DESC:Cross-reference of applications
This complete specification claims priority from Indian Patent Application No. 202041003595 filed as provisional patent application on 27th January 2020. This complete specification also claims priority from Indian Patent Application No. 202141001569 filed as provisional patent application on 13th January 2021.

Field of The Invention
The present invention relates to an improved process for the preparation of Liraglutide having the sequence chemical formula (I).

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Glu-Palmitoyl)-Glu-Phe-lle-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
Formula-I
The present invention also relates to novel fragments-2 and -4 which are useful in the preparation of Liraglutide.
Fragment-2: Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH
Fragment-4: Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH

Background of the Invention

Liraglutide is a long-acting glucagon like peptide agonist developed by Novo Nordisk for the treatment of type-2 diabetes. Liraglutide is marketed under brand name “Victoza” in the form of injection in United States and Europe. Liraglutide is an injectable drug which reduces sugar levels in blood. It is also used to treat obesity.

Liraglutide and its process for the preparation is first disclosed in US 6268343. This process leads to the formation of impurities and additional purification techniques required to get pure Liraglutide. This process is highly expensive and commercially not viable.

US 7273921 B2, US 6268343 B2, US 6451974 B2, US 9260474 B2, WO 2020074583 A1, WO 2019170918 A1, CN 102875665 and CN 103145828 also disclose the process for the preparation of Liraglutide.

Chemical peptide synthesis was mainly approached by solid phase peptide synthesis (SPPS), liquid phase peptide synthesis (LPPS) or a hybrid approach.

In SPPS, an amino acid anchored by its C-terminus to an insoluble polymer resin. Protected amino acids are sequentially assembled on resin. The growing chain is bound to the insoluble support, the excess of reagents can be removed by simple filtration. However, during the synthesis of peptides, side products can accumulate in addition to side products formed during deprotection. The purification of the final product obtained by SPPS is very difficult and commercially not viable to meet stringent regulatory requirements.

In LPPS, the synthesis takes place in a homogeneous reaction medium and it is not suitable for the production of larger peptides at commercial scale.

In hybrid approach, both SPPS and LPPS are employed at appropriate places, targeted peptide is assembled by fragment condensation in solution phase whereas the fragments are generated through conventional solid phase peptide synthesis. This approach yields the targeted peptide with good purity and high yields. As the short peptide fragment on solid phase exhibits high coupling efficiency, which makes fragment synthesis scalable to the commercial production. Hybrid approach does not require any advanced equipment for the same.
Most of prior-art processes of Liraglutide have reported with solid phase peptide synthesis and having several disadvantages associated with generation of new impurities and not suitable for large scale production due to complex purification methods.

In view of all these disadvantages, there is a significant need to develop a cost effective, stable, commercially viable, large scale and robust process for the preparation of highly pure Liraglutide with good yield.

Summary of The Invention
The present invention provides an improved process for the preparation of Liraglutide by a hybrid approach.

The present invention provides a cost effective, novel and an efficient process for the preparation of Liraglutide by making appropriate fragments in a solid phase approach followed by condensing these fragments using solution phase approach with higher yields and purity.

In one embodiment, the present invention relates to an improved process for the preparation of Liraglutide by using three, four or five fragments through hybrid approach. The process will involve the coupling of appropriate fragments in a required sequence, deprotection and condensing them in solution phase, followed by purification on reverse phase HPLC, freeze drying and isolation to get pure liraglutide.

In another embodiment, the present invention provides a solid phase peptide synthesis for the preparation of Liraglutide compound of formula-I.
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Glu-Palmitoyl)-Glu-Phe-lle-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
Formula-I
which comprises:
a) synthesis of fragments-1,-2,-3,-4 and -5 on solid support
Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1)
Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2)
Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3)
Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH (Fragment-4)
H-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu (Fragment-5);
b) anchoring H-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu (Fragment-5) to a resin followed by selective deprotection in presence of a base;
c) condensing Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH (Fragment-4) with a resin obtained in stage-b) in presence of a coupling agent and solvent followed by deprotection in presence of a base;
d) condensing Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3) with a resin obtained in stage-b) in presence of a coupling agent and solvent followed by deprotection in presence of a base;
e) condensing Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2) with a resin obtained in stage-b) in presence of a coupling agent and solvent followed by deprotection in presence of a base;
f) condensing Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1) with a resin obtained in stage-b) in presence of a coupling agent to obtain protected Liraglutide;
g) cleaving the protected Liraglutide from resin using a reagent to obtain crude Liraglutide;
h) purifying crude Liraglutide by preparative HPLC to obtain pure Liraglutide.

In yet another embodiment, the present invention provides a hybrid approach for the preparation of Liraglutide compound of formula-I.
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Glu-Palmitoyl)-Glu-Phe-lle-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
Formula-I
which comprises:
a) synthesis of fragments-1, -2, -3, -4 and -5 on solid support
Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1)
Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2)
Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3)
Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH (Fragment-4)
H-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu (Fragment-5);
b) condensing Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp (Boc)-OH (Fragment-4) with Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu (Fragment-5) in presence of coupling agent and solvent followed by deprotection to obtain Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu;
c) condensing Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3) with peptide obtained in stage-b) in presence of coupling agent and solvent followed by deprotection to obtain Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu;
d) condensing Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2) with peptide obtained in stage-c) in presence of a coupling agent followed by deprotection to obtain Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu;
e) condensing Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1) with peptide obtained in step-d) in presence of a coupling agent to obtain protected Liraglutide;
f) cleaving the protected Liraglutide using a reagent to obtain crude Liraglutide;
g) purifying the crude Liraglutide by preparative HPLC to obtain pure Liraglutide.

In yet another embodiment, the present invention relates to novel fragments-2 and -4 which are useful in the preparation of Liraglutide.
Fragment-2: Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH
Fragment-4: Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH

In yet another embodiment, the present invention relates to an improved process for the preparation of novel fragments using solid phase peptide synthesis approach which are useful in the preparation of Liraglutide.

In yet another embodiment, the present invention provides a solid phase peptide synthesis for the preparation of Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH of fragment-2
which comprises:
a) anchoring Fmoc-Val-Ser(Oxa)-OH to a resin in presence of a coupling agent;
b) selective deprotection of amino acid using a base;
c) coupling of Fmoc-Ser(tBu)-OH to a resin obtained in step-a) in presence of coupling agent in a solvent to obtain dipeptide resin;
d) sequential coupling of Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH to the obtained resin in step-a) in presence of a coupling agent;
e) cleaving of protected peptide from solid support resin in presence of a reagent to get fragment-2.

In yet another embodiment, the present invention provides a solid phase peptide synthesis for the preparation of Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH of fragment-4
which comprises:
a) anchoring Fmoc-Trp(Boc)-OH to a resin in presence of a coupling agent;
b) selective deprotection of amino acid using a base;
c) coupling of Fmoc-Ala-OH to a resin obtained in step-a) in presence of coupling agent in a solvent to obtain dipeptide resin;
d) sequential coupling of Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys[Pal(Glu-OtBu)]-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH to the obtained resin in step-a) in presence of a coupling agent;
e) cleaving of protected peptide from solid support resin in presence of a reagent to get fragment-4.

In yet another embodiment, the present invention provides a hybrid approach for the preparation of Liraglutide compound of formula-I.
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Glu-Palmitoyl)-Glu-Phe-lle-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
Formula-I
which comprises:
a) synthesis of fragments-3, -6 and -7 on solid support;
Boc-His(Trt)-Ala-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-7)
Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3)
H-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu (Fragment-6);
b) condensing H-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu (Fragment-6) with Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3) in presence of coupling agent and solvent in in-situ manner followed by deprotection in presence of base to obtain Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu (OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu;
c) condensing Boc-His(Trt)-Ala-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp (OtBu)-Val-Ser(Oxa)-OH (Fragment-7) with peptide obtained in step-b) in presence of a coupling agent to obtain protected Liraglutide;
d) cleaving the protected Liraglutide using a reagent to obtain crude Liraglutide;
e) purifying the crude Liraglutide by preparative HPLC to obtain pure Liraglutide.

In yet another embodiment, the present invention provides a hybrid approach for the preparation of Liraglutide compound of formula-I.
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Glu-Palmitoyl)-Glu-Phe-lle-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
Formula-I
which comprises:
a) Synthesis of fragments-1, -2, -3 and -6 on solid support;
Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1)
Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2)
Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3)
H-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg (Pbf)-Gly-Arg(Pbf)-Gly-OtBu (Fragment-6);
b) condensing H-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu (Fragment-6) with Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3) in presence of coupling agent and solvent in in-situ manner followed by deprotection in presence of base to obtain H-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu (OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu;
c) condensing Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2) with peptide obtained in step-b) in presence of a coupling agent in in-situ manner followed by deprotection in presence of base to obtain H-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu;
d) condensing Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1) with peptide obtained in step-c) in presence of a coupling agent in in-situ manner followed by deprotection in presence of base to obtain protected Liraglutide;
e) cleaving the protected Liraglutide using a reagent to obtain crude Liraglutide;
f) purifying the crude Liraglutide by preparative HPLC to obtain pure Liraglutide.

