Methods for producing glucagon

Abstract

The present invention relates to a method for producing glucagon, which comprises condensing fragment 1 (a 24-mer providing amino acid residues 6-29 of glucagon) with fragment 2 (a 5-mer providing amino acid residues 1-5 of glucagon) in the presence of a coupling agent to obtain protected glucagon, deprotecting the protected glucagon with a cocktail mixture to obtain crude glucagon, followed by isolating pure glucagon by RP-HPLC purification.

Description

[Technical Field]

[0001] Related Applications: This application claims priority to our Indian patent application IN 202341005417, filed on January 27, 2023, which is incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention relates to a compound of formula-I H2N-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-COOH The present invention relates to a method for producing glucagon represented by the formula: The present invention relates to a method for producing glucagon by solid phase peptide synthesis. Further, the present invention relates to a method for producing glucagon by coupling fragments in the desired sequence, deprotecting, and condensing them on solid phase, followed by RP-HPLC purification to obtain pure glucagon. [Background technology]

[0003] Background of the Invention Glucagon is a linear peptide hormone consisting of 29 amino acid residues secreted from the alpha cells of the pancreas. Glucagon shares the same precursor, proglucagon, with GLP-1 and GLP-2. Through tissue-specific post-translational processing, glucagon is secreted from the pancreatic alpha cells, while GLP-1 and GLP-2 are secreted from the intestinal L cells. All of these peptides share considerable sequence homology and form the glucagon family, a subfamily of the secretin-glucagon superfamily.

[0004] Among the members of the glucagon family, the primary structure of glucagon is the most highly conserved among vertebrates. Glucagon is the major hyperglycemic hormone and functions as an insulin-balancing hormone. Glucagon generally increases blood glucose levels by promoting gluconeogenesis and glycogenolysis. Glucagon affects various organs in the body, including adipose tissue, pancreas, brain, and kidney, but its greatest influence is on the liver.

[0005] U.S. Pat. No. 3,642,763 describes the synthesis of glucagon by condensation of the [aa1-6] and [aa7-29] peptide fragments in the presence of N-hydroxysuccinimide or N-hydroxyphthalimide, followed by cleavage of the protecting groups in the presence of trifluoroacetic acid.

[0006] Japanese Patent Application No. 1995-146255 describes the Fmoc solid phase peptide synthesis of glucagon by sequential coupling of amino acids based on the peptide backbone of glucagon.

[0007] PCT Application Publication No. 2020254479 describes the synthesis of glucagon by condensation of the [aa1-4] and [aa5-29] peptide fragments, where the C-terminal [aa5-29] fragment consists of at least one pseudoproline dipeptide.

[0008] Chinese Patent No. CN103333239B describes an Fmoc solid phase peptide synthesis of glucagon in which coupling is carried out at elevated temperatures.

[0009] The inventors of the present invention have used existing synthetic methods to produce glucagon, and have found that the prior art has technical problems, such as many synthetic steps, long synthetic cycles, low purity and yield, high production costs, and is not suitable for large-scale production. For this purpose, the inventors have conducted research into the synthetic methods of glucagon, and have thereby obtained the technical solution of the present invention.

[0010] The present invention provides a method for producing glucagon by coupling the appropriate fragments in the required order, deprotecting them, and condensing them on solid phase, followed by purification to obtain glucagon. Summary of the Invention [Problem to be solved by the invention]

[0011] Summary of the Invention A first aspect of the present invention relates to a method for producing glucagon as shown in Scheme 1, the method comprising the steps of: a. condensing fragment 1 with fragment 2 in the presence of a coupling agent to obtain protected glucagon; b. Deprotecting the protected glucagon with a cocktail mixture to obtain crude glucagon; and c. RP-HPLC purification to isolate pure glucagon

[0012] [Table 1]

[0013] [Table 2]

[0014] [Table 3]

[0015] In a first aspect of the first embodiment, a method for producing (Fragment 1) comprises the following steps: a) anchoring the first protected terminal amino acid to a Wang resin; b) selectively deprotecting amino groups; c) coupling the carboxyl terminus of the next N-protected amino acid to the amine group; d) Repeating steps b) and c) to form the peptide sequence [aa6-29] of glucagon, where the peptide sequence is attached to the resin.

[0016] In a second aspect of the first embodiment, a method for producing (Fragment 2) comprises the steps of: a) anchoring a first protected terminal amino acid to a 2-chlorotrityl chloride resin; b) selectively deprotecting amino groups; c) coupling the carboxyl terminus of the next N-protected amino acid to the amine group; d) repeating steps b) and c) to form the peptide sequence [aa1-5] of glucagon; and e) Cleavage of fragment 2 from the resin.

