key: cord-0747309-uyq8c5sm
authors: Laurent, Quentin; Martinent, Rémi; Lim, Bumhee; Pham, Anh-Tuan; Kato, Takehiro; López-Andarias, Javier; Sakai, Naomi; Matile, Stefan
title: Thiol-Mediated Uptake
date: 2021-05-03
journal: JACS Au
DOI: 10.1021/jacsau.1c00128
sha: dc1356b5db4b50ece00b0ae63bd3a525637dc134
doc_id: 747309
cord_uid: uyq8c5sm

[Image: see text] This Perspective focuses on thiol-mediated uptake, that is, the entry of substrates into cells enabled by oligochalcogenides or mimics, often disulfides, and inhibited by thiol-reactive agents. A short chronology from the initial observations in 1990 until today is followed by a summary of cell-penetrating poly(disulfide)s (CPDs) and cyclic oligochalcogenides (COCs) as privileged scaffolds in thiol-mediated uptake and inhibitors of thiol-mediated uptake as potential antivirals. In the spirit of a Perspective, the main part brings together topics that possibly could help to explain how thiol-mediated uptake really works. Extreme sulfur chemistry mostly related to COCs and their mimics, cyclic disulfides, thiosulfinates/-onates, diselenolanes, benzopolysulfanes, but also arsenics and Michael acceptors, is viewed in the context of acidity, ring tension, exchange cascades, adaptive networks, exchange affinity columns, molecular walkers, ring-opening polymerizations, and templated polymerizations. Micellar pores (or lipid ion channels) are considered, from cell-penetrating peptides and natural antibiotics to voltage sensors, and a concise gallery of membrane proteins, as possible targets of thiol-mediated uptake, is provided, including CLIC1, a thiol-reactive chloride channel; TMEM16F, a Ca-activated scramblase; EGFR, the epithelial growth factor receptor; and protein-disulfide isomerase, known from HIV entry or the transferrin receptor, a top hit in proteomics and recently identified in the cellular entry of SARS-CoV-2.

"Thiol-mediated uptake" refers to the observation that (i) cellular uptake reliably and often dramatically increases in the presence of chalcogenides (or mimics) capable of dynamic covalent exchange, usually disulfides, and that (ii) this uptake can be inhibited with thiol-reactive agents ( Figure 1 ). 1−7 Thiol-mediated uptake has been around for a long time, with scattered observations coming from different fields. 1, 3, 4 Recently, it received more attention with the emergence of privileged scaffolds such as cell-penetrating poly(disulfide)s (CPDs) 5 or cyclic oligochalcogenides (COCs) 6 and the renewed interest in relation to viral entry 8 and antivirals, possibly including SARS-CoV-2. 7 Considering the overwhelming evidence for usefulness in practice, it is surprising that little is known about how thiolmediated uptake really works (vide infra). 7, 4 It is understood that thiol-mediated uptake can be coupled to different uptake pathways. Dynamic covalent sulfur exchange cascades before or during direct translocation across the plasma membrane into the cytosol appear to dominate with synthetic delivery systems. 5, 6 HIV entry is an established example for inhibitable dynamic covalent sulfur exchange on cell surfaces followed by membrane fusion, 8 while dynamic covalent sulfur exchange with proteases in endosomes and lysosomes, such as cathepsins, can, perhaps, be considered as arguably borderline examples for thiol-mediated uptake combined with endocytosis. 9 More precise mechanistic information is often missing, particularly for direct translocation, also because dynamic covalent chemistry 10−13 dramatically complicates, and discourages, target identification. However, it is also this dynamic covalent chemistry that makes thiol-mediated uptake so unique and attractive, particularly from the viewpoint of supramolecular chemistry. The objective of this Perspective is, after a summary of the uptake facts available today, to map out this tantalizing chemical space surrounding thiol-mediated uptake. Topics covered are extreme sulfur chemistry, adaptive networks, thiol affinity chromatography, molecular walkers, and templated ring-opening polymerization, followed by more biological aspects such as micellar pores and a gallery of possible protein targets. Whereas only time will tell us about the direct relevance of the individual topics for thiol-mediated uptake, they are, in the spirit of a Perspective, brought up in the following to arouse curiosity and inspire progress.

The first explicit observation of thiol-mediated uptake was made by Ryser and co-workers in 1990 ( Figure 2 ). 1 In a systematic study of uptake of disulfide-bridged drug− polylysine conjugates, inhibition by Ellman's reagent, i.e., DTNB 1, led to the conclusion that "the reductive properties of the cell surface may be important to maintain membrane protein sulf hydryl groups, which appear to be required for active membrane transport and for endocytosis of immune complexes". 1 This breakthrough was quickly adapted to show that the entry of HIV is initiated by thiol-mediated uptake followed by fusion and can be inhibited with DTNB (see section 5.6). 8 The same was found early on for diphtheria toxin, followed by many examples for various microbe−host interactions (section 5.6). 14 Arguably the first example for the use of thiol-mediated uptake for delivery was reported by Behr and co-workers in 1995. 2, 15 The delivery of lipoplexes was shown to increase with various thiol-reactive agents, including disulfides, but maleimide 2 was performing best (Figure 2 ). Thiol-mediated uptake through endocytosis was explicitly considered as a mode of action, although inhibition was not reported. Such an inhibition with DTNB 1 was shown by Sagan and co-workers in 2009 to apply to the uptake of peptides 3 equipped with an activated disulfide. 3 Other scattered examples followed, mostly other peptides 16, 17 and contrast agents, 18, 19 together with a growing arsenal of probes to image and quantify the presence of thiols on cell surfaces. 20−30 However, it would be incorrect to limit thiol-mediated uptake to thiols on cell surfaces; disulfides on cell surfaces exchanging with arriving thiols are equally plausible, although clearly less considered (Figure 1a) . 4 This also includes the possibility of exofacial disulfides in dynamic covalent exchange with substrates other than thiols. This combination is beyond the term "thiol-mediated uptake", which is however maintained for historical reasons and the lack of convincing alternatives. 4 Possible contributions from glutathione (GSH) pumped from the cytosol to the cell surface, and other extracellular thiols, should not be ignored either. 22, 26, 30, 31 Besides these explicit early studies on thiol-mediated uptake, often supported by inhibition experiments, there is a remarkably rich literature that simply mentions the fact that uptake increases in the presence of chalcogenides, usually disulfides, without further explanations. 4,32−51 In gene transfection, disulfide polymers have been heavily explored but not with the intention to enable uptake but to degrade the transporters after uptake by reductive depolymerization in the cytosol. 52−60 In their seminal opinion piece in 2012, Gait and co-workers collected many of the early examples to formulate the concept of thiol-mediated uptake. 4 We entered the field in 2014 by introducing cell-penetrating poly(disulfide)s (CPDs) 4, 5 quickly followed by cellpenetrating cyclic oligochalcogenides (COCs) such as 5 in 2015. 6 More recently, COCs were found not only to activate but also to competitively inhibit thiol-mediated uptake, with thiosulfonates 6 emerging as the current best to inhibit the entry of SARS-CoV-2 lentivirus models. 7 

Coming from cell-penetrating peptides (CPPs) 7 61 and the self-organizing surface-initiated polymerization (SOSIP) of photosystems, 62−65 CPDs 4 have been introduced with the intention to replace their peptidic backbone with a disulfide polymer without changing the cationic side chains ( Figure  3 ). 5, 66 This substitution was expected, and confirmed, to minimize toxicity and endosomal capture, resulting in efficient cytosolic delivery, where the transporter is depolymerized upon arrival. Thiol-mediated uptake was supported by DTNB inhibition experiments. CPDs 4 are readily accessible by ringopening polymerization of COC monomers, usually derivatives of lipoic acid such as 8. In the presence of initiators such as 9− 13 (Figures 3 and 4) and terminators 14 like iodoacetamide, their polymerization into CPDs 4 can occur within 5 min in neutral aqueous buffer. Interestingly, the thiolate reacts selectively with the "secondary" sulfur in monomer 8. 67 Instantaneous migration to the primary sulfur proceeds until equilibrium is reached at 40% conversion, which implies that the monomers in CPDs are incorporated in a secondary/ primary ratio of 6:4. With single-channel current recordings of thiolated transmembrane pores, ring-opening polymerization as well as the reverse ring-closing depolymerization could be directly observed and characterized on the single-molecule level ( Figure 3 ). 68 Further progress with CPDs focused mostly on initiators, while monomers 5 and terminators 69 received comparably little attention ( Figure 4 ). Thiol conjugates with biotin 9, NTA 10, tetrazine 11, hydroxylamine 12, or cyclodextrin 13, mostly pioneered by Yao and co-workers, have all been introduced to interface the resulting CPDs with substrates of free choice using standard streptavidin, His-tag, IEDDA, or oxime technologies, respectively. 70−77 In a recent elegant study, Liu, Moore, and co-workers showed that aryl thiols 15 provide access to cyclic CPDs 16 because of the reactivity of the initial disulfide. 78 Traceless linkers 17 have been integrated to release native amines instead of thiols upon reductive depolymerization. 77 Most importantly, Lu and co-workers have shown very recently that under cryopolymerization conditions CPDs can be grown efficiently and under biorelevant conditions directly on proteins such as thiolated GFP, providing access to conjugate 18 in situ. 79 CPDs have been used extensively to deliver a broad variety of substrates of biological interest, including probes, proteins, and so on ( Figure 5 ). Among the more advanced systems is an artificial metalloenzyme 19 that turns on a gene switch in the cytosol for, ultimately, metabolic engineering. 80 Among the largest systems are quantum dots (QDs) 20, covered with streptavidin and loaded with functional nanobodies that are otherwise notoriously difficult to deliver directly to the cytosol. 81 The mobility analysis and the poor colocalization between QDs and fluorescently labeled dextrans demonstrated their cytosolic localization, while intracellular formation of aggregates 21 via GFP and the anti-GFP nanobody attached on the QDs showed intact functionality of the nanobodies (Figure 5a ). Highlights among the extensive, creative, and highly successful studies by Yao and co-workers arguably are mesoporous silica nanoparticles (MPSNPs). 71, 72, 75, 76, 82 The most impressive examples include glutathione-responsive MPSNPs 22 loaded with functional antibodies and decorated with mitochondria-targeting triphenylphosphonium moieties besides CPDs 75 or MPSNPs 23 loaded with fluorophores and decorated with antigen/antibody complexes and fluorescence quenchers besides CPDs. 72 Gene transfection with CPDs obtained by templated polymerization has also been confirmed in a most elegant study by Yang, Li, and co-workers (see section 5.5). 83 

