Thiol-based Michael-type addition. A systematic evaluation of its controlling factors

Abstract This paper is about the factors controlling kinetics and product stability of this popular bioconjugation reaction. We demonstrate that a) thiol pKa, i.e. the amount of thiolates, is the only determinant of the reaction kinetics for the nucleophile; b) product degradation occurs primarily via hydrolysis (not thiol exchange), and is more prominent for the most rapidly reacting electrophiles. In terms of molecular design, acrylamides and low pKa thiols appear as the reaction partners that provide the best compromise for stability and reaction rate.