WO2021007701A1

WO2021007701A1 - A method for preparing liraglutide via a solid phase peptide synthesis

Info

Publication number: WO2021007701A1
Application number: PCT/CN2019/095781
Authority: WO
Inventors: Ivan Di BONAVENTURA; Runze HE
Original assignee: Shanghai Space Peptides Pharmaceutical Co Ltd
Current assignee: Shanghai Space Peptides Pharmaceutical Co Ltd
Priority date: 2019-07-12
Filing date: 2019-07-12
Publication date: 2021-01-21
Anticipated expiration: 2022-01-12

Abstract

Provided is a method for preparing Liraglutide via a solid phase peptide synthesis.

Description

A METHOD FOR PREPARING LIRAGLUTIDE VIA A SOLID PHASE PEPTIDE SYNTHESIS

TECHNIAL FIELD

The present invention pertains to the field of biomedicine and is directed to a green and environment friendly synthetic method of liraglutide. Specifically, the present invention is directed a method for preparing liraglutide via a solid phase peptide synthesis.

BACKGROUND OF THE INVENTION

Liraglutide is a long-acting, fatty acylated glucagon-like peptide-1 (GLP-1) analog administered subcutaneously, with antihyperglycemic activity. It is mainly used as a hypoglicemic agent and supplemental therapy in the treatment of DIABETES MELLITUS. Liraglutide's prolonged action and half-life of 11-15 hours are attributed to the attachment of the fatty acid palmitic acid to GLP-1 that reversibly binds to albumin. Albumin binding protects liraglutide from immediate degradation and elimination and causes GLP-1 to be released from albumin in a slow and consistent manner. As a widely used active pharmaceutical ingredient, Liraglutide is a derivative of human incretin glucagon-like peptide-1 (GLP-1) that is used as a long-acting glucagon-like peptide-1 receptor agonist.

The Active Pharmaceutical Ingredient (API) Liraglutide is a 33 amino acid peptide with a chemical formula of C ₁₇₂H ₂₆₅N ₄₃O ₅₁ and a molecular weight of 3751.262 g/mol. Liraglutide is also known as Victoza.

Solid Phase Peptide Synthesis (SPPS) is a fast method for polypeptides synthesis. However, conventional SPPS methods for producing large quantity of long peptides like Liraglutide are time-consuming, producing large amount of toxic solvents and are not able to be achieved in satisfying yield in large scale. Therefore those skilled in the art are committed to develop new Liraglutide synthetic methods, which are efficient and economical to allow the manufacturing of Liraglutide in large quantity and high purity.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a new method for preparing liraglutide.

According to the present invention, there is provided a method for preparing liraglutide by using a solid phase peptide synthesis (SPPS) , which comprises the following steps:

a) According to the peptide sequence of the main chain of liraglutide, successively coupling of Fmoc-Gly-resin with amino acids with N-ends protected by Fmoc and side chains protected by protective groups and the amino acids comprise a branching lysine, wherein the branching lysine is used in a form selected from the group consisting of Fmoc-Lys (Alloc) -OH, Alloc-Lys (Fmoc) -OH, Fmoc-Lys (Mtt) -OH, Mtt-Lysine (Fmoc) -OH, Fmoc-Lysine (Dde) -OH, Dde-Lys (Fmoc) -OH, Fmoc-Lys (ivDde) -OH and ivDde-Lys (Fmoc) -OH;

b) Removing Fmoc, Alloc, Mtt, ivDde and Dde from the side chain of the branching lysine;

c) Coupling the side chain or main chain of the branching lysine with Fmoc-Glu-OtBu and then palmitic acid;

d) Removing the protection groups and the resin via a cleavage reaction to obtain a crude liraglutide;

e) Purifying the crude liraglutide to obtain a purified liraglutide solution;

f) Lyophilizing the purified liraglutide solution to obtain a lyophilized Liraglutide powder.

According to a preferred embodiment of the invention, the Fmoc-Gly-resin is selected from the group consisting of Fmoc-Gly-wang resin, Fmoc-Gly-2-CTC resin, Fmoc-Gly-Rink amide ProTide (LL) resin and Fmoc-Gly-ChemMatrix wang resin.

According to a preferred embodiment of the invention, the coupling reagents in step a) are selected from the group consisting of ethyl 2-oxime cyanoacetate, N, N'diisopropylcarbodiimide, N, N'-dicyclohexylcarbodiimide, N, N-diisopropylethylamine, Benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, Dicyclohexylcarbodiimide, and O- (7-Azabenzotriazol-1-yl) -N, N, N', N`-Tetramethyluronium Hexafluorophosphate, and any combinations thereof.

