WO2023105497A1

WO2023105497A1 - Synthesis of glp-1 analogues

Info

Publication number: WO2023105497A1
Application number: PCT/IB2022/062039
Authority: WO
Inventors: Atul Kasturchand Godha; Sathya Vanganur NARAYANASWAMY; Areti Venkata Bhagyaraj; Aslam Jahangir Bavadekar; Rudresh LAKSHMANAPPA; Sheiksyedali NAGOOR; Ranjithkumar MURUGAN; Amol Vasantrao Gadakh; Ganesh Sambasivam
Original assignee: Anthem Biosciences Pvt Ltd
Current assignee: Anthem Biosciences Pvt Ltd
Priority date: 2021-12-10
Filing date: 2022-12-12
Publication date: 2023-06-15
Anticipated expiration: 2024-06-10

Abstract

The present invention provides a method of preparation of GLP-1 peptides by using different peptide fragments having amino acid sequences SEQ ID NO:1 (Fragment IG), SEQ ID NO:2 (Fragment SG), Fragment BG, SEQ ID NO:3, and SEQ ID NO:4 5 and combinations thereof. The method of preparation is environmentally benign, simple, straightforward, and efficient and provides the end product in high yield and purity. The purification method of the peptides is also described.

Description

SYNTHESIS OF GLP-1 ANALOGUES

FIELD OF INVENTION:

The present invention relates generally to field of synthetic organic chemistry and preparation of peptides of pharmaceutical significance. More particularly, the disclosure provides synthesis method of glucagon like peptides via fragment approach and sequential amino acids couplings.

BACKGROUND OF THE INVENTION

Glucagon-like peptide- 1 is a naturally occurring 30 or 31 amino acid long peptide hormone deriving from the tissue-specific posttranslational processing of the proglucagon peptide, which stimulates insulin release and decreases the level of the anti-insulin hormone glucagon in response to increases in blood sugar levels. Glucagon-like peptide- 1 is typically produced by yeast through recombinant gene technology.

Commonly known GLP- 1 analogues include Exenatide, Liraglutide, Lixisenatide, Albiglutide, Dulaglutide, Semaglutide and Taspoglutide.

The synthesis strategies adopted in the prior art for the glucagon-like peptide- 1 peptide includes different approaches such as uses of different/multiple resins, coupling reagents, fragments, different protecting group for amino acids.

Liraglutide is an analogue of glucagon-like peptide- 1 containing a backbone of 31 amino acids, wherein the Lys-20 is condensed with Glu-Pal group. Liraglutide is produced by covalently linking glucagon-like peptide- 1 to a fatty acid. It is a once- daily injectable and has the effects of lowering blood sugar level, reducing body weight, promoting islet cell regeneration, as well as protecting cardiovascular system.

Semaglutide is a next generation glucagon-like peptide- 1 analogue as a once- weekly injection. Semaglutide shares a similar backbone to Liraglutide, with Ala2 being substituted by Aib, and Lys20 is side modification moiety derivatized with N-(17-carboxy-loxoheptadecyl)-L-y-glutamyl-2-[2-(2-aminoethoxy] ethoxy] acetyl-2-[2-(2-aminoethoxy) ethoxy] acetyl.

In the solid phase synthesis of GLP-1 peptides, conventional coupling reagent and additives such as ethyl cyano (hydroxyimino) acetate (Oxyma)/Diisopropylcarbodiimide (DIC) and HOBt anhydrous play a versatile role but, HOBt anhydrous is explosive in nature. However, it is an efficient combination for amide coupling in peptide synthesis. Nevertheless, some articles recently were published mentioning evolution of very lethal hydrogen cyanide (HCN) during amino acid activation under ambient conditions (20 °C) in N, N- dimethylformamide (DMF) (Org. Process Res. Dev. 2019, 23, 2099-2105 and Org. Process Res. Dev. 2020, 24, 7, 1341-1349). Concentration versus time profiles obtained by 1H NMR spectroscopy in the presence and absence of amino acids indicate that HCN is formed upon addition of DIC to the reaction mixture and that HCN evolution continues to occur even after amino acid activation is complete when Oxyma and DIC are used in excess amounts relative to the amino acid.

Due to massive demand of these peptides in the market, it is very crucial to develop methods that provides more efficient industrially scalable method with reduced time cycle, easy purification and method which avoids the use of toxic or otherwise undesirable reagents.

In W02006097537A2 synthesis of Semaglutide is reported wherein coupling of linear peptide backbone with linker (branched moiety) using Rink acid, Trityl resin and Wang Resin and for branching purpose Fmoc-Lys(Mmt)-OH, Fmoc-Lys(Mtt)- OH, Fmoc-Lys(Dde)-OH, FmocLys(ivDde)-OH is provided. After deprotection of 8 protecting group of different Lysine, branching is carried out with different kind of activated chains of linkers such as anhydride, OSu and amide coupling.

CN106478806A provides uses of Dde-Lys(Fmoc)-OH for branching after sequential synthesis of peptide backbone. After deprotection of -Fmoc protecting group of Lysine branching is carried out, Dde of lysine is de -protected with hydrazine hydrate in DMF and further amino acids are coupled.

CN105753964A describes uses of different resins for linear synthesis such as Cl- trityl resin,

HMPB-AM resin, Wang Resin in combination with Dde-Lys(Fmoc)-OH, Fmoc- Lys(Alloc)OH as branching points on the linear backbone. For branching, methyl ester of linker is synthesized using resin. Also, dipeptide Ala-Ala and Ser-Ser fragments are used to avoid defective peptide formation. Methyl ester of linker is hydrolysed by LiOH to afford pure peptide.

CN103848910A describes sequential synthesis of backbone peptide which includes Fmoc-Lys(Mmt)-OH and Boc-His-OH for branching.

WO 2019/069274 Al provides uses of different fragments (1-19) with a fragment (20-31), or a fragment (1-18) with a fragment (19-31). Another preferred coupling of fragment (1-17) with a fragment (18-31). In addition, with carboxyl terminal amino acid of the first fragment is a Leu residue coupling a fragment (1-14) with a fragment (15-31).

US 2021/0009631 Al discloses use of Alloc-Lys(Fmoc)-OH forbranching coupling followed by sequential synthesis of peptide backbone. After deprotection of-Fmoc protecting group of Lysine branching is carried out and Alloc of lysine is deprotected with Pd(PPh3)4 in DMF and further amino acids are coupled.

In the step-by-step solid-phase synthesis of GLP-1 analogues, Liraglutide and Semaglutide, in the above referred patent applications, are known to contain positions in their sequences where the coupling and deprotection steps become difficult. This results in the formation of several undesirable by products which lower the yield and make purification more difficult. One particular difficulty with the synthesis lies in the incorporation of Lys (20) into the growing peptide chain of Liraglutide and Semaglutide due to presence of its side chain part, which often results in racemization and the formation of unwanted peptide. To avoid such problems, the above prior arts disclose opting out synthesis of main peptide backbone in the initial step followed by incorporation of side chain at Lys (20) position in the second step to form the desired GLP-1 peptide.

CN111732650A discloses a method for preparing Semaglutide by combining a continuous flow solid-phase synthesis system. The method comprises the following steps of firstly, introducing Octadecanedioic(OtBu) acid-gamma-Glu-(OtBu)- AEEA-AEEA into a side chain of Lys20, and then completing a strategy of a main chain, wherein Fmoc-Gly-resin is adopted as a main chain structure, and continuous flow solid-phase synthesis is adopted for polypeptide fragment and Semaglutide peptide chain growth. The method further involves purification of peptide by mixing the crude peptide in a solution of acetonitrile and water, carrying out purification by using a C18 or C8 preparation column to remove main impurities before and after the product, followed by fine purification to remove micro impurities, and carrying out salt conversion and freeze-drying to obtain the product with the purity of 99.62%. Although CN111732650A discloses obtaining pure Semaglutide, it uses pseudo serine in the synthesis of one of the peptide fragments, which is not only expensive from industrial point of view, also not amenable from scale up point of view.

US20190177392A1 discloses methods for synthesis of GLP-1 peptides, such as Liraglutides and Semaglutides, and a method for purifying Liraglutide where crude Liraglutide has 56.5% purity, which after several HPLC purification cycles reaches to the 98.5% purity level.

W02020190757A1 discloses methods for the preparation and purification of Semaglutide and intermediates, where the crude Semaglutide obtained have 60.1% purity. The purification of Semaglutide was done by 3 purification systems on preparative HPLC column, which resulted in 98.9% purity. At the final purification stage RP HPLC was used to obtain 99.7% purity.

US20200317721A1 discloses a method for preparing GLP-1 and GLP-2 peptides, which comprises following multiple steps of purification involving HPLC for obtaining more than 99% purity in peptides. US20210009631A1 discloses methods and compounds for the solid phase synthesis of Semaglutide which results in 42.1% pure crude Semaglutide which after more than one step of dimensional purification by RP-HPLC.

WO2022018748A1 discloses improved and effective purification methods for Semaglutide by following RP-HPLC purification at three stages under varying conditions. First purification is followed in basic condition, followed by second in acidic and then third again in basic condition. After multiple RP-HPLC approaches Semaglutide obtained had more than 98% purity.

Costs of peptide largely depends on the purification approaches and as discussed above such purified peptides are obtained only after multiple steps of HPLCs. HPLC is a costly approach and using such purification technique for more than one time adds up to the costing value of the peptide. Therefore, there remains a need to develop an economical and cost effective method of synthesizing GLP-1 peptides. Accordingly, the present inventors have developed novel peptide fragments for the preparation of GLP-1 peptide, which allows introduction of the side chain part at Lys at an early stage of the synthesis by using fairly safe reagents thus rendering the method economical.

OBJECT OF THE INVENTION

It is an object of the present invention is to provide an improved and industrially advantageous method for preparation of peptides of pharmaceutical and commercial significance via fragment approach.

It is another object of the present invention to provide a synthetic approach allowing introduction of N-(l 7-carboxy-l -oxoheptadecyl)-L-Y-glutamyl-2-[2-(2- aminoethoxyjethoxy] acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl modified Lys at an early stage of the synthesis of GLP-1 peptide.

It is a yet another object of the present invention to provide novel peptide fragments which allows introduction of modified Lys at an early stage of the synthesis by using fairly safe reagents. BRIEF DESCRIPTION OF ABBREVIATIONS

Abbreviations used in the specification and claims are listed in the table below:

Fmoc 9-fluorenylmethyloxycarbonyl

HBTU O-Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate

COMU l-Cyano-2-ethoxy-2oxoethylidenaminooxy)dimethylamino-morpholino- carbeniu-m hexafluorophosphate

HATU O-(7-Aza-benzotriazole-l-oxy)-N,N,N',N'-tetramethyluronium hexafluorophosphate

DIC Diisopropylcarbodiimide

HOBt 1-hydroxybenzotrizole

HO At 1 -hydroxy-7-azabenzotriazole

DIEA N,N-diisopropylethylamine

Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl Trt Trityl tBu Tert-butyl

Boc- tert-Butyloxycarbonyl

DMF N,N-dimethyl formamide

DCM Dichloromethane

2-CTC 2-chlorotrityl resin

TFA Trifluoroacetic acid

NMP N-methyl pyrrolidone

DMSO Dimethyl sulfoxide

EDT Ethanedithiol

DODT 3,6-dioxa-l,8-octanedithiol

TIPS Triisopropyl silane

BRIEF DESCRIPTION OF FIGURES

Figure 1 illustrates synthesis of peptide fragment of SEQ ID NO: 1 (Fragment IG)

Figure 2 illustrates synthesis of peptide fragment of SEQ ID NO: 2 (Fragment SG)

Figure 3 illustrates synthesis of peptide fragment Fragment BG

Figure 4 illustrates synthesis of peptide fragment of SEQ ID NO: 3 (Fragment YF)

Figure 5 illustrates synthesis of peptide fragment of SEQ ID NO: 4 (Fragment YA) Figure 6 illustrates synthesis of Semaglutide involving coupling of peptide fragments of SEQ ID NO: 1, i.e., P-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-OH; SEQ ID NO: 2, i.e., P-Ser (tBu) - Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH and Fragment BG P-Aib-Glu (OtBu)-Gly-OH.

