BOC-D-ALA-OME

White powder

To a solution of D-alanine methyl ester hydrochloride (5 g, 35.8 mmol) and sodium hydrogen carbonate (9.0 g, 107 mmol) in water (100 mL) was added di-tert-butyl dicarbonate (11.7 g, 53.7 mmol) and the resulting solution was stirred at RTovernight. The reaction mixture was extracted with DCM (3 x 100 mL) and the combined organics were dried over MgSO4, filtered and concentrated under reduced pressure. Crude NMR revealed large amounts of un-reacted di-tert-butyl dicarbonate, hence the material was taken up in DCM (50 mL) and treated with N, N-dimethyl ethylenediamne (5 mL), and stirred for 30 mins. The solution waswashed with 1M HCI (50 mL), dried over MgSO4, filtered and concentrated under reduced pressure to afford 5.35 g (73percent yield) of the title compound as a colourless oi I.1H NMR (500 MHz, Chloroform-d) 6 5.04 (5, 1 H), 4.31 (m, 1 H), 3.73 (5, 3H), 1 .44 (5, 9H), 1.37 (d, J = 7.2 Hz, 3H).Intermediate 57 Boc-D-AlaOMe Boc-D-AlaOH (10.0 g, 52.9 mmol) was dissolved in dry DMF (60 mL), then potassium carbonate (8.0 g, 58.0 mmol) was added, the mixture was stirred at rt for 15 min, and methyl iodide (6.58 mL, 106.0 mmol) was added dropwise. The stirring was maintained for 18h. Then, the mixture was concentrated in vacuo and the residue was dissolved in EtOAc (lOOmL). The organic layer was washed with water, brine, dried over MgSCU, filtered and concentrated. The crude product was purified by flash column chromatography (silica gel, DCM/MeOH 0 to 1percent) to give 10.36 g (51.0 mmol, 96percent yield) of the title compound. *Η NMR (DMSO-dExample 452-tert-Butoxycarbonylamino-propionic acid methyl ester Boc-D-Ala-OH (4.0 g, 21 mmol) and potassium carbonate (11.7 g, 84.6 mmol) was dissolved in dimethylformamide (90 mL) and iodomethane (1.6 mL, 25 mmol) was added to the reaction mixture. The reaction was allowed to stir at room temperature. overnight. The reaction mixture was partitioned between ethyl acetate and water. The organic layer was washed with portions of water and brine, dried over anhydrous sodium sulfate, filtered and concentrated to give the title compound as a colorless oil (3.53 g, 82percent).General procedure: The soil samples were collected from various locations inChina, and then the strains were screened by utilizing N-Boc-2-aminobutyrate as a sole carbon source and selective platewith bromocresol purple indicator. Isolated strains weretransferred to 150 mL fermentation medium and cultivatedat 200 rpm, 30 °C for 48 h. After the cells were harvested, 1 g wet cell were washed and suspended in 5 mL 0.1 M, pH 7.0 potassium phosphate buffer (KPB). Cell suspensionswere mixed with 50 μL substrate solution (N-Boc-2-aminobutyrate:acetone = 1:4, V/V) and kept at 200 rpm, 30 °Cfor 6 h reaction, the enantioselective hydrolysis of the substratewas determined by GC-MS. The screening mediumcontained (per liter, pH 7) 0.5 g KCl, 0.5 g MgSO47H2O, 1 g K2HPO43H2O, 3 g NaNO3, 0.01 g FeSO4, 0.25 g Bromocresolpurple, 20 g agar. Fermentation medium contained(per liter, pH 7) 0.5 g KCl, 0.5 g MgSO47H2O, 1 gK2HPO43H2O, 3 g NaNO3, 0.01 g FeSO4, 20 g sucrose.Enantioselective hydrolysis was performed on Boc-dl-Ala-OMe by adding both a substrate with a concentration range of 0.1 to 4.0M and a 500mg lyophilized cell of B. amyloliquefaciens WZZ002 in a 10mL (50mL flask) phosphate buffer solution (0.2M, pH6.0–12.0) at 20°C to 60°C. The solution was stirred at 400rpm. The pH level was controlled through automatic titration using different alkali solutions (2M). The samples were withdrawn at regular intervals and were immediately acidified with HCl (2M) to stop the reaction and to enhance the extractability of Boc-dl-Ala. The sample was extracted using ethyl acetate, whereas the organic phase was isolated and dried using anhydrous Na

Molecular Weight:203.24
XLogP3:0.8
Hydrogen Bond Donor Count:1
Hydrogen Bond Acceptor Count:4
Rotatable Bond Count:5
Exact Mass:203.11575802
Monoisotopic Mass:203.11575802
Topological Polar Surface Area:64.6
Heavy Atom Count:14
Complexity:219
Defined Atom Stereocenter Count:1
Covalently-Bonded Unit Count:1
Compound Is Canonicalized:Yes