb 2þ are GSH and metallothioneins, which cause perturbations of essential metal ion homeostasis and are likely to be involved in human lead poisoning. 207 Pb NMR signals for the thiol-rich binding sites are expected to be shifted downfield to oxygen-and nitrogen-rich ones, thus allowing to distinguish {PbS 3 } versus {PbS 3 O} coordination environments. 109 In this regard, Pecoraro and coworkers presented the first report on 207 Pb NMR spectroscopy used to directly probe the binding of Pb 2þ to a Cys 3 -motif in thiolate-rich metallopeptide by utilizing three-strand coiled-coil peptides, showing the preferred {PbS 3 } homoleptic trigonal pyramidal geometry for Pb 2þ . 110 Such evidence has been recently confirmed by performing 207 Pb NMR experiments aimed at revealing the coordination behavior of Pb 2þ toward GSH and zinc-finger proteins. 111 Claudio and coworkers 112 have reported on the first 2D [ 1 H, 207 Pb] heteronuclear multiple quantum coherence (HMQC) spectrum (Fig. 14) and demonstrated that this experiment can provide useful information about the lead coordination environment in aqueous Pb(II) complexes. This technique allows 207 Pb-1 H couplings through up to three bonds to be identified and could prove useful for the investigation of Pb 2þ ions in more complex systems, such as biological and environmental samples. Despite arsenic reputation as a highly toxic substance, this element may actually be necessary for good health. Studies on animals such as chickens, rats, goats, and pigs showed that it is necessary for proper growth, development, and reproduction. In these reports, the main symptoms of arsenic deficiency were retarded growth and development. Arsenic compounds occur naturally in marine organisms, such as arsenobetaines in flat fish, and are used as growth promoters for farm animals. It is suspected, but not yet proven, that arsenic may be an essential element necessary for the functioning of the nervous system and for growth. Arsenic trioxide (As 2 O 3 , Trisenox) has been approved by the Food and Drug Administration to treat a rare and deadly form of leukemia called acute promyelocytic leukemia. 113 Antimony appears to have no known role in the body. Sb(III) compounds cause damage to the liver and are used in some cases to induce vomiting and sweating. Some Sb(V) derivatives are used to treat the parasitic disease leishmaniasis. 114 Bismuth has no known natural biological role and is relatively nontoxic. However, it has been used for some time as a medicine (e.g., as tripotassium dicitratobismuthate) for treatment of gastrointestinal disorders. Nowadays, it is used for the treatment of stomach ulcers since it is effective against the bacterium Helicobacter pylori 115a and can be added in antihemorrhoid creams, such as Anusol and Hemocane, as bismuth oxide and in Anusol ointment as bismuth subgallate. 115b Finally, it was recently reported that bismuth complexes may inhibit the severe acute respiratory syndrome coronavirus. 116 75 As, 121/123 Sb, and 209 Bi are the corresponding NMR-active isotopes and are characterized by a high sensitivity and large quadrupole moments, which make their linewidths very sensitive to the environmental symmetry of the nuclei. Although both their natural abundances and receptivities are favorable, recording NMR spectra of these nuclei is known to be very difficult due to their very broad signals, especially for 209 Bi. As far as we are aware of, no NMR studies concerning the involvement of As, Sb, or Bi in biologically relevant systems have been reported to date.

Relative receptivity to 13 Tellurium is a noble metalloid that can act as either a Lewis acid or a Lewis base. It has no known biological role and most tellurium compounds are highly toxic. Organotellurium derivatives are potent immunomodulators (both in vitro and in vivo) with a variety of potential therapeutic applications. For example, ammonium trichloro(dioxoethylene-O,O 0 )tellurate (AS101) is known to be effective in the treatment of AIDS and cancer. It confers protection against side effects of both radiotherapy and chemotherapy by protecting the bone marrow and preventing from alopecia. It also exhibits synergistic effects with a variety of other drugs and is reported to be effective against systemic lupus erythematosus and psoriasis. 117 Tellurium has two naturally occurring NMR-active isotopes, 123 Te and 125 Te, both having I ¼ 1/2. 125 Te has higher receptivity and natural abundance, thus making it the more favorable isotope for direct NMR observations. The use of 125 Te NMR spectroscopy to probe the ligand chemistry of tellurium has been widely reviewed. 118 Tellurium coordination chemistry is dominated by sulfur ligands. Owing to the strong affinity of Te(IV) for thiols, tellurium has been investigated as a selective cysteine protease inhibitor. In this regard, AS101 was shown to inhibit cysteine proteases in a timeand concentration-dependent way but no inhibitory activity of serine, metalloprotease, or aspartic proteases was observed. Remarkably, the capability of Te(IV) to interact with cysteine thiol groups is believed to correlate with its inhibitory activity. 125 Te NMR has been used to follow the interaction between cysteine and Te(IV) or Te(VI) compounds. 119 Although the resonances are highly sensitive to the environment (e.g., solvent, concentration, and temperature), they can be divided into well-defined frequency ranges corresponding to the tellurium oxidation state. Data reported in this study demonstrated a clear distinction in reactivity of Te(IV) and Te(VI) complexes toward thiol nucleophiles. Whereas the latter do not interact with the thiols, all the investigated Te(IV) derivatives exhibited significant shifts upon interaction with cysteine. For example, AS101 experienced a downfield shift from þ1700 to þ1807 ppm, respectively, attributable to the formation of [Te(Cys) 4 ].