According to a preferred embodiment of the invention, the Fmoc protective group of the Fmoc-Gly-resin in step a) is deprotected in the presence of a first mixture of piperidine or piperazine in DMF and γ-valerolactone for 10-20 minutes at 50℃-90℃ to obtain H-Gly-resin.

Further, in the above Fmoc deprotection reaction, a mixture of piperidine or piperazine in DMF and γ-valerolactone (preferably 1/4 volume of piperidine in γ-valerolactone and DMF) is added.

γ-valerolactone is environment friendly green organic solvent which is widely used in food industry. The combination reagents of the solvent mixture of γ-valerolactone and DMF are suitable for peptides stable at a high temperature such as 90℃. Otherwise the peptides are easy to be degraded under such a high temperature.

According to a preferred embodiment of the invention, the above first mixture has a volume ratio of piperidine or piperazine in DMF and γ-valerolactone of between 1: 3 to 1: 5, preferably 1: 4. Further, in the mixture of DMF and γ-valerolactone, DMF and γ-valerolactone can be mixed with any volume ratio, preferably with a ratio range from 1: 4 to 4: 1.

According to a preferred embodiment of the invention, the H-Gly-resin is further washed by a second mixture of DMF and γ-valerolactone.

According to a preferred embodiment of the invention, γ-valerolactone presents 60 to 90 v/v %of the second mixture of DMF and γ-valerolactone. In other words, in the present invention, γ-valerolactone is used to substitute 60%-90%of DMF, thereby reducing a large amount of DMF consumption in manufacture of liraglutide.

According to a preferred embodiment of the invention, the step a) comprises the following sub-steps:

a1) Deprotecting the Fmoc protective group of the Fmoc-Gly-resin in the presence of a mixture of piperidine or piperazine in DMF and γ-valerolactone for 10-20 minutes at 50℃-90℃ to obtain H-Gly-resin;

a2) Washing the H-Gly-resin obtained in step a1) by a mixture of DMF and γ-valerolactone; and a3) Successively coupling with the amino acids according to the peptide sequence of the main chain of liraglutide by repeating the steps a1) and a2) .

According to a preferred embodiment of the invention, after the completion of the deprotecting reaction of the Fmoc protective groups, each of the reactions in the solid phase peptide synthesis of the liraglutide is washed with the mixture of DMF and γ-valerolactone for 2-3 times.

According to a preferred embodiment of the invention, in step b) , the protective groups of Fmoc, Alloc, Mtt, ivDde and Dde are deprotected from the side chain of the branching lysine by washing with specific organic solvents. Specifically, the protective group Alloc of the side chain or the main chain of lysine is removed by washing with 0.1-0.4 fold of Pd (PPh ₃) ₄ and 10-30 folds of phenyl silane or borane dimethylamine under inert conditions for 45 min and repeated twice; the protective group Mtt of the side chain of the lysine is removed by washing with a solution of TFA/TIS/DCM (1: 2: 97) for 30 minutes, then the solution is filtered, washed with: DCM, MeOH, DCM, 1%DIEA in DMF, and DMF. This method is repeated three times totally. The protective group ivDde of the side chain of the lysine is removed by washing with a solution of 10%hydrazine monohydrate in DMF for 10 minutes and repeated three times with DMF. The protective group Dde on the main chain of the lysine is removed by washing with a solution of 2%of hydrazine monohydrate in DMF for 3 minutes and this method is repeated 3 times.

According to a preferred embodiment of the invention, the coupling reaction of the amino acid in the solid phase peptide synthesis of the liraglutide on the resin is carried out at a temperature of 50 to 90℃ for 10 to 30 minutes depending on the temperature.

According to a preferred embodiment of the invention, the resin is blown off with nitrogen before the cleavage reaction of step d) is carried out.

According to a preferred embodiment of the invention, the cleavage reaction of step d) is carried out at a temperature between 10 to 50℃ for 2-3 hours. Further, the purity of the trifluoroacetic acid added in the reaction is 95%.

According to a preferred embodiment of the invention, in the coupling reaction of the amino acid in the solid phase peptide synthesis of the liraglutide on the resin, the equivalents of the amino acids used are 2-3 times of the molar amount of the resin.

According to a preferred embodiment of the invention, in the step e) , the crude liraglutide is purified by Pre-HPLC with a specific buffer, preferably acetonitrile and water with sodium dihydrogen phosphate buffer.

