Mechanisms of Alkyl and Aryl Thiol Addition to <i>N</i>‑Methylmaleimide

A mechanistic study was undertaken to elucidate the reaction pathways for thiol addition to N-methylmaleimide in water. We used linear free energy relationships, solvent kinetic isotope effects (SKIEs), activation parameters, and ionic strength effects to probe the nature of the rate-limiting transition states. Calculations were also employed and assisted in illuminating three possible mechanistic pathways: (1) stepwise addition with rate-limiting nucleophilic attack, (2) stepwise addition with rate-limiting proton transfer, and (3) concerted addition with nucleophilic attack and proton transfer occurring concurrently. Alkyl thiolate addition exhibits βnucRS−= 0.4, small negative ΔS‡ values, prominent ionic strength effects, and no evidence of general acid catalysis, consistent with pathway 1. Aryl thiolate addition exhibited βnucArS− = 1.0, large negative ΔS‡ values, normal primary SKIEs, general acid catalysis, and negligible sensitivity to ionic strength, consistent with pathways 2 and 3. The experimental and computational data depict an energy surface where ground state effects, namely the energy of the alkyl/aryl thiolate, play a major role in shaping the governing pathway. Application of these findings to bioconjugation chemistry is also discussed.