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

Abstract

This work addresses the need for chemical tools that can selectively form cross-links. Contemporary thiol-selective cross-linkers, for example, modify all accessible thiols, but only form cross-links between a subset. The resulting terminal "dead-end" modifications of lone thiols are toxic, confound cross-linking-based studies of macromolecular structure, and are an undesired, and currently unavoidable, byproduct in polymer synthesis. Using the thiol pair of Cu/Zn-superoxide dismutase (SOD1), we demonstrated that cyclic disulfides, including the drug/nutritional supplement lipoic acid, efficiently cross-linked thiol pairs but avoided dead-end modifications. Thiolate-directed nucleophilic attack upon the cyclic disulfide resulted in thiol-disulfide exchange and ring cleavage. The resulting disulfide-tethered terminal thiolate moiety either directed the reverse reaction, releasing the cyclic disulfide, or participated in oxidative disulfide (cross-link) formation. We hypothesized, and confirmed with density functional theory (DFT) calculations, that mono- S-oxo derivatives of cyclic disulfides formed a terminal sulfenic acid upon ring cleavage that obviated the previously rate-limiting step, thiol oxidation, and accelerated the new rate-determining step, ring cleavage. Our calculations suggest that the origin of accelerated ring cleavage is improved frontier molecular orbital overlap in the thiolate-disulfide interchange transition. Five- to seven-membered cyclic thiosulfinates were synthesized and efficiently cross-linked up to 10-fold faster than their cyclic disulfide precursors; functioned in the presence of biological concentrations of glutathione; and acted as cell-permeable, potent, tolerable, intracellular cross-linkers. This new class of thiol cross-linkers exhibited click-like attributes including, high yields driven by the enthalpies of disulfide and water formation, orthogonality with common functional groups, water-compatibility, and ring strain-dependence.

Nucleophilic substitution reactions of cyclic thiosulfinates are accelerated by hyperconjugative interactions

“…[13][14][15] Cyclic disuldes are one of the rst known crosslinking-specic molecules; Agar and co-workers showed that they could selectively cross-link cysteine pairs while reversibly modifying lone cysteines in vivo. 16 Our groups recently introduced a six-membered cyclic thiosulnate (1,2-dithiane-1-oxide) capable of cross-linking free cysteine pairs up to 10 4 -fold faster than a sixmembered cyclic disulde (1,2-dithiane), by circumventing the rate-determining oxidation step. 16 A covalent cross-link is efficiently formed between the sulfenic acid intermediate and a second thiolate.…”

“…16 Our groups recently introduced a six-membered cyclic thiosulnate (1,2-dithiane-1-oxide) capable of cross-linking free cysteine pairs up to 10 4 -fold faster than a sixmembered cyclic disulde (1,2-dithiane), by circumventing the rate-determining oxidation step. 16 A covalent cross-link is efficiently formed between the sulfenic acid intermediate and a second thiolate. Whitesides and coworkers augmented their experiments with MM2 17 calculations to show that the reactivities of cyclic disuldes towards biological thiolate-based nucleophiles were strain-promoted.…”

Molecular and pharmacological chaperones for SOD1

“…Similarly, cyclic disulphides have been shown to tether opposing Cys111 residues, prevent dead-end single thiol modifications and are cell penetrable. Mono-S-oxo derivatives including 1,2-dithiane-1-oxide were able to bind and tether 95% of SOD1 monomers as dimers within cells over 30 min with an EC 50 of 5 μM [82]. Taking another approach, cisplatin increases metal-free SOD1 thermal stability and is able to prevent or reverse SOD1 aggregation within neuron-like cells without subunit tethering [83].…”

Nucleophilic Substitution Reactions of Cyclic Thiosulfinates Are Accelerated by Hyperconjugative Interactions

“…[13][14][15] Cyclic disulfides are one of the first known cross-linking-specific molecules; Agar and co-workers showed that they could selectively cross-link cysteine pairs while reversibly modifying lone cysteines in vivo. 16 Our groups recently introduced a six-membered cyclic thiosulfinate (1,2-dithiane-1-oxide) capable of cross-linking free cysteine pairs up to 10 4 -fold faster than a six-membered cyclic disulfide (1,2-dithiane), by circumventing the rate-determining oxidation step. 16 A covalent cross-link is efficiently formed between the sulfenic acid intermediate and a second thiolate.…”

“…16 Our groups recently introduced a six-membered cyclic thiosulfinate (1,2-dithiane-1-oxide) capable of cross-linking free cysteine pairs up to 10 4 -fold faster than a six-membered cyclic disulfide (1,2-dithiane), by circumventing the rate-determining oxidation step. 16 A covalent cross-link is efficiently formed between the sulfenic acid intermediate and a second thiolate. Whitesides and coworkers augmented their experiments with MM2 17 calculations to show that the reactivities of cyclic disulfides towards biological thiolate-based nucleophiles were strain-promoted.…”