Platform for Orthogonal N-Cysteine-Specific Protein Modification Enabled by Cyclopropenone Reagents.

Abstract

Protein conjugates are valuable tools for studying biological processes or producing therapeutics, such as antibody-drug conjugates. Despite the development of several protein conjugation strategies in recent years, the ability to modify one specific amino acid residue on a protein in the presence of other reactive side chains remains a challenge. We show that monosubstituted cyclopropenone (CPO) reagents react selectively with the 1,2-aminothiol groups of N-terminal cysteine residues to give a stable 1,4-thiazepan-5-one linkage under mild, biocompatible conditions. The CPO-based reagents, all accessible from a common activated ester CPO-pentafluorophenol (CPO-PFP), allow selective modification of N-terminal cysteine-containing peptides and proteins even in the presence of internal, solvent-exposed cysteine residues. This approach enabled the preparation of a dual protein conjugate of 2×cys-GFP, containing both internal and N-terminal cysteine residues, by first modifying the N-terminal residue with a CPO-based reagent followed by modification of the internal cysteine with a traditional cysteine-modifying reagent. CPO-based reagents enabled a copper-free click reaction between two proteins, producing a dimer of a de novo protein mimic of IL2 that binds to the β-IL2 receptor with low nanomolar affinity. Importantly, the reagents are compatible with the common reducing agent dithiothreitol (DTT), a useful property for working with proteins prone to dimerization. Finally, quantum mechanical calculations uncover the origin of selectivity for CPO-based reagents for N-terminal cysteine residues. The ability to distinguish and specifically target N-terminal cysteine residues on proteins facilitates the construction of elaborate multilabeled bioconjugates with minimal protein engineering.