[0017] A second embodiment provides that novel fragments 1 and 2 are as follows: a)H2N-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Leu-Asp(OtBu)-Ser(tBu)-Arg(P bf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)-Thr(tBu)-Wang resin [Fragment 1] b) Boc-His(Trt)-Ser(tBu)-Gln(Trt)-Gly-Thr(tBu)-OH [Fragment 2]

[0018] A third aspect of the present invention relates to a method for producing glucagon as shown in Scheme 2, comprising the steps of: a. condensing fragment 3 with fragment 4 in the presence of a coupling agent to obtain protected glucagon; b. Deprotecting the protected glucagon with a cocktail mixture to obtain crude glucagon; and c. Purifying by RP-HPLC to isolate pure glucagon.

[0019] In a fourth aspect of the present invention, PG-His(PG)-Ser(PG)-Gln(PG)-Gly-Thr(PG)-OH Provides for the use of where PG is a suitable amino protecting group.

[0020] [Table 4]

[0021] [Table 5]

[0022] [Table 6]

[0023] In a first aspect of the third embodiment, a method for producing (Fragment 3) comprises the steps of: a) anchoring the first protected terminal amino acid to a Wang resin; b) selectively deprotecting amino groups; c) coupling the carboxyl terminus of the next N-protected amino acid to the amine group; d) repeating steps b) and c) to form the peptide sequence [aa7-29] of glucagon, which is attached to the resin.

[0024] In a second aspect of the third embodiment, a method for producing (Fragment 4) comprises the steps of: a) anchoring a first protected terminal amino acid to a 2-chlorotrityl chloride resin; b) selectively deprotecting amino groups; c) coupling the carboxyl terminus of the next N-protected amino acid to the amine group; d) repeating steps b) and c) to form the peptide sequence [aa1-6] of glucagon; and e) Cleavage of fragment 2 from the resin.

[0025] A fourth embodiment provides that novel fragments 3 and 4 are as follows: a) H2N-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Leu-Asp(OtBu)-Ser(tbu)-Arg(Pbf)-Arg(Pbf)-Ala-Gln (Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu_met-Asn(Trt)-Thr(tBu)-Wang resin [fragment 3] b) Boc-His(Trt)-Ser(tBu)-Gln(Trt)-Gly-Thr(tBu)-Phe-OH [Fragment 4]

[0026] A fifth aspect provides for the purification of glucagon as follows. a) subjecting crude glucagon to reversed-phase high performance liquid chromatography (RP-HPLC purification) in gradient elution mode using an aqueous mobile phase containing formic acid and an acetonitrile / isopropanol mixture; b) collecting fractions containing purified glucagon with an HPLC purity of greater than 98%;

[0027] A sixth aspect provides for the purification of a GLP-1 analogue selected from the group consisting of liraglutide, semaglutide, glucagon and teduglutide, comprising a step of RP-HPLC purification using a mobile phase comprising formic acid. DETAILED DESCRIPTION OF THE INVENTION

[0028] Detailed Description of the Invention The best mode for carrying out the invention is illustrated by the following examples, which are provided for illustrative purposes only and therefore should not be construed as limiting the scope of the invention.

[0029] Abbreviation: Boc tert-butyloxycarbonyl tBu tert-butyl DCM dichloromethane MDC dichloride DIC or DIPC N,N'-diisopropylcarbodiimide DMF N,N'-dimethylformamide DMAP dimethylaminopyrimidine DIPEA or DIEA Diisopropylethylamine HOBt Hydroxybenzotriazole HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) TFA trifluoroacetic acid MTBE tert-butyl methyl ether HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) TIS Triisopropylsilane DTT Dithioteritol NH4I Ammonium iodide Met L-Methionine Trp L-tryptophan DIPC N,N'-Diisopropylcarbodiimide

[0030] DMAP 4-dimethylaminopyridine 2-CTC Resin 2-Chlorotrityl Chloride Resin Fmoc 9-Fluorenylmethoxycarbonyl HOBt N-hydroxybenzotriazole MTBE Methyl tert-butyl ether Histidine Ser Serine Gln glutamine Gly glycine Thr Threonine Phe phenylalanine Asp aspartic acid Tyr Tyrosine Lys Lysine Leu leucine Arg Arginine Ala Alanine Gln glutamine Val Balin Trp tryptophan Met methionine Asn Asparagine

[0031] Fmoc deprotection of the loaded amino acid according to the present invention is carried out using 0.05-0.5 M Oxymapure in 5-15% piperidine in DMF, or 0.05-0.5 M formic acid in 5-15% piperidine in DMF, 0.05-0.5 M HOBt in 5-15% piperidine in DMF, or 1-5% DBU / 0.1-1 M Oxymapure in 5-15% piperidine in DMF, or 1-5% DBU / 0.05-0.5 M HOBt in 5-15% piperidine in DMF. More preferably, 10% piperidine in DMF is used.

