# US 2020/0399339 A1 · Process for the preparation of high purity glucagon > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2020/0399339 A1 - Document ID: US20200399339A1 - Status: Published application - Kind: A1 - Application number: 16/905,474 - Filed: 2020-06-18 - Published: 2020-12-24 - Earliest claimed: 2019-06-18 - Assignees: Fresenius Kabi Ipsum S.R.L. - Applicants: Fresenius Kabi Ipsum S.R.L. - Inventors: Andrea ORLANDIN; Antonio RICCI; Walter CABRI; Steve MCINTYRE; Alex SAUNDERS - Web page: https://patentgrep.com/patent/US20200399339A1 ## CPC - C07K 14/605: Glucagons - C07K 1/063: {for alpha-amino functions} - C07K 14/001: {by chemical synthesis} - A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) - 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) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract The present invention provides an improved process for the preparation of high purity glucagon comprising the use of Xmb-protected amino acids, wherein may Xmb include, e.g., 2,4,6-trimethoxybenzyl, 2,4-dimethoxybenzyl, or 2-hydroxy-4-methoxybenzyl. The process also comprises the use of building blocks such as pseudoprolines to avoid aggregation and obtain the product in high yield and purity. ## Claims ### Claim 1 A process for the preparation of glucagon of formula I (SEQ ID NO: 1) (I) 1 5 10 His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- 15 20 Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln- 25 29 Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr, the process comprising the use of at least one Xmb-protected amino acid and at least one pseudoproline dipeptide, wherein Xmb is 2,4,6-trimethoxybenzyl (Tmb), 2,4-dimethoxybenzyl (Dmb), or 2-hydroxy-4-methoxybenzyl group (Hmb). ### Claim 2 (depends on 1) The process according to claim 1, wherein the at least one Xmb-protected amino acid is glycine, alanine, or leucine. ### Claim 3 (depends on 2) The process according to claim 2, wherein the at least one Xmb-protected amino acid is a Tmb-protected amino acid. ### Claim 4 (depends on 3) The process according to claim 3, wherein the Tmb-protected amino acid is (Tmb)Gly. ### Claim 5 (depends on 1) The process according to claim 1, wherein the process is performed by solid phase peptide synthesis (SPPS). ### Claim 6 (depends on 1) The process according to claim 1, wherein the pseudoproline dipeptide is selected from: Fmoc-Asp(P)-Ser[psi(R1, R1)pro]-OH (Fmoc-pDS) Fmoc-Asn(P)-Thr[psi(R1, R1)pro]-OH (Fmoc-pNT) Fmoc-Tyr(P)-Ser[psi(R1, R1)pro]-OH (Fmoc-pYS) Fmoc-Phe-Thr[psi(R1, R1)pro]-OH (Fmoc-pFT) and Fmoc-Thr(P)-Ser[psi(R1, R1)pro]-OH (Fmoc-pTS), wherein P is a protecting group or is absent, and R1is hydrogen or methyl. ### Claim 7 (depends on 6) The process according to claim 6, wherein the pseudoproline dipeptide is selected from: Fmoc-Asp(OtBu)-Ser[psi(Me, Me)pro]-OH Fmoc-Asn(Trt)-Thr[psi(Me, Me)pro]-OH Fmoc-Tyr(tBu)-Ser[psi(Me, Me)pro]-OH Fmoc-Phe-Thr[psi(Me, Me)pro]-OH and Fmoc-Thr(tBu)-Ser[psi(Me, Me)pro]-OH. ### Claim 8 (depends on 6) The process according to claim 6, wherein one of the following C-terminal