Single-Molecule Observation of Intermediates in Bioorthogonal 2-Cyanobenzothiazole Chemistry.

Yujia Qing,William J. Ramsay,Mira D. Liu,Denis Hartmann,Linna Zhou,Hagan Bayley

doi:10.1002/anie.202005729

Abstract

We report a single‐molecule mechanistic investigation into 2‐cyanobenzothiazole (CBT) chemistry within a protein nanoreactor. When simple thiols reacted reversibly with CBT, the thioimidate monoadduct was approximately 80‐fold longer‐lived than the tetrahedral bisadduct, with important implications for the design of molecular walkers. Irreversible condensation between CBT derivatives and N‐terminal cysteine residues has been established as a biocompatible reaction for site‐selective biomolecular labeling and imaging. During the reaction between CBT and aminothiols, we resolved two transient intermediates, the thioimidate and the cyclic precursor of the thiazoline product, and determined the rate constants associated with the stepwise condensation, thereby providing critical information for a variety of applications, including the covalent inhibition of protein targets and dynamic combinatorial chemistry.

Highlights

Orthogonal chemistry that proceeds rapidly at mild temperatures in aqueous solution is desired for the chemical manipulation of biological systems
We envision that CBT chemistry will be a strong addition to the dynamic covalent chemistry toolbox and that the rate constants derived here will be key in achieving fine control of such systems.[36]
A multi-cysteine track can potentially interact with CBT molecules through consecutive dynamic covalent reactions, for which the association and dissociation kinetics determine the processivity of the mobile molecules.[22,37]

Summary

Introduction

Orthogonal chemistry that proceeds rapidly at mild temperatures in aqueous solution is desired for the chemical manipulation of biological systems. Individual bond-making and bondbreaking events within the nanoreactor caused characteristic time-dependent changes in ionic current flow under an applied potential.[15] The nanoreactor approach is ideal for studying CBT chemistry because of the high molecular sensitivity and sub-millisecond temporal resolution,[16] and because of the biocompatible reaction conditions, which resemble the environment that prevails for actual applications of CBT reagents. By using this approach, we have, for example, differentiated photochemically and thermally interconverting isomers[17,18] and characterized a transient tetrahedral intermediate.[19]

Results and Discussion

Conclusion

Conflict of interest