Realizing the power of thiol-mediated uptake with CPDs, we quickly shifted our attention toward COCs. 6 The general aim was to vary speed, selectivity, and the very nature of dynamic covalent exchange chemistry on the way into cells. COCs were selected because the released thiolates remain nearby, ready for further exchange, thus providing access to exchange cascades, adaptive networks, and so on (see sections 5.1−5.5). Initial studies suggested that increasing ring tension in COCs correlates with increasing uptake activity. 6 In the relaxed disulfide 24, an acyclic oligochalcogenide (AOC), the CSSC dihedral angle is 90°. This dihedral angle is best to minimize lone pair repulsion and maximize hyperconjugation ( Figure  6 ). 84−86 In 1,2-dithiolanes 5 derived from asparagusic acid (AspA), the angle decreases to 27°, and in epidithiodiketopiperazines (ETPs) 25 and 1,2-diselenolanes (DSLs) 26, the CXXC dihedral angle is around 0°. 87 Recent studies by Raines, Movassaghi, and co-workers clarified that n−π* interactions to the carbonyls stabilize ETPs sufficiently to exist under the extreme tension at 0°, 88 while in DSLs, it is the longer Se−Se bond that makes the tension at 0°bearable. 87 Like uptake activity, exchange kinetics with thiols increase with increasing tension. In the resulting ring-opened AspA 5, the intramolecular thiol has the standard acidity of pK a 8.2 and is only marginally deprotonated in neutral water. In ringopened ETP 25 89 and DSL 26, 87 the obtained thiol and selenol are much more acidic. This acidity keeps reactivity high in neutral water, including possible ring closure and release of the external thiol. This increased dynamic nature is likely to contribute to their high uptake activity (see sections 5.1−5.6).

As the name implies, AspA is a natural product found in asparagus of essentially unknown function except that the metabolites add a characteristic malodor to the urine. 6 ETPs also occur in natural products such as gliotoxin 27, 91,92 which, interestingly, has been reported to have antiviral activity. 93 Benzopolysulfanes (BPS) 28 are also present in natural products. 91, 94, 95 Varacin 29, from tunicates, an early target in total synthesis, 96 is arguably the most popular. BPS 30 emerged at the top of a giant library screening for kinase inhibitors involved in neurodegenerative disorder and is active in mice. 97 Thiol-mediated uptake of BPS 28 exceeded all other COCs (see section 5.3). 90, 98 Elegant studies by Wu and co-workers demonstrated that the transannular Prelog strain in γ-turns 99−101 31 correlates with efficient cellular uptake ( Figure 7) . 22, 102 The best was a Cys-Gly-Cys-motif (R′ = H) attached to the N-terminus of peptides, and Cys-Gly-Gly-Cys and Cys-Cys-Gly controls were less active. 102 While relaxed single AOCs 24 showed poor activity, 6 Abe and co-workers introduced phosphoramidite 32 for use in automated DNA and RNA synthesis. 103−105 Dependent on their positioning, the presence of 5−10 AOCs 32 resulted in efficient cytosolic oligonucleotide delivery. Peptide-based oligomers of COCs, together with the introduction of cell-penetrating streptavidin, also showed that multivalency of COCs strongly increases uptake. 106 The complementary AspA 33 for automated peptide synthesis has been introduced as well, and already one AspA per peptide was confirmed to suffice for efficient delivery. 107 1,2-Dithiolane probes, such as 34, that turn on fluorescence (or release drugs) upon reductive opening as outlined in 35, have been quite extensively explored by Fang and co-workers. 108−110 The puzzling relation of specific thioredoxin labeling with 34 and thiol-mediated uptake is being addressed by Thorn-Seshold and co-workers. 111 Like CPDs, COCs have been used to deliver a broad variety of substrates, including also artificial enzymes and quantum dots. 107, 112 However, the largest substrates delivered in intact form to the cytosol are liposomes and polymersomes 36 of up to 400 nm diameter, loaded with internal fluorescent probes and externally added cationic COC amphiphiles. 113 Documenting the power and practical usefulness of thiol-mediated uptake, the different biological applications of CPDs, COCs, and AOCs have been covered in more detail in several recent reviews. 70 

As mentioned in the Introduction, inhibition with Ellman's reagent 1 is commonly accepted as evidence for thiol-mediated uptake ( Figure 2 ). However, it has been noticed by several groups that DTNB inhibition is neither efficient nor reliable. The product of exchange with exofacial thiols is an activated disulfide which easily continues to exchange. We thus considered that the newly available collection of COCs could, besides containing top transporters for thiol-mediated uptake, also contain competitive inhibitors of the same process. According to the inhibition of the uptake of fluorescently labeled ETP 25 and BPS 28, COCs are up to 5000 times better inhibitors than DTNB, with minimum inhibitory concen- trations (MICs) reaching into the nanomolar range ( Figure  8 ). 7 This result provided exhaustive support, if needed, that thiol-mediated uptake exists and is unrelated to simple passive diffusion.

MICs against ETP 25 (E) and BPS 28 (B) differed for every inhibitor. The resulting selectivity of inhibition can be described as the E/B ratio of the respective MICs. Different E/B values for different inhibitors supported that different uptake pathways exist for thiol-mediated uptake and that not only reactivity but also molecular recognition of and within adaptive dynamic covalent networks contribute to activity (see section 5.7). These influences beyond differences in reactivity were consistent with observations from proteomics with thiolreactive probes. 120−123 About 60% of all proteins targeted by new probes differ from those targeted by established controls. 120 ETP uptake was best inhibited by the expanded ETP tetrasulfide 37, which is a relatively poor transporter. 7,90 Selfinhibition by ETP disulfides was almost as efficient, as was inhibition with BPS. Not surprisingly, thiol-mediated BPS uptake was generally more difficult to inhibit; i.e., E/B values were generally <1. The best inhibitors with E/B > 1 values were γ-turns 31, followed by the cyclic thiosulfonate 6. Removing oxygens from 6 to thiosulfinates 38 and DTT/DTE disulfides 39 (stereochemistry irrelevant) gradually removed inhibitory activity. The acyclic thiosulfinate allicin 40, present in garlic, was inactive. 124, 125 Irreversible thiol-reactive agents such as the recent 41 were moderately active inhibitors, with activity increasing with reactivity. 126 Ebselens 42 inhibited uptake of both probes.

Thiosulfonates 6 also inhibited the cellular uptake of SARS-CoV-2 lentivectors. 7 The identified IC 50 's were significantly better than those measured for ebselens 42, which have received recent attention as SARS-CoV-2 inhibitors. 127−130 The cytosolic delivery by thiol-mediated uptake of CPDs or COCs is usually insensitive to inhibitors of various forms of endocytosis. 5,6,74,87,89 Insensitivity toward wortmannin or cytochalasin B excludes significant macropinocytosis, chlorpromazine clathrin-mediated endocytosis, methyl-β-cyclodextrin caveolae-mediated endocytosis, and so on.

Thiol-mediated uptake is attractive because it is so powerful in practice, as outlined above. Thiol-mediated uptake is tantalizing because so little is known beyond the occurrence of inhibitable dynamic covalent sulfur exchange chemistry. This poor understanding of the "sulfur magic" opens wonderful perspectives in different directions. They are outlined in the following.