Abbreviations:
Fmoc: 9-fluorenylmethoxycarbonyl
Boc: Di tert-butyl dicarbonate
DCM: dichloromethane
DMF: N, N-dimethyl formamide
DIC: N, N’-diisopropyl carbodiimide
DIEA: Diisopropylethylamine
DCC: Dicyclohexyl carbodiimide
HOBt: N-hydroxy benzotriazole
HOAt: 1-Hydroxy-7-azabenzotriazole
CTC resin: 2-Chlorotrityl chloride resin
SPPS: Solid phase peptide synthesis
LPPS: Liquid phase peptide synthesis
TFA: Trifluoroacetic acid
TIPS: Triisopropylsilane
TIS: Triisopropyl silane
EDC.HCl: 1-(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
DTT: Diothreitol

Detailed Description of the Invention

The present invention provides an improved process for the preparation of Liraglutide by making appropriate fragments on solid support, followed by condensing these fragments using solution phase approach with higher yields and purity.

Peptide fragments which are used in the preparation of Liraglutide are as follows.
Fragment-1: Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH
Fragment-2: Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH
Fragment-3: Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH
Fragment-4: Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH
Fragment-5: H-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu
Fragment-6: H-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu
Fragment-7: Boc-His(Trt)-Ala-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp (OtBu)-Val-Ser(Oxa)-OH

Peptide fragments are prepared by using solid phase peptide synthesis through linear approach.
Solid phase peptide synthesis is carried out on an insoluble polymer which is acid sensitive. Acid sensitive resin selected from the group consisting of chloro trityl resin (CTC), sasrin, wang resin, 4-methyltrityl chloride, rink acid resin. Preferably using CTC resin. The resin used for the synthesis of Liraglutide undergoes swelling in presence of a solvent selected from the group consisting of dichloromethane (DCM), N,N-dimethylformamide (DMF) and N-methyl-2-pyrrolidone or mixture.

The coupling agent used in the reaction can be selected from the group consisting of Ethylcyano (hydroxyimino)acetate-02)-tri-(1-pyrrolidinyl)-Phosphonium hexa fluorophosphate (PyOxim), ethyl-2-cyano-2-(hydroxy amino) acetate (Oxyma pure), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), diisopropyl carbodiimide (DIC), 1,3-dicyclohexylcabodiimide (DCC), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), 1-(dimethyl aminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), O-(benzotriazol-1-yl)-1,1,3,3-tetra methyluronium hexafluorophosphate (HBTU), 1-Hydroxybenzotriazole (HOBt), Isopropyl chloro formate (IPCF), Benzotriazol-1-yl-oxy-tris(dimethyl-amino)-phosphonium hexa fluorophosphate (BOP), benzotriazole-1-yloxytri(pyrrolidino)phosphonium hexa fluoro phosphate (PyBOP), N,N-bis-(2-oxo-3-oxazolidinyl)phosphonic dichloride (BOP-Cl), bromotri(pyrrolidino)phosphonium hexa fluoro phosphate (PyBrOP), O-(6-Chloro-1-hydrocibenzotriazol-1-yl)-1,1,3,3-tetramethyl uranium tetra fluoroborate (TCTU), chlorotri (pyrrolidino)phosphonium hexafluorophosphate (PyClOP), Ethyl 1,2-dihydro-2-ethoxyquinoline-carboxylate(EEDQ), isobutyl chloro formate (IBCF), 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate(TSTU), 1-Cyano-2-ethoxy-2-oxo ethylidene aminooxy) dimethyl amino morpholino-carbeniumhexafluorophosphate (COMU), 2-(5-norbornen-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), propane phosphonic acid anhydride (PPAA), 3-(diethoxy phosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) or its mixture.

Coupling of amino acid to a resin is carried out in presence of a base. The base is organic or inorganic base. The inorganic base is selected from the group consisting of potassium carbonate, lithium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, ammonium hydroxide and mixture thereof; the organic base is selected from the group consisting of diisopropyl amine, N,N-diisopropyl ethylamine, triethylamine, dimethylamine, tri methyl amine, isopropyl ethylamine, pyridine, N-methyl morpholine and mixture thereof.

Solvents used in this coupling reaction is selected from the group consisting of DMF, DCM, tetrahydrofuran, NMP, DMAC, methanol, ethanol, isopropanol, dichloroethane, 1,4-dioxane, ethyl acetate, acetonitrile, acetone or a mixture thereof.

The base used in the deprotection reaction can be selected from group consisting of tert-butyl amine, 20% of 4-methyl piperidine in Dimethyl formamide, 20% of piperidine in Dimethyl formamide and 20% of piperazine in Dimethyl formamide. Preferably using tert-butylamine.

According to the present invention, the cleavage and global deprotection of the peptide is carried out with a cocktail mixture. The cleavage of peptide from resin involves treating the protected peptide anchored to a resin with an acid having at least a scavenger. The acid used in the cleavage is tri fluoro acetic acid. The scavengers used are selected from the group consisting of TIPS, phenol, thioanisole, water or mixture thereof. Preferably using a cocktail mixture of TFA, TIPS, water and DTT (90%: 5%: 5%: 2.5%).

In the present invention, isolation of Liraglutide is carried out by precipitating with ether solvent. Ether solvent used in this reaction is selected from the group consisting of methyl tert-butyl ether, di ethyl ether, t-butyl methyl ether, diisopropyl ether or mixtures thereof. Finally, lyophilization was carried out to get pure Liraglutide.

Accordingly, the present invention provides solid phase peptide synthesis for the preparation of Liraglutide of compound of formula-I.

Scheme-I

In one embodiment, the present invention provides a solid phase peptide synthesis for the preparation of Liraglutide compound of formula-I.

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Glu-Palmitoyl)-Glu-Phe-lle-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
Formula-I

which comprises:
a) synthesis of fragments-1, -2, -3, -4 and -5 on solid support
Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1)
Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2)
Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3)
Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH (Fragment-4)
H-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu (Fragment-5);
b) Anchoring Fmoc-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu (Fragment-5) to a resin followed by selective deprotection in presence of a base;
c) condensing Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp (Boc)-OH (Fragment-4) with a resin obtained in stage-b) in presence of a coupling agent in solvent followed by deprotection in presence of a base;
d) condensing Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3) with a resin obtained in stage-b) in presence of a coupling agent followed by deprotection in presence of a base;
e) condensing Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2) in presence of a coupling agent followed by deprotection in presence of a base;
f) condensing Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1) in presence of a coupling agent to obtain protected Liraglutide;
g) cleaving the protected Liraglutide from resin using a reagent to obtain crude Liraglutide;
h) purifying by preparative HPLC to obtain pure Liraglutide.

In step-b), CTC resin was taken in a SPPS reactor and dichloromethane was added to it. Deprotection the Fmoc group is carried out in presence of a base, preferably using 20% piperidine in dimethylformamide.

In step-c), condensation of peptide resin obtained in step-b) with Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH (Fragment-4) in presence of a coupling agent, preferably using diisopropyl carbodiimide (DIC).

The reaction temperature may range from 25°C to 30°C. The duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.

Selective deprotection of peptide resin obtained from step-c) using a base, preferably using 20% of piperidine in Dimethyl formamide.

The reaction temperature may range from 25°C to 30°C.

In step-d) Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3) was condensed with peptide resin obtained from step-b) in presence of a coupling agent followed by deprotection using a base. Coupling agent used preferably is DIC and oxyma pure in DMF. Preferable base for deprotection step is 20% of piperidine in Dimethyl formamide.

In step-e) Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2) was condensed with peptide resin obtained from step-b) in presence of a coupling agent followed by deprotection using a base.

Coupling agent used preferably is DIC and oxyma pure in DMF. Preferable base for deprotection step is 20% of piperidine in Dimethyl formamide.

In step-f) condensation of Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1) with peptide resin obtained in step-b) in presence of a coupling agent to obtain protected Liraglutide. Coupling agent used preferably is DIC and oxyma pure in DMF

In step-g) cleavage of protected Liraglutide from solid support resin using a reagent to obtain crude Liraglutide. The preferably used reagent in cleavage step is cocktail mixture of TFA, TIPS, water and DTT.

The cleaving of peptide from the resin involves treating the protected peptide anchored to the resin with an acid and at least one scavenger. The peptide cleavage reagent used in the process of the present invention is a cocktail mixture of acid, scavengers and solvents.

The reaction temperature may range from 5°C to 30°C, preferably 10-15°C. The duration of the reaction may range from 2 to 6 hours, preferably for the period of 3-4 hours.
In step-h), the obtained crude Liraglutide was purified on reverse phase HPLC using a buffer and a solvent followed by freeze drying to obtain Liraglutide.

