Exocyclic Olefinic Maleimides: Synthesis and Application for Stable and Thiol-Selective Bioconjugation.

Michael addition reactions between biological thiols and endocyclic olefinic maleimides are extensively used for site-specific bioconjugation. The resulting thio-succinimidyl linkages, however, lack stability because of their susceptibility to thiol exchange. Reported herein is that in contrast to their endocyclic counterparts, exocyclic olefinic maleimides form highly stable thio-Michael adducts which resist thiol exchange at physiological conditions. A high-yielding approach for synthesizing a variety of exocyclic olefinic maleimides, by 4-nitrophenol-catalyzed solvent-free Wittig reactions, is reported. Mechanistic studies reveal that the catalyst facilitates the formation of the Wittig ylide intermediate through sequential proton donation and abstraction. Overall, this report details an improved thiol bioconjugation approach, a facile method for synthesizing exocyclic olefinic maleimides, and demonstrates that phenolic compounds can catalyze ylide formation.