# US 5,348,973 A · Resolution of hydroxychroman-2-carboxylic acid esters by enantiomeric hydrolysis > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,348,973 A - Document ID: US5348973A - Status: Granted - Kind: A - Application number: 07/594,317 - Filed: 1990-10-09 - Granted: 1994-09-20 - Earliest claimed: 1990-10-09 - Assignees: Rhone-Poulenc Chimie - Inventors: Muppala S. Raju; Namdol Huh - Web page: https://patentgrep.com/patent/US5348973A ## CPC - C12P 17/06: containing a six-membered hetero ring, e.g. fluorescein - C07D 311/66: with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2 - C12P 41/005: {by esterification of carboxylic acid groups in the enantiomers or the inverse reaction} - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08) ## Abstract Hydroxychroman-2-carboxylic acid esters can be resolved by enantiospecific hydrolysis by the use of liver enzymes, particularly solvent treated (acetone) enzymes. Liver enzymes from calf, cat, dog, eel, goat, lungfish, mouse, rat and seal have been found to be enantiospecific. ## Claims ### Claim 1 A process for resolving by enantiospecific hydrolysis carboxylic acid esters which are the methyl or ethyl esters of 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid comprising the step of hydrolyzing said esters with an animal liver enzyme derived from an animal selected from the group consisting of calf, cat, dog, eel, goat, lungfish, mouse, rat and seal; wherein percent acid produced upon hydrolysis is at least about 26% and percent enantiomeric excess of said resolved acid is at least about 8%. ### Claim 2 (depends on 1) The method as recited in claim 1 wherein the hydroxychroman carboxylic acid esters are the methyl and ethyl esters of 6-hydroxy-2,5,7,8-tetramethyl chroman-2-carboxylic acid. ### Claim 3 (depends on 1) The method recited in claim 1 wherein the liver enzyme is in crude form, solvent treated liver enzyme or a purified liver enzyme. ### Claim 4 (depends on 3) The method as recited in claim 3 wherein the liver enzyme is a solvent treated liver enzyme and the solvent is acetone. ### Claim 5 (depends on 1) The method as recited in claim 1 wherein the liver enzyme is immobilized on a solid support. ### Claim 6 (depends on 5) The method as recited in claim 5 wherein the enzyme is immobilized by absorbing the enzyme on an ion exchange resin followed by contacting the so absorbed enzyme with a polyaziridine prepolymer in the presence of a fatty acid. ### Claim 7 (depends on 1) The method as recited in claim 1 wherein the hydrolysis is conducted in an aqueous phase in the presence of an organic solvent for the ester. ### Claim 8 (depends on 7) The method as recited in claim 7 wherein the solvent is a hydrocarbon solvent, an aromatic hydrocarbon solvent or an aprotic polar organic solvent. ### Claim 9 (depends on 7) The method as recited in claim 7 wherein the solvent is an aliphatic or aromatic ether. ### Claim 10 (depends on 9) The method as recited in claim 9 wherein the ether is selected from the group consisting of propyl ether, isopropyl ether, butyl ether, isobutyl ether and diphenyl ether. ### Claim 11 (depends on 7) The method as recited in claim 7 wherein the organic solvent is toluene or xylenes. ### Claim 12 (depends on 1) The process of claim 1 additionally comprising stabilizing the enzyme with a protease inhibitor. ### Claim 13 (depends on 1) The method of claim 1 additionally comprising immobilizing the enzyme with a prepolymer of at least one of polyazetidine, carboxymethyl cellulose, polymethylene isocyanate, polyurethane hydrogel and polyfunctional aziridine. ### Claim 14 (depends on 1) The method of claim 1 wherein said hydrolyzing is carried out at a temperature of below about 50.degree. C. ### Claim 15 (depends on 1) The method of claim 1 wherein said hydrolyzing is carried out at a temperature as low as about 10.degree. C. ### Claim 16 (depends on 1) The method of claim 1 wherein said hydrolyzing is carried out at a temperature of about 25.degree. C. to about 50.degree. C. ### Claim 17 (depends on 1) The method of claim 1 wherein said hydrolyzing is carried out at a temperature of about 30.degree. C. to about 40.degree. C. ### Claim 18 (depends on 1) The method of claim 1 wherein said hydrolyzing is carried out at a pH of from about 5 to about 8.5. ### Claim 19 (depends on 18) The method of claim 18 wherein said pH is from about 6.5 to about 7.5. ### Claim 20 (depends on 1) The method of claim 1 wherein said hydrolyzing ranges from about one half hour to about several days. ### Claim 21 (depends on 1) The method of claim 1 wherein said hydrolyzing is carried out in an aqueous solution or in a mixed aqueous solution-organic solvent system. ### Claim 22 (depends on 21) The method of claim 21 additionally comprising separating the resolved acid from solution by salting