# US 4,400,465 A · Semi-synthesis of human insulin > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 4,400,465 A - Document ID: US4400465A - Status: Granted - Kind: A - Application number: 06/336,071 - Filed: 1981-12-31 - Granted: 1983-08-23 - Earliest claimed: 1979-04-13 - Assignees: Shionogi & Co., Ltd. - Inventors: Kazuyuki Morihara; Tatsushi Oka; Hiroshige Tsuzuki - Web page: https://patentgrep.com/patent/US4400465A ## CPC - C07K 14/62: Insulins - Y02P 20/55: Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups - Y10S 435/824: Achromobacter - Y10S 435/886: Streptomyces - Y10S 435/896: Streptomyces fradiae - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS - Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS ## Abstract A process for preparing a B30-threonine-insulin which comprising reacting a des-B30-insulin with an excess amount of threonine derivative in the presence of an enzyme specifically acting on the basic-amino-acid carbonyl in peptide bondings. ## Claims ### Claim 1 A process for preparing a B30-threonine-insulin which comprises converting an animal insulin to a des-B30-insulin, reacting the des-B30-insulin with a threonine derivative of the formula. Thr(R.sup.1)(R.sup.2) wherein Thr is an L-threonine residue, R.sup.1 is hydrogen or a hydroxy-protecting group and R.sup.2 is a carboxyl-protecting group, in a molar ratio of the threonine derivative to the des-30-insulin of 5:1 to 1000:1, and in the presence of an enzyme specifically acting on the basic-amino-acid carbonyl in peptide bondings selected from the group consisting of trypsin, a trypsin-like enzyme produced by Streptomyces fradiae ATCC 3535 and a trypsin-like enzyme produced by Streptomyces erythreus, and subjecting the resultant product to one or more reactions to remove all of the protecting groups. ### Claim 2 (depends on 1) The process according to claim 1, wherein the reaction is carried out in the presence of trypsin. ### Claim 3 (depends on 1) The process according to claim 1, wherein the molar ratio of the threonine derivative to the des-B30-insulin is about 25:1 to 200:1. ### Claim 4 (depends on 1) The process according to claim 1, wherein the molar ratio of the threonine derivative to the des-B30-insulin is about 20:1 to 100:1. ### Claim 5 (depends on 1) The process according to claim 1, wherein the B30-threonine-insulin is human insulin, and the animal insulin is porcine insulin. ### Claim 6 (depends on 1, 2) The process according to claim 1 or 2, wherein the reaction is effected in a medium containing one or more water-miscible organic solvents. ### Claim 7 (depends on 6) The process according to claim 6, wherein the organic solvent is selected from the group consisting of methanol, ethanol, dimethylformamide, dimethyl sulfoxide and glycerol. ### Claim 8 (depends on 7) The process according to claim 7, wherein the organic solvent is selected from the group consisting of ethanol and dimethylformamide. ### Claim 9 A process for preparing a protected B30-threonine-insulin which comprises converting an animal insulin to a des-B30-insulin, and reacting the des-B30-insulin with a threonine derivative of the formula Thr(R.sup.1)(R.sup.2) wherein Thr is an L-threonine residue, R.sup.1 is hydrogen or a hydroxy-protecting group and R.sup.2 is a carboxyl-protecting group, in a molar ratio of the threonine derivative to the des-B30-insulin of 5:1 to 1000:1, and in the presence of an enzyme specifically acting on the basic-amino-acid carbonyl in peptide bondings selected from the group consisting of trypsin, a trypsin-like enzyme produced by Streptomyces fradiae ATCC 3535 and a trypsin-like enzyme produced by Streptomyces erythreus. ### Claim 10 (depends on 9) The process according to claim 9, wherein the reaction is carried out in the presence of trypsin. ### Claim 11 (depends on 9) The process according to claim 9, wherein the molar ratio of the threonine derivative to the des-B30-insulin is about 25:1 to 200:1. ### Claim 12 (depends on 9) The process according to claim 9, wherein the molar ratio of the threonine derivative to the des-B30-insulin is about 20:1 to 100:1. ### Claim 13 (depends on 9) The process according to claim 9, wherein the protected B30-threonine-insulin is a protected human insulin, and the animal insulin is porcine insulin. ### Claim 14 (depends on 1, 9) The process according to claim 1 or 9, wherein the des-B30-insulin is des-B30-porcine-insulin. ### Claim 15 (depends on 1, 9) The process according to claim 1 or 9, wherein the des-B30-insulin is des-B30-bovine insulin. ### Claim 16 (depends on 1, 9) The process according to claim 1 or 9, wherein the threonine derivative is L-threonine t-butyl ester. ### Claim 17 (depends on 1, 9) The process according to claim 1 or 9, wherein the reaction is effected at pH 5 to pH 9. ### Claim 18 (depends on 17) The process according to claim 17, wherin the reaction is effected at pH 6 to pH 7. ### Claim 19 (depends on 1, 9) The process according to claim 1 or 9, wherein the reaction is effected at a temperature of 0.degree. to 50.degree. C. ### Claim 20 (depends on 19) The process according to claim 19, wherein the reaction is effected at a temperature of 20.degree. to 40.degree. C.