According to a preferred embodiment of the invention, in the step f) , the purified Liraglutide solution is lyophilized at -50℃ to -70℃ for 18-48 hours.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic of liraglutide molecular structure.

Figure 2 is an HPLC analysis of Liraglutide prepared in Example 1.

Figure 3 is a Mass spectrum of Liraglutide prepared in Example 1.

Figure 4 is an HPLC analysis of Liraglutide prepared in Example 2.

Figure 5 is an HPLC analysis of Liraglutide prepared in Example 3.

Figure 6 is an HPLC analysis of Liraglutide prepared in Example 4.

Figure 7 is an HPLC analysis of Liraglutide prepared in Example 5.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for producing Liraglutide and is directed to the synthetic method of Liraglutide by solid phase peptide synthesis using Fmoc-Gly-Wang resin as a carrier. The invention has the advantages of high yield, less by-product formation and simple separation and purification, and it is suitable for pilot and industrial production.

Liraglutide shown in Figure 1 is 33 amino acids synthetic peptide. The invention will now be described with reference to specific embodiments. It must be understood that these examples are merely illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise stated, the percentages are by volume. The experimental materials and reagents used in the examples below are available from commercially available sources unless otherwise specified.

Table 1: List of amino acid abbreviations

Table 2: List of chemical reagent abbreviations

Table 3: List of intermediates and Fmoc protected amino acids

The present invention provides a synthetic method of Liraglutide using Fmoc-Gly-wang resin by solid phase peptide synthesis comprising the following steps of:

Step 1: Fluorenylmethoxycarbonyl-Glycine-Wang resin can be purchased directly from SigmaAldrich;

Step 2: The resin is swollen for 5 to 15 minutes under nitrogen in a mixture of DMF and γ-valerolactone;

Step 3: Fluorenylmethoxycarbonyl chloride protecting group is removed under the following conditions: 20%of piperidine in DMF and γ-valerolactone mixture for 10 to 20 minutes at 50℃-90℃.

Step 4: Preparation of fluorenylmethoxycarbonyl-Arginine (Pbf) -Glycine-Wang resin: the fluorenylmethoxycarbonyl-glycine-Wang resin obtained in step 1 is deprotected, washed with the mixture of DMF and γ-valerolactone and Fmoc-L-Arg- (Pbf) -OH is subjected to a condensation reaction in the presence of the polypeptide coupling reagent to give fluorenylmethoxycarbonyl-Arginine (Pbf) -glycine-Wang resin;

Step 5: Preparation of fluorenylmethoxycarbonyl-Glycine-Arginine (Pbf) -Glycine-Wang resin: The Fmoc-dipeptide obtained in step 4 is deprotected and washed, and then reacted with Fmoc-Glycine-OH in the presence of the peptide coupling reagent to give fluorenylmethoxycarbonyl-glycine-Arginine (Pbf) -glycine-Wang resin;

Step 6: Preparation of fluorenylmethoxycarbonyl-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. The resulting fluorenylmethoxycarbonyl-Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and Fmoc-L-Arginine (Pbf) -OH is added and then reacted in the presence of the peptide coupling reagent to give fluorenylmethoxycarbonyl-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 7: Preparation of fluorenylmethoxycarbonyl-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Fluorenylmethoxycarbonyl-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and then reacted with fluorenylmethoxycarbonyl-valine-OH in the presence of the polypeptide coupling reagent to obtain fluorenylmethoxycarbonyl-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin.

Step 8: Preparation of fluorenylmethoxycarbonyl-Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Fluorenylmethoxycarbonyl-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-leucine-OH is added in the presence of the peptide coupling reagent to give fluorenylmethoxycarbonyl-Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 9: Preparation of fluorenylmethoxycarbonyl-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. To fluorenylmethoxycarbonyl-Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin obtained in step 8, is deprotected, washed and Fmoc-L-Trp (Boc) -OH is added and reacted in the presence of a peptide coupling reagent conditions to give fluorenylmethoxycarbonyl-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 10: Preparation of fluorenylmethoxycarbonyl-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. To fluorenylmethoxycarbonyl-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin obtained in step 9 Fmoc-L-Alanine-OH is added and subjected to a condensation reaction in the presence of a polypeptide coupling reagent to give fluorenylmethoxycarbonyl-Alanine-Tryptophane (Boc) - Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 11: Preparation of fluorenylmethoxycarbonyl-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. The peptide obtained in step 10 fluorenylmethoxycarbonyl-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin was deprotected, washed and reacted with fluorenylmethoxycarbonyl-Isoleucine-OH in the presence of the peptide coupling reagent to give fluorenylmethoxycarbonyl-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 12: Preparation of fluorenylmethoxycarbonyl-phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. The peptide obtained in step 11 is deprotected, washed and treated with fluorenylmethoxycarbonyl-Phenilalanine-OH to give fluorenylmethoxycarbonyl-phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 13: Preparation of fluorenylmethoxycarbonyl-glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. The peptide obtained in step 12 is deprotected, washed and treated with fluorenylmethoxycarbonyl-Glu (OtBu) -OH in the presence of a polypeptide coupling reagent to give fluorenylmethoxycarbonyl-glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 14: Preparation of Alloc-Lysine (fluorenylmethoxycarbonyl) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. The peptide obtained in step 13 is deprotected, washed and treated with Alloc-Lysine (fluorenylmethoxycarbonyl) -OH in the presence of the peptide coupling reagent to give Alloc-Lysine (fluorenylmethoxycarbonyl) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

