TW202348620A - Process for preparing glucagon-like peptide-1 - Google Patents
Process for preparing glucagon-like peptide-1 Download PDFInfo
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- TW202348620A TW202348620A TW112118592A TW112118592A TW202348620A TW 202348620 A TW202348620 A TW 202348620A TW 112118592 A TW112118592 A TW 112118592A TW 112118592 A TW112118592 A TW 112118592A TW 202348620 A TW202348620 A TW 202348620A
- Authority
- TW
- Taiwan
- Prior art keywords
- peptide
- glu
- gly
- boc
- glucagon
- Prior art date
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- 238000004519 manufacturing process Methods 0.000 title abstract description 7
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 title description 3
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Abstract
Description
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人類胰高血糖素樣肽-1（以下簡稱為GLP-1）受體激動劑包括艾塞那肽（exenatide）、利拉魯肽（liraglutide）、度拉糖肽（dulaglutide）、利司那肽（lixisenatide）及索馬魯肽（semaglutide）。其中，索馬魯肽（商品名Ozempic®）及利拉魯肽（商品名Victoza®）被指定為飲食及運動的輔助藥物（adjunct），以改進第2型糖尿病成人患者的血糖控制。Human glucagon-like peptide-1 (hereinafter referred to as GLP-1) receptor agonists include exenatide, liraglutide, dulaglutide, and lixisenatide (lixisenatide) and semaglutide. Among them, semaglutide (trade name: Ozempic®) and liraglutide (trade name: Victoza®) are designated as adjuncts to diet and exercise to improve blood sugar control in adults with type 2 diabetes.
索馬魯肽為具有SEQ ID NO: 1的胜肽，其已經工程化而與原始人類GLP-1具有94%的同源性，其係藉由在第8位置處以α-胺基異丁酸（Aib）取代丙胺酸且在第34位置處以精胺酸取代離胺酸。索馬魯肽係進一步衍生自已知的工程化胜肽，其係藉由在第26位置之離胺酸殘基處通過PEG修飾之麩胺酸親水性區段連接C-18脂肪二酸。據此，具有已知的基於原始人類GLP-1之修飾的索馬魯肽結構如下所示： Semaglutide is a peptide with SEQ ID NO: 1 that has been engineered to be 94% homologous to original human GLP-1 by adding α-aminoisobutyric acid at position 8 (Aib) replaces alanine and arginine in place of lysine at position 34. Semaglutide is further derived from a known engineered peptide by linking a C-18 fatty diacid via a PEG-modified glutamic acid hydrophilic segment at the lysine residue at position 26. Accordingly, the structure of semaglutide with known modifications based on original human GLP-1 is as follows:
索馬魯肽之分子式為C 187H 291N 45O 59且分子量為4,113.58道耳吞。其最初由Novo Nordisk開發，並於2017年12月獲得美國食品和藥物管理局（FDA）批准，商品名為「Ozempic®」。 The molecular formula of semaglutide is C 187 H 291 N 45 O 59 and the molecular weight is 4,113.58 Daltons. It was originally developed by Novo Nordisk and was approved by the U.S. Food and Drug Administration (FDA) in December 2017 under the trade name "Ozempic®".
利拉魯肽為具有SEQ ID NO: 2的胜肽，其已經工程化而與原始人類GLP-1具有97%的同源性，其係藉由在第34位置處以精胺酸取代離胺酸。利拉魯肽係進一步衍生自已知的工程化胜肽，其係藉由在第26位置之離胺酸殘基處通過麩胺酸區段連接C-16脂肪酸（軟脂酸）。據此，具有已知的基於原始人類GLP-1之修飾的利拉魯肽結構如下所示： Liraglutide is a peptide with SEQ ID NO: 2 that has been engineered to be 97% homologous to original human GLP-1 by replacing lysine with arginine at position 34 . Liraglutide is further derived from a known engineered peptide by linking a C-16 fatty acid (palmitic acid) through a glutamic acid segment at the lysine residue at position 26. Accordingly, the structure of liraglutide with known modifications based on original human GLP-1 is as follows:
利拉魯肽之分子式為C 172H 265N 43O 51且分子量為3,751.2道耳吞。其最初由Novo Nordisk開發，並於2010年1月獲得美國食品和藥物管理局（FDA）批准，商品名為「Victoza®」。 The molecular formula of liraglutide is C 172 H 265 N 43 O 51 and the molecular weight is 3,751.2 Daltons. It was originally developed by Novo Nordisk and was approved by the U.S. Food and Drug Administration (FDA) in January 2010 under the trade name "Victoza®".
固相胜肽合成（SPPS）相對簡單且適用於胜肽之合成。胺基酸藉由酸側上的連接基團連接至固相，並連接至胺側上的保護基。可移除保護基，使得第二個胺基酸可偶合至原始胺基酸上的胺側。第二個（及後續的）胺基酸最初亦受保護，因此一般過程為去保護（保護基）、偶合及重複循環，直至完成所需之胜肽，隨後從固相將完成的胜肽切下。Solid-phase peptide synthesis (SPPS) is relatively simple and suitable for peptide synthesis. The amino acid is linked to the solid phase via a linking group on the acid side and to a protecting group on the amine side. The protecting group can be removed so that a second amino acid can be coupled to the amine side of the original amino acid. The second (and subsequent) amino acid is also initially protected, so the general process is deprotection (protecting group), coupling and repeating the cycle until the desired peptide is completed. The completed peptide is then cleaved from the solid phase. Down.
美國專利第9,040,480B2號揭露了一種製造GLP-1或GLP-1激動劑胜肽之方法，其中使用胺基酸及偽脯胺酸雙胜肽而在固相上合成胜肽，但未揭露用於利拉魯肽或索馬魯肽之Lys側鏈的合成方法。U.S. Patent No. 9,040,480B2 discloses a method for manufacturing GLP-1 or GLP-1 agonist peptides, in which amino acid and pseudoproline dual peptides are used to synthesize the peptide on a solid phase, but does not disclose the use of Synthesis method of Lys side chain of liraglutide or semaglutide.
中國專利第109311961B號揭露了一種合成索馬魯肽之方法。該方法包含：將雙胜肽片段及三胜肽片段與具有N端Fmoc保護的胺基酸偶合以獲得索馬魯肽，但未揭露製備索馬魯肽所用的偽脯胺酸雙胜肽。Chinese Patent No. 109311961B discloses a method of synthesizing semaglutide. The method includes: coupling a bi-peptide fragment and a tri-peptide fragment with an amino acid with N-terminal Fmoc protection to obtain semaglutide, but does not disclose the pseudo-proline bi-peptide used to prepare semaglutide.
中國專利申請公開第110922470A號揭露了一種製造索馬魯肽之方法，其中Lys側鏈依序偶合Fmoc-AEEA-OH、Fmoc-AEEA-OH及（γ-Glu-OtBu）-十八碳二酸單三級丁基酯，但在索馬魯肽之說明書中未揭露任何特定具體實施例。Chinese Patent Application Publication No. 110922470A discloses a method for manufacturing semaglutide, in which the Lys side chain is sequentially coupled to Fmoc-AEEA-OH, Fmoc-AEEA-OH and (γ-Glu-OtBu)-octadecanedioic acid Single tertiary butyl ester, but the description of semaglutide does not disclose any specific embodiment.
儘管有上述製造方法，但仍需要開發更有效及改進的方法來製備GLP-1激動劑胜肽。本揭露內容解決此需求並亦提供相關優點。Despite the above manufacturing methods, there is still a need to develop more efficient and improved methods to prepare GLP-1 agonist peptides. The present disclosure addresses this need and also provides related advantages.
本申請案主張美國暫時專利申請第63/348,003號於2022年6月1日提申之內容，其出於所有目的全部併入本文。This application claims the contents of U.S. Provisional Patent Application No. 63/348,003, filed on June 1, 2022, which is fully incorporated herein for all purposes.
在一態樣中，本揭露內容提供一種製備由式(1)表示之胰高血糖素樣肽-1激動劑胜肽的方法， H-His 1-X 2-Glu 3-Gly 4-Thr 5-Phe 6-Thr 7-Ser 8-Asp 9-Val 10-Ser 11-Ser 12-Tyr 13-Leu 14-Glu 15-Gly 16-Gln 17-Ala 18-Ala 19-Lys 20(R)-Glu 21-Phe 22-Ile 23-Ala 24-Trp 25-Leu 26-Val 27-Arg 28-Gly 29-Arg 30-Gly 31-OH (1) 其中： X為Ala或Aib； R為-γ-Glu-十六酸或-AEEA-AEEA-γ-Glu-十八碳二酸； 其包含下列步驟： (a) 藉由逐步偶合經保護之胺基酸及經保護之雙胜肽而在固相上合成經保護之胜肽，經保護之雙胜肽包含偽脯胺酸雙胜肽及非偽脯胺酸雙胜肽； (b) 從固相切割經保護之胜肽，並將經保護之胜肽去保護。 In one aspect, the present disclosure provides a method for preparing a glucagon-like peptide-1 agonist peptide represented by formula (1), H-His 1 -X 2 -Glu 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Val 10 -Ser 11 -Ser 12 -Tyr 13 -Leu 14 -Glu 15 -Gly 16 -Gln 17 -Ala 18 -Ala 19 -Lys 20 (R)-Glu 21 -Phe 22 -Ile 23 -Ala 24 -Trp 25 -Leu 26 -Val 27 -Arg 28 -Gly 29 -Arg 30 -Gly 31 -OH (1) Where: X is Ala or Aib; R is -γ-Glu -Hexadecanoic acid or -AEEA-AEEA-γ-Glu-octadecanoic acid; which includes the following steps: (a) on the solid phase by stepwise coupling of protected amino acids and protected bis-peptides Synthesize protected peptides. The protected bis-peptides include pseudo-proline bis-peptides and non-pseudo-proline bis-peptides; (b) Cleave the protected peptides from the solid phase, and convert the protected peptides into Peptide deprotection.