Figure 7 illustrates synthesis of Semaglutide involving coupling of peptide fragments of SEQ ID NO: 1, i.e., P-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-OH; SEQ ID NO: 3, i.e., P-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala- Lys(AEEAc-AEEAc -y-Glu-17-Carboxyheptadecanoyl)-Glu(OtBu)-Phe-OH; and Fragment BG, i.e., P-Aib-Glu (OtBu)-Gly-OH.

Figure 8 illustrates synthesis of Semaglutide involving coupling of peptide fragments of SEQ ID NO: 1, i.e., P-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-OH; SEQ ID NO: 4, i.e., P-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-OH and Fragment BG, i.e., P-Aib-Glu (OtBu)-Gly-OH.

SUMMARY OF THE INVENTION

Accordingly, in an aspect of the present invention there is provided a peptide fragment having amino acids sequence selected from sequences SEQ ID NO:1 (Fragment IG), SEQ ID NO:2 (Fragment SG), Fragment BG, SEQ ID NOG (Fragment YF), and SEQ ID NO:4 (Fragment YA),

In another aspect of the present invention there is provided a method of preparing a GLP-1 peptide, comprising the steps of: a. providing a first peptide fragment having amino acid sequence namely P -Arg (Pbf)-Gly-resin; b. deprotecting said first peptide fragment in presence of deprotecting agent and coupling deprotected first peptide fragment with peptide fragment having amino acid sequence SEQ ID NO:1 to form peptide fragment having amino acid sequence SEQ ID NOG in presence of a coupling agent, a base and a solvent, c. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 5 in presence of deprotecting agent and coupling deprotected peptide fragment having amino acid sequence SEQ ID NO:5 with amino acids P-Phe-OH and P-Glu(OtBu)-OH to form peptide fragment having amino acid sequence SEQ ID NO:6 in presence of a coupling agent, a base and a solvent, d. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 6 in presence of deprotecting agent and coupling deprotected peptide fragment having amino acid sequence SEQ ID NO: 6 with a modified amino acid Lys(W) to form peptide fragment having amino acid sequence SEQ ID NO:7 in presence of a coupling agent, a base and a solvent, e. deprotecting said peptide fragment having amino acid sequence SEQ ID NO:7 in presence of deprotecting agent and coupling deprotected peptide fragment having amino acid sequence SEQ ID NO:7 with amino acids P-Ala-OH, P-Ala-OH and P-Gln(trt)-OH respectively to form peptide fragment having amino acid sequence SEQ ID NO: 8 in presence of a coupling agent, a base and a solvent, f. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 8 in presence of deprotecting agent and coupling deprotected peptide fragment having amino acid sequence SEQ ID NO:8 with peptide fragment having amino acid sequence SEQ ID NO:2 to form peptide fragment having amino acid sequence SEQ ID NO:9, in presence of a coupling agent, a base and a solvent, g. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 9 in presence of deprotecting agent and coupling deprotected peptide fragment having amino acid sequence SEQ ID NO:9 with amino acids P-Val-OH, P-Asp(OtBu)-OH, P-Ser(tBu)-OH, P-Thr(tBu)-OH, P- (Phe-OH, P-Thr(tBu)-OH respectively to form peptide fragment having amino acid sequence SEQ ID NO: 10, in presence of a coupling agent, a base and a solvent, h. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of deprotecting agent and coupling deprotected peptide fragment having amino acid sequence SEQ ID NO: 10 with peptide fragment Fragment BG to form peptide fragment having amino acid sequence SEQ ID NO: 11 in presence of a coupling agent, a base and a solvent, i. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 11 in presence of deprotecting agent and coupling deprotected peptide fragment having amino acid sequence SEQ ID NO: 11 with amino acid Boc-His(Boc)-OH to form peptide fragment having amino acid sequence SEQ ID NO: 12 in presence of a coupling agent, a base and a solvent, j. deprotecting the whole peptide sequence having amino acid sequence SEQ ID NO: 12 followed by filtration to obtain crude GLP-1 peptide; wherein said P is a N-terminal protecting group; and said resin is an acid sensitive polymeric resin.

In another aspect there is provided a method of preparing GLP- 1 peptide, comprising the steps of: i. providing a first peptide fragment having amino acid sequence namely P-Arg (Pbf)-Gly-resin, ii. deprotecting said first peptide fragment in presence of deprotecting agent and coupling deprotected first peptide fragment with peptide fragment having amino acid sequence SEQ ID NO:1 to form peptide fragment having amino acid sequence SEQ ID NO:5 in presence of a coupling agent, a base and a solvent, iii. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 5 in presence of deprotecting agent and coupling deprotected peptide fragment having amino acid sequence SEQ ID NO:5 with peptide fragment having amino acid sequence SEQ ID NO: 3 to form peptide fragment having amino acid sequence SEQ ID NO: 13 in presence of a coupling agent, a base and a solvent, iv. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 13 in presence of deprotecting agent and coupling deprotected peptide fragment having amino acid sequence SEQ ID NO: 13 with P- Ser(tBu)-OH , P-Ser(tBu)-OH, P-Val-OH, P-Asp(OtBu)-OH, P-Ser(tBu)- OH, P-Thr(tBu)-OH, P-Phe-OH, and P-Thr(tBu)-OH respectively to form peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of a coupling agent, a base and a solvent, v. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of deprotecting agent and coupling deprotected peptide fragment having amino acid sequence SEQ ID NO: 10 with peptide fragment having amino acid sequence SEQ ID NO:3 to form peptide fragment having amino acid sequence SEQ ID NO: 9 in presence of a coupling agent, a base and a solvent, vi. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 9 in presence of deprotecting agent and coupling deprotected peptide fragment having amino acid sequence SEQ ID NO: 9 with P-Val- OH, P-Asp(OtBu)-OH, P-Ser(tBu)-OH, P-Thr(tBu)-OH, P-(Phe-OH, P- Thr(tBu)-OH respectively to form peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of a coupling agent, a base and a solvent, vii. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of deprotecting agent and coupling deprotected peptide fragment having amino acid sequence SEQ ID NO: 10 with peptide fragment Fragment BG to form peptide fragment having amino acid sequence SEQ ID NO: 11 in presence of a coupling agent, a base and a solvent, viii. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 11 in presence of deprotecting agent and coupling deprotected peptide fragment having amino acid sequence SEQ ID NO: 11 with Boc- His(Boc)-OH to form peptide fragment having amino acid sequence SEQ ID NO: 12 in presence of a coupling agent, a base and a solvent, ix. deprotecting the whole peptide sequence having amino acid sequence SEQ ID NO: 12 followed by filtration to obtain crude GLP-1 peptide; wherein said P is a N-terminal protecting group; and said resin is an acid sensitive polymeric resin.

In other aspect there is provided a method of preparing a GLP-1 peptide, comprising the steps of: a. . providing a first peptide fragment having amino acid sequence namely P - Arg (Pbf)-Gly-resin, b. . deprotecting said first peptide fragment in presence of deprotecting agent and coupling deprotected first peptide fragment with peptide fragment having amino acid sequence SEQ ID NO: 1 to form peptide fragment having amino acid sequence SEQ ID NO:5 in presence of a coupling agent, a base and a solvent, c. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 5 in presence of deprotecting agent and coupling deprotected peptide fragment having amino acid sequence SEQ ID NO:5 with P-Phe-OH and P- Glu(OtBu)-OH to form peptide fragment having amino acid sequence SEQ ID NO: 6 in presence of a coupling agent, a base and a solvent, d. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 6 in presence of deprotecting agent and coupling deprotected peptide fragment having amino acid sequence SEQ ID NO: 6 with modified lysine, P-Lys (W)-OH to form peptide fragment having amino acid sequence SEQ ID NO:7 in presence of a coupling agent, a base and a solvent, e. deprotecting said peptide fragment having amino acid sequence SEQ ID NO:7 in presence of deprotecting agent and coupling deprotected peptide fragment having amino acid sequence SEQ ID NO:7 with peptide fragment having amino acid sequence SEQ ID NO:4 to form peptide fragment having amino acid sequence SEQ ID NO: 14 in presence of a coupling agent, a base and a solvent, f. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 14 in presence of deprotecting agent and coupling deprotected peptide fragment having amino acid sequence SEQ ID NO: 14 with P-Ser(tBu)-OH, P-Ser(tBu)-OH, P-Val-OH, P-Asp(OtBu)-OH, P-Ser(tBu)-OH, P-Thr(tBu)- OH, P-Phe-OH and P-Thr(tBu)-OH respectively to form peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of a coupling agent, a base and a solvent, g. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of deprotecting agent and coupling deprotected peptide fragment having amino acid sequence SEQ ID NO: 10 with peptide fragment Fragment BG to form peptide fragment having amino acid sequence SEQ ID NO: 11 in presence of a coupling agent, a base and a solvent, h. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of deprotecting agent and coupling deprotected peptide fragment having amino acid sequence SEQ ID NO: 11 with amino acid Boc- His(Boc)-OH to form peptide fragment having amino acid sequence SEQ ID NO: 12 in presence of a coupling agent, base and solvent, i. deprotecting followed by resin cleavage of the peptide sequence having amino acid sequence SEQ ID NO: 12 followed by filtration to obtain crude GLP-1 peptide; wherein said P is a N-terminal protecting group; and said resin is an acid sensitive polymeric resin. DETAILED DESCRIPTION OF THE INVENTION

The following detailed description of the invention is provided to aid those skilled in the art in practicing the present invention. Even so, the detailed description should not be construed to unduly limit the present invention as modifications and verifications in the embodiments discussed herein may be made by those of skill in the art with the help of the foregoing description and accompanying figures without departing from the spirit or scope of the present invention.

The following description with reference to the accompanying drawings is provided to assist in a comprehensive understanding of various embodiments of the present disclosure as defined by the claims and their equivalents. It is to be understood that the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a component surface" includes reference to one or more of such surfaces.

The terminology used herein is for the purpose of describing particular various embodiments only and is not intended to be limiting to various embodiments. It will be further understood that the terms "comprises" and/or "comprising" used herein specify the presence of stated features, integers, steps, operations, members, components, and/or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, operations, members, components, and/or groups thereof. Also, expressions such as "at least one of," when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

Described herein are the novel peptide fragments having amino acid sequences and methods for synthesizing GLP-1 peptide by using those peptides.

