# US 5,350,871 A · Method of preparing optically active cyanohydrins > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,350,871 A - Document ID: US5350871A - Status: Granted - Kind: A - Application number: 07/986,784 - Filed: 1992-12-08 - Granted: 1994-09-27 - Earliest claimed: 1991-12-11 - Assignees: Duphar International Research B.V. - Inventors: Hendrik W. Geluk; Wybrand T. Loos - Web page: https://patentgrep.com/patent/US5350871A ## CPC - C12P 13/004: {Cyanohydrins} - C12P 17/04: containing a five-membered hetero ring, e.g. griseofulvin {, vitamin C} - 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)} ## Abstract The invention relates to a method of preparing an optically active cyanohydrin by addition of hydrogen cyanide to a carbonyl compound, selected from aldehydes and ketones, in the presence of hydroxynitrile lyase, in a biphasic solvent system, comprising a homogeneous aqueous solution of said enzyme, and a suitable organic solvent which is at least substantially immiscible with water. Said method is performed in that said homogeneous aqueous solution is buffered with an acetate buffer having a buffer concentration of between 0.005 and 0.1 mole per liter or with a non-acetate buffer, that the volume ratio organic phase:aqueous phase is between approx. 5:1 and approx. 1:5, and that a solution of hydrogen cyanide and said carbonyl compound in said organic solvent is properly mixed during the reaction period with said homogeneous aqueous solution of hydroxynitrile lyase. ## Claims ### Claim 1 A method of preparing an optically active cyanohydrin by addition of hydrogen cyanide to a carbonyl compound, selected from aldehydes and ketones which allow addition of hydrogen cyanide, in a biphasic solvent system, comprising a homogeneous aqueous solution of hydroxynitrile lyase, and a suitable organic solvent which is at least substantially immiscible with water, said method being characterized in that said homogeneous aqueous solution is buffered with a buffer solution having a pH between 3 and 6, said buffer solution being selected from an acetate buffer and a non-acetate buffer, wherein said acetate buffer has a buffer concentration of between 0.005 and 0.1 mole per liter and wherein said non-acetate buffer is selected from citrate, succinate, glutamate and phthalate buffers, that the volume ratio organic phase:aqueous phase is between approx. 5:1 and approx, 1:5, and that a solution of hydrogen cyanide and said carbonyl compound in said organic solvent is properly mixed to ensure sufficient interfacial area between the phases during the reaction period with said homogeneous aqueous solution of hydroxynitrile lyase. ### Claim 2 (depends on 1) A method as claimed in claim 1, characterized in that the volume ratio organic phase:aqueous phase varies between approx 3:1 and approx 1:3. ### Claim 3 (depends on 1, 2) A method as claimed in claim 1 or 2, characterized in that the concentration of the non-acetate buffer is between 0.005 and 0.5 mole per liter and that the pH of the buffer solution is between approx. 4 and approx. 5.5. ### Claim 4 (depends on 3) A method as claimed in claim 3, characterized in that the buffer is selected from the group consisting of citrate, succinate, glutamate and phthalate buffer. ### Claim 5 (depends on 4) A method as claimed in claim 4, characterized in that to a solution of hydrogen cyanide in said organic solvent are successively added said homogeneous buffered solution of hydroxynitrile lyase and said carbonyl compound, after which the biphasic reaction system, thus obtained, is properly mixed during the reaction period. ### Claim 6 (depends on 1) A method as claimed in claim 1 wherein the organic solvent is selected from the group consisting of di(C.sub.1 -C.sub.6)alkyl ethers, (C.sub.1 -C.sub.5)carboxylic-(C.sub.1 -C.sub.5)alkyl esters, di(C.sub.1 -C.sub.5)alkyl ketones, (C.sub.4 -C.sub.8)aliphatic alcohols, and mixtures of these solvents with each other or with apolar diluents. ### Claim 7 (depends on 6) A method as claimed in claim 6, wherein the organic solvent is selected from diethyl ether, di-n-propyl ether, di-isopropyl ether, di-n-butyl ether, di-isobutyl ether, methyl-t-butyl ether, n-propyl acetate, ethyl acetate, isopropyl acetate, isomeric butyl acetates, isomeric amyl acetates, methylethylketone, diethylketone, methylisobutylketone, and a mixture of these solvents with each other or with an apolar diluent selected from aromatic hydrocarbons, aliphatic hydrocarbons and chlorinated aromatic or aliphatic hydrocarbons. ### Claim 8 (depends on 7) A method as claimed in claim 7, wherein the organic solvent is selected from n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, sec.-butyl acetate and amyl acetate. ### Claim 9 (depends on 6, 7, 8) A method as claimed in claim 6, 7 or 8, characterized in that the concentration of said carbonyl compound in said organic solvent is more than 5% wt./vol. ### Claim 10 (depends on 1) A method as claimed in claim 1, characterized in that after the reaction period the aqueous phase is separated from the organic phase and is reused at least once by properly mixing said aqueous phase with a fresh solution of hydrogen cyanide and said carbonyl compound in said organic solvent during another reaction period. ### Claim 11 (depends on 3) A method as claimed in claim 3, wherein the concentration of the non-acetate buffer is between 0.01 and 0.2 mole per liter. ### Claim 12 A method of preparing an optically active cyanohydrin by addition of hydrogen cyanide to a carbonyl compound, selected from aldehydes and ketones which allow addition of hydrogen cyanide, in a biphasic solvent system, comprising a homogeneous aqueous solution of hydroxynitrile lyase, and a suitable organic solvent which is at least substantially immiscible with water, said method being characterized in that said homogeneous aqueous solution is buffered with a buffer solution having a pH between 3 and 6, said buffer solution being selected from an acetate buffer and non-acetate buffer, wherein said acetate buffer has a buffer concentration of between 0.005 and 0.1 mole per liter and wherein said non-acetate-buffer is selected from citrate, succinate, glutamate and phthalate buffers, that the volume ratio organic phase:aqueous phase is between approx. 3:1 and 1:3, and that a solution of hydrogen cyanide and said carbonyl compound in said organic solvent is properly mixed to ensure sufficient interfacial area between the phase during the reaction period with said homogeneous aqueous solution of hydroxynitrile lyase.