[0032] The coupling of amino acids according to the present invention is carried out in the presence of a coupling agent, a coupling additive, an inorganic salt, and a base. The coupling agent is selected from the group consisting of HBTU, HATU, COMU, DEPBT, or DIC. The coupling additive is selected from the group consisting of Oxymapure or HOBt. The base is selected from the group consisting of DIPEA, NMM, or TMP. The inorganic salt is selected from the group consisting of MgCl2, CuCl2, or ZnCl2.

[0033] The method for producing (Fragment 1) comprises the following steps: a) anchoring the first protected terminal amino acid to a Wang resin; b) selectively deprotecting amino groups; c) coupling the carboxyl terminus of the next N-protected amino acid to the amine group; d) repeating steps b) and c) to form the peptide sequence [aa6-29] of glucagon; and e) Cleavage of fragment 1 from the resin.

[0034] Example 1: Synthesis of Fragment 1 (a 24-mer providing amino acid residues 6-29 of glucagon) Stage 1: Swelling of Wang resin Wang resin with a substituent group of 0.50–0.90 mmol / g was placed in a reaction vessel, and a sufficient amount of DCM (10–15 volumes) was added. The swelling treatment was carried out at room temperature for 1–3 hours, and the liquid was then drained.

[0035] Stage 2: Preparation of Fmoc-Thr(tBu)-Wang resin The required amount of Fmoc-Thr(tBu)-OH (2.0–7.0 equiv. relative to resin loading) was dissolved in DCM (3–6 volumes). The amino acid solution was charged into the reaction vessel containing the resin, followed by DIPC (3–9 equiv.) in DCM (3–5 volumes) and DMAP (0.01–0.1 equiv.) in DCM (0.1–2 volumes). The reaction was kept stirring at room temperature for 1–3 h. After completion of the coupling, the mixture was washed repeatedly with DCM and DMF 3–5 times (5–15 volumes), respectively.

[0036] Stage 3: Capping Capping was performed to block unreacted sites. A solution of 5-10% acetic anhydride and 10-20% DIPEA in DMF (5-10 volumes) was prepared and added to the reaction vessel containing the resin loaded with the first amino acid. The reaction was stirred at room temperature for 15-60 minutes and then drained. The capping process was repeated for an additional 30 minutes and then drained. This was followed by 4-6 DMF washes (5-15 volumes).

[0037] Stage 4: Fmoc deprotection 5–15% piperidine in DMF (5–15 volumes) was added to the reaction vessel, stirred at 15–30°C for 5 minutes, and drained. The resin was again treated with 5–15% piperidine in DMF (5–15 volumes) at 15–30°C for 5–15 minutes and drained. Two washes with HOBt.HO (0.1–1.0 M) in DMF (5–15 volumes) were performed. Two–five DMF washes (5–15 volumes) were performed. A Kaiser test was performed to confirm Fmoc deprotection before coupling the next amino acid.

[0038] After adding Fmoc-Asp(OtBu)-OH to the sequence, deprotection continued with 0.05–0.5 M HOBt.HO in 5–15% piperidine in DMF (5–15 volumes), agitated for 5–15 min, and drained. The resin was again treated with 0.05–0.5 M HOBt in 5–15% piperidine in DMF (5–15 volumes) for 5–15 min and drained. Two washes with 0.1–1.0 M HOBt.HO in DMF (5–15 volumes) were performed. Four washes with DMF (5–15 volumes) were performed. A Kaiser test was performed to confirm Fmoc deprotection before coupling the next amino acid.

[0039] Stage 5: Fmoc-amino acid coupling: The required amounts of Fmoc amino acid (1.0–5.0 equivalents), 1-hydroxybenzotriazole monohydrate (HOBtHO) (1–5 equivalents), and MgCl2 / CuCl2 / ZnCl2 (0.5–1.0 equivalents) were weighed out and dissolved in 1–5 volumes of DMF. The solution was added to the peptide resin. The required amount of HBTU / HATU (1.5–5.0 equivalents) was weighed out and dissolved in 1–5 volumes of DMF. The solution was added to the peptide resin, followed by the addition of diisopropylethylamine (DIPEA) (3–8 equivalents). The mixture was stirred for approximately 5 hours. A Kaiser test was performed to confirm the completion of the coupling reaction. If the test was positive, recoupling was performed.