out or precipitation. ### Claim 23 (depends on 1) The method of claim 1 additionally comprising stabilizing the resolved acid against oxidation during the course of hydrolysis by adding a reducing agent. ### Claim 24 (depends on 23) The method of claim 23 wherein said reducing agent is selected from at least one of ascorbic acid, sodium metabisulfite and sodium sulfite. ### Claim 25 (depends on 23) The method of claim 23 additionally comprising adding said reducing agent in an amount ranging from about 1 mg/ml to 5 mg/ml. ### Claim 26 (depends on 22) The method of claim 22 wherein said mixed aqueous solution-organic solvent system comprises from about zero to about 99% by volume water-miscible organic solvent. ### Claim 27 (depends on 26) The method of claim 26 wherein said water-miscible solvent is selected from at least one of alcohols, glycols, glycol ethers, triols, cyclic oxides, ketones and nitrogen-containing compounds. ### Claim 28 (depends on 27) The method of claim 27 wherein said water-miscible solvent is selected from at least one of C.sub.1 -C.sub.3 alcohols, 1-methoxy-2-propanol, propylene glycol, polyethylene glycol, polypropylene glycol, dimethyl ether of ethylene glycol, dimethyl ether of propylene glycol, dimethyl ether of diethylene glycol, dimethyl ether of tetraethylene glycol, glycerol, tetrahydrofuran, dioxane, acetone, acetonitrile and dimethyl formamide. ### Claim 29 (depends on 22) The method of claim 22 wherein said mixed aqueous solution--organic solvent system comprises from about zero to about 50% by volume aqueous component and from about 100% to about 50% water immiscible organic solvent component to form a two phase system. ### Claim 30 (depends on 29) The method of claim 29 wherein said water-immiscible solvent is selected from at least one of hydrocarbons, chlorinated hydrocarbons, esters, ethers and alcohols. ### Claim 31 (depends on 30) The method of claim 30 wherein said water-immiscible solvent is selected from at least one of hexane, heptone, isooctane, decane, hexadecane, kerosene, petroleum ether, toluene, xylenes, methylene chloride, chloroform, ethyl acetate, propyl ether, isopropyl ether, butyl ether, isobutyl ether, diphenyl ether, 2-ethyl-1-hexanol and 2-octanol. ### Claim 32 (depends on 1) The method of claim 1 wherein said enantiomeric excess of said resolved acid is at least about 28%. ### Claim 33 (depends on 1) The process of claim 1 wherein said liver enzyme is derived from an animal selected from the group consisting of goat, mouse and rat, the percent acid produced upon hydrolysis is at least about 36%, and the percent enantiomeric excess of said resolved acid is at least about 28%. ### Claim 34 (depends on 1) The process of claim 1 wherein said esters are hydrolyzed with acetone powder containing said animal liver enzyme. ### Claim 35 (depends on 1) The process of claim 1 wherein methyl ester of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid is hydrolyzed. ### Claim 36 (depends on 1) The process of claim 1 wherein ethyl ester of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid is hydrolyzed. ### Claim 37 (depends on 36) The process of claim 36 wherein the animal liver enzyme is derived from goat. ### Claim 38 (depends on 37) The process of claim 37 wherein the enzyme is treated with a solvent selected from the group consisting of isopropyl ether, propyl ether, diphenyl ether, toluene, butyl ether and isobutyl ether, the percent acid produced upon hydrolysis is at least about 31%, and the percent enantiomeric excess of said resolved acid is at least about 24%. ### Claim 39 (depends on 37) The process of claim 37 wherein said enzyme is initially purified by loading upon a DEAE-Sepharose column, equilibrating with buffer, and stepwise eluting with salt gradients, and the resulting enantiomeric excess of resolved acid is at least about 66%. ### Claim 40 (depends on 37) The process of claim 37 wherein said enzyme is initially immobilized by adsorbing on a resin followed by contacting with XAMA-2 solution in the presence of a fatty acid, and the enantiomeric excess of resolved acid is at least about 98%. ### Claim 41 (depends on 37) The process of claim 37 wherein hydrolysis is conducted in the presence of a reducing agent selected from the group consisting of mercaptoethanol, ascorbic acid, sodium metabisulfite and sodium sulfite. ### Claim 42 (depends on 1) The method of claim 1 comprising the additional step of recovering at least one resolved enantiomer of said acid. ### Claim 43 (depends on 1) The method of claim 1 additionally comprising initially esterifying a racemic mixture of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid. ### Claim 44 (depends on 1) The method of claim 1 wherein said esters are C.sub.1 to C.sub.4 alkyl esters of 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid.