The roles of Cysteines in living organisms are essential. As mentioned previously, thiol-disulfide exchange helps to maintain protein structures. Cysteine derivatives such as glutathione protect cells from oxidative stress. The presence of Cysteines in enzymes is critical for their functions. In addition, Cysteines participate in complexing various metals in a broad range of proteins.169–171 Since the functions of Cysteine are mostly based on the nucleophilicity of the sulfur atom at the side chain, a wide variety of thiol-reactive reagents has been developed in the last decades to selectively and efficiently target sulfhydryl group of Cysteines. These probes have been extensively used to detect and modulate Cysteines which resulted various useful protein inhibitors, sensors or chemical proteomic probes. Numerous reactions have been employed to access Cysteine reactivities in mildest conditions, for instances, Michael addition,
65 cyclization, disulfide cleavage, metal complexation or oxidations.170 Some popular probes are listed in Figure 38. Since the utilization of such probes has been review recently,169–171 In this section, only some probes related to the content of this thesis would be discussed.
Figure 38. Some common thiol-reactive reagents. Adapted from reference.171
As an attempt to find a less toxic reagent for Cysteines cross-linking in proteins which could avoid “dead-end” modification of lone thiols, Agar et al. introduced cyclic thiosulfinates in 2018.172 A thiolate could trigger ring cleavage in cyclic thiosulfinate 146 to yield a disulfide
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bond and a sulfenic acid moiety (Figure 39b). The resulting terminal sulfenic acid could participate in further disulfide formation after condensation with another thiolate in close proximity. In comparison with the more popular cyclic disulfide 147, the cross-linking process with the alternative thiosulfinate was much favored. For 147, after ring opening, oxidation of sulfhydryl group to sulfenic acid was required for disulfide formation (Figure 39a).
Figure 39. Reaction of (a) cyclic disulfide 147 and (b) cyclic thiosulfinate 146 with two adjacent Cysteines. Adapted from reference.172
Since the oxidation step was the rate-determining step and extremely slow (t1/2 = 10 days), cyclic thiosulfinate was expected to be more than 104 times efficient to cross-link adjacent Cysteines. Applying this novel thiol-reactive probe, the authors were successfully to cross-linked Cu/Zn-superoxide dismutase (SOD1) monomers to form dimeric structure with minimal side reactions. This method could be used even in living cells. The most important advantage
67 for cyclic thiosulfinates was that the new covalent bridges were formed based on disulfide bonds which could easily participate in thiol-disulfide exchange. Thus, the initial structure could be restored and led to low cytotoxicity.172
Moving from cyclic thiosulfinate to cyclic thiosulfonate – the more oxidized derivative of cyclic disulfide, Law et al. have shown that they were excellent probes for disturbing disulfide bonds in proteins.173 Similar to the less oxidized alternatives, cyclic thiosulfonates like 148 could react with thiolate to form a disulfide bond and release sulfinate functionality (Figure 40). The nucleophilicity of sulfinate group allows it to attack nearby disulfide bonds to yield a linear thiosulfonate and a free thiolate which could continue to exchange with other disulfides. Anticancer activity of sulfinate derivatives was believed to relate to its ability to cleave disulfide bonds in Epidermal Growth Factor Receptor (EGFR) which disrupted EGFR structure.173
Figure 40. Reaction of cyclic thiosulfonate 148 and thiolate results sulfinate group which could disrupt disulfide bonds in proteins. Adapted from reference.173
Apart from reversible thiol-reactive reagents, irreversible thiol-blocking agents have also been actively used to modify Cysteines in proteins. Even though “dead-end” modification of proteins usually leads to high toxicity in living systems, the great reactivity of such probes is
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undeniable. Most of them show activity at concentrations much lower than toxic concentration.