[0040] Stage 6: Fmoc deprotection The resin was treated with 0.05–0.5 M HOBt.HO in 5–15% piperidine in DMF (5–15 volumes) and stirred at 20–40°C for 5–15 min, then drained. The resin was again treated with 0.05–0.5 M HOBt.HO in 5–15% piperidine in DMF (10–20 volumes) for 5–15 min at 20–40°C, then drained. Washes were performed with 2 × 0.1–1.0 M HOBt.HO in DMF (5–10 volumes). DMF washes (4 × 5–10 volumes) were performed. A Kaiser test was performed to confirm Fmoc deprotection before coupling the next 1–5mer fragment.

[0041] The method for producing (Fragment 2) comprises the following steps: a) anchoring a first protected terminal amino acid to a 2-chlorotrityl chloride resin; b) selectively deprotecting amino groups; c) coupling the carboxyl terminus of the next N-protected amino acid to the amine group; d) repeating steps b) and c) to form the peptide sequence [aa1-5] of glucagon; and e) Cleavage of fragment 2 from the resin.

[0042] In a preferred embodiment, cleavage of fragment 2 from the resin is performed softly using 0.5-5% TFA or 10-20% trifluoroethanol. In this soft cleavage, only fragment 2 is cleaved from the attached resin, and no deprotection of the side chains occurs within the fragment.

[0043] Example 2: Synthesis of Fragment 2 (a 5-mer providing amino acid residues 1-5 of glucagon) Stage 1: Swelling of CTC resin CTC resin with 1.0-1.5 mmol / g of substituents was placed in a reaction vessel and a sufficient amount of DCM (5-15 volumes) was added. The swelling process was carried out at 20-30°C for 1-3 hours. The DCM was drained using a vacuum.

[0044] Stage 2: Preparation of Fmoc-Thr(tBu)-CTC resin The required amount of Fmoc-Thr(tBu)-OH (1.0–4.0 equivalents relative to resin loading) and the required amount of DIPEA (3.0–8.0 equivalents relative to resin loading) were dissolved in DCM (5–15 volumes). This solution was filled into the reaction vessel containing the resin, and the reaction mixture was kept at 20 to 35°C for 1 to 5 hours with stirring.

[0045] Stage 3: Capping Capping was performed to block unreacted sites. A solution of 1-20% DIPEA in 1-20% methanol in DCM (5-20 volumes) was prepared and added to the reaction vessel containing the resin loaded with the first amino acid. The reaction was stirred at 20-35 °C for 10-60 minutes and drained. The capping process was repeated for an additional 10-60 minutes and drained. DMF washes (4x 5-10 volumes) were performed. The post-capping samples were submitted for loading estimation.

[0046] Stage 4: Fmoc deprotection 5–20% piperidine in DMF (5–20 volumes) was added to the reaction vessel, stirred for 5 minutes, and drained. The resin was again treated with 5–20% piperidine in DMF (5–20 volumes) for 5–20 minutes and drained. Two washes with HOBt.HO (0.1–1.0 M) in DMF (5–20 volumes) were performed. Four DMF washes (5–20 volumes) were performed. A Kaiser test was performed to confirm Fmoc deprotection before coupling the next amino acid.

[0047] Stage 5: Fmoc-amino acid coupling: The required amounts of Fmoc amino acid (1–5 equivalents), 1-hydroxybenzotriazole monohydrate (HOBtHO) (1–5 equivalents), and MgCl2, ZnCl2, or CuCl2 (0.5 equivalents) were weighed out and dissolved in 1–5 volumes of DMF. The solution was added to the peptide resin (after deblocking). The required amount of HBTU / HATU (1–5 equivalents) was weighed out and dissolved in 1–5 volumes of DMF. The solution was added to the peptide resin, followed by the addition of diisopropylethylamine (DIPEA) (3–8 equivalents). The mixture was stirred for 1.0–3.0 hours. A Kaiser test was performed to confirm the completion of the coupling reaction. If the test was positive, recoupling was performed.

[0048] Stage 6a: Soft cleavage with trifluoroacetic acid A 0.5-5% TFA solution in DCM (10-50 V) was prepared and added to the dried peptidyl resin in 0.5-5% TFA in DCM (5-20 V). The mixture was stirred for approximately 5 minutes and then drained. The effluent was collected and neutralized with DIPEA. This cycle of adding TFA to the peptidyl resin and draining was repeated 1-5 times, and all effluents were collected in one flask. The effluent was concentrated to 10-40% of the volume of the cocktail mixture using a rotary evaporator. Pre-chilled MTBE was slowly added to the concentrate while stirring. The precipitated solid was filtered and washed with MTBE. or

[0049] Stage 6b: Soft cleavage using trifluoroethanol. A 10-20% solution of trifluoroethanol (TFE) in DCM (10-50 V) was prepared and added to the dried peptidyl resin. The mixture was stirred for approximately 1.0-3.0 hours and then drained. The effluent was collected and concentrated to 10-40% of the volume of the cleavage mixture using a rotary evaporator. Pre-chilled MTBE, hexane, or heptane was slowly added to the concentrate while stirring. The precipitated solid was filtered and washed with MTBE, hexane, or heptane.

