| 12.8 g |
Stage #1: Tert-butyl (5S)-6-chloro-5-hydroxy-3-oxohexanoate With diethyl methoxy borane In tetrahydrofuran; methanol at -70℃; for 0.333333h;
Stage #2: With sodium tetrahydridoborate In tetrahydrofuran; methanol at -70℃; for 3h;
Stage #3: With dihydrogen peroxide In tetrahydrofuran at 0 - 20℃; for 0.5h; Further stages.; |
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| 17 g |
Stage #1: Tert-butyl (5S)-6-chloro-5-hydroxy-3-oxohexanoate With diethyl methoxy borane In tetrahydrofuran; methanol at 25℃; for 1h; Inert atmosphere;
Stage #2: With sodium tetrahydridoborate In tetrahydrofuran; methanol at -78℃; for 4h; Inert atmosphere; |
3 Example 3: Process for synthesis of (3R,5S)-tert-butyl 6-chloro-3,5- dihydroxyhexanoate [VIII] using methoxy diethyl borane and sodium borohydride in THF
To a solution of (S -tert-butyl 6-chloro-5-hydroxy-3-oxohexanoate (16 g) in THF (160 mL) was added methanol (16 mL) under inert atmosphere at 25°C. Methoxy diethyl borane in THF (25 mL) was added slowly at 25°C to the above reaction mixture and stirred for 1 h at 25°C. After cooling to -78 °C, sodium borohydride (2.83 g) was added in 5-6 portions at -78 °C and stirred for 4 h at this temperature. Reaction was monitored on TLC for complete conversion of starting material, after which reaction was quenched by slow addition of 30% aqueous solution of hydrogen peroxide (65 mL) at -78 °C and stirred for 1 h. Organic volatile material was removed under reduced pressure and residue was extracted with ethyl acetate (3 X 50 mL). Combined organic layer was washed with 10% aqueous solution of sodium bisulfite (170 mL) at 0 °C. The organic layer was separated and dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to obtain (JR,55)-tert-butyl 6-chloro-3,5-dihydroxyhexanoate (17 g, 97% de) as light brown oil. Diastereomeric excess was determined by converting it to corresponding acetonide as per procedure given in example 10. NMR of R,5S)-tert- ut 6-chloro-3,5-dihydroxyhexanoate: H NMR (400MHz, CDC13): δ.47 (9H), 1.72 (t, 2H), 2.4(d, 2H), 3.52-3.65 (m, 2H), 4.10-4.13 (t, 1H), 4.29-4.32 (t, 1H). 13C-NMR: δ 28.0, 39.3, 42.0, 49.0, 68.1, 70.4, 81.6, 172.3 |
| 97 % de |
With glucose dehydrogenase; alpha-D-glucopyranose; BgADH5 enzyme; β-nicotinamide adenine dinucleotide phosphate, oxidized form In lithium hydroxide monohydrate at 30℃; Enzymatic reaction; diastereoselective reaction; |
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With sodium tetrahydridoborate; lithium hydroxide monohydrate; sodium dioctyl sulfosuccinate at 0℃; for 14h; diastereoselective reaction; |
Reduction of 3 in aqueous micellar solution obtained from dioctyl sodium sulfosuccinate
To a mixture of (S)-tert-butyl 6-chloro-5-hydroxy-3-oxohexanoate (7.0 g; 29.66 mmol) in water (95 mL )was added dioctyl sodium sulfosuccinate (2.8 g; 40%w/w of compound 3). After cooling to 0°C, sodium borohydride (1.4 g; 36.84 mmol) was added in portions and stirred for 14 h at 0°C. Completion of reaction was monitored on TLC and after completion of reaction, ethyl acetate (200 mL) was added and stirred for 10 min. Organic layer was separated and washed with 10%aqueous solution of hydrochloric acid (40 mL). Organic layer was separated and dried over anhydrous magnesium sulphate, filtered and concentrated under reduced pressureto give crude product (3R,5S)-tert-butyl 6-chloro-3,5-dihydroxyhexanoate (6.2 g, 88% isolated yield and92% de) as yellow oil (diastereomeric excess was determined by chiral GC by converting to compound 5). |