COCs have been introduced for thiol-mediated uptake to open access to dynamic covalent exchange cascades. 6, 67, 112 With strained cyclic disulfides as substrate 5, tension-releasing ring opening by exchange with a thiol on a cell surface, an "exofacial" 4 thiol, affords a new thiol in the tethered substrate next to the new disulfide bridge between the substrate and the cell. This thiol can react with a nearby exofacial disulfide (A, Figure 9a ). In the resulting two-step exchange cascade, cyclic disulfides thus consume one thiol and one disulfide on the cell surface (A), build two disulfide bridges to the cell surface, and produce one new thiol (B, Figure 9a ). With an additional exofacial thiol near the first disulfide formed, the exchange cascade can continue toward the next proximal disulfide (B, Figure 9a , section 5.4). 67 Resulting from the reduction of exofacial disulfide bridges, the neighboring thiols required to trigger continuing cascades are ubiquitous on cell surfaces. 21−30 The local protein misfolding caused by the exchange cascade can be expected to introduce local disorder and flexibility, which can be easily repaired after uptake via a reverse exchange cascade. Possible long-distance coupling in exchange cascades from exofacial thiols have been indicated in pioneering early model studies on transmembrane signaling. 132 In this context, disulfide COCs as in substrate 5 can be considered as hyperreactive mimics of disulfide bridges in protein substrates, with their respective selectivities added by molecular recognition. Reduction could afford 1,3-dithiols 43 already before uptake. Cascade exchange with two proximal disulfides on the cell surface (C, Figure 9b ) could then yield a dynamic network that is identical with the one produced by disulfide 5 (B, Figure 9a ). Such a "reversed" thiol-mediated uptake mechanism is conceivable ( Figure 1a ) and of interest, also considering, for example, the facile cellular uptake of phosphorothioate oligonucleotides 44 (Figure 9b ). 48−50 On the nondynamic sulfide level, redox switching to sulfoxide and sulfone is quite well appreciated for the rational control of structure and function. 133−136 On the disulfide level, the same redox switching opens attractive perspectives toward more extreme sulfur chemistry, that is, thiosulfinates 38, thiosulfonates 6, and beyond. Reactivity toward thiols increases with every oxygen added. Cyclic thiosulfinates 38 have been shown to selectively target neighboring thiols (D), yielding, through a sulfenate intermediate (E), two disulfide bridges 45 between the cell and substrate (F, Figure 9c ). 137, 138 The opening of cyclic thiosulfonate substrates 6 with exofacial thiols (G) affords intermediate 46 with one disulfide bridge plus one proximal sulfinate in the substrate, which can continue to exchange with an exofacial disulfide (H, Figure  9d ). 139 The net result of the full cascade is the consumption of one exofacial thiol and one disulfide, yielding macrocycle 47 with one disulfide and one thiosulfonate bridge from substrate to cell plus one new exofacial thiol (I, Figure 9d ). This outcome identifies thiosulfonate COCs 6 as functional analogues of disulfide COCs 5, including the possibility to extend exchange cascades in the likely presence of an exofacial thiol next to the disulfide bridge in I (Figure 9d ). Their reactivity, however, differs significantly, according to the different functional groups involved. In small-molecule models, the formation of macrocycles 45, sulfinates 46, and both disulfide-and sulfonate-bridged cascade products 47 has been confirmed. 137, 139 These selected examples on the unfolding extreme sulfur chemistry illustrate intriguing perspectives as the COC family expands. Their development in the context of the topics that follow, from adaptive networks to templated ring-opening polymerization, promises access to diverse functions that may or may not include thiol-mediated uptake and antivirals.

The best performing BPS 28 have been introduced to drive the notion of adaptive dynamic covalent networks to the extreme (Figures 6 and 10) . 90 Spontaneous ring contraction and expansion of the pentasulfide 28, probably triggered by traces of thiols in the solution, have been reported by several groups and studied in more detail by Greer, Kawamura, and coworkers. 95 Among the identified BPS 48−53, pentasulfide 28 is preferred, followed by trisulfide 48 and other odd-numbered rings up to nonasulfide 53.

In the presence of thiolates and disulfides, a rich dynamic network unfolds, including besides the above BPS 48−53 also acyclic monomers 54 as well as cyclic oligomers 55. 90 The largest rings observed by LC-MS are heptamers 55 7 (m = 6) with up to 19 sulfur atoms in the giant macrocycle (Σn x = 5). From such dynamic covalent networks, cells are expected to select the best for uptake, which then could be amplified by reequilibration of the network. This concept is as appealing as it is difficult to validate. Especially, amplification in response to uptake or different templates, including metals, remains as a most daunting challenge to tackle in future studies. The same is true for the application of the lessons learned to other COCs and the integration of techniques developed with dynamic covalent libraries. 10, 11 These fundamental studies have provided most impressive examples for the complexity of oligomer structures that can emerge already from disulfide exchange networks. 10, 11 Expansion into alternative reaction pathways also deserves much attention, including redox and radical chemistry, triggered by oxygen, light, and more.

Thiol affinity chromatography has been considered as a convenient empirical tool to fingerprint exchange cascades and possibly mimic thiol-mediated uptake. 87 For fluorescently labeled 1,2-dithiolane 5, a first fraction was directly eluted without retention (A, Figure 11a,b) . A second fraction of 5 was eluted only upon addition of reduced DTT to the mobile phase. This partial retention was consistent with operational dynamic covalent exchanges on the thiolate surface of the resin, with the COC presumably being eluted in reduced form 43 (B, Figure 11a,b) .

In contrast, DSL 26 was poorly retained (Figure 11c ,d) 87 and the first fraction eluted with a slightly longer retention Figure 10 . Adaptive dynamic covalent network produced by BPS 28, with cells expected to select the best for uptake, which then will be amplified by re-equilibration. time compared to that of AspA 5. These findings supported that ring opening by thiol/diselenide exchange can be followed by ring closure, with the selenophilicity of the reactive selenolate overruling the less pronounced ring tension (C, Figures 11c and 6 ). Although meaningful, this lack of retention obviously failed to demonstrate the occurrence of ring opening by operational dynamic covalent exchange (which was however evinced otherwise, e.g., the catalysis of DTT oxidation or trapping open reactive intermediates). 67,87 ETP 25 was also poorly retained on thiol affinity columns, a finding that nicely illustrates the similarity with DSL 26 with regard to the reactivity of the released thiol and possible ring closure.

Retention of BPS 28 was exceptionally strong and full elution with DTT nearly impossible, causing permanent damage to the column. 90 This behavior was consistent with the "sticky" adaptive network produced by BPS and differed nicely from the clean retention and release of AspA 5 on the one hand and the lack of retention with DSL 26 and ETP 25 on the other hand. Overall, thiol affinity column fingerprinting emerges as promising tool to identify the nature of different exchange cascades, which in turn are likely to provide access to different mechanisms of thiol-mediated uptake.

The dynamic covalent exchange cascades accessible with COCs can be reminiscent of molecular walkers or hoppers. The hopping of disulfide 56 along six thiols on a β-sheet track inside a transmembrane β-barrel pore 57 has been realized by Bayley and co-workers (Figure 12a ). 140 Equipped with a voltage-sensitive bulky DNA polyanion, the dynamic covalent exchange of disulfide 56 with the first thiol of track 57 was reported as change of the current flowing through a single pore (A, B) . Exchange of the resulting disulfide with the next thiol makes the DNA hop one step forward (B, C). The directionality of further hopping along the thiol track across the pore was assured by the applied voltage (C, D, Figure 12a ).

Walking instead of hopping along the same transmembrane thiol track was realized by merging dynamic covalent chalcogen and pnictogen chemistry. 141 Dialkyl phenylarsonodithioites 58 are well-known to covalently recognize neighboring thiols (E−G, Figure 12b ). To initiate the dynamic covalent exchange cascade needed to walk along the transmembrane thiol track 57, thiol 59, reminiscent of a molecular crutch, was added. Thiolate exchange on the pnictogen center allows the crutch-supported walker to lift one foot (H), exchange with the next thiolate on the track to make one step forward (I), and so on (I, J).

This combination of dynamic covalent chalcogen and pnictogen chemistry has been used in other functional systems, such as artificial vesicles made from monomers containing three proximal thiols or polymer nanoparticles stabilized with monomers containing up to four proximal thiols. 142 Most significant in the present context are fluorescent arsonodithioite exchangers to dynamically label two to four neighboring thiols, the latter introduced by Tsien and co-workers as FLASH probes for the intracellular targeting of proteins with an engineered (Cys) 4 motif. 143, 144 Exofacial neighboring thiols have been imaged with such fluorescent arsonodithioite exchangers. 23−25 Phenyl arsine oxides were also shown early on to inhibit the entry of HIV virus about as efficiently as DTNB. 8 In clear contrast, thiol-mediated uptake and its inhibition remain a fascinating topic to be explored with dynamic covalent pnictogen chemistry, including possible contributions of turned-on noncovalent pnictogen bonds into deepened σ holes. 145 Complementary to COC networks, the dynamic covalent networks accessible with pnictogens are arguably best illustrated with arsphenamine. 146 Ehrlich's "magic bullet", often considered the first drug developed by dedicated SAR, has long been considered to be the azobenzene homologue 60 (Figure 12c) . Only rather recent studies have indicated that 60 evolves into a collection of cyclic oligomers 61 and 62, which then release 63, which is considered to be the active form of the antisyphilis drug and further cyclizes into larger rings such as tetramer 64.