Buffer used in the reaction is selected from the group consisting of Glacial acetic acid, ammonia solution, Trifluoroacetic anhydride in water, Purified water, Orth phosphoric acid in water, acetonitrile, ethanol, methanol, ethyl acetate, triethylamine in water, ammonium acetate in water, ammonium bicarbonate in water or its mixture.

The Fmoc protected amino acids are commercially available or may be prepared according to procedures known in the literature.

The coupling reactions may be monitored by Kaiser test. The cleavage of the peptide from the solid support may be accomplished by any conventional methods well known in the art.
Accordingly, the present invention provides solution phase peptide synthesis for the preparation of Liraglutide of compound of formula-I.

Scheme-II

In other embodiment, the present invention provides a hybrid approach for the preparation of Liraglutide compound of formula-I.
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Glu-Palmitoyl)-Glu-Phe-lle-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
Formula-I
which comprises:
a) synthesis of fragments-1, -2, -3, -4 and -5 on solid support
Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1)
Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2)
Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3)
Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH (Fragment-4)
Fmoc-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu (Fragment-5);
b) condensing Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp (Boc)-OH (Fragment-4) with Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu (Fragment-5) in presence of coupling agent and solvent followed by deprotection to obtain Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu;
c) condensing Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3) with peptide obtained in stage-b) in presence of coupling agent followed by deprotection to obtain Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu;
d) condensing Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2) with peptide obtained in stage-c) in presence of a coupling agent followed by deprotection to obtain Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu;
e) condensing Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1) with peptide obtained in step-d) in presence of a coupling agent to obtain protected Liraglutide;
f) cleaving the protected Liraglutide using a reagent to obtain crude Liraglutide;
g) purifying the crude Liraglutide by preparative HPLC to obtain pure Liraglutide.

In step-b), Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH (Fragment-4) was condensed with Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu (Fragment-5) in presence of coupling agent to obtain protected Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu.
Coupling agent used preferably in this step is EDC.HCl and HOBt in DCM.

The reaction temperature may range from 25°C to 30°C. The duration of the reaction may range from 10 to 30 minutes, preferably for the period of 15-20 minutes.

Deprotection of Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu was carried out by using a base. The base used in the reaction is preferably using tert-butyl amine.

In step-c), Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3) was condensed with peptide obtained from step-b) in presence of a coupling agent to obtain Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu.

Coupling agent used preferably in this step is EDC.HCl and HOBt in DCM.

The reaction temperature may range from 25°C to 30°C. The duration of the reaction may range from 10 to 30 minutes, preferably for the period of 15-20 minutes.

Deprotection of peptide was carried out by using a base. The base used preferably in this reaction is tert-butyl amine.

In step-d), Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2) was condensed with peptide obtained from step-c) in presence of a coupling agent to obtain Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg (pbf) Gly-Arg (pbf)-Gly-OtBu peptide.

Coupling agent used preferably in this step is EDC.HCl and HOBt in DCM.

The reaction temperature may range from 25°C to 30°C. The duration of the reaction may range from 10 to 30 minutes, preferably for the period of 15-20 minutes.

Deprotection of obtained peptide was carried out by using a base. The base used preferably in this reaction is tert-butyl amine.

In step-e), Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1) was condensed with peptide obtained in step-d) in presence of a coupling agent to obtain protected Liraglutide.

Coupling agent used preferably in this step is EDC.HCl and HOBt in DCM.

The reaction temperature may range from 25°C to 30°C. The duration of the reaction may range from 10 to 30 minutes, preferably for the period of 15-20 minutes.

In step-f), protected Liraglutide obtained from step-e) was deprotected using a reagent to obtain crude Liraglutide.

The preferably used reagent in cleavage step is cocktail mixture of TFA, TIPS, water and DTT.

The deprotection of protected peptide carried out by treating with an acid and at least one scavenger. The peptide cleavage reagent used in the process of the present invention is a cocktail mixture of acid, scavengers and solvents.

The reaction temperature may range from 5°C to 30°C, preferably 10-15°C. The duration of the reaction may range from 2 to 6 hours, preferably for the period of 3-4 hours.

In step-g), the obtained crude Liraglutide was purified on reverse phase HPLC using a buffer and a solvent, followed by freeze drying to obtain Liraglutide.

Buffer used in this reaction is selected from the group consisting of Glacial acetic acid, ammonia solution, Trifluoroacetic anhydride in water, Purified water, Orth phosphoric acid in water, acetonitrile, Triton-X-100, ethanol, methanol, ethyl acetate, triethyl amine in water, ammonium acetate in water, ammonium bicarbonate in water or its mixture.

The Fmoc protected amino acids are commercially available or may be prepared according to procedures known in the literature.

The coupling reactions may be monitored by Kaiser test. The cleavage of the peptide from the solid support may be accomplished by any conventional methods well known in the art.

In yet another embodiment, the present invention relates to novel fragments-2 and -4 which are useful in the preparation of Liraglutide.
Fragment-2: Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH
Fragment-4: Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH

In yet another embodiment, The present invention provides a solid phase peptide synthesis for the preparation of Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH of fragment-2
which comprises:
a) anchoring Fmoc-Val-Ser(Oxa)-OH to a resin in presence of a coupling agent;
b) selective deprotection of amino acid using a base;
c) coupling of Fmoc-Ser(tBu)-OH to a resin obtained in step-a) in presence of coupling agent in a solvent to obtain dipeptide resin;
d) sequential coupling of Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH to the obtained resin in step-a) in presence of a coupling agent;
e) cleaving of protected peptide from solid support resin in presence of a reagent to get fragment-2.

In step-a), CTC resin was taken in a SPPS reactor and dichloromethane was added to it.
In step-b) deprotection was carried out in presence of a base. The base preferably used in this step is 20% piperidine in dimethylformamide.

In step-c), condensation of peptide resin obtained in step-a) with Fmoc-Ser(tBu)-OH in presence of a coupling agent. The coupling agent preferably used in the reaction is diisopropyl carbodiimide (DIC).

The reaction temperature may range from 25°C to 30°C. The duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.
Selective deprotection of peptide resin obtained from step-c) using a base. The base preferably used in this reaction is 20% of piperidine in Dimethyl formamide.
The reaction temperature may range from 25°C to 30°C.

In step-d) Sequential addition of Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH to the obtained resin in step-a) in presence of a coupling agent. Coupling agent preferably used in this step is DIC, oxyma pure in DMF. The base preferable used in deprotection reaction is this step is 20% of piperidine in Dimethyl formamide.

In step-e) cleavage is carried out for protected peptide from solid support resin using a reagent to obtain crude Liraglutide.

The preferably used reagent in cleavage step is cocktail mixture of TFA, TIPS, water and DTT.

In yet another embodiment, The present invention also provides a solid phase peptide synthesis for the preparation of Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH of fragment-4
which comprises:
a) anchoring Fmoc-Trp(Boc)-OH to a resin in presence of a coupling agent;
b) selective deprotection of amino acid using a base;
c) coupling of Fmoc-Ala-OH to a resin obtained in step-a) in presence of coupling agent in a solvent to obtain dipeptide resin;
d) sequential coupling of Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys[Pal(Glu-OtBu)]-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH to the obtained resin in step-a) in presence of a coupling agent;
e) cleaving of protected peptide from solid support resin in presence of a reagent to get fragment-4.

In step-a), CTC resin was taken in a SPPS reactor and dichloromethane was added to it. Deprotecting the Fmoc group in presence of a base, preferably using 20% piperidine in dimethylformamide.

In step-c), condensation of peptide resin obtained in step-b) with Fmoc-Ala-OH in presence of a coupling agent. Coupling agent used preferably is DIC, oxyma pure in DMF.

The reaction temperature may range from 25°C to 30°C. The duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.
In step-d), sequential addition of Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys[Pal(Glu-OtBu)]-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH to the obtained resin in step-a) in presence of a coupling agent. Coupling agent used in preferable in this step is DIC and oxyma pure in DMF.

The base used in deprotection step is preferably using 20% of piperidine in Dimethyl formamide.
In step-e) cleavage is carried out for protected peptide from solid support resin using a reagent to obtain crude Liraglutide.

The preferably used reagent in cleavage step is cocktail mixture of TFA, TIPS, water and DTT.

In yet another embodiment, the present invention provides a solid phase peptide synthesis for the preparation of Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu (Fragment-6)
Which comprises:
a) anchoring Fmoc-Arg(Pbf)-OH to a resin in presence of a coupling agent;
b) selective deprotection of amino acid using a base;
c) coupling of Fmoc-Gly-OH to a resin obtained in step-a) in presence of coupling agent in a solvent to obtain dipeptide resin;
d) sequential coupling of Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Cbz)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH to the obtained resin in step-a) in presence of a coupling agent and solvent;
e) partial deprotection of peptide obtained in step-d) in presence of a reagent to obtain 14 amino acid chain peptide;
f) Coupling of H-Gly-OtBu.HCl to 14 amino acid chain peptide obtained from step-e) in presence of coupling agent to obtain protected 15 amino acid peptide chain;
g) deprotection of protected peptide obtained from step-f) in presence of a reagent to get 15 amino acid peptide chain;
h) coupling of Pal-Glu(OSu)-OtBu to 15 amino acid peptide chain in step-g) in presence of a base to obtain protected 15 amino acid peptide chain;
i) deprotection of protected 15 amino acid peptide chain in step-h) in presence of reagent to obtain fragment-6.

