DESC:FIELD OF THE INVENTION
The present invention relates to a process for the preparation of Liraglutide of formula (1) by fragment condensation by solid phase peptide synthesis.

BACKGROUND OF THE INVENTION
The following discussion of the prior art is intended to present the invention in an appropriate technical context and allows its significance to be properly appreciated. Unless clearly indicated to the contrary, reference to any prior art in this specification should not be construed as an expressed or implied admission that such art is widely known or forms part of common general knowledge in the field.

Liraglutide developed by Novo Nordisk, glucagon-like peptide-1 (GLP-1) receptor agonist, as a
subcutaneous formulation, can play a good role in lowering blood glucose. Liraglutide is marketed under the brand names Victoza and Saxenda.

The peptide sequence of the Liraglutide can be represented in terms of chemical formula (1) as
follows:
H-1His-Ala-3Glu-Gly-5Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-16Gly-17Gln-Ala-Ala-Lys(Pal-Glu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-31Gly-OH

Further, it is structurally represented as:

Liraglutide is a synthetic analog of human glucagon-like peptide-1(GLP-1) and acts as a GLP-1 receptor agonist. Liraglutide is 97% like native human GLP-1, differing primarily by substituting arginine for lysine at position 34. Liraglutide is made by attaching a C-16 fatty acid (palmitic acid) with a glutamic acid spacer on the remaining lysine residue at position 26 of the peptide precursor.

Liraglutide increases intracellular cyclic AMP (cAMP), leading to insulin release in the presence of elevated glucose concentrations. This insulin secretion subsides as blood glucose concentrations decrease and approach euglycemia. Liraglutide also decreases glucagon secretion in a glucose-dependent manner. The mechanism of blood glucose lowering also involves a delay in gastric emptying. GLP-1 (7-37) has a half- life of 1.5-2 minutes due to degradation by the ubiquitous endogenous enzymes, dipeptidyl peptidase IV (DPP- IV) and neutral endopeptidases (NEP). Unlike native GLP-1, Liraglutide is stable against metabolic degradation by both peptidases and has a plasma half-life of 13 hours after subcutaneous administration. The pharmacokinetic profile of Liraglutide, which makes it suitable for once daily administration, is a result of self-association that delays absorption, plasma protein binding and stability against metabolic degradation by DPP-IV and NEP.

Liraglutide was first disclosed in US6268343B1, in which Liraglutide was prepared by solid-liquid synthetic method, wherein the intermediate GLP-l (7-37)-OH required reverse phase HPLC purification; followed by reaction with Na-hexadecanoyl-Glu (ONSu)-OtBu under liquid phase condition to obtain Liraglutide.

US9260474 discloses a process of preparation of Liraglutide by solid phase synthesis, involving sequential addition of amino acids to the supported resin including Fmoc-Lys (Alloc)-OH is employed for lysine; removal of Alloc protecting group of lysine side chain and coupling with Palmitoyl-Glu-OtBu; followed by de-protection and cleavage of resin to obtain crude Liraglutide.

CN103864918B discloses fragment based solid phase synthesis of Liraglutide, involving sequential addition of 1-10 amino acids to the supported resin and coupling with Palmitoyl-Glu OtBu; followed by coupling of 11-19 amino acid and 20-31 amino acid sequences, de-protection, and cleavage of resin to obtain crude Liraglutide.
There remains a need to provide efficient process for preparation of Liraglutide, which is high yielding, scalable, cost effective, environment friendly and commercially viable.
The present invention relates to a process for the preparation of Liraglutide by using two or more suitable fragments (protected) by solid phase peptide synthesis, coupling of the fragments on solid support, concurrent cleavage from the solid support and deprotection of peptide to give Liraglutide.

SUMMARY OF THE INVENTION
The present invention provides a solid phase peptide synthesis for the preparation of Liraglutide compound of formula (1),
H-1His-Ala-3Glu-Gly-5Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-16Gly-17Gln-Ala-Ala-Lys(Pal-Glu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-31Gly-OH
which comprises:
a) preparing X-17Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-31Gly-Resin (Fragment P), X-5Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr (Y)-Leu-Glu(Y)-16Gly-OH (Fragment I), X-Glu(Y)-Gly-OH (Fragment R), X-Ala-OH (Fragment M) and X-His(Y)-OH (Fragment L) by solid phase synthesis in a solvent;
b) condensing (Fragment P) with (Fragment I) in presence of a coupling agent and in a solvent to get X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
c) condensing the peptide obtained in step (b) with a dipeptide Fragment R in presence of a coupling agent and in a solvent to get X-(3)Glu(Y)-Gly-Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
d) condensing the peptide obtained in step (c) with a Fragment M in presence of a coupling agent and in a solvent to get X-(2)Ala-Glu(Y)-Gly-Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
e) condensing the peptide obtained in step (d) with a Fragment L in presence of a coupling agent and in a solvent to get protected Liraglutide; and
f) deprotecting the peptide obtained in step (e) to get Liraglutide.

wherein, X represents amino protecting group, Y represents carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group.