Isotope A (%) I g (Â10 7 rad T À1 s À1 ) Q (fm 2 ) Relative receptivity to 13 Scandium has no known biological role. It is relatively nontoxic, although there have been suggestions that some of its compounds might be carcinogenic and can cause lung embolisms, especially upon long-term exposure. 21 Yttrium is not normally found in human tissues and plays no known biological role. Targeted radionuclide therapy of cancer using the high-energy b-emitting isotope 90 Y is now in advanced clinical trials (using the conjugates of somatostatin receptor binding peptides), with promising results, at least at a palliative level. 120 Both 45 Sc and 89 Y are NMR-active nuclides with 100% natural abundance. 45 Sc has high resonance frequency, high receptivity, and a relatively small quadrupole moment but, despite these favorable features, 45 Sc NMR spectroscopy has been scarcely exploited for biological studies of scandium derivatives to date. As already shown for 27 Al (see text earlier), 45 Sc NMR can be used to probe metal-binding sites in large proteins. The solution chemistry of scandium is based entirely on Sc 3þ , which forms almost exclusively six-coordinate complexes. The ionic radius of Sc 3þ is only slightly larger than Fe 3þ (0.75 vs. 0.65 Å), thus making it a suitable probe for the Fe(III)-binding sites of transferrins in which the metal ion is coordinated by six donor atoms, four from the side chains of four protein residues and two from the synergistic anion (i.e., carbonate), in a distorted octahedral geometry. In this regard, the binding of Sc 3þ to chicken ovotransferrin has been investigated by 45 Sc and 13 C NMR spectroscopy. 121 In the presence of carbonate, both 45 Sc and 13 C NMR spectra show two signals assigned to the bound 45 Sc 3þ and 13 CO 3 2À groups in the N-and C-binding sites of the protein (Fig. 15 ). Several properties of the transferrin-bound 45 Sc signals, such as their dependence on pulse length, magnetic field, protein size, and temperature, are consistent with the detection of only the central transition of the quadrupolar nucleus in conditions far away from the extreme narrowing ones. Additionally, from the 45 Sc chemical shift and linewidth data recorded at four magnetic fields, the quadrupolar coupling constants and the rotational correlation time have been calculated for the bound metal ion in each site of the protein. Remarkably, this work represented the first, and so far the only, 45 Sc NMR study of a metalloprotein. More recent relevant reports have been dealing with the use of 45 Sc NMR spectroscopy for the characterization of Sc(III) derivatives as potential PET agents and radiopharmaceuticals. 122 89 Y has I ¼ 1/2 and, as such, it should be attractive for NMR study. Nevertheless, it is not routinely used in the characterization of yttrium complexes owing to the low receptivity and resonance frequency, and long relaxation times, which lead to problems with detection and to the necessity for lengthy experiments. The use of 89 Y NMR spectroscopy for the characterization of organometallic and coordination compounds containing yttrium has been previously reported. 123 On the contrary, fewer biologically related reports exist on the exploitation of this technique. For example, it was shown that yttrium complexes can be successfully hyperpolarized, 124,125 so as to overcome the intrinsic detectability issues of 89 Y itself. As such, owing to the long longitudinal relaxation time, 89 Y derivatives seem promising as MRI agents. 125 Moreover, yttrium and gadolinium have similar ionic radii and coordination chemistry, so the former can be used as model for gadolinium-based contrast agents (see text later). In this regard, a recent paper reports on the use of hyperpolarized 89 Y NMR to monitor slow complexation processes. 126 Titanium Isotope A (%) I g (Â10 7 rad T À1 s À1 ) Q (fm 2 ) Relative receptivity to 13 Titanium is believed to be a nonessential element, although some observations indicate that Ti 4þ may have a variety of biological roles. In medicine, titanium is used to make hip and knee replacements, pacemakers, bone plates, and screws and cranial plates for skull fractures, and it has been also used to attach false teeth. Two Ti(IV) derivatives (budotitane and titanocene dichloride) have been investigated as potential metallodrugs. 79c,127 In particular, titanocene dichloride, [TiCl 2 Cp 2 ] (Cp ¼ cyclopentadienyl), entered phase I clinical trials in 1993 and, although nephrotoxicity was identified as a major issue, the absence of any effect on the proliferative activity of the bone marrow suggested that titanocene dichloride might have significant potential for possible use in combination therapy. Subsequently, two phase II clinical trials involving patients with advanced renal cell carcinoma and breast metastatic carcinoma have been reported. However, outcomes were not sufficiently promising compared to other treatment regimes to warrant further studies, and titanocene dichloride was discontinued from further clinical trials. 128 Titanium has two naturally occurring quadrupolar NMR-active isotopes, 47 Ti and 49 Ti, the latter having slightly higher receptivity and narrower linewidths, thus usually making it the more favorable isotope for direct NMR observations in spite of the lower natural abundance. The 47/49 Ti chemical shifts of titanocene dichloride were determined over 30 years ago, 129 but, to the best of our knowledge, no further biological investigations have been performed by means of Ti NMR spectroscopy. Vanadium exists in a variety of oxidation states, from À3 to þ5. In vivo, owing to the constraints of standard physiological conditions (pH 3-7, aerobic aqueous solution, room temperature), oxidation states þ4 and þ5 prevail, with thermodynamically plausible species including vanadate, a mixture of [HVO 4 ] 2À and [H 2 VO 4 ] À , and vanadyl, [VO] 2þ . Vanadium is normally present at very low concentrations (<10 À8 M) in virtually all cells in plants and animals. In the oxidation states þ3, þ4, and þ5, it readily forms VÀ O bonds and binds N-and S-donors as well, forming robust coordination compounds. From a coordination chemistry point of view, vanadium is remarkably flexible. For example, V(V) has nonrigid stereochemical requirements and can form coordination complexes in geometries ranging from tetrahedral and octahedral to trigonal and pentagonal bipyramidal. On the contrary, V(IV) is much less flexible, with square pyramidal or, if a sixth position is occupied, distorted octahedral geometries. Vanadium derivatives readily undergo redox reactions under physiological conditions and form both cationic and anionic complexes. The interplay between V(V) and V(IV) represents the key redox process of vanadium complexes in vivo, and the two oxidation states coexist in equilibrium both intra-and extracellularly. 130 The biological relevance of vanadium was confirmed by the discovery of two naturally occurring vanadium proteins, a V-bromoperoxidase and a V-nitrogenase. 131 In addition, vanadium has received much attention in recent years due to the discovery of many therapeutic properties. Several vanadium salts and their complexes have shown insulin-mimetic pharmacological properties, including stimulation of glucose transport into the cells and its oxidation via glycolysis, glycogen synthesis, and lipogenesis, and inhibition of gluconeogenesis and glycogenolysis. The antidiabetic properties of vanadium compounds, demonstrated both in vitro and in vivo, have attracted much interest as potential therapeutic agents for diabetes mellitus. They can be administered orally and promote glucose uptake in animal models of type 1 and type 2 diabetes. Phase I clinical trial of bis(ethylmaltolato)oxovanadium(IV) was completed in 2000, and the results of the phase II clinical trial were first published in 2009. 79b,132 Moreover, the anticancer potential of, for example, vanadocene derivatives and a vanadium(IV)-aspirin complex is currently being investigated. 133 Coordination compounds of vanadium, which may have pharmacological relevance, include not only vanadate [VO x L y ] and vanadyl [VOL z ] derivatives but also the peroxovanadates [VO(O 2 )(H 2 O)(L-L 0 )] nÀ (n ¼ 0, 1) and [VO (O 2 ) 2 (L-L 0 )] nÀ (n ¼ 1, 2, 3). 134 51 V NMR is a powerful and selective probe of vanadium in biological systems. Unlike many other biologically relevant isotopes, the NMR receptivity of the quadrupolar 51 V nucleus is rather high due to a large magnetic moment, a small quadrupole moment, a relatively large g, a high natural abundance, and a rapid quadrupolar relaxation in solution. 51 V chemical shifts are quite sensitive to changes in the nature of the ligands, thereby providing an excellent diagnostic tool for detailed investigations of vanadium speciation and binding to macromolecules. 135 In particular, the speciation of vanadate and vanadium(V)-based coordination compounds under physiologically relevant conditions and in presence of selected biomolecules has been robed by Pettersson and coworkers, and results were reviewed recently. 136 Inorganic vanadium salts were the first compounds for which insulin-enhancing behavior was detected in vivo, but several vanadium complexes with organic ligands were found to be more potent and less toxic than vanadium salts, likely owing to the increased selectivity. In this regard, speciation studies of vanadate-ligand and peroxovanadate-ligand systems in aqueous media and under physiological conditions have been carried out by means of 51 V NMR spectroscopy in order to provide insights into their insulin-mimetic behavior. 137 51 V NMR spectroscopy has been successfully employed to probe the solution chemistry and stability under physiologically relevant conditions of several vanadium-based chemotherapeutic agents, such as the leading compound bis(maltolato)oxovanadium(IV), 138a its kojato analog, 138b and a prodrug of peroxovanadate insulin-mimetic hexakis-(benzylammonium)decavanadate(V) dihydrate. 138c An example of the suitability of this technique is illustrated in Fig. 16 , in which the variable pH 51 V NMR spectra of a solution of [VO (maltolate) 2 ] (Fig. 16A ) and of genuine [VO 2 (maltolate) 2 ] À (Fig. 16B) are reported. As shown in Fig. 16A , in basic solution, only one peak (V 1 ) at À537 ppm was observed and assigned to mixed protonation states of vanadate. As the pH was lowered, peaks V 2 ([VO 2 (maltolate) 2 ] À ) and V 3 ([VO 2 (maltolate)(OH)(H 2 O)] À ) appeared at À496 and À510 ppm, respectively. As the acidity of the solution increased, peak V 1 shifted downfield and disappeared below pH 5.5. By comparison with the variable pH 51 V NMR spectra of NH 4 [VO 2 (maltolate) 2 ] (Fig. 16B) , it was proved that some V(V) complexes form in a solution of [V IV O (maltolato) 2 ], even under relatively acidic conditions.

Other applications of the favorable spectroscopic properties of 51 V include: the use of 51 V NMR to probe the interaction of vanadium derivatives with proteins, such as transferrins 139 and actin, 140 the study of the uptake, intracellular reduction, and binding of the aerobic oxidation products of oxovanadium(IV) compounds in human erythrocytes, 141 and the interactions of vanadium(V)-citrate complexes with the sarcoplasmic reticulum calcium pump 142 have been reported.

Vanadium derivatives are currently under intense investigation for their potential medicinal applications other than insulin mimetics, including their anticancer, 143a,143b antimoebic, 143c antiparasitic 143d and biological catalytic (peroxidase mimicking) 143e activities. In most cases, 51 V NMR proved useful not only to characterize the novel vanadium-based derivatives and study their stability in aqueous systems but also to probe their binding to DNA. 143a In recent years, VO 2 films have been attracting growing attention as thermochromic materials for applications in intelligent windows. 144a Although most studies focus on the optimization of preparation methods and enhancement of energy-saving efficiency, metal oxide nanomaterials might be toxic to human health and the environment. 144b In this regard, Jin and coworkers have successfully exploited 51 V NMR to evaluate the potential toxicity of VO 2 films. 144c Spectra recorded on extracts of VO 2 films in PBS solution showed the formation over time of a 51 V peak at À560 ppm (corresponding to mononuclear tetrahedral vanadate) and, subsequently, the appearance of a signal at À580 ppm (associated with the corresponding divanadate species). The study demonstrated the release of vanadium under physiological-like conditions as V(V) and this was linked to the potential toxic mechanism of VO 2 -based nanomaterials due to the vanadate disturbing ATP synthesis as a phosphate analogue through a vanadatephosphate antagonism.

