Highly Stable trans-Cyclooctene Amino Acids for Live-Cell Labeling

Area of Science:

• Biochemistry
• Organic Chemistry
• Chemical Biology

Background:

• Non-canonical amino acids (ncAAs) functionalized with trans-cyclooctene are valuable tools for bioorthogonal chemistry.
• Efficient synthesis and characterization of these ncAAs are crucial for their application in protein labeling.

Purpose of the Study:

• To improve the synthetic route for axially and equatorially linked trans-cyclooctene isomers.
• To compare the stability and reactivity of these isomers for bioorthogonal applications.
• To evaluate their performance in labeling proteins on intact cells.

Main Methods:

• Improved organic synthesis for trans-cyclooctene isomers.
• Stopped-flow kinetics to determine reaction rates with tetrazines.
• Cell-based assays to assess protein labeling efficiency.

Main Results:

• A highly improved synthetic access to both axial and equatorial trans-cyclooctene isomers was achieved.
• The axial isomer exhibits a 10-fold longer half-life and significantly faster reaction kinetics with tetrazines compared to the equatorial isomer.
• Differential labeling performance of the insulin receptor on intact cells was observed using the two isomers.

Conclusions:

• The axial trans-cyclooctene isomer offers superior stability and reactivity for bioorthogonal chemistry.
• These findings provide enhanced tools for protein modification and imaging in biological systems.
• Improved ncAAs facilitate more effective site-specific protein labeling in cellular environments.