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.

π-Clamp-Mediated Homo- and Heterodimerization of Single-Domain Antibodies via Site-Specific Homobifunctional Conjugation

“…Post-translational protein–protein conjugation strategies are alternatives that permit greater diversity in the resulting conjugates, such as N-to-N, C-to-C, or even internally linked conjugates . Strategies to achieve this include chemically installed click handles, − enzymatic conjugation, tag-based and noncanonical amino acid-based methods, ,− heterobifunctional linking, − and homobifunctional linking. − The latter of these has an inherently simple workflow (Figure ); the linkers are easily synthesized, and diverse linker motifs can be incorporated into protein–protein conjugates.…”

oxSTEF reagents are tunable and versatile electrophiles for selective disulfide-rebridging of native proteins

“…Collectively, this data demonstrates that oxSTEF reagents can perform disulfide rebridging, but, interestingly, might also be used for site-selective N-Cys-modification of peptides. 11,43,44 oxSTEF reagents selectively rebridge disulfides in native proteins Next, we tested the reagents for disulfide rebridging in native proteins using human growth hormone (hGH) as a model system. This protein has two naturally occurring disulfide bonds.…”

“…1,2,3 Furthermore, protein modifications that enhance stability towards thermal denaturation, proteases or which modulate pharmacokinetic properties are of high importance within both biopharma and biocatalysis. 4 , 5 , 6 , 7 Achieving preferential reactivity among numerous (nucleophilic) amino acid residues 3 is a substantial selectivity challenge which in recent years has inspired the development of a collection of (electrophilic) reagents that enable selective functionalization to be performed at intrachain residues 8,9 or the chain termini 10,11,12,13 . Still, the ability to generate homogenous bioconjugates in any setting remains a significant challenge.…”

A stable α-lactam reagent for bioconjugation and proteomic profiling

“…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 many others, [16][17][18][19][20][21][22][23] have been recently pursuing.…”