[0050] 1. A method for producing glucagon as shown in Scheme 1, comprising the steps of: a. condensing fragment 1 with fragment 2 in the presence of a coupling agent to obtain protected glucagon; b. deprotecting the protected glucagon with a cocktail mixture to obtain crude glucagon; and c. RP-HPLC purification to isolate pure glucagon; A method comprising:

[0051] Example 3: Synthesis of glucagon Stage 1: Swelling of fragment 1: Fragment 1 on Wang resin from Example 1 (ie, a 24-mer providing amino acid residues 6-29 of glucagon) was swollen in 5-20 volumes of DMF, stirred for 1-3 hours, and drained.

[0052] Stage 2: Coupling of fragment 2: The required amount of fragment 2 from Example 2, 3 equivalents (i.e., a 5-mer providing amino acid residues 1–5 of glucagon), 1-hydroxybenzotriazole monohydrate (HOBt HO) (1–5 equivalents), and MgCl2, ZnCl2, or CuCl2 (0.5–30 equivalents) were weighed out. These were dissolved in 1–5 volumes of DMF. The solution was added to the peptide resin (after deblocking). The required amount of HATU / HBTU (1–5 equivalents) was weighed out. These were dissolved in 1–5 volumes of DMF. The solution was added to the peptide resin, followed by diisopropylethylamine (DIPEA) (3–8 equivalents), and the mixture was stirred for 1–5 hours. A Kaiser test was performed to confirm the completion of the coupling reaction. If the coupling reaction was complete, it should be negative. If the test is positive, recoupling is performed.

[0053] Stage 3: Complete amputation A thoroughly dried, preserved peptidyl resin was loaded with a 5-20-fold volume mixture of TFA:TIS:phenol:DTT:NH4I:Met:Trp (81.5:5:5:2.5:2:2:2) relative to the volume of the peptidyl resin. The reaction mixture was stirred at room temperature (10-40°C) for 1-6 hours and then filtered through a coarse filter funnel. The filtrate was concentrated to 20-60% of the volume of the cocktail mixture using a rotary evaporator. Pre-chilled MTBE was slowly added to the concentrate while stirring. The precipitated solid was filtered and washed with MTBE to obtain crude glucagon. Molar yield: 90-95% HPLC purity: 50~60% The isolated crude glucagon was dried in a VTD until constant weight was reached and subjected to RP-HPLC purification.

[0054] The method for producing (Fragment 3) comprises the following steps: a) anchoring the first protected terminal amino acid to a Wang resin; b) selectively deprotecting amino groups; c) coupling the carboxyl terminus of the next N-protected amino acid to the amine group; d) repeating steps b) and c) to form the peptide sequence [aa7-29] of glucagon; and e) Cleavage of fragment 3 from the resin.

[0055] Example 4: Synthesis of Fragment 3 (a 23-mer providing amino acid residues 7-29 of glucagon) Stage 1: Swelling of Wang resin Wang resin with a substituent group of 0.50–0.90 mmol / g was placed in a reaction vessel, and a sufficient amount of DCM (8–15 volumes) was added. The swelling treatment was carried out at 20–30°C for 1–4 hours, and the liquid was then drained.

[0056] Stage 2: Preparation of Fmoc-Thr(tBu)-Wang resin The required amount of Fmoc-Thr(tBu)-OH (3–7.0 equiv. relative to resin loading) was dissolved in DCM (2–5 volumes). The amino acid solution was charged into the reaction vessel containing the resin, followed by DIPC (5–10 equiv.) in DCM (2–5 volumes) and DMAP (0.01–0.25 equiv.) in DCM (0.1–1.5 volumes). The reaction was kept at 20–35 °C with stirring for 1–5 h. After completion of the coupling, washing with DCM and DMF was performed three times each (5–20 volumes).

[0057] Stage 3: Capping Capping was performed to block unreacted sites. A 5-10% acetic anhydride solution containing 10-25% DIPEA in DMF (5-10 volumes) was prepared and added to the reaction vessel containing the resin loaded with the first amino acid. The reaction was stirred at 20-35°C for approximately 30 minutes and then drained. The capping process was repeated for another 30 minutes and then drained. DMF washing (4x 5-20 volumes) was performed.

