Synthesis of fluorogenic substrates for continuous assay of phosphatidylinositol-specific phospholipase C

  • Bioconjug Chem. 2001 Mar-Apr;12(2):307-13. doi: 10.1021/bc0001138.
T O Zaikova  1 ,  A V Rukavishnikov ,  G B Birrell ,  O H Griffith ,  J F Keana
Affiliations
  • 1. Department of Chemistry and Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA.
PMID: 11312693 DOI: 10.1021/bc0001138
Abstract

An improved synthesis of fluorogenic substrate analogues for phosphatidylinositol-specific Phospholipase C (PI-PLC) is described. The water-soluble substrates, which are derived from fluorescein, are not fluorescent until cleaved by the enzyme, and provide a convenient means to continuously monitor PI-PLC activity. The improvement in the synthesis lies in the method used to protect the hydroxyl groups of the inositol portion of the substrate molecule and allows a milder deprotection procedure to be used. The result is a much more reproducible synthesis of the substrate. The improved procedure has been employed to synthesize a series of fluorogenic substrates, which differ in the length of the aliphatic tail attached to the fluorescein portion of the molecule. The length of the tail was found to have a significant effect on the rate of cleavage of these substrates.

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