Design and Synthesis of AX7574: A Microcystin-Derived, Fluorescent Probe for Serine/Threonine Phosphatases

The design and synthesis of AX7574, a microcystin-derived probe for serine/threonine phosphatases, is described. A key step in the synthesis was the conjugation under basic conditions of a tetramethylrhodamine 1,3-diketone derivative to the arginine side chain present in microcystin-LR. The resultin...

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Bibliographic Details
Published in:	Bioconjugate chemistry Vol. 15; no. 4; pp. 790 - 798
Main Authors:	Shreder, Kevin R, Liu, Yongsheng, Nomanhboy, Tyzoon, Fuller, Stacy R, Wong, Melissa S, Gai, Wen Zhi, Wu, Jiangyue, Leventhal, Phillip S, Lill, Jennie R, Corral, Sergio
Format:	Journal Article
Language:	English
Published:	United States American Chemical Society 01-07-2004
Subjects:	Chemical synthesis Chemistry Design Drug Design Enzyme Inhibitors - pharmacology Enzymes Fluorescence Fluorescent Dyes Humans Inhibitory Concentration 50 Jurkat Cells Kinetics Mass Spectrometry Microcystins Molecular Probes - analysis Molecular Probes - antagonists & inhibitors Molecular Probes - chemical synthesis Molecular Probes - chemistry Molecular Structure Oxazoles - pharmacology Peptides, Cyclic - analysis Peptides, Cyclic - antagonists & inhibitors Peptides, Cyclic - chemical synthesis Peptides, Cyclic - chemistry Phosphoprotein Phosphatases - analysis Phosphoprotein Phosphatases - metabolism Proteome - drug effects Proteome - metabolism Staining and Labeling
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Summary:	The design and synthesis of AX7574, a microcystin-derived probe for serine/threonine phosphatases, is described. A key step in the synthesis was the conjugation under basic conditions of a tetramethylrhodamine 1,3-diketone derivative to the arginine side chain present in microcystin-LR. The resulting conjugate specifically labeled the active site of protein phosphatases 1 (PP-1) with a 1:1 stoichiometry and IC50 of 4.0 nM. AX7574 was used to isolate and identify PP-1, PP-2A, PP-4, and PP-6 in Jurkat cells. Finally, AX7574 was able to record changes in the phosphatase activity levels of calyculin A treated Jurkat cells versus untreated control cells.
Bibliography:	ark:/67375/TPS-7D47C265-R istex:0CB80052C164616CCFD6F3491DF3A67789143E49
ISSN:	1043-1802 1520-4812
DOI:	10.1021/bc0499580