[0058] Stage 4: Fmoc deprotection 5-20% piperidine in DMF (5-20 volumes) was added to the reaction vessel, stirred at 20-35°C for 5-10 minutes, and drained. The resin was again treated with 5-20% piperidine in DMF (5-20 volumes) at 20-35°C for 5-20 minutes and drained. Two washes with HOBt.HO (0.1-1.0 M) in DMF (5-20 volumes) were performed. DMF washes (4x 5-20 volumes) were performed. A Kaiser test was performed to confirm Fmoc deprotection before coupling the next amino acid.

[0059] After adding Fmoc-ASP(OtBu)-OH to the sequence, deprotection continued with 0.05–0.5 M HOBt in 5–20% piperidine in DMF (5–20 volumes), agitated for 5–10 min, and drained. The resin was again treated with 0.1–1 M HOBt in 5–20% piperidine in DMF (5–20 volumes) for 5–20 min and drained. Two washes with 0.5 M HOBt.HO in DMF (5–20 volumes) were performed. DMF washes (4x 5–20 volumes) were performed. A Kaiser test was performed to confirm Fmoc deprotection before coupling the next amino acid.

[0060] Stage 5: Fmoc-amino acid coupling: The required amounts of Fmoc amino acid (2-5 equivalents), 1-hydroxybenzotriazole monohydrate (HOBtHO) (2-5 equivalents), and MgCl2, ZnCl2, or CuCl2 (0.1-1.0 equivalents) were weighed out and dissolved in 2-5 volumes of DMF. The solution was added to the peptide resin (after deblocking). The required amount of HBTU / HATU (2-5 equivalents) was weighed out and dissolved in 2-5 volumes of DMF. The solution was added to the peptide resin, followed by diisopropylethylamine (DIPEA) (2-9 equivalents). The mixture was stirred for 1.0-4 hours. A Kaiser test was performed to confirm the completion of the coupling reaction. If the test was positive, recoupling was performed.

[0061] Stage 6: Fmoc deprotection 0.05–0.5 M HOBt.HO was dissolved in 5–20% piperidine in DMF (5–20 volumes), stirred at 20–35°C for approximately 5 min, and drained. The resin was again treated with 0.05–0.5 M HOBt.HO in 5–20% piperidine in DMF (5–20 volumes) for 5–20 min at 10–35°C and drained. Two washes with 0.1–1.0 M HOBt.HO in DMF (5–10 volumes) were performed. Four DMF washes (8 volumes) were performed. A Kaiser test was performed to confirm Fmoc deprotection before coupling the next 1–5-mer fragment.

[0062] The method for producing (Fragment 4) comprises the following steps: a) anchoring a first protected terminal amino acid to a 2-chlorotrityl chloride resin; b) selectively deprotecting amino groups; c) coupling the carboxyl terminus of the next N-protected amino acid to the amine group; d) repeating steps b) and c) to form the peptide sequence [aa1-6] of glucagon; and e) Excision of fragment 4 from the resin.

[0063] In a preferred embodiment, cleavage of fragment 4 from the resin is performed softly using 0.5-5% TFA or 10-20% trifluoroethanol, in which only fragment 4 is cleaved from the resin to which it is attached, without deprotection of the side chains within the fragment.

[0064] Example 5: Synthesis of fragment 4 (a 6-mer providing amino acid residues 1-6 of glucagon) Stage 1: Swelling of CTC resin CTC resin with 1.0–1.5 mmol / g of substituents was placed in a reaction vessel and a sufficient amount of DCM (5–20 volumes) was added. The swelling process was carried out at 20–35°C for 1–3 hours. The DCM was then drained using a vacuum.

[0065] Stage 2: Preparation of Fmoc-Phe-O-CTC resin The required amount of Fmoc-Phe-OH (1.5-3.0 equivalents relative to resin loading) and DIPEA (3-7 equivalents relative to resin loading) was dissolved in DCM (5-20 volumes). This solution was charged into the reaction vessel containing the resin, and the reaction was maintained at 20-35 °C with stirring for 1-4 hours. The reaction was then drained, and the resin was washed with DMF (10 volumes x 3).

[0066] Stage 3: Capping Capping was performed to block unreacted sites. A solution of 5-20% methanol containing 2-7% DIPEA in DCM (5-20 volumes) was prepared and added to the reaction vessel containing the resin loaded with the first amino acid. The reaction was stirred at 20-35 °C for approximately 30 minutes and then drained. The capping process was repeated and drained. DMF washes (4 x 10 volumes) were performed.

[0067] After capping, the loading coefficient of the Fmoc-Phe-O-CTC resin thus prepared was estimated and found to be in the range of 1.2 mmol / gm to 1.6 mmol / gm.

