Chemical tools for imaging phospholipase D activity

A method for detecting phospholipase D(PLD) activity in a cell, the method comprising:

• (i) providing a cell containing endogenous PLD;
• (ii) contacting said cell with an exogenous alcohol of the formula HO—(CH2)n—Y, wherein Y is a first functional group and n is an integer of 1-12;
• (iii) stimulating the endogenous PLD in said cell by contacting the cell with phorbol 12-myristate-13-acetate (PMA) or platelet-derived growth factor (PDGF) while said cell is in contact with said exogenous alcohol, whereby the stimulated PLD catalyzes a transphosphatidylation reaction in the cell between phosphatidylcholine or a derivative thereof and said exogenous alcohol to form a phosphatidyl alcohol reaction product containing said first functional group in unreacted form, wherein the transphosphatidylation reaction catalyzed by PLD in the cell in step (iii) proceeds according to the following reaction scheme:
• wherein R1 and R2 are independently selected from acyl fatty acid groups —C(O)R, wherein R is independently selected from alkyl or alkenyl groups having at least 12 carbon atoms;
• and R3 is a H atom, hydrocarbon group having 1-3 carbon atoms, or a cationic species;
• (iv) reacting said phosphatidyl alcohol reaction product with a detectable label of the formula D-Y′ wherein D is a detectable group and Y′ is a second functional group reactive with the first functional group Y on said phosphatidyl alcohol reaction product, to form a labeled phosphatidyl alcohol reaction product containing said detectable label, wherein step (iv) proceeds according to the following reaction scheme:
• wherein Y—Y′ represents the linkage formed by reaction between Y and Y′;
• and
• (v) detecting said labeled phosphatidyl alcohol reaction product.