In an embodiment there are provided peptide fragments having amino acid sequences SEQ ID NO: 1 (Fragment IG), SEQ ID NO:2 (Fragment SG), Fragment BG, SEQ ID NO: 3 (Fragment YF) and SEQ ID NO: 4 (Fragment YA) for the synthesis of GLP-1 peptides. The present invention provides

SEQ ID NO: 1 (Fragment IG): P-He-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-OH

SEQ ID NO: 2 (Fragment SG): P-Ser (tBu) - Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)- Gly-OH

SEQ ID NO: (Fragment BG): P-Aib-Glu (OtBu)-Gly-OH

SEQ ID NO: 3 (Fragment YF): P-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala- Eys(AEEAc-AEEAc -y-Glu-17-Carboxyheptadecanoyl)-Glu(OtBu)-Phe-OH

SEQ ID NO: 4 (Fragment YA): P-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala- OH wherein P is N terminal protecting group.

To prevent the unwanted reactions among the peptides and amino acids, the amino acids and the peptide fragments are provided with a protecting group.

The protecting groups for preventing reaction with N terminal group of amino acids are selected from Fmoc, Boc, Cbz, Bpoc.

According to another embodiment, the present invention provides method of preparation of GEP-1 peptide involving one or more steps of coupling of peptide fragments with amino acids.

The present invention provides a method of preparation of GEP-1 peptide which comprises at least one of said peptide fragment sequences selected from SEQ ID NO: 1 (Fragment IG), SEQ ID NO: 2 (Fragment SG), (Fragment BG), SEQ ID NOG (Fragment YF), and SEQ ID NO: (Fragment YA) or combinations thereof couple with one or more amino acids to form GLP-1 peptide.

The GLP-1 peptides formed by the coupling of one or more peptide fragments sequences selected from the above group with amino acids are found to follow less complicated and economical purification procedures, which do not involve multiple steps of HPLC.

The GLP-1 peptides formed by the above method are selected from Exenatide, Liraglutide, Lixisenatide, Albiglutide, Dulaglutide, Semaglutide and Taspoglutide. In an embodiment the present invention provides a method of preparing a GLP-1 peptide, comprising the steps of: a. providing a first peptide fragment having amino acid sequence namely P -Arg (Pbf)-Gly-resin; b. deprotecting said first peptide fragment in presence of a deprotecting agent and coupling the deprotected first peptide fragment with peptide fragment having amino acid sequence SEQ ID NO:1 to form peptide fragment having amino acid sequence SEQ ID NO:5 in presence of a coupling agent, a base and a solvent, c. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 5 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:5 with amino acids P-Phe-OH and P-Glu(OtBu)-OH to form peptide fragment having amino acid sequence SEQ ID NO:6 in presence of a coupling agent, a base and a solvent, d. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 6 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 6 with a modified amino acid Lys(W) to form peptide fragment having amino acid sequence SEQ ID NO:7 in presence of a coupling agent, base and solvent, e. deprotecting said peptide fragment having amino acid sequence SEQ ID NO:7 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:7 with amino acids P-Ala-OH, P-Ala-OH and P-Gln(trt)-OH respectively to form peptide fragment having amino acid sequence SEQ ID NO: 8 in presence of a coupling agent, a base and a solvent, f. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 8 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:8 with peptide fragment having amino acid sequence SEQ ID NO:2 to form peptide fragment having amino acid sequence SEQ ID NO:9, in presence of a coupling agent, a base and a solvent g. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 9 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:9 with amino acids P-Val-OH, P-Asp(OtBu)-OH, P-Ser(tBu)-OH, P-Thr(tBu)-OH, P- (Phe-OH, P-Thr(tBu)-OH respectively to form peptide fragment having amino acid sequence SEQ ID NO: 10, in presence of a coupling agent, a base and a solvent, h. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 10 with peptide fragment Fragment BG to form peptide fragment having amino acid sequence SEQ ID NO: 11 in presence of a coupling agent, a base and a solvent, i. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 11 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 11 with amino acid Boc-His(Boc)-OH to form peptide fragment having amino acid sequence SEQ ID NO: 12 in presence of a coupling agent, a base and a solvent, j. deprotecting followed by resin cleavage of the peptide sequence having amino acid sequence SEQ ID NO: 12 followed by filtration to obtain crude GLP-1 peptide;

The side chain W attached to Lys 20 can be N-e(y Glu[N-a- hexadecanoyl]) for Liraglutide or AEEAc-AEEAc-y -Glul7- Carboxyheptadecanoyl for Semaglutide. The deprotection of the protecting groups of peptide fragment sequences or amino acids are carried out by deprotecting agent which is a mixture of reagents selected from the group comprising of TFA, TIS, TIPS, DTT, EDT, ammonium iodide, 2,2'-(ethylenedioxy)diethane and acetyl cysteincysteine, DMS, phenol, cresol and thiocresol.

The coupling of peptide fragments and amino acids are carried out by using coupling agent, base and organic solvents.

The coupling agent used for coupling of peptide fragments are selected from the group comprising of hydroxybenzotriazole (HOBt); O-(7- azabenzotriazol- 1 -yl)- 1 , 1 ,3 ,3tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazol-l-yl)-N,N,N',N'tetramethyluronium tetrafluoroborate (TBTU), 1,3-dicyclohexylcarbodlimide (DCC), l(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HC1), diisopropylcarbodiimide (DIC), isopropylchloroformate (IPCF), O- (benzotriazol- 1 -yl) 1 , 1 ,3 ,3-tetramethyluronium hexafluorophosphate

(HBTU), benzotriazol- l-yl-oxytris(dimethyl-amino)-phosphonium hexafluorophosphate (BOP), N,N-bis-(2-oxo-3-oxazolidinyl)phosphonic dichloride (BOP-CI), benzotriazo lyloxytri(pyrolidino)phosphoniumhexafluorophosphate(PyB OP), bromotri(pyrrolidino)phosphonium hexafluorophosphate (PyBrOP), chlorotri(pynolidino)phosphonium hexafluorophosphate (PyClOP), ethyl-2-cyano-2(hydroxyimino) acetate (Oxyma Pure), O-(6-Chloro-l- hydrocibenzotriazol- 1 -yl)- 1 , 1 ,3 ,3tetramethyluronium tetrafluoroborate (TCTU), 245-norbomen-2,3-dicarboximido)-l,l,3,3tetramethyluronium tetrafluoroborate (TNTU), propane phosphonic acid anhydride (PPAA), 2-succinimido-l,l,3,3-tetramethyluronium tetrafluoro borate (TSTU), bromo-tris-pyrrolidinophosphonium hexafluorophosphate (PyBroP), iso- butylchloroformate (IBCF), Ethyl l,2dihydro-2-ethoxyquinoline-l- carboxylate (EEDQ), l-Cyano-2-ethoxy-

2oxoethylidenaminooxy)dimethylamino-morpholino-carbeniu- m hexafluorophosphate (COMU), 3-(diethoxyphosphoryloxy)-l,2,3- benzotriazin-4(3H)-one (DEPBT) or mixtures thereof.

The base used for coupling of peptide fragments are selected from a group comprising but not limiting to di-Cl-ClO -alkyl sulphides, alkyl phenyl sulphides, piperidine, 1,2-aminothiol of cysteine, a 1,2-aminoethanol of serine, a 1,2-aminoethanol of threonine, an aminooxyacetyl functional group, a mono-hydrazine succinyl functional group, ammonia, hydrazine, an alkoxide, a 4-hydrazinobenzoyl functional group, diisopropylamine, N,N-diisopropylethylamine triethylamine, dimethylamine, trimethyl amine, isopropyl ethylamine, pyridine, N- methyl morpholine and mixtures thereof.

The organic solvent used for coupling of peptide fragments are selected from a group comprising of DMF, DCM, THF, NMP, DMAC methanol, ethanol, isopropanol, dichloroethane, 1,4-dioxane, 2-methyl tetrahydrofuran ethyl acetate, acetonitrile, acetone.

The whole peptide sequence having amino acid sequence SEQ ID NO: 12 so obtained from the method of preparation of GLP-1 peptide is further deprotected followed by resin cleavage in presence of cleavage mixture of TFA: Phenol: DODT: Thioanisole: H2O having ratio of 82.5 : 5.0 : 2.5 : 5.0 : 5.0.

The resin cleaved peptide having amino acid sequence SEQ ID NO: 12 is further filtered to obtain the crude GLP-1 peptide.

In a preferred embodiment, the whole peptide sequence having amino acid sequence SEQ ID NO: 12 is Semaglutide.

In another embodiment there is provided a method of preparation of GLP- 1 peptide comprising one or more coupling steps with peptide fragments having amino acid SEQ ID NO: 1, 3 and Fragment BG with other amino acid fragments, wherein the steps comprises i. providing a first peptide fragment having amino acid sequence namely P -Arg (Pbf)-Gly-resin, ii. deprotecting said first peptide fragment in presence of a deprotecting agent and coupling the deprotected first peptide fragment with peptide fragment having amino acid sequence SEQ ID NO:1 to form peptide fragment having amino acid sequence SEQ ID NO:5 in presence of a coupling agent, a base and a solvent, iii. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 5 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:5 with peptide fragment having amino acid sequence SEQ ID NOG to form peptide fragment having amino acid sequence SEQ ID NO: 13 in presence of a coupling agent, a base and a solvent, iv. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 13 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 13 with P-Ser(tBu)-OH , P-Ser(tBu)-OH, P-Val-OH, P-Asp(OtBu)-OH, P- Ser(tBu)-OH, P-Thr(tBu)-OH, P-Phe-OH, and P-Thr(tBu)-OH respectively to form peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of a coupling agent, a base and a solvent, v. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 10 with peptide fragment having amino acid sequence SEQ ID NOG to form peptide fragment having amino acid sequence SEQ ID NO: 9 in presence of a coupling agent, a base and a solvent, vi. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 9 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 9 with P-Val- OH, P-Asp(OtBu)-OH, P-Ser(tBu)-OH, P-Thr(tBu)-OH, P-(Phe-OH, P- Thr(tBu)-OH respectively to form peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of a coupling agent, a base and a solvent, vii. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 10 with peptide fragment Fragment BG to form peptide fragment having amino acid sequence SEQ ID NO: 11 in presence of a coupling agent, a base and a solvent, viii. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 11 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 11 with Boc-His(Boc)-OH to form peptide fragment having amino acid sequence SEQ ID NO: 12 in presence of a coupling agent, a base and a solvent, wherein P is a N-terminal protecting group; and resin is an acid sensitive polymeric resin.