Further expanding from molecular walkers that operate with dynamic−covalent chalcogen and pnictogen chemistry, divalent reversible Michael acceptors have also been considered to generate directional exchange cascades. Leigh and co-workers have realized molecular walkers 65 that walk with conjugate addition along a polyamine track 66 ( Figure  13a ). 147 Initial conjugate addition triggers the elimination of trimethylamine to yield a second conjugate acceptor (A, Figure  13a ). The second amine on the track then adds to this second alkene, releasing the first amine and regenerating another alkene at the same time (B). To move forward, the third amine then adds to this alkene, and so on (C, D).

Michael cascades for thiol addition have received the most attention for the fluorescence imaging of neighboring thiols 67, also on cell surfaces (Figure 13b) . 20 Conjugate addition of the first thiolate to cascade probe 68 (E) produces a new acceptor (F, G) which reacts with the second thiol (G) to end up with two sulfide bridges 69 from probe to protein. The reversibility required for dynamic adaptive networks and, perhaps, cellular uptake depends on the acidity of the α hydrogens. Increasing reversibility has been obtained with cyano acceptors and πacidic aromatics as in 70, also in lipid bilayer membranes. 148−151 Conjugate addition is ubiquitous in medicinal chemistry, arguably leading to the renaissance of covalent drugs. For conjugate addition to membrane proteins, activation of the spice receptor TRPA1 with cinnamaldehyde, mustard oil, or the synthetic superspice 71 is a most spectacular example (see section 5.7). 152 Reversible cascade Michael acceptors remain to be explored for thiol-mediated uptake, its inhibition, and the related topics outlined above.

In light of the different expressions of the dynamic covalent chemistry of COCs described thus far, their reversible polymerization into CPDs can be considered as a special expression of cascade exchange. From this perspective, adaptive networks translate into the templated ring-opening polymerization (TROP) of COCs ( Figure 14 ). For TROP, COCs are first covalently or noncovalently interfaced with a template using a linker L (A); then the polymerization is initiated with a thiol (A, B); and then the template is detached to liberate polymers such as CPDs 4 (C, Figures 14 and 3) . Templated polymerization would be of interest to control length and, at best, also sequence of the resulting polymer. While TROP has been used regularly to build functional systems with 1,2-dithiolanes (Figure 15 ), studies on template release (Figure 14 , B, C), sequence selectivity, and eventually also self-replication remain to be realized. 153 ROP and TROP with other COCs are unexplored, as it is appealing not only with regard to new CPDs (Figures 3−5) but also for materials applications, dynamer plastics, and beyond.

The first systematic study on TROP with 1,2-dithiolanes was reported by Regen and co-workers, who attached lipoic acids at the end of lipid tails (Figure 15 ). 154 These artificial lipids 72 were assembled into vesicles. Their TROP was then initiated with the addition of hydrophobic thiols (Figure 15, A, B) . The obtained poly(disulfide)s stabilized vesicles and were not further characterized.

Templated ROP of cationic lipoic acid derivatives on oligonucleotides has been considered early on as a strategy for gene transfection (without consideration of thiol-mediated uptake). 58 Monomers 8 developed for CPDs ( Figure 3) were 

pubs.acs.org/jacsau Perspective studied more recently by Yang, Li, and co-workers for templated polymerization on DNA templates and gene transfection by thiol-mediated uptake (Figure 15 , C, D). 83 Whereas uptake was excellent, separation from the template and characterization of the polymers obtained by TROP were beyond the context of the study. Templated ROP of COC dimers such as 73 has emerged as the method of choice to grow ordered and oriented doublechannel photosystems with antiparallel redox gradients on conducting surfaces (Figure 15 , E). 62−65 Hydrogen-bonding assisted π stacks of the central aromatics serve to template ROP of the COCs at both sides. Evidence for operational templation has been secured by self-sorting. The resulting selforganizing surface-initiated polymerization (SOSIP) has been developed comprehensively.

Macrocycles 74 can be considered as unusual templates for ROP of lipoic acid 8 into catenated polymers 75. 155 In the materials sciences, nontemplated ROP of 1,2-dithiolanes is attracting increasing attention in the context of responsive dynamic−covalent materials, including "green plastics". 78,156−163 COCs other than 1,2-dithiolanes were much less considered thus far for ROP, not to speak of TROP. The cyclic oligomers 55 in adaptive networks produced by BPS 28 provide an inspiring illustration for what could possibly be achieved with TROP of COCs other than 1,2-dithiolanes ( Figure 10 ). Already TROP of 1,2-diselenolane homologues 26 will be intriguing considering their preference to stay closed. Only dimers 76 have so far been trapped during efforts to detect and characterize the opening of 1,2-diselenolanes 26. 67 Poly(diselenides)s have been prepared by other methods that involve neither ROP nor dynamic covalent chalcogenide exchange chemistry and shown to penetrate cells efficiently. 164 

A central question with thiol-mediated uptake is how large substrates, from proteins to nanoparticles and liposomes, are delivered to the cytosol by mechanisms different from endocytosis, thus bypassing endosomal capture (sections 3.1 and 3.2). With CPPs, this question has arguably been answered with transient micellar pores. 165 It is thus not unlikely that they contribute also to thiol-mediated uptake by direct translocation across the plasma membrane into the cytosol.

Micellar pores, referred to also as lipid ion channels or toroidal pores, can form spontaneously in lipid bilayer membranes. Their saddle-shaped surface is the result of a combination of negative and positive curvature, obtained by the combination of normal (or hexagonal 1) and inversed (or hexagonal 2) micellar fragments as outlined in Figure 16 .

Observed as early as 1974, 166 micellar pores were characterized in detail by Heimburg and co-workers, including voltage dependence. 167 Colombini and co-workers reported that the presence of ceramides favors the formation of micellar pores. 168 Many peptides are known to induce the formation of micellar pores, including helical toxins and antibiotics.

Matsuzaki and co-workers demonstrated that magainininduced micellar pores coincide with lipid flip-flop and can be characterized with the respective assays. 169 Micellar pores have also been proposed to explain ion channel formation by transmembrane B-DNA helices. 170 The formation of micellar pores by CPPs has been demonstrated with single-channel conductance experiments and supported by computational models. 171, 172 MacKinnon and co-workers implied similar micellar defects to account for lipid-mediated voltage gating of neuronal potassium channels. The elastic and transient nature of micellar pores is consistent with the translocation of also CPPs that carry large, even giant, substrates. Sealed throughout without any content leakage, such pores can conceivably expand temporarily to match the diameter of the substrate and contract and self-heal once they have passed (Figure 16 ). For the remarkably efficient, endocytosis-and fusionindependent cytosolic delivery with CPDs and COCs, contributions from transient micellar pores offer a plausible explanation. Contrary to the repulsion-driven ion pairing 165 that binds CPPs to anionic membrane surfaces, micellar pore formation during thiol-mediated uptake would coincide with the unfolding of adaptive networks by dynamic covalent chalcogenide exchange chemistry and local protein denaturation, as described in the preceding chapters ( Figure 16 ). Possible protein targets participating in these events will be briefly portrayed in the following.

While micellar pores may or may not contribute to direct translocation during thiol-mediated uptake, the cellular targets of dynamic covalent sulfur exchange most likely do. However, their involvement beyond dynamic covalent exchange remains to be determined, and their nature, with a few exceptions, remains to be identified.