According to the present invention, the Alloc-Lysine (Fmoc) -OH can be replaced with Fmoc-Lys (Alloc) -OH, Fmoc-Lys (Mtt) -OH, Mtt-Lysine (Fmoc) -OH, Fmoc-Lysine (Dde) -OH, Dde-Lys (Fmoc) -OH and Fmoc-Lys (ivDde) -OH, so as to obtain side chains of lysine having different protecting groups.

Step 15: Preparation of Alloc-Lysine (fluorenylmethoxycarbonyl-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. The peptide obtained in step 14 is deprotected, washed and subjected to a condensation reaction with fluorenylmethoxycarbonyl-glutamic acid (OtBu) under conditions of a polypeptide coupling reagent to give Alloc-Lysine (fluorenylmethoxycarbonyl-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 16: Preparation of Alloc-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Alloc-Lysine (fluorenylmethoxycarbonyl-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and Palmitic acid was added in the presence of the peptide coupling reagent to give Alloc-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 17: Preparation of NH2-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Alloc-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is washed and subjected to a Alloc deprotection reaction to generate a free amine group on the branching lysine.

Step 18: Preparation of fluorenylmethoxycarbonyl-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine- Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. NH2-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is washed and subjected to a condensation reaction with fluorenylmethoxycarbonyl-alanine-OH in the presence of a polypeptide coupling reagent to give fluorenylmethoxycarbonyl-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 19: Preparation of fluorenylmethoxycarbonyl-alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Fluorenylmethoxycarbonyl-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-alanine-OH is added in a polypeptide coupling reagent conditions, to give fluorenylmethoxycarbonyl-alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 20: Preparation of fluorenylmethoxycarbonyl-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Fluorenylmethoxycarbonyl-alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-Gln (Trt) -OH is added in a peptide coupling reagent conditions, to give fluorenylmethoxycarbonyl-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 21: Preparation of fluorenylmethoxycarbonyl-Gly-Gln (Trt) -alanine-alanine- Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Fluorenylmethoxycarbonyl-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-glycine-OH is added in a peptide coupling reagent conditions, to give fluorenylmethoxycarbonyl-Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 22: Preparation of fluorenylmethoxycarbonyl-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Fluorenylmethoxycarbonyl-Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-Glu (OtBu) -OH is added in a peptide coupling reagent conditions, to give fluorenylmethoxycarbonyl-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 23: Preparation of Fluorenylmethoxycarbonyl-Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Fluorenylmethoxycarbonyl-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-Leu-OH is added in a peptide coupling reagent conditions, to give Fluorenylmethoxycarbonyl-Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine- Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 24: Preparation of Fluorenylmethoxycarbonyl-Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Fluorenylmethoxycarbonyl-Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-Tyr (tBu) -OH is added in a peptide coupling reagent conditions, to give Fluorenylmethoxycarbonyl-Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 25: Preparation of Fluorenylmethoxycarbonyl-Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Fluorenylmethoxycarbonyl-Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-Ser (tBu) -OH is added in a peptide coupling reagent conditions, to give Fluorenylmethoxycarbonyl-Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 26: Preparation of Fluorenylmethoxycarbonyl-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Fluorenylmethoxycarbonyl-Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine- Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-Ser (tBu) -OH is added in a peptide coupling reagent conditions, to give Fluorenylmethoxycarbonyl-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 27: Preparation of Fluorenylmethoxycarbonyl-Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Fluorenylmethoxycarbonyl-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-Val-OH is added in a peptide coupling reagent conditions, to give Fluorenylmethoxycarbonyl-Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 28: Preparation of Fluorenylmethoxycarbonyl-Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Fluorenylmethoxycarbonyl-Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-Asp (OtBu) -OH is added in a peptide coupling reagent conditions, to give Fluorenylmethoxycarbonyl-Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine- Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 29: Preparation of Fluorenylmethoxycarbonyl-Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Fluorenylmethoxycarbonyl-Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-Ser (tBu) -OH is added in a peptide coupling reagent conditions, to give Fluorenylmethoxycarbonyl-Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 30: Preparation of Fluorenylmethoxycarbonyl-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Fluorenylmethoxycarbonyl-Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-Thr (tBu) -OH is added in a peptide coupling reagent conditions, to give Fluorenylmethoxycarbonyl-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 31: Preparation of Fluorenylmethoxycarbonyl-Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine- Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Fluorenylmethoxycarbonyl-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-Phe-OH is added in a peptide coupling reagent conditions, to give Fluorenylmethoxycarbonyl-Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 32: Preparation of Fluorenylmethoxycarbonyl-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Fluorenylmethoxycarbonyl-Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-Thr (tBu) -OH is added in a peptide coupling reagent conditions, to give Fluorenylmethoxycarbonyl-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 33: Preparation of Fluorenylmethoxycarbonyl-Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Fluorenylmethoxycarbonyl-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-Gly-OH is added in a peptide coupling reagent conditions, to give Fluorenylmethoxycarbonyl-Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 34: Preparation of Fluorenylmethoxycarbonyl-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Se r (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Fluorenylmethoxycarbonyl-Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-Glu (OtBu) -OH is added in a peptide coupling reagent conditions, to give Fluorenylmethoxycarbonyl-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Se r (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 35: Preparation of Fluorenylmethoxycarbonyl-Ala-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Va l-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Fluorenylmethoxycarbonyl-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Se r (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-Ala-OH is added in a peptide coupling reagent conditions, to give Fluorenylmethoxycarbonyl-Ala-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Va l-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 36: Preparation of Fluorenylmethoxycarbonyl-His (Trt) -Ala-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin. Fluorenylmethoxycarbonyl-Ala-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Va l-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-His (Trt) -OH is added in a peptide coupling reagent conditions, to give Fluorenylmethoxycarbonyl-His (Trt) -Ala-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -alanine-alanine-Lysine (Palmitic acid-Glutamic acid (Otbu) ) -glutamic acid (OtBu) -phenylalanine-Isoleucine-Alanine-Tryptophane (Boc) -Leucine-Valine-Arginine (Pbf) -Glycine-Arginine (Pbf) -Glycine-Wang resin;

Step 37: The peptide anchored to the solid support is cleaved with a mixture of TFA/H ₂O/TIS 95: 2.5: 2.5. The cleavage solution is precipitated with diethyl ether and centrifuged at 3500-5000 rpm to give a white precipitate. The precipitate is washed and centrifuged with diethyl ether for another three times to give a final precipitate;

Step 38: The crude Liraglutide crude is purified by Preparative-HPLC;

Step 39: The purified Liraglutide solution is lyophilized by lyophilizer at a temperature between -50℃ to -70℃ for 18-48 hours to give prepared liraglutide more than 98%purity.

ADVANTAGES OF THE PRESENT IINVENTION

1. The conventional method of SPPS for the synthesis of Liraglutide API produces commonly large amounts of by-products difficult to separate and large amount of solvent waste that is expensive to dispose. The present method produces less amount of hazardous solvent waste and allows obtaining Liraglutide with a high yield.

2. The conventional method of SPPS for the synthesis of liraglutide is generally carried out under a lower temperature of 20 to 30℃. The present method is carried out under a higher temperature of 50 to 90℃ and thus has a faster reaction rate than the conventional SPPS process.

3. Unlike the conventional method of SPPS for the synthesis of liraglutide, the present method is a one-pot method and is continuously carried out in a reactor.

EXAMPLES

Example 1: Synthesis of Liraglutide using Fmoc-Gly-wang resin with a loading degree of 0.47 mmol/g and Alloc-Lysine (Fmoc) -OH as a branching lysine.

The synthesis scale was 100 mmol and 250 mmol amino acids were used.

400G of Fmoc-Gly-wang resin with a substitution degree of 0.47mmol/g was weighted and added to the solid-phase reactor. Subsequently, the resin was swelled for 50 minutes with a mixture of DMF and γ-valerolactone and Fmoc deprotection was performed by a mixture of piperidine/DMF and γ-valerolactone (DMF and γ-valerolactone were mixed in a volume ratio of 1: 4) at 90℃ for 10 minute and washed twice with a 5L mixture of DMF and γ-valerolactone (mixed in a volume ratio of 1: 4) . Fmoc-Arg (Pbf) -OH, coupling reagent mixture were added in the reactor. The above Fmoc de-protection step was repeated as mentioned above and corresponding amino acid couplings were performed twice at 90℃ for 10 minutes. The following amino acids were used: Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Alloc-Lys (Fmoc) -OH. Fmoc deprotection was performed as mentioned above and Fmoc-Glu-OtBu and palmitic acid were coupled one by one to the lysine. For the palmitic acid a tetra coupling performed at 75℃ was chosen. After this first branch, Alloc deprotection was performed as described before and Liraglutide backbone was grown to the main chain of the lysine with the following amino acids: Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH and Fmoc-His (Trt) -OH were sequentially coupled at 90℃ with a double coupling. The linear liraglutide wang resin was washed for 3 times by DMF and γ-valerolactone mixture for subsequent synthesis reaction. The peptide was cleaved from the solid support with a mixture of TFA/TIS/H ₂O 95: 2.5: 2.5 for 3 hours and precipitated in cold TBME. The precipitate was washed with TBME for 5 times and centrifuged with a speed of 3500 rpm for 10 minutes each centrifugation. The precipitate was dried under vacuum and dissolved in water/acetonitrile at pH 7.8. The crude yield was analyzed by LC-MS with a final yield of 67%. The crude Liraglutide crude is purified by Preparative-HPLC; then the purified Liraglutide solution is lyophilized by lyophilizer at a temperature of -50℃ for 18 hours to give prepared liraglutide having a high purity, as shown in Figure 2.

EXAMPLE 2: Synthesis of Liraglutide using Fmoc-Gly-2-CTC Resin with a Substitution Degree of 0.47 mmol/g and Dde-Lys (Fmoc) -OH as a branching lysine.

The synthesis scale was 100 mmol and 250 mmol amino acids were used.

400 G of Fmoc-Gly-2-CTC resin with a substitution degree of 0.47mmol/g was swelled for 50 minutes with DMF and γ-valerolactone and Fmoc deprotection was performed by a mixture of piperidine/DMF and γ-valerolactone (DMF and γ-valerolactone were mixed in a volume ratio of 1: 4) at 90℃ for 10 minute and washed twice with a 5L mixture of DMF and γ-valerolactone (mixed in a volume ratio of 1: 4) . Fmoc-Arg (Pbf) -OH, coupling reagents were added to the reactor vessel and let to react. The above Fmoc de-protection steps were repeated as mentioned above and corresponding amino acid coupling were performed twice at 90℃ for 2 minutes. The following amino acids were used: Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Dde-Lys (Fmoc) -OH. Fmoc deprotection was performed as mentioned above and Fmoc-Glu-OtBu and palmitic acid were coupled one by one to the lysine. For the palmitic acid a tetra coupling performed at 75℃ was chosen. After this first branch, Dde deportection was performed with a solution of 2%of hydrazine monohydrate in DMF and this method was repeated 3 times.

Liraglutide backbone was grown to the main chain of the lysine with the following amino acids: Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH and Fmoc-His (Trt) -OH were sequentially coupled at 90℃ with a double coupling. The linear Liraglutide resin was washed for 3 times by DMF and γ-valerolactone mixture for subsequent synthesis reaction. The peptide was cleaved from the solid support with a mixture of TFA/TIS/H ₂O 95: 2.5: 2.5 for 3 hours and precipitated in cold TBME. The precipitate was washed with TBME for 5 times and centrifuged with a speed of 3500 rpm for 10 minutes each centrifugation. The precipitate was washed with TBME for 5 times and centrifuged with a speed of 3500 rpm for 10 minutes each centrifugation. The precipitate was dried under vacuum and dissolved at pH 7.8. The crude yield was analyzed by LC-MS with a final yield of 71%. The crude Liraglutide crude is purified by Preparative-HPLC; then the purified Liraglutide solution is lyophilized by lyophilizer at a temperature of -70℃ for 48 hours to give prepared liraglutide having a high purity, as shown in Figure 4.

EXAMPLE 3. Synthesis of Liraglutide using Fmoc-Gly-wang resin Resin with a Substitution Degree of 0.47 mmol/g and Fmoc-Lysine (Mtt) -OH as a branching lysine.

The synthesis scale was 100 mmol and 250 mmol amino acids were used.

400 G of Fmoc-Gly-wang resin with a substitution degree of 0.47mmol/g was swelled as mentioned above and Fmoc deprotection was performed with a mixture mentioned in previous examples Fmoc-Arg (Pbf) -OH and coupling reagents were added to the reactor vessel and reacted at as mentioned above. The above Fmoc de-protection steps were repeated as mentioned above and corresponding amino acid coupling were coupled to the resin. The following amino acids were used: Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Mtt) -OH. Fmoc deprotection was performed as mentioned above and Liraglutide backbone was grown to the main chain of the lysine with the following amino acids: Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH and Fmoc-His (Trt) -OH were sequentially coupled with a double coupling. Mtt was deprotected with a mixture of TFA/TIS/DCM and Fmoc-Glu-OtBu and palmitic acid were coupled one by one to the lysine. The peptide was cleaved from the solid support with a mixture of TFA/TIS/H ₂O 95: 2.5: 2.5 for 3 hours and precipitated in cold TBME. The precipitate was washed with TBME for 5 times and centrifuged with a speed of 3500 rpm for 10 minutes each centrifugation. The precipitate was dried under vacuum and dissolved at pH 7.8. The crude yield was analysed by LC-MS with a final yield of 51%. The crude Liraglutide crude is purified by Preparative-HPLC; then the purified Liraglutide solution is lyophilized by lyophilizer at a temperature of -60℃ for 30 hours to give prepared liraglutide having a high purity, as shown in Figure 5.

EXAMPLE 4. Synthesis of Liraglutide using Fmoc-Gly-wang resin Resin with a Substitution Degree of 0.47 mmol/g and Fmoc-Lysine (ivDde) -OH as a branching lysine.

The synthesis scale was 100 mmol and 250 mmol amino acids were used.

400 G of Fmoc-Gly-wang resin with a substitution degree of 0.47mmol/g was swelled and Fmoc deprotection was performed as mentioned above. Fmoc-Arg (Pbf) -OH and coupling reagents were let to react with the same procedure described above. The above Fmoc de-protection steps were repeated as mentioned above and corresponding amino acid coupling were performed twice at. The following amino acids were used: Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (ivDde) -OH. Fmoc deprotection was performed as mentioned above and Liraglutide backbone was grown to the main chain of the lysine with the following amino acids: Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH and Fmoc-His (Trt) -OH were sequentially coupled at 90℃ with a double coupling. IvDde was deprotected as mentioned above and Fmoc-Glu-OtBu and palmitic acid were coupled one by one to the lysine. The linear Liraglutide wang resin was washed for 3 times. The peptide was cleaved from the solid support with a mixture of TFA/TIS/H ₂O 95: 2.5: 2.5 for 3 hours and precipitated in cold TBME. The precipitate was washed with TBME for 5 times and centrifuged with a speed of 3500 rpm for 10 minutes each centrifugation. The precipitate was dried under vacuum and dissolved at pH 7.8. The crude yield was analyzed by LC-MS with a final yield of 45%. The crude Liraglutide crude is purified by Preparative-HPLC; then the purified Liraglutide solution is lyophilized by lyophilizer at a temperature of -55℃ for 24 hours to give prepared liraglutide having a high purity, as shown in Figure 6.

EXAMPLE 5. Synthesis of Liraglutide using Fmoc-Gly-wang resin Resin with a loading Degree of 0.47 mmol/g and Dde-Lysine (Fmoc) -OH as a branching lysine.

The synthesis scale was 100 mmol and 250 mmol amino acids were used.

400 G of Fmoc-Gly-wang resing with a substitution degree of 0.47mmol/g was weighted and was swelled and Fmoc deprotection was performed as mentioned above. Fmoc-Arg (Pbf) -OH and coupling reagents were added to the reactor vessel and let to react. The above Fmoc de-protection steps were repeated as mentioned above and corresponding amino acid coupling were performed twice at 90℃. The following amino acids were used: Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Dde-Lys (Fmoc) -OH. Fmoc deprotection was performed as mentioned above and Fmoc-Glu-OtBu and palmitic acid were coupled one by one to the lysine. After this first branch, Dde deportection was performed with a solution of 5%of hydrazine monohydrate in DMF f and this method was repeated 3 times.

Liraglutide backbone was grown to the main chain of the lysine with the following amino acids: Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH and Fmoc-His (Trt) -OH were sequentially coupled at 90℃ with a double coupling. The linear Liraglutide wang resin was washed for 3 times with the same mixture used above. The peptide was cleaved from the solid support with a mixture of TFA/TIS/H ₂O 95: 2.5: 2.5 for 3 hours and precipitated in cold TBME. The precipitate was washed with TBME for 5 times and centrifuged. The precipitate was dried under vacuum and dissolved at pH 7.8. The crude yield was analyzed by LC-MS with a final yield of 73%. The crude Liraglutide crude is purified by Preparative-HPLC; then the purified Liraglutide solution is lyophilized by lyophilizer at a temperature of -65℃ for 36 hours to give prepared liraglutide having a high purity, as shown in Figure 7.

Claims

A method for preparing Liraglutide via a solid phase peptide synthesis, comprising the following steps:

a) According to the peptide sequence of the main chain of Liraglutide, successively coupling a Fmoc-Gly-resin with amino acids with N-ends protected by Fmoc and side chains protected by protective groups in the presence of at least one coupling reagents, and the amino acids comprise a branching lysine, wherein the branching lysine is used in a form selected from the group consisting of Fmoc-Lys (Alloc) -OH, Alloc-Lys (Fmoc) -OH, Fmoc-Lys (Mtt) -OH, Mtt-Lysine (Fmoc) -OH, Fmoc-Lys (Dde) -OH, Dde-Lys (Fmoc) -OH, Fmoc-Lys (ivDde) -OH and ivDde-Lys (Fmoc) -OH;

b) Removing Fmoc, Alloc, Mtt, ivDde and Dde from the side chain of the branching lysine;

c) Coupling the side chain or main chain of the branching lysine with Fmoc-Glu-OtBu and then palmitic acid;

d) Removing the protection groups and the resin via a cleavage reaction to obtain a crude liraglutide;

e) Purifying the crude Liraglutide to obtain a purified liraglutide solution; and

f) Lyophilizing the purified Liraglutide solution to obtain a lyophilized Liraglutide.
The method according to claim 1, wherein the Fmoc-Gly-resin is selected from the group consisting of Fmoc-Gly-wang resin, Fmoc-Gly-2-CTC resin, Fmoc-Gly-Rink amide ProTide (LL) resin and Fmoc-Gly-ChemMatrix wang resin.
The method according to claim 1, wherein the coupling reagents in step a) are selected from the group consisting of ethyl 2-oxime cyanoacetate, N, N'diisopropylcarbodiimide, N, N'-dicyclohexylcarbodiimide, N, N-diisopropylethylamine, Benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, Dicyclohexylcarbodiimide, and O- (7-Azabenzotriazol-1-yl) -N, N, N', N`-Tetramethyluronium Hexafluorophosphate, and any combinations thereof.
The method according to claim 1, wherein the Fmoc protective group of the Fmoc-Gly-resin in step

a) is deprotected in the presence of a mixture of piperidine or piperazine in DMF and γ-valerolactone to obtain H-Gly-resin.
The method according to claim 4, wherein the H-Gly-resin is further washed by a mixture of DMF and γ-valerolactone.
The method according to claim 5, wherein γ-valerolactone presents 60 to 90 v/v %of the mixture of DMF and γ-valerolactone.
The method according to claim 1, wherein the step a) comprises the following sub-steps:

a1) Deprotecting the Fmoc protective group of the Fmoc-Gly-resin in the presence of a mixture of piperidine or piperazine in DMF and γ-valerolactone;

a2) Washing the H-Gly-resin obtained in step a1) by a mixture of DMF and γ-valerolactone;

a3) Successively coupling with the amino acids according to the peptide sequence of the main chain of Liraglutide by repeating the steps a1) and a2) .
The method according to claim 7, after the completion of the deprotecting reaction of the Fmoc protective groups, each of the reactions in the solid phase peptide synthesis of the Liraglutide is washed with the mixture of DMF and γ-valerolactone for 2-3 times.
The method according to claim 1, wherein in step b) , the protective groups of Fmoc, Alloc, Mtt, ivDde and Dde are deprotected from the side chain of the branching lysine by washing with organic solvents.
The method according to claim 1, wherein the coupling of the amino acids in the solid phase peptide synthesis of the Liraglutide on the resin is carried out at a temperature of 50 to 90℃ for 10 to 30 minutes depending on the temperature.
The method according to claim 1, wherein the cleavage reaction of step d) is carried out at a temperature between 10 to 50℃ for 2-3 hours.
The method according to claim 1, wherein in the coupling of the amino acid in the solid phase peptide synthesis of the liraglutide on the resin, the equivalents of the amino acids used are 2-3 times of the molar amount of the resin.
The method according to claim 1, wherein in the step f) , the purified Liraglutide solution is lyophilized at -50 to -70℃ for 18-48 hours.