According to another embodiment there is provided a method for preparation of GLP-1 peptide comprising one or more coupling steps with peptide fragments having amino acid SEQ ID NO: l(Fragment IG), SEQ ID NO: 4 (Fragment YF)and Fragment BG with other amino acid fragments, wherein the steps comprises p. providing a first peptide fragment having amino acid sequence namely P -Arg (Pbf)-Gly-resin, q. deprotecting said first peptide fragment in presence of a deprotecting agent and coupling the deprotected first peptide fragment with peptide fragment having amino acid sequence SEQ ID NO:1 to form peptide fragment having amino acid sequence SEQ ID NO:5 in presence of a coupling agent, a base and a solvent, r. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 5 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 5 with P-Phe- OH and P-Glu(OtBu)-OH to form peptide fragment having amino acid sequence SEQ ID NO: 6 in presence of a coupling agent, a base and a solvent, s. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 6 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:6 with modified amino acid namely, P-Lys (W)-OH to form peptide fragment having amino acid sequence SEQ ID NO:7 in presence of a coupling agent, a base and a solvent, t. deprotecting said peptide fragment having amino acid sequence SEQ ID NO:7 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:7 with peptide fragment having amino acid sequence SEQ ID NO:4 to form peptide fragment having amino acid sequence SEQ ID NO: 14 in presence of a coupling agent, a base and a solvent, u. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 14 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 14 with P-Ser(tBu)-OH, P-Ser(tBu)-OH, P-Val-OH, P-Asp(OtBu)-OH, P- Ser(tBu)-OH, P-Thr(tBu)-OH, P-Phe-OH and P-Thr(tBu)-OH respectively to form peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of a coupling agent, a base and a solvent, v. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 10 with peptide fragment Fragment BG to form peptide fragment having amino acid sequence SEQ ID NO: 11 in presence of a coupling agent, a base and a solvent, w. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 11 with Boc-His(Boc)-OH to form peptide fragment having amino acid sequence SEQ ID NO: 12 in presence of a coupling agent, a base and a solvent,

The method for preparation of GLP-1 peptide after the steps of coupling comprises the step of global deprotection of the long peptide sequence so formed by one or more coupling steps in presence of a cleavage mixture comprising TFA: Phenol: DODT: Thioanisole: H2O having ratio 82.5: 5.0: 2.5 : 5.0 : 5.0 to obtain the crude GLP-1 peptide which is further filtered to obtain the final pure GLP-1 peptide.

The crude GLP- 1 peptide as obtaining in accordance with the present invention is isolated by lyophilisation and is further purified by reverse phase HPLC using solvents.

The solvents used in reverse HPLC purification is selected from TFA in water, acetic acid, acetonitrile, orthophosphoric acid in water, triethylamine in water, ammonium acetate in water and ammonium bicarbonate in water.

The present method involving the coupling of one or more peptide fragments selected from the peptide fragment sequence SEQ ID NO: 1 (Fragment IG), SEQ ID NO: 2(Fragment SG), Fragment BG, SEQ ID NO: 3(Fragment YF) and SEQ ID NO: 4(Fragment YA) results in preparation of crude GLP-1 peptides, which excludes multiple steps of HPLC purification to obtain the pure peptide.

In a preferred embodiment crude Semaglutide obtained by using the peptide fragments having amino acid sequences SEQ ID NO: l(Fragment IG), SEQ ID NO: 2(Fragment SG), Fragment BG, SEQ ID NO: 3(Fragment YF), and SEQ ID NO: 4(Fragment YA) involves HP 20 resin treatment followed by single step of HPEC, which results in formation of Semaglutide having >99.5% purity.

The present invention is now illustrated by way of non-limiting examples.

Example 1: Synthesis of Peptide Fragment having amino add SEQ ID NO: 1 (Fragment IG): Fmoc-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-OH:-

2-CTC resin (250 g, loading capacity 1.51 mmol/g, 100 -200 mesh) charged in a clean dry peptide synthesizer vessel. Resin was swelled in DCM (10 Vol.) for 30 minutes, two times each.

1^st amino acid coupling: To a pre-swelled resin, previously prepared solution of first amino acid Fmoc-Gly-OH (561.15 g, 5.0 eq) in DCM (10 Vol.) along with DIPEA (657.52 mL, 10.0 eq) was added and agitated for 12 hour at room temperature. After 12 hours the solvent was drained and resin was washed with DCM (2 x 10 Vol.). For capping, mixture of DCM: MeOH: DIPEA (85:10:5, 10 Vol.) was added to resin and agitated for 30 minutes. Solvent was drained and resin was washed with DCM (2 x 10 Vol.), followed by DMF (2 xlO Vol.).

Fmoc deprotection: Fmoc deprotection is performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. The solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

2^nd amino acid coupling: Pre-activated amino acid solution Fmoc-Arg (Pbf)-OH (734.65 g, 3.0 eq) in DMF (10 Vol.), HOBt monohydrate (173.85 g, 3.0 eq) and DIC (177.40 mF, 3.0 eq) was added to above Fmoc deprotected resin. Coupling continued for 6 hours. Solvent was drained and peptide resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 xlO Vol.) respectively. Fmoc deprotection and coupling procedure were followed for following amino acids as per sequence of amino acids, Fmoc-Arg (Pbf)-OH, Fmoc-Val-OH (384.35 g, 3.0 eq), Fmoc-Leu-OH (400.22 g, 3.0 eq), Fmoc-Trp (Boc)-OH (596.37 g, 3.0 eq), Fmoc-Ala-OH (352.5 g, 3.0 eq), Fmoc-Ile-OH (400.33 g, 3.0 eq) were performed similar to the above procedure.

After each coupling and deprotection the peptide resin was washed with DMF (2 xlO Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

Each coupling and deprotection was monitored by Bromophenol blue test.

After completion of synthesis, peptide resin was washed with 20% Methanol in DCM (lx 10 Vol.), DCM (2 x 10 Vol.) and MTBE (2 x 10 Vol.) respectively.

Resin cleavage: Peptide resin was washed with DCM (1 x 10 Vol.) for 2-3 minutes and then filtered. Peptide was cleaved from the resin using 1% TFA/ DCM (3 x 10 Vol.), 5 minutes for each time. Cleavage fractions were collected and neutralized with pyridine (1:1 Volume ratio to TFA in each cleavage). Combined cleavage fractions were concentrated under reduced pressure at 30-35 °C. Resulting solution was co-evaporated with ethanol (2 x 5 Vol.) up to minimum volume and slurry was added to purified water (50 Vol.), stirred for 18h. Solid was filtered over Buchner funnel, washed with purified water (2 x 5 Vol.) and n-Heptane (2 x 5 Vol.). Solid was dried for 8 hrs under reduced pressure at 38 - 42°C to afford white solid. Desired peptide fragment formation was confirmed by LCMS; m/z =1389[M+H]⁺with a reported yield of 471.91 g and 90% purity (Figure 1).

Example 2: Synthesis of Peptide Fragment having amino acid SEQ ID NO: 2 (Fragment SG): Fmoc-Ser (tBu) - Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH Peptide fragment having amino acid sequence SEQ ID NO: 2 was synthesized by using 2-CTC resin by Fmoc solid phase peptide strategy.

2-CTC resin (50.0 g, loading capacity 1.7 mmol/g, 100 -200 mesh) was charged in a clean dry peptide synthesizer vessel. Resin was swelled by DCM (10 Vol.) for 30 minutes, 2 times each.

1^st amino acid coupling: To a pre-swelled resin, freshly prepared solution of first amino acid, Fmoc-Gly-OH (126.35 g, 5.0 eq) in DCM (10 Vol.) with DIPEA (144.76 mL, 10.0 eq) was added and agitated for 12 hour at room temperature. After 12 hour, solvent was drained and resin was washed with DCM (2 x 10 Vol.). Capping mixture, DCM: MeOH: DIPEA (85:10:5, 10 Vol.) was added to resin and agitated for 30 minutes. Solvent was drained and resin was washed with DCM (2 x 10 Vol.) followed by DMF (2 xlO Vol.).

Fmoc deprotection: Fmoc deprotection was performed by adding 20 % piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. The solvent was drained and resin was washed by using DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

2^nd amino acid coupling: The pre-activated solution of Fmoc-Glu(OtBu)-OH (112.9 g, 3.0 eq), HOBt monohydrate (39.14 g, 3.0 eq) and DIC ( 40.22 mF, 3.0 eq) in DMF (10 Vol.) was added to above resin. Coupling was continued for 6 hours. Solvent was drained and peptide resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 xlO Vol.) respectively.

Fmoc deprotection and coupling procedure were followed for following amino acids as per sequence of amino acids, Fmoc-Leu-OH (90.0 g, 3.0 eq), Fmoc- Tyr(tBu)-OH (117.1 g, 3.0 eq), Fmoc-Ser(tBu)-OH (97.76 g, 3.0 eq), Fmoc- Ser(tBu)-OH (97.6 g, 3.0 eq) were performed similar to the above procedure.

After each coupling and deprotection the peptide resin was washed DMF (2 xlO Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

Each coupling and deprotection was monitored by Bromophenol blue test.

After completion of synthesis, peptide resin was washed with 20 % Methanol in DCM (lx 10 Vol.), DCM (2 x 10 Vol.) and MTBE (2 x 10 Vol.) respectively.

Resin cleavage: Peptide resin was washed with DCM (1 x 10 Vol) for 2-3 minutes and then filtered. Peptide fragment was cleaved from the resin using 1% TFA/ DCM (3 x 10 Vol.), 5 minutes for each time. Cleavage fractions were collected and neutralized with pyridine (1:1 Volume ratio to TFA in each cleavage), cleaved fractions were concentrated under reduced pressure at 30-35°C. Resulting solution was co-evaporated with ethanol (2 x 5 Vol.) up to minimum volume and slurry was added to purified water (50 Vol.), stirred for 18hours. Solid was filtered over Buchner funnel, washed with purified water (2 x5 Vol.). Solid was washed with n- heptane (2 x 5 Vol.) and dried in VTD under reduced pressure at 38-42°C to afford white solid. Desired peptide fragment formation was confirmed by LCMS, m/z =1101[M+H]⁺with a reported yield of 74.9 g and 80.1% purity (Figure 2).

Example 3: Synthesis of Peptide Fragment Fragment BG : Fmoc-Aib-Glu (OtBu)-Gly-OH:-

Peptide fragment Fragment BG was synthesized on 2-CTC resin by Fmoc solid phase peptide strategy.

2-CTC resin (250.0 g, loading capacity 1.8 mmol/g, 100 -200 mesh) was charged in a clean dry peptide synthesizer vessel. Resin was swelled in DCM (lOVol.) for 30 minutes, 2 times each.

1^st amino acid coupling: To a pre-swelled resin, freshly prepared solution of first amino acid Fmoc-Gly-OH (670.0 g, 5.0 eq) in DCM (10 Vol.) along with DIPEA (783.0 mL, 10.0 eq) was added and agitated for 12 hours at room temperature. After 12 hours, solvent was drained and resin was washed with DCM (2 x 10 Vol.). Capping solution mixture of DCM: MeOH: DIPEA (85:10:5, 10 Vol.) was added to resin and agitated for 30 minutes. After 30 minutes, solvent was drained and resin was washed with DCM (2 x 10 Vol.) followed by DMF (2 xlO Vol.).

Fmoc deprotection: Fmoc deprotection was performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by lOminutes respectively. The solvent was drained and, resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

2^nd amino acid coupling: The pre-activated Fmoc-Glu (OtBu)-OH (574.42 g, 3.0 eq) with HOBt monohydrate (206.95 g, 3.0 eq) and DIC (211.37 mL, 3.0 eq) in DMF (10 Vol.) were added to Fmoc deprotected resin. Coupling continued for 4 hours. After 4 hours, solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 xlO Vol.) respectively.