在另一態樣中，本揭露內容提供一種由式(1)表示之胰高血糖素樣肽-1激動劑胜肽， H-His 1-X 2-Glu 3-Gly 4-Thr 5-Phe 6-Thr 7-Ser 8-Asp 9-Val 10-Ser 11-Ser 12-Tyr 13-Leu 14-Glu 15-Gly 16-Gln 17-Ala 18-Ala 19-Lys 20(R)-Glu 21-Phe 22-Ile 23-Ala 24-Trp 25-Leu 26-Val 27-Arg 28-Gly 29-Arg 30-Gly 31-OH (1) 其中： X為Ala或Aib； R為-γ-Glu-十六酸或-AEEA-AEEA-γ-Glu-十八碳二酸；且 在胰高血糖素樣肽-1激動劑胜肽之第2位置處的X缺失含量為小於0.10%。 In another aspect, the present disclosure provides a glucagon-like peptide-1 agonist peptide represented by formula (1), H-His 1 -X 2 -Glu 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Val 10 -Ser 11 -Ser 12 -Tyr 13 -Leu 14 -Glu 15 -Gly 16 -Gln 17 -Ala 18 -Ala 19 -Lys 20 (R)-Glu 21 - Phe 22 -Ile 23 -Ala 24 -Trp 25 -Leu 26 -Val 27 -Arg 28 -Gly 29 -Arg 30 -Gly 31 -OH (1) Where: X is Ala or Aib; R is -γ-Glu-ten hexanoic acid or -AEEA-AEEA-γ-Glu-octadecanoic acid; and the X deletion content at the second position of the glucagon-like peptide-1 agonist peptide is less than 0.10%.
在另一態樣中，本揭露內容提供一種根據製備由式(1)表示之胰高血糖素樣肽-1激動劑胜肽之方法獲得的胰高血糖素樣肽-1激動劑胜肽， H-His 1-X 2-Glu 3-Gly 4-Thr 5-Phe 6-Thr 7-Ser 8-Asp 9-Val 10-Ser 11-Ser 12-Tyr 13-Leu 14-Glu 15-Gly 16-Gln 17-Ala 18-Ala 19-Lys 20(R)-Glu 21-Phe 22-Ile 23-Ala 24-Trp 25-Leu 26-Val 27-Arg 28-Gly 29-Arg 30-Gly 31-OH (1) 其中： X為Ala或Aib； R為-γ-Glu-十六酸或-AEEA-AEEA-γ-Glu-十八碳二酸； 其包含下列步驟： (a) 藉由逐步偶合經保護之胺基酸及經保護之雙胜肽而在固相上合成經保護之胜肽，經保護之雙胜肽包含偽脯胺酸雙胜肽及非偽脯胺酸雙胜肽； (b) 從固相切割經保護之胜肽，並將經保護之胜肽去保護； 在胰高血糖素樣肽-1激動劑胜肽之第2位置處的X缺失含量為小於0.10%。 In another aspect, the present disclosure provides a glucagon-like peptide-1 agonist peptide obtained according to a method for preparing a glucagon-like peptide-1 agonist peptide represented by formula (1), H-His 1 -X 2 -Glu 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Val 10 -Ser 11 -Ser 12 -Tyr 13 -Leu 14 -Glu 15 -Gly 16 - Gln 17 -Ala 18 -Ala 19 -Lys 20 (R) -Glu 21 -Phe 22 -Ile 23 -Ala 24 -Trp 25 -Leu 26 -Val 27 -Arg 28 -Gly 29 -Arg 30 -Gly 31 -OH ( 1) Among them: The protected peptides are synthesized on solid phase using amino acids and protected bis-peptides. The protected bis-peptides include pseudo-proline bis-peptides and non-pseudo-proline bis-peptides; (b) The protected peptide is cleaved from the solid phase and the protected peptide is deprotected; the X deletion content at position 2 of the glucagon-like peptide-1 agonist peptide is less than 0.10%.
表徵本發明新穎性之各種特徵在所附且構成本揭露內容之一部分的申請專利範圍中被特別指出。為了更好的理解本發明、其操作優點及通過其用途達成的具體目的，應參考說明及描述本發明之較佳具體實施例的描述內容。The various features characterizing the novelty of the invention are particularly pointed out in the appended patent claims which form a part of this disclosure. For a better understanding of the invention, its operating advantages and the specific objects accomplished by its use, reference should be made to the description which illustrates and describes preferred embodiments of the invention.
I. 總則I. GENERAL PROVISIONS
本揭露內容提供一種製備胰高血糖素樣肽-1激動劑胜肽之改進方法。具體而言，本發明係有關一種在利拉魯肽或索馬魯肽之製備中用於減少雜質及增加產率和成本平衡的製造方法。特別是，本方法在製備步驟中使用偽脯胺酸雙胜肽及非偽脯胺酸雙胜肽，其在利拉魯肽或索馬魯肽之製備中提供更好的產率及更少的雜質。The present disclosure provides an improved method for preparing glucagon-like peptide-1 agonist peptide. Specifically, the present invention relates to a manufacturing method for reducing impurities and increasing yield and cost balance in the preparation of liraglutide or semaglutide. In particular, this method uses pseudoproline bispeptides and non-pseudoproline bispeptides in the preparation step, which provides better yields and less cost in the preparation of liraglutide or semaglutide. of impurities.
下表1總結了本揭露內容之具體實施例相較於本領域中報導之方法的優點或特徵。
表1
SEQ ID NO: 1意指索馬魯肽中工程化胜肽的胺基酸序列。序列表示為： H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH（SEQ ID NO: 1）。 SEQ ID NO: 1 refers to the amino acid sequence of the engineered peptide in semaglutide. The sequence is represented as: H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys - Glu-Phe- Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (SEQ ID NO: 1).
SEQ ID NO: 2意指利拉魯肽中工程化胜肽的胺基酸序列。序列表示為： H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH（SEQ ID NO: 2）。 III. 具體實施例之描述 SEQ ID NO: 2 refers to the amino acid sequence of the engineered peptide in liraglutide. The sequence is represented as: H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys - Glu-Phe- Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (SEQ ID NO: 2). III. Description of Specific Embodiments
為了清楚起見，且有助於理解本揭露內容，說明書及申請專利範圍中使用的術語及縮寫定義在表2中。
表2
在第一個態樣中，本發明提供一種製備由式(1)表示之胰高血糖素樣肽-1激動劑胜肽， H-His 1-X 2-Glu 3-Gly 4-Thr 5-Phe 6-Thr 7-Ser 8-Asp 9-Val 10-Ser 11-Ser 12-Tyr 13-Leu 14-Glu 15-Gly 16-Gln 17-Ala 18-Ala 19-Lys 20(R)-Glu 21-Phe 22-Ile 23-Ala 24-Trp 25-Leu 26-Val 27-Arg 28-Gly 29-Arg 30-Gly 31-OH (1) 其中： X為Ala或Aib； R為-γ-Glu-十六酸或-AEEA-AEEA-γ-Glu-十八碳二酸； 其包含下列步驟： (a) 藉由逐步偶合經保護之胺基酸及經保護之雙胜肽而在固相上合成經保護之胜肽，經保護之雙胜肽包含偽脯胺酸雙胜肽及非偽脯胺酸雙胜肽； (b) 從固相切割經保護之胜肽，並將經保護之胜肽去保護以獲得胜肽。 In a first aspect, the present invention provides a method for preparing a glucagon-like peptide-1 agonist peptide represented by formula (1), H-His 1 -X 2 -Glu 3 -Gly 4 -Thr 5 - Phe 6 -Thr 7 -Ser 8 -Asp 9 -Val 10 -Ser 11 -Ser 12 -Tyr 13 -Leu 14 -Glu 15 -Gly 16 -Gln 17 -Ala 18 -Ala 19 -Lys 20 (R)-Glu 21 -Phe 22 -Ile 23 -Ala 24 -Trp 25 -Leu 26 -Val 27 -Arg 28 -Gly 29 -Arg 30 -Gly 31 -OH (1) Where: X is Ala or Aib; R is -γ-Glu- Hexadecanoic acid or -AEEA-AEEA-γ-Glu-octadecanoic acid; which includes the following steps: (a) Synthesized on solid phase by stepwise coupling of protected amino acids and protected bis-peptides Protected peptides, the protected bis-peptides include pseudo-proline bis-peptides and non-pseudo-proline bis-peptides; (b) Cleave the protected peptide from the solid phase, and separate the protected peptides Go to protect to get peptides.
在一些具體實施例中，偽脯胺酸雙胜肽為Fmoc-Val-Ser(Psi (Me,Me)pro)-OH。Fmoc-Val-Ser(Psi (Me,Me)pro)-OH係用於合成利拉魯肽或索馬魯肽，其可減少Des-Thr 5雜質並增加產率。 In some specific embodiments, the pseudoproline bispeptide is Fmoc-Val-Ser(Psi (Me,Me) pro)-OH. Fmoc-Val-Ser(Psi (Me,Me) pro)-OH is used in the synthesis of liraglutide or semaglutide, which can reduce Des-Thr 5 impurities and increase yields.
在一些具體實施例中，非偽脯胺酸雙胜肽係選自由Boc-His(Boc)-Aib-OH、Boc-His(Boc)-Ala-OH、Boc-His(Trt)-Aib-OH及Boc-His(Trt)-Ala-OH組成之群組。Boc-His(Boc)-Aib-OH、Boc-His(Boc)-Ala-OH、Boc-His(Trt)-Aib-OH或Boc-His(Trt)-Ala-OH係用於合成利拉魯肽或索馬魯肽，其可增加粗胜肽之純度。較佳地，非偽脯胺酸雙胜肽係選自由Boc-His(Trt)-Aib-OH及Boc-His(Trt)-Ala-OH組成之群組。Boc-His(Trt)-Aib-OH或Boc-His(Trt)-Ala-OH係用於合成利拉魯肽或索馬魯肽，其可減少Des-Ala 2或Des-Aib 2雜質並增加產率（轉化速率）。特別是，偽脯胺酸雙胜肽（諸如Fmoc-Val-Ser(Psi (Me,Me)pro)-OH）及非偽脯胺酸雙胜肽（諸如Boc-His(Trt)-Aib-OH或Boc-His(Trt)-Ala-OH），其可一起用於合成利拉魯肽或索馬魯肽，具有協同效果，其可極大地減少Des-Thr 5、Des-Gly 4、Des-Ala 2或Des-Aib 2雜質並增加產率。 In some embodiments, the non-pseudoproline bispeptide system is selected from the group consisting of Boc-His(Boc)-Aib-OH, Boc-His(Boc)-Ala-OH, and Boc-His(Trt)-Aib-OH. and the group consisting of Boc-His(Trt)-Ala-OH. Boc-His(Boc)-Aib-OH, Boc-His(Boc)-Ala-OH, Boc-His(Trt)-Aib-OH or Boc-His(Trt)-Ala-OH are used to synthesize liraru peptide or semaglutide, which can increase the purity of the crude peptide. Preferably, the non-pseudoproline bis-peptide is selected from the group consisting of Boc-His(Trt)-Aib-OH and Boc-His(Trt)-Ala-OH. Boc-His(Trt)-Aib-OH or Boc-His(Trt)-Ala-OH is used to synthesize liraglutide or semaglutide, which can reduce Des-Ala 2 or Des-Aib 2 impurities and increase Yield (conversion rate). In particular, pseudoproline bispeptides such as Fmoc-Val-Ser(Psi (Me,Me) pro)-OH and non-pseudoproline bispeptides such as Boc-His(Trt)-Aib-OH or Boc-His(Trt)-Ala-OH), which can be used together to synthesize liraglutide or semaglutide, which has a synergistic effect and can greatly reduce Des-Thr 5 , Des-Gly 4 , Des- Ala 2 or Des-Aib 2 impurities and increase yield.
在一些具體實施例中，偶合試劑DEPBT / DIPEA係用於偶合非偽脯胺酸雙胜肽Boc-His(Trt)-Aib-OH或Boc-His(Trt)-Ala-OH。偶合試劑DEPBT / DIPEA係用於在利拉魯肽或索馬魯肽之合成中偶合Boc-His(Trt)-Aib-OH或Boc-His(Trt)-Ala-OH，其可增加粗胜肽之純度及產率。In some specific embodiments, the coupling reagent DEPBT/DIPEA is used to couple the non-pseudoproline bispeptide Boc-His(Trt)-Aib-OH or Boc-His(Trt)-Ala-OH. The coupling reagent DEPBT/DIPEA is used to couple Boc-His(Trt)-Aib-OH or Boc-His(Trt)-Ala-OH in the synthesis of liraglutide or semaglutide, which can increase the crude peptide the purity and yield.
在一些具體實施例中，非偽脯胺酸雙胜肽進一步包含選自由Boc-Arg(Pbf)-Gly-OH及Boc-Glu(OtBu)-Gly-OH所組成群組之至少一者。In some specific embodiments, the non-pseudoproline bispeptide further includes at least one selected from the group consisting of Boc-Arg(Pbf)-Gly-OH and Boc-Glu(OtBu)-Gly-OH.
在一些具體實施例中，使用不超過3種的非偽脯胺酸雙胜肽。較佳地，使用不超過2種的非偽脯胺酸雙胜肽。最佳地，使用不超過1種的非偽脯胺酸雙胜肽。In some embodiments, no more than 3 non-pseudoproline bipeptides are used. Preferably, no more than two non-pseudoproline bipeptides are used. Optimally, no more than one non-pseudoproline bipeptide is used.
在一些具體實施例中，Fmoc-Lys(Alloc)-OH或Fmoc-Lys(ivDde)-OH係用作Lys之經保護胺基酸。In some embodiments, Fmoc-Lys(Alloc)-OH or Fmoc-Lys(ivDde)-OH is used as the protected amino acid for Lys.
在一些具體實施例中，胜肽為利拉魯肽。In some embodiments, the peptide is liraglutide.
在一些具體實施例中，胜肽為索馬魯肽。In some embodiments, the peptide is semaglutide.
在一些具體實施例中，R為-AEEA-AEEA-γ-Glu-十八碳二酸，且在步驟(a)後使用AEEA-AEEA-(γ-Glu-OtBu)-十八碳二酸單三級丁基酯與Lys之ε-胺基側鏈偶合。In some embodiments, R is -AEEA-AEEA-γ-Glu-octadecanedioic acid, and after step (a) AEEA-AEEA-(γ-Glu-OtBu)-octadecacarbondioic acid mono The tertiary butyl ester is coupled to the ε-amine side chain of Lys.
在一些具體實施例中，R為-AEEA-AEEA-γ-Glu-十八碳二酸，且在步驟(a)後使用Fmoc-AEEA-OH、Fmoc-AEEA-OH、(γ-Glu-OtBu)-十八碳二酸單三級丁基酯依序與Lys之ε-胺基側鏈偶合。In some embodiments, R is -AEEA-AEEA-γ-Glu-octadecanedioic acid, and after step (a), Fmoc-AEEA-OH, Fmoc-AEEA-OH, (γ-Glu-OtBu )-Octadecanedioic acid monotertiary butyl ester is sequentially coupled with the ε-amino side chain of Lys.
在一些具體實施例中，該方法進一步包含在步驟(b)後的純化步驟。In some embodiments, the method further comprises a purification step after step (b).
在一些具體實施例中，固相可為（但不限於）聚苯乙烯系4-烷氧基芐基醇（Wang）樹脂、聚合二苯基重氮甲烷（PDDM）樹脂、4-（2',4'-二甲氧基苯基-Fmoc-胺基甲基）-苯氧基甲基-聚苯乙烯（Rink）樹脂、2-甲氧基-4-烷氧基芐基醇（Sasrin）樹脂、及2-氯三苯甲基氯（CTC）樹脂。此外，可使用適用於SPPS之任何其他樹脂。In some specific embodiments, the solid phase can be (but is not limited to) polystyrenic 4-alkoxybenzyl alcohol (Wang) resin, polymerized diphenyldiazomethane (PDDM) resin, 4-(2' ,4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxymethyl-polystyrene (Rink) resin, 2-methoxy-4-alkoxybenzyl alcohol (Sasrin) resin, and 2-chlorotrityl chloride (CTC) resin. Additionally, any other resin suitable for SPPS can be used.
在一些具體實施例中，經保護之胺基酸為包含N端α胺保護基之胺基酸。N端α胺保護基可為（但不限於）Fmoc、Boc及Cbz。較佳地，N端α胺保護基為Fmoc或Boc，且更佳為Fmoc。在任何後續的偶合步驟中，在下一個經保護之胺基酸或經保護之雙胜肽偶合之前，移除先前偶合步驟中形成的胜肽之N端α胺保護基，例如藉由與切割試劑反應，該切割試劑例如在Fmoc情況下的鹼（諸如哌啶），或在Boc情況下的酸（諸如TFA）。In some embodiments, the protected amino acid is an amino acid containing an N-terminal alpha amine protecting group. The N-terminal α-amine protecting group can be (but is not limited to) Fmoc, Boc and Cbz. Preferably, the N-terminal α-amine protecting group is Fmoc or Boc, and more preferably, it is Fmoc. In any subsequent coupling step, the N-terminal α-amine protecting group of the peptide formed in the previous coupling step is removed before coupling to the next protected amino acid or protected bipeptide, for example by coupling with a cleavage reagent reaction with a cleavage reagent such as a base (such as piperidine) in the case of Fmoc, or an acid (such as TFA) in the case of Boc.
在一些具體實施例中，SPPS方法係基於Fmoc合成策略。Fmoc保護基在溶劑中以包含鹼的切割試劑切割（deFmoc）。鹼可選自二級胺，諸如哌啶及4-甲基哌啶。溶劑可選自由DMF、NMP、DMSO、DCM、THF、乙腈、甲苯及其混合物組成之群組。反應通常在環境溫度下，例如在15至30℃之溫度範圍內進行。鹼不穩定及酸穩定的Fmoc可在短時間內（諸如2至15分鐘）被切除。In some embodiments, the SPPS method is based on the Fmoc synthesis strategy. The Fmoc protecting group is cleaved with a base-containing cleavage reagent in a solvent (deFmoc). The base may be selected from secondary amines such as piperidine and 4-methylpiperidine. The solvent may be selected from the group consisting of DMF, NMP, DMSO, DCM, THF, acetonitrile, toluene and mixtures thereof. The reaction is usually carried out at ambient temperature, for example in the temperature range of 15 to 30°C. Base-labile and acid-stable Fmoc can be cleaved in a short time, such as 2 to 15 minutes.
在一些具體實施例中，切割試劑係選自由含有5-50%（w/w）哌啶或4-甲基哌啶的DMF、含有5-50%（w/w）哌啶或4-甲基哌啶的NMP、含有1-5%（w/w）DBU的DMF、及含有50%（w/w）嗎啉的DMF組成之群組。在移除Fmoc後小心洗去切割試劑。使用DMF清洗，直至pH值呈中性。為了確保完全移除鹼基，在隨後的清洗循環中添加少量的HOBt可能是有利的。In some embodiments, the cleavage reagent is selected from DMF containing 5-50% (w/w) piperidine or 4-methylpiperidine, DMF containing 5-50% (w/w) piperidine or 4-methylpiperidine. A group consisting of NMP based on piperidine, DMF containing 1-5% (w/w) DBU, and DMF containing 50% (w/w) morpholine. Carefully wash away the cleavage reagent after removing Fmoc. Wash with DMF until pH is neutral. To ensure complete base removal, it may be advantageous to add a small amount of HOBt in subsequent wash cycles.
在一些具體實施例中，經保護之胺基酸為進一步包含胺側鏈保護基的胺基酸。胺側鏈保護基可為（但不限於）Fmoc、Boc、Mtt、Mmt、Trt、Pbf、OtBu、tBu、OMpe、Pmc、ivDde及Alloc。In some embodiments, the protected amino acid is an amino acid further comprising an amine side chain protecting group. Amine side chain protecting groups may be (but are not limited to) Fmoc, Boc, Mtt, Mmt, Trt, Pbf, OtBu, tBu, OMpe, Pmc, ivDde and Alloc.
在一些具體實施例中，經保護之胺基酸可為（但不限於）Fmoc-Aib-OH、Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pmc)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Asn(Mtt)-OH、Fmoc-Asp(tBu)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Asp(OMpe)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Cys(Mmt)-OH、Fmoc-Gly-OH、Fmoc-Gln(Mtt)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Glu(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-His(l-Trt)-OH、Fmoc-Ile-OH、Fmoc-Leu-OH、Fmoc-Lys(Alloc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(ivDde)-OH、Fmoc-Met-OH、Fmoc-Phe-OH、Fmoc-Pro-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Trp(Boc)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Val-OH及Boc-His(Boc)-OH。In some specific embodiments, the protected amino acid can be (but is not limited to) Fmoc-Aib-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Mtt)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OMpe)-OH, Fmoc-Cys(Trt )-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Gly-OH, Fmoc-Gln(Mtt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu( OtBu)-OH, Fmoc-His(l-Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys( ivDde)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH and Boc-His(Boc)-OH.
在一些具體實施例中，典型上含有經保護之胺基酸殘基的利拉魯肽或索馬魯肽的合成較佳為使用His殘基中的酸不穩定N端α胺保護基（諸如Boc），使得可在一個步驟中移除（可選地連同任何固相，例如Wang樹脂）經保護之利拉魯肽或索馬魯肽序列的N端α胺保護基及胺酸側鏈保護基。舉例而言，His之N端α胺Boc基可與酸不穩定胺側鏈保護基及Wang樹脂一起被移除，其係藉由切割雞尾酒處理（典型上切割雞尾酒包含TFA，且可為TFA與含有DTT的DCM的混合物），從而產生利拉魯肽或索馬魯肽。In some embodiments, the synthesis of liraglutide or semaglutide, which typically contains protected amino acid residues, is preferably accomplished using an acid-labile N-terminal alpha-amine protecting group in the His residue, such as Boc), allowing the N-terminal alpha-amine protecting group and the amino acid side chain protection of the protected liraglutide or semaglutide sequence to be removed (optionally together with any solid phase, such as Wang resin) in a single step base. For example, the N-terminal α-amine Boc group of His can be removed together with the acid-labile amine side chain protecting group and Wang resin by processing a cleavage cocktail (a typical upper cleavage cocktail contains TFA, and can be TFA and mixture of DCM containing DTT), resulting in liraglutide or semaglutide.
在一些具體實施例中，經保護之胺基酸或經保護之雙胜肽的偶合係於作為溶劑的DMF中進行，例如將經保護之胺基酸或經保護之雙胜肽、偶合試劑及可選地添加劑溶解於DMF中並混合。DIC可用作偶合試劑，並與作為添加劑的HOBt組合。或者，TBTU或DEPBT可用於在鹼（較佳為DIPEA）存在下將Fmoc胺基酸轉化為活性酯。偶合試劑/添加劑混合物係選自由DIC / HOBt、TBTU / DIPEA、TATU / DIPEA、DEPBT / DIPEA、HBTU / DIPEA及HATU / DIPEA組成之群組。經保護之胺基酸或經保護之雙胜肽的偶合係於一段時間內進行，諸如1至74小時。可選地，進行一或多個重新偶合步驟以完成胺基的轉化可能是有利的。In some embodiments, the coupling of protected amino acids or protected bis-peptides is carried out in DMF as a solvent, for example, the protected amino acids or protected bis-peptides, coupling reagents and Optionally the additives are dissolved in DMF and mixed. DIC can be used as a coupling reagent and combined with HOBt as an additive. Alternatively, TBTU or DEPBT can be used to convert Fmoc amino acids into active esters in the presence of a base, preferably DIPEA. The coupling reagent/additive mixture is selected from the group consisting of DIC/HOBt, TBTU/DIPEA, TATU/DIPEA, DEPBT/DIPEA, HBTU/DIPEA and HATU/DIPEA. Coupling of the protected amino acid or protected bipeptide is performed over a period of time, such as 1 to 74 hours. Alternatively, it may be advantageous to perform one or more recoupling steps to complete the conversion of the amine group.
可進行封端，以在後續合成步驟中阻斷未經反應之胜肽的N端α胺，並避免缺失變體的形成。其可藉由以大量過量的高度反應性未受阻的酸衍生物（諸如N-羥基琥珀醯亞胺、乙酸酐或苯甲醯氯）及鹼（諸如吡啶、三甲基吡啶（collidine）或DIPEA）對樹脂結合的胜肽進行短期處理來達成。封端典型上將產生一個截斷的序列，其通常不同於最終胜肽並可完全分離。在進行下一個去保護步驟之前，典型上濾除封端步驟中使用的試劑，並以DMF及可選地甲醇仔細清洗樹脂結合的胜肽。Endcapping can be performed to block the N-terminal α-amine of the unreacted peptide in subsequent synthetic steps and avoid the formation of deletion variants. This can be accomplished by adding a large excess of a highly reactive unhindered acid derivative (such as N-hydroxysuccinimide, acetic anhydride or benzyl chloride) and a base (such as pyridine, collidine or DIPEA ) is achieved by short-term treatment of resin-bound peptides. Endcapping will typically result in a truncated sequence that is usually different from the final peptide and completely separable. The reagents used in the capping step are typically filtered off and the resin-bound peptide is carefully washed with DMF and optionally methanol before proceeding to the next deprotection step.
在一些具體實施例中，從樹脂結合的胜肽中移除Lys之Alloc或ivDde保護基可藉由使用催化劑（諸如[Pd(PPh 3) 4]=Pd(Ph 3P) 4、Pd 2(dba) 3.CHC1 3、Pd(dba) 2、Pd(Ph 3P) 2Cl 2、(Ph 3P) 2NiCl 2或Pd(OAc) 2來進行。較佳地，可使用[Pd(PPh 3) 4]。 In some embodiments, the Alloc or ivDde protecting group of Lys can be removed from the resin-bound peptide by using a catalyst such as [Pd(PPh 3 ) 4 ]=Pd(Ph 3 P) 4 , Pd 2 ( dba) 3.CHC1 3 , Pd(dba) 2 , Pd(Ph 3 P) 2 Cl 2 , (Ph 3 P) 2 NiCl 2 or Pd(OAc) 2. Preferably, [Pd(PPh) can be used 3 ) 4 ].
為了從樹脂上切下胜肽，可使用包含TFA的組成物。較佳地，組成物包含超過50%（v/v）TFA，更佳為超過75%（v/v）TFA，特別為至少80%（v/v）或甚至至少90%（v/v）TFA。組成物亦可包含水及/或一或多種清除劑。較佳地，清除劑為硫醇清除劑，諸如EDT、DTT及DTE；以及矽烷清除劑，諸如TIPS及TES。To cleave peptides from the resin, compositions containing TFA can be used. Preferably, the composition contains more than 50% (v/v) TFA, more preferably more than 75% (v/v) TFA, especially at least 80% (v/v) or even at least 90% (v/v) TFA. The composition may also include water and/or one or more scavengers. Preferably, the scavengers are thiol scavengers such as EDT, DTT and DTE; and silane scavengers such as TIPS and TES.
在從樹脂上切下胜肽之後，通常藉由過濾來分離樹脂。將反溶劑與所得濾液混合，隨後沉澱出粗胜肽。反溶劑可選自由二乙基醚、IPE、MTBE、ACN及其混合物組成之群組。After cleaving the peptide from the resin, the resin is usually isolated by filtration. The antisolvent is mixed with the resulting filtrate, and the crude peptide is subsequently precipitated. The antisolvent may be selected from the group consisting of diethyl ether, IPE, MTBE, ACN, and mixtures thereof.
