Bioorthogonal Cycloadditions with Sub-Millisecond Intermediates.

Area of Science:

• Bioconjugation Chemistry
• Chemical Biology
• Single-Molecule Biophysics

Background:

• Tetrazine and sydnone click reactions are vital bioconjugation tools, known for rapid kinetics and benign byproducts (N2 or CO2).
• Previous mechanistic studies primarily investigated the initial cycloaddition steps, with N2 or CO2 release from intermediates largely explored computationally, predicting femtosecond lifetimes.

Purpose of the Study:

• To experimentally investigate the N2 or CO2 extrusion step in bioorthogonal click reactions at the single-molecule level.
• To determine the experimental lifetimes of bicyclic intermediates in these reactions and compare them with computational predictions.

Main Methods:

• Utilized single-molecule experiments within a protein nanoreactor to observe bioorthogonal cycloadditions.
• Applied high-resolution single-molecule techniques to probe reaction kinetics and intermediate lifetimes.

Main Results:

• Observed bioorthogonal cycloadditions involving N2 or CO2 extrusion at the single-molecule level.
• The reactions, at this resolution, appeared to proceed in a single step.
• Established an upper limit for the lifetimes of the bicyclic intermediates at approximately 80 microseconds.

Conclusions:

• Experimental single-molecule observations provide crucial insights into the kinetics of N2 or CO2 extrusion in bioorthogonal click reactions.
• The experimentally determined intermediate lifetimes are consistent with prior computational predictions, validating theoretical models.
• This work bridges the gap between computational predictions and experimental reality for key steps in bioorthogonal chemistry.