Thia-Michael addition: the route to promising opportunities for fast and cysteine-specific modification.

Over the last few decades, design and discovery of chemical reactions that enable modification of proteins at pre-determined sites have been the focus of synthetic organic chemists. As an invaluable tool, the site-and chemoselective functionalization of peptides and proteins offers an exciting opportunity for creating high-value multicomponent conjugates with diverse applications in life sciences and pharmacology. In recent years, multiple strategies have emerged that target natural amino acids directly or convert them into other reactive species for further ligations. However, reactivity and selectivity are still key issues in the current state of chemical modification methodologies. Cysteine is one of the least abundant amino acids and exhibits unique chemistry of the thiol or thiolate group which makes it susceptible to a series of post-translational modifications. The thia-Michael "click" addition reactions, which can proceed under facile conditions provide a promising way for thiol-selective modification of cysteine-containing proteins. In this review, we summarize various reactions for cysteine-selective peptide and protein modification, focus on thia-Michael "click" addition reactions, elaborate on their historical perspective and mechanism, and highlight their applications in modifying biomolecules in a site-specific way.

N-Vinyl Acrylamides: Versatile Heterobifunctional Electrophiles for Thiol-Thiol Bioconjugations.

Herein we report the first examples of thiol-selective heterobifunctional electrophiles, N-vinyl acrylamides, that enable efficient highly selective thiol-thiol bioconjugations and cysteine modification of peptides. We demonstrate that these new classes of thiol-selective scaffolds can readily undergo a thia-Michael addition and an orthogonal radical induced thiol-ene "click" reaction under biocompatible conditions. Furthermore, the formation of an unexpected Markovnikov N,S-acetal hydrothiolation was explained using computational studies. We also reveal that N-methylation of the N-vinyl acrylamide scaffold changes the regioselectivity of the reaction. We demonstrate that use of N-vinyl acrylamides shows promise as an efficient, mild, and exquisite cysteine-selective protocol for facile construction of fluorophore-labeled peptides and proteins and that the resultant conjugates are resistant to degradation and thiol exchange, thus significantly improving their biophysical properties.

Photo-induced radical thiol-ene chemistry: a versatile toolbox for peptide-based drug design.

While the global market for peptide/protein-based therapeutics is witnessing significant growth, the development of peptide drugs remains challenging due to their low oral bioavailability, poor membrane permeability, and reduced metabolic stability. However, a toolbox of chemical approaches has been explored for peptide modification to overcome these obstacles. In recent years, there has been a revival of interest in photoinduced radical thiol-ene chemistry as a powerful tool for the construction of therapeutic peptides.