peptides is used as an intermediate: (SEQ ID NO: 2) (Xmb)Gly-Thr(P)-Phe-Thr(P)-Ser(P)-Asp(P)-Tyr(P)- Ser(P)-Lys(P)-Tyr(P)-Leu-pDS-Arg(P)-Arg(P)-Ala- Gln(P)-Asp(P)-Phe-Val-Gln(P)-Trp(P)-Leu-Met- Asn(P)-Thr(P), (SEQ ID NO: 3) (Xmb)Gly-Thr(P)-Phe-Thr(P)-Ser(P)-Asp(P)-pYS- Lys(P)-Tyr(P)-Leu-Asp(P)-Ser(P)-Arg(P)-Arg(P)- Ala-Gln(P)-Asp(P)-Phe-Val-Gln(P)-Trp(P)-Leu-Met- Asn(P)-Thr(P), (SEQ ID NO: 4) (Xmb)Gly-Thr(P)-Phe-pTS-Asp(P)-Tyr(P)-Ser(P)- Lys(P)-Tyr(P)-Leu-Asp(P)-Ser(P)-Arg(P)-Arg(P)- Ala-Gln(P)-Asp(P)-Phe-Val-Gln(P)-Trp(P)-Leu-Met- Asn(P)-Thr(P), (SEQ ID NO: 5) (Xmb)Gly-Thr(P)-Phe-pTS-Asp(P)-Tyr(P)-Ser(P)- Lys(P)-Tyr(P)-Leu-pDS-Arg(P)-Arg(P)-Ala-Gln(P)- Asp(P)-Phe-Val-Gln(P)-Trp(P)-Leu-Met-Asn(P)- Thr(P), (SEQ ID NO: 6) (Xmb)Gly-Thr(P)-Phe-Thr(P)-Ser(P)-Asp(P)-pYS- Lys(P)-Tyr(P)-Leu-pDS-Arg(P)-Arg(P)-Ala-Gln(P)- Asp(P)-Phe-Val(P)-Gln(P)-Trp(P)-Leu-Met-Asn(P)- Thr(P), (SEQ ID NO: 7) pTS-Asp(P)-Tyr(P)-Ser(P)-Lys(P)-Tyr(P)-(Xmb)Leu- Asp(P)-Ser(P)-Arg(P)-Arg(P)-Ala-Gln(P)-Asp(P)- Phe-Val-Gln(P)-Trp(P)-Leu-Met-Asn(P)-Thr(P), (SEQ ID NO: 8) pYS-Lys(P)-Tyr(P)-(Xmb)Leu-Asp(P)-Ser(P)-Arg(P)- Arg(P)-Ala-Gln(P)-Asp(P)-Phe-Val-Gln(P)-Trp(P)- Leu-Met-Asn(P)-Thr(P), (SEQ ID NO: 9) pDS-Arg(P)-Arg(P)-(Xmb)Ala-Gln(P)-Asp(P)-Phe- Val-Gln(P)-Trp(P)-Leu-Met-Asn(P)-Thr(P), (SEQ ID NO: 10) pYS-Lys(P)-Tyr(P)-Leu-Asp(P)-Ser(P)-Arg(P)- Arg(P)-(Xmb)Ala-Gln(P)-Asp(P)-Phe-Val-Gln(P)- Trp(P)-Leu-Met-Asn(P)-Thr(P), and (SEQ ID NO: 11) pTS-Asp(P)-Tyr(P)-Ser(P)-Lys(P)-Tyr(P)-Leu- Asp(P)-Ser(P)-Arg(P)-Arg(P)-(Xmb)Ala-Gln(P)- Asp(P)-Phe-Val-Gln(P)-Trp(P)-Leu-Met-Asn(P)- Thr(P), wherein Xmb is selected from Tmb, Dmb, and Hmb, and P is a side-chain protecting group or is absent. ### Claim 9 (depends on 8) The process according to claim 8, wherein the intermediate C-terminal peptide is: (SEQ ID NO: 2) (Tmb)Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)- Asp(tBu)-Tyr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)- Leu-pDS-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)- Asp(tBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met- Asn(Trt)-Thr(tBu), wherein pDS is Asp(OtBu)-Ser[psi(Me, Me)pro]. ### Claim 10 (depends on 8) The process according to claim 8, wherein the intermediate C-terminal peptide is attached to a solid support at the C-terminal end. ### Claim 11 (depends on 10) The process according to claim 10, wherein the solid support is a Wang resin. ### Claim 12 (depends on 1) The process according to claim 1, wherein at least one coupling reagent selected from diisopropylcarbodiimide, dicyclohexylcarbodiimide, (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, and ethyl-dimethylaminopropyl carbodiimide is used. ### Claim 13 (depends on 12) The process according to claim 12, wherein the coupling reagent is diisopropylcarbodiimide, which is used in the presence of ethyl 2-cyano-2-hydroxyimino-acetate. ### Claim 14 (depends on 6) The process according to claim 6, wherein the side-chain protecting group P is tertbutyloxycarbonyl, tert-butyl, trityl, or 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl. ### Claim 15 (depends on 8) The process according