John Scheigetz

3,6-Fluorescein Diphosphate: A Sensitive Fluorogenic and Chromogenic Substrate for Protein Tyrosine Phosphatases

A highly sensitive and continuous protein tyrosine phosphatase (PTPase) assay using 3,6-fluorescein diphosphate (FDP) is described. Leukocyte phosphatase CD45 (leukocyte common antigen), protein tyrosine phosphatase-lB, and leukocyte common antigen-related protein LAR preferentially hydrolyze FDP to fluorescein monophosphate (FMP) with Vmax and Km values comparable with those of phosphotyrosine peptide substrates. Further hydrolysis of FMP to fluorescein was less efficient because of increased Km values compared with those of FDP. FMP absorbs strongly at 445 nm and fluoresces intensely near 515 nm, both of which are insensitive to pH perturbations above pH 6. Its high catalytic efficiency, coupled with the highly sensitive dual detection in the visible wavelength region and wider pH operating range, make FDP the substrate of choice for PTPase inhibitor screening in HTS format and assay miniaturization.

FLUORESCEIN MONOPHOSPHATES AS FLUOROGENIC SUBSTRATES FOR PROTEIN TYROSINE PHOSPHATASES

A series of novel fluorescein monophosphates aimed as substrates for protein tyrosine phosphatases (PTPs) were synthesized and evaluated against fluorescein diphosphate (FDP), the currently used fluorescent substrate for PTPs. In contrast to FDP, which is dephosphorylated to monophosphate and then to fluorescein in a sequential reaction, these monophosphates are dephosphorylated in a single step. This eliminates the complication in assaying PTPs due to the cleavage of the second phosphate group. The kinetic studies of these substrates with PTPs were performed and Michaelis-Menten parameters were obtained. These designed substrates have Km 0.03-0. 35 mM, kcat/Km of 3-100 mM-1 s-1 with CD45 and PTP1B. The results showed that the substrates with negative charge groups on the fluorescein have higher affinities for PTP1B, which are consistent with other observations. In this series, fluorescein monosulfate monophosphate (FMSP) was the best substrate observed. Since FMSP showed large increases in both absorption and fluorescence upon dephosphorylation by PTPs at pH>6.0, it is one of the most sensitive, stable and high affinity substrates reported for PTPs.

Formation of a novel thiopyranoindole ring system

The novel thiopyran[2,3,4-c,d]indole ring system (e.g.3) has been prepared from a substituted indole by intramolecular cyclisation of a t-BuS group onto an allyl substituent. The reaction occurs in high yield under the influence of protic acids, Lewis acids or electrophiles (Br2, I2, Hg(OAc)2)

Novel caged fluorescein diphosphates as photoactivatable substrates for protein tyrosine phosphatases.

We have characterized some novel caged fluorescein diphosphates as photoactivatable, cell-permeable substrates for protein tyrosine phosphatases and explored their usefulness in identifying inhibitors of tyrosine phosphatases. 1-(2-Nitrophenyl)ethyl protected fluorescein diphosphate (NPE-FDP) undergoes rapid photolysis to release FDP upon irradiation with a 450-W UV immersion lamp and its by-product does not inactivate protein tyrosine phosphatase 1B (PTP1B) or alters the viability of cells. The generated FDP from photolysis of NPE-FDP was shown to have exactly the same properties as FDP, which can be used as a PTP substrate in pure enzyme assays. We have also demonstrated that the PTP activity can be measured using NPE-FDP in small droplets. Its advantage as an inert substrate before photolysis allows the possibility of applying nanospray technology in screening and optimizing PTP inhibitors through a large chemical library. Like other caged bioeffectors such as nucleotide and inositol trisphosphate, NPE-FDP is cell-permeable. The NPE-FDP can be photolyzed to generate FDP inside cells, and then can be hydrolyzed by phosphatases to produce fluorescein monophosphate and subsequently to fluorescein. Although Jurkat cells contain high concentrations of CD45, it has not been possible to use FDP as a substrate to measure CD45 activity in the intact cell. This is due to the hydrolysis of FDP by several other cellular phosphatases. However, NPE-FDP can be useful as a cell-permeable substrate for overexpressed phosphatases such as alkaline phosphatase.

Investigation of ketone warheads as alternatives to the nitrile for preparation of potent and selective cathepsin K inhibitors

Amino ketone warheads were explored as alternatives to the nitrile group of a potent and selective cathepsin K inhibitor. The resulting compounds were potent and selective inhibitors of cathepsin K and these nitrile replacements had a significant effect on metabolism and pharmacokinetics.

Synthesis of 5-Methanesulfonamido-6-(2,4-Difluorophenylthio)-1-Indanone

Abstract The ethylene ketal of 5-nitro-6-bromo-1-indanone 6 was prepared via oxidation of N-(6-Bromo-5-indanyl)-acetamide 1. A subsequent mild displacement and elaboration to 6-thiosubstituted-5-amino indanone derivative 10 is also described.

Synthesis of Fluorescein Phosphorotriesters Using Photolabile Protecting Groups

Abstract Fluorescein phosphorotriesters 5, each having four identical photoactivatable adducts have been synthesized to investigate their applications as intracellular fluorescence indicators.