Fmoc deprotection: Fmoc deprotection was performed by adding 20 % piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by lOminutes respectively. The solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

3^rd amino acid coupling: The pre-activated Fmoc-Aib-OH (439.42 g, 3.0 eq) with HOBt monohydrate (206.95 g, 3.0 eq) and DIC (211.37 mL, 3.0 eq) in DMF (10 Vol.) was added to Fmoc deprotected resin. Coupling was continued for 3 hours. After 3 hours, solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 xlO Vol.) respectively.

After each coupling and deprotection peptide resin was washed DMF (2 xlO Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

Each coupling and deprotection was monitored by Bromophenol blue test.

Resin cleavage: Peptide Resin was washed with DCM (1 x 10 Vol.) for 2-3 minutes. Resin was cleaved by using 10 % TFE in DCM (8 x 10 Vol.), each wash was carried for 45 minutes. Combined fractions were concentrated under reduced pressure at 30-35°C. Resulting solution was co-evaporated with MTBE (2 x 5 Vol.), n-heptane (1 x 10 Vol.), dried under reduced pressure for 2-3 hours.

Desired peptide fragment formation was confirmed by LCMS, m/z = 568.6[M+H]⁺., with a reported yield of 229.89 g and 90 % purity (Figure 3).

Example 4: Synthesis of Peptide Fragment having amino acid SEQ ID NO: 3 (Fragment YF):-Fmoc-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala- Lys(AEEAc-AEEAc-y-Glu-17-Carboxyheptadecanoyl)-Glu(OtBu)-Phe-OH:-

Peptide fragment having amino acid sequence SEQ ID NO: 3 (Fragment YF) was synthesized using 2-CTC resin by Fmoc solid phase peptide strategy.

2-CTC resin (5.0 g, Loading capacity- 1.51 mmol/g) was charged in a clean dry peptide synthesizer vessel. Resin was swelled with DCM (10 Vol for30 minutes, 2 times each.

1^st amino acid coupling: First amino acid, Fmoc-Phe-OH (8.76 g, 5.0 eq) with DIPEA (13.15 mL, 10.0 eq) in DCM (10 Vol.) was added to pre-swelled resin and agitated for 12 hours. The solvent was drained and resin was washed with DCM (2 x 10 Vol.). Capping solution, DCM: MeOH: DIPEA (85%: 10%:5%, 10 Vol.) was charged to above resin, agitated for 30 min for capping the resin. Solvent was drained and resin was washed with DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

Fmoc deprotection: Fmoc deprotection was performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. The solvent was drained and resin was washed by DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

2^nd amino acid coupling: The pre-activated solution of Fmoc-Glu(OtBu)-OH (9.63 g, 3.0 eq), HOBt monohydrate (3.47 g, 3.0 eq) and DIC (3.54 mF, 3.0 eq) in DMF (10 Vol.) were added to resin and agitated for 6 hours. After 6 hours, solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Fmoc deprotection: Fmoc deprotection was performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. The solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

3^rd amino acid coupling: The pre-activated solution of Fmoc-Lys(AEEAc-AEEAc- y-Glul7-Carboxyheptadecanoyl)-OH (17.94 g, 2.0 eq) with HBTU (5.68 g, 2.0 eq) and HOBt monohydrate (2.04 g, 2.0 eq), DIPEA(2.6 mb, 2.0eq ) in DMF (10 Vol.) were added to resin, agitated for 5 hours. The solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Sequential coupling and Fmoc deprotection method was followed for rest of the amino acids, Fmoc-Ala-OH (7.05g, 3.0 eq), Fmoc-Ala-OH (7.05 g, 3.0 eq), Fmoc- Gln(Trt)-OH (13.83g, 3.0 eq), Fmoc-Gly-OH (6.68 g, 3.0 eq), Fmoc-Glu(OtBu)- OH (9.52 g, 3.0 eq), Fmoc-Leu-OH (7.93 g, 3.0 eq), Fmoc-Tyr(tBu)-OH (10.37 g, 3.0 eq) were performed similar to the above procedure.

Each coupling and deprotection was monitored by Bromophenol blue test. After completion of synthesis, peptide resin was washed with 20 % Methanol in DCM (2 x 5 Vol.), DCM (2 xlO Vol.), MTBE (2 x 10 Vol.) and DCM (2 xlO Vol.) respectively.

Resin cleavage: Peptide was cleaved from the resin using 1% TFA / DCM (3 x 10 Vol.), 5 minutes for each time. Cleavage fractions were collected and neutralized with pyridine (1:1 Volume ratio to TFA in each cleavage). Combined cleavage fractions were concentrated under reduced pressure at 30-35 °C. Resulting solution was co-evaporated with ethanol (2 x 5 Vol.) up to minimum volume and slurry was added to purified water (50 Vol.), stirred for 18hours. Solid was filtered over Buchner funnel, and washed with purified water (2 x 5 Vol.). Solid was washed with n-heptane (2 x 5 Vol.) and dried at 35-40 °C to afford white solid.

Desired peptide fragment formation was confirmed by LCMS, m/z = 2616 (M/2= 1308), with a reported yield of 13.92 g and 70.9% purity (Figure 4).

Example 5: Synthesis of Peptide Fragment having amino acid SEQ ID NO: 4 (Fragment YA): Fmoc-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-OH

Peptide fragment having amino acid sequence SEQ ID NO: 4 (Fragment YA) was synthesized on 2-CTC resin by Fmoc solid phase peptide strategy.

2-CTC resin (250 g, loading capacity 1.8 mmol/g, 100 -200 mesh) was charged in a clean dry peptide synthesizer vessel. Resin was swelled in DCM (lOVol.) for 30 min, two times each.

1^st amino acid coupling: To a pre-swelled resin, previously prepared solution of first amino acid Fmoc-Ala-OH (700.0 g, 5.0 eq) in DCM (10 Vol.) along with DIPEA (783.5 mL, 10.0 eq) was added and agitated for 12 hours at room temperature. After 12 hours, solvent was drained and resin was washed with DCM (2 x 10 Vol.). Capping solution mixture of DCM: MeOH: DIPEA (85:10:5, 10 Vol.) was added to resin and agitated for 30 min. Solvent was drained and resin was washed with DCM (2 x 10 Vol.) followed by DMF (2 xlO Vol.).

2^nd amino acid coupling: The pre-activated Fmoc-Ala-OH (420.0 g, 3.0 eq) with HOBt monohydrate (206.95 g, 3.0 eq) and DIC (211.35 mL, 3.0 eq) in DMF (10 Vol.) was added to Fmoc deprotected resin. Coupling was continued for 3 hours. After 3 hours, solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 xlO Vol.) respectively.

Fmoc deprotection and coupling procedure were followed for following amino acids as per sequence of amino acids, Fmoc-Gln(Trt)-OH (673.5 g, 3.0 eq), Fmoc- Gly-OH (401.35 g, 3.0 eq), Fmoc-Glu(OtBu)-OH (574.4 g, 3.0 eq), Fmoc-Leu-OH (477.05 g, 3.0 eq), Fmoc-Tyr(tBu)-OH (620.35 g, 3.0 eq) were performed similar to the above procedure.

Each coupling and deprotection was monitored by Bromophenol blue test.

Resin cleavage: Peptide resin was washed with DCM (1 x 10 Vol.) for 2-3 minutes. Peptide product was cleaved from resin using 1% TFA in DCM (3 x 10 Vol.), 5 minutes for each time. Cleaved fractions were collected and neutralized with pyridine (1:1 Volume ratio to TFA for each cleavage). Combined cleavage fractions were concentrated under reduced pressure at 30-35°C. Resulting solution was coevaporated with ethanol (2 x 5 Vol.) up to minimum volume and slurry was added to purified water (50 Vol.), stirred for 18 hours. Solid was filtered over buchner funnel, washed with purified water (2 x 5 Vol.), and n-Heptane (2 x 5 Vol) and well dried to afford white solid. Desired peptide fragment formation was confirmed by LCMS, m/z = 1327.56[M+H]⁺ with a reported yield of 476.1 g and 80.0 % purity (Figure 5). Example 6: Preparation of Semaglutide involving coupling of peptide fragments having amino acid sequences SEQ ID NO: KFragment IG), SEQ ID NO:3 (Fragment YE), Fragment BG and amino acids.

Synthesis of Fmoc-Arg(Pbf)-Gly-Wang resin: -

Synthesis was carried out using Wang resin by Fmoc solid phase peptide strategy. Fmoc-Gly-Wang resin (5.0 g, loading capacity - 0.3 mmol/g) was charged in a clean dry peptide synthesizer vessel. Resin swelled with DMF (10 Vol.) for 30 minutes, two times each.

Fmoc deprotection: Fmoc deprotection was performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15 minutes respectively. The solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

1^st amino acid coupling: The pre-activated Fmoc-Arg(Pbf)-OH (2.91 g, 3.0 eq) with HOBt monohydrate (0.69 g, 3.0 eq) and DIC (0.70 mL, 3.0 eq) in DMF (10 Vol.) added to above resin, agitated for 3hours. The solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Synthesis of peptide fragment having amino acid sequence SEQ ID NO: 5, Fmoc- Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin:-

Fmoc deprotection: Fmoc deprotection was performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. The solvent was drained and washed the resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

Coupling of peptide fragment having amino acid sequence SEQ ID NO: 1 Fmoc- Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-OH with NH2-Arg(Pbf)-Gly-Wang resin:- Pre-activated peptide fragment having amino acid sequence SEQ ID NO: 1, Fmoc-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-OH(6.25 g, 3.0 eq) with HBTU (1.7g, 3.0 eq), HOBt monohydrate (0.69g, 3.0 eq) and DIPEA (0.78 mL, 3.0 eq) in 20 % DMSO/ DMF (10 Vol.) were added to above Fmoc deprotected resin, agitated for 5 hours. After 5 hours the solvent was drained and peptide resin was washed with, DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Synthesis of peptide fragment having amino acid sequence SEQ ID NO: 13, Fmoc- Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(AEEAc-AEEAc-y -Glul7- Carboxyheptadecanoyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly- Arg(Pbf)-Gly-wang resin:-

Coupling of peptide fragment having amino acid sequence SEQ ID NO: 3, Fmoc- Tyr(tBu)-Eeu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Eys (AEEAc-AEEAc-y-Glul7- Carboxyheptadecanoyl)-Glu(OtBu)-Phe-OH with peptide fragment having amino acid sequence SEQ ID NO: 5, NH2-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly- Arg(Pbf)-Gly-Wang Resin :- The pre-activated solution of peptide fragment having amino acid sequence SEQ ID NO: 3, Fmoc-Tyr(tBu)-Leu-Glu(OtBu)-Gly- Gln(Trt)-Ala-Ala-Lys(AEEAc-AEEAc-y-Glul7-Carboxyheptadecanoyl)- Glu(OtBu)-Phe-OH (7.84 g, 2.0 eq) with HOBt monohydrate (0.459 g, 2.0 eq), HBTU (1.14 g, 2.0 eq) and DIPEA (0.52 mL, 2.0 eq) in 20 % DMSO/ DMF (10 Vol.) was added to above peptide resin and agitated for 5 hours. The solvent was drained and resin was washed with DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Synthesis of peptide fragment having amino acid sequence SEQ ID NO: 10, Fmoc- Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)- Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(AEEAc-AEEAc-Y-Glul7- Carboxyhept adecanoyl)-Glu(OtBu)-Phe-He-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)- Gly-Arg(Pbf)-Gly-Wang resin

Fmoc deprotection: Fmoc deprotection was performed by adding 20% piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15 minutes respectively. The solvent was drained and resin was washed with DMF (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

Fmoc-Ser(tBu)-OH coupling:- The pre-activated Fmoc-Ser(tBu)-OH (1.72 g, 3.0 eq) with HOBt monohydrate (0.69 g, 3.0 eq) and DIC (0.70 mL, 3.0 eq) in DMF (10 Vol.) was added to above resin. Coupling continued for 6 hours. Solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 xlO Vol.) respectively.