In step-a), CTC resin was taken in a SPPS reactor and dichloromethane was added to it. Fmoc-Arg(Pbf)-OH was added to the resulting reaction mixture in presence of diisopropyl ethylamine.

In step-b), Deprotecting the Fmoc group in presence of a base, preferably using 20% piperidine in dimethylformamide. The reaction temperature may range from 25°C to 30°C.

In step-c), condensation of peptide resin obtained in step-a) with Fmoc-Gly-OH in presence of coupling agent. The coupling agent preferable used in this step is DIC and oxyma pure in DMF.

In step-d) Sequential addition of Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys[Pal(Glu-OtBu)]-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH to the obtained resin in step-a) in presence of a coupling agent. The coupling agent preferable used in this step is DIC and oxyma pure in DMF

The reaction temperature may range from 25°C to 30°C. The duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.
The base used in deprotection step is preferably using 20% of piperidine in Dimethyl formamide.

In step-e) partial deprotection is carried out for protected peptide from solid support resin using a reagent to obtain Fragment-6.

Reagent used in partial deprotection is selected from the group consisting of TFA, TIPS, Water, DTT, Thioanisole, EDT, DMS, cresol, phenol, thiocresol, ammonium iodide, 2,2'-(ethylene dioxy)diethane or its mixture. Preferably using TFA in dichloromethane.

In step-f) coupling of H-Gly-OtBu.HCl to the 14 amino acid peptide chain obtained in step-e) in presence of coupling agent. Coupling agent preferably used in this step is EDC.HCl, HOAt in DMF.

In step-g), reagent used for deprotection of peptide obtained in step-f) is preferably using palladium on carbon.

In step-h) coupling of Pal-Glu(OSu)-OtBu to 15 amino acid peptide chain in step-g) in presence of a base like sodium carbonate solution or potassium carbonate solution to obtain protected 15 amino acid peptide chain

In step-i) deprotection of protected 15 amino acid peptide chain in step-h) is carried out in presence of tert-butyl amine.

In yet another embodiment, the present invention provides a solid phase peptide synthesis for the preparation of Boc-His(Trt)-Ala-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-7)
Which comprises:
a) anchoring Fmoc-Val-Ser(Oxa)-OH to a resin in presence of a coupling agent;
b) selective deprotection of amino acid using a base;
c) coupling of Fmoc-Ser(tBu)-OH to a resin obtained in step-a) in presence of coupling agent in a solvent to obtain dipeptide resin;
d) sequential coupling of Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Boc-His(Trt)-OH to the obtained resin in step-a) in presence of a coupling agent;
e) cleaving of protected peptide from solid support resin in presence of a reagent to get fragment-7.

In step-a), CTC resin was taken in a SPPS reactor and dichloromethane was added to it. Fmoc-Val-Ser(Oxa)-OH was added to the resulting reaction mixture in presence of coupling agent and diisopropyl ethylamine. The coupling agent preferably used in this step is DIC and oxyma pure in DMF.

In step-b), Deprotecting the Fmoc group in presence of a base. The base used in this step is preferably using 20% piperidine in dimethylformamide.

The reaction temperature may range from 25°C to 30°C. The duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.
In step-c) condensation of peptide resin obtained in step-b) with Fmoc-Ser(tBu)-OH in presence of coupling agent. The coupling agent preferably used in this step is DIC and oxyma pure in DMF.
In step-d) Sequential addition of Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Boc-His(Trt)-OH to the obtained resin in step-a) in presence of a coupling agent. The coupling agent preferably used in this step is DIC and oxyma pure in DMF.

The reaction temperature may range from 25°C to 30°C. The duration of the reaction may range from 1 to 4 hours, preferably for the period of 2-3 hours.
Deprotection of obtained peptide was carried out using base, preferably using 20% of piperidine in Dimethyl formamide.

In step-e) cleavage is carried out for protected peptide from solid support resin using a reagent to obtain Fragment-7.

The preferably used reagent in cleavage step is TFA in dichloromethane.

Accordingly, the present invention provides solution phase peptide synthesis for the preparation of Liraglutide of compound of formula-I by using three fragment approach.

Scheme-III

Accordingly, the present invention provides solution phase peptide synthesis for the preparation of Liraglutide of compound of formula-I by using four fragment approach.

Scheme-IV
In yet another embodiment, the present invention provides a hybrid approach for the preparation of Liraglutide compound of formula-I.
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Glu-Palmitoyl)-Glu-Phe-lle-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
Formula-I
which comprises:
a) synthesis of fragments-3, -6 and -7 on solid support;
Boc-His(Trt)-Ala-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-7)
Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3)
H-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu (Fragment-6);
b) condensing Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu (Fragment-6) with Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3) in presence of coupling agent in solvent in in-situ manner followed by deprotection in presence of a base to obtain H-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu;
c) condensing Boc-His(Trt)-Ala-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp (OtBu)-Val-Ser(Oxa)-OH (Fragment-7) with peptide obtained in step-b) in presence of a coupling agent to obtain protected Liraglutide;
d) cleaving the protected Liraglutide using a reagent to obtain crude Liraglutide;
e) purifying the crude Liraglutide by preparative HPLC to obtain pure Liraglutide.

In step-a) fragments-3, -6 and -7 are prepared by solid phase peptide synthesis.
In step-b), Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)- Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu (Fragment-6) was condensed with Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3) in presence of coupling agent obtain protected Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu in in-situ manner. Further, it is deprotected in presence of a base to obtain Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu.
The coupling agent preferably used in this reaction is EDC.HCl and HOAt in DCM.
The reaction temperature may range from 25°C to 30°C. The duration of the reaction may range from 10 to 30 minutes, preferably for the period of 15-20 minutes.
Deprotection of Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu was carried out by using a base in in-situ manner, The base preferably used is tert-butylamine.
In step-c), Boc-His(Trt)-Ala-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp (OtBu)-Val-Ser(Oxa)-OH (Fragment-7) was condensed with peptide obtained from step-b) in presence of a coupling agent to obtain protected Liraglutide.
Coupling agent preferably used in this reaction is EDC.HCl and HOAt in DCM.
The reaction temperature may range from 25°C to 30°C. The duration of the reaction may range from 1 to 3 hours, preferably for the period of 1-2 hours.
In step-d), protected Liraglutide obtained from step-c) was deprotected using a reagent to obtain crude Liraglutide.
The preferably used reagent in cleavage step is cocktail mixture of TFA, TIPS, water and DTT.

The deprotection of protected peptide carried out by treating with an acid and at least one scavenger. The peptide cleavage reagent used in the process of the present invention is a cocktail mixture of acid, scavengers and solvents.
The reaction temperature may range from 5°C to 30°C, preferably 10-15°C. The duration of the reaction may range from 2 to 6 hours, preferably for the period of 3-4 hours.
In step-e), the obtained crude Liraglutide was purified on reverse phase HPLC using a buffer and a solvent, followed by freeze drying to obtain Liraglutide.
where the buffer used in the reaction is selected from the group consisting of Glacial acetic acid, ammonia solution, Trifluoroacetic anhydride in water, Purified water, Orth phosphoric acid in water, acetonitrile, Triton-X-100, ethanol, methanol, ethyl acetate, triethyl amine in water, ammonium acetate in water, ammonium bicarbonate in water or its mixture.

The Fmoc protected amino acids and Fmoc-Lys(Pal-?-Glu-OtBu)-OH are commercially available or may be prepared according to procedures known in the prior art literature.
The coupling reactions may be monitored by Kaiser test. The cleavage of the peptide from the solid support may be accomplished by any conventional methods well known in the art.
In yet another embodiment, the present invention provides a hybrid approach for the preparation of Liraglutide compound of formula-I.
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Glu-Palmitoyl)-Glu-Phe-lle-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
Formula-I
which comprises:
a) Synthesis of fragments-1, -2, -3 and -6 on solid support;
Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1)
Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2)
Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3)
H-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg (Pbf)-Gly-Arg(Pbf)-Gly-OtBu (Fragment-6);
b) condensing Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu (Fragment-6) with Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3) in presence of coupling agent and solvent in in-situ manner followed by deprotection in presence of base to obtain H-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu (OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu;
c) condensing Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2) with peptide obtained in step-b) in presence of a coupling agent in in-situ manner followed by deprotection in presence of base to obtain H-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu;
d) condensing Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1) with peptide obtained in step-c) in presence of a coupling agent in in-situ manner followed by deprotection in presence of base to obtain protected Liraglutide;
e) cleaving the protected Liraglutide using a reagent to obtain crude Liraglutide;
f) purifying the crude Liraglutide by preparative HPLC to obtain pure Liraglutide.

