Single‐Molecule Observation of Intermediates in Bioorthogonal 2‐Cyanobenzothiazole Chemistry

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.

CBT‐Cys click reaction for optical bioimaging in vivo

“…Moreover, owing to its programmable controllability (e.g., pH, reduction, or enzyme), CBT‐Cys click reaction could be used to fabricate smart imaging probes with self‐assembling behaviors in physiological context. Therefore, on one hand, CBT‐Cys click reaction can be exploited as a highly biocompatible biorthogonal tool for chemists and biologists to enjoy chemistry in physiological environment, such as site‐specific protein labeling or intracellular nanostructure fabrication 84,85 . On the other hand, it confers a programmable and adaptable in vivo self‐assembly strategy: by rational molecular designs of stimuli‐activatable CBT‐Cys probes, imaging agent cargos could be selectively accumulated and retained at stimuli‐rich pathological lesions of interest through CBT‐Cys click reaction‐mediated in situ self‐assembly, thus enabling enhanced and precise imaging of tumors/diseases.…”

“…Therefore, on one hand, CBT-Cys click reaction can be exploited as a highly biocompatible biorthogonal tool for chemists and biologists to enjoy chemistry in physiological environment, such as site-specific protein labeling or intracellular nanostructure fabrication. 84,85 On the other hand, it confers a programmable and adaptable in vivo self-assembly strategy: by rational molecular designs of stimuli-activatable CBT-Cys probes, imaging agent cargos could be selectively accumulated and retained at stimuli-rich pathological lesions of interest through CBT-Cys click reaction-mediated in situ self-assembly, thus enabling enhanced and precise imaging of tumors/ diseases. Despite of the encouraging and inspiring breakthroughs summarized in this mini-review, some challenges (or chances) still remain to be addressed.…”

Single-Molecule Interconversion between Chiral Configurations of Boronate Esters Observed in a Nanoreactor

“…Biological nanopores can be engineered as “nanoreactors” to observe dynamic chemical processes at a single-molecule level. They possess a submillisecond temporal resolution, so that chemical reactions of single molecules, including formation and breaking of chemical bonds, − the capture of the intermediate state of a reaction, − the motion of a molecular walker along a protein track, , and the growth and the degradation of a single polymer chain , can be clearly observed. Previously, Bayley et al reported observation of dynamic isomer interconversion, exemplified by pyrimidal inversion of a chiral As center or photoisomerization of an azobenzene, with engineered alpha-hemolysin (α-HL) nanopores.…”

Formation of Molecular Junctions by Single-Entity Collision Electrochemistry

“…Beyond molecular sensing and sequencing, nanopores have great potential to be engineered as nanoreactors to investigate chemical reactions at the single-molecule level. , Acknowledging its geometrical advantages and high temporal resolution, transient dynamics of chemical reactions, such as monitoring a chemical reaction process, , capturing the intermediate state of a reaction, , recording formation and breaking of chemical bonds, , and identifying selective and chiral reactions, − have been clearly observed. Bayley’s group confined a polymer substrate within the α-hemolysin (αHL) protein pore to promote site-selective and regioselective chemistry.…”