Relative receptivity to 13 Chromium may be an essential trace element for mammals and be involved in the maintenance of proper carbohydrate and lipid metabolism, and its mild dietary deficiency is sometimes associated with possible heart disease. Recent studies revealed that the chromium-binding oligopeptide chromodulin may play a role in the autoamplification of insulin signaling. Attempts to develop chromium-containing nutritional supplements and therapeutics have been made. 145 Nevertheless, in anything other than trace amounts, chromium compounds are regarded as highly toxic. The health effects of chromium are at least partially related to the oxidation state of the metal at the time of exposure. Cr(III) and Cr(VI) compounds are thought to be the most biologically significant. Cr(VI) is generally considered much more toxic than Cr(III), and prolonged exposure to Cr(VI) has been associated with increased incidence of lung cancer. 2 Molybdenum is an essential trace element for virtually all life forms. In humans, upon binding to a unique pterin, molybdenum functions as a cofactor that, in different variants, is the active compound at the catalytic site of two families of molybdenum containing enzymes, that is, sulfite oxidase and xanthine oxidase. 146 As a metallopharmaceutical, molybdocene dichloride, an analog of titanocene dichloride (see preceding text), and its derivatives are still under investigation as anticancer agents, 147, 148 and tetrathiomolybdate has been developed as an effective anticopper therapy for the initial treatment of Wilson's disease, an autosomal recessive disorder that leads to abnormal copper accumulation. 128 Opinions are mixed about the need for tungsten in plant and animal life processes. It is not known whether humans need tungsten for good health although it was proved necessary for certain bacteria. Additionally, polyoxotungstate clusters have been evaluated as insulin mimetics in animal models. 148 There are several practical problems involved in the routine chromium NMR spectroscopy. 53 Cr, the only NMR-active isotope, has a small magnetic moment, resulting in low Larmor frequency and sensitivity, and a relatively large quadrupole moment, which leads to short nuclear relaxation times and large experimental linewidths. The low natural isotope abundance combined with the small overall receptivity results in extremely low sensitivity to detection by NMR spectroscopy. To date, only a few 53 Cr solution NMR measurements have been reported, and none is of any biological interest. 149 Molybdenum has two naturally occurring NMR-active isotopes, 95 Mo and 97 Mo, both quadrupolar and characterized by low sensitivity to detection by NMR and low natural abundance. 183 W is the only nonquadrupolar NMR-active nucleus in group 6 family, but it is also characterized by low natural abundance and extremely low receptivity. Thus, despite the potentially interesting biological properties of molybdenum and tungsten, only a few biologically relevant data on the application of 95/97 Mo and 183 W NMR spectroscopy have been reported. For example, 95 Mo NMR was used to detect the binding of tetraoxo-and tetrathiomolybdate to BSA by exploiting 95 Mo-enriched species. 150 More recently, the cleavage of the phosphoester in a DNA model promoted by a polyoxomolybdate was probed by 95 Mo NMR spectroscopy. 151 As far as 183 W NMR is concerned, such technique was exploited to characterize some heteropolytungstates tested as anti-HIV 152 and anticancer 153 agents' and some organoantimony(III)-containing heteropolytungstates with potential applications as antimicrobial agents. 154

Manganese

Relative receptivity to 13 Manganese has many roles in biological systems. It can exist in 11 oxidation states (from þ7 to À3), more than any other element, but its aqueous chemistry is dominated by Mn(II) complexes. Both Mn(II) and Mn(III) ions are found in enzymes. The ionic radius of 0.9 Å places Mn 2þ in between Mg 2þ and Ca 2þ , so that it is not surprising that there is overlap in function with these two ions in providing structural charge stabilization of enzymes and, in some cases, substrates, such as Mn-ATP. In this regard, manganese has been useful in substituting Mg 2þ and Ca 2þ as a probe of divalent metal ion binding and function in enzymes 155 and ribozymes. 47 Manganese also acts as a superacid catalyst in several hydrolytic enzyme-catalyzed reactions. 156 The classes of enzymes that have manganese cofactors are very broad and include oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, lectins, and integrins. The Mn superoxide dismutase enzyme is probably one of the most ancient, as nearly all aerobic organisms use it to deal with the toxic effects of superoxide. 157 In addition to its important biological role, manganese may have several therapeutic uses, including the treatment of arthritis, cancer, cardiovascular diseases, and HIV. 158 Consequently, to the biological relevance of manganese, NMR spectroscopy might be a potential analytic tool to investigate its behavior. 55 Mn is quadrupolar and has a natural abundance of 100%, a Larmor frequency close to 13 C, and a chemical shift range of approximately 3500 ppm, but because of the large Q, 55 Mn NMR spectroscopy has found limited applications and mainly for Mn (I)-carbonyl derivatives, 159 owing to their potential use as CO-releasing molecules (CORMs). Moreover, both Mn(II) and Mn(III) ions are paramagnetic, thus preventing the direct detection of these nuclei in solution. On the other hand, their paramagnetism can be used to probe indirectly the active sites of metallobiomolecules via paramagnetic shifts and line broadening analysis. 160

Relative receptivity to 13 C Reference sample 99 Tc a "100" b 9/2 6.046 Technetium does not occur naturally on Earth and it was the first element to be produced artificially. 99 Tc is a b-emitting radionuclide with a long half-life (2.13 Â 10 5 years) and is generated as a by-product of nuclear power plants and atomic weapon tests. Interest in technetium chemistry arises from the application in nuclear medicine. Radiopharmaceuticals containing 99m Tc linked to a variety of carrier biomolecules are in clinical use. 79b Therefore, such extensive use of this radionuclide in medicine calls for more detailed structural information to design more effective and selective radiopharmaceuticals. 99 Tc chemical shifts and linewidths may provide insights into technetium oxidation states and into the composition and structure of its derivatives. The 99 Tc nuclide is extremely convenient for NMR spectroscopy due to its high receptivity and to the 100% abundance. Although 99 Tc has a significant quadrupole moment, the effect of line broadening in solution is attenuated by a large spin. In fact, its resonances are among the narrowest for quadrupolar nuclei. However, due to the presence of unpaired electrons in Tc(II), Tc(IV), and Tc(VI) compounds, the metal cannot be detected directly by 99 Iron is the fourth most abundant element in the Earth's crust, but only a trace element in biological systems, making up only 0.004% of the body's mass. Yet, it is an essential component or cofactor of numerous metabolic reactions. Approximately 70% of this iron in man is contained in hemoglobin, and the remaining 30% is stored in the bone marrow, spleen, liver, and muscles. Myoglobin and enzymes contain about 15% of the iron, and ferritin almost as much (14%), whereas only about 1% is in transit in serum. Iron distribution is heavily regulated in mammals and a major component of this regulation is the protein transferrin, which binds iron absorbed from the duodenum and carries it in the blood to cells. Biologically relevant iron may exist in low-and highspin configurations in both þ2 and þ3 oxidation states. The energy difference between the two spin configurations for given oxidation state can be very small, accounting for the facile interconversion and the subsequent important biochemical implications. 21 Despite the importance of iron in biological chemistry, only limited studies of Fe NMR have been reported. 57 Fe, the only isotope of iron suitable for NMR study, has favorable I ¼ 1/2 nuclear spin but also a very low sensitivity when investigated at natural abundance. Fe(III) and high-spin Fe(II) centers are paramagnetic, so the diamagnetic low-spin ferrous ion 57 Fe 2þ is the only NMRobservable iron isotope. However, use of 57 Fe-enriched material and high fields combined with the very large chemical shift range have yielded useful information especially about heme proteins. Ferrocenes and porphyrins have been used as model compounds to obtain chemical shift data and to provide insights into the relaxation mechanisms. 162 57 Fe NMR spectra are available for myoglobins and cytochrome c (Fig. 17) , 163 and, in general, 57 Fe NMR proved powerful to probe iron-ligand interactions in these proteins, particularly for porphyrin derivatives. 162a,164 

Isotope A (%) I g (Â10 7 rad T À1 s À1 ) Q (fm 2 ) Relative receptivity to 13 Both ruthenium and osmium have no known natural biological function. However, during the last decades, ruthenium and osmium complexes have aroused great interest for their potential use as anticancer agents. Two Ru(III) complexes, namely, trans-[RuCl 4 (Im)(DMSO)]ImH (NAMI-A) and trans-[RuCl 4 (Ind) 2 ]IndH (KP1019), are currently undergoing phases I and II clinical trials, respectively, thus boosting the development of other ruthenium-based metallopharmaceuticals. 165 In this regard, also the activity of Ru(II) complexes is currently being explored. In particular, since arenes are known to stabilize ruthenium in the þ2 oxidation state, the potential of Ru(II)-(Z 6 -arene) derivatives as anticancer agents is under investigation. 166 Additionally, owing to the strict similarity with Ru(II), the design of Os(II)-(Z 6 -arene) chemotherapeutics has been also taken into account. 167 Among the two quadrupolar ruthenium NMR-active isotopes, despite the slightly lower natural abundance, 99 Ru nuclide is favored for NMR spectroscopy due to the smaller quadrupole moment. Anyway, a major issue of 99 Ru NMR is the low resonance frequency, resulting in acoustic ringing that causes distorted baselines. Very little is known about the dependence of d( 99 Ru) on structure and temperature; thus, it is not surprising that few 99 Ru NMR spectroscopic studies have been reported. 168 In addition, due to the presence of unpaired electrons, Ru(III) compounds cannot be directly detected by 99 Ru NMR spectroscopy, so 99 Ru(II) is the only NMR-observable ruthenium isotope. To date, 99 Ru NMR spectroscopy has been successfully employed for Ru(II) complexes having highly symmetrical environments around the metal nucleus. 169 Like ruthenium, also for osmium only one of its two magnetically receptive nuclei is useful for NMR measurements, the other possessing a large quadrupole moment. Nevertheless, although having I ¼ 1/2, the 187 Os nuclide has very low natural abundance of only 1.96% and is the most insensitive nucleus in the periodic table, making its observation by conventional NMR techniques extremely difficult. In fact, for many years, the only known chemical shift was that of the reference compound OsO 4 . 170 However, the advent of polarization transfer techniques, which require a nonzero scalar n J(M-X) coupling, has improved the detection of lowg nonquadrupolar nuclei. 187 Os NMR data have been reported for some Os(II)-(Z 6 -arene) and sandwich-type complexes, using inverse 2D [ 31 Despite the low biological abundance, cobalt plays a unique role in the metabolism of several living organisms. Cobalt derivatives contain the metal ion in the oxidation states þ1, þ2, and þ3. A major acknowledged biological role of cobalt is its participation in the vitamin B 12 family of compounds, whose active forms are responsible for catalyzing a wide variety of processes related to nucleic acid, protein, and lipid syntheses and for maintaining the normal function of epithelial and nervous cells. Cobalamins also stand out as nature's most complex nonpolymeric structures and the first discovered biomolecule containing a metal-carbon bond. In addition to vitamin B 12 and its derivatives, several different types of cobalt-containing enzymes have been also identified. 172 The NMR detection of 59 Co nucleus should be, in principle, easy. It is 100% naturally abundant, it possesses a relatively high magnetogyric ratio and, owing to the magnetic mixing of its occupied and excited d orbitals, it may experience substantial paramagnetic deshieldings that can reveal even subtle changes in chemical environments. On the other hand, the I ¼ 7/2 is associated with a quadrupole moment that provides an efficient relaxation mechanism, thus leading to broadened resonances in solution. 173 59 Co NMR spectroscopy has been used to study naturally occurring cobalamins. Targets of these investigations included vitamin B 12 , B 12 coenzyme (adenosylcobalamin), methylcobalamin, and dicyanocobyrinic acid heptamethylester. 174 Illustrative 59 Co NMR spectra are shown in Fig. 18 . Solid-state 59 Co NMR spectroscopy was used in similar studies and, owing to a favorable combination of different factors, the resulting spectra were of much higher quality than their solution counterparts. 174 Solid-state 59 Co NMR spectroscopy has been successfully employed also to study a single crystal of vitamin B 12 and in the analysis of vitamin B 12 in its different polymorphic forms. 175 In contrast to cobalt, rhodium has no known biological function. Recently, rhodium complexes have been investigated for their possible applications in medicine as antitumor, antibacterial, and antiparasitic agents. 176 As a monoisotopic nonquadrupolar species with a wide chemical shift range, the 103 Rh nucleus is attractive for NMR studies. Unfortunately, low receptivity, negative magnetogyric ratio, and very long relaxation times (>50 s) are major drawbacks. A recent survey of 103 Rh NMR spectroscopy has been published, 177 but, to the best of our knowledge, no studies of biological relevance have been reported. 

