# US 5,352,590 A · Process for the preparation of .sup.13 C-labelled compounds > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,352,590 A - Document ID: US5352590A - Status: Granted - Kind: A - Application number: 07/846,478 - Filed: 1992-03-06 - Granted: 1994-10-04 - Earliest claimed: 1991-03-08 - Assignees: Tokyo Gas Co., Ltd. - Inventors: Nobuo Kato; Yoshiyuki Sato; Kunihiko Shibata - Web page: https://patentgrep.com/patent/US5352590A ## CPC - C12N 11/18: Multi-enzyme systems - C12P 1/00: Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes - C12P 19/14: produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - 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 A process for preparing a .sup.13 C-labelled compound which comprises reacting an alcohol .sup.13 C-labelled carbon source compound and a substrate in the presence of an enzyme system consisting of oxidase belonging to EC1 group and capable of converting the alcohol .sup.13 C-labelled carbon source compound to an aldehyde .sup.13 C-labelled carbon source compound, lyase belonging to EC4 group and capable of synthesizing a carbon-carbon bond and at least one of isomerases belonging to EC5 group and capable of isomerizing substrates to obtain a .sup.13 C-labelled compound specifically labelled with .sup.13 C in a specific carbon position. ## Claims ### Claim 1 A process for preparing a .sup.13 C-labelled compound which comprises reacting a substrate and an aldehyde .sup.13 C-labelled carbon source compound at a C-1 position in the presence of an enzyme system consisting of lyase specific to the substrate belonging to EC4 group synthesizing a carbon-carbon bond and at least one of isomerases capable of isomerizing substrates to obtain a .sup.13 C-labelled compound specifically labelled with .sup.13 C, only in a carbon position C-1. ### Claim 2 (depends on 1) A process as claimed in claim 1, which process comprises reacting ribose 5-phosphate with .sup.13 C-labelled formaldehyde in the presence of a formaldehyde-fixing enzyme system consisting of phosphoriboisomerase, hexulose phosphate synthase and phosphohexuloisomerase to obtain .sup.13 C-labelled fructose 6-phosphate specifically with .sup.13 C, only in a carbon position C-1. ### Claim 3 (depends on 1) A process as claimed in claim 1, which process comprises reacting ribose 5-phosphate with .sup.13 C-labelled formaldehyde in the presence of a formaldehyde-fixing enzyme system consisting of phosphoriboisomerase, hexulose phosphate synthase, phosphohexuloisomerase and phosphoglucoisomerase to obtain .sup.13 C-labelled glucose 6-phosphate specifically with .sup.13 C, only in a carbon position C-1. ### Claim 4 A process for preparing a .sup.13 C-labelled compound which comprises isomerizing a substrate in the presence of a first isomerase to form an isomerized substrate, reacting the isomerized substrate in the presence of lyase with an aldehyde .sup.13 C-labelled carbon source compound at a C-1 position to obtain a condensate specifically labelled with .sup.13 C, only in a carbon position C-1, and isomerizing the condensate in the presence of a second isomerase to obtain a first isomer specifically labelled with .sup.13 C, only in a carbon position C-1. ### Claim 5 (depends on 4) A process as claimed in claim 4, which process further comprises isomerizing the first isomer in the presence of a third isomerase to form a second isomer. ### Claim 6 (depends on 5) A process as claimed in claim 5, which process comprises isomerizing ribose 5-phosphate in the presence of phosphoriboisomerase to form riburose 5-phosphate, reacting the riburose 5-phosphate with .sup.13 C-labelled formaldehyde in the presence of hexulose phosphate synthase to form .sup.13 C-labelled hexulose 6-phosphate, isomerizing .sup.13 C-labelled hexulose 6-phosphate in the presence of phosphohexuloisomerase to form .sup.13 C-labelled fructose 6-phosphate, and isomerizing .sup.13 C-labelled fructose 6-phosphate in the presence of phosphoglucoisomerase to form .sup.13 C-labelled glucose 6-phosphate specifically labelled with .sup.13 C, only in a carbon position C-1. ### Claim 7 (depends on 4) A process as claimed in claim 4, which process comprises isomerizing ribose 5-phosphate in the presence of phosphoriboisomerase to form riburose 5-phosphate, reacting the riburose 5-phosphate with .sup.13 C-labelled formaldehyde in the presence of hexulose phosphate synthase to form .sup.13 C-labelled hexulose 6-phosphate, and isomerizing .sup.13 C-labelled hexulose 6-phosphate in the presence of phosphohexuloisomerase to form .sup.13 C-labelled fructose 6-phosphate specifically labelled with .sup.13 C, only in a carbon position C-1. ### Claim 8 (depends on 6, 7) A process as claimed in any one of claims 6-7, wherein hexulose phosphate synthase and phosphohexuloisomerase are prepared by a process which comprises incubating and culturing Methylomonas aminofaciens 77a strain with a liquid medium containing methanol as a sole carbon source to obtain a cell-free extract, and separating and purifying the cell-free extract. ### Claim 9 A process for preparing a .sup.13 C-labelled compound which process comprises reacting ribose 5-phosphate with .sup.13 C-labelled methanol in the presence of alcohol oxidase and of a formaldehyde-fixing enzyme system consisting of phosphoriboisomerase, hexulose phosphate synthase and phosphohexuloisomerase to obtain .sup.13 C-labelled fructose 6-phosphate specifically labelled with .sup.13 C, only in a carbon position C-1. ### Claim 10 A process for preparing a .sup.13 C-labelled compound, which process comprises isomerizing ribose 5-phosphate in the presence of phosphoriboisomerae to form riburouse 5-phosphate, reacting the riburose 5-phosphate in the presence of hexulose phosphate synthase with .sup.13 C-labelled formaldehyde to form .sup.13 C-labelled hexulose 6-phosphate, and isomerizing .sup.13 C-labelled hexulose 6-phosphate in the presence of phosphohexuloisomerase to form .sup.13 C-labelled fructose 6-phosphate specifically labelled with .sup.13 C, only in a carbon position C-1, said .sup.13 C-labelled formaldehyde being formed by oxidizing .sup.13 C-labelled methanol in the presence of a alcohol oxidase. ### Claim 11 A process for preparing a .sup.13 C-labelled compound, which process comprises reacting ribose 5-phosphate with .sup.13 C-labelled methanol in the presence of alcohol oxidase and of a formaldehyde-fixing enzyme system consisting of phosphoriboisomerase, hexulose phosphate synthase, phosphohexuloisomerase and phosphoglucoisomerase to obtain .sup.13 C-labelled glucose 6-phosphate specifically labelled with .sup.13 C, only in a carbon position C-1. ### Claim 12 A process for preparing a .sup.13 C-labelled compound, which process comprises isomerizing ribose 5-phosphate in the presence of phosphoriboisomerase to form riburose 5-phosphate, reacting the riburose 5-phosphate in the presence of hexulose phosphate synthase with .sup.13 C-labelled formaldehyde to form .sup.13 C-labelled hexulose 6-phosphate, isomerizing .sup.13 C-labelled hexulose 6-phosphate in the presence of phosphohexuloisomerase to form .sup.13 C-labelled fructose 6-phosphate specifically labelled with .sup.13 C, only in a carbon position C-1, and isomerizing .sup.13 C-labelled fructose 6-phosphate in the presence of phosphoglucoisomerase to form .sup.13 C-labelled glucose 6-phosphate specifically labelled with .sup.13 C, only in a carbon position C-1, said .sup.13 C-labelled formaldehyde being formed by oxidizing .sup.13 C-labelled methanol in the presence of alcohol oxidase. ### Claim 13 (depends on 9, 10, 11) A process as claimed in claim 9, 10, 11, or 12 wherein said oxidase is used in combination with catalase and hydrogen peroxide. ### Claim 14 (depends on 2, 3, 9, 11) A process as claimed in claim 2, 3, 9 or 11 wherein the formaldehyde-fixing enzyme system is a cell-free extract prepared by incubating and culturing Methylomonas aminofaciens 77a strain with a liquid medium containing methanol as a sole carbon source and containing phosphoriboisomerase, hexulose phosphate synthase, phosphohexuloisomerase and phosphoglucoisomerase.