Yield:88.7 % ee

Reaction Conditions:

with D-glucose;ethyl acetate;NADP at 30; pH=6.5; for 24 h;Conversion of starting material;Aqueous phosphate buffer;

Steps:

12

First, 5 mg of the acetone dried microbial cells obtained in Example 11, 2 mg of each type of the ethyl 2-formylalkanoates shown in Table 4, 10 mg of glucose, 1 mg of NADP, 0.5 ml of 0.1M phosphate buffer (pH=6.5), and 0.5 ml of ethyl acetate were added to each test tube with a cap and shaken at 30°C for 24 hours. After the reaction, the reaction solution was extracted with ethyl acetate of the same volume. The amounts of the substrate and the product in the extract were analyzed by gas chromatographic (GC) method to obtain the conversion rate (%). The product was derivatized with a HPLC labeling agent (3,5-dinitrobenzoyl chloride), and purified by thin-layer chromatography (TLC), and thereafter analyzed by HPLC to determine the optical purity. The results are shown in Table 4. The analysis conditions are shown below. GC analysis conditions: Column: HP-5, 30 m×0.32 mm I.D.(manufactured by J&W Scientific Inc.), Carrier gas: He=125 kPa, Detection: FID, Column temperature: 120°C, Detection time: 6.9 minutes for ethyl 2-formylhexanoate and 9.8 minutes for ethyl 2-hydroxymethylhexanoate; HPLC analysis condition:Column: Chiralcel OD-H, 0.46x25 cm I.D. (manufactured by Daicel Chemical Industries, Ltd.), Eluant: hexane:isopropanol=95:5, Flow rate: 0.5 ml/min. Detection: 210 nm, Column temperature: 40°C, Detection time: 3.06 minutesfor a (R)-2-hydroxymethylhexanoic ethyl derivative and 37.4 minutes for a (S)-2-hydroxymethyl hexanoic ethyl derivative

References:

EP1568679,2005,A1 Location in patent:Page/Page column 14-15