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

Area of Science:

• Bioconjugation Chemistry
• Protein Engineering
• Medicinal Chemistry

Background:

• Protein conjugates are crucial for therapeutics and biological studies.
• Selective modification of specific amino acid residues on proteins remains a significant challenge.
• Existing methods often struggle to differentiate between various reactive sites within a protein.

Purpose of the Study:

• To develop a novel method for selective protein modification at N-terminal cysteine residues.
• To create versatile cyclopropenone (CPO) reagents for targeted bioconjugation.
• To demonstrate the utility of CPO reagents in constructing complex protein conjugates and mimics.

Main Methods:

• Utilized monosubstituted cyclopropenone (CPO) reagents for selective reaction with N-terminal cysteine 1,2-aminothiol groups.
• Developed CPO-based reagents from a common activated ester, CPO-pentafluorophenol (CPO-PFP).
• Employed quantum mechanical calculations to elucidate the selectivity mechanism of CPO reagents.

Main Results:

• Achieved stable 1,4-thiazepan-5-one linkage formation under mild, biocompatible conditions.
• Demonstrated selective modification of N-terminal cysteine in peptides and proteins, even with internal cysteines present.
• Successfully prepared a dual-labeled protein conjugate (2×cys-GFP) and a protein mimic dimer of IL2 with low nanomolar affinity for the β-IL2 receptor.
• Confirmed compatibility of CPO reagents with dithiothreitol (DTT).

Conclusions:

• CPO reagents offer a powerful and selective method for targeting N-terminal cysteine residues in proteins.
• This approach facilitates the construction of complex, multilabeled bioconjugates with enhanced precision.
• The developed strategy holds significant potential for advancing protein-based therapeutics and research tools.