Structural and mechanistic effects in one-electron oxidation of model disulfides

Abstract Disulfide bonds are abundant and critical structural elements that stabilize approximately 21% of all proteins, yet their oxidative degradation pathways remain significantly less characterized than their thiol precursor, cysteine, and the thioether methionine. This study employed nanosecond laser flash photolysis (LFP) and high-resolution LC-MS/MS to elucidate the mechanism of the one-electron photooxidation of two model disulfides - the cyclic species trans-4,5-dihydroxy-1,2-dithiane (DHDT) and the linear disulfide cystamine (CA) - sensitized by the excited triplet state of 3-carboxybenzophenone (3CB). Kinetic analysis reveals that the quenching rate constant for DHDT ( k q = 8.5×10 9 M −1 s − 1 ) is significantly higher than that for linear CA ( k q = 3.9×10 9 M −1 s − 1 ) mirroring structural reactivity trends previously observed with both 1 O 2 and two-electron oxidants. In contrast to previous studies, direct spectroscopic identification of primary intermediates, including the disulfide radical-cation (− S ●+ −S−; identified as a transient absorption at ~ 380 nm) and the 3CB radical anion (broad absorption at ~ 600 nm) was achieved on a nanosecond timescale. Stable products arising from decay of the disulfide radical-cation were determined by LC-MS/MS, with three competing mechanistic pathways identified: (i) deprotonation yielding carbon-centred α-thioalkyl radicals and subsequent αS-αS dimers; (ii) disulfide (S-S) bond cleavage leading to products containing a single sulfur atom; and (iii) carbon-sulfur (C-S) bond cleavage which fragments the molecule whilst leaving the disulfide bridge intact. These findings provide a comprehensive map of the early events and subsequent free-radical cascades of disulfide oxidation, offering essential insights into the light-induced degradation of disulfide-containing proteins.