Application of Intramolecular O-to-N Phosphoryl Transfer Reaction to Design Fluorogenic Probes to Detect Activities of Enzymes That Metabolize Short Peptides and Acylamino Acids

ACS Sens. 2025 Feb 28;10(2):664-670. doi: 10.1021/acssensors.4c03402. Epub 2025 Feb 4.

Abstract

We propose the design strategy of fluorogenic probes of proteases/peptidases and acylamino acid hydrolases utilizing an intramolecular O-to-N phosphoryl transfer reaction, in which the main chain of peptides or amino acids is retained from the natural substrate but the side chain was designed to attach the fluorophore. The strategy is useful to design fluorogenic probes for peptidases/proteases that do not prefer the main chain modification and acylamino acid hydrolases. We have developed the fluorogenic substrates for GGT5, GGCT, and PM20D1 and have performed the screening of PM20D1 inhibitors/activators to characterize the compounds that modify the activity of PM20D1.

Keywords: chemical biology; enzymes; enzymomics; fluorogenic substrates; high-throughput screening.

Publication types

  • Research Support, Non-U.S. Gov't
  • Letter

MeSH terms

  • Amino Acids* / chemistry
  • Amino Acids* / metabolism
  • Enzyme Assays / methods
  • Fluorescent Dyes* / chemical synthesis
  • Fluorescent Dyes* / chemistry
  • Fluorescent Dyes* / metabolism
  • Peptide Hydrolases* / metabolism
  • Peptides* / chemistry
  • Peptides* / metabolism

Substances

  • Fluorescent Dyes
  • Peptides
  • Amino Acids
  • Peptide Hydrolases