Compounds, reagents, and uses thereof
A compound, deuteration technology, applied in the field of compounds, reagents and their uses, can solve problems such as unknown structure and limited practicability
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Embodiment 1
[0313] Example 1. Structural Elucidation of Plasma and Urine Metabolite Compound A
[0314] Sample Preparation
[0315] Add 6 mL of pooled human plasma and 30 mL of methanol to a 50 mL plastic centrifuge tube. The mixture was vortexed for about 1 minute and centrifuged at 4000 rpm for 15 minutes at 4°C. The supernatant was transferred to a 96-well plate at 500 μL per well and dried at 40° C. under a gentle flow of nitrogen. The residues in 5 wells were reconstituted sequentially in water (200 mL) under vortexing (1 min each). The mixture was then transferred to a 1.5 mL Eppendorf tube and centrifuged at 14,000 rpm for 10 minutes at room temperature. The supernatant was then transferred to vials for LC / MS analysis. Urine samples were centrifuged at 14,000 rpm for 10 minutes at room temperature, and the supernatant (diluted with water if necessary) was transferred to vials for LC / MS analysis. Regarding the retention time comparison of Compound A in plasma and urine samples,...
Embodiment 2
[0356] Example 2. Synthesis of (2R,3R)-2,3-dihydroxy-5-methylthio-trans-4-pentenoic acid (trans-DMTPA)
[0357] method 1.
[0358] Wittig reaction
[0359] (Methylthiomethyl)triphenylphosphonium chloride (500 mg, 1.39 mmol, 2.2 equiv) was suspended in tetrahydrofuran in a 25 mL round bottom flask under argon atmosphere. The mixture was cooled to 0° C., and sodium bis(trimethylsilyl)amide (1.0 M in tetrahydrofuran, 1.39 mL, 1.39 mmol, 2.2 eq.) was added slowly while the mixture was stirred. The resulting mixture was stirred at 0 °C for another 30 minutes, and 2,3-cyclohexylene-L-erythuronic acid (136 mg in 1.0 mL tetrahydrofuran, 0.633 mmol, 1.0 equiv) was added slowly. After 5 minutes, the reaction mixture was warmed to room temperature and stirred overnight. The reaction mixture was transferred to NaH 2 PO 4 aqueous solution (1.0 M, 25 mL), and the resulting mixture (pH~6.0) was extracted with ethyl acetate (2 x 40 mL). The combined organic extracts were dried (MgSO 4 ...
Embodiment 3
[0373] Example 3. LC-MS / MS Measurement of Compound A
[0374] Reversed-phase columns (Waters ACQUITY BEH C18, 1.7 μm, 2.1 x 100 mm) liquid chromatography. Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. Linear gradient elution was performed with an initial condition of 2% mobile phase B (hold it for 3.00 minutes). Mobile phase B was then increased to 98% over 0.40 minutes and held for 0.50 minutes. Return mobile phase B to 2% over 0.10 minutes for the next injection to equilibrate. The total run time is 5.00 minutes. For each sample, inject a 5.0 µL ring-fixed aliquot of the final sample solution. The flow rate was 350 μL / min and the eluent was introduced directly into the electrospray source of the mass spectrometer. The strong needle wash (200 μL) is pure methanol, and the weak needle wash (600 μL) is a mixture of methanol and water (0.5:99.5). The seal wash solution is a mixture of methanol and water (10:90).
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