Fmoc deprotection and coupling procedure were followed for following amino acids as per sequence of amino acid, Fmoc- Ser(tBu)-OH (1.72 g, 3.0 eq), Fmoc- Val-OH (1.52 g, 3.0 eq), Fmoc- Asp(OtBu)-OH (1.92 g, 3.0 eq), Fmoc-Ser(tBu)- OH (1.72 g, 3.0 eq), Fmoc- Thr(tBu)-OH (1.78 g, 3.0 eq), Fmoc-Phe-OH (1.74 g, 3.0 eq), Fmoc-Thr(tBu)-OH (1.78 g, 3.0 eq) were performed similar to the above procedure.

Synthesis of peptide fragment having amino acid sequence SEQ ID NO: 11, Fmoc- Aib-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)-Ala-Ala-Lys(AEEAc-AEEAc-y- Glul7-Carboxyheptadecanoyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val- Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin:-

Fmoc deprotection: Fmoc deprotection was performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minute followed by 15minute respectively. Drained the solvent, washed the resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

Coupling of peptide fragment Fragment BG, Fmoc-Aib-Glu(OtBu)-Gly-OH:- The pre-activated solution of Fmoc-Aib-Glu(OtBu)-Gly-OH (2.55 g, 3.0 eq) with HBTU (1.7 g, 3.0 eq) HOBt monohydrate(0.69 g, 3.0 eq), and DIPEA (0.78 mL, 3.0 eq) in 20 % DMSO/ DMF (10 Vol.) added to above peptide resin, agitated for 5 hrs. Drained solvent and peptide resin washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Synthesis of peptide fragment having amino acid sequence SEQ ID NO: 12, Boc- His(Trt)-Aib-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)-Ala-Ala-Lys(AEEAc- AEEAc-y-Glul7-Carboxyheptadecanoyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu- Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin:-

Fmoc deprotection: Fmoc deprotection was performed by adding 20 % piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15 minutes respectively. Drained the solvent, washed the resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

Coupling of Boc-His(Trt)-OH The pre-activated solution of Boc-His(Trt)- OH(2.23 g, 3.0 eq) with HOBt monoh ydrate(0.69 g, 3.0 eq), HBTU (1.7 g, 3.0 eq) and DIPEA (0.78 mF, 3.0 eq) in DMF (10 Vol.) was added to above peptide resin, agitated for 5 hours. Drained the solvent and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Instead of Boc-His(trt)-OH, Boc-His(Boc)-OH was also used.

Coupling of Boc-His(Boc)-OH The pre-activated solution of Boc-His(Boc)-OH( 2.41g, 3.0 eq) with HOBt monohydrate (0.69 g, 3.0 eq), HBTU (1.7 g, 3.0 eq) and DIPEA (0.78 mF, 3.0 eq) in DMF (10 Vol.) was added to above peptide resin, agitated for 5 hours. Drained the solvent and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Each coupling and deprotection was monitored by Bromophenol blue test.

After completion of synthesis, peptide resin was washed with 20 % Methanol in DCM (2 x lOVol.) and MTBE (1 x 10 Vol.) to afford dried peptide resin (12.2 g).

Synthesis of peptide fragment having amino acid sequence SEQ ID NO: 12 His- Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala- Lys(AEEAc-AEEAc-Y-Glul7-Carboxyheptadecanoyl)-Glu-Phe-Ile-Ala-Trp-Leu- Val-Arg-Gly-Arg-Gly-OH (Semaglutide, crude) (Figure -7)

Deprotection and Resin Cleavage of whole peptide: To a pre-cooled cleavage cocktail of TFA: Phenol: DODT: Thioanisol: H2O (82.5:5:2.5:5:5 %, 20 Vol.) was added to fully protected peptide resin (12.2 g) at 5-10 °C. Resulting reaction mixture was agitated for 5 hours, at 20-25 °C. After completion of cleavage, resin was filtered and filtrate was concentrated up to 30 % and poured into pre-cooled MTBE (100 Vol.). Solid was stirred for 25-30 minutes and filtered over Buchner funnel, washed with MTBE (2 x 5 Vol.). Crude peptide was dissolved in H2O (10 Vol.) and lyophilized to afford off white solid. Yield: 6.2 g, crude with55.0 % HPLC purity.

HP 20 resin treatment: HP20 resin (120.0 g) is swelled with Methanol (14 Vol.) for 10 min, filtered, washed with purified water (3 x 14 Vol.). Crude Semaglutide (6 g, assay 44.62%) was dissolved in 0.25M Ammonium bicarbonate (200 Vol.) and loaded to above resin, agitated for 2 hrs. After 2 hrs, resin was loaded in glass column and washed with H2O (3 x 14 Vol.) followed by 2.5, 5.0, 7.5, 10.0 and 40.0 of ACN/H2O. Fractions were collected and lyophilized to afford off white solid (4.1 g, HPLC purity 63.4 %, 58.7 % assay).

To achieve desired purity, impure Semaglutide was further purified by preparative RP-HPLC. Desired purity fractions were pooled and lyophilized to afford white solid (1.34 g, HPLC Purity 99.52 %).

Example 7: Preparation of Semaglutide involving coupling of peptide fragments having amino acid sequences SEQ ID NO: 1 (Fragment IG), SEQ ID NO: 2 (Fragment SG), (Fragment BG) and amino acids

Synthesis of peptide fragment having amino acid sequence SEQ ID NO: 9, Fmoc- Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(AEEAc- AEEAc-y-Glul7-Carboxyheptadecanoyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu- Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resim-Synthesis of peptide fragment having amino acid sequence SEQ ID NO: 5, Fmoc-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)- Gly-Arg(Pbf)-Gly-Wang Resin was carried out as mentioned in Example 6.

Fmoc-Phe-OH coupling :- The pre-activated Fmoc-Phe-OH(1.74 g, 3.0 eq) with HOBt monohydrate(0.69 g, 3.0 eq) and DIC (0.70 mL, 3.0 eq) in DMF (10 Vol.) added to above resin. Coupling was continued for 6 hours. Solvent was drained and peptide resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 xlO Vol.) respectively. Fmoc deprotection and coupling procedure were followed for following amino acid as per sequence which are Fmoc-Glu (OtBu)-OH (1.91 g, 3.0 eq), were performed similar to the above procedure.

Synthesis of peptide fragment having amino acid sequence SEQ ID NO: 7, Fmoc- Lys(AEEAc-AEEAc-y-Glul7-Carboxyheptadecanoyl)-Glu(OtBu)-Phe-He-Ala- Trp(Boc)-Leu- Vai- Arg(Pbf)-Gly-Arg(Pbf)-Gly -Wang Resin

Fmoc deprotection: Fmoc deprotection performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. Drained the solvent, washed the resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

Coupling of Fmoc-Lys(W)-OH:-The pre-activated Fmoc- Lys(AEEAc-AEEAc-y- Glul7-Carboxyheptadecanoyl)-OH (3.58 g, 2.0 eq), with HBTU(1.13 g, 2.0 eq) and HOBt monohydrate (0.46 g, 2.0 eq) and DIPEA (0.52 mL , 2.0 eq)in 20% DMSO/DMF (10 Vol.) was added to the above peptide resin, agitated for 5hrous. After 5 hours the solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Fmoc deprotection and coupling procedure were followed for following amino acid as per sequence, which are, Fmoc-Ala-OH (1.40 g, 3.0 eq), Fmoc-Ala-OH (1.40 g, 3.0 eq), Fmoc-Gln(Trt)-OH (2.77 g, 3.0 eq) were performed similar to the above Fmoc-Phe-OH coupling procedure.

Coupling of peptide fragment having amino acid sequence SEQ ID NO: 2:-The preactivated solution of peptide fragment having amino acid sequence SEQ ID NO: 2, Fmoc-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly -OH (4.95 g, 3.0 eq) with HBTU(1.7 g, 3.0 eq) and HOBt monohydrate (0.69 g, 3.0 eq) and DIPEA (0.78, 3.0 eq)in 20% DMSO/DMF (10 Vol.) was added to above peptide resin, agitated for 5hours. After 5 hours, the solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Synthesis of peptide fragment having amino acid sequence SEQ ID NO: 10, Fmoc- Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)- Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(AEEAc-AEEAc-y-Glul7- Carboxyheptadecano yl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu- Vai- Arg(Pbf)-Gly- Arg(Pbf)-Gly-Wang Resin

Fmoc deprotection: Fmoc deprotection was performed by adding 20% Piperidine in DMF (2 x 10 Vol.) for 10 min. followed by 15 min respectively. Drained the solvent, washed resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

Coupling of Fmoc-Val-OH: The pre-activated solution of Fmoc-Val-OH (1.52 g, 3.0 eq) with HOBt monohydrate (0.69 g, 3.0 eq) and DIC (0.70 mL, 3.0 eq) in DMF (10 Vol.) added to above resin. Coupling was continued for 3 hrs. Solvent drained and peptide resin washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

Fmoc deprotection and coupling procedure were followed for following amino acids as per sequential, which are Fmoc-Asp (OtBu)-OH (1.85 g, 3.0 eq), Fmoc- Ser(tBu)-OH (1.72 g, 3.0 eq), Fmoc- Thr(tBu)-OH (1.78 g, 3.0 eq), Fmoc-Phe-OH (1.74 g, 3.0 eq), Fmoc-Thr(tBu)-OH (1.78 g, 3.0 eq), were performed similar to the above procedure.

Fmoc deprotection: Fmoc deprotection performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. Drained the solvent, washed the resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively. peptide fragment Fragment BG coupling:- The pre-activated solution of peptide fragment Fragment BG, Fmoc-Aib-Glu(OtBu)-Gly-OH (2.55 g, 3.0 eq) with HBTU(1.7 g, 3.0 eq) and HOBt monohydrate (0.69 g, 3.0 eq) and DIPEA (0.78, 3.0 eq) in 20% DMSO/DMF (10 Vol.) added to above peptide resin, agitated for 5h. Drained solvent and resin washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Synthesis of peptide fragment having amino acid sequence SEQ ID NO: 11, Boc- His(Boc)-Aib-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)-Ala-Ala-Lys(AEEAc- AEEAc-y-Glul7-Carboxyheptadecanoyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu- Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resin: -

Fmoc deprotection: Fmoc deprotection was performed by adding 20% Piperidine in DMF (2 x 10 Vol.) for 10 minutes, followed by 15 minutes respectively. Drained the solvent, washed the resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

Boc-His(Boc)-OH coupling:- The pre-activated solution of Boc-His(Boc)-OH (2.23 g, 3.0 eq) with HBTU(1.7 g, 3.0 eq), HOBt monohydrate (0.69 g, 3.0 eq) and DIPEA (0.78 mL, 3.0 eq) in DMF (10 Vol.) added to above peptide resin. Resin was agitated for 4h. Drained solvent and resin washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Each coupling and deprotection monitored by Bromophenol blue test.