Target identification for thiol-mediated uptake is complicated by the dynamic nature of the process. While proteomics analysis is, of course, possible, 173 it is difficult to tell if the detected proteins are really relevant for thiol-mediated uptake. The most reliable feedback can be obtained by suppression and overexpression of interesting candidates. 173, 50, 139 How-ever, laborious suppression−overexpression approaches evaluating one target after the other cope poorly with the high number of possible targets. The most developed example for multiple targets in thiol-mediated uptake is the transferrin receptor (TFRC), identified top in proteomics analysis and validated by suppression−overexpression for AspA 5 but less relevant for ETP 25 (Figure 6 ). 173 These proteomics results and protein data bank analysis reveal an overwhelming number of possible targets for thiol-mediated uptake. In the following, a few short portraits of the most inspiring candidates are provided ( Figure 17 ). TFRC (Figure 17a) , the transferrin receptor, is a highly abundant transmembrane protein on the cell surface. It enters cells by clathrin-mediated endocytosis after binding to the transferrin−iron complex and returns to the cell surface after releasing iron. Many drug delivery systems have been developed to profit from this system. 174 Various viruses, including SARS-CoV-2, also use TFRC binding for their entry into cells. 175, 176 Proteomics studies using fluorescently labeled AspA 5 identified TFRC as the top candidate to mediate its cellular uptake. Reduced uptake of AspA upon mutations of a pair of Cys 556/558 demonstrated their critical importance. 173 Thus, the first step of the thiol-mediated uptake through TFRC should be a ring-opening exchange between one of these cysteine thiolates and a COC (Figures 17a, A, B and 9a) . The resulting TFRC−chalcogenide conjugate B could enter the cell by endocytosis, but to reach the cytosol, the chalcogenide has to dissociate from TFRC and cross the membrane. The formed thiolate (or selenolate) might undergo further exchange reactions with disulfides near the membrane water interface stapling two TFRC units, which would require a significant protein conformational change (B, C). Cleavage of the two disulfide bonds could allow the two protein units to dissociate and expose space for the COC to slip through (C, D). The covalent bonds between TFRC and chalcogenides could be cleaved by reacting with an intracellular exchanger, e.g., glutathione (D−F), or with the thiols on the other TFRC unit (D, A) to release the chalcogenide inside of the cell and recover TFRC in the original state.

Contrary to AspA 5, thiol-mediated uptake of ETP 25 is much less sensitive to TFRC knockdown ( Figure 6 ). 89 This observation supports insights from inhibitor selectivity and proteomics that more than one pathway is available for thiolmediated uptake (Figure 8 ). 7 siRNA−CPD polyplexes conjugated to transferrin have been shown to enter deep tissue by transcytosis, but contributions from dynamic covalent exchange chemistry have not been considered. 60 CLIC1, the chloride intracellular channel protein 1, is a cytosolic protein that exists in both soluble (G) and membrane-bound forms (H, Figure 17b ). 177−179 Conductance measurements show that soluble CLIC1 G added to one side (cis) of a bilayer membrane inserts spontaneously in the membrane and forms poorly selective anion channels H. 180 Importantly, the conductance can be regulated by the addition of thiol-reactive agents, such as N-ethylmaleimide (NEM), to the opposite side (trans) of the membrane. Thus, the reactive Cys24 is accessible to the aqueous medium at the other side of the membrane, which is, if extrapolated to in vivo, the extracellular side. The same Cys24 is also the critical residue for CLIC1's glutathione S-transferase like activity in the soluble form G. 181 Although the structure of the membrane-bound form H is not known, it is believed to consist of helix bundles forming barrel-stave like structure. The presence of different conductance levels implies that the number of monomer units and thus the channel diameter can vary. 178 CLIC1 was one of the top five proteins identified by the proteomics study with AspA 5. 173 The reaction of COCs with Cys24 of CLIC1 in the membrane-bound form H, followed by the disulfide exchange with another Cys24 of another monomer unit (I), might release COCs in the adaptable CLIC1 channels to pass through. TMEM16F (Figure 17c) , a Ca 2+ -activated scramblase, mediates bidirectional flip-flop of phospholipids in an energyindependent manner. 182−184 Upon activation, the hydrophilic micellar-type pores open within the protein to allow the translocation of hydrophilic lipid head groups across the hydrophobic core of the membrane (J). Its sensitivity to thiolreactive agents is implied by its involvement in the formation of giant plasma membrane vesicles (GPMVs) by the action of NEM and paraformaldehyde. 185 Moreover, several cysteine residues are located in the transmembrane helices near the micellar hydrophilic pore. Thus, the reaction of these cysteines with COCs followed by its passage through micellar pores might be possible (J, K). TMEM16F arguably comes closest to the hypothesis combining micellar pores with adaptive sulfur networks, at best molecular walkers (Figures 9a and 16) . TRPA1 (Figure 17d) , the transient receptor potential cation channel, also called the "wasabi receptor", is activated by a wide variety of thiolate reactive electrophiles, including the wasabi and mustard component, allyl isocyanate, cinnamaldehyde, and the more electrophilic superspice 71 (Figures 17d  and 13 ). 152, 186 The binding sites Cys 415/422/622 are in the cytosol and thus not easily accessible to a hydrophilic COC. 152 However, other cysteine residues present within the transmembrane helices might contribute to the passage of COCs via thiolate−dichalcogenide exchange (L, M, Figure 17d ).

Ca v 1.1 (Figure 17e) , a voltage-gated calcium channel, contains many cysteine residues in the transmembrane helices (N). Its redox sensitivity was shown by the oxidative (GSSG or DTNB) or reductive (GSH or DTT) treatment to result in the increase or decrease of the open probability of the ion channels, respectively. 187 Similar modulations of transport activities by thiol-reactive agents were also noted for a cystine/ glutamate antiporter 188 and organic anion transporter. 189 These highly reactive cysteines are thus located near the transport pathways and might contribute to the thiol-mediated uptake in the manner similar to that of CLIC1. EGFR (Figure 17f ), the epidermal growth factor receptor, is a transmembrane receptor protein circulating between the plasma membrane and the endosomes, similarly to TFRC. 190 The extracellular part of this protein (O) is particularly rich in cysteine/cystine residues and is known to be targeted by dithiolanes and thiosulfinates. 139, 191 In the elegant work of Castellano and co-workers, EGFR has been proposed to be the target of cyclic thiosulfonates similar to 6 ( Figure 9d ). 139 Cascade opening converting one exofacial thiol and one disulfide into one disulfide and one thiosulfonate bridge plus one new exofacial thiol has been suggested to ultimately account for antitumor activity. Crooke and co-workers have demonstrated that EGFR directly interacts with phosphorothioate antisense oligonucleotides 44 during their uptake into the cytosol and that EGFR suppression and overexpression reduce and enhance uptake via clathrin-mediated endocytosis (Figure 9b ). Although the lack of cysteines in the transmembrane helices may (or may not) be incompatible with disulfide tracks for walking, this protein might capture COCs, pass them on to the other proteins to be transported across the membrane, or find other ways to mediate translocation.

Such roles can possibly be played by many other cysteinerich proteins, such as integrins, 192 mucins, 193 or scavenger receptors. Scavenger receptor class B type 1 (SCARB1), for instance, is attractive because it is a membrane protein responsible for cholesteryl ester uptake, a proteomics target of AspA 5, 173 and involved in the uptake of phosphorothioate RNA 44 49 and hepatitis virus 194 (which is known to be thiolmediated). 195 PDIs (Figure 18 ), protein disulfide isomerases, are soluble thiol oxidoreductases of a thioredoxin superfamily. They locate mainly in the ER and assist the oxidative folding of nascent proteins. However, they are also found in the cytosol, in mitochondria, in nuclei, and on cell surfaces. 196, 197 Extracellular PDIs associate to the cell surface through binding to the other surface-confined proteins, such as galectin-9, which in turn binds to glycosylated membrane proteins. 198 The cell surface PDIs are involved in, for example, cell adhesion, nitrosyl transfer, and viral entry. 8, 199 JACS Au pubs.acs.org/jacsau Perspective

As already outlined in the introduction, HIV entry has been one of the earliest observed examples of thiol-mediated uptake ( Figure 2 ). 1, 8 The developed mechanism suggests that HIV attaches to the cells by the binding of gp120 and CD4 ( Figure  18 , A). Then, PDI bound to CD4 reduces disulfides in gp120 to induce the conformational change needed to expose the Nterminus of gp41, which spontaneously inserts into the membrane (B) to prepare for fusion controlled by the popular helix bundle formation (C). This mechanism is supported by inhibition with Ellman's reagent 1 ( Figure 2 ) and arsine oxides similar to Ehrlich's magic bullet 60 ( Figure 12 ). 8, 146 By now, it is known that many other viruses use thiol− disulfide exchange to initiate cell entry, including Sindbis 200, 201 and the LDV virus, 202 some coronaviruses, 203−205 murine leukemia virus, 206, 207 baculovirus, 208 Newcastle disease, 209 or hepatitis. 195, 210 With toxins, dynamic covalent exchange on cell surfaces (diphteria) 14,211,212 is less common than enzymatic reduction after endocytosis (ricin, 14,213 cholera, 214 pseudonomas exotoxin, 215 and anthrax 216 ). Many bacteria and parasites require thiols for attachment to mammalian cells. 217−223 The involvement of thiol-mediated uptake in the entry of SARS-CoV-2 has received little support so far. The best established model is centered around the molecular recognition between the spike protein of the virus and ACE2, the angiotensin-converting enzyme 2. 9, 224, 225 The recognition can be followed by clathrin-dependent endocytosis, with the cathepsin cysteine proteases essential for endosomal escape. Alternatively, the serine protease TMPRSS2 on the cell surface can activate the spike/ACE2 complex for clathrin-independent membrane fusion. The understanding of these mechanisms is under constant evolution. The cell surface proteins neuropilin-1 and the serine protease furin have been added recently. 226 The most recent confirmation that the transferrin receptor is involved in SARS-CoV-2 entry 176 is particularly intriguing because it is a proteomics-confirmed target of thiol-mediated uptake 173 and overexpressed on the blood−brain barrier endothelial cells for iron transport by transcytosis. 60 Also very recently, we have identified inhibitors that are active against both thiol-mediated uptake and SARS-CoV-2 entry. 7 The best so far are several cyclic thiosulfonates 6, better than the much discussed ebselens 42 (Figure 8) . The future will tell if this is more than a coincidence. The same holds for TFRC as a common target, 60, 173, 176 EGFR as an interesting candidate, 50, 139 and possibly also SCARB1 173, 194, 195 and other candidates outlined in section 5.7. Other targets have been proposed for all active compounds, including zinc fingers 227 and cysteine proteases 129,130 that are presumably not involved with cellular uptake, and multitarget mechanisms are quite likely. 128 6. CONCLUSIONS Thiol-mediated uptake is fascinating because it works so well in practice, while the modes of action are essentially unknown. Dynamic covalent chalcogenide exchange chemistry appears to account for both sides of the coin. In the spirit of a Perspective, we spend only a little time summarizing the many successful applications of thiol-mediated uptake in biology that have already been reviewed elsewhere. 70, [115] [116] [117] 119 Instead, we tried to map out the chemical space that enriches and complicates the elucidation of possible modes of action.

