Mechanisms of chemical protein 19F-labeling and NMR-based biosensor construction in vitro and in cells using self-assembling ligand-directed tosylate compounds

Yousuke Takaoka, Yedi Sun, Shinya Tsukiji, Itaru Hamachi

Research output: Contribution to journalArticlepeer-review

31 Citations (Scopus)


Chemical labeling methods that convert a specific endogenous protein into a semisynthetic biosensor offer numerous new opportunities for biological research and drug discovery. We recently developed a novel protein labeling scheme, termed ligand-directed tosyl (LDT) chemistry, which can sitespecifically introduce a synthetic probe to a protein with the concomitant release of the affinity ligand. In previous work, we demonstrated that LDT reagent 1 can be used to modify carbonic anhydrase I (CAI) with a 19F probe, converting it into a 19F NMR-based biosensor for CAI inhibitors either in vitro or in red blood cells (RBCs). We herein report the chemical properties of 1, and the mechanisms controlling biosensor construction. It was revealed that the LDT reagent forms self-assembled aggregates in the absence of the target protein. In the aggregated state, nonproductive hydrolysis of the reagent was significantly suppressed, which suggests the potential utility of self-assembly in the design of labeling reagents that have increased stability. In the presence of the target protein, the aggregates were disrupted to form a noncovalent protein-reagent complex, and protein 19F-labeling proceeded to generate 19F-labeled CAI. The ligand-binding pocket of the labeled CAI retained the cleaved ligand fragment in vitro, whereas the pocket was vacant in RBC. Further biochemical studies suggested that an anion transporter might play a role in eliminating the cleaved ligand from the interior to the exterior of the cells. The findings provide a fundamental basis for the rational design of reagents applicable to selective protein labeling and biosensor construction in biological contexts.

Original languageEnglish
Pages (from-to)511-520
Number of pages10
JournalChemical Science
Issue number3
Publication statusPublished - 2011 May 10
Externally publishedYes

ASJC Scopus subject areas

  • Chemistry(all)

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