In step-b), Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)- Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu (Fragment-6) was condensed with Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3) in presence of coupling agent obtain protected Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu in in-situ manner. Further, it is deprotected in presence of a base to obtain Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu.

The coupling agent preferably used in this step is EDC.HCl and HOAt in DCM.

The reaction temperature may range from 25°C to 30°C. The duration of the reaction may range from 1 to 3 hours, preferably for the period of 1-2 hours.

Deprotection of Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu was carried out by using a base in in-situ manner. The base used in the reaction is preferably using tert-butylamine.

In step-c), Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2) was condensed with peptide obtained from step-b) in presence of a coupling agent to obtain Fmoc protected Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu in in-situ manner.

The coupling agent preferably used in this step is EDC.HCl and HOAt in DCM.

The reaction temperature may range from 25°C to 30°C. The duration of the reaction may range from 1 to 3 hours, preferably for the period of 1-2 hours.

Deprotection of Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu was carried out by using a base in in-situ manner, preferably base used in deprotection reaction is tert-butylamine.
In step-d), Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH was condensed with the peptide obtained in step-c) in presence of a coupling agent and solvent to obtain protected Liraglutide. The coupling agent preferably used in this step is EDC.HCl and HOAt in DCM.

The reaction temperature may range from 25°C to 30°C. The duration of the reaction may range from 1 to 3 hours, preferably for the period of 1-2 hours.
In step-e) protected Liraglutide obtained from step-d) was deprotected using a reagent to obtain crude Liraglutide.
The deprotection of protected peptide carried out by treating with an acid and at least one scavenger. The peptide cleavage reagent used in the process of the present invention is a cocktail mixture of acid, scavengers and solvents.
The preferably used reagent in cleavage step is cocktail mixture of TFA, TIPS, water and DTT.

The reaction temperature may range from 5°C to 30°C, preferably 10-15°C. The duration of the reaction may range from 2 to 6 hours, preferably for the period of 3-4 hours.
In step-f), the obtained crude Liraglutide was purified on reverse phase HPLC using a buffer and a solvent, followed by freeze drying to obtain Liraglutide.

Preparative HPLC method for purification of Liraglutide:

Orthophosphoric acid purification:
Sample preparation: Liraglutide was dissolved in 50 ml of water and 25% aqueous ammonia solution added dropwise to get the clear solution.
Column: YMC Triart (50×250 mm, 10 µm)
Mobile phase-A: Ortho phosphoric acid (5 mL) + water (5 mL)
Mobile phase-B: water (1 mL) + Acetonitrile (4 mL) + Ortho phosphoric acid (5 mL)
Equilibrate the column with 2% mobile phase B at a flow rate of 50 mL/min.

S. No. Time Flow Mobile phase-B %
1 0.01 50 5
2 10 50 25
3 150 50 60
4 300 50 stop

Collect the fractions as 25 mL/vial

Ammonium acetate purification process:
Fraction obtained from the above purification process is diluted with water.
Mobile phase-A: water (5 Ltr) + Ammonium acetate (3.85 gms); pH adjusted to 7.5 with 25% aqueous ammonia solution.
Mobile phase-B: Acetonitrile: Isopropyl alcohol (1:1)
Equilibrate the column with 5% mobile phase-B with a flow rate of 50mL/min.

S. No. Time Flow Mobile phase-B %
1 0.01 50 5
2 10 50 25
3 150 50 60
4 300 50 stop

Collect the fractions as 25mL/vial and pooled fraction was lyophilized to get the pure Liraglutide.
Purity: 98.87%

EXPERIMENTAL PORTION

The details of the invention are given in the examples provided below, which are given to illustrate the invention only and therefore should not be construed to limit the scope of the invention.

Example-1: Process for the preparation of Liraglutide by using soild phase peptide synthesis approach

Stage-1: Synthesis of Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH [Fragment-1]

Step-A:
CTC resin (50 grams) was taken in a SPPS reactor and dichloromethane was added and allowed it to swell for 10 minutes.

Step-B:
A solution of Fmoc-Gly-OH (47.57 grams) and Diisopropylethylamine (51.69 grams) in dry dichloromethane was added to the resin obtained from step-A and stirred for 2 hours at room temperature.
The above resin was deblocked with 20% piperidine in DMF for 10-15 minutes and washed with DMF.

Step-C:
Fmoc-Glu(OtBu)-OH (76.5 grams) was dissolved in DMF and stirred for 10 minutes. DIC (22.72 grams) and oxyma (25.56 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF for 10 minutes and washed with DMF.

Step-D:
Fmoc-Ala-OH (56.03 grams) was dissolved in DMF and stirred for 10 minutes. DIC (22.72 grams) and oxyma (25.56 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.

Step-E:
Boc-His(Trt)-OH (89.46 grams) was dissolved in DMF and stirred for 10 minutes. DIC (22.72 grams) and oxyma (25.56 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was washed with DMF, isopropanol and dichloromethane.

Step-F:
Selective cleavage of CTC-resin from Boc-His(Trt)-Ala-Glu(OtBu)-Gly-CTC resin was performed with a mixture of 1% Trifluoroacetic acid in dichloromethane. The crude protected peptide was isolated by precipitating with ether.

Stage-2: Synthesis of Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH [Fragment-2]
Step-A:
CTC resin (50 grams) was taken in a SPPS reactor and dichloromethane was added and allowed it to swell for 10 minutes.

Step-B:
A solution of Fmoc-Val-Ser(Oxa)-OH (41 grams) and Diisopropylethylamine (20.77 grams) in dry dichloromethane was added to the resin obtained from step-A and stirred for 2 hours at room temperature.
The above resin was deblocked with 20% piperidine in DMF for 10-15 minutes and washed with DMF.

Step-C:
Fmoc-Ser(tBu)-OH (18.49 grams) was dissolved in DMF and stirred for 10 minutes. DIC (6 grams) and oxyma (6.82 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF for 10 minutes and washed with DMF.

Step-D:
Fmoc-Thr(tBu)-OH (19.08 grams) was dissolved in DMF and stirred for 10 minutes. DIC (6 grams) and oxyma (6.82 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.

Step-E:
Fmoc-Phe-OH (18.60 grams) was dissolved in DMF and stirred for 10 minutes. DIC (6.0 grams) and oxyma (6.82 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was washed with DMF, isopropanol and dichloromethane.

Step-F:
Fmoc-Thr(tBu)-OH (19.08 grams) was dissolved in DMF and stirred for 10 minutes. DIC (6.0 grams) and oxyma (6.82 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was washed with DMF, isopropanol and dichloromethane.

Step-G:
Selective cleavage of CTC-resin from Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-CTC resin was performed with a mixture of 1% Trifluoroacetic acid in dichloromethane. The crude protected peptide was isolated by precipitating with ether.

Stage-3: Synthesis of Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH [Fragment-3]

Step-A:
CTC resin (50 grams) was taken in a SPPS reactor and dichloromethane was added and allowed it to swell for 10 minutes.

Step-B:
A solution of Fmoc-Gly-OH (71.4 grams) and Diisopropylethylamine (51.72 grams) in dry dichloromethane was added to the resin obtained from step-A and stirred for 2 hours at room temperature.
The above resin was deblocked with 20% piperidine in DMF for 10-15 minutes and washed with DMF.

Step-C:
Fmoc-Gly(OtBu)-OH (63.82 grams) was dissolved in DMF and stirred for 10 minutes. DIC (18.92 grams) and oxyma (21.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF for 10 minutes and washed with DMF.

Step-D:
Fmoc-Leu-OH (53.01 grams) was dissolved in DMF and stirred for 10 minutes. DIC (18.92 grams) and oxyma (21.30 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.

Step-E:
Fmoc-Tyr(tBu)-OH (69 grams) was dissolved in DMF and stirred for 10 minutes. DIC (18.92 grams) and oxyma (21.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was washed with DMF, isopropanol and dichloromethane.

Step-F:
Fmoc-Ser(tBu)-OH (57.51 grams) was dissolved in DMF and stirred for 10 minutes. DIC (18.92 grams) and oxyma (21.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was washed with DMF, isopropanol and dichloromethane.

Step-G:
Selective cleavage of CTC-resin from Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-CTC resin was performed with a mixture of Trifluoroacetic acid in dichloromethane. The crude protected peptide was isolated by precipitating with ether.

Stage-4: Synthesis of Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH [Fragment-4]

Step-A:
CTC resin (50 grams) was taken in a SPPS reactor and dichloromethane was added and allowed it to swell for 10 minutes.

Step-B:
A solution of Fmoc-Trp(Boc)-OH (16.84 grams) and Diisopropylethylamine (10.35 grams) in dry dichloromethane was added to the resin obtained from step-A and stirred for 2 hours at room temperature.
The above resin was deblocked with 20% piperidine in DMF for 10-15 minutes and washed with DMF.