[0068] Stage 4: Fmoc deprotection 5-20% piperidine in DMF (5-20 volumes) was added to the reaction vessel, stirred for approximately 5 minutes, and drained. The resin was treated with 5-20% piperidine in DMF (5-20 volumes) for 5-20 minutes and drained. Washes were performed with HOBt.HO (2x 0.05-0.50 M) in DMF (5-20 volumes). DMF washes (4x 5-20 volumes) were performed. A Kaiser test was performed to confirm Fmoc deprotection before coupling the next amino acid.

[0069] Stage 5: Fmoc-amino acid coupling The required amounts of Fmoc amino acid (1.5-5.0 equivalents), 1-hydroxybenzotriazole monohydrate (HOBt.HO) (2-5 equivalents), and MgCl2, ZnCl2, or CuCl2 (0.1-1.0 equivalents) were weighed and dissolved in 2-5 volumes of DMF. The solution was added to the peptide resin (after deblocking). The required amount of HBTU / HATU (2-5 equivalents) was weighed and dissolved in 2-5 volumes of DMF. The solution was added to the peptide resin, followed by diisopropylethylamine (DIPEA) (4-8 equivalents). The mixture was stirred for 1.0-4.0 hours. A Kaiser test was performed to confirm the completion of the coupling reaction. If the test was positive, recoupling was performed.

[0070] Stage 6a: Soft cleavage with trifluoroacetic acid A 0.5-2% TFA solution in DCM (30-60 volumes) was prepared. 0.5-2.0% TFA in DCM (5-20 volumes) was added to the dried peptidyl resin, stirred for approximately 2 minutes, and drained. The effluent was collected and neutralized with DIPEA. This cycle of adding TFA to the peptidyl resin and draining was repeated 3-5 times, and all effluents were collected in one flask. The effluent was concentrated to 10-40% of the volume of the cocktail mixture using a rotary evaporator. Pre-chilled MTBE was slowly added to the concentrate while stirring. The precipitated solid was filtered and washed with MTBE. or

[0071] Stage 6b: Soft cleavage with trifluoroethanol A 10% to 20% solution of trifluoroethanol (TFE) in DCM (10 to 50 V) was prepared and added to the dried peptidyl resin. The mixture was stirred for approximately 1.0 to 3.0 hours and then drained. The effluent was collected and concentrated to 10 to 40% of the volume of the cleavage mixture using a rotary evaporator. Pre-chilled MTBE, hexane, or heptane was slowly added to the concentrate while stirring. The precipitated solid was filtered and washed with MTBE, hexane, or heptane.

[0072] 1. A method for producing glucagon as shown in Scheme 1, comprising the steps of: a. condensing fragment 3 with fragment 4 in the presence of a coupling agent to obtain protected glucagon; b. deprotecting the protected glucagon with a cleavage mixture to obtain crude glucagon; and c. Purify by RP-HPLC if necessary to isolate pure glucagon.

[0073] Example 6: Synthesis of glucagon Stage 1: Swelling of fragment 3 Fragment 3 on Wang resin from Example 4 (ie, a 23-mer providing amino acid residues 7-29 of glucagon) was swollen in 10-20 volumes of DMF, stirred for 1-3 hours, and drained.

[0074] Stage 2a: Coupling of fragment 4 The required amounts of 1-6mer fragments (2-5 equivalents), 1-hydroxybenzotriazole monohydrate (HOBt.HO) (2-5 equivalents), and MgCl2, ZnCl2, or CuCl2 (0.1-1.0 equivalents) were weighed out and dissolved in 3-6 volumes of DMF. The solution was added to the peptide resin (after deblocking). The required amount of HATU (1.5-5.0 equivalents) was weighed out and dissolved in 3-6 volumes of DMF. The solution was added to the peptide resin, followed by diisopropylethylamine (DIPEA) (4-8 equivalents), and the mixture was stirred for 2-3 hours. A Kaiser test was performed to confirm the completion of the coupling reaction. If the coupling reaction was complete, it should be negative. If the test is positive, recoupling is performed. or

[0075] Stage 2b: Coupling of fragment 4 The required amounts of 1-6mer fragments (2-5 equivalents), 1-hydroxybenzotriazole monohydrate (HOBt.HO) (2-5 equivalents), and diisopropylcarbodiimide (2-5 equivalents) were weighed out. They were dissolved in 3-6 volumes of DMF. The solution was added to the peptide resin, and the mixture was stirred for 2-3 hours. A Kaiser test was performed to confirm the completion of the coupling reaction. If the coupling reaction was complete, it should be negative. If the test was positive, recoupling was performed.