所得粗胜肽可藉由一或多種層析方法來進行進一步純化。層析方法包含凝膠滲透層析術（GPC）、粒徑排除層析術（SEC）、離子交換層析術（IEC）、高效能液相層析術（HPLC）、逆相HPLC（RP-HPLC）、超效液相層析術（UPLC）及逆相UPLC（RP-UPLC）。The resulting crude peptide can be further purified by one or more chromatography methods. Chromatography methods include gel permeation chromatography (GPC), particle size exclusion chromatography (SEC), ion exchange chromatography (IEC), high performance liquid chromatography (HPLC), reverse phase HPLC (RP- HPLC), ultra performance liquid chromatography (UPLC) and reverse phase UPLC (RP-UPLC).
在第二個態樣中，本發明提供一種由式(1)表示之胰高血糖素樣肽-1激動劑胜肽， H-His 1-X 2-Glu 3-Gly 4-Thr 5-Phe 6-Thr 7-Ser 8-Asp 9-Val 10-Ser 11-Ser 12-Tyr 13-Leu 14-Glu 15-Gly 16-Gln 17-Ala 18-Ala 19-Lys 20(R)-Glu 21-Phe 22-Ile 23-Ala 24-Trp 25-Leu 26-Val 27-Arg 28-Gly 29-Arg 30-Gly 31-OH (1) 其中： X為Ala或Aib； R為-γ-Glu-十六酸或-AEEA-AEEA-γ-Glu-十八碳二酸；且 在胰高血糖素樣肽-1激動劑胜肽之第2位置處的X缺失含量為小於0.10%。 In a second aspect, the present invention provides a glucagon-like peptide-1 agonist peptide represented by formula (1), H-His 1 -X 2 -Glu 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Val 10 -Ser 11 -Ser 12 -Tyr 13 -Leu 14 -Glu 15 -Gly 16 -Gln 17 -Ala 18 -Ala 19 -Lys 20 (R)-Glu 21 - Phe 22 -Ile 23 -Ala 24 -Trp 25 -Leu 26 -Val 27 -Arg 28 -Gly 29 -Arg 30 -Gly 31 -OH (1) Where: X is Ala or Aib; R is -γ-Glu-ten hexanoic acid or -AEEA-AEEA-γ-Glu-octadecanoic acid; and the X deletion content at the second position of the glucagon-like peptide-1 agonist peptide is less than 0.10%.
在一些具體實施例中，由式(1)表示之胰高血糖素樣肽-1激動劑胜肽，其中胰高血糖素樣肽-1激動劑胜肽之第2位置處的X缺失含量小於0.09%，較佳為小於0.08%，最佳為小於0.07%。In some embodiments, the glucagon-like peptide-1 agonist peptide represented by formula (1), wherein the X deletion content at position 2 of the glucagon-like peptide-1 agonist peptide is less than 0.09%, preferably less than 0.08%, most preferably less than 0.07%.
在一些具體實施例中，由式(1)表示之胰高血糖素樣肽-1激動劑胜肽，其中胰高血糖素樣肽-1激動劑胜肽之第5位置處的Thr缺失含量小於0.06%，較佳為小於0.05%。In some embodiments, the glucagon-like peptide-1 agonist peptide represented by formula (1), wherein the Thr deletion content at position 5 of the glucagon-like peptide-1 agonist peptide is less than 0.06%, preferably less than 0.05%.
在一些具體實施例中，由式(1)表示之胰高血糖素樣肽-1激動劑胜肽，其中胰高血糖素樣肽-1激動劑胜肽之第4位置處的Gly缺失含量小於0.01%。In some embodiments, the glucagon-like peptide-1 agonist peptide represented by formula (1), wherein the Gly deletion content at position 4 of the glucagon-like peptide-1 agonist peptide is less than 0.01%.
在第三個態樣中，本發明提供一種根據製備由式(1)表示之胰高血糖素樣肽-1激動劑胜肽之方法獲得的胰高血糖素樣肽-1激動劑胜肽， H-His 1-X 2-Glu 3-Gly 4-Thr 5-Phe 6-Thr 7-Ser 8-Asp 9-Val 10-Ser 11-Ser 12-Tyr 13-Leu 14-Glu 15-Gly 16-Gln 17-Ala 18-Ala 19-Lys 20(R)-Glu 21-Phe 22-Ile 23-Ala 24-Trp 25-Leu 26-Val 27-Arg 28-Gly 29-Arg 30-Gly 31-OH (1) 其中： X為Ala或Aib； R為-γ-Glu-十六酸或-AEEA-AEEA-γ-Glu-十八碳二酸； 其包含下列步驟： (a) 藉由逐步偶合經保護之胺基酸及經保護之雙胜肽而在固相上合成經保護之胜肽，經保護之雙胜肽包含偽脯胺酸雙胜肽及非偽脯胺酸雙胜肽； (b) 從固相切割經保護之胜肽，並將經保護之胜肽去保護以獲得胜肽； 在胰高血糖素樣肽-1激動劑胜肽之第2位置處的X缺失含量為小於0.10%。 IV. 實施例 In a third aspect, the present invention provides a glucagon-like peptide-1 agonist peptide obtained according to a method for preparing a glucagon-like peptide-1 agonist peptide represented by formula (1), H-His 1 -X 2 -Glu 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Val 10 -Ser 11 -Ser 12 -Tyr 13 -Leu 14 -Glu 15 -Gly 16 - Gln 17 -Ala 18 -Ala 19 -Lys 20 (R) -Glu 21 -Phe 22 -Ile 23 -Ala 24 -Trp 25 -Leu 26 -Val 27 -Arg 28 -Gly 29 -Arg 30 -Gly 31 -OH ( 1) Among them: The protected peptides are synthesized on solid phase using amino acids and protected bis-peptides. The protected bis-peptides include pseudo-proline bis-peptides and non-pseudo-proline bis-peptides; (b) The protected peptide is cleaved from the solid phase and the protected peptide is deprotected to obtain the peptide; the X deletion content at position 2 of the glucagon-like peptide-1 agonist peptide is less than 0.10% . IV. Examples
藉由HPLC測定粗胜肽及最終胜肽的純度及雜質含量。具體而言，雜質計算如下所述。分別收集HPLC層析圖中每一尖峰，並將其全部注入HPLC-ESI（電噴灑離子化）-MS系統以進行質量鑑定。針對HPLC-ESI-MS系統，使用填充有C18基質的逆相管柱在高水性流動相中結合胜肽，洗去鹽及緩衝液，並使用高有機流動相溶析胜肽。在TIC（總離子流）層析圖中，除了空白以外的訊號將被識別並提取以提供EIC（提取的離子層析圖）。每一EIC的強度將被計算為一比率，該比率表示為100的分數，隨後乘以HPLC層析圖中尖峰的RA%（相對面積百分比），從中所收集樣本可提供雜質的最終值。以下述比較例1中的Des-Aib
2作為實例：
利用下列原則評估最終胜肽的純度（尖峰面積百分比）： ○：純度 ≧ 98% △：97% ≦ 純度 ＜ 98% ×：96% ≦ 純度 ＜ 97% Evaluate the purity of the final peptide (% peak area) using the following principles: ○：Purity ≧ 98% △: 97% ≦ Purity ＜ 98% ×: 96% ≦ Purity ＜ 97%
利用下列公式計算轉化速率： 轉化速率 = [所得最終胜肽的重量] / [所用空白樹脂的重量] Calculate conversion rate using the following formula: Conversion rate = [weight of final peptide obtained] / [weight of blank resin used]
利用下列公式計算估計總回收率： 胜肽含量[%（w/w）]：最終胜肽減去水及鹽的含量 Calculate the estimated total recovery rate using the following formula: Peptide content [% (w/w)]: final peptide minus water and salt content
比較例1Comparative example 1
Fmoc-Gly-Wang樹脂之製備Preparation of Fmoc-Gly-Wang resin
稱取30 g的乾燥Wang樹脂（具有1.04 mmol/g之取代程度），並添加至SPPS反應器中。樹脂首先以DMF清洗兩次，再以DMF溶脹30分鐘，體積為樹脂床的2-3倍，再以DMF清洗3次。將18.912 g的Fmoc-Gly-OH（2.0當量）及9.559 g的HOBt.H 2O（2.0當量）溶解於DMF及DCM的混合溶劑中。在胺基酸溶解之後，將溶液倒入SPPS反應器中，隨後添加9.8 ml的DIC（2.0當量）及0.762g的DMAP（0.2當量）。所得反應混合物在環境溫度下吹氮並攪拌24小時。在反應完成之後，添加適量的乙酸酐及吡啶混合溶液（體積比率：Ac 2O/DIPEA=1/2），以阻斷反應30分鐘或以上，隨後以DMF清洗3次，以甲醇收縮2次，並減壓乾燥。在反應停止之後，獲得取代程度為0.999 mmol/g的Fmoc-Gly-Wang樹脂。 Weigh 30 g of dry Wang resin (with a degree of substitution of 1.04 mmol/g) and add to the SPPS reactor. The resin is first washed twice with DMF, then swollen with DMF for 30 minutes, the volume is 2-3 times of the resin bed, and then washed three times with DMF. Dissolve 18.912 g of Fmoc-Gly-OH (2.0 equivalents) and 9.559 g of HOBt.H 2 O (2.0 equivalents) in a mixed solvent of DMF and DCM. After the amino acid was dissolved, the solution was poured into the SPPS reactor, followed by the addition of 9.8 ml of DIC (2.0 equiv) and 0.762 g of DMAP (0.2 equiv). The resulting reaction mixture was purged with nitrogen and stirred at ambient temperature for 24 hours. After the reaction is completed, add an appropriate amount of acetic anhydride and pyridine mixed solution (volume ratio: Ac 2 O/DIPEA = 1/2) to block the reaction for 30 minutes or more, then wash with DMF 3 times and shrink with methanol 2 times. , and dried under reduced pressure. After the reaction was stopped, Fmoc-Gly-Wang resin with a degree of substitution of 0.999 mmol/g was obtained.