Step-C:
Fmoc-Ala-OH (9.33 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF for 10 minutes and washed with DMF.

Step-D:
Fmoc-Ile-OH (10.60 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.

Step-E:
Fmoc-Phe-OH (11.61 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was washed with DMF, isopropanol and dichloromethane.

Step-F:
Fmoc-Glu(OtBu)-OH (12.76 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF, isopropanol and dichloromethane.

Step-G:
Fmoc-Lys[Pal(Glu-OtBu)]-OH (11.88 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.

Step-H:
Fmoc-Ala-OH (9.33 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF, isopropanol and dichloromethane.

Step-I:
Fmoc-Ala-OH (9.33 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF, isopropanol and dichloromethane.

Step-J:
Fmoc-Gln(Trt)-OH (18.32 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF, isopropanol and dichloromethane.

Step-K:
Selective cleavage of CTC-resin from Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-CTC resin was performed with a mixture of Trifluoroacetic acid in dichloromethane. The crude protected peptide was isolated by precipitating with ether.

Stage-5: Synthesis of H-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu [Fragment-5]
Step-A:
CTC resin (50 grams) was taken in a SPPS reactor and dichloromethane was added and allowed it to swell for 10 minutes.

Step-B:
A solution of Fmoc-Arg(Pbf)-OH (129.76 grams) and Diisopropylethylamine (69.85 grams) in dry dichloromethane was added to the resin obtained from step-A and stirred for 2 hours at room temperature.
The above resin was deblocked with 20% piperidine in DMF for 10-15 minutes and washed with DMF.

Step-C:
Fmoc-Gly-OH (44.59 grams) was dissolved in DMF and stirred for 10 minutes. DIC (23.2 grams) and oxyma (21.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF for 10 minutes and washed with DMF.

Step-D:
Fmoc-Arg(Pbf)-OH (97.3 grams) was dissolved in DMF and stirred for 10 minutes. DIC (23.2 grams) and oxyma (21.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.

Step-E:
Fmoc-Val-OH (50.9 grams) was dissolved in DMF and stirred for 10 minutes. DIC (23.2 grams) and oxyma (21.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF, isopropanol and dichloromethane.

Step-F:
Fmoc-Leu-OH (53 grams) was dissolved in DMF and stirred for 10 minutes. DIC (23.2 grams) and oxyma (21.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF, isopropanol and dichloromethane.

Step-G:
Selective cleavage of CTC-resin from Fmoc-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-CTC resin was performed with a mixture of Trifluoroacetic acid and TIPS in dichloromethane. The crude protected peptide was isolated by precipitating with ether.

Step-H:
Resin and peptide obtained from step-G were taken in a SPPS reactor and N,N-Dimethyl formamide was added and allowed it to swell for 10 minutes. Gly-OtBu. HCl (6.57 grams) is added in presence of EDC.HCl (7.59 grams) and NMM (3.48 grams) at 25-30°C and stirred for 2-3 hours at the same temperature. Cooled the resulting reaction mixture and water was added to it. Filtered the precipitated solid and washed with water.

Step-I:
Selective cleavage of resin from protected peptide resin obtained from step-H was performed with tert-butyl amine (35.1 grams) in n-heptane. The crude peptide was extracted with ethyl acetate and washed with water followed by brine solution. Filtered the precipitated peptide.

Stage-6: Synthesis of Liraglutide by solid phase peptide fragment condensation:

Step-A:
CTC resin was taken in a SPPS reactor and dichloromethane was added and allowed it to swell for 10 minutes. H-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu [Fragment-5] and Diisopropyl ethylamine in dry dichloromethane were added to the resin and stirred for 2 hours at 25-30°C. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resulting resin was deblocked with 20% piperidine in DMF.

Step-B:
Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH (Fragment-4) was dissolved in DMF and stirred for 10 minutes. DIC and oxyma were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF. The resulting resin was deblocked with 20% piperidine in DMF followed by washing with DMF.

Step-C:
Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3) was dissolved in DMF and stirred for 10 minutes. DIC and oxyma were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF. The resulting resin was deblocked with 20% piperidine in DMF followed by washing with DMF.

Step-D:
Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2) was dissolved in DMF and stirred for 10 minutes. DIC and oxyma were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF. The resulting resin was deblocked with 20% piperidine in DMF followed by washing with DMF.

Step-E:
Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1) was dissolved in DMF and stirred for 10 minutes. DIC and oxyma were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF.

Step-F:
Selective cleavage of resin from protected peptide resin obtained from step-E was performed with a cocktail mixture of TFA, TIPS, water and DTT in presence od DCM at 10-15°C and stirred for 3-6 hours at the same temperature. Chilled DIPE was added to the resulting mixture and stirred for 2 hours. The precipitated solid was filtered and washed with DCM followed by DIPE to get crude Liraglutide.

Stage-7: Preparative HPLC purification of Liraglutide
Crude Liraglutide obtained in step-F was dissolved in 0.5 M ammonium formate and loaded on to preparative C8 column (50 X 250 mm, 100 A°). The peptide was purified using a linear gradient of aqueous TFA (0.1%) and acetonitrile: methanol (8:1, 0.1% TFA) from 40% to 90% over 60 minutes. The pure fraction containing the Liraglutide was pooled. Volatiles were removed under reduced pressure and aqueous layer was lyophilized to give Liraglutide as a powder. The resulting peptide was analysed by RP-HPLC and confirmed by MALDI or LC-MS.

Example-2: Process for the preparation of Liraglutide by using solution phase peptide synthesis approach

Step-A:
Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH (Fragment-4) obtained from stage-4 of example-1 was dissolved in DMF and stirred for 10 minutes at 25-30°C. H-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu [Fragment-5] obtained from stage-5 of example-1, EDC.HCl and HOBT in DCM were added to the resulting reaction mixture at 25-30°C and stirred for 15-20 minutes at the same temperature. Precipitated solid was filtered and washed with water and hexane. The resulting protected peptide was deprotected with tert-butyl amine and n-heptane in DMF. Filtered the precipitated solid and washed with water, hexane and methanol to get Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu.

Step-B:
Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3) obtained from stage-3 of example-1 was dissolved in DMF and stirred for 10 minutes. H-Protected 16 amino acid peptide obtained in step-A was added in presence of EDC.HCl and HOBT in DCM at 25-30°C and stirred for 15-20 minutes at the same temperature. Precipitated solid was filtered and washed with water and hexane followed by dried under vacuum for 2 hours. The resulting protected peptide was deprotected with tert-butyl amine and n-heptane in DMF. Filtered the precipitated solid and washed with water, hexane and methanol to get Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu.

Step-C:
Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2) obtained from stage-2 of example-1 was dissolved in DMF and stirred for 10 minutes. Peptide obtained in step-B was added in presence of EDC.HCl and HOBT in DCM at 25-30°C and stirred for 15-20 minutes at the same temperature. Precipitated solid was filtered and washed with water and hexane followed by dried under vacuum for 2 hours. The resulting protected peptide was deprotected with tert-butyl amine and n-heptane in DMF. Filtered the precipitated solid and washed with water, hexane and methanol to get Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu.

Step-D:
Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1) obtained from stage-1 of example-1 was dissolved in DMF and stirred for 10 minutes. Peptide obtained in step-C was added in presence of EDC.HCl and HOBT in DCM at 25-30°C and stirred for 15-20 minutes at the same temperature. Precipitated solid was filtered and washed with water and hexane followed by dried under vacuum for 2 hours to get Boc-protected peptide.
The resulting protected peptide was cleaved with a cocktail mixture of TFA, TIPS, water and DTT in presence of DCM at 10-15°C and stirred for 3-6 hours at the same temperature. Chilled DIPE was added to the resulting mixture and stirred for 2 hours. The precipitated solid was filtered and washed with DCM followed by DIPE to get crude Liraglutide.

Example-3: Process for the preparation of Liraglutide by using hybrid approach [three fragment approach]
Stage-1: solid phase peptide synthesis of Boc-His(Trt)-Ala-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-7)
Step-A:
CTC resin (20 grams) was taken in a SPPS reactor and dichloromethane (120 mL) was added and allowed it to swell for 10 minutes.

Step-B:
A solution of Fmoc-Val-Ser(Oxa)-OH (41 grams) and Diisopropylethylamine (20.77 grams) in dry dichloromethane was added to the resin obtained from step-A and stirred for 2 hours at room temperature. The above resin was deblocked with 20% piperidine in DMF for 10-15 minutes and washed with DMF.

Step-C:
Fmoc-Ser(tBu)-OH (18.49 grams) was dissolved in DMF and stirred for 10 minutes. DIC (6 grams) and oxyma (6.82 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF for 10 minutes and washed with DMF.