OBJECTIVE OF INVENTION
An objective of the present invention is to provide a process for preparing Liraglutide, which is simple, industrially applicable, and robust.

BRIEF DESCRIPTION OF ABBREVIATIONS
ACN: Acetonitrile
Ala: Alanine
Arg: Arginine
Asp: Aspartic acid.
Boc: t-butyloxycarbonyl
Bpoc: 2-(4-biphenyl)-2-propyloxycarbonyl
Cbz: benzyloxycarbonyl
DCM: Dichloromethane
DIC: Diisoprpopylcarbodiimide
DIPEA: N,N-Diisopropylethylamine
DMF: N,N-Dimethylformamide
DMT: dimethoxy trityl
DCC: N,N-Dicyclohexylcarbodiimide
DIEA: N,N-Diisopropylethylamine
eq : equivalents
Fmoc: 9-fluorenylmethoxycarbonyl
Gln: Glutamine
Glu: Glutamic acid
Gly: Glycine
His: Histidine
HOBt: N-hydroxybenzotriazole
HPLC: High-performance liquid chromatography
Ile: Isoleucine
Leu: Leucine
Lys: Lysine
LiOH: Lithium Hydroxide
Li2CO3: Lithium Carbonate
MeOH: Methanol
MMT: Methoxytrityl
NMP: N-methyl Pyrrolidone
OtBu: t-butyl ester
HOSu: N-Hydroxy Succinamide
Pbf: 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl
Pmc: 2,2,5,7,8-pentamethylchroman-6-sulfonyl
Phe: Phenylalanine
Pal-Glu: Palmitoyl Glutamyl
RT: Room or Ambient temperature;
Ser: Serine
SPPS: Solid Phase Peptide Synthesis
tBu: t-butyl
TFA: Trifluoroacetic acid
THF: Tetrahydrofuran
TIPS, TIS: Triisopropyl silane
Trp: Tryptophan
Trt: Trityl or Triphenylmethyl
Tyr: Tyrosine
Thr: Threonine
TFE: 2,2,2-Trifluoroethanol
Val: Valine
TFE: 2,2,2-Trifluoroethanol

DETAILED DESCRIPTION OF THE INVENTION
Before the present invention is described, it is to be understood that this invention is not limited to methodologies and materials described, as these may vary as per the person skilled in the art. It is also to be understood that the terminology used in the description is for describing the embodiments only and is not intended to limit the scope of the present invention.
Before the present invention is described, it is to be understood that unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Further, it is to be understood that the present invention is not limited to the methodologies and materials similar, equivalent to those described herein can be used in the practice, or testing of the present invention, the preferred methods and materials are described, as these may vary within the specification indicated. Unless stated to the contrary, any use of the words such as "including," "containing," "comprising," "having" and the like, means "including without limitation" and shall not be construed to limit any general statement that it follows to the specific or similar items or matters immediately following it. Embodiments of the invention are not mutually exclusive but may be implemented in various combinations. The described embodiments of the invention and the disclosed examples are given for the purpose of illustration rather than limitation of the invention as set forth the appended claims. Further, the terms disclosed embodiments are merely exemplary methods of the invention, which may be embodied in various forms.
The present invention relates to a process for the preparation of Liraglutide by coupling of 3 or more suitable protected fragments on solid support.
In one embodiment of the present invention is to provide a process for the preparation of Liraglutide, which comprises:
a) synthesis of suitable fragments by Solid phase peptide synthesis (SPPS);
b) coupling of the suitable fragments in presence of coupling agents and solvent; and
c) deprotecting the peptide to get Liraglutide.

According to the present invention, the suitable fragments are prepared by solid phase synthesis in a solvent. These fragments are coupled in presence of coupling reagents and solvent to get protected Liraglutide and further deprotected to get Liraglutide.

The suitable fragments selected for the preparation of Liraglutide are as follows:
? X-17Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-31Gly-Resin = Fragment P
? X-5Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-16Gly-OH = Fragment I
? X-Glu(Y)-Gly-OH = Fragment R
? X-Ala-OH = Fragment M
? X-His(Y)-OH = Fragment L

In other embodiment the present invention is to provide a process for the preparation of Liraglutide, which comprises:
a) preparing X-17Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-31Gly-Resin (Fragment P), X-5Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr (Y)-Leu-Glu(Y)-16Gly-OH (Fragment I), X-Glu(Y)-Gly-OH (Fragment R), X-Ala-OH (Fragment M) and X-His(Y)-OH (Fragment L) by solid phase synthesis in a solvent;
b) condensing (Fragment P) with (Fragment I) in presence of a coupling agent and in a solvent to get X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
c) condensing the peptide obtained in step (b) with a dipeptide Fragment R in presence of a coupling agent and in a solvent to get X-(3)Glu(Y)-Gly-Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
d) condensing the peptide obtained in step (c) with a Fragment M in presence of a coupling agent and in a solvent to get X-(2)Ala-Glu(Y)-Gly-Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
e) condensing the peptide obtained in step (d) with a Fragment L in presence of a coupling agent and in a solvent to get protected Liraglutide; and
f) deprotecting the peptide obtained in step (e) to get Liraglutide.

wherein, X represents amino protecting group, Y represents carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group.

