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

Abstract

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.

α‐Lactam Electrophiles for Covalent Chemical Biology**

“…[4,[11][12][13] As a separate area of application, many bioconjugation methods also rely on electrophilic agents with the aim of achieving selective biomolecular labelling with residue-or sequence-specific resolution. The introduction of new electrophilic motifs can therefore have broad impact, and this is an area that we, [14,15] as well as others, [16][17][18][19][20][21][22][23] have been recently pursuing.…”

One-Pot Sequence of Staudinger/aza-Wittig/Castagnoli–Cushman Reactions Provides Facile Access to Novel Natural-like Polycyclic Ring Systems

“…This compound was obtained according to a slightly modified literature procedure [ 31 ] . Sodium azide (5.85 g, 90 mmol, 3.0 eq.)…”

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

“…Recently, Kavianinia and Brimble reported the first examples of thiol-selective heterobifunctional electrophiles, N -vinyl acrylamides 85 , that enable the execution of thiol–thiol conjugation in a site-selective manner to afford stable, irreversible conjugate addition adducts when exposed to GSH rendering N -vinyl acrylamides as a valuable alternative to commonly used maleimides (Scheme 25). 90 These new classes of thiol-selective scaffolds possess both an α,β-unsaturated Michael acceptor that can readily undergo a thia-Michael addition and a vinyl amide functionality that is amenable to radical induced thiol–ene “click” reaction (Scheme 25a). In the course of this work, it was discovered that the enamide functionality of the N -vinyl acrylamide linker is highly reactive towards nucleophilic addition of thiols and can participate in a rapid, green, and unusual Markovnikov-selective hydrothiolation reaction in less than one minute (Scheme 25b).…”

“…The utility of the N -methyl- N -vinyl acrylamide linker for facile construction of fluorophore-labelled peptides and proteins was also demonstrated. 90…”