反应详情

温度

35 °C

D(+)-glucose monoydrate (300 g) and magnesium chloride hexahydrate (1.0 g) were dissolved in 10 mM MES buffer pH 6.5 (2.4 L; Sigma M3671). After addition of 4-oxo-6-aza-spiro[2.5]octane-6-carboxylic acid tert-butyl ester (300 g; 1.33 mmol) and β-NAD (3.0 g; free acid; Roche Diagnostics Cat. No. 10 004 626) the pH was re-adjusted and the suspension heated to 35° C. The reaction was started by adding ketoreductase KRED-NADH-117 (3.0 g; former Biocatalytics, now Codexis) and glucose dehydrogenase GDH-102 (300 mg; Biocatalytics). The suspension was vigorously stirred at 35° C. keeping the pH constant at 6.5 by the controlled addition (pH-stat) of 1.0 M aq. sodium hydroxide solution. After a consumption of 1.307 L (corresponding to 98% conversion; after 17 h) the reaction mixture was extracted with ethyl acetate (10 L). The organic phase was dried over sodium sulfate and concentrated in vacuo (200 mbar/45° C.) until evaporation fell off. Upon cooling the oily residue (411 g) started to crystallize and was stirred with heptane (1 L) for 2 h. The crystals were filtered off and the filtrate evaporated to dryness, redissolved in ethyl acetate (150 ml) and concentrated in vacuo as described above. The crystal suspension formed again upon cooling was stirred with heptane (200 ml; 2 h) and the crystals filtered off. Both crops of crystals were washed with heptane and dried under high vacuum to yield the title compound in 93% yield (250.77 g and 34.60 g white crystals), each having a purity of >98.5% GC and 99.8% ee. [α]D=−44.97° (C=1.00, CHCl3).

1. customAfter a consumption of 1.307 L (corresponding to 98% conversion; after 17 h) the reaction mixture
2. extractionwas extracted with ethyl acetate (10 L)
3. dry with materialThe organic phase was dried over sodium sulfate
4. concentrationconcentrated in vacuo (200 mbar/45° C.) until evaporation
5. temperatureUpon cooling the oily residue (411 g)
6. customto crystallize
7. stirringwas stirred with heptane (1 L) for 2 h
8. filtrationThe crystals were filtered off
9. customthe filtrate evaporated to dryness
10. dissolutionredissolved in ethyl acetate (150 ml)
11. concentrationconcentrated in vacuo
12. customThe crystal suspension formed again
13. temperatureupon cooling
14. filtrationthe crystals filtered off
15. washBoth crops of crystals were washed with heptane
16. customdried under high vacuum