The amino protecting groups are selected from Fmoc, Boc, Cbz or Bpoc.

The present invention the carboxyl, phenolic and alcoholic groups are protected with groups selected from DMT, MMT, Trt, tert-butyl or t-butoxy carbonyl.

The guanidine protecting groups are selected from a group comprising of Pbf and Pmc.

The coupling agents in the process are selected from the group comprising of hydroxybenzotriazole (HOBt); O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), 1,3-dicyclohexylcarbodiimide (DCC), 1-(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), diisopropylcarbodiimide (DIC), isopropylchloroformate (IPCF), O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), benzotriazol-1-yl-oxy-tris(dimethyl-amino)-phosphonium hexafluorophosphate (BOP), N,N-bis-(2-oxo-3-oxazolidinyl)phosphonic dichloride (BOP—Cl), benzotriazol-1-yloxytri(pyrrolidino) phosphonium hexafluorophosphate (PyBOP), bromotri(pyrrolidino)phosphonium hexafluoro- phosphate (PyBrOP), chlorotri(pyrrolidino)phosphonium hexafluorophosphate (PyClOP), ethyl-2-cyano-2-(hydroxyimino) acetate (Oxyma Pure), O-(6-Chloro-1-hydrocibenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU), 2-(5-norbornen-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), propane phosphonic acid anhydride (PPAA), 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoro borate (TSTU), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP), iso-butylchloroformate (IBCF), Ethyl 1,2-dihydro-2-ethoxyquinoline-1-carboxylate (EEDQ), 1-Cyano-2-ethoxy-2-oxoethyli- denaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) or mixtures thereof.

The coupling takes place in one of the solvents selected from the group comprising of DMF, DCM, THF, NMP, DMAC methanol, ethanol, isopropanol, dichloroethane, 1,4-dioxane, 2-methyl tetrahydrofuran ethyl acetate, acetonitrile, acetone or a mixture thereof.

The coupling reaction is carried out in presence of a base. The base is organic or inorganic base. The inorganic base is selected from the group comprising of potassium carbonate, lithium carbonate, sodium carbonate, sodium ethoxide, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, ammonium hydroxide, and mixtures thereof; the organic base is selected from the group comprising of diisopropylamine, N,N-diisopropylethylamine triethylamine, dimethylamine, trimethyl amine, isopropyl ethylamine, pyridine, N-methyl morpholine and mixtures thereof.

In the present invention, the solid phase synthesis is carried out on an insoluble polymer which is acid sensitive. Acid sensitive resin selected from the group comprising Chlorotrityl resin (CTC), Sasrin, Wang Resin, 4-methytrityl chloride, TentaGel S, TentaGel TGA, Rink acid resin, NovaSyn TGT resin, HMPB-AM resin, 4-(2-(amino methyl)-5-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA, 4-(4-(amino methyl)-3-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA and 4-(2-(amino methyl)-3,3-dimethoxy)phenoxy butyric acid anchored to polymeric resin MBHA.

Accordingly, the reagents used in the present invention for removal of the protection group comprises 15% to 25% of the organic base prepared in an organic solvent. Preferably, 20% of the organic base is prepared in an organic solvent is employed in the deprotection of the bound peptide chain to the resin.

The protected peptide is cleaved from the peptide resin and deprotected, simultaneously to obtain Liraglutide.

In the present process for solid phase synthesis provides deprotection of the peptide using a combination of Trifluoroacetic acid (TFA) and radical scavengers. Accordingly, one or more radical scavengers are selected from the group comprising triisopropylsilane (TIS), dithiothreitol (DTT), 1,2-ethanedithiol (EDT), Phenol, cresol, anisole, thioanisole, ammonium iodide, DMS and water.

Simultaneous deprotection of all the protecting groups was carried out by the treatment of either of the cocktail cleavage mixtures stated below:
TFA:TIS:Water:Anisole:Phenol (86.2:5:5:2:1 v/v).

