Fluorescein monophosphates as fluorogenic substrates for protein tyrosine phosphatases

A series of novel fluorescein monophosphates aimed as substrates for protei n tyrosine phosphatases (PTPs) were synthesized and evaluated against fluor escein diphosphate (FDP), the currently used fluorescent substrate for PTPs . In contrast to FDP, which is dephosphorylated to monophosphate and then t o fluorescein in a sequential reaction, these monophosphates are dephosphor ylated in a single step. This eliminates the complication in assaying PTPs due to the cleavage of the second phosphate group. The kinetic studies of t hese substrates with PTPs were performed and Michaelis-Menten parameters we re obtained. These designed substrates have K-m, 0.03-0.35 mM, k(cat)/K-m, of 3-100 mM(-1) s(-1) with CD45 and PTP1B. The results showed that the subs trates with negative charge groups on the fluorescein have higher affinitie s for PTP1B, which are consistent with other observations. In this series, fluorescein monosulfate monophosphate (FMSP) was the best substrate observe d. Since FMSP showed large increases in both absorption and fluorescence up on dephosphorylation by PTPs at pH>6.0, it is one of the most sensitive, st able and high affinity substrates reported for PTPs. (C) 1999 Elsevier Scie nce B.V. All rights reserved.