1812 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 Rose et al. Figure 4. Structures of the N-oxalylamino acids investigated in this study. DCMS screen exploited the thiol side chain of N-oxalyl- D-cysteine 1b, which is predicted to project into the subpocket of JMJD2E as a “support” ligand (Figure 3). In this method, a compound that binds to the active site (the support ligand) which contains a thiol is allowed to react (either on the enzyme in the active site, or in solution) with a set of thiols to form a mixture of disulfides. Nondenaturing ESI-MS is then used to analyze which disulfides bind preferentially to the enzyme. A mixture of five thiols, each thiol at 15 μM, including thiophenol, 3-bromothiophenol, 3-methoxythiophenol, pro- panethiol, and 2-naphthalenethiol, was mixed with an equi- molar amount of 1b (15 μM) and JMJD2E (15 μM) in ammonium acetate (15 mM, pH 7.5) under aerobic condi- tions. The mixture was then analyzed for binding with JMJD2E (mass 41264 Da) by nondenaturing ESI-MS. New complexes with peaks at 41587 and 41557 Da were observed (Figure S1, Supporting Information); these peaks correspond to the addition of disulfides 1b 3 3-methoxythiophenol (2a) and 1b 3 thiophenol (2b) to JMJD2E, respectively. This result was confirmed by deletion experiments in which specific thiols were removed from the mixture. Because disulfides are not necessarily stable under the FDH coupled assay conditions, the carbon analogues, 3a and 3b, respectively, wherein the disulfide bond is replaced with a C-S bond, were then synthesized (Figure 2). Both 3a and 3b bind to JMJD2E, as shown by nondenaturing ESI-MS. The FDH coupled assay results indicate that both carbon analogues 3a (IC50 = 204 μM) and 3b (IC50 = 300 μM) are less potent as JMJD2E