Isotope A (%) I g (Â10 7 rad T À1 s À1 ) Q (fm 2 ) Relative receptivity to 13 Often found in the þ2 oxidation state, nickel in biological systems plays a role acknowledged only since 1975, when urease was shown to be a nickel enzyme. Since then, other nickel-containing enzymes have been discovered in bacteria and/or Archaea, including hydrogenase, methyl-S-coenzyme-M-reductase, carbon monoxide dehydrogenase, nickel superoxide dismutase, glyoxylase I, and a putative nickel cis-trans isomerase. 178 61 Ni is the only naturally occurring NMR-active isotope of nickel. It is a quadrupolar nucleus characterized by low sensitivity to detection by NMR and low natural abundance. The 61 Ni chemical shifts of several Ni(0) organometallic derivatives have been recently reported, 179 whereas octahedral and tetrahedral Ni(II) derivatives cannot be studied directly by NMR because they are paramagnetic. Palladium is a nonessential element for life. Pharmaceutical interest in Pd(II) compounds is driven by analogy to antitumor Pt(II) complexes (see text later) and antiviral, antifungal, and antimicrobial metallotherapeutics. 180 Palladium has one naturally occurring NMR-active isotope, 105 Pd. Although it has high natural abundance, this quadrupolar nucleus is characterized by a low sensitivity to detection by NMR, low resonance frequency, and extremely fast relaxation times (<10 À5 s). As far as we are aware of, no 105 Pd NMR studies in solution of biological relevance have been reported.

A (%) I g (Â10 7 rad T À1 s À1 ) Q (fm 2 ) Relative receptivity to 13 Platinum is a relatively inert metal and has no natural biological role. Nevertheless, platinum complexes are now among the most widely used drugs for the treatment of cancer. 181 NMR methods proved useful in the investigation of platinum drugs from the time that cisplatin was first introduced into the clinic over 40 years ago. 195 Pt is a reasonably sensitive nucleus for NMR detection having high natural abundance and relative receptivity. However, the detection limit in the millimolar range makes the observation of natural abundance 195 Pt signals in physiological fluids rather difficult, although Bachert and coworkers successfully used in vivo 195 Pt NMR (at 2.0 T) in rats to monitor local disposition kinetics of carboplatin in intact tissue following a subcutaneous injection. 182 An attractive feature of 195 Pt NMR for studies of platinum anticancer drugs is the very large chemical shift range, which allows ready differentiation between Pt(II) and Pt(IV), having chemical shifts at the high-field and low-field ends of the range, respectively. The resonances for square planar Pt(II) complexes span some 4000 ppm and ligand substitutions produce predictable chemical shift ranges. The 195 Pt chemical shifts of some platinum antitumor complexes and various adducts are listed in Table 5 with the 195 Pt chemical shift ranges illustrated pictorially in Fig. 19 .

Both 1D 195 Pt and 2D [ 1 H, 15 N] NMR techniques have been providing a major contribution in the understanding of the molecular mechanism of action of platinum-based anticancer drugs, including kinetic studies on the interaction with biomolecules by using 195 Pt isotopically enriched platinum complexes. The overall topic has been extensively reviewed elsewhere. 79, 183 Moreover, the use of state-of-the-art computational methods, allowed the prediction of 195 Pt NMR chemical shifts for a number of Pt(II) and Pt(IV) cytotoxic agents, and the subsequent use of 195 Pt NMR parameters to generate reliable quantitative structureactivity relationship (QSAR) models for platinum-based antitumor compounds. 184 Notwithstanding 195 Pt NMR is commonly used as a routine analytical tool to characterize novel platinum anticancer agents, recent papers also reported on its use to probe platinum binding to glutathione and plasma, 185a to DNA 185b,185c and the HIV NCp7 protein, 185d and even the release of cisplatin encapsulated into carbon nanotubes as drug delivery systems. 185e d-Block: Group 11 (Cu, Ag, and Au)

A (%) I g (Â10 7 rad T À1 s À1 ) Q (fm 2 ) Relative receptivity to 13 Copper is the third most abundant transition metal element in biological systems after iron and zinc. The bioinorganic chemistry of copper is dominated by þ1 and þ2 oxidation states. Almost all of the copper in the human body is bound to transport proteins (ceruloplasmin and copper-albumin), storage proteins (metallothioneins), or copper-containing enzymes, whereas unbound free copper is not found in large quantities. Copper is essential for the proper functioning of copper-dependent enzymes, including cytochrome c oxidase (energy production), superoxide dismutase (antioxidant protection), tyrosinase (pigmentation), dopamine hydroxylase (catecholamine production), lysyl oxidase (collagen and elastin formation), clotting factor V (blood clotting), and ceruloplasmin (antioxidant protection, iron metabolism, and copper transport). In addition to its enzymatic roles, copper is used for biological electron transport. The "blue copper" proteins (named after their intense blue color arising from a ligand-to-metal charge-transfer absorption band at $600 nm) that participate in electron transport include azurin and plastocyanin. 21 Copper is a redox active nucleus and may be also involved in the formation of reactive oxygen species. In this regard, the interaction of copper complexes with nucleic acids and their capability to damage DNA have been investigated. 186 In addition, some artificial DNA enzymes were shown to have Cu 2þ -dependent activity. 187 Finally, some copper complexes are currently being investigated for their potential anticancer and anti-inflammatory activity. 188 The NMR-active copper isotopes 63 Cu and 65 Cu are quadrupolar and couple strongly to local EFGs. Since the quadrupole moments cause a significant line broadening when the charge is unevenly distributed, only copper derivatives with regular tetrahedral or cubic symmetry may be observable by direct NMR detection. 189 63 Cu is by far most used in copper NMR spectroscopy because of both the higher receptivity and natural abundance compared to 65 Cu.

For the past decades, the structures, electronic states, and reactivity of Cu(II) complexes have been widely investigated by various spectroscopic methods, such as UV-vis, Raman, and electron paramagnetic resonance spectroscopy because of their characteristic absorptions resulting from d-d transitions, ligand-to-metal charge transfers, and unpaired electrons on the copper(II) ion. On the other hand, these analytic techniques have not been applied extensively to Cu(I) derivatives because of featureless spectroscopic properties resulting from the closed shell d 10 electron configuration. For diamagnetic Cu(I) complexes, 63 Cu NMR spectroscopy appears to have the greatest potential for characterizing their structures and electronic states, because most Cu(I) complexes prefer a tetrahedral configuration, thus giving rise to relatively sharp resonance lines, 190 whereas the paramagnetism of Cu(II) center prevents its detection by 63 Cu NMR spectroscopy.

