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>

Development and Recent Advances in Lysine and N-Terminal Bioconjugation for Peptides and Proteins

“…After that, Bernardes and co-workers discovered a selective reaction between biocompatible and stable monosubstituted cyclopropenones 62 (CPOs) and the 1,2-aminothiol of N-terminal cysteine to afford a heterocyclic 1,4-thiazepa-5-none linker under mild conditions in 2020 (Figure 7f) [137,138]. Notably, cyclopropenones showed diverse structural advantages including the aromatic character rendering their stability, ring strain, and high dipole moment, allowing the occurrence of cycloaddition and ring-opening reactions, and α,β-unsaturated ketones acting as electrophiles in 1,2-and 1,4-nucleophilic addition reactions.…”

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

“…In addition, the reaction between 2-cyanobenzothiazole (CBT) and Cys, which is the last step of the synthesis of d -luciferin, has been developed into a useful ligation strategy for protein labeling and functional macrocycle construction. ,, Recently, N -hydroxysuccinimide activated acrylamide has been designed and synthesized for both the functionalization of N-terminal Cys and the cyclization of in-chain or C-terminal Cys and nearby lysine (Lys) residues . 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 CBT-cysteine reaction (9.19 M –1 s –1 ). , 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 . Particularly, this reaction has also been exploited for the construction of cyclic peptide libraries displayed on the phage surface .…”