Direct anabolic metabolism of three-carbon propionate to a six-carbon metabolite occurs in vivo across tissues and species

Synthesis was conducted as previously described with modifications (Valenzano C.R. You Y.O. Garg A. Keatinge-Clay A. Khosla C. Cane D.E. Stereospecificity of the dehydratase domain of the erythromycin polyketide synthase.). Briefly, diisopropylethylamine (0.12 ml, 0.70 mmol) was added to a solution of 2M2PE (30.6 ml, 0.26 mmol) in dichloromethane (2 ml) cooled to 0 °C. Isobutyl chloroformate (64 μl, 0.65 mmol) was added at 0 °C with stirring. The resulting solution was allowed to warm to 22 °C and stirred for an additional 2 h, after which, volatiles were removed under reduced pressure to provide a colorless residue. Tetrahydrofuran (THF, 1 ml) was added to the residue resulting in the precipitation of diisopropylethylammonium chloride. This mixture was transferred to a 1.6 ml conical tube and centrifuged to pellet the solid. The THF solution was added to a solution of coenzyme A (30 mg, 0.039 mmol) in aqueous bicarbonate solution (1 ml, 500 mM, pH 8). The biphasic mixture was stirred vigorously at 22 °C. After 3 h, stirring was stopped and THF was evaporated under a stream of N2. The remaining aqueous solution was neutralized with trifluoroacetic acid (20 ml) and immediately purified by C18-reversed phase-silica chromatography. Method: (solvents - A: 0.05% trifluoroacetic acid in water, B: acetonitrile) gradient - 0–2 column volumes (CV), 0% B; 2–12 CV, 0%–50% B; 12–13 CV, 50%–100% B; and 13–25 CV, 100% B. Fractions containing the desired product were pooled and lyophilized to provide a fluffy white powder (17.7 mg, 52.5% yield). 1H NMR (500 MHz, D2O) δ 8.63 (s, 1H), 8.42 (d, J = 3.2 Hz, 1H), 6.77 (t, J = 7.2 Hz, 1H), 6.18 (d, J = 5.7 Hz, 1H), 4.91 (s, 1H), 4.89–4.84 (m, 1H), 4.60 (s, 1H), 4.35–4.19 (m, 2H), 4.01 (s, 1H), 3.89 (d, J = 9.1 Hz, 1H), 3.65 (d, J = 9.2 Hz, 1H), 3.44 (t, J = 6.8 Hz, 2H), 3.33 (t, J = 6.5 Hz, 2H), 2.99 (t, J = 6.3 Hz, 2H), 2.43 (q, J = 6.3 Hz, 2H), 2.19 (p, J = 7.5 Hz, 2H), 1.77 (s, 2H), 0.99 (t, J = 7.6 Hz, 3H), 0.94 (s, 3H), 0.82 (s, 3H). HRMS (ESI) calculated for C27H44N7O17P3S [M + H]+ 864.1800 found 864.1811. 2M2PE-CoA was added as Lipidmaps LMFA07050487 and updated in PubChemID Compound CID: 121225619.

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