Isotope A (%) I g (Â10 7 rad T À1 s À1 ) Q (fm 2 ) Relative receptivity to 13 Both silver and gold have no known natural biological role in humans, but interest in the medicinal applications of silver and gold derivatives has increased in recent years. Historical treatments with Ag(I) salts as antiseptic agents are well documented. Although the introduction of modern antibiotics greatly reduced their use in antimicrobial drugs, the increasing development of bacterial resistance toward antibiotics currently marketed has renewed the interest in novel and more efficient silver-based antimicrobial agents. 191 Chrysotherapy (i.e., the therapeutic use of gold compounds) has been established for centuries for the treatment of rheumatoid arthritis, owing to the known immunosuppressive and anti-inflammatory properties of some gold salts. In particular, late-stage disease was treated with various Au(I) drugs, such as solganal, myocrisin, sanocrysin, allocrysin, and auranofin, and some are still in clinical use. 192 In addition, both Au(I) and Au(III) derivatives are emerging as a new class of metal-based anticancer agents. 193 All naturally occurring silver is found in two NMR-active isotopic forms, 107 Ag and 109 Ag, both having I ¼ 1/2. Despite its lower natural abundance, 109 Ag is normally preferred for NMR studies because of the slightly higher sensitivity. Difficulties in obtaining 109 Ag NMR spectra with good signal-to-noise ratio are due to the extremely long T 1 and relatively poor receptivity, both stemming from the very low g. 109 Ag chemical shift is strongly affected by several factors, including the type and number of coordinating atoms, the number of bridging versus terminal donor atoms, bond distances and bond angles, the solvent and the nature of the counterion, and also the concentration. For example, the 109 Ag chemical shift differs from about 50 ppm for 1 and 9 M (nearly saturated) AgNO 3 aqueous solutions, 194 and since aqueous AgNO 3 is frequently used as reference and often its concentration is not stated, this makes the direct comparison of 109 Ag NMR chemical shifts reported in the literature rather difficult.

Notwithstanding all the limitations, 109 Ag NMR spectroscopy is a potentially useful tool to characterize silver-containing compounds since both the chemical shifts and the coupling constants are very sensitive to the coordination geometry of the Ag(I) center. 195 For example, 109 Ag NMR spectroscopy was successfully employed to characterize silver-based antimicrobial and antifungal agents 196 and to investigate the solution behavior of the anticancer agent [Ag(d2pype) 2 ]NO 3 (d2pype ¼ 1,2-bis (di-2pyridylphosphino)ethane), the latter by exploiting 2D [ 31 P, 109 Ag] HMQC technique. 197 NMR spectroscopy occupies a major role in probing protein metal-binding sites where the native quadrupolar metal ions, for example, Zn(II), Ca(II), Fe(II), Mg(II), Cu(II), and Cu(I), can be replaced by I ¼ 1/2 metal isotopes. In spite of the attractiveness of Ag(I) as a redox stable analog for Cu(I), 109 Ag NMR has so far found limited applications in biological systems due to the poor sensitivity of the nucleus, thus making direct detection impractical for most biological systems. Relatively few biologically relevant examples have been reported to date, 198 and the only known metalloprotein studied by 2D [ 1 H, 109 Ag] HMQC spectroscopy to date is the 109 Ag-substituted yeast metallothionein (Fig. 20) . 199 Since the interaction between Ag(I) ions and cysteine-rich proteins seems to play a key role in bacterial inactivation, an in-depth investigation of the behavior of Ag(I) in the presence of cysteine, GSH, and penicillamine was carried out by means of 109 Ag NMR spectroscopy, demonstrating the strong tendency of the thiolate sulfur atoms to form bridges between silver ions. 200 Ag(I) may be also used to induce metal-mediated base pairs in nucleic acids, that is, two opposite nucleobases (either artificial or natural) coordinated to a metal ion incorporated in the core of the helix. Nucleic acids have interesting applications in nanotechnology, their potential relying on their iterative structural motifs and self-assembly properties, together with the possibility to incorporate specific nucleosides designed on purpose. Modified nucleic acids resulting from the site-specific incorporation of metal ions may be exploited as nanomaterials, for example, by increasing the conductive properties of the natural analogues. 201 In this regard, a recent paper reports on the use of the NMR properties of silver to confirm the incorporation of Ag(I) ions in a DNA duplex bearing three imidazole (Im) moieties by forming stable Im-Ag þ -Im base pairs. Intriguingly, although no 109 Ag signal could be detected in the [ 1 H, 109 Ag] HMQC spectrum, a direct coupling between silver and the coordinated Im nitrogen atoms was observed, thus confirming the occurrence of a strong binding of Ag(I) ions to the artificial nucleobases. 202 Similarly, 109 Ag NMR (in conjunction with DFT calculations) confirmed the formation of the Ag þ -mediated cytosine-cytosine base pair within a DNA duplex in solution. 203 In fact, the 109 Ag chemical shift recorded at 442 ppm, together with the observation of Naturally occurring gold has a single isotope, 197 Au, characterized by a large quadrupole moment. As a result of fast quadrupole relaxation, the resonances are extremely broad and weak. Due to the low NMR receptivity of 197 Zinc is an essential element in humans and, under physiological conditions, exists in the þ2 oxidation state. It has catalytic, structural, or regulatory functions in more than 200 zinc metalloenzymes, and it is estimated that around 3000 proteins in the human body contain zinc prosthetic groups. In particular, zinc plays a structural role in the formation of the so-called zinc fingers, proteins exploited by transcription factors for interacting with DNA and regulating gene activity. Another function of zinc is in the maintenance of the integrity of biological membranes resulting in their protection against oxidative injury. In addition, there are over a dozen types of cells in the human body that secrete zinc ions, and the consequences of these secreted zinc signals in medicine and health are now being actively studied. Over 95% of the total body zinc is bound to proteins within cells and cell membranes. Most of the zinc in blood is found in the RBC bound to the metalloenzyme carbonic anhydrase, whereas plasma contains only 0.1% of the total zinc (of which approximately 18% bound to a 2 -macroglobulin, 80% to albumin, and 2% to such proteins as transferrin and ceruloplasmin). 21 67 Zn, the only naturally occurring NMR-active isotope of zinc, has I ¼ 5/2 and a large quadrupole moment responsible for high relaxation rates. This quadrupolar nucleus is characterized by low sensitivity and low resonance frequency. The required concentration for Zn 2þ to be detected by NMR is typically high (>0.1 M) but lower concentrations can be utilized following isotopic enrichment. Anyway, due to the quadrupolar nature of the nuclide, even if solution NMR methods afforded observable zinc resonances in a metalloprotein, the resulting linewidths would likely obscure the determination of site-specific isotropic chemical shifts. Consequently, it is not surprising that the literature of 67 Zn NMR data in solution is scarce and, among those reports, only a very few are biologically relevant. In this regard, 67 Zn NMR has been used to study the environment and/or behavior of Zn 2þ interacting with proteins, such as concanavalin, calmodulin, thermolysin, and S100. 205 Given the unfavorable NMR properties of 67 Zn, the method of choice for the study of zinc in biological systems in solution remains its replacement with 113 Cd (see succeeding text). On the other hand, the exploitation of solid-state techniques may represent a reliable alternative. Historically, solid-state 67 Zn NMR spectroscopy has been undesirable due to broad quadrupolar lineshapes and low sensitivity. In recent years, however, dramatic improvements in the solid-state NMR of quadrupolar nuclides have occurred. A general strategy for the observation of low-g half-integer quadrupolar nuclides has been developed, involving a combination of low temperature (4-100 K) with cross-polarization experiments employing specific spin-echo sequences. 206 Nevertheless, although such methodology affords sufficient sensitivity to examine Zn 2þ -binding sites, as far as we are aware of, still few reports are available to date in the literature describing the use of solid-state 67 Zn NMR for the study of zinc in metalloproteins or in zinc complexes as models for active sites of metalloproteins. 207

A (%) I g (Â10 7 rad T À1 s À1 ) Q (fm 2 ) Relative receptivity to 13 Cadmium belongs to a category of heavy metal ions that have increasingly attracted attention over the years, due to their toxic effect toward the environment and various living organisms, including man. 208 Although nonessential in the human physiology, Cd(II) is largely associated with Zn(II), and Cd/Zn competition in binding to biomolecules is associated with cadmium-related toxicity. 21 In spite of the similar magnetic properties and natural occurrence of the two I ¼ 1/2 cadmium isotopes, the large majority of the biological NMR studies have used 113 Cd due to its slightly higher relative sensitivity compared to 111 Cd. 209 The sensitivity of 113 Cd at natural abundance is about eight times that of 13 C, putting it on the fringe of accessibility for biological applications using modern high-field spectrometers. However, an approximate eightfold enhancement can be obtained by using 113 Cd-enriched starting materials, thus allowing reasonable quality spectra to be acquired, for example, on as little as 0.5 mM of a 113 Cd-substituted protein sample in a few hours of data accumulation.

The adaptable ligand coordination number and geometry of Cd(II) are the most commonly cited reasons why cadmium can be used to replace an extensive range of biologically relevant metal ions and mimic essential elements such as Zn(II) and Cu(II) in metallothioneins and the natural cofactor Mg(II) in nucleic acids.

Metallothioneins (MTs) are small cysteine-rich proteins that are present in several living organisms, including vertebrate, bacteria, fungi, and plants. Due to the high content of cysteines, they bind preferentially soft metal ions, forming metal-thiolate clusters. In particular, they show high affinity toward the toxic metal ions Zn(II), Cu(I), Cd(II), and Hg(II) and are believed to be involved in metal detoxification. The suitable NMR properties of 113 Cd make it a reliable probe to study these metal cluster arrangements. In particular, it can be used to mimic the tetrahedral coordination geometry of Zn(II), whose NMR properties are less favorable (see earlier text). 6b 113 Cd chemical shifts span a 900 ppm range and are extremely sensitive to number, nature, and coordination geometry of the ligands, as clearly summarized in Fig. 21 . Remarkably, the broad chemical shift dispersion of the 113 Cd resonances allows to not only provide information about the ligand type(s) at a particular metal site but also assess the selectivity in the presence of multiple metal-binding sites with identical ligand coordination.