In other embodiment the present invention is to provide a process for the preparation of Liraglutide, which comprises:
a) preparing Fmoc-17Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu -Val-Arg(Pbf)-Gly-Arg(Pbf)-31Gly-Wang resin (Fragment P), Fmoc-5Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-Ser(PsiMe,MePro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-16Gly-OH (Fragment I), Fmoc-Glu(OtBu)-Gly-OH (Fragment R), Fmoc-Ala-OH (Fragment M) and Boc-His(Trt)-OH (Fragment L) by solid phase synthesis in a solvent;
b) condensing (Fragment P) with (Fragment I) in presence of a coupling agent and in a solvent to get Fmoc-(5)Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-Ser(PsiMe,MePro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-(31)Gly-Wang resin;
c) condensing the peptide obtained in step (b) with a dipeptide Fragment R in presence of a coupling agent and in a solvent to get Fmoc-(3)Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-Ser(PsiMe,MePro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-(31)Gly-Wang resin;
d) condensing the peptide obtained in step (c) with a Fragment M in presence of a coupling agent and in a solvent to get Fmoc-(2)Ala-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-Ser(PsiMe,MePro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-(31)Gly-Wang resin;
e) condensing the peptide obtained in step (d) with a Fragment L in presence of a coupling agent and in a solvent to get protected Liraglutide; and
f) deprotecting the peptide obtained in step (e) to get Liraglutide.
In yet another embodiment of the present invention in step (b), the coupling agent used is a mixture of hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide (DIC) in NMP solvent.
In yet another embodiment of the present invention in step (c), the coupling agent used is a mixture of hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide (DIC) in DMF solvent.
In yet another embodiment of the present invention in step (d), the coupling agent used is a mixture of hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide (DIC) in DMF solvent.
In yet another embodiment of the present invention in step (e), the coupling agent used is a mixture of hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide (DIC) in DMF solvent.
In yet another embodiment of the present invention in step (f), the deprotection of all the protecting groups was carried out by the treatment of the cocktail cleavage mixtures TFA: TIS:Water:Anisole:Phenol (86.2:5:5:2:1 v/v).

Within the context of the present invention, the preparation of Liraglutide is described in the following scheme-I:
Synthesis of Liraglutide
Fmoc-Gly-Wang resin

20% Piperidine/DMF
Fmoc-Arg(Pbf)-OH

Fmoc-Arg(Pbf)-31Gly-Wang resin
20% Piperidine/DMF
Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH
Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH
Fmoc-Ile-OH, Fmoc-Phe-OH
Fmoc-Glu(OtBu)-OH

Fmoc-21Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-31Gly-Wang resin
20% Piperidine/DMF
Fmoc-Lys(Pal-Glu-OtBu)-OH

Fmoc-20Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-31Gly-Wang resin

20% Piperidine/DMF
Fmoc-Ala-OH,
Fmoc-Ala-OH
Fmoc-Gln-OH

Fmoc-17Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-31Gly-Wang resin

20% Piperidine/DMF
Fmoc-5Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)- Asp(tBu)-Val-Ser(PsiMe, MePro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-16Gly-OH


Fmoc-5Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-Ser(PsiMe, MePro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-16Gly-17Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-31Gly-Wang resin (5 to 31)

20% Piperidine/DMF
Fmoc-Glu(OtBu)-Gly-OH

Fmoc-3Glu(OtBu)-Gly-5Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-Ser(PsiMe, MePro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-16Gly-17Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-31Gly-Wang resin

20% Piperidine/DMF
Fmoc-Ala-OH, Boc-His(Trt)-OH

Boc-1His(Trt)-Ala-3Glu(OtBu)-Gly-5Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-Ser(PsiMe, MePro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-16Gly-17Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-31Gly-Wang resin

Cleavage of peptide from resin &
Side chain deprotection

H-1His-Ala-3Glu-Gly-5Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-16Gly-17Gln-Ala-Ala-Lys(Pal-Glu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-31Gly-OH
Liraglutide

Scheme 1

Within the context of the present invention, Fragment P is prepared by solid phase synthesis as
the process described in the scheme-II:

Fmoc-Gly-Wang resin
20% Piperidine/DMF
Fmoc-Arg(Pbf)-OH

Fmoc-Arg(Pbf)-31Gly-Wang resin
20% Piperidine/DMF
Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH
Fmoc-Val-OH, Fmoc-Leu-OH,
Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH
Fmoc-Ile-OH, Fmoc-Phe-OH
Fmoc-Glu(OtBu)-OH

Fmoc-21Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-31Gly-Wang resin
20% Piperidine/DMF
Fmoc-Lys(Pal-Glu-OtBu)-OH

Fmoc-20Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-31Gly-Wang resin 20 to 31

20% Piperidine/DMF
Fmoc-Ala-OH,
Fmoc-Ala-OH
Fmoc-Gln-OH,

Fmoc-17Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-31Gly-Wang resin

Scheme-2
Within the context of the present invention, Fragment I is prepared by solid phase synthesis as the process described in the Scheme 3:
2-CTC resin
Loading of Fmoc-Gly-OH
DCM/DIEA/MeOH

Fmoc-Gly-OCTC resin
20% Piperidine/DMF
Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH

Fmoc-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OCTC resin
20% Piperidine/DMF
Fmoc-Val-Ser(PsiMe, MePro)-OH

Fmoc-Val-Ser(PsiMe, MePro))-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OCTC resin

20% Piperidine/DMF
Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH
Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,
Fmoc-Thr(tBu)-OH

Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-Ser(PsiMe, MePro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OCTC resin

Protected cleavage using 20% TFE/DCM
MTB Ether/Hexane

Fmoc-5Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-Ser(PsiMe, MePro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-16Gly-OH

Scheme -3
In the present invention, the process for preparing Fragment I employs a linear sequential synthesis, using an Fmoc-pseudoproline dipeptide unit at the relevant position to prepare the Val-Ser segment of the peptide chain. The remaining sequence is then prepared by stepwise sequential synthesis. Pseudoprolines are artificially created dipeptides that minimize aggregation during Fmoc solid phase synthesis of peptides.
Further in an embodiment, the present invention provides following advantages:
1. the time and cost of material required for convergent coupling was reduced by performing the coupling at an elevated temperature instead of room temperature;
2. aggregation in the growing chain was reduced by introducing 0.25M HOBt.H2O in DMF and Piperidine washings;
3. the presence of Wang resin-based impurity in the peptide was minimized by modifying the cleavage cocktail; and
4. the process robustness and quality of the crude peptide were enhanced.