Step-D:
Fmoc-Thr(tBu)-OH (19.08 grams) was dissolved in DMF and stirred for 10 minutes. DIC (6 grams) and oxyma (6.82 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.

Step-E:
Fmoc-Phe-OH (18.60 grams) was dissolved in DMF and stirred for 10 minutes. DIC (6.0 grams) and oxyma (6.82 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.

Step-F:
Fmoc-Thr(tBu)-OH (19.08 grams) was dissolved in DMF and stirred for 10 minutes. DIC (6.0 grams) and oxyma (6.82 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.

Step-G:
Fmoc-Gly-OH (47.57 grams) was dissolved in DMF and stirred for 10 minutes. DIC (6.0 grams) and oxyma (6.82 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.

Step-H:
Fmoc-Glu(OtBu)-OH (76.5 grams) was dissolved in DMF and stirred for 10 minutes. DIC (22.72 grams) and oxyma (25.56 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF for 10 minutes and washed with DMF.

Step-I:
Fmoc-Ala-OH (56.03 grams) was dissolved in DMF and stirred for 10 minutes. DIC (22.72 grams) and oxyma (25.56 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.

Step-J:
Boc-His(Trt)-OH (89.46 grams) was dissolved in DMF and stirred for 10 minutes. DIC (22.72 grams) and oxyma (25.56 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was washed with DMF, isopropanol and dichloromethane.

Step-K:
Selective cleavage of CTC-resin from Boc-His(Trt)-Ala-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-CTC resin was performed with a mixture of 1% Trifluoroacetic acid in dichloromethane. The crude protected peptide was isolated by precipitating with ether.

Stage-2: Solid phase peptide synthesis of Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu (Fragment-6)

Step-A:
CTC resin (50 grams) was taken in a SPPS reactor and dichloromethane was added and allowed it to swell for 10 minutes.

Step-B:
A solution of Fmoc-Arg(Pbf)-OH (129.76 grams) and Diisopropylethylamine (69.85 grams) in dry dichloromethane was added to the resin obtained from step-A and stirred for 2 hours at room temperature.
The above resin was deblocked with 20% piperidine in DMF for 10-15 minutes and washed with DMF.

Step-C:
Fmoc-Gly-OH (44.59 grams) was dissolved in DMF and stirred for 10 minutes. DIC (23.2 grams) and oxyma (21.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF for 10 minutes and washed with DMF.

Step-D:
Fmoc-Arg(Pbf)-OH (97.3 grams) was dissolved in DMF and stirred for 10 minutes. DIC (23.2 grams) and oxyma (21.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.

Step-E:
Fmoc-Val-OH (50.9 grams) was dissolved in DMF and stirred for 10 minutes. DIC (23.2 grams) and oxyma (21.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.

Step-F:
Fmoc-Leu-OH (53 grams) was dissolved in DMF and stirred for 10 minutes. DIC (23.2 grams) and oxyma (21.3 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.

Step-G:
A solution of Fmoc-Trp(Boc)-OH (16.84 grams) and Diisopropylethylamine (10.35 grams) in dry dichloromethane was added to the resin obtained from step-A and stirred for 2 hours at room temperature. The above resin was deblocked with 20% piperidine in DMF for 10-15 minutes and washed with DMF.

Step-H:
Fmoc-Ala-OH (9.33 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the room temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser tests. After completion of the reaction, the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF for 10 minutes and washed with DMF.

Step-I:
Fmoc-Ile-OH (10.60 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.

Step-J:
Fmoc-Phe-OH (11.61 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction the resin was washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.

Step-K:
Fmoc-Glu(OtBu)-OH (12.76 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.

Step-L:
Fmoc-Lys(Cbz)-OH (11.88 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was drained and washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF. The resulting resin was deblocked with 20% piperidine in DMF.

Step-M:
Fmoc-Ala-OH (9.33 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.

Step-N:
Fmoc-Ala-OH (9.33 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF, isopropanol and dichloromethane. The resulting resin was deblocked with 20% piperidine in DMF.

Step-O:
Fmoc-Gln(Trt)-OH (18.32 grams) was dissolved in DMF and stirred for 10 minutes. DIC (3.79 grams) and oxyma (4.26 grams) were added to the resulting reaction mixture and stirred for 5-10 minutes at the same temperature. It was added to the resin obtained in step-A and stirred for 2-3 hours at room temperature. The progress of coupling was monitored by Kaiser test. After completion of reaction, the resin was washed with DMF, isopropanol and dichloromethane.

Step-P:
H-Gly-OtBu.HCl was dissolved in DMF and stirred for 10 minutes. EDC.HCl and HOAt were added to the resulting reaction mixture and stirred for 1-2 hours at the same temperature. It was added to the resin obtained in step-A and stirred for 1-2 hours. Protected 15 amino acid peptide chain was deprotected with 5% palladium on carbon to obtain 15 amino acid peptide chain.

Step-Q:
Pal-Glu(OSu)-OtBu was dissolved in DMF and added to 15 amino acid peptide chain in presence of sodium carbonate solution and stirred for 4-5 hours at room temperature. The resulting peptide is precipitated with water, hexane and dried under vacuum at 45° C for 2-3 h to obtain protected 15 Amino acid peptide chain. The obtained peptide was dissolved in DMF and cooled to 5-10°C. Tert-butyl amine was added to the resulting solution. Water was added to the resulting reaction mixture to obtain Fragment-6.

Stage-4: Synthesis of Liraglutide by solution phase peptide fragment condensation:

Step-A:
Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu (3.7 grams) (Fragment-6) was dissolved in DMF and stirred for 10 minutes at 25-30°C. Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (1.37 grams) [Fragment-3]obtained from stage-3 of example-1, EDC.HCl and HOAT in DCM were added to the resulting reaction mixture at 25-30°C and stirred for 15-20 minutes at the same temperature. Precipitated solid was filtered and washed with water and hexane. The resulting protected peptide was deprotected with tert-butylamine. Filtered the precipitated solid and washed with water and hexane to get Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu.

Step-B:
Boc-His(Trt)-Ala-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-7) was dissolved in DMF and stirred for 10 minutes. H-Protected 20 amino acid peptide obtained in step-A was added in presence of EDC.HCl and HOAT in DCM at 25-30°C and stirred for 1-2 hours at the same temperature. Precipitated solid was filtered and washed with water and hexane followed by dried under vacuum at 40-45°C for 2 hours. The resulting protected peptide was cleaved with a cocktail mixture of TFA, TIPS, water and DTT in presence of DCM at 25-30°C and stirred for 3-6 hours at the same temperature. Chilled DIPE was added to the resulting mixture and stirred for 2 hours. The precipitated solid was filtered and washed with DCM followed by DIPE to get crude Liraglutide.

Stage-C: Preparative HPLC purification of Liraglutide

Crude Liraglutide obtained in step-B was dissolved in purified water and 25% aqueous ammonia and loaded on to preparative YMC Triart (50 × 250 mm, 10 µm). The peptide was purified using a linear gradient of aqueous orthophosphoric acid and acetonitrile: water with flow rate of 50 mL/minute. The pure fraction containing the Liraglutide was pooled.
It is diluted with purified water and purified using a linear gradient of water, ammonium acetate and aqueous ammonia [adjusted to pH = 7.5]; acetonitrile: isopropyl alcohol with a flow rate of 50 mL/minute. Volatiles were removed under reduced pressure and aqueous layer was lyophilized to give Liraglutide as a powder. The resulting peptide was analysed by RP-HPLC and confirmed by MALDI or LC-MS.

Purity: 98.87 %

Example-4: Process for the preparation of Liraglutide by using hybrid approach [four fragment approach]

Stage-1: Synthesis of Liraglutide by solution phase peptide fragment condensation:
Step-A:
Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu (Fragment-6) obtained from stage-2 of example-3 was dissolved in DMF and stirred for 10 minutes at 25-30°C. Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3) obtained from stage-3 of example-1, EDC.HCl and HOAT in DCM were added to the resulting reaction mixture at 25-30°C and stirred for 15-20 minutes at the same temperature. Precipitated solid was filtered and washed with water and hexane. The resulting protected peptide was deprotected with tert-butylamine. Filtered the precipitated solid and washed with water and hexane to get Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu.

Step-B:
Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2) was dissolved in DMF and stirred for 10 minutes. H-Protected 20 amino acid peptide obtained in step-A was added in presence of EDC.HCl and HOAT in DCM at 25-30°C and stirred for 15-20 minutes at the same temperature. Precipitated solid was filtered and washed with water and hexane. The resulting protected peptide was deprotected with tert-butylamine. Filtered the precipitated solid and washed with water and hexane to get Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu.