Owing to the capability of the Cd 2þ ion to replace a number of metal ions in a variety of metalloproteins and to the sensitivity of the 113 Cd resonances to changes in the chemical environment around the metal center, 113 Cd NMR spectroscopy is regarded as a powerful and straightforward method to identify the metal-binding sites and to define the coordination geometry. In this regard, a number of data have been reported providing insights into cadmium coordination in metalloproteins, 210 in protein active core model compounds, 211 and in de novo designed metalloproteins and polypeptides. 212 Cd(II) is regarded as "softer" than Mg(II) and it is normally used in thio-rescue experiments to identify metal ion-binding sites within large RNAs 47 and, owing to its coordination properties, it can be used as mimic of Mg(II) to study its binding properties in RNA-metal ion interactions. 213 Direct 113 Cd NMR detection was used to study the interaction of Cd(II) with a small ribozyme. The addition of an excess of Cd(NO 3 ) 2 caused a downfield shift and broadening of 113 Cd signal, indicating a fast exchange between free and bound cadmium. 214 Several papers report on the indirect observation of cadmium bound to RNA. For example, upon titration of a hammerhead ribozyme metal-binding site model with Cd(II), strong perturbations in 1 H, 13 C, and 15 N resonances were observed, in particular a strong upfield shift (c. 20 ppm) of a 15 N signal, resulting from the direct coordination of Cd(II) to a specific guanine N (7). The authors also investigated the one-bond 113 Cd-15 N scalar coupling, but no coupling could be detected due to the fast exchange between free and bound Cd(II). Nevertheless, according to literature data, if the chemical exchange could be lowered down, a 1 J( 113 Cd, 15 N) coupling of 78-216 Hz should be observed in the NMR spectra. 215 113 Cd chemical shifts are strongly influenced by the concentration and the nature of the counterion and care should be taken when choosing the Cd(II) salt to be used for titration studies. A detailed multinuclear NMR study of the CdCl 2 -ATP and the Cd(ClO 4 ) 2 -ATP systems as a function of pH has been reported, showing how the affinity of the counterion to Cd(II) influences the nature of cadmium-ATP interaction. In fact, the different behavior of Cd 2þ and CdCl þ toward ATP has a direct strong effect also on the observed 113 Mercury has no known natural biological role. Hg(0) is volatile and relatively nontoxic, whereas Hg(II) is toxic to most living organisms because of its avid coordination to thiol groups within biological systems. Hg(II) is known to undergo biological methylation to methylmercury and dimethylmercury, both of which are extremely toxic to humans. 217 Mercury has two NMR-active isotopes, 199 Hg and 201 Hg, the latter being quadrupolar. The I ¼ 1/2 nuclide 199 Hg has a chemical shift range of 2500 ppm and a relative sensitivity 5.9 times that of 13 C, which should make it, at least in principle, an excellent probe for direct NMR detection of organomercury adducts, but its inherent insensitivity is a major drawback for biologically related studies. In fact, by means of conventional NMR direct detection techniques, including large (10-15 mm) sample tubes and long accumulation times, concentrations exceeding the solubility limits of most proteins and nucleic acids would be necessary to obtain reasonable 199 Hg NMR spectra. Consequently, a variety of indirect detection techniques have been devised to improve the observation of this insensitive nucleus, 218 together with the use of 199 Hg-enriched mercury precursors. 113 Cd NMR is still the method of choice to probe metal-binding sites in metalloproteins (see preceding text) but 199 Hg NMR spectroscopy has been increasingly employed. In fact, although the number of applications where the Hg(II) ion can replace isostructurally the native metal ions is limited owing to the 1.10 Å ionic radius and the preference for either linear or trigonal coordination geometry, 199 Hg NMR spectra of 199 Hg-substituted metalloproteins, for example, carbonic anhydrase, azurin, plastocyanin, rusticyanin, rubredoxin, Gal4, MerR and MerP, and a number of mercury model complexes, have been reported. 219 Much of these data are a result of the studies of O'Halloran and coworkers and have revealed 199 Hg chemical shifts indicative of certain coordination environments. For example, two-coordinate aliphatic Hg(II) thiolates {HgS 2 } show chemical shifts at c. À800 ppm, whereas the range for three-coordinate {HgS 3 } species is reported to be from À80 to À160 ppm for the trigonalplanar geometry and around À360 ppm for the distorted trigonal one. The chemical shifts of Hg(II) bound to four cysteine thiolates {HgS 4 } fall within the range À300 to À500 ppm, whereas coordination by four histidines {HgN 4 } is observed at the other extreme of chemical shift at about À1200 ppm. In addition, 199 Hg exhibits scalar couplings to 1 H, 13 C, and 15 N that are slightly larger than those observed with 113 Cd, thus allowing heteronuclear correlation experiments to be carried out to confirm the ligand type(s) and stoichiometry.

Recently, Pecoraro and coworkers extended such studies to probe the metal ion coordination in other native proteins and in de novo designed polypetides. 212b,220 For example, it was shown by 199 Hg NMR spectroscopy that at pH 7.5 Hg(II) is bound to a monomeric human copper chaperone (HAH1) as a two-coordinate linear complex {HgS 2 }, whereas upon increasing, the pH Hg 2þ promotes HAH1 association, leading to formation of {HgS 3 } and {HgS 4 } complexes, which are in exchange on the ms-ns time scale. 221 199 Hg NMR spectroscopy was also shown to be a useful tool to assess the capability of different molecules to act as mercury detoxification agents. Recent reports include the formation of Hg(II) adducts with penicillamine in alkaline solution and with GSH at alkaline and physiological pH and with cysteine-containing pseudopeptides to be used as possible mercury-sequestering agents in water. 222 Mercury can interact with nucleic acids, and the study of these interactions may shed light on the genotoxicity of this heavy metal. Several groups in the past used NMR spectroscopy to characterize mercury adducts with model nucleotides, polynucleotides, and oligonucleotides, but the effect of Hg(II) binding was probed only indirectly by evaluating its impact on 1 H, 13 C, and 15 N chemical shifts. 223 On the other hand, 199 Hg NMR spectroscopy was used to follow the interaction of Hg 2þ with the model nucleobase 1,3-dimethyluracil (1,3-DimeU). 224 Starting from the species [(1,3-DimeU-C (5))Hg(OAc)], the replacement of the acetate with different ligands was investigated in terms of the overall effect on both the 199 Hg chemical shifts and 3 J( 1 H-199 Hg) coupling constants. It was found that both parameters decrease as a function of the ligand according to the following order NO 3 À > OAc À > Cl À $ Br À > I À > SCN À > CN À > 1, 3 À DimeU À C 5 ð Þ. The last species of this series is [Hg(1,3-DimeU-C (5)) 2 ], in which the central mercury is coordinated to two model nucleobases and can be regarded as a metal-modified base pair.

As previously reminded (see text earlier), the study of metal-modified base pairs represents the starting point to obtain nucleic acids functionalized with metal ions (including Hg 2þ ) to be used as nanomaterials. In this regard, NMR was used to probe indirectly the presence of linear {T-Hg(II)-T} base pairs in DNA duplexes containing mismatched thymidine residues (T). The authors treated DNA duplexes containing differently 15 N-labeled mismatched thymidines with natural abundance Hg(II) showing that, in addition to a strong shift of the 15 N resonances upon metallation (Dd > 30 ppm), a mercury-mediated coupling was clearly observed between the two metal-coordinated N (3) atoms of the opposite thymidine involved in the artificial base pairing ( 2 J( 15 N, 15 N) ¼ 2.4 Hz). 225 Similarly, Hg(II) was reacted with RNA duplexes containing up to 20 uridine mismatches, leading to the formation of mercury-mediated base pairs of the type {U-Hg(II)-U}. 6b, 226 The authors focused on a 22-nucleotide long palindromic sequence containing six successive uracil residues and could prove the incorporation of Hg(II) by means of a combination of NMR methods, including 1 H, 15 N, 199 Hg, and 1 H diffusion-ordered spectroscopy NMR experiments. Interestingly, the only detectable 199 Hg was that originated by free Hg(II) ions in solution, most likely accounting for the kinetically labile metal binding and the strong line broadening owing to 199 Hg chemical shift anisotropy relaxation. Anyway, the last examples showed that even if direct observation of 199 Hg may be somehow elusive, the incorporation of Hg 2þ in nucleic acids can be confidently assessed by a combination of different more classical NMR techniques.