The invention is further illustrated by the following examples which are provided to be exemplary of the invention, and do not limit the scope of the invention. While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.
EXAMPLES
Example 1:

Preparation of Fmoc-(17)Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-(31)Gly–Wang Resin (Fragment P)

Stage-1: Synthesis of Fmoc–(30)Arg(Pbf)-Gly-Wang Resin:
Fmoc-Gly-Wang resin with a Loading of ~0.27 mmole/gram (about 37.03 g resin, 10 mmol) was swelled in twice by using DMF. Fmoc-deprotection of the Fmoc-Gly-Wang resin was carried out by washing the resin using 20 % Piperidine, HOBt (0.25M) in DMF solution two times for 2 and 10 min, followed by washing the resin four times with DMF. The coupling of the second amino acid Fmoc-Arg(Pbf)-OH (20 mmol, 2.0 eq), was carried out by addition of HOBt (20 mmol, 2.0 eq) and DIC (20 mmol, 2.0 eq) in DMF solvent. The coupling mixture was agitated under nitrogen for 120 minutes, followed by decanting the solvent. The resin was then washed and stirred with nitrogen at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
Stage-2 : Synthesis of Fmoc-(29)Gly-Arg(Pbf)-Gly–Wang Resin
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % Piperidine, HOBt (0.25M) in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Gly-OH (20 mmole, 2.0 eq) was coupled using HOBt (20 mmole, 2.0 eq) and DIC (20 mmole, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 minutes at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-3 : Synthesis of Fmoc-(28)Arg(Pbf)-Gly-Arg(Pbf)-Gly–Wang Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % Piperidine, HOBt (0.25M) in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Arg(Pbf)-OH (20 mmole, 2.0 eq) was coupled using HOBt (20 mmole, 2.0 eq) and DIC (20 mmole, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 minutes at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-4 : Synthesis of Fmoc-(27)Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly–Wang Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % Piperidine, HOBt (0.25M) in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Val-OH (20 mmole, 2.0 eq) was coupled using HOBt (20 mmole, 2.0 eq) and DIC (20 mmole, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 minutes at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-5 : Synthesis of Fmoc-(26)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly–Wang Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % Piperidine, HOBt (0.25M) in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Leu-OH (20 mmole, 2.0 eq) was coupled using HOBt (20 mmole, 2.0 eq) and DIC (20 mmole, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 minutes at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin six times by DMF.

Stage-6: Synthesis of Fmoc-(25)Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly–Wang Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % Piperidine, HOBt (0.25M) in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Trp(Boc)-OH (20 mmole, 2.0 eq) was coupled using HOBt (20 mmole, 2.0 eq) and DIC (20 mmole, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 minutes at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-7: Synthesis of Fmoc-(24)Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly–Wang Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % Piperidine, HOBt (0.25M) in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Ala-OH (20 mmole, 2.0 eq) was coupled using HOBt (20 mmole, 2.0 eq) and DIC (20 mmole, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 minutes at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-8: Synthesis of Fmoc-(23)Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly–Wang Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % Piperidine, HOBt (0.25M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt (0.25M) in DMF. The Fmoc-Ile-OH (20 mmole, 2.0 eq) was coupled using HOBt (20 mmole, 2.0 eq) and DIC (20 mmole, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 minutes at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin six times by DMF.

Stage-9: Synthesis of Fmoc-(22)Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly–Wang Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % Piperidine, HOBt (0.25M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt (0.25M) in DMF. The Fmoc-Phe-OH (20 mmole, 2.0eq) was coupled using HOBt (20 mmole, 2.0 eq) and DIC (20 mmole, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 minutes at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-10: Synthesis of Fmoc-(21)Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly–Wang Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % Piperidine, HOBt (0.25M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt (0.25M) in DMF. The Fmoc-Glu(OtBu)-OH (20 mmole, 2.0 eq) was coupled using HOBt (20 mmole, 2.0 eq) and DIC (20 mmole, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 minutes at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-11:
Synthesis of Fmoc-(20)Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly–Wang Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % Piperidine, HOBt (0.25M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt 0.25M) in DMF. The Fmoc-Lys(Pal-Glu-OtBu)-OH (30 mmole, 3.0 eq) was coupled using HOBt (30 mmole, 3.0 eq) and DIC (30 mmole, 3.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 180 minutes at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-12:
Synthesis of Fmoc-(19)Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly–Wang Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % Piperidine, HOBt (0.25M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt (0.25M) in DMF. The Fmoc-Ala-OH (20 mmole, 2.0 eq) was coupled using HOBt (20 mmole, 2.0 eq) and DIC (20 mmole, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 minutes at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-13:
Synthesis of Fmoc-(18)Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly–Wang Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % Piperidine, HOBt (0.25M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt (0.25M) in DMF. The Fmoc-Ala-OH (20 mmole, 2.0eq) was coupled using HOBt (20 mmole, 2.0 eq) and DIC (20 mmole, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 minutes at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-14:
Synthesis of Fmoc-(17)Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-(31)Gly–Wang Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % Piperidine HOBt (0.25M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt (0.25M) in DMF. The Fmoc-Gln-OH (20 mmole, 2.0eq) was coupled using HOBt (20 mmole, 2.0 eq) and DIC (20 mmole, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 minutes at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Example 2:

Preparation of Fmoc-5Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-Ser(PsiMe, MePro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-16Gly-OH (Fragment I)
Fmoc-Gly-OH (26.2 g) was charged to a glass beaker and dissolved in DCM (8 vol), added 1.5 eq. of DIEA (N,N-Diisopropylethyl amine) and stirred solution for 5 min. 2-CTC resin (62.5 g) with functionality 1.6 mmole/g was taken in PP Bottle, added the activated amino acid solution to PP bottle having resin, the bottle was manually shaken for next 5 minutes and then added 4 equivalents of DIEA/DCM mixture in PP bottle, the PP bottle is shaken on mechanical shaker/ Tumbler for 40-60 min. After 50 min added Dry Methanol (0.8 vol) for capping purpose and again Mixture placed on stirring for 20 min. After 20 min mixture poured in solid phase vessel and washed resin with solvent mixture DCM:DIEA:Methanol (85:5:10) followed by DMF washings. The obtained Fmoc protecting group in Fmoc-Gly-2-CTC resin was removed with 20% Piperidine twice for 15 min and 10 min to obtain H-Gly-2-CTC resin, which was then washed with DMF (6 times).
Fmoc-Glu(OtBu)-OH (0.15 mol), HOBt.H2O (0.15 mol) were dissolved in cooled DMF (150 ml) at 0±2°C and while stirring DIC (0.15 mol) was added and stirred for 50 sec. It was added to the above solid phase vessel and stirred for 3 h at room temperature (the reaction end point is detected by the Ninhydrin method, if the resin is colorless and transparent, the reaction is complete, and the resin develops blue color, indicating that the reaction is incomplete, and the coupling reaction is required for another reactivation/Recoupling force) to obtain Fmoc-Glu(OtBu) -Gly-2-CTC resin. According to the amino acid sequence from the C-terminal to the N-terminal, repeat the above steps of removing the protecting group and adding the corresponding amino acid for coupling to complete Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH and use of Pseudoproline coupling of Fmoc-(10)Val-(11)Ser(PsiMe,MePro)-OH. After the completion of sequence, it was shrunk with methanol/Ether, the resin was vacuum-dried overnight, and weighed to obtain polypeptide fragment (5-16; 123 g) without 2-CTC resin removal.
Peptidyl resin was suspended in cleavage reagent (200 ml of 20% TFE in dichloromethane solution) and stirred for 2 h. Then the resin was filtered and washed resin twice with 20% TFE:DCM mixture, collect the filtrate, filtrate evaporated completely using rotavapour, Observed thick sticky mass which was treated with Hexane: MTB Ether (80:20) to obtain white solid, washed twice with Hexane: MTB ether mixture, and dried in vacuum to obtain 48.5g of polypeptide fragment 5-16, Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-Ser(PsiMe,MePro)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH. [Purity: 98.4 %; Yield: 88.0%].

Example 3: Coupling of Fragment P and Fragment I

Synthesis of Fmoc-(5)Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-Ser(PsiMe,MePro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-(31)Gly-Wang resin

Fmoc-deprotection of the peptidyl resin (example 1) was carried out by washing the resin using 20% Piperidine, HOBt (0.25M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt (0.25M) in DMF. The Protected fragment (example 2) (12 mmole,1.2 eq) was coupled using HOBt (24 mmole,2.4 eq) and DIC (24 mmole, 2.4 eq) in NMP solvent. The mixture was stirred via Nitrogen for 180 min at 60°C. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Example 4:

Preparation of Fragment R

Step-1: PFP esterification of Fmoc-Glu(OtBu)-OH:

Weigh 150gm (1.0 eq) of Fmoc-Glu(OtBu)-OH and dissolved in 1500 ml (10 ml /gm) Ethyl acetate in 3L RB flask, after complete dissolution added solution of 71.4 gm (1.1 eq) Pentafluorophenol dissolved in Ethyl acetate (72 ml). The mixture was then cooled to 5°C using ordinary ice bath, and then added portion wise the solution of DCC/Ethyl acetate [109.1 gm (1.5 eq) DCC dissolved in 110 ml Ethyl acetate]. Stirred the reaction mixture in cooling 2-8°C & monitor progress of reaction on HPLC. After reaction completion reaction taken for workup, filtered the reaction mixture & discarded the residual DCU. Distilled off the ethyl acetate layer using Rotavapour & Solid white material treated with 50:50 (Ethanol: Water mixture) filtered the mixture & allow material on drying.
White solid powder 208 gm (100% Yield) analysed on HPLC showed 95.5% pure, material taken for next step.
Step-2: Synthesis of Fmoc-Glu(OtBu)-Gly-OH:

