Site-Specific Chemoselective Cyclization and Fluorogenic Modification of Protein Cysteine Residues: From Side-Chain to Backbone
The selective modification of natural protein templates has emerged as a powerful tool for investigating the protein structure and function as well as for designing therapeutic bioconjugates. While significant progress has been made in modifying protein side chains and terminal groups, backbone modi...
محفوظ في:
| المؤلف الرئيسي: | |
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| مؤلفون آخرون: | , , , , , |
| منشور في: |
2025
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| الموضوعات: | |
| الوسوم: |
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| الملخص: | The selective modification of natural protein templates has emerged as a powerful tool for investigating the protein structure and function as well as for designing therapeutic bioconjugates. While significant progress has been made in modifying protein side chains and terminal groups, backbone modifications remain underexplored due to the inherent inertness of amide bonds and the challenge of achieving site specificity. Despite the critical role of the backbone in the protein function, its selective chemical modification under physiological conditions has proven to be difficult. With this research, we introduce a site-specific, chemoselective, and two-step strategy for protein backbone modification via thiol/amine coupling and cyclization reactions driven by the release of volatile methyl mercaptans via a small-molecular conjugate acceptor, operating on small-molecule cysteine mimics, peptides, and proteins. This approach leverages the unique reactivity of the conjugate acceptor to first couple a cysteine residue, followed by intramolecular cyclization with an adjacent amide, forming a five-membered heterocyclic unit under aqueous conditions without requiring catalysts or heating. Additionally, molecular dynamics simulations reveal that the resulting rigid structure induces a local backbone torsion, hydrogen bond disruption, and altered side-chain orientation, thereby influencing protein folding. Preliminary investigations further explore the consequent changes in the protein thermal stability and enzymatic activity induced by backbone modification. More importantly, the process is accompanied by a fluorescence turn-on signal, enabling the real-time in situ monitoring of the modification process. Thus, this unique strategy offers a new platform for backbone-specific chemical modifications, paving the way for potential protein and peptide functional studies, fluorogenic labeling, and the development of novel bioconjugates. |
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