Step-C:
Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1) was dissolved in DMF and stirred for 10 minutes. H-Protected amino acid peptide obtained in step-B was added in presence of EDC.HCl and HOAT in DCM at 25-30°C and stirred for 15-20 minutes at the same temperature. Precipitated solid was filtered and washed with water and hexane followed by dried under vacuum for 2 hours. The resulting protected peptide was cleaved with a cocktail mixture of TFA, TIPS, water and DTT in presence of DCM at 10-15°C and stirred for 3-6 hours at the same temperature. Chilled DIPE was added to the resulting mixture and stirred for 2 hours. The precipitated solid was filtered and washed with DCM followed by DIPE to get crude Liraglutide. ,CLAIMS:1. A fragment-based hybrid approach for the preparation of Liraglutide of formula-I.
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Glu-Palmitoyl)-Glu-Phe-lle-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
Formula-I
which comprises:
a) synthesis of fragments-3, -6 and -7 on solid support;
Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3)
H-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu (Fragment-6)
Boc-His(Trt)-Ala-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-7);

b) condensing Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu (Fragment-6) with Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3) in presence of coupling agent in solvent in-situ manner followed by deprotection in presence of a base to obtain H-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu;
c) condensing Boc-His(Trt)-Ala-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp (OtBu)-Val-Ser(Oxa)-OH (Fragment-7) with peptide obtained in step-b) in presence of a coupling agent to obtain protected Liraglutide;
d) cleaving the protected Liraglutide using a reagent to obtain crude Liraglutide;
e) purifying the crude Liraglutide by preparative HPLC to obtain pure Liraglutide.

2. A fragment-based hybrid approach for the preparation of Liraglutide of formula-I.
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala- Lys(Glu-Palmitoyl)-Glu-Phe-lle-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
Formula-I
which comprises:
a) Synthesis of fragments-1, -2, -3 and -6 on solid support;
Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1)
Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2)
Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3)
H-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg (Pbf)-Gly-Arg(Pbf)-Gly-OtBu (Fragment-6);
b) condensing Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu (Fragment-6) with Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3) in presence of coupling agent and solvent in in-situ manner followed by deprotection in presence of base to obtain H-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu (OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu;
c) condensing Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2) with peptide obtained in step-b) in presence of a coupling agent in in-situ manner followed by deprotection in presence of base to obtain H-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu;
d) condensing Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1) with peptide obtained in step-c) in presence of a coupling agent in in-situ manner followed by deprotection in presence of base to obtain protected Liraglutide;
e) cleaving the protected Liraglutide using a reagent to obtain crude Liraglutide;
f) purifying the crude Liraglutide by preparative HPLC to obtain pure Liraglutide.

3. A fragment-based hybrid approach for the preparation of Liraglutide of formula-I
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Glu-Palmitoyl)-Glu-Phe-lle-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
Formula-I
which comprises:
a) synthesis of fragments-1, -2, -3, -4 and -5 on solid support
Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1)
Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2)
Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3)
Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH (Fragment-4)
Fmoc-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu (Fragment-5);
b) condensing Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp (Boc)-OH (Fragment-4) with Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu (Fragment-5) in presence of coupling agent followed by deprotection to obtain Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu;
c) condensing Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3) with peptide obtained in stage-b) in presence of coupling agent followed by deprotection to obtain Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu;
d) condensing Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2) with peptide obtained in stage-c) in presence of a coupling agent followed by deprotection to obtain Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg (pbf)-Gly-OtBu;
e) condensing Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1) with peptide obtained in step-d) in presence of a coupling agent to obtain protected Liraglutide;
f) cleaving the protected Liraglutide using a reagent to obtain crude Liraglutide;
g) purifying the crude Liraglutide by preparative HPLC to obtain pure Liraglutide.

4. A solid phase peptide synthesis for the preparation of Liraglutide of formula-I
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Glu-Palmitoyl)-Glu-Phe-lle-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
Formula-I
which comprises:
a) synthesis of fragments-1, -2, -3, -4 and -5 on solid support
Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1)
Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2)
Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3)
Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH (Fragment-4)
Fmoc-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu (Fragment-5);
b) anchoring Fmoc-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OtBu (Fragment-5) to a resin followed by selective deprotection in presence of a base;
c) condensing Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH (Fragment-4) with a resin obtained in stage-b) in presence of a coupling agent and solvent followed by deprotection in presence of a base;
d) condensing Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH (Fragment-3) with a resin obtained in stage-b) in presence of a coupling agent and solvent followed by deprotection in presence of a base;
e) condensing Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH (Fragment-2) with a resin obtained in stage-b) in presence of a coupling agent followed by deprotection in presence of a base;
f) condensing Boc-His(Trt)-Ala-Glu(OtBu)-Gly-OH (Fragment-1) with a resin obtained in stage-b) in presence of a coupling agent to obtain protected Liraglutide;
g) cleaving the crude Liraglutide from resin using a reagent to obtain crude Liraglutide;
h) purifying the crude Liraglutide by preparative HPLC to obtain pure Liraglutide.

5. A solid phase peptide synthesis for the preparation of Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH of fragment-2, which comprises:
a) anchoring Fmoc-Val-Ser(Oxa)-OH to a resin in presence of a coupling agent;
b) selective deprotection of amino acid using a base;
c) coupling of Fmoc-Ser(tBu)-OH to a resin obtained in step-a) in presence of coupling agent in a solvent to obtain dipeptide resin;
d) sequential coupling of Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH to the obtained resin in step-a) in presence of a coupling agent;
e) cleaving of protected peptide from solid support resin in presence of a reagent to get fragment-2.

6. A solid phase peptide synthesis for the preparation of Fmoc-Gln(Trt) Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH of fragment-4, which comprises:
a) anchoring Fmoc-Trp(Boc)-OH to a resin in presence of a coupling agent;
b) selective deprotection of amino acid using a base;
c) coupling of Fmoc-Ala-OH to a resin obtained in step-a) in presence of coupling agent in a solvent to obtain dipeptide resin;
d) sequential coupling of Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys[Pal(Glu-OtBu)]-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH to the obtained resin in step-a) in presence of a coupling agent;
e) cleaving of protected peptide from solid support resin in presence of a reagent to get fragment-4.

7. The process as claimed in claim 1, claim 2, claim 3, claim 4, claim 5 and claim 6, wherein said coupling agent is selected from the group consisting of Ethylcyano (hydroxyimino)acetate-02)-tri-(1-pyrrolidinyl)-Phosphonium hexa fluorophosphate (PyOxim), ethyl-2-cyano-2-(hydroxy amino) acetate (Oxyma pure), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), diisopropyl carbodiimide (DIC), 1,3-dicyclohexylcabodiimide (DCC), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), 1-(dimethyl aminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), O-(benzotriazol-1-yl)-1,1,3,3-tetra methyluronium hexafluorophosphate (HBTU), 1-Hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), Isopropyl chloro formate (IPCF), Benzotriazol-1-yl-oxy-tris(dimethyl-amino)-phosphonium hexa fluorophosphate (BOP), benzotriazole-1-yloxytri(pyrrolidino)phosphonium hexa fluoro phosphate (PyBOP), N,N-bis-(2-oxo-3-oxazolidinyl)phosphonic dichloride (BOP-Cl), bromotri(pyrrolidino)phosphonium hexa fluoro phosphate (PyBrOP), O-(6-Chloro-1-hydrocibenzotriazol-1-yl)-1,1,3,3-tetramethyl uranium tetra fluoroborate (TCTU), chlorotri (pyrrolidino)phosphonium hexafluorophosphate (PyClOP), Ethyl 1,2-dihydro-2-ethoxyquinoline-carboxylate(EEDQ), isobutyl chloro formate (IBCF), 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate(TSTU), 1-Cyano-2-ethoxy-2-oxo ethylidene aminooxy) dimethyl amino morpholino-carbeniumhexafluorophosphate (COMU), 2-(5-norbornen-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), propane phosphonic acid anhydride (PPAA), 3-(diethoxy phosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) or its mixture.

8. The process as claimed in claims 1 to 6, wherein said base used for deprotection is selected from the group consisting of tert-butyl amine, 20% of 4-methyl piperidine in Dimethyl formamide, 20% of piperidine in Dimethyl formamide and 20% of piperazine in Dimethyl formamide.

9. The process as claimed in claims 1 to 6, wherein said reagent used in cleavage step is selected from the group consisting of TFA, TIPS, Water, DTT, Thioanisole, EDT, DMS, cresol, phenol, thiocresol, ammonium iodide, 2,2'-(ethylene dioxy)diethane or its mixture. Preferably using cocktail mixture of TFA, TIPS, water and DTT.

10. The process as claimed in claims 1 to 6, wherein said solvent used is selected from the group consisting of DMF, DCM, tetrahydrofuran, NMP, DMAC, methanol, ethanol, isopropanol, dichloroethane, 1,4-dioxane, ethyl acetate, acetonitrile, acetone or a mixture thereof.

11. Novel fragment-2 and fragment-4 used the preparation of GLP-1 peptides.
Fragment-2: Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(Oxa)-OH
Fragment-4: Fmoc-Gln(Trt)-Ala-Ala-Lys(palmityl-?-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH