Site-Selective Cysteine-Cyclooctyne Conjugation.

Area of Science:

• Bioconjugation Chemistry
• Chemical Biology
• Molecular Biology

Background:

• Site-selective modification of peptides and proteins is crucial for developing bioconjugates.
• Traditional cysteine modification methods can lack selectivity, especially in proteins with multiple cysteine residues.
• Thiol-yne click chemistry offers a bioorthogonal reaction for bioconjugation but can be slow in aqueous environments.

Purpose of the Study:

• To develop a peptide tag that accelerates site-selective cysteine-cyclooctyne conjugation.
• To demonstrate the utility of this tag for labeling peptides and proteins with various functional molecules.
• To enable regioselective modification of proteins containing multiple endogenous cysteines.

Main Methods:

• A seven-residue peptide tag (DBCO-tag) containing a cysteine residue was designed.
• The conjugation efficiency and reaction rate of the DBCO-tag with aza-dibenzocyclooctyne (DBCO) reagents were evaluated.
• The DBCO-tag was fused to peptides and proteins, including green fluorescent protein and the antibody trastuzumab, to assess its performance in complex biological systems.

Main Results:

• The DBCO-tag increased the rate of the thiol-yne reaction by 220-fold compared to a cysteine peptide control.
• Selective conjugation of the DBCO-tag to DBCO-linked fluorescent probes, affinity tags, and cytotoxic drug molecules was achieved.
• Regioselective cysteine modification was successfully demonstrated on proteins with multiple endogenous cysteines, such as green fluorescent protein and trastuzumab.

Conclusions:

• Short peptide tags can significantly accelerate slow bond-forming reactions in aqueous media.
• The DBCO-tag facilitates site-selective and regioselective bioconjugation via thiol-yne click chemistry.
• This approach provides a versatile tool for labeling peptides and proteins for various applications in chemical biology and drug development.