19F NMR (470.52 MHz, CDCl3) C173.92. of quantifying released fluoride and thus AMACR activity. These sensors generally suffered from high background signal and lacked reproducibility under the assay conditions. In summary, the elimination reaction can be used to characterise inhibitors, but it was not possible to develop a convenient colorimetric or fluorescent assay using 3-fluoro-2-methylacyl-CoA substrates. Introduction -Methylacyl-CoA racemase (AMACR, P504S; E.C. 5.1.99.4) catalyses a key step in the degradation of branched-chain fatty acids.1C3 The enzyme catalyses the conversion of either epimer of a 2-methylacyl-CoA ester into a 1?:?1 mixture of 2and are derived from dietary fatty acids.3 Thus, AMACR enables metabolism of models.21 Other approaches have also been used to develop a convenient assay for AMACR activity. The use of acyl-CoA oxidase as a coupling enzyme enables a colorimetric assay to be performed.22 This enzyme is not commercially available and rationally designed acyl-CoA inhibitors of AMACR are also likely to inhibit the coupling enzyme, complicating the analysis. Coupled enzyme assays for other racemases/epimerases have also been reported, 23C27 but these are not readily adaptable to measuring AMACR activity. Direct measurement of racemisation by MCR (the bacterial homologue of AMACR from 50% of substrate 1 after 1 h incubation. See ESI Table 1 for absolute substrate conversion levels in the presence of inhibitors and positive controls 50% of substrate 1 Tectoridin was converted into unsaturated product 2 by active AMACR. Negative controls containing heat-inactivated enzyme showed <5% conversion of 1 1 to 2 2, levels of which did not change over the incubation period. The presence Mouse monoclonal to SMAD5 of each inhibitor (at 100 M final concentration) resulted in a reduction in the level of conversion of 1 1 (Table 1). Compounds 3C7 showed moderate levels of inhibition in most cases, with the most significant reduction in activity occurring with Fenoprofenoyl-CoA 3 and Ibuprofenoyl-CoA 5. Modest levels of inhibition are expected with compounds 3C7, as the concentration of substrate 1 (100 M) is significantly above its reported an SN2 mechanism with inversion of stereochemistry. This loss of stereochemistry is probably due to an SN1 reaction occurring, Tectoridin with consequent addition of fluoride to both faces of the stabilised benzylic carbocation. Conversion of 14 to the methyl ester 16 followed by treatment with DAST also resulted in significant loss of stereochemistry on conversion to 17, suggesting that steric hindrance by the chiral auxiliary was not the deciding factor. Open in a separate window Scheme 2 Synthesis of 15 and 17. Reagents and conditions: i: Bu2BOTf, i-Pr2EtN, DCM, C78 C, 99%; ii: NaOMe, MeOH, 0 C, 36%; iii. DAST, DCM, C78 C. Stereochemical course of reaction iii: 14 to 15, 74%, 53% de; 16 to 17, 53%, 50% de. Synthesis of the 4-nitrophenyl- derivative was investigated (Scheme 3) in order to destabilise the carbocation intermediate and hence improve diastereoselectivity. Condensation of 4-nitrobenzaldehyde 18 with values reported Tectoridin to 0.1 Hz. Multiplicities are described as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. Stock concentrations of acyl-CoA esters for assays were determined using 1H NMR.35 Mass spectra were recorded by ESI TOF at the University of Bath Mass Spectrometry Service. High resolution mass spectra were recorded in ES mode. Aqueous solutions for biological experiments were prepared in 18.2 M cmC1 Nanopure water and pH-adjusted with aq. HCl or NaOH. Syntheses were carried out at ambient temperature, unless otherwise specified. Solutions in organic solvents were dried over anhydrous magnesium sulfate and evaporated under reduced pressure. Synthesis of 8.62 (1H, Tectoridin s), 8.35 (1H, s), 6.15 (1H, d, = 6.0 Hz), 4.57C4.48 (1H, m), 4.23C4.13 (2H, m), 3.97 (1H, s), 3.84 (3H, s), 3.81C3.76 (1H, m), 3.53C3.47 (1H, m), 3.39 (2H, t, = 6.5 Hz), 3.35C3.22 (3H, m), 2.95C2.82 (4H, m), 2.38 (2H, t, = 6.5 Hz), 1.55C1.35 (2H, m), 1.25C1.05 (18H, m), 0.87 (3H, s), 0.76 (3H, t, = 7.0 Hz), 0.72 (3H, s); HRMS (ES) [M + 2Na C 3H]C Calcd. For C35H60N8Na2O17P3S: 1035.2805, found 1035.3050. Attempted synthesis of (Major diastereomer (selected isolated peaks) 7.43C7.16 (10H, m), 5.63 (1H,.