Selective N-Terminal Cysteine Protein Modification with Cyclopropenones

Abstract

<div><div><div><p>Protein conjugates are valuable tools to create therapeutics, such as antibody-drug conjugates, or to study biological processes. Despite a number of protein conjugation strategies having been developed over recent years, the ability to modify one specific amino acid on a protein in the presence of other side chains with similar reactivity remains a challenge. We used the reaction between a monosubstituted cyclopropenone (CPO) probe and the 1,2-aminothiol of an N-terminal cysteine to give a stable 1,4-thiazepa-5-none linkage under mild, biocompatible conditions. The method relies on the ability of cyclopropenones to ring-open after sequential sulfhydryl and α-amine conjugation to be truly specific for N-terminal cysteine. We show that our CPO probes selectively label N-terminal cysteine containing peptides and proteins even in the presence of internal, solvent-exposed cysteines, which can be subsequently modified by using conventional cysteine modification reagents. The ability to distinguish and specifically target N-terminal cysteine residues on a protein will facilitate the construction of elaborate multi-labelled bioconjugates.</p></div></div></div>

Recent advances in protein modifications techniques for the targeting N‐terminal cysteine

“…[60] Recently, Bernardes et al developed a chemical approach that utilizes monosubstituted cyclopropenone. [78] This compound reacts with 1,2-aminothiols of cysteine, peptides, and proteins in base-containing buffers, to generate a heterocyclic 1,4-thiazepa-5-none bridge (Figure 10). The reagent also exhibits an exceptional stability profile, as evidenced by incubation in phosphate buffers (50 mM, pH 7-8) at 37 C for days.…”

Fast and Selective Reaction of 2-Benzylacrylaldehyde with 1,2-Aminothiol for Stable N-Terminal Cysteine Modification and Peptide Cyclization

“…30 Monosubstituted cyclopropenone (CPO) has also been exploited for selective N-terminal Cys modification with a reaction rate (3.0 M −1 s −1 ) comparable to that for the CBTcysteine reaction (9.19 M −1 s −1 ). 20,31 In addition, the reaction of 2-((alkylthio)(aryl)methylene) malononitrile (TAMM) and 1,2-aminothiol have been developed recently as a novel bioorthogonal reaction (4.2 M −1 s −1 ) for site-specific protein modifications and peptide cyclization. 22 Particularly, this reaction has also been exploited for the construction of cyclic peptide libraries displayed on the phage surface.…”