Tetrazine-Enabled Peptide Macrocyclization and Bioorthogonal Cell Penetration Profiling

Area of Science:

• Medicinal Chemistry
• Chemical Biology
• Peptide Therapeutics

Background:

• Macrocyclic peptides offer enhanced stability and permeability compared to linear peptides.
• Cysteine-selective conjugation is crucial for developing targeted peptide therapeutics.
• Bioorthogonal chemistry allows for precise molecular modifications in biological systems.

Purpose of the Study:

• To develop a cysteine-selective macrocyclization strategy using tetrazines for unprotected peptides.
• To synthesize and evaluate novel cancer-associated fibroblast activation protein (FAP)-targeting peptides.
• To create a bioorthogonal cell penetration assay (BCPA) for quantitative peptide internalization studies.

Main Methods:

• Employed a chloroethyl tetrazine motif for cysteine-selective alkylation of unprotected peptides.
• Synthesized rofapitide tetraxetan analogues, including novel peptide 2o.
• Developed a "click-to-release" assay using tetrazine-peptide conjugates and TCO-caged Nile Blue dye.

Main Results:

• Efficiently produced cyclic peptides with bioorthogonal tetrazine handles.
• Identified peptide 2o with potent in vitro and in vivo binding to FAP.
• Demonstrated quantitative evaluation of peptide internalization using the BCPA.

Conclusions:

• The tetrazine-mediated macrocyclization strategy enables efficient synthesis of functional cyclic peptides.
• This approach facilitates the discovery of targeted therapeutics, such as FAP inhibitors.
• The developed BCPA offers a novel tool for assessing peptide uptake in drug discovery and research.