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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