In a beaker dissolved Fmoc-Glu(OtBu)-OPFP 56.0gm (1.0 eq) in 2240 ml (40 ml/gm) of 1,4-dioxane, stirred it to clear and cooled to 5°C using ordinary ice bath. In another beaker, 1120 ml (20 ml/gm) water, dissolved 7.95 g LiOH.H2O (2.0 eq) and 5.24gm Li2CO3 (0.75 eq) stirred it to get the clear solution, then added 21.31 g (3 eq) glycine. The clear glycine solution added portion wise to Fmoc-Glu(OtBu)-OPFP solution, after complete addition stirred reaction mixture at room temperature for 14 hours, Monitored reaction progress on HPLC. Reaction mixture taken for workup after 14 hours, distilled off dioxane and water layer washed twice with ethyl acetate. Water layer cooled to 5°C and acidified using 10% HCl solution, white solid material filtered after 10 hours of aging at cooling washed with water and dried in hot air oven.
Yield: 32gm (70.06%); Purity (by HPLC): 98.63%.

Example 5: Coupling of Fragment R with peptidyl resin of example 3.

Synthesis of Fmoc-(3)Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-Ser(PsiMe,MePro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-(31)Gly-Wang resin:
Fmoc-deprotection of the peptidyl resin (example 3) was carried out by washing the resin using 20 % Piperidine, HOBt (0.25M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt (0.25M) in DMF. The Fmoc-Glu(OtBu)-Gly-OH (20 mmole, 2.0 eq) was coupled using HOBt (20 mmole, 2.0 eq) and DIC (20 mmole, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 minutes at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Example 6: Coupling of Fragment M with peptidyl resin of example 5.

Synthesis of Fmoc-(2)Ala-lu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-Ser(PsiMe,MePro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-(31)Gly-Wang resin:
Fmoc-deprotection of the peptidyl resin (example 5) was carried out by washing the resin using 20% Piperidine, HOBt (0.25M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt (0.25M) in DMF. The Fmoc-Ala-OH (20 mmole, 2.0 eq) was coupled using HOBt (20 mmole, 2.0 eq) and DIC (20 mmole, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 minutes at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Example 7: Coupling of Fragment L with peptidyl resin of 2-31.

Synthesis of Boc-(1)His(Trt)-Ala-lu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-Ser(PsiMe,MePro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-(31)Gly-Wang resin:
Fmoc-deprotection of the peptidyl resin (example 6) was carried out by washing the resin using 20% Piperidine, HOBt (0.25M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt (0.25M) in DMF. The Boc-His(Trt)-OH (20 mmole, 2.0 eq) was coupled using HOBt (20 mmole, 2.0 eq) and DIC (20 mmole, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 minutes at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Post completion of the synthesis, the resin was thoroughly washed with methanol and Et2O and drying of resin in desiccator.
Weight of the peptidyl resin: 89 gm

Example 8:
Preparation of Crude Liraglutide: H-His1-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Pal-Glu)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Ala27-Arg28-Gly29-Arg30-Gly31-OH:
Simultaneous deprotection of all the protecting groups was carried out by the treatment of the cocktail cleavage mixtures stated below: TFA:TIS:Water:Anisole:Phenol (86.2:5:5:2:1V/v) cocktail mixture.
The cleavage was carried out using cleavage cocktail 10 ml/gm of peptidyl resin at -10°C for initial 15 minutes followed by the stirring of the peptidyl resin for 3 hours at ambient temperature. The crude cleavage mixture was then filtered, the resin washed thoroughly with TFA. The filtrate was dropped on to 12 mL of cold dry MTB ether per mL of cocktail and further 6 additional washing with 0.5 litre of MTB ether were done to the product. Product was dried under vacuum for 16 hours.
The isolated yield of the crude peptide: 37 gm (100% process yield)
The Purity of the crude peptide: 61.93%
,CLAIMS:We claim:
1. A process for the preparation of Liraglutide, which comprises:
a) preparing X-17Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-31Gly-Resin (Fragment P), X-5Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr (Y)-Leu-Glu(Y)-16Gly-OH (Fragment I), X-Glu(Y)-Gly-OH (Fragment R), X-Ala-OH (Fragment M) and X-His(Y)-OH (Fragment L) by solid phase synthesis using a resin in a solvent;
b) condensing (Fragment P) with (Fragment I) in presence of a coupling agent and in a solvent to get X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
c) condensing the peptide obtained in step (b) with a dipeptide Fragment R in presence of a coupling agent and in a solvent to get X-(3)Glu(Y)-Gly-Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
d) condensing the peptide obtained in step (c) with a Fragment M in presence of a coupling agent and in a solvent to get X-(2)Ala-Glu(Y)-Gly-Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
e) condensing the peptide obtained in step (d) with a Fragment L in presence of a coupling agent and in a solvent to get protected Liraglutide; and
f) deprotecting the peptide obtained in step (e) to get Liraglutide.

