Method for enzymatic resolution of chiral substances

Patent metadata

Field	Value
Publication number	US-12319952-B2
Application number	US-201917311835-A
Country	US
Kind code	B2
Filing date	Oct 25, 2019
Priority date	Dec 10, 2018
Publication date	Jun 3, 2025
Grant date	Jun 3, 2025

Abstract

The invention belongs to the field of bioengineering and food technology, and discloses a method for enzymatic resolution of chiral substances, including the following steps: (1) preparing an enzyme solution with a lipase concentration of 1-3000 U/mL, and adding a soluble salt, a hydrophilic solvent and a hydrophobic solvent to the enzyme solution to form a three-liquid phase system; the hydrophobic solvent contains esters or amide compounds composed of racemic chiral compounds; (2) subjecting the three-liquid phase system to enzyme-catalyzed reaction under stirring condition; after the reaction is completed, standing or centrifuging the three-liquid phase system to divide it into three layers, which are a upper liquid layer, a middle liquid layer and a lower liquid layer from top to bottom. The optically pure chiral product after hydrolysis is mainly rich in the middle liquid layer or the lower liquid layer, while the upper liquid layer product is another ester or amide product containing an optically pure chiral product. The method has the advantages of low energy consumption, high raw material utilization rate, and mild reaction conditions, and solves the problems of low chiral resolution efficiency, poor chiral selectivity, low recovery rate, and difficulty in industrialization in the existing enzymatic method.

First claim

The invention claimed is:

1. A method for enzymatic resolution of chiral substances, characterized in that it comprises the following steps:
(1) preparing an enzyme solution with a lipase concentration of 1-3000 U/mL, adding one or more soluble salts, one or more hydrophilic solvents and one or more hydrophobic solvents to the enzyme solution to form a three-liquid phase system;
wherein the mass ratios of the soluble salts, the hydrophilic solvents and the hydrophobic solvents to the enzyme solution are 0.1-0.9, 0.1-5 and 0.1-10, respectively, and wherein the hydrophobic solvents contain one or more ester or amide compounds composed of one or more racemic chiral compounds;
(2) subjecting the three-liquid phase system to enzyme-catalyzed reaction by stirring to hydrolyze one enantiomer of the racemic chiral compounds of each of the one or more esters or amides; and then
(3) standing or centrifuging the three-liquid phase system until it is divided into three layers, which are an upper liquid layer, a middle liquid layer and a lower liquid layer, wherein the chiral product of the hydrolyzed enantiomer of the each ester or amide is concentrated in the middle liquid layer or the lower liquid layer, and the other enantiomer of each of the one or more esters or amides is concentrated in the upper liquid layer.

2. The method according to claim 1 , characterized in that the hydrophilic solvents in step (1) are one or more of polyethylene glycol, polypropylene glycol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, ethylene glycol, acetone, 1-Butyl-3-methylimidazolium bromide ([BMIM]Br), 1-Butyl-3-methylimidazolium tetrafluoroborate ([BMIM]BF 4 ), 1-Ethyl-3-methylimidazolium ethyl sulfate ([EMIM]ETSO 4 ), and 1-methyl-3-octylmidazolium chloride ([OMIM]Cl).

3. The method according to claim 2 , characterized in that the reaction conditions of step (2) are: a temperature of 30-45° C. and a reaction time of 20 min-4 h.

4. The method according to claim 3 , characterized in that the pH value of the three-liquid phase system in step (1) is 5-9.

5. The method according to claim 2 , characterized in that the ester or amide compounds composed of the racemic chiral compounds are one or more of racemic methyl mandelate, racemic Naproxen methyl ester, racemic (4-methoxy-phenyl)-1-ethanol acetate, racemic 1-(4-methoxyphenyl) ethanol acetate, and racemic 6-methyl-5-heptenyl-2-ol acetate.

6. The method according to claim 2 , characterized in that the ester or amide compounds composed of the racemic chiral compounds accounts for 0.1%-10% of the mass of the hydrophobic solvents.

7. The method according to claim 1 , characterized in that the soluble salts in step (1) are one or more of sodium citrate, sodium chloride, ammonium sulfate, sodium carbonate, potassium phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate.

8. The method according to claim 7 , characterized in that the reaction conditions of step (2) are: a temperature of 30-45° C. and a reaction time of 20 min-4 h.

9. The method according to claim 7 , characterized in that the ester or amide compounds composed of the racemic chiral compounds are one or more of racemic methyl mandelate, racemic Naproxen methyl ester, racemic (4-methoxy-phenyl)-1-ethanol acetate, racemic 1-(4-methoxyphenyl) ethanol acetate, and racemic 6-methyl-5-heptenyl-2-ol acetate.

10. The method according to claim 7 , characterized in that the ester or amide compounds composed of the racemic chiral compounds accounts for 0.1%-10% of the mass of the hydrophobic solvents.

11. The method according to claim 1 , characterized in that the hydrophobic solvents in step (1) are one or more of n-hexane, diethyl ether, isopropyl ether, ethyl acetate, cyclohexanol, petroleum ether, isooctane, benzene and toluene.

12. The method according to claim 11 , characterized in that the reaction conditions of step (2) are: a temperature of 30-45° C. and a reaction time of 20 min-4 h.

13. The method according to claim 11 , characterized in that the ester or amide compounds composed of the racemic chiral compounds are one or more of racemic methyl mandelate, racemic Naproxen methyl ester, racemic (4-methoxy-phenyl)-1-ethanol acetate, racemic 1-(4-methoxyphenyl) ethanol acetate, and racemic 6-methyl-5-heptenyl-2-ol acetate.

14. The method according to claim 11 , characterized in that the ester or amide compounds composed of the racemic chiral compounds accounts for 0.1%-10% of the mass of the hydrophobic solvents.

15. The method according to claim 1 , characterized in that the reaction conditions of step (2) are a temperature of 30-45° C., and a reaction time of 20 min-4 h.

16. The method according to claim 15 , characterized in that the pH value of the three-liquid phase system in step (1) is 5-9.

17. The method according to claim 16 , characterized in that the lipase concentration in step (1) is 5-2000 U/mL.

18. The method according to claim 17 , characterized in that the mass ratios of the soluble salts, hydrophilic solvents and hydrophobic solvents to the enzyme solution are 0.2-0.8, 0.2-0.8 and 0.2-4, respectively.

19. The method according to claim 1 , characterized in that the ester or amide compounds composed of the racemic chiral compounds are one or more of racemic methyl mandelate, racemic Naproxen methyl ester, racemic (4-methoxy-phenyl)-1-ethanol acetate, racemic 1-(4-methoxyphenyl) ethanol acetate, and racemic 6-methyl-5-heptenyl-2-ol acetate.

20. The method according to claim 1 , characterized in that the ester or amide compounds composed of the racemic chiral compounds accounts for 0.1%-10% of the mass of the hydrophobic solvents.

Assignees

• Univ South China Tech

Inventors

• Li Zhigang
• Su Jinfen
• Yang Bo
• Wang Yonghua
• Li Zhengcheng
• Chen Huayong

Classifications

• C12P13/02

  Amides, e.g. chloramphenicol {or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes (peptides C12P21/00 or C07K)} · CPC title

• C12Y301/01003 Primary

  Triacylglycerol lipase (3.1.1.3) · CPC title

• C12P41/004

  by esterification of alcohol- or thiol groups in the enantiomers or the inverse reaction · CPC title

• C12P41/005

  by esterification of carboxylic acid groups in the enantiomers or the inverse reaction · CPC title

• C12P7/42

  Hydroxy-carboxylic acids · CPC title