Although considered as nonessential elements for life, lanthanides are biologically active and have several important medicinal applications in both diagnosis and therapy, 227 the most successful being the use of Gd(III) complexes as MRI contrast agents. 228 The latest positive outcomes are mainly related to gadolinium derivatives of polydentate aminocarboxylate. Gadolinium also has potential for use in neutron capture therapy with the advantage that its uptake can be monitored by MRI. There are two main goals for future development. One is to control the biological behavior (cellular uptake and retention, tissue targeting, and in vivo stability) by incorporating Gd(III) into bioconjugates, such as lipids with acid labile bonds. The other is to improve the efficiency with which the complexes induce spin relaxation in protons of water molecules, by designing chelators to control exchange rates of coordinated, or hydrogen-bonded water molecules, and by controlling the mobility and rotation (e.g., through molecular size) of complexes. These two goals can be merged by designing agents whose spin relaxation properties are dependent on the physiological environment, so that MRI scans can provide biochemical/physiological and structural information. 229 The paramagnetic trivalent lanthanide cations (Ln(III)) have been exploited as shift reagents in NMR spectroscopy for a long time. 230 Applications have been both qualitative, to simplify the spectrum, and quantitative, by comparison of the lanthanideinduced shift and relaxation rate enhancements with values calculated for a proposed structure. Currently, the main application of Ln(III) complexes as shift reagents include the NMR separation of enantiomers (chiral shift reagents), the identification of NMR resonances from intra-and extracellular alkali metal ions (see preceding text), and their exploitation as paramagnetic probes tagging metalloproteins to determine their 3D structure in solution by NMR spectroscopy. 231 The overall more favorable nuclear properties of the I ¼ 1/2 15 N isotope, together with the development of 15 N-enrichment methods, have prevented the use of 14 N NMR spectroscopy from becoming a routine technique to investigate nitrogen-containing compounds. 14 N NMR spectra may be acquired directly due to the high natural abundance and the rapid relaxation of this quadrupolar nucleus. On the other hand, 14 N peaks are often broad due to the large quadrupole moment. In addition, the low resonance frequency of 14 N can result in "ringing" or acoustic resonance within the probe, a consequence of which is rolling baselines in the spectra, making broad signals even harder to detect. 5 Although the development of antiringing pulse sequences can greatly improve baselines and dramatically reduce the time needed for acquisition of good quality spectra, 14 N NMR spectroscopy still remains poorly exploited.

To the best of our knowledge, biologically related 14 N NMR studies have been only reported to follow reactions of cisplatin in the blood plasma and cell culture media 232 and, more recently, to investigate the structure and bonding of Pt(IV)-azido complexes, which are being explored as potential photoactivatable anticancer agents, 233 One of the main reasons for studying oxygen is its ubiquity in biology. Indeed, oxygen controls or participates in nearly every biological process, especially those involving aerobic metabolism. Oxygen occupies a key position at both structural and physiological level. In all macromolecules, including peptides, proteins, nucleic acids, and carbohydrates, oxygen plays a major role in the observed molecular conformation, owing to its involvement in H-bond formation. As such, oxygen atoms are involved in triggering, signaling, and activation mechanisms. 235 17 O is the only NMR-active oxygen isotope and shows several unfavorable properties. It has a small gyromagnetic ratio, so that the resonance frequency is about one-seventh that of protons, and low natural abundance, thus making isotopic enrichment often necessary. Finally, it is quadrupolar and frequently presents large EFGs. 17 239 Recently, it was shown that ligand-protein interaction can be studied by means of quadruple central transition (QCT) spectroscopy. This approach relies on the multiexponential relaxation behavior of half-integer quadrupolar nuclei and on the possibility to detect a narrow signal for the central transition in the slow motion conditions. A detailed description of the theory and applications of 17 O QCT spectroscopy to the study of the interaction between 17 O-labeled oxalate, biotin, and palmitic acid with model proteins has been reported. 240 Protein hydration dynamics is crucial, since it is strictly connected to correct folding, stability, and functioning of proteins. This phenomenon was studied by magnetic relaxation dispersion (MRD) of 17 O in water molecules. 241 Moreover, protein conformational motions have been investigated by analyzing 17 O MRD of internal water molecules for two model proteins. 242 The use of 17 O NMR to monitor the interactions between water and proteins has also been previously reported. 243 Similarly, 17 O relaxation dispersion has been used to evaluate the residence time of water in minor and major grooves of DNA duplexes. 244 The use of 17 O NMR spectroscopy to assess the structure and dynamics of small molecules has been widely exploited. Examples related to biological systems include recent studies on internal carboxylate dynamics of DTPA (diethylenetriamine pentaacetic acid) and DOTA (tetraazacyclododecane tetraacetic acid) complexes of lanthanides, in particular Gd(III) analogues due to their use as MRI contrast agents (see text earlier). 245 Moreover, owing to the possibility of using Mn(II) derivatives as contrast agents, detailed studies on Mn 2þ -containing macrocycles have been performed by means of different techniques, including 17 O relaxation experiments. 246 Finally, 17 O NMR was used on 17 O-labeled nucleobases to study the interactions within the bases themselves and with the solvents 247 and also to evaluate ATP-metal ion interactions. 248 A two-part comprehensive review on the theoretical principles and applications of 17 Sulfur is essential for all living cells and may also serve as a chemical food source for some primitive organisms, such as some forms of bacteria using hydrogen sulfide in place of water as the electron donor in a primitive photosynthesis-like process. Sulfide forms a part of iron-sulfur clusters and the bridging ligand in the Cu A site of cytochrome c oxidase, involved in the utilization of oxygen by all aerobic life. In plants and animals, sulfur is found in all peptides, proteins, and enzymes containing the amino acids cysteine and methionine. Homocysteine and taurine are other sulfur-containing acids that are similar in structure but which are not coded for by DNA and are not part of the primary structure of proteins. GSH is an important sulfur-containing tripeptide that plays a role in cells as a source of chemical reduction potential through its sulfhydryl group. Many important cellular enzymes use prosthetic groups ending with sulfhydryl groups to handle reactions involving acyl-containing biochemicals: two common examples from basic metabolism are coenzyme A and a-lipoic acid. Disulfide (S-S) bonds formed between cysteine residues in peptide chains are very important in protein assembly and structure. These strong covalent bonds between peptide chains give proteins a great deal of extra toughness and resilience. 251 33 S is a quadrupolar nucleus characterized by a low g and scarce natural abundance, resulting in very low receptivity and low resonance frequency. These properties can increase the effects of spurious signals in the first part of the free induction decay signal, leading to severe distortions in the FT NMR spectrum. The moderate Q leads to an efficient quadrupolar relaxation and to broad NMR signals (from few to thousands hertz). Reasonably narrow resonances can be obtained only for small molecules in which the sulfur atom is located at sites with high electronic symmetry, such as the tetrahedral SO 4 2À anion and low-molecular-weight sulfones and sulfonates. In thiols, sulfides, sulfoxides, and other organic and inorganic functional groups with low symmetry, 33 S linewidths are often larger than 10 000 Hz, and the signals are observable only in a limited number of cases. Large linewidths prevent the accurate measurement of chemical shifts, may cause the overlap of resonances when more than one type of sulfur atom is present, and have a significant influence on the achievable signal-to-noise ratio, thus consequently affecting the experimental times needed to obtain reasonable spectra. Consequently to these limitations, direct detection of 33 S signals is very challenging, although applications of 33 S NMR in solution exist and have been recently reviewed. 252 To the best of our knowledge, the only exploitation of 33 S NMR spectroscopy for biochemical investigations is related to the detection of taurine in biological tissues. 253 Taurine (2-aminoethanesulfonic acid) is a naturally occurring b-amino acid widely distributed in the biosphere. Despite the intensive studies, many mechanisms of the biochemical reactions involving taurine remain unknown or uncertain, probably because of the difficulty in detecting taurine in intact tissues. In this work, 33 S NMR spectra of biological tissues were reported for the first time. As shown in Fig. 22 , the 33 S spectrum of Lithophaga lithophaga homogenates exhibits a single signal that was assigned to the À 33 SO 3 À group of taurine on the basis of its chemical shift value (À6.8 ppm).

Remarkably, in the spectral range examined, no 33 S NMR signals were detected from other sulfur-containing biological molecules, for instance, cystine, cysteine, methionine, and hypotaurine. Vale and coworkers carried out a detailed multinuclear NMR characterization of the antitubercular drug ethionamide. 254 Notwithstanding the low symmetry of the thioamide sulfur atom, they succeeded in recording the corresponding 33 S peak of the drug at 190 ppm in DMSO-d 6 (relative to external 2 M aqueous solution of Cs 2 SO 4 ) but only at 2.5 M concentration and after 14 days of data accumulation. On the other hand, Gale et al. developed a new techniques based on 33 S NMR to monitor the sulfate transport capability of synthetic transporters mimicking the lipid bilayer of cell membrane. 255 The method relies on the use of 33 Slabeled sulfate anion and the exploitation of the relaxation properties of paramagnetic agents, such as Mn 2þ and Fe 3þ , to discriminate between intra-and extravescicular sulfate. The corresponding 33 S peaks were observed in 162 mM solutions of 33 SO 4 2À and after only 3200 scans with a relaxation delay of 250 ms.

Currently, the increasing availability of high-field NMR spectrometers and the development of hardware and probe technology can improve the detection of 33 S signals. In this regard, by developing a 10 mm 33 S cryogenic NMR probe operating at 9-26 K, sensitivity was increased up to 9.8 times compared to conventional 5 mm broadband probes. 256 This 33 S cryogenic probe was applied to biological samples, such as human urine, bile, chondroitin sulfate, and scallop tissue, allowing easy detection of sulfur compounds having ÀSO 4 2À and ÀSO 3 À groups.