One class of highly selective irreversible thiol-blocking reagents for proteins modification is heteroaromatic sulfones which was first reported by Xian et al. in 2012.174 In biologically relevant conditions, heteroaromatic sulfone such as methylsulfonyl benzothiazole (MSBT) selectively reacts with free thiol via nucleophilic aromatic substitution. Reactions could be completed within minutes without signs of any side reactions. Other functional groups which are popular in protein structure like amino or hydroxy groups remained inactive. In addition, the resulting product shows excellent stability toward different reductants and oxidants over a long time. MSBT was also shown to be extremely efficient in blocking free thiol residues in Glyceraldehyde 3-phosphate dehydrogenase (GAPDH).174 Following this study, Barbas et al.
extended the family of heteroaromatic sulfones by varying structures of the heterocycles.175 Replacing benzothiazole ring in MSBT by phenyltetrazine or phenyloxadiazole greatly enhanced the reactivity of the probe. They were applied to couple fluorophore or polyethylene glycol (PEG) into protein structure. More detailed studies concerned other factors which could affect heteroaromatic sulfones properties such as substituents at the aromatic ring, nature of the sulfone leaving group were addressed by Martin et al.176 Different modifications were utilized to access a big library of heteroaromatic sulfones with reactivity differences spread across several order of magnitudes. The best probes could be used in Cysteine proteomic studies.
Even though thiol-reactive reagents have been considered as a very important tool in chemical biology, in the context of thiol-mediated uptake, thiol-reactive reagents have been scarcely explored for developing new transporters. Their roles as universal thiol-mediated uptake inhibitors are almost untouched. Therefore, it could be of interest to repurpose existed highly reactive probes for inhibiting thiol-mediated uptake or use them for other applications.
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CHAPTER 2:
OBJECTIVES
For π catalysis project, the first objective is to apply peptide secondary structure to anion-π catalyst design. The rigid helical structure of α-helices will create precise relative positions between amino acid residues in peptide sequence and anion-π catalyst. A high level of conformation control will provide better understanding of structure activity relationship. On the other hand, the chiral environment of peptides around anion-π catalyst will contribute to asymmetric anion-π catalysis. For this purpose, NDI will be used as covalent linker to stabilize one turn of an α-helix. Introduction of a tertiary amine to a side chain of one amino acid residue in peptide sequence will turn on anion-π catalysis above NDI surface. Modifications in peptide backbone will give access to a library of catalysts. The structure of catalysts will be studied by various spectroscopic techniques. Their catalytic activity will be evaluated using MAHT reaction (a benchmark reaction to probe anion-π catalysis). Information obtained for catalyst structures and their catalytic activities will allow rationalizing catalyst design.
To provide a more versatile stapling method to integrate anion-π catalysts into peptide structure, PDI will also be chosen to stabilize one helix peptide turn. A larger PDI surface will enable anion-π catalysis for peptide sequences that could not operate effectively with NDI.
Besides, planar chirality created by twisting PDI surface could be advantageous to improve enantioselectivity of the systems. A library of PDI-peptide conjugate catalysts will be
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synthesized following the developed method for NDI. Their secondary structures will be studied by different spectroscopic techniques. To get a direct comparison with NDI catalysts, PDI catalysts will be tested using MAHT reaction. Their catalytic performance together with catalyst secondary structure will again allow rationalizing structure reactivity relationship.
Finally, highly electron-deficient additives will be added to catalytic system to examine the influence of induced polarization to catalyst activities.
For thiol-mediated uptake inhibitor project, a big library of reversible and irreversible inhibitors will be developed. A wide range of thiol-reactive reagents will be chosen for the library. For reversible inhibitors, the focus will be kept on thiosulfonates and disulfide bridged γ-turn peptides because of their selective reactivity toward free thiol and low cytotoxicity.
Various modifications will be applied for these scaffolds to tun their reactivity. Among irreversible inhibitors, heteroaromatic sulfones will be the major class since their reactivity could be change significantly by simply utilizing different heteroaromatic cycles. The potential application of thiosulfonates as thiol-mediated uptake transporters will be addressed by synthesizing fluorescently labelled derivatives.
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