胜肽之合成Synthesis of peptides
稱取具有0.999 mmol/g之取代程度的25.869 g的Fmoc-Gly-Wang樹脂（19.977 mmol），並以DMF溶脹30分鐘，隨後根據SEQ ID NO: 1之胺基酸序列依序偶合，以2-3當量的胺基酸進料並以DIC + A或B + C進行縮合，直至樹脂以氯醌（chloranil）檢測呈透明，其中A為HOBt.H 2O或（羥基亞胺基）氰基乙酸乙酯；B為HATU或DEPBT；C為DIPEA；且溶劑選自DMF或DCM。所有偶合的胺基酸皆為市售的Fmoc單胺基酸，除了最後一個His殘基被偶合為Boc-His(Boc)-OH.DCHA殘基以外。藉由含有5%（w/w）哌啶的DMF且隨後的DMF清洗來完全移除鹼試劑，達成Fmoc去保護。藉由氯醌測試評估清洗效率，重複清洗直至偶合前不再觀察到藍色。所有偶合皆進行得很好，且不需要重新偶合。 Weigh 25.869 g of Fmoc-Gly-Wang resin (19.977 mmol) with a substitution degree of 0.999 mmol/g, and swell with DMF for 30 minutes, and then sequentially couple according to the amino acid sequence of SEQ ID NO: 1, with 2 -3 equivalents of amino acid are fed and condensed with DIC + A or B + C until the resin is transparent when detected by chloranil, where A is HOBt.H 2 O or (hydroxyimino)cyano. Ethyl acetate; B is HATU or DEPBT; C is DIPEA; and the solvent is selected from DMF or DCM. All coupled amino acids were commercially available Fmoc monoamino acids, except for the last His residue, which was coupled as Boc-His(Boc)-OH.DCHA residue. Fmoc deprotection was achieved by complete removal of the alkaline reagent by DMF containing 5% (w/w) piperidine and subsequent DMF washes. Cleaning efficiency was assessed by the chloroquinone test and the cleaning was repeated until no more blue color was observed before coupling. All couplings went well and no recoupling was required.
在Lys側鏈偶合之前，Alloc保護基以含有0.2至0.3當量的Pd(PPh 3) 4及10當量的嗎啉的DCM溶液移除1小時。在第二次以tBuOSteGlu(AEEA-AEEA)OtBu片段完成側鏈上剩餘胺基酸的偶合。使用5.1683 g的樹脂結合的胜肽，且在Alloc-去保護及偶合完成後獲得約5.5609 g的樹脂結合的胜肽。 Prior to Lys side chain coupling, the Alloc protecting group was removed with a solution containing 0.2 to 0.3 equivalents of Pd( PPh3 ) 4 and 10 equivalents of morpholine in DCM for 1 hour. In the second time, the coupling of the remaining amino acids on the side chain is completed with the tBuOSteGlu(AEEA-AEEA)OtBu fragment. 5.1683 g of resin-bound peptide was used, and approximately 5.5609 g of resin-bound peptide was obtained after Alloc-deprotection and coupling were completed.
將32 mL的切割試劑按照TFA : EDT : PPW = 90 : 13.6 : 5之體積比率添加至100 mL反應釜中，並預冷至0至10℃。取上述獲得的4.00 g的樹脂結合的胜肽，並緩慢添加至反應釜中，使得在室溫下反應2小時。在反應完成之後，濾除樹脂，並收集濾液。樹脂以少量TFA清洗，合併濾液，隨後冷卻至0至10℃。將冰冷的MTBE添加至濾液中，粗胜肽在0至10℃下沉澱、過濾、以MTBE清洗並真空乾燥，以獲得2.29 g的粗胜肽。粗胜肽以美國專利公開第20210206800號揭露的HPLC方法進一步純化，以獲得550 mg的最終胜肽索馬魯肽。Add 32 mL of cutting reagent into the 100 mL reaction kettle at a volume ratio of TFA:EDT:PPW=90:13.6:5, and pre-cool to 0 to 10°C. Take 4.00 g of the resin-bound peptide obtained above and slowly add it to the reaction kettle, allowing it to react at room temperature for 2 hours. After the reaction was completed, the resin was filtered off, and the filtrate was collected. The resin was washed with a small amount of TFA and the filtrates were combined and then cooled to 0 to 10°C. Ice-cold MTBE was added to the filtrate, and the crude peptide was precipitated at 0 to 10°C, filtered, washed with MTBE, and dried under vacuum to obtain 2.29 g of crude peptide. The crude peptide was further purified using the HPLC method disclosed in US Patent Publication No. 20210206800 to obtain 550 mg of the final peptide semaglutide.
比較例2Comparative example 2
胜肽之合成Synthesis of peptides
稱取6 g比較例1中合成的具有0.999 mmol/g之取代程度的Fmoc-20mer-Wang樹脂（1.4 mmol），並以DMF溶脹30分鐘，隨後根據SEQ ID NO: 1之胺基酸序列依序偶合，以2至3當量的胺基酸進料並以DIC + A或B + C進行縮合，直至樹脂以氯醌檢測呈透明，其中A為HOBt.H 2O或（羥基亞胺基）氰基乙酸乙酯；B為HATU或DEPBT；C為DIPEA；且溶劑選自DMF或DCM。所有偶合的胺基酸皆為市售的Fmoc單胺基酸，除了Val 10-Ser 11殘基被偶合為偽脯胺酸雙胜肽Fmoc-Val-Ser(Ψ (Me,Me)pro)-OH殘基以外；最後一個His被偶合為Boc-His(Boc)-OH.DCHA殘基以外。藉由含有20%（w/w）哌啶的DMF且隨後的DMF清洗來完全移除鹼試劑，達成Fmoc去保護。藉由氯醌測試評估清洗效率，重複清洗直至偶合前不再觀察到藍色。所有偶合皆進行得很好，且不需要重新偶合。 Weigh 6 g of the Fmoc-20mer-Wang resin (1.4 mmol) with a substitution degree of 0.999 mmol/g synthesized in Comparative Example 1, and swell with DMF for 30 minutes, and then follow the amino acid sequence of SEQ ID NO: 1 Sequential coupling, feed 2 to 3 equivalents of amino acid and perform condensation with DIC + A or B + C until the resin is transparent when detected by chloroquinone, where A is HOBt.H 2 O or (hydroxyimine group) Ethyl cyanoacetate; B is HATU or DEPBT; C is DIPEA; and the solvent is selected from DMF or DCM. All coupled amino acids are commercially available Fmoc monoamino acids, except that Val 10 -Ser 11 residues are coupled as pseudoproline double peptide Fmoc-Val-Ser(Ψ (Me,Me) pro)- Except for the OH residue; the last His is coupled as Boc-His(Boc)-OH.Except for the DCHA residue. Fmoc deprotection was achieved by complete removal of the alkaline reagent by DMF containing 20% (w/w) piperidine and subsequent DMF washes. Cleaning efficiency was assessed by the chloroquinone test and the cleaning was repeated until no more blue color was observed before coupling. All couplings went well and no recoupling was required.
在Lys側鏈偶合之前，Alloc保護基以含有0.2至0.3當量的Pd(PPh 3) 4及10當量的嗎啉的DCM移除1小時。側鏈上剩餘胺基酸的偶合係依次與Fmoc-AEEA-OH（3.0當量）偶合兩次且tBuSte-Glu-tBu.DCHA（3.0當量）偶合一次，並以DIC + A或B + C進行縮合，直至樹脂以氯醌檢測呈透明，其中A為HOBt.H 2O或（羥基亞胺基）氰基乙酸乙酯；B為HATU或DEPBT；C為DIPEA；且溶劑選自DMF或DCM。藉由含有20%（w/w）哌啶的DMF且隨後的DMF清洗來完全移除鹼試劑，達成Fmoc去保護。藉由氯醌測試評估清洗效率，重複清洗直至偶合前不再觀察到藍色。所有偶合皆進行得很好，且不需要重新偶合。使用2.09 g的樹脂結合的胜肽，且在Alloc-去保護及偶合完成後獲得約2.21 g的樹脂結合的胜肽。 Prior to Lys side chain coupling, the Alloc protecting group was removed in DCM containing 0.2 to 0.3 equivalents of Pd( PPh3 ) 4 and 10 equivalents of morpholine for 1 hour. The coupling system of the remaining amino acids on the side chain was sequentially coupled twice with Fmoc-AEEA-OH (3.0 equivalents) and once with tBuSte-Glu-tBu.DCHA (3.0 equivalents), and condensation was carried out with DIC + A or B + C , until the resin is transparent when detected by chloroquinone, where A is HOBt.H 2 O or (hydroxyimino) ethyl cyanoacetate; B is HATU or DEPBT; C is DIPEA; and the solvent is selected from DMF or DCM. Fmoc deprotection was achieved by complete removal of the alkaline reagent by DMF containing 20% (w/w) piperidine and subsequent DMF washes. Cleaning efficiency was assessed by the chloroquinone test and the cleaning was repeated until no more blue color was observed before coupling. All couplings went well and no recoupling was required. 2.09 g of resin-bound peptide was used, and approximately 2.21 g of resin-bound peptide was obtained after Alloc-deprotection and coupling were completed.
