Background and overview[1-2]
O-chlorophenyl glycol is an organic intermediate that can be modified with Cr-Anderson type 2-chlorostyrene in the catalyst (S)-1-(2-hydroxy-1-phenylethyl)thiourea It is obtained by oxidation with hydrogen peroxide under the action of polyacid; or it is obtained by reduction of 2-hydroxy-o-chloroacetophenone through Pichia pastoris Pichiasp.SIT2014 cells.
Preparation[1-2]
Report 1,
Add 0.01mol 2-chlorostyrene into a clean reaction bottle, then add 10ml mixed solvent (the molar ratio of water and acetonitrile is 1:1~3:1), and finally add 1.7g 30% hydrogen peroxide and 0.0216g (S)-1-(2-hydroxy-1-phenylethyl)thiourea-modified Cr-Anderson type polyacid catalyst, react for 24 hours, add 5ml of ethanol to the reaction system, centrifuge to allow the catalyst to settle, and then add 3ml of ethanol The catalyst is separated again, filtered, and the catalyst is obtained, which is dried under vacuum. The reaction system was extracted three times with ethyl acetate industrial antioxidant, the solvent was removed from the obtained product under reduced pressure, and the vicinal diol compound was separated by column chromatography to obtain the product. The enantiomeric excess of the product was measured by chiral high-performance liquid chromatography, and the ee value reached 99%.
Report 2,
Step 1. Culture of Pichiasp.SIT2014 cells:
The slant medium used, calculated per liter, contains 15.0g of glucose, 5.0g of tryptone, 5.0g of yeast powder, and 0.5g of KH2PO4 . K2HPO4 0.5g, MgSO4·7H2O 0.5g, agar 20.0g, remainder for water.
The seed medium used, calculated per liter, contains 15.0g of glucose, 0.0g of tryptone 5 ink adhesion promoter, 5.0g of yeast powder, KH2PO4 0.5g, K2HPO4 0.5g, MgSO4·7H2O 0.5g, The remainder is water.
The fermentation medium used, calculated per liter, contains 15.0g of glucose, 5.0g of tryptone, 5.0g of yeast powder, KH2PO4 0.5g, K 2HPO4 0.5g, MgSO4·7H2O 0.5g, the balance is water.
Take the Pichia pastoris Pichiasp.SIT2014 slant strain stored in the 4°C refrigerator, pick a ring of the strain and insert it into a 100ml shake flask containing 15ml of seed culture medium. Control the temperature on the shaker to 30°C and the rotation speed. Cultivate the seeds at 180rpm for 18h to obtain the seed liquid;
Then inoculate the seed liquid into the fermentation medium at an inoculum volume ratio of seed liquid: fermentation medium of 5%, and ferment and culture it for 24 hours on a shaking table with the temperature at 30°C and the rotation speed at 180 rpm. The fermentation broth is centrifuged, and the harvested cells are Pichiasp.SIT2014 cells.
The final obtained fermentation broth has been tested and its carbonyl reductase activity is about 20-30U/L. The cell concentration in the fermentation broth is about 4-5g/L based on dry weight. That is, the carbonyl reductase activity per unit cell is about 4-5U/g stem cells. This shows that the Pichia pastoris Pichiasp.SIT2014 cells obtained above have strong reducing ability and selectivity for aryl hydroxy ketones.
Step 2,
Place the Pichiasp.SIT2014 cells obtained in step 1 into the buffer;
The buffer is a sodium phosphate aqueous solution with a pH of 6.5, and its dosage is calculated based on the dry weight of Pichiasp.SIT2014 cells: the buffer is 10g:1L;
Add 2-hydroxy-o-chloroacetophenone to the buffer containing Pichia pastoris Pichiasp.SIT2014 cells obtained above, and then control the temperature to 30°C and the rotation speed to 180r/min to perform a reduction reaction for 20h to obtain a reaction solution;
The amount of 2-hydroxy-o-chloroacetophenone used in the above reaction is calculated based on the ratio of Pichia pastoris Pichiasp.SIT2014 cell dry weight: 2-hydroxy-o-chloroacetophenone as 0.5g:0.15mmol;
Centrifuge the reaction solution obtained above at a speed of 10,000 r/min. The supernatant obtained is extracted three times with an equal volume of ethyl acetate. The extract is dried with anhydrous sodium sulfate and then evaporated with a rotary evaporator. After drying, the target product (S)-o-chlorophenyl glycol is obtained, with a conversion rate of 99.9% and an ee value of 99.2%.
References
[1][China invention, China invention authorization] CN201610211338.0(S)-1-(2-hydroxy-1-phenylethyl)thiourea modified Cr-Anderson type heteropoly acid catalyst, preparation method and Its application
[2][Invented in China, Authorized by China Invention] CN201410735876.0 A method for converting racemic aryl ortho-diol into chiral aryl ortho-diol