The result is a collection of classics in dynamic covalent chemistry, combining adaptive networks with templated polymerization, molecular walkers with micellar pores and affinity chromatography, and a gallery of membrane proteins as possible partners. The covered topics all relate to dynamic covalent processes, which means that they are hard to detect and characterize. This does not mean that they are absent or less significant. Quite the contrary, they all stand for translational supramolecular chemistry at its best, supporting the general expectation that focusing on different, underrecognized, perhaps elusive, and at best new ways to get into contact on the molecular level will ultimately allow us to tackle some of the more persistent challenges in science and society, here to unravel new ways to get into cells, and, perhaps, to stop viruses to do the same.

The relevance of most of this collection of classics in supramolecular chemistry for thiol-mediated uptake and the discovery of antivirals remain to be seen. Illustrating the breadth, complexity, and beauty of the topic, they are developed in this Perspective to outline inspirational possibilities that wait to be explored, with the objective to ultimately understand all the different expressions of thiolmediated uptake, to understand how it really works.

Cleavage of Disulfide Bonds in Endocytosed Macromolecules. A Processing Not Associated with Lysosomes or Endosomes

Efficient Gene Delivery with Neutral Complexes of Lipospermine and Thiol-Reactive Phospholipids

Cell-Surface Thiols Affect Cell Entry of Disulfide-Conjugated Peptides

Exploiting Cell Surface Thiols to Enhance Cellular Uptake

Cellular Uptake of Substrate-Initiated Cell-Penetrating Poly(disulfide)s

Ring Tension Applied to Thiol-Mediated Cellular Uptake

Progress in Targeting HIV-1 Entry. Drug Discovery Today

Inhibitors of SARS-CoV-2 Entry: Current and Future Opportunities

Dynamic Combinatorial Libraries: From Exploring Molecular Recognition to Systems Chemistry

Evolution of Dynamic Combinatorial Chemistry

Dynamic Covalent Chemistry as a Facile Route to Unusual Main-Group Thiolate Assemblies and Disulfide Hoops and Cages

Complex Molecules That Fold Like Proteins Can Emerge Spontaneously

Cell Surface Sulfhydryls Are Required for the Cytotoxicity of Diphtheria Toxin but Not of Ricin in Chinese Hamster Ovary Cells

Targeted Transfection of Human Hepatoma Cells with a Combination of Lipospermine and Neo-Galactolipids

Enhanced Cytosolic Delivery of Plasmid DNA by a Sulfhydryl-Activatable Listeriolysin O/ Protamine Conjugate Utilizing Cellular Reducing Potential

CyLoP-1: A Novel Cysteine-Rich Cell-Penetrating Peptide for Cytosolic Delivery of Cargoes

Exofacial Protein Thiols as a Route for the Internalization of Gd(III)-Based Complexes for Magnetic Resonance Imaging Cell Labeling

Melanoma Tumor Xenograft with a Thiol-Reactive Gadolinium Based MRI Contrast Agent

A β-Allyl Carbamate Fluorescent Probe for Vicinal Dithiol Proteins

Thiol−Disulfide Exchange Reactions in the Mammalian Extracellular Environment

Biscysteine-Bearing Peptide Probes To Reveal Extracellular Thiol− Disulfide Exchange Reactions Promoting Cellular Uptake

Development of a Red Fluorescent Light-up Probe for Highly Selective and Sensitive Detection of Vicinal Dithiol-Containing Proteins in Living Cells

Site-Selective Tagging of Proteins by Pnictogen-Mediated Self-Assembly

Decrease of Protein Vicinal Dithiols in Parkinsonism Disclosed by a Monoarsenical Fluorescent Probe

Thioether-Bonded Fluorescent Probes for Deciphering Thiol-Mediated Exchange Reactions on the Cell Surface

Highly Selective Fluorescent Probe for Vicinal-Dithiol-Containing Proteins and in Situ Imaging in Living Cells

Selective and Ratiometric Fluorescent Trapping and Quantification of Protein Vicinal Dithiols and in Situ Dynamic Tracing in Living Cells

A Fluorescent Probe for Stimulated Emission Depletion Super-Resolution Imaging of Vicinal-Dithiol-Proteins on Mitochondrial Membrane

Tracking the Bioreduction of Disulfide-Containing Cationic Dendrimers

Discovery of a Macropinocytosis-Inducing Peptide Potentiated by Medium-Mediated Intramolecular Disulfide Formation

Activation of Cell-Penetrating Peptides by Disulfide Bridge Formation of Truncated Precursors

Effective Incorporation of 2′-O-Methyl-Oligoribonuclectides into Liposomes and Enhanced Cell Association through Modification with Thiocholesterol

Biophysical and Biological Studies of End-Group-Modified Derivatives of Pep-1

A Novel Cell-Penetrating Peptide Derived from Human Eosinophil Cationic Protein

Structural Parameters Modulating the Cellular Uptake of Disulfide-Rich Cyclic Cell-Penetrating Peptides: MCoTI-II and SFTI-1

Cysteine and Arginine-Rich Peptides as Molecular Carriers

Roseltide RT7 Is a Disulfide-Rich, Anionic, and Cell-Penetrating Peptide That Inhibits Proteasomal Degradation

Intraneuronal Delivery of Protein Kinase C Pseudosubstrate Leads to Growth Cone Collapse

Design of Carrier Peptide-Oligonucleotide Conjugates with Rapid Membrane Translocation and Nuclear Localization Properties

Cell Penetrating PNA Constructs Regulate Galanin Receptor Levels and Modify Pain Transmission in Vivo

Antisense Inhibition of P-Glycoprotein Expression Using Peptide−Oligonucleotide Conjugates

Inhibition of PRb Phosphorylation and Cell Cycle Progression by an Antennapedia-P16INK4A Fusion Peptide in Pancreatic Cancer Cells

Delivery and Release of Small-Molecule Probes in Mitochondria Using Traceless Linkers

Targeted Subcellular Protein Delivery Using Cleavable Cyclic Cell-Penetrating Peptides

EGF-like and Other Disulfide-Rich Microdomains as Therapeutic Scaffolds

Temperature-, Concentrationand Cholesterol-Dependent Translocation of L-and D-Octa-Arginine across the Plasma and Nuclear Membrane of CD34+ Leukaemia Cells

A Cytoplasmic Pathway for Gapmer Antisense Oligonucleotide-Mediated Gene Silencing in Mammalian Cells

The Interaction of Phosphorothioate-Containing RNA Targeted Drugs with Proteins Is a Critical Determinant of the Therapeutic Effects of These Agents

Cellular Uptake Mediated by Epidermal Growth Factor Receptor Facilitates the Intracellular Activity of Phosphorothioate-Modified Antisense Oligonucleotides

Glutathione-Responsive Selenosulfide Prodrugs as a Platform Strategy for Potent and Selective Mechanism-Based Inhibition of Protein Tyrosine Phosphatases

Bioreducible Polycations in Nucleic Acid Delivery: Past, Present, and Future Trends

The Role of the Disulfide Group in Disulfide-Based Polymeric Gene Carriers

Bioreducible Polymers for Gene Delivery

Bioreducible Polymers for Gene Silencing and Delivery

Multifunctional Dendronized Peptide Polymer Platform for Safe and Effective SiRNA Delivery

Examination of the Effect of Increasing the Number of Intra-Disulfide Amino Functional Groups on the Performance of Small Molecule Cyclic Polyamine Disulfide Vectors