After completion of synthesis, peptide resin was washed with 20% Methanol in DCM (2 x lOVol.), MTBE (1 x 10 Vol) and well dried to afford 12.0 g of peptide resin.

Synthesis of peptide fragment having amino acid sequence SEQ ID NO: 12, Boc- His-Aib-Glu-Gly-Thr- Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln- Ala- Ala- Lys(AEEAc-AEEAc-y-Glul7-Carboxyheptadecanoyl)-Glu-Phe-Ile-Ala-Trp- Leu-Val-Arg-Gly-Arg-Gly-OH, Semaglutide, crude

Deprotection and Resin Cleavage of whole peptide: To a pre-cooled cleavage cocktail of TFA: Phenol: DODT: Thioanisole: H2O (82.5 : 5.0 : 2.5 : 5.0 : 5.0, 20 Vol.) was added to protected peptide resin (12.0 g) at 5-10 °C. Resulting mixture was agitated for 5 hrs at 20-25 °C. After completion of cleavage, resin was filtered and filtrate was concentrated up to 30 % and poured into pre-cooled MTBE (20 Vol.), solid was stirred for 15-30 minutes and filtered over Buchner funnel, washed with MTBE (2 x 5 Vol.). Crude peptide was dissolved in H2O (10 Vol.) and lyophilized to afford off white solid. Yield: 5.82 g, 61.2 % HPLC purity (Figure 6).

HP 20 resin treatment: HP20 resin (100.0 g) is swelled with Methanol (14 Vol.) for 10 min, filtered, washed with purified water (3 x 14 Vol.). Crude Semaglutide (5 g, purity 61.2%) was dissolved in 0.25M Ammonium bicarbonate (200 Vol.) and loaded to above resin, agitated for 2 hrs. After 2 hrs, resin was loaded in glass column and washed with H2O (3 x 14 Vol.) followed by 2.5, 5.0, 7.5, 10.0 and 40.0 of ACN/H2O. Fractions were collected and lyophilized to afford off white solid (3.3 g)-

To achieve desired purity, impure Semaglutide was further purified by preparative RP-HPFC. Desired purity fractions were pooled and lyophilized to afford white solid (0.91 g, HPLC Purity 99.63 %).

Example 8: Preparation of Semaglutide involving coupling of peptide fragments having amino acid sequences SEQ ID NO: 1 (Fragment IG), SEQ ID NO:4 (Fragment YA), Fragment BG and amino acids

Synthesis of peptide fragment having amino acid sequence SEQ ID NO: 6, Fmoc- Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Eeu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resin: peptide fragment having amino acid sequence SEQ ID NO: 5, Fmoc-Ile-Ala- Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-Wang resin was synthesized as mentioned above.

Fmoc deprotection: Fmoc deprotection was performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. Drained the solvent, washed the resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

Fmoc-Phe-OH coupling: The pre-activated solution of Fmoc-Phe-OH(1.74 g, 3.0 eq) with HOBt monohydrate(0.69 g, 3.0 eq) and DIC (0.70 mF, 3.0 eq) in DMF (10 Vol.), added to Fmoc-deprotected resin and agitated for 5 hours. After 5 hours solvent was drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

The above Fmoc deprotection and coupling procedure was followed for Fmoc-Glu (OtBu)-OH (1.91 g, 3.0 eq)

Coupling of Fmoc-Lys(W)-OH:-The pre-activated Fmoc- Lys(AEEAc-AEEAc-y- Glul7-Carboxyheptadecanoyl)-OH (3.58 g, 2.0 eq), with HBTU(1.13 g, 2.0 eq) and HOBt monohydrate (0.46 g, 2.0 eq) and DIPEA (0.52 mL , 2.0 eq)in 20% DMSO/DMF (10 Vol.) was added to above peptide resin, agitated for 5hours. After 5 hours., drained the solvent and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Synthesis of peptide fragment having amino acid sequence SEQ ID NO: 13, Fmoc- Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(AEEAc-AEEAc-y-Glul7- Carboxyheptadecanoyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly- Arg(Pbf)-Gly-Wang resin

Fmoc deprotection: Fmoc deprotection was performed by adding 20 % piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. Drained the solvent, washed the resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively. peptide fragment having amino acid sequence SEQ ID NO: 4, Fmoc-Tyr(tbu)-Leu- Glu(otbu)-Gly-Gln(trt)-Ala-Ala-OH coupling:- The pre- activated solution of peptide fragment having amino acid sequence SEQ ID NO: 4, Fmoc-Tyr(tBu)-Leu- Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-OH (5.97 g, 3.0 eq) with HBTU (1.7 g, 3.0eq) HOBt monohydrate(0.69 g, 3.0 eq) and DIPEA (0.78 mL, 3.0eq) in 20 % DMSO/DMF (10 Vol.), DIC (0.70 mL, 3.0 eq) added to resin and agitated for 5 hours. After 5 hours, solvent drained and peptide resin washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Synthesis of peptide fragment having amino acid sequence SEQ ID NO: 10, Fmoc- Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr (tBu)- Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(AEEAc-AEEAc-y-Glul7- Carboxyheptade canoyl)-Glu(OtBu)-Phe-He-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)- Gly-Arg(Pbf)-Gly-Wang resin

Fmoc deprotection: Fmoc deprotection performed by adding 20% piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15 minutes respectively. Drained the solvent, washed resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

Fmoc-Ser (tBu)-OH of coupling: The pre-activated solution of Fmoc-Ser (tBu)-OH (1.72 g, 3.0 eq) with HOBt monohydrate (0.69 g, 3.0 eq) and DIC (0.70 mL, 3.0 eq) in DMF (10 Vol.), was added to resin and agitated for 5 hrs. After 5 hrs, solvent drained and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Fmoc deprotection and coupling procedure were followed for following amino acids as per sequential. Which are Fmoc-Ser(tBu)-OH (1.72 g, 3.0 eq), Fmoc-Val- OH (1.52 g, 3.0 eq), Fmoc-Asp(OtBu)-OH (1.85 g, 3.0 eq), Fmoc-Ser(tBu)-OH (1.72 g, 3.0 eq), Fmoc- Thr(tBu)-OH (1.78 g, 3.0 eq), Fmoc-Phe-OH (1.74 g, 3.0 eq), Fmoc-Thr(tBu)-OH (1.78 g, 3.0 eq) were performed similar to the above procedure.

Synthesis of peptide fragment having amino acid sequence SEQ ID NO: 11, Fmoc- Aib-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)-Ala-Ala-Lys(AEEAc-AEEAc-y- Glul7-Carboxyheptadecanoyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val- Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resin: -

Fmoc deprotection: Fmoc deprotection performed by adding 20 % piperidine in DMF (2 x 10 Vol.) for 10 minutes followed by 15minutes respectively. Drained the solvent, washed resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively. peptide fragment Fragment BG, Fmoc-Aib-Glu (OtBu)-Gly-OH coupling:- The pre-activated solution of peptide fragment Fragment BG, Fmoc-Aib-Glu(OtBu)- Gly-OH (2.55 g, 3.0 eq) with HBTU (1.7 g, 3.0eq) HOBt monohydrate(0.69 g, 3.0 eq) and DIPEA (0.78 mL, 3.0eq) in 20 % DMSO/DMF (10 Vol.), DIC (0.70 mL, 3.0 eq) was added to resin and agitated for 5 hrs. After 5 hours, solvent was drained and peptide resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Synthesis of peptide fragment having amino acid sequence SEQ ID NO: 12, Boc- His(Trt)-Aib-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)-Ala-Ala-Lys(AEEAc- AEEAc-y-Glul7-Carboxyheptadecanoyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu- Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resin: -

Boc-His(Trt)-OH coupling:-The pre-activated solution of Boc-His (Trt)-OH (2.41 g, 3.0 eq) with HBTU (1.7 g, 3.0eq) HOBt monohydrate(0.69 g, 3.0 eq) and DIPEA (0.78 mL, 3.0eq) in 20 % DMSO/DMF (10 Vol.), DIC (0.70 mL, 3.0 eq) added to resin and agitated for 5 hrs. After 5 hrs, solvent drained and peptide resin washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Each coupling and deprotection was monitored by Bromophenol blue test.

After completion of synthesis, peptide resin was washed with 20% Methanol in DCM (2 x lOVol.), MTBE (1 x 10 Vol) and dried well to afford 13.25 g of peptide resin.

Synthesis of peptide fragment having amino acid sequence SEQ ID NO: 12, His- Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala- Lys(AEEAc-AEEAc-y-Glul7-Carboxyheptadecanoyl)-Glu(otbu)-Phe-Ile-Ala- Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (Semaglutide, crude)

Deprotection and Resin Cleavage of whole peptide: To a pre-cooled cleavage cocktail TFA: Phenol: DODT: Thioanisol: H₂O (82.5 % : 5 % : 2.5 % : 5 % : 5 %, 20 Vol.) was added to peptide resin (13.25 g) at 5-10 °C. Resulting mixture was agitated for 5 hrs at 20-25 °C. After completion of cleavage, resin was filtered and filtrate was concentrated up to 30 % and poured into pre-cooled MTBE (20 Vol.), solid was stirred for 15-30 minutes. Solid filtered over Buchner funnel, washed with MTBE (2 x 5 Vol.). Crude peptide was dissolved in H₂O (20 Vol.) and lyophilized to afford white solid, Yield, 4.58 g (Figure 8).

Crude peptide was purified by RP-HPLC to afford pure Semaglutide > 99.5 %.

Claims

44 CLAIMS:

1. A peptide fragment having amino acids sequence selected from sequences SEQ ID NO: 1 (Fragment IG), SEQ ID NO:2 (Fragment SG), Fragment BG:, SEQ ID NO: 3 (Fragment YF), and SEQ ID NO: 4 (Fragment YA).

2. The peptide fragment as claimed in claim 1, wherein at least one of said peptide fragment having amino acid sequence selected from sequences SEQ ID NO:1, SEQ ID NO:2, Fragment BG, SEQ ID NOG, and SEQ ID NO:4 or combinations thereof couple with one or more amino acids to form a GLP-1 peptide.

3. The peptide fragment as claimed in claim 2, wherein said GLP-1 peptide is selected from Exenatide, Liraglutide, Lixisenatide, Albiglutide, Dulaglutide, Semaglutide and Taspoglutide.