取上述獲得的2.21 g的樹脂結合的胜肽，並添加至50 mL反應釜中。將17.84 mL的切割試劑按照TFA : EDT : PPW = 83 : 12.5 : 4.5之體積比率調配，並預冷至0至10℃，隨後倒入50 mL反應釜中，使得在室溫下反應2小時。在反應完成之後，濾除樹脂，並收集濾液。樹脂以少量TFA清洗，合併濾液，隨後冷卻至0至10℃。將冰冷的MTBE添加至濾液中，粗胜肽在0至10℃下沉澱、過濾、以MTBE清洗並真空乾燥，以獲得1.26 g的粗胜肽。900 mg的粗胜肽以美國專利公開第20210206800號揭露的HPLC方法進一步純化，以獲得319 mg的最終胜肽索馬魯肽。Take 2.21 g of the resin-bound peptide obtained above and add it to the 50 mL reaction kettle. Prepare 17.84 mL of cutting reagent according to the volume ratio of TFA: EDT: PPW = 83: 12.5: 4.5, pre-cool to 0 to 10°C, then pour into a 50 mL reaction kettle, and allow to react at room temperature for 2 hours. After the reaction was completed, the resin was filtered off, and the filtrate was collected. The resin was washed with a small amount of TFA, and the filtrates were combined and then cooled to 0 to 10°C. Ice-cold MTBE was added to the filtrate, and the crude peptide was precipitated at 0 to 10°C, filtered, washed with MTBE, and dried under vacuum to obtain 1.26 g of crude peptide. 900 mg of the crude peptide was further purified using the HPLC method disclosed in US Patent Publication No. 20210206800 to obtain 319 mg of the final peptide semaglutide.
比較例3Comparative example 3
胜肽之合成Synthesis of peptides
稱取6 g比較例1中合成的具有0.999 mmol/g之取代程度的Fmoc-20mer-Wang樹脂（1.4 mmol），並以DMF溶脹30分鐘，隨後根據SEQ ID NO: 1之胺基酸序列依序偶合，以2至3當量的胺基酸進料並以DIC + A或B + C進行縮合，直至樹脂以氯醌檢測呈透明，其中A為HOBt.H 2O或（羥基亞胺基）氰基乙酸乙酯；B為HATU或DEPBT；C為DIPEA；且溶劑選自DMF或DCM。所有偶合的胺基酸皆為市售的Fmoc單胺基酸，除了Val 10-Ser 11殘基被偶合為偽脯胺酸雙胜肽Fmoc-Val-Ser(Ψ (Me,Me)pro)-OH殘基以外；最後一個His被偶合為Boc-His(Boc)-OH.DCHA殘基以外。藉由含有20%（w/w）哌啶的DMF且隨後的DMF清洗來完全移除鹼試劑，達成Fmoc去保護。藉由氯醌測試評估清洗效率，重複清洗直至偶合前不再觀察到藍色。所有偶合皆進行得很好，且不需要重新偶合。 Weigh 6 g of the Fmoc-20mer-Wang resin (1.4 mmol) with a substitution degree of 0.999 mmol/g synthesized in Comparative Example 1, and swell with DMF for 30 minutes, and then follow the amino acid sequence of SEQ ID NO: 1 Sequential coupling, feed 2 to 3 equivalents of amino acid and perform condensation with DIC + A or B + C until the resin is transparent when detected by chloroquinone, where A is HOBt.H 2 O or (hydroxyimine group) Ethyl cyanoacetate; B is HATU or DEPBT; C is DIPEA; and the solvent is selected from DMF or DCM. All coupled amino acids are commercially available Fmoc monoamino acids, except that Val 10 -Ser 11 residues are coupled as pseudoproline double peptide Fmoc-Val-Ser(Ψ (Me,Me) pro)- Except for the OH residue; the last His is coupled as Boc-His(Boc)-OH.Except for the DCHA residue. Fmoc deprotection was achieved by complete removal of the alkaline reagent by DMF containing 20% (w/w) piperidine and subsequent DMF washes. Cleaning efficiency was assessed by the chloroquinone test and the cleaning was repeated until no more blue color was observed before coupling. All couplings went well and no recoupling was required.
在Lys側鏈偶合之前，Alloc保護基以含有0.2至0.3當量的Pd(PPh 3) 4及10當量的嗎啉的DCM移除1小時。在第二次以tBuOSteGlu(AEEA-AEEA)OtBu片段完成側鏈上剩餘胺基酸的偶合。使用5.1683 g的樹脂結合的胜肽，且在Alloc-去保護及偶合完成後獲得約5.5609 g的樹脂結合的胜肽。 Prior to Lys side chain coupling, the Alloc protecting group was removed in DCM containing 0.2 to 0.3 equivalents of Pd( PPh3 ) 4 and 10 equivalents of morpholine for 1 hour. In the second time, the coupling of the remaining amino acids on the side chain is completed with the tBuOSteGlu(AEEA-AEEA)OtBu fragment. 5.1683 g of resin-bound peptide was used, and approximately 5.5609 g of resin-bound peptide was obtained after Alloc-deprotection and coupling were completed.
取上述獲得的2.21 g的樹脂結合的胜肽，並添加至50 mL反應釜中。將17.84 mL的切割試劑按照TFA : EDT : PPW = 83 : 12.5 : 4.5之體積比率調配，並預冷至0至10℃，隨後倒入50 mL反應釜中，使得在室溫下反應2小時。在反應完成之後，濾除樹脂，並收集濾液。樹脂以少量TFA清洗，合併濾液，隨後冷卻至0至10℃。將冰冷的MTBE添加至濾液中，粗胜肽在0至10℃下沉澱、過濾、以MTBE清洗並真空乾燥，以獲得1.26 g的粗胜肽。粗胜肽以美國專利公開第20210206800號揭露的HPLC方法進一步純化，以獲得400 mg的最終胜肽索馬魯肽。Take 2.21 g of the resin-bound peptide obtained above and add it to the 50 mL reaction kettle. Prepare 17.84 mL of cutting reagent according to the volume ratio of TFA: EDT: PPW = 83: 12.5: 4.5, pre-cool to 0 to 10°C, then pour into a 50 mL reaction kettle, and allow to react at room temperature for 2 hours. After the reaction was completed, the resin was filtered off, and the filtrate was collected. The resin was washed with a small amount of TFA and the filtrates were combined and then cooled to 0 to 10°C. Ice-cold MTBE was added to the filtrate, and the crude peptide was precipitated at 0 to 10°C, filtered, washed with MTBE, and dried under vacuum to obtain 1.26 g of crude peptide. The crude peptide was further purified using the HPLC method disclosed in US Patent Publication No. 20210206800 to obtain 400 mg of the final peptide semaglutide.
實施例1Example 1
Fmoc-Gly-Wang樹脂之製備Preparation of Fmoc-Gly-Wang resin
稱取40 g的乾燥Wang樹脂（具有1.07 mmol/g之取代程度），並添加至SPPS反應器中。樹脂首先以DMF清洗兩次，再以DMF溶脹30分鐘，體積為樹脂床的2-3倍，再以DMF清洗3次。將38.18 g的Fmoc-Gly-OH（3.0當量）及19.667 g的HOBt.H 2O（3.0當量）溶解於DMF中。在胺基酸溶解之後，將溶液倒入SPPS反應器中，隨後添加20 ml的DIC（3.0當量）及1.5693 g的DMAP（0.3當量）。所得反應混合物在環境溫度下吹氮並攪拌16小時。在反應完成之後，添加適量的乙酸酐及吡啶混合溶液（體積比率：Ac 2O/DIPEA = 1/2），以阻斷反應30分鐘或以上，隨後以DMF清洗3次，以甲醇收縮2次，並減壓乾燥。在反應停止之後，獲得取代程度為1.061 mmol/g的Fmoc-Gly-Wang樹脂。 40 g of dry Wang resin (with a degree of substitution of 1.07 mmol/g) was weighed and added to the SPPS reactor. The resin is first washed twice with DMF, then swollen with DMF for 30 minutes, the volume is 2-3 times of the resin bed, and then washed three times with DMF. Dissolve 38.18 g of Fmoc-Gly-OH (3.0 equiv) and 19.667 g of HOBt.H 2 O (3.0 equiv) in DMF. After the amino acids were dissolved, the solution was poured into the SPPS reactor, followed by the addition of 20 ml of DIC (3.0 equiv) and 1.5693 g of DMAP (0.3 equiv). The resulting reaction mixture was purged with nitrogen and stirred at ambient temperature for 16 hours. After the reaction is completed, add an appropriate amount of acetic anhydride and pyridine mixed solution (volume ratio: Ac 2 O/DIPEA = 1/2) to block the reaction for 30 minutes or more, then wash with DMF 3 times and shrink with methanol 2 times , and dried under reduced pressure. After the reaction was stopped, Fmoc-Gly-Wang resin with a degree of substitution of 1.061 mmol/g was obtained.