Lipoic Acid-Derived Amphiphiles for Redox-Controlled DNA Delivery

Reductively Cleavable Nanocaplets for siRNA Delivery by Template-Assisted Oxidative Polymerization

Transferrin-Appended Nanocaplet for Transcellular SiRNA Delivery into Deep Tissues

Anion-Mediated Transfer of Polyarginine across Liquid and Bilayer Membranes

Self-Organizing Surface-Initiated Polymerization: Facile Access to Complex Functional Systems

Lateral Self-Sorting on Surfaces: A Practical Approach to Double-Channel Photosystems

Stack Exchange Strategies for the Synthesis of Covalent Double-Channel Photosystems by Self-Organizing Surface-Initiated Polymerization

Zipper Assembly of Photoactive Rigid-Rod Naphthalenediimide π-Stack Architectures on Gold Nanoparticles and Gold Electrodes

Substrate-Initiated Synthesis of Cell-Penetrating Poly-(disulfide)s

The Opening of 1,2-Dithiolanes and 1,2-Diselenolanes: Regioselectivity, Rearrangements, and Consequences for Poly(disulfide)s, Cellular Uptake and Pyruvate Dehydrogenase Complexes

Ethynyl Benziodoxolones: Functional Terminators for Cell-Penetrating Poly(disulfide)s

Bypassing Endocytosis: Direct Cytosolic Delivery of Proteins

Simultaneous Imaging of Endogenous Survivin mRNA and On-Demand Drug Release in Live Cells by Using a Mesoporous Silica Nanoquencher

Nanoquencher-Based Selective Imaging of Protein Glutathionylation in Live Mammalian Cells

Intracellular Delivery of Native Proteins Using Biodegradable Silica Nanoparticles

Intracellular Delivery of Functional Proteins and Native Drugs by Cell-Penetrating Poly-(disulfide)s

Intracellular Delivery of Functional Native Antibodies under Hypoxic Conditions by Using a Biodegradable Silica Nanoquencher

Cell-Penetrating Poly(disulfide) Assisted Intracellular Delivery of Mesoporous Silica Nanoparticles for Inhibition of miR-21 Function and Detection of Subsequent Therapeutic Effects

Intracellular Delivery of Native Proteins Facilitated by Cell-Penetrating Poly(disulfide)s

Architecture-Controlled Ring-Opening Polymerization for Dynamic Covalent Poly(disulfide)s

Cryopolymerization of 1,2-Dithiolanes for the Facile and Reversible Grafting-from Synthesis of Protein−Polydisulfide Conjugates

A Cell-Penetrating Artificial Metalloenzyme Regulates a Gene Switch in a Designer Mammalian Cell

Efficient Delivery of Quantum Dots into the Cytosol of Cells Using Cell-Penetrating Poly(disulfide)s

Intracellular Delivery of Native Proteins Using Biodegradable Silica Nanoparticles

Self-Assembled and Size-Controllable Oligonucleotide Nanospheres for Effective Antisense Gene Delivery through an Endocytosis-Independent Pathway

Rabbit-Ears Hybrids, VSEPR Sterics, and Other Orbital Anachronisms

Pleading for a Dual Molecular-Orbital/Valence-Bond Culture

Predicting the Stability of Cyclic Disulfides by Molecular Modeling: Effective Concentrations in Thiol-Disulfide Interchange and the Design of Strongly Reducing Dithiols

Diselenolane-Mediated Cellular Uptake

n→π* Interactions Modulate the Disulfide Reduction Potential of Epidithiodiketopiperazines

Strain-Promoted Thiol-Mediated Cellular Uptake at the Highest Tension

Cell-Penetrating Dynamic-Covalent Benzopolysulfane Networks

Metabolites from Marine Organisms

Biogenetically-Inspired Total Synthesis of Epidithiodiketopiperazines and Related Alkaloids

The Chemistry of Organic Polysulfanes R−Sn−R (n > 2)

Synthesis, Characterization, Mechanism of Decomposition, and Antiproliferative Activity of a Class of PEGylated Benzopolysulfanes Structurally Similar to the Natural Product Varacin

Total Synthesis of the Novel Benzopentathiepin Varacinium Trifluoroacetate: The Viability of "Varacin-Free Base

Inhibitor of the Tyrosine Phosphatase STEP Reverses Cognitive Deficits in a Mouse Model of Alzheimer's Disease

Cell-Penetrating Streptavidin: A General Tool for Bifunctional Delivery with Spatiotemporal Control, Mediated by Transport Systems Such as Adaptive Benzopolysulfane Networks

Stabilization of γ-Turn Conformations in Peptides by Disulfide Bridging

Structure and Function of Bacillus Subtilis YphP, a Prokaryotic Disulfide Isomerase with a CXC Catalytic Motif

Hallberg, A. Design, Synthesis, and Biological Activities of Four Angiotensin II Receptor Ligands with γ-Turn Mimetics Replacing Amino Acid Residues 3−5

CXC-Mediated Cellular Uptake of Miniproteins: Forsaking "Arginine Magic

Design, Synthesis, and Cellular Uptake of Oligonucleotides Bearing Glutathione-Labile Protecting Groups

Disulfide-Unit Conjugation Enables Ultrafast Cytosolic Internalization of Antisense DNA and siRNA

Intracellular Delivery of Antisense DNA and siRNA with Amino Groups Masked with Disulfide Units

Oligomers of Cyclic Oligochalcogenides for Enhanced Cellular Uptake

Peptide Targeting of an Intracellular Receptor of the Secretory Pathway

Highly Selective Off−On Fluorescent Probe for Imaging Thioredoxin Reductase in Living Cells

Selective Activation of a Prodrug by Thioredoxin Reductase Providing a Strategy to Target Cancer Cells

Combination of Chemotherapy and Oxidative Stress to Enhance Cancer Cell Apoptosis

Thorn-Seshold, O. Cyclic 5-Membered Disulfides Are Not Selective Substrates of Thioredoxin Reductase, but Are Opened Nonspecifically by Thiols

Diselenolane-Mediated Cellular Uptake: Efficient Cytosolic Delivery of Probes, Peptides, Proteins, Artificial Metalloenzymes and Protein-Coated Quantum Dots

Strain-Promoted Thiol-Mediated Cellular Uptake of Giant Substrates: Liposomes and Polymersomes

Growing Prospects of Dynamic Covalent Chemistry in Delivery Applications

From Endocytosis to Nonendocytosis: The Emerging Era of Gene Delivery

Rational Design of Nanocarriers for Intracellular Protein Delivery

Improving Cellular Uptake of Therapeutic Entities through Interaction with Components of Cell Membrane

Dynamic Covalent Polymers for Biomedical Applications

Cell-Penetrating Peptides and Polymers for Improved Drug Delivery

Proteome-Wide Profiling of Targets of Cysteine Reactive Small Molecules by Using Ethynyl Benziodoxolone Reagents

Bicyclobutane Carboxylic Amide as a Cysteine-Directed Strained Electrophile for Selective Targeting of Proteins

Chemical Proteomic Identification of Functional Cysteines with Atypical Electrophile Reactivities

Characterization of Redox-Active Proteins on Cell Surface

Polysulfides as Biologically Active Ingredients of Garlic

The Effect of Allium Sativum (Garlic) Extract on Infectious Bronchitis Virus in Specific Pathogen Free Embryonic Egg

Tunable Heteroaromatic Sulfones Enhance In-Cell Cysteine Profiling

Structure of Mpro from SARS-CoV-2 and Discovery of Its Inhibitors

Multi-Targeting of Functional Cysteines in Multiple Conserved SARS-CoV-2 Domains by Clinically Safe Zn-Ejectors

Potential Therapeutic Use of Ebselen for COVID-19 and Other Respiratory Viral Infections

Molecular Characterization of Ebselen Binding Activity to SARS-CoV-2 Main Protease

Extreme Sulfur Chemistry

Redox Control of Secondary Structure in a Designed Peptide

A Chiral and Colorful Redox Switch: Enhanced π Acidity in Action

Dithienothiophenes at Work: Access to Mechanosensitive Fluorescent Probes, Chalcogen-Bonding Catalysis, and Beyond

Functional Modification of Thioether Groups in Peptides, Polypeptides, and Proteins

Cyclic Thiosulfinates and Cyclic Disulfides Selectively Cross-Link Thiols While Avoiding Modification of Lone Thiols

Nucleophilic Substitution Reactions of Cyclic Thiosulfinates Are Accelerated by Hyperconjugative Interactions

Novel Agents That Downregulate EGFR, HER2, and HER3 in Parallel

Directional Control of a Processive Molecular Hopper

Continuous Observation of the Stochastic Motion of an Individual Small-Molecule Walker

Self-Assembly of Reversed Bilayer Vesicles through Pnictogen Bonding: Water-Stable Supramolecular Nanocontainers for Organic Solvents

Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells

Rotamer-Restricted Fluorogenicity of the Bis-Arsenical ReAsH

Pnictogen-Bonding Catalysis: Brevetoxin-Type Polyether Cyclizations

The Composition of Ehrlich's Salvarsan: Resolution of a Century-Old Debate

One-Dimensional Random Walk of a Synthetic Small Molecule toward a Thermodynamic Sink

Studies of Reversible Conjugate Additions

Template Effects of Vesicles in Dynamic Covalent Chemistry

Quantitative Imaging of Glutathione in Live Cells Using a Reversible Reaction-Based Ratiometric Fluorescent Probe

Small Molecule Inhibitors of Dynamin I GTPase Activity: Development of Dimeric Tyrphostins

Noxious Compounds Activate TRPA1 Ion Channels through Covalent Modification of Cysteines

Replication of Sequence Information in Synthetic Oligomers

Polymerized Liposomes Formed under Extremely Mild Conditions

Ring-Opening Polymerization of Lipoic Acid and Characterization of the Polymer

1,2-Dithiolane-Derived Dynamic, Covalent Materials: Cooperative Self-Assembly and Reversible Cross-Linking

Formation of Polymeric Nanocubes by Self-Assembly and Crystallization of Dithiolane-Containing Triblock Copolymers

Structurally Dynamic Hydrogels Derived from 1,2-Dithiolanes

Assembling a Natural Small Molecule into a Supramolecular Network with High Structural Order and Dynamic Functions

Dual Closed-Loop Chemical Recycling of Synthetic Polymers by Intrinsically Reconfigurable Poly(disulfides)

Toughening a Self-Healable Supramolecular Polymer by Ionic Cluster-Enhanced Iron-Carboxylate Complexes

Polymerized Surface Micelles Formed under Mild Conditions

Poly-Lipoic Ester-Based Coacervates for the Efficient Removal of Organic Pollutants from Water and Increased Point-of-Use Versatility

Selenium-Containing Polymers: Promising Biomaterials for Controlled Release and Enzyme Mimics

Activation of Cell-Penetrating Peptides with Ionpair−π Interactions and Fluorophiles

Spontaneous Conductance Changes, Multilevel Conductance States and Negative Differential Resistance in Oxidized Cholesterol Black Lipid Membranes

Lipid Ion Channels and the Role of Proteins

An Antimicrobial Peptide, Magainin 2, Induced Rapid Flip-Flop of Phospholipids Coupled with Pore Formation and Peptide Translocation

Ion Channels Made from a Single Membrane-Spanning DNA Duplex

Arginine-Rich Peptides Destabilize the Plasma Membrane, Consistent with a Pore Formation Translocation Mechanism of Cell-Penetrating Peptides

Phospholipids and the Origin of Cationic Gating Charges in Voltage Sensors

Strained Cyclic Disulfides Enable Cellular Uptake by Reacting with the Transferrin Receptor

The Transferrin Receptor Part II: Targeted Delivery of Therapeutic Agents into Cancer Cells

Crossing the Iron Gate: Why and How Transferrin Receptors Mediate Viral Entry

Transferrin Receptor Is Another Receptor for SARS-CoV-2 Entry

The Enigma of the CLIC Proteins: Ion Channels, Redox Proteins, Enzymes, Scaffolding Proteins?

Two Decades with Dimorphic Chloride Intracellular Channels (CLICs)

Emerging Biological Roles of

Redox Regulation of CLIC1 by Cysteine Residues Associated with the Putative Channel Pore

Members of the Chloride Intracellular Ion Channel Protein Family Demonstrate Glutaredoxin-Like Enzymatic Activity

Atomistic Insight into Lipid Translocation by a TMEM16 Scramblase

Cryo-EM Structures and Functional Characterization of the Murine Lipid Scramblase TMEM16F

Cryo-EM Studies of TMEM16F Calcium-Activated Ion Channel Suggest Features Important for Lipid Scrambling

Chemically Induced Vesiculation as a Platform for Studying TMEM16F Activity

Structure of the TRPA1 Ion Channel Suggests Regulatory Mechanisms

Evidence for Redox Sensing by a Human Cardiac Calcium Channel

Thiol Modification of Cysteine 327 in the Eighth Transmembrane Domain of the Light Subunit XCT of the Heteromeric Cystine/Glutamate Antiporter Suggests Close Proximity to the Substrate Binding Site/Permeation Pathway

Cysteine Residues in the Organic Anion Transporter MOAT1

EGF Receptor Trafficking: Consequences for Signaling and Cancer

Lead Generation of 1,2-Dithiolanes as Exon 19 and Exon 21 Mutant EGFR Tyrosine Kinase Inhibitors

Advancement in Integrin Facilitated Drug Delivery

Thiolated Polymers: Bioinspired Polymers Utilizing One of the Most Important Bridging Structures in Nature

Initiation of Hepatitis C Virus Infection Is Dependent on Cholesterol and Cooperativity between CD81 and Scavenger Receptor B Type I

Hepatitis C Virus (HCV) Envelope Glycoproteins E1 and E2 Contain Reduced Cysteine Residues Essential for Virus Entry

Proteins of the PDI Family: Unpredicted Non-ER Locations and Functions

Protein Disulfide Isomerases: Redox Connections in and out of the Endoplasmic Reticulum

Galectin-9 Binding to Cell Surface Protein Disulfide Isomerase Regulates the Redox Environment to Enhance T-Cell Migration and HIV Entry

Role of Oxidative Stress on SARS-CoV (SARS) and SARS-CoV-2 (COVID-19) Infection: A Review

Sindbis Virus Membrane Fusion Is Mediated by Reduction of Glycoprotein Disulfide Bridges at the Cell Surface

The Role of Low PH and Disulfide Shuffling in the Entry and Fusion of Semliki Forest Virus and Sindbis Virus

Disulfide Bonds between Two Envelope Proteins of Lactate Dehydrogenase-Elevating Virus Are Essential for Viral Infectivity

Murine Coronavirus Membrane Fusion Is Blocked by Modification of Thiols Buried within the Spike Protein

Significant Redox Insensitivity of the Functions of the SARS-CoV Spike Glycoprotein -Comparison with HIV Envelope

Cell Entry by Enveloped Viruses: Redox Considerations for HIV and SARS-Coronavirus

Localization of the Labile Disulfide Bond between SU and TM of the Murine Leukemia Virus Envelope Protein Complex to a Highly Conserved CWLC Motif in SU That Resembles the Active-Site Sequence of Thiol-Disulfide Exchange Enzymes

Kinetic Analyses of the Surface-Transmembrane Disulfide Bond Isomerization-Controlled Fusion Activation Pathway in Moloney Murine Leukemia Virus

Membrane Fusion Mediated by Baculovirus Gp64 Involves Assembly of Stable Gp64 Trimers into Multiprotein Aggregates

Thiol/Disulfide Exchange Is Required for Membrane Fusion Directed by the Newcastle Disease Virus Fusion Protein

Entry of Hepatitis Delta Virus Requires the Conserved Cysteine Residues of the Hepatitis B Virus Envelope Protein Antigenic Loop and Is Blocked by Inhibitors of Thiol-Disulfide Exchange

Inhibition of a Reductive Function of the Plasma Membrane by Bacitracin and Antibodies against Protein Disulfide-Isomerase

Cell-Mediated Reduction and Incomplete Membrane Translocation of Diphtheria Toxin Mutants with Internal Disulfides in the A Fragment

Protein Disulphide-Isomerase Reduces Ricin to Its A and B Chains in the Endoplasmic Reticulum

Protein-Disulfide Isomerase-Mediated Reduction of the A Subunit of Cholera Toxin in a Human Intestinal Cell Line

Reduction of Furin-Nicked Pseudomonas Exotoxin A: An Unfolding Story

The Disulfide Bond Cys255-Cys279 in the Immunoglobulin-Like Domain of Anthrax Toxin Receptor 2 Is Required for Membrane Insertion of Anthrax Protective Antigen Pore

Structural Analysis and Demonstration of the 29 KDa Antigen of Pathogenic Entamoeba Histolytica as the Major Accessible Free Thiol-Containing Surface Protein

Surface Accessibility of the 70-Kilodalton Chlamydia Trachomatis Heat Shock Protein Following Reduction of Outer Membrane Protein Disulfide Bonds

Protein Disulfide Isomerase, a Component of the Estrogen Receptor Complex

Neospora Caninum Protein Disulfide Isomerase Is Involved in Tachyzoite-Host Cell Interaction

Attachment to Mammalian Cells Requires Protein Disulfide Isomerase

An ERp57-Mediated Disulphide Exchange Promotes the Interaction between

KDa Thioredoxin Protein Disulfide Isomerase of Toxoplasma Gondii during Infection to Human Cells

Cell Entry Mechanisms of SARS-CoV-2

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Neuropilin-1 Facilitates SARS-CoV-2

Inhibitors of HIV Nucleocapsid Protein Zinc Fingers as Candidates for the Treatment of AIDS