Isotope A (%) I g (Â10 7 rad T À1 s À1 ) Q (fm 2 ) Relative receptivity to 13 Selenium is a trace element essential for mammals. Low-molecular-weight selenium compounds present in the human body include selenocysteine (or selenocystine) and selenomethionine, with much lower abundance of their metabolic counterparts. Diseases associated with selenium deficiency include asthma, Keshan disease, and HIV. Therefore, selenium-based agents are currently being investigated for their potential therapeutic applications. Moreover, selenium compounds have been shown to be of value as cancer chemoprotective agents. 257 Selenium displays many similarities with its congener sulfur, that is, they have rather similar electronegativity and atom size and share the same major oxidation states. For these reasons, there are many sulfur compounds that have selenium analogs. However, in spite of these similarities, there are clearly differences between the two elements, and substitution for one another results in compounds with quite diverse chemical properties. 258 Selenium has six natural isotopes, but only one, 77 Se, is NMR-active with I ¼ 1/2, thus allowing high-resolution NMR spectroscopy to be carried out. 77 Se is approximately three times more sensitive than 13 C and, taking into account that longitudinal relaxation times are in the range of seconds and that nuclear Overhauser enhancement (NOE) effects are nearly always absent, the sensitivity of 13 C and 77 Se are comparable in routine NMR experiments. 259 77 Se NMR spectroscopy has been successfully employed in several cases, including the characterization of selenium-based potential drugs 260 the characterization of selenoproteins, 261 and their interaction with drugs. 262 Interestingly, owing to the unfavorable nuclear properties of 33 S, 77 Se may be used as a surrogate in order to gain insights into the multifaceted roles of sulfur in biology. Unfortunately, in the past, such NMR studies were hindered by the low availability of selenium-rich proteins (in particular selenocysteine) and the low sensitivity in the absence of isotopic enrichment. In this regard, recent advances in 77 Se labeling of proteins, including selectivity and fine-tuning the percent of selenium incorporation, allowed the identification of multiple selenocysteine and selenomethionine residues in the sulfhydryl oxidase augmenter of liver regeneration 263 and the visualization of disulfide bonds through diselenide proxies in a 37-residue spider toxin (k-ACTX-Hv1c) containing four disulfide bonds, including a rare and functionally critical vicinal disulfide bridge between the adjacent cysteine residues Cys13 and Cys14. 264 77 Se NMR has been also used to study the interaction between the nonenzymatic carbohydrate-binding proteins lectins and selenium-labeled glycosides, 265 and to identify the 2-selenouridine moieties in the wobble position of the anticodon stem loop of three tRNA species. 266 p-Block: Halogens (F, Cl, Br, and I)

Isotope A (%) I g (Â10 7 rad T À1 s À1 ) Q (fm 2 ) Relative receptivity to 13 Fluorine is an essential trace element and is present in the human body mostly in the form of solid fluorides in the bones and teeth.

The 19 F nucleus is a I ¼ 1/2 species exiting in 100% natural abundance and possessing a magnetogyric ratio that is 83% that of the proton. The large g translates into both high sensitivity in 1D 19 F NMR spectroscopy and strong dipolar couplings, allowing for the measurement of 19 F-19 F and 19 F-1 H NOEs for distance restraints and the study of topology and contact with solvent. 267 Fluorine chemistry is an expanding area of research that is attracting increasing interest, due to the impact of fluorine in the life sciences. Therefore, owing to the high receptivity and the inherent sensitivity of the fluorine resonances to the local environment, coupled with the virtual absence of background fluorine signals, it is not surprising that 19 F NMR spectroscopy (a somewhat neglected technique within the bio-NMR community) in recent years has been emerging as a powerful tool in biological, pharmaceutical, and medicinal chemistry, as proved by the several publications continuously appearing in the literature.

The following applications of 19 F NMR spectroscopy, exploiting several parameters such as chemical shift changes, relaxation rates, intermolecular magnetization transfer, and diffusion data, have been extensively reviewed 268 and will not be further discussed in this article:

• Metabolic studies monitoring the biotransformation, pharmacokinetics, and metabolism of fluorinated xenobiotics and drugs 269

• Binding studies involving protein-protein, protein-DNA, small molecule-protein, and small molecule-DNA interactions, crucial for drug screening 269b,270

• Structural analysis of protein structure and dynamics by incorporating fluorine-containing probes 271 and of protein cavities 272 • Fluorine-based MRI contrast agents 273 Among its uses, in recent years, 19 F NMR spectroscopy has been successfully applied to RNA research. RNA is involved in many biological processes and is nowadays an acknowledged potential drug target. 274 In order to be active, RNA must adopt specific secondary and tertiary structures, and RNA-based biological processes often rely on the ability of RNA to interconvert between several structures. In this regard, 19 F NMR proved useful to study such conformational changes by incorporating a fluorine label either into a nucleobase, for example, by using 5-fluorouracil or 5-fluorocytosine, 275 or at the sugar moiety. 276 Micura and coworkers showed that the 2 0 -19 F labeling of the ribose can be used to discriminate between double helical and single-stranded regions in RNAs, 276 and to study RNA hairpin/duplex equilibriums, providing accurate melting temperature values. 277 More recently, they also reported on the use of 19 F NMR spectroscopy for the evaluation of the ligand-induced conformational change of a riboswitch, that is, an RNA element located in the 5 0 untranslated region of mRNA that controls gene expression upon conformational change induced by ligand binding. 278 In general, when a fluorine label is chosen, care should be taken in its positioning along the RNA structure to avoid modifications in the physicochemical properties of the RNA itself. In order to overcome this possible drawback, Tisné and coworkers have recently proposed the use of fluorinated diaminocyclopentanes as external probes for RNA structure. 279 Besides its use in structure and conformational dynamics of RNA, 19 F NMR spectroscopy has been successfully employed also to evaluate the binding properties of small molecules toward RNA, crucial for the design of RNA-targeting drugs. For example, by incorporating a fluorinated label into the RNA itself, it was possible to identify site-specific RNA binding molecules. 280a Moreover, it was recently shown that binding competition studies involving fluorinated probes may open up new perspectives in the suitability of 19 F NMR to screen RNA-drug interaction. 280b Overall, these studies have been opening up new perspectives in the use of 19 F NMR spectroscopy to study both the RNA-ligand interactions and the structure and dynamics of RNAs as already done currently with proteins (see text earlier).

A (%) I g (Â10 7 rad T À1 s À1 ) Q (fm 2 ) Relative receptivity to 13 Chlorine is an essential element for all living organisms largely as chloride, but hypochlorite is produced in some cell compartments to destroy invading organisms. Together with Na þ , Cl À is a major extracellular electrolyte. In conjunction with H þ and monovalent alkali metal ions, it controls transmembrane potentials and regulates the equilibrium of cellular electrolytes and osmotic pressures. A defect in Cl transport causes cystic fibrosis, a genetic disorder resulting in a defect in the transmembrane chloride channel. 21 All naturally occurring chlorine is found in two NMR-active isotopic forms, 35 Cl and 37 Cl, both quadrupolar. Owing to the higher natural abundance and receptivity, 35 Cl is the preferred isotope for NMR spectroscopic observation. Its relaxation is normally dominated by the contributions from interaction of the quadrupole moments with time-dependent EFG at the nucleus. Although both the natural abundance and receptivity are favorable, recording 35 Cl NMR spectra is often challenging as it gives rise to rather broad signals. Therefore, a very few papers report on the use of 35 Cl NMR techniques for specific biological purposes. 35 Cl NMR spectroscopy has been used in the past to monitor the chloride binding to proteins, 281 the anion distribution and transport in membranes, 282 also in presence of shift reagents to discriminate intra-and extracellular chloride signals, 283 and the interaction between drugs and membranes. 284 An interesting application involved the use of 35 Cl NMR spectroscopy to assess the mechanism through which chloride ions activate oxygen evolution in photosystem II of green plants. 285 Although beyond the scope of this article, it is worth mentioning recent publications reporting on the use of solid-state 35 Cl NMR to study the polymorphism in hydrochloride pharmaceuticals, 286a and active pharmaceutical ingredients. 286b

Isotope A (%) I g (Â10 7 rad T À1 s À1 ) Q (fm 2 ) Relative receptivity to 13 Bromine is currently not thought to be an essential element for man, although it is present in blood at micromolar concentrations. As hypobromite, it has a potential role in the destruction of pathogenic organisms. Iodine is an essential micronutrient for humans, where it is required for incorporation into the thyroid hormones thyroxine and triiodothyronine. 21

Br and 127 I are the magnetically active quadrupolar isotopes and are characterized by a high sensitivity to detection by NMR spectroscopy. Although 79 Br has a slightly higher natural abundance, the preferred isotope for bromine NMR spectroscopic observation is 81 Br, due to its higher receptivity. Conversely, 127 I is the only NMR-active isotope of iodine at 100% natural abundance.

The use of 81 Br in biological systems has been poorly explored, with only few dated examples in the field of protein research. 287 127 I NMR spectroscopy has only rarely been utilized for studies of fluid systems partly because, for covalent environments, the 127 I signals are generally broadened beyond detection. On the other hand, the iodide ion may be conveniently detected. For example, 127 I has been used as a probe for the anion-binding properties of human serum albumin, 288 and analogous studies have been performed to investigate the binding of iodide to some peroxidases. 289 The application of 127 I NMR spectroscopy to study the enzymatic degradation of k-carrageenan has been recently reported. 290 

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Financial support by the National University of Ireland Galway (Millennium Fund to LR; Hardiman Research Scholarship to EF) and the Irish Research Council (Postgraduate Scholarship to EF) is gratefully acknowledged.