| 6.4 g |
With sodium tetrahydridoborate In tetrahydrofuran at -40 - 30℃; for 6h; Inert atmosphere; |
3 preparation of (3R,5S)-tert-butyl 6-chloro-3,5-dihydroxyhexanoate
(S) -tert-butyl 6-chloro-5-hydroxy-3-oxohexanoate was added to a three-necked flask under a nitrogen atmosphere.After diluting with 200 mL of tetrahydrofuran, the internal temperature was cooled to -40 to -30 DEG C, and 4.8 gr of sodium borohydride was added to 10Respectively. When the sodium borohydride is added, the internal temperature of the reaction solution rises,And the mixture was stirred at the same temperature for 6 hours. After completion of the reaction, 5 mL of acetone was added, followed by stirring for 30 minutes.Min of sodium borohydride and then acetic acid was added to the reaction solution to maintain the pH of the reaction solution at 5.0 to 6.0Respectively. After adjusting the pH, 100 mL of water and 200 mL of ethyl acetate were added, and the mixture was stirred for 30 minutes, and the product was extracted and separatedThe obtained organic layer was treated with anhydrous magnesium sulfate, followed by filtration and concentration under reduced pressure to obtain an oil,Purification by gel column chromatography (developing solvent condition: heptane / ethyl acetate = 3/1) afforded 6.4 g of the title compound as an oil |
| > 99.5 % de |
With Klebsiella oxytoca alcohol dehydrogenase overexpressed in Escherichia coli Rosetta (DE3); NADPH; nicotinamide adenine dinucleotide In aq. buffer at 30℃; for 24h; Enzymatic reaction; stereoselective reaction; |
2.7. Substrate specificity
General procedure: First, two different cofactors NADH and NADPH were tested usingwhole cells biocatalysts to see which one could improve the activity ofKleADH. Reactions were conducted in 1 mL of triethanolamine buffer(100 mM, pH7.0) containing 10 mM substrates, 10 mM cofactors, and400 mg/mL recombinant wet cells at 30 °C with vigorous shaking for24 h. Then, a wide range of potential substrates were investigated toexplore the substrate specificity of KleADH. The substrates used in thisstudy were listed in Table. 2. Reactions were conducted in 1 mL oftriethanolamine buffer (100 mM, pH7.0) containing 10 mM substratesand 400 mg/mL recombinant wet cells at 30 °C with vigorous shakingfor 24 h. Then the aqueous phase was extracted by ethyl acetate andanalyzed by chiral HPLC or GC [30,31]. |
| > 99.5 % de |
With aldo-keto reductase from thermotolerant yeast Kluyveromyces marxianus ZJB14056 mutant with W297H/Y296W/K29H; NADPH In aq. phosphate buffer at 35℃; Enzymatic reaction; stereoselective reaction; |
2.2.3. Enzyme activity assay and kinetics characterization
General procedure: The activities of Variant III and its mutants were assayed at 35 °C,500 rpm, in a total of 1.0 mL reaction mixture, composed of pH 7.0,100 mM sodium phosphate buffer supplemented with 2 mM (5R)-1, and0.25 mM NADPH together with appropriate amounts of the purifiedKmAKRs. An aliquot of 500 μL was withdrawn after reaction for 5 min,which was quenched by adding 2 μL, 6.0 mol L-1 HCl. The resultantmixture was centrifuged at 12,000 rpm for 3 min. The supernatant wascombined, and subjected to microfiltration with 0.22 μm PTFE organicmembranes. The resulting filtrate was subsequently analyzed by HighPerformance Liquid Chromatography (HPLC). |
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