thiolspecific

Thiol-specific describes reagents, probes, and reactions that selectively target sulfhydryl groups (-SH) of thiols, most notably the cysteine residues in proteins and low-molecular-weight thiols such as glutathione. The term is used in chemical biology, proteomics, and bioconjugation to covalently modify, label, or block thiols while minimizing reaction with other nucleophiles.

The chemistry relies on the thiolate form of a thiol being a nucleophile, often generated by mild base or by the intrinsic pKa of cysteine around 8.3. Reactions include alkylation (iodoacetamide, iodoacetic acid), Michael-type additions to maleimides (N-ethylmaleimide, other maleimide derivatives), and disulfide exchange or formation. These approaches enable stable covalent linkages or reversible interactions depending on reagent choice and conditions.

Common reagents and their outcomes include iodoacetamide and iodoacetic acid, which alkylate thiols to form stable thioether linkages; N-ethylmaleimide and related maleimide reagents, which form thioether conjugates with thiols. Fluorescent tags or affinity handles are frequently introduced via maleimide or related thiol-reactive handles for selective labeling of cysteine-containing targets.

Applications span proteomic sample preparation, fluorescent labeling of proteins, affinity tagging and immobilization, and the selective blocking of free thiols to prevent disulfide reshuffling or unwanted oxidation during experiments.

Considerations include the pH dependence of thiol reactivity, local protein environment, and thiol pKa, which influence selectivity and rate. Some thiol reagents can react with amines at higher pH, and over-alkylation or hydrolysis can occur; in some contexts, reversible thiol-reactive chemistries are used for dynamic labeling.