2. The process as claimed in claim 1, wherein, Y represents amino protecting group, X represents carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group.
3. The process as claimed in claim 2, wherein the amino protecting groups are selected from a group comprising of Fmoc, Boc, Cbz, Bpoc; wherein the carboxyl, phenolic and alcoholic protecting groups are selected from DMT, MMT, Trt, tert-butyl, t-butoxy carbonyl; and the guanidine protecting groups are selected from a group comprising of Pbf and Pmc.
4. The process as claimed in claim 1, wherein the coupling agents used in steps (b), (c), (d) and (e) are selected from the group comprising of hydroxybenzotriazole (HOBt); O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), 1,3-dicyclohexylcarbodiimide (DCC), 1-(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), diisopropylcarbodiimide (DIC), isopropylchloroformate (IPCF), O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), benzotriazol-1-yl-oxy-tris(dimethyl-amino)-phosphonium hexafluorophosphate (BOP), N,N-bis-(2-oxo-3-oxazolidinyl)phosphonic dichloride (BOP—Cl), benzotriazol-1-yloxytri(pyrrolidino) phosphonium hexafluorophosphate (PyBOP), bromotri(pyrrolidino)phosphonium hexafluoro- phosphate (PyBrOP), chlorotri(pyrrolidino)phosphonium hexafluorophosphate (PyClOP), ethyl-2-cyano-2-(hydroxyimino) acetate (Oxyma Pure), O-(6-Chloro-1-hydrocibenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU), 2-(5-norbornen-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), propane phosphonic acid anhydride (PPAA), 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoro borate (TSTU), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP), iso-butylchloroformate (IBCF), Ethyl 1,2-dihydro-2-ethoxyquinoline-1-carboxylate (EEDQ), 1-Cyano-2-ethoxy-2-oxoethyli- denaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) or mixtures thereof.
5. The process as claimed in claim 1, wherein the solvent used in steps (b), (c), (d) and (e) are selected from the group comprising of DMF, DCM, THF, NMP, DMAC methanol, ethanol, isopropanol, dichloroethane, 1,4-dioxane, 2-methyl tetrahydrofuran ethyl acetate, acetonitrile, acetone or a mixture thereof.
6. The process as claimed in claim 1, wherein the resin used in the solid phase synthesis is selected from the group comprising Chlorotrityl resin (CTC), Sasrin, Wang Resin, 4-methytrityl chloride, TentaGel S, TentaGel TGA, Rink acid resin, NovaSyn TGT resin, HMPB-AM resin, 4-(2-(amino methyl)-5-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA, 4-(4-(amino methyl)-3-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA and 4-(2-(amino methyl)-3,3-dimethoxy)phenoxy butyric acid anchored to polymeric resin MBHA.
7. The process as claimed in claim 1, wherein the deprotection of the peptide and all protecting groups in step (f) is carried out in the presence of combination of Trifluoroacetic acid (TFA) and radical scavengers.
8. The process as claimed in claim 7, wherein the radical scavengers are selected from the group comprising triisopropylsilane (TIS), dithiothreitol (DTT), 1,2-ethanedithiol (EDT), Phenol, cresol, anisole, thioanisole, ammonium iodide, DMS and water.
9. The process as claimed in claim 7 or claim 8, wherein the deprotection of the peptide and all protecting groups is carried out using cocktail cleavage mixture TFA:TIS:Water:Anisole:Phenol (86.2:5:5:2:1 v/v).
10. A process for the preparation of Liraglutide, which comprises:
a) preparing Fmoc-17Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu -Val-Arg(Pbf)-Gly-Arg(Pbf)-31Gly-Wang resin (Fragment P), Fmoc-5Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-Ser(PsiMe,MePro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-16Gly-OH (Fragment I), Fmoc-Glu(OtBu)-Gly-OH (Fragment R), Fmoc-Ala-OH (Fragment M) and Boc-His(Trt)-OH (Fragment L) by solid phase synthesis in a solvent;
b) condensing (Fragment P) with (Fragment I) in presence of mixture of hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide (DIC) in NMP to get Fmoc-(5)Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-Ser(PsiMe,MePro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-(31)Gly-Wang resin;
c) condensing the peptide obtained in step (b) with a dipeptide Fragment R in presence of mixture of hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide (DIC) in DMF to get Fmoc-(3)Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-Ser(PsiMe,MePro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-(31)Gly-Wang resin;
d) condensing the peptide obtained in step (c) with a Fragment M in presence of mixture of hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide (DIC) in DMF to get Fmoc-(2)Ala-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Val-Ser(PsiMe,MePro)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln-Ala-Ala-Lys(Pal-Glu-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-(31)Gly-Wang resin;
e) condensing the peptide obtained in step (d) with a Fragment L in presence of mixture of hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide (DIC) in DMF to get protected Liraglutide; and
f) deprotecting the peptide obtained in step (e) to get Liraglutide.