4. A method of preparing a GLP-1 peptide, comprising the steps of: a. providing a first peptide fragment having amino acid sequence of P - Arg (Pbf)-Gly-resin; b. deprotecting said first peptide fragment in presence of a deprotecting agent and coupling the deprotected first peptide fragment with peptide fragment having amino acid sequence SEQ ID NO:1 to form peptide fragment having amino acid sequence SEQ ID NOG in presence of a coupling agent, a base and a solvent, c. deprotecting said peptide fragment having amino acid sequence SEQ ID NOG in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NOG with amino acids P-Phe-OH and P-Glu(OtBu)-OH to form peptide fragment having amino acid sequence SEQ ID NOG in presence of a coupling agent, a base and a solvent, 45 d. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 6 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 6 with a modified amino acid Lys(W) to form peptide fragment having amino acid sequence SEQ ID NO:7 in presence of a coupling agent, a base and a solvent, e. deprotecting said peptide fragment having amino acid sequence SEQ ID NO:7 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:7 with amino acids P-Ala-OH, P-Ala-OH and P-Gln(trt)-OH respectively to form peptide fragment having amino acid sequence SEQ ID NO: 8 in presence of a coupling agent, a base and a solvent, f. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 8 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:8 with peptide fragment having amino acid sequence SEQ ID NO:2 to form peptide fragment having amino acid sequence SEQ ID NO:9, in presence of a coupling agent, a base and a solvent, g. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 9 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:9 with amino acids P-Val-OH, P-Asp(OtBu)-OH, P-Ser(tBu)-OH, P-Thr(tBu)-OH, P- (Phe-OH, P-Thr(tBu)-OH respectively to form peptide fragment having amino acid sequence SEQ ID NO: 10, in presence of a coupling agent, a base and a solvent, h. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 10 with peptide fragment Fragment BG to form peptide fragment having 46 amino acid sequence SEQ ID NO: 11 in presence of a coupling agent, a base and a solvent, i. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 11 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 11 with amino acid fragment Boc-His(Boc)-OH to form peptide fragment having amino acid sequence SEQ ID NO: 12 in presence of a coupling agent, a base and a solvent, j. deprotecting followed by resin cleavage of the peptide sequence having amino acid sequence SEQ ID NO: 12 followed by filtration to obtain crude GLP-1 peptide; wherein said P is a N-terminal protecting group; and said resin is an acid sensitive polymeric resin.

5. A method of preparing GLP-1 peptide, comprising the steps of: i. providing a first peptide fragment having amino acid sequence of P - Arg (Pbf)-Gly-resin, ii. deprotecting said first peptide fragment in presence of a deprotecting agent and coupling the deprotected first peptide fragment with peptide fragment having amino acid sequence SEQ ID NO:1 to form peptide fragment having amino acid sequence SEQ ID NO:5 in presence of a coupling agent, a base and a solvent, iii. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 5 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:5 with peptide fragment having amino acid sequence SEQ ID NOG to form peptide fragment having amino acid sequence SEQ ID NO: 13 in presence of a coupling agent, a base and a solvent, iv. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 13 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 13 with P-Ser(tBu)-OH , P-Ser(tBu)-OH, P-Val-OH, P-Asp(OtBu)-OH, P- Ser(tBu)-OH, P-Thr(tBu)-OH, P-Phe-OH, and P-Thr(tBu)-OH respectively to form peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of a coupling agent, a base and a solvent, v. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 10 with peptide fragment having amino acid sequence SEQ ID NO:3 to form peptide fragment having amino acid sequence SEQ ID NO: 9 in presence of a coupling agent, a base and a solvent, vi. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 9 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 9 with P-Val- OH, P-Asp(OtBu)-OH, P-Ser(tBu)-OH, P-Thr(tBu)-OH, P-(Phe-OH, P- Thr(tBu)-OH respectively to form peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of a coupling agent, a base and a solvent, vii. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 10 with peptide fragment Fragment BG to form peptide fragment having amino acid sequence SEQ ID NO: 11 in presence of a coupling agent, a base and a solvent, viii. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 11 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 11 with Boc-His(Boc)-OH to form peptide fragment having amino acid sequence SEQ ID NO: 12 in presence of a coupling agent, a base and a solvent, ix. deprotecting followed by resin cleavage of the peptide sequence having amino acid sequence SEQ ID NO: 12 followed by filtration to obtain crude GLP-1 peptide. wherein said P is a N-terminal protecting group; and said resin is an acid sensitive polymeric resin.

6. A method of preparing a GLP-1 peptide, comprising the steps of: i. providing a first peptide fragment having amino acid sequence of P -Arg (Pbf)-Gly-resin, ii. deprotecting said first peptide fragment in presence of a deprotecting agent and coupling the deprotected first peptide fragment with peptide fragment having amino acid sequence SEQ ID NO: 1 to form peptide fragment having amino acid sequence SEQ ID NO:5 in presence of a coupling agent, a base and a solvent, iii. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 5 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 5 with P-Phe- OH and P-Glu(OtBu)-OH to form peptide fragment having amino acid sequence SEQ ID NO:6 in presence of a coupling agent, a base and a solvent, iv. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 6 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:6 with modified amino acid, P-Lys (W)-OH to form peptide fragment having amino acid sequence SEQ ID NO:7 in presence of a coupling agent, a base and a solvent, v. deprotecting said peptide fragment having amino acid sequence SEQ ID NO:7 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:7 with peptide fragment having amino acid sequence SEQ ID NO:4 to form peptide fragment having amino acid sequence SEQ ID NO: 14 in presence of a coupling agent, a base and a solvent, vi. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 14 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 14 with P- Ser(tBu)-OH, P-Ser(tBu)-OH, P-Val-OH, P-Asp(OtBu)-OH, P-Ser(tBu)- OH, P-Thr(tBu)-OH, P-Phe-OH and P-Thr(tBu)-OH respectively to form peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of a coupling agent, a base and a solvent, vii. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 10 with peptide fragment Fragment BG to form peptide fragment having amino acid sequence SEQ ID NO: 11 in presence of a coupling agent, a base and a solvent, viii. deprotecting said peptide fragment having amino acid sequence SEQ ID NO: 10 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO: 11 with Boc- His(Boc)-OH to form peptide fragment having amino acid sequence SEQ ID NO: 12 in presence of a coupling agent, a base and a solvent, ix. deprotecting followed by resin cleavage of the peptide sequence having amino acid sequence SEQ ID NO: 12 followed by filtration to obtain crude GLP-1 peptide, wherein said P is a N-terminal protecting group; and said resin is an acid sensitive polymeric resin. 50

7. The method of preparing a GLP-1 peptide, as claimed in any of claims 4, 5 and 6, wherein said GLP-1 peptide is Semaglutide or Liraglutide.

8. The method of preparing a GLP-1 peptide as claimed in any of claims 4, 5 and 6, wherein said W is selected from N-e(y Glu[N-a- hexadecanoyl]) and AEEAc-AEEAc-y -Glul7-Carboxyheptadecanoyl.

9. The method of preparing a GLP-1 peptide as claimed in any of claims 4, 5 and 6, wherein said P is selected from Fmoc, Boc, Cbz, and Bpoc.

10. The method of preparing a GLP-1 peptide as claimed in any of claims 4, 5 and 6, wherein said acid sensitive polymeric resin is selected from the group Chlorotrityl resin (CTC), Sasrin, Wang Resin, 4- methytrityl chloride, TentaGel S, TentaGel TGA, Rink acid resin, NovaSyn TGT resin, HMPB-AM resin, 4-(2-(amino methyl)-5- methoxy)phenoxy butyric acid anchored to polymeric resin MBHA, 4-(4- (amino methyl)-3-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA and 4-(2-(amino methyl)-3,3-dimethoxy)phenoxy butyric acid anchored to polymeric resin MBHA.

11. The method of preparing a GLP- 1 peptide as claimed in any of claims

4, 5 and 6, wherein said coupling agent is selected from the group comprising of hydroxybenzotriazole (HOBt); O-(7-azabenzotriazol-l- yl)-l,l,3,3tetramethyluronium hexafluorophosphate (HATU), O- (benzotriazol- 1 -yl)-N,N,N',N'tetramethyluronium tetrafluoroborate

(TBTU), 1,3-dicyclohexylcarbodlimide (DCC), l(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HC1), diisopropylcarbodiimide (DIC), isopropylchloroformate (IPCF), O- (benzotriazol- 1 -yl) 1 , 1 ,3 ,3 -tetramethyluronium hexafluorophosphate

(HBTU), benzotriazol- l-yl-oxytris(dimethyl-amino)-phosphonium hexafluorophosphate (BOP), N,N-bis-(2-oxo-3-oxazolidinyl)phosphonic 51 dichloride (BOP-CI), benzotriazo lyloxytri(pyrolidino)phosphoniumhexafluorophosphate(PyB OP), bromotri(pyrrolidino)phosphonium hexafluorophosphate (PyBrOP), chlorotri(pynolidino)phosphonium hexafluorophosphate (PyClOP), ethyl-2-cyano-2(hydroxyimino) acetate (Oxyma Pure), O-(6-Chloro-l- hydrocibenzotriazol- 1 -yl)- 1 , 1 ,3 ,3tetramethyluronium tetrafluoroborate (TCTU), 245-norbomen-2,3-dicarboximido)-l,l,3,3tetramethyluronium tetrafluoroborate (TNTU), propane phosphonic acid anhydride (PPAA), 2-succinimido-l,l,3,3-tetramethyluronium tetrafluoro borate (TSTU), bromo-tris-pyrrolidinophosphonium hexafluorophosphate (PyBroP), iso- butylchloroformate (IBCF), Ethyl l,2dihydro-2-ethoxyquinoline-l- carboxylate (EEDQ), l-Cyano-2-ethoxy-

2oxoethylidenaminooxy)dimethylamino-morpholino-carbeniu- m hexafluorophosphate (COMU), 3-(diethoxyphosphoryloxy)-l,2,3- benzotriazin-4(3H)-one (DEPBT) and mixtures thereof.

12. The method of preparing a GLP-1 peptide as claimed in any of claims 4, 5 and 6, wherein said solvent is selected from a group comprising of DMF, DCM, THF, NMP, DMAC methanol, ethanol, isopropanol, dichloroethane, 1,4-dioxane, 2-methyl tetrahydrofuran ethyl acetate, acetonitrile, acetone and mixtures thereof.

13. The method of preparing a GEP-1 peptide as claimed in any of claims 4, 5 and 6, wherein said base is selected from a group comprising but not limiting to di-Cl-ClO -alkyl sulphides, alkyl phenyl sulphides, piperidine, 1,2-aminothiol of cysteine, a 1,2-aminoethanol of serine, a 1,2-aminoethanol of threonine, an aminooxyacetyl functional group, a mono-hydrazine succinyl functional group, ammonia, hydrazine, an alkoxide, a 4-hydrazinobenzoyl functional group, diisopropylamine, N,N-diisopropylethylamine triethylamine, dimethylamine, trimethyl amine, isopropyl ethylamine, pyridine, N- methyl morpholine and mixtures thereof. 52

14. The method of preparing a GLP-1 peptide as claimed in any of claims 4, 5 and 6, wherein the step of deprotection is carried out in presence of reagents selected from the group comprising of TFA, TIS, TIPS, DTT, EDT, ammonium iodide, 2,2'-(ethylenedioxy)diethane and acetyl cysteincysteine, DMS, phenol, cresol and thiocresol.

15. The method of preparing a GLP-1 peptide as claimed in any of claims 4, 5 and 6, wherein the step of deprotecting the peptide followed by resin cleavage is carried in presence of a cleavage mixture of TFA: Phenol: DODT: Thioanisole: H2O having ratio 82.5 : 5.0 : 2.5 : 5.0 : 5.0.

16. The method of preparing a GLP-1 peptide as claimed in any of the preceding claims 4 to 15, further comprising the step of treating the crude GLP-1 peptide in HP20 resin followed by a single step RP-HPLC.

17. The method of preparing a GLP-1 peptide as claimed in any of the preceding claims 4 to 15, wherein the purity of said GLP-1 peptide is >99.5%.