Claims:
1. A process for the preparation of side chain Pal-Glu (Y)-OR of Liraglutide comprising the following steps:
a) Activation of palmitic acid in presence of an activating agent to form Palmitic acid active ester Pal-Y;
b) Acylation of L-Glutamate- ester with Pal-Y in presence of an inorganic base to form Pal-Glu-OR;
c) Activation of Pal–Glu-OR in presence of an activating agent to form Pal-Glu(Y)-OR
d) Wherein R is C1-6 alkyl group and activating agent Y is selected from N-hydroxysuccinimide or 4-nitrophenol or 2,4,6-trichlorophenol or penta-fluorophenol and the like.

2. A process for the preparation of side chain Pal-Glu(ONSu)-OR of Liraglutide comprising the following steps:
a. Coupling of palmitic acid, N-hydroxysuccinimide in presence of coupling agent to form Pal-ONSu
b. Acylation of L-Glutamate- ester with Pal-ONSu in presence of an inorganic base to form Pal-Glu-OR
c. Coupling of Pal–Glu-OR with N-hydroxysuccinimide in presence of a coupling agent to form Pal-Glu(ONSu)-OR
Wherein R is C1-6 alkyl group

3. The process as claimed in preceding claims, wherein the inorganic base is selected from sodium bicarbonate, potassium bicarbonate and the like.

4. The process as claimed in preceding claims, wherein the coupling agent is selected from DIPC, DCC and EDCI.

5. A process for the preparation of Liraglutide comprising the following steps:
a. Acylation of linear peptide component of Liraglutide in a solvent using Pal-Glu(Y)-OR in presence of a base to form R-protected Liraglutide;
b. De-protection of R group using an acid in presence of an amino acid to form Liraglutide.
Wherein R is C1-6 alkyl and activating agent Y is selected from N-hydroxysuccinimide or 4-nitrophenol or 2,4,6-trichlorophenol or penta-fluorophenol and the like.

6. A process for the