Protein tyrosine phosphatases (PTPs) are important targets of the H(2)O(2) that is produced during mammalian signal transduction. H(2)O(2)-mediated inactivation of PTPs also may be important in various pathophysiological conditions involving oxidative stress. Here we review the chemical and structural biology of redox-regulated PTPs. Reactions of H(2)O(2) with PTPs convert the catalytic cysteine thiol to a sulfenic acid. In PTPs, the initially generated sulfenic acid residues have the potential to undergo secondary reactions with a neighboring amide nitrogen or cysteine thiol residue to yield a sulfenyl amide or disulfide, respectively. The chemical mechanisms by which formation of sulfenyl amide and disulfide linkages can protect the catalytic cysteine residue against irreversible overoxidation to sulfinic and sulfonic oxidation states are described. Due to the propensity for back-door and distal cysteine residues to engage with the active-site cysteine after oxidative inactivation, differences in the structures of the oxidatively inactivated PTPs may stem, to a large degree, from differences in the number and location of cysteine residues surrounding the active site of the enzymes. PTPs with key cysteine residues in structurally similar locations may be expected to share similar mechanisms of oxidative inactivation.

Model reactions offer a chemical mechanism by which formation of a sulfenyl amide residue at the active site of the redox-regulated protein tyrosine phosphatase PTP1B protects the cysteine redox switch in this enzyme against irreversible oxidative destruction. The results suggest that "overoxidation" of the sulfenyl amide redox switch to the sulfinyl amide in proteins is a chemically reversible event, because the sulfinyl amide can be easily returned to the native cysteine thiol residue via reactions with cellular thiols.Intracellular concentrations of hydrogen peroxide (H 2 O 2 ) increase under conditions of oxidative stress and during some normal signal transduction processes. [1][2][3] An important mechanism by which cells issue temporary responses to such transitory increases in H 2 O 2 levels involves reversible oxidation of cysteine residues on critical "sensor" proteins.4 ,5 The ability of cysteine residues to serve as reversible redox switches relies upon the unique ability of the γ-sulfur atom in this amino acid to cycle easily between (at least) two oxidation states under physiological conditions. Specifically, oxidation of a cysteine thiol by H 2 O 2 yields a sulfenic acid residue (reaction i, Scheme 1A) that can, over time, be returned to the native thiol by reactions with biological thiols (reaction ii, Scheme 1A). [4][5][6][7][8] Protein sulfenic acid residues also have the potential to undergo further reaction with hydrogen peroxide to generate the corresponding sulfinic acid (reaction iii, Scheme 1A). [4][5][6][7][8][9] This reaction is irreversible, except in the case of some peroxiredoxins 8 and, therefore, yields an overoxidized, "broken" redox switch. Alternatively, in some proteins, the initially-formed sulfenic acid intermediate undergoes reaction with a neighboring "back door" cysteine thiol to generate a disulfide linkage (reaction ii, Scheme 1B). [10][11][12][13] Rudolph and Sohn provided evidence that, at least in the context of the phosphatase Cdc25B, disulfide formation protects the enzyme against irreversible overoxidation. 10,11 There are at least two possible mechanisms underlying this protection. First, the disulfide may be relatively resistant to further oxidation (reaction iii, Scheme 1B). 10,11 Second, if "overoxidation" does occur, the resulting thiosulfinate likely could be converted cleanly back to the native cysteine residues by reactions with biological thiols (reaction v, Scheme 1B). 14 © 2009 Elsevier Ltd. All rights reserved. * Corresponding author. Tel.: +1-573-882-6763; fax: +1-573-882-2754 gatesk@missouri.edu.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaim...

Protein tyrosine phosphatase 1B (PTP1B) is a validated drug target, but it has proven difficult to develop medicinally useful, reversible inhibitors of this enzyme. Here we explored covalent strategies for the inactivation of PTP1B using a conjugate composed of an active site-directed 5-aryl-1,2,5-thiadiazolidin-3-one 1,1-dioxide inhibitor connected via a short linker to an electrophilic α-bromoacetamide moiety. Inhibitor-electrophile conjugate 5a caused time-dependent loss of PTP1B activity consistent with a covalent inactivation mechanism. The inactivation occurred with a second-order rate constant of (1.7 ± 0.3) × 10 M min. Mass spectrometric analysis of the inactivated enzyme indicated that the primary site of modification was C121, a residue distant from the active site. Previous work provided evidence that covalent modification of the allosteric residue C121 can cause inactivation of PTP1B [Hansen, S. K., Cancilla, M. T., Shiau, T. P., Kung, J., Chen, T., and Erlanson, D. A. (2005) Biochemistry 44, 7704-7712]. Overall, our results are consistent with an unusual enzyme inactivation process in which noncovalent binding of the inhibitor-electrophile conjugate to the active site of PTP1B protects the nucleophilic catalytic C215 residue from covalent modification, thus allowing inactivation of the enzyme via selective modification of allosteric residue C121.

Many 1,2,4-benzotriazine 1,4-dioxides display the ability to selectively kill the oxygen-poor cells found in solid tumors. As a result, there is a desire for synthetic routes that afford access to substituted 1,2,4-benzotriazine 1-oxides that can be used as direct precursors in the synthesis of 1,2,4-benzotriazine 1,4-dioxides. Here we describe the use of Suzuki-Miyaura and Buchwald-Hartwig cross-coupling reactions for the construction of various 1,2,4-benzotriazine 1-oxide analogs bearing substituents at the 3-, 6-, and 7-positions.