[0076] Stage 3: Complete amputation A thoroughly dried protected peptidyl resin was loaded with a 5-20-fold volume mixture of TFA:TIS:phenol:DTT:NH4I:Met:Trp (81.5:5:5:2.5:2:2:2) relative to the volume of the peptidyl resin. The reaction mixture was stirred at room temperature (15-35°C) for approximately 3 hours and then filtered through a coarse filter funnel. The filtrate was concentrated to 20-50% of the volume of the cocktail mixture using a rotary evaporator. Pre-chilled MTBE was slowly added to the concentrate while stirring. The precipitated solid was filtered and washed with MTBE to obtain crude glucagon. Molar yield: 90-95% RP-HPLC purity: 50-60% The isolated crude glucagon was dried in vacuo and subjected to RP-HPLC purification.

Claims

1. 1. A method for producing glucagon, comprising: a. Condensing fragment 1 (a 24-mer providing amino acid residues 6-29 of glucagon) with fragment 2 (a 5-mer providing amino acid residues 1-5 of glucagon) in the presence of a coupling agent to obtain protected glucagon; b. Deprotecting the protected glucagon with a cocktail mixture to obtain crude glucagon; and c. Purifying by RP-HPLC to isolate pure glucagon The manufacturing method comprising:

2. 2. The method of claim 1, wherein the coupling agent in step a) is selected from the group consisting of HATU, DIPEA, HOBt, and DIC.

3. Step b) is a step of adding TFA, TIS, phenol, DTT, Met, Trp and NH ４ The method of claim 1, wherein the method is carried out using a cocktail mixture of I.

4. Fragment 1 is H 2 N-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Leu-Asp(OtBu)-Ser(tBu)-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)-Thr(tBu)-Wang resin The method of claim 1, wherein

5. Fragment 2 is Boc-His(Trt)-Ser(tBu)-Gln(Trt)-Gly-Thr(tBu)-OH The method of claim 1, wherein

6. Use of Boc-His(Trt)-Ser(tBu)-Gln(Trt)-Gly-Thr(tBu)-OH in the manufacture of glucagon.

7. below: a) anchoring the first protected terminal amino acid to a Wang resin; b) selectively deprotecting the amino groups; c) coupling the carboxyl terminus of the next N-protected amino acid to the amine group; and d) repeating steps b) and c) to form the peptide sequence [aa6-29] of glucagon, which is attached to the resin; 10. The method of claim 1, further comprising producing Fragment 1 comprising:

8. below: a) anchoring a first protected terminal amino acid to a 2-chlorotrityl chloride resin; b) selectively deprotecting the amino groups; c) coupling the carboxyl terminus of the next N-protected amino acid to the amine group; d) repeating steps b) and c) to form the peptide sequence [aa1-5] of glucagon; and e) Cleavage of fragment 2 from the resin.

2. The method of claim 1, further comprising producing fragment 2, comprising:

9. 1. A method for producing glucagon, comprising: a. condensing fragment 3 (a 23-mer providing amino acid residues 7-29 of glucagon) with fragment 4 (a 6-mer providing amino acid residues 1-6 of glucagon) in the presence of a coupling agent to obtain protected glucagon; b. Deprotecting the protected glucagon with a cleavage mixture to obtain crude glucagon; and c. Optionally, purify by RP-HPLC to isolate pure glucagon. The manufacturing method comprising:

10. Fragment 3 is H 2 N-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Leu-Asp(OtBu)-Ser(tBu)-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)-Thr(tBu)-Wang resin The method of claim 9, wherein

11. Fragment 4 is Boc-His(Trt)-Ser(tBu)-Gln(Trt)-Gly-Thr(tBu)-Phe-OH The method of claim 9, wherein

12. Use of Boc-His(Trt)-Ser(tBu)-Gln(Trt)-Gly-Thr(tBu)-Phe-OH in the manufacture of glucagon.

13. below: a) anchoring the first protected terminal amino acid to a Wang resin; b) selectively deprotecting the amino groups; c) coupling the carboxyl terminus of the next N-protected amino acid to the amine group; and d) repeating steps b) and c) to form the peptide sequence [aa7-29] of glucagon, which is attached to the resin; 10. The method of claim 9, further comprising producing a fragment 3 comprising:

14. below: a) anchoring a first protected terminal amino acid to a 2-chlorotrityl chloride resin; b) selectively deprotecting the amino groups; c) coupling the carboxyl terminus of the next N-protected amino acid to the amine group; d) repeating steps b) and c) to form the peptide sequence [aa1-6] of glucagon; and e) cleaving fragment 4 from the resin; 10. The method of claim 9, further comprising producing a fragment 4 comprising:

15. 15. The method of claim 14, wherein fragment 4 is cleaved from the resin using trifluoroethanol.

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