to claim 8, wherein the side-chain protecting group P is tertbutyloxycarbonyl, tert-butyl, trityl, or 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl. ### Claim 16 A protected glucagon sequence selected from: (SEQ ID NO: 12) His(P)-Ser(P)-Gln(P)-(Xmb)Gly-Thr(P)-Phe-Thr(P)- Ser(P)-Asp(P)-Tyr(P)-Ser(P)-Lys(P)-Tyr(P)-Leu- pDS-Arg(P)-Arg(P)-Ala-Gln(P)-Asp(P)-Phe-Val- Gln(P)-Trp(P)-Leu-Met-Asn(P)-Thr(P), (SEQ ID NO: 13) His(P)-Ser(P)-Gln(P)-(Xmb)Gly-Thr(P)-Phe-Thr(P)- Ser(P)-Asp(P)-pYS-Lys(P)-Tyr(P)-Leu-Asp(P)- Ser(P)-Arg(P)-Arg(P)-Ala-Gln(P)-Asp(P)-Phe-Val- Gln(P)-Trp(P)-Leu-Met-Asn(P)-Thr(P), (SEQ ID NO: 14) His(P)-Ser(P)-Gln(P)-(Xmb)Gly-Thr(P)-Phe-pTS- Asp(P)-Tyr(P)-Ser(P)-Lys(P)-Tyr(P)-Leu-Asp(P)- Ser(P)-Arg(P)-Arg(P)-Ala-Gln(P)-Asp(P)-Phe-Val- Gln(P)-Trp(P)-Leu-Met-Asn(P)-Thr(P), (SEQ ID NO: 15) His(P)-Ser(P)-Gln(P)-(Xmb)Gly-Thr(P)-Phe-pTS- Asp(P)-Tyr(P)-Ser(P)-Lys(P)-Tyr(P)-Leu-pDS- Arg(P)-Arg(P)-Ala-Gln(P)-Asp(P)-Phe-Val-Gln(P)- Trp(P)-Leu-Met-Asn(P)-Thr(P), (SEQ ID NO: 16) His(P)-Ser(P)-Gln(P)-(Xmb)Gly-Thr(P)-Phe- Thr(P)-Ser(P)-Asp(P)-pYS-Lys(P)-Tyr(P)-Leu-pDS- Arg(P)-Arg(P)-Ala-Gln(P)-Asp(P)-Phe-Val(P)- Gln(P)-Trp(P)-Leu-Met-Asn(P)-Thr(P), (SEQ ID NO: 17) His(P)-Ser(P)-Gln(P)-Gly-Thr(P)-Phe-pTS- Asp(P)-Tyr(P)-Ser(P)-Lys(P)-Tyr(P)-(Xmb)Leu- Asp(P)-Ser(P)-Arg(P)-Arg(P)-Ala-Gln(P)- Asp(P)-Phe-Val-Gln(P)-Trp(P)-Leu-Met-Asn(P)- Thr(P), (SEQ ID NO: 18) His(P)-Ser(P)-Gln(P)-Gly-Thr(P)-Phe-Thr(P)- Ser(P)-Asp(P)-pYS-Lys(P)-Tyr(P)-(Xmb)Leu- Asp(P)-Ser(P)-Arg(P)-Arg(P)-Ala-Gln(P)-Asp(P)- Phe-Val-Gln(P)-Trp(P)-Leu-Met-Asn(P)-Thr(P), (SEQ ID NO: 19) His(P)-Ser(P)-Gln(P)-Gly-Thr(P)-Phe-Thr(P)- Ser(P)-Asp(P)-Tyr(P)-Ser(P)-Lys(P)-Tyr(P)- Leu-pDS-Arg(P)-Arg(P)-(Xmb)Ala-Gln(P)-Asp(P)- Phe-Val-Gln(P)-Trp(P)-Leu-Met-Asn(P)-Thr(P), (SEQ ID NO: 20) His(P)-Ser(P)-Gln(P)-(Xmb)Gly-Thr(P)-Phe- Thr(P)-Ser(P)-Asp(P)-pYS-Lys(P)-Tyr(P)-Leu- Asp(P)-Ser(P)-Arg(P)-Arg(P)-(Xmb)Ala-Gln(P)- Asp(P)-Phe-Val-Gln(P)-Trp(P)-Leu-Met-Asn(P)- Thr(P), and (SEQ ID NO: 21) His(P)-Ser(P)-Gln(P)-Gly-Thr(P)-Phe-pTS- Asp(P)-Tyr(P)-Ser(P)-Lys(P)-Tyr(P)-Leu- Asp(P)-Ser(P)-Arg(P)-Arg(P)-(Xmb)Ala- Gln(P)-Asp(P)-Phe-Val-Gln(P)-Trp(P)-Leu-Met- Asn(P)-Thr(P), wherein Xmb is selected from Tmb, Dmb, and Hmb, pDS is Asp(P)-Ser[psi(R1, R1)pro]-OH, pYS is Tyr(P)-Ser[psi(R1, R1)pro]-OH, pTS is Thr(P)-Ser[psi(R1, R1)pro]-OH, P is a side-chain protecting group or is absent, and R1 is hydrogen or methyl. ### Claim 17 (depends on 16) The protected glucagon sequence according to claim 16, which is: (SEQ ID NO: 12) His(tBu)-Ser(tBu)-Gln(Trt)-(Xmb)Gly-Thr(tBu)- Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Tyr(tBu)-Ser(tBu)- Lys(Boc)-Tyr(tBu)-Leu-pDS-Arg(Pbf)-Arg(Pbf)-Ala- Gln(Trt)-Asp(tBu)-Phe-Val-Gln(Trt)-Trp(Boc)- Leu-Met-Asn(Trt)-Thr(tBu). ### Claim 18 (depends on 16) The protected glucagon sequence according to claim 16, which is attached to a solid support. ### Claim 19 (depends on 18) The protected glucagon sequence according to claim 18, wherein the solid support is a Wang resin. ### Claim 20 (depends on 17) The protected glucagon sequence according to claim 17, which is attached to a solid support.