胜肽之合成Synthesis of peptides
稱取具有1.061 mmol/g之取代程度的39.433 g的Fmoc-Gly-Wang樹脂（31.8 mmol），並以DMF溶脹30分鐘，隨後根據SEQ ID NO: 1之胺基酸序列依序偶合，以2至3當量的胺基酸進料並以DIC + A或B + C進行縮合，直至樹脂以氯醌檢測呈透明，其中A為HOBt.H 2O或（羥基亞胺基）氰基乙酸乙酯；B為HATU或DEPBT；C為DIPEA；且溶劑選自DMF。所有偶合的胺基酸皆為市售的Fmoc單胺基酸，除了Val 10-Ser 11殘基被偶合為偽脯胺酸雙胜肽Fmoc-Val-Ser(Ψ (Me,Me)pro)-OH殘基以外，並以DIC +（羥基亞胺基）氰基乙酸乙酯進行縮合；最後一個His 1-Aib 2被偶合為Boc-His(Trt)-Aib-OH殘基以外，並以DEPBT + DIPEA進行縮合。藉由含有20%（w/w）哌啶的DMF且隨後的DMF清洗來完全移除鹼試劑，達成Fmoc去保護。藉由氯醌測試評估清洗效率，重複清洗直至偶合前不再觀察到藍色。所有偶合皆進行得很好，且不需要重新偶合。 Weigh 39.433 g of Fmoc-Gly-Wang resin (31.8 mmol) with a substitution degree of 1.061 mmol/g, and swell with DMF for 30 minutes, and then sequentially couple according to the amino acid sequence of SEQ ID NO: 1, with 2 Feed 3 equivalents of amino acid and perform condensation with DIC + A or B + C until the resin is transparent when detected by chloroquinone, where A is HOBt.H 2 O or ethyl (hydroxyimino)cyanoacetate ; B is HATU or DEPBT; C is DIPEA; and the solvent is selected from DMF. All coupled amino acids are commercially available Fmoc monoamino acids, except that Val 10 -Ser 11 residues are coupled as pseudoproline double peptide Fmoc-Val-Ser(Ψ (Me,Me) pro)- Except for the OH residue, and condensed with DIC + (hydroxyimino) ethyl cyanoacetate; the last His 1 -Aib 2 was coupled to Boc-His(Trt)-Aib-OH residue, and condensed with DEPBT + DIPEA for condensation. Fmoc deprotection was achieved by complete removal of the alkaline reagent by DMF containing 20% (w/w) piperidine and subsequent DMF washes. Cleaning efficiency was assessed by the chloroquinone test and the cleaning was repeated until no more blue color was observed before coupling. All couplings went well and no recoupling was required.
在Lys側鏈偶合之前，Alloc保護基以含有0.05至0.1當量的Pd(PPh 3) 4及10當量的嗎啉的甲苯溶液移除6小時。在第二次以tBuOSteGlu(AEEA-AEEA)OtBu片段完成側鏈上剩餘胺基酸的偶合。在Alloc-去保護及偶合完成後獲得約26.11 g的樹脂結合的胜肽。 Prior to Lys side chain coupling, the Alloc protecting group was removed with a toluene solution containing 0.05 to 0.1 equivalents of Pd( PPh3 ) 4 and 10 equivalents of morpholine for 6 hours. In the second time, the coupling of the remaining amino acids on the side chain is completed with the tBuOSteGlu(AEEA-AEEA)OtBu fragment. After Alloc-deprotection and coupling were completed, approximately 26.11 g of resin-bound peptide was obtained.
將173 mL的切割試劑按照TFA : EDT : PPW = 90 : 13.6 : 5之體積比率添加至500 mL反應釜中，並預冷至0-10℃。取上述獲得的25.213 g的樹脂結合的胜肽，並緩慢添加至反應釜中，使得在0-10℃下反應6小時。在反應完成之後，濾除樹脂，並收集濾液。樹脂以少量TFA清洗，合併濾液，隨後冷卻至-30-0℃。將冰冷的MTBE添加至濾液中，粗胜肽在-30-0℃下沉澱、過濾、以MTBE清洗並真空乾燥，以獲得14.229 g的粗胜肽。粗胜肽以美國專利公開第20210206800號揭露的HPLC方法進一步純化，以獲得5.84 g的最終胜肽索馬魯肽。Add 173 mL of cutting reagent into the 500 mL reaction kettle at a volume ratio of TFA: EDT: PPW = 90: 13.6: 5, and pre-cool to 0-10°C. Take 25.213 g of the resin-bound peptide obtained above and slowly add it to the reaction kettle to react at 0-10°C for 6 hours. After the reaction was completed, the resin was filtered off, and the filtrate was collected. The resin was washed with a small amount of TFA, the filtrates were combined, and then cooled to -30-0°C. Ice-cold MTBE was added to the filtrate, and the crude peptide was precipitated at -30-0°C, filtered, washed with MTBE and dried under vacuum to obtain 14.229 g of crude peptide. The crude peptide was further purified using the HPLC method disclosed in US Patent Publication No. 20210206800 to obtain 5.84 g of the final peptide semaglutide.
實施例1及比較例1-3之分析結果總結於表3中。
表3.
基於比較實施例1及比較例1-3之分析結果，偽脯胺酸雙胜肽（諸如Fmoc-Val-Ser(Psi (Me,Me)pro)-OH）及非偽脯胺酸雙胜肽（諸如Boc-His(Trt)-Aib-OH）係一起用於索馬魯肽之合成中，具有協同效果，可顯著降低Des-Thr 5及Des-Aib 2雜質，並增加產率（轉化速率及估計總回收率）。同時，基於比較比較例2-3之分析結果，全側鏈方法優於依序偶合的側鏈方法。 Based on the analysis results of Comparative Example 1 and Comparative Examples 1-3, pseudo-proline bis-peptides (such as Fmoc-Val-Ser(Psi (Me,Me) pro)-OH) and non-pseudo-proline bis-peptides (such as Boc-His(Trt)-Aib-OH) are used together in the synthesis of semaglutide, which has a synergistic effect and can significantly reduce Des-Thr 5 and Des-Aib 2 impurities, and increase the yield (conversion rate and estimated total recovery rate). At the same time, based on the analysis results of Comparative Examples 2-3, the full side chain method is superior to the sequentially coupled side chain method.
實施例2Example 2
合成方法與實施例1中之描述類似。The synthesis method is similar to that described in Example 1.
實施例3Example 3
合成方法與實施例1中之描述類似，除了以Boc-His(Boc)-Aib-OH用於替代Boc-His(Trt)-Aib-OH以外。The synthesis method was similar to that described in Example 1, except that Boc-His(Boc)-Aib-OH was used instead of Boc-His(Trt)-Aib-OH.
實施例4Example 4
合成方法與實施例1中之描述類似，除了依序偶合側鏈以外，並與比較例3中之描述類似。The synthesis method was similar to that described in Example 1, except that the side chains were coupled sequentially, and similar to that described in Comparative Example 3.
實施例5Example 5
合成方法與實施例1中之描述類似，除了以Boc-His(Boc)-Aib-OH用於替代Boc-His(Trt)-Aib-OH以外，且依序偶合側鏈。The synthesis method was similar to that described in Example 1, except that Boc-His(Boc)-Aib-OH was used instead of Boc-His(Trt)-Aib-OH, and the side chains were coupled sequentially.
實施例2-5之分析結果總結於表4中。
表4.
基於比較來自實施例2-5之粗胜肽純度的分析結果，Boc-His(Trt)-Aib-OH在合成索馬魯肽過程中優於Boc-His(Boc)-Aib-OH，不論是全側鏈方法或依序偶合的側鏈方法。Based on the analysis results comparing the purity of the crude peptides from Examples 2-5, Boc-His(Trt)-Aib-OH is superior to Boc-His(Boc)-Aib-OH in the synthesis of semaglutide, regardless of Full side chain approach or sequentially coupled side chain approach.
實施例6Example 6
合成方法與實施例1中之描述類似。The synthesis method is similar to that described in Example 1.
實施例7Example 7
合成方法與實施例1中之描述類似，除了以HATU /DIPEA 用作偶合試劑以外。The synthesis method was similar to that described in Example 1, except that HATU/DIPEA was used as the coupling reagent.
實施例8Example 8
合成方法與實施例1中之描述類似，除了以DIC用作偶合試劑以外。The synthesis method was similar to that described in Example 1, except that DIC was used as the coupling reagent.
實施例6-8之分析結果總結於表5中。
表5.
基於比較來自實施例6-8之粗胜肽純度及轉化速率的分析結果，偶合試劑DEPBT/DIPEA在非偽脯胺酸雙胜肽Boc-His(Trt)-Aib-OH之偶合過程中優於HATU/DIPEA或DIC。Based on the analysis results comparing the purity and conversion rate of the crude peptides from Examples 6-8, the coupling reagent DEPBT/DIPEA is superior to the non-pseudoproline bis-peptide Boc-His(Trt)-Aib-OH in the coupling process HATU/DIPEA or DIC.
本發明不限於上述僅作為實例呈現的具體實施例，而是可在所附專利申請專利範圍所界定之保護範圍內以各種方式進行修改。The invention is not limited to the specific embodiments presented above merely as examples, but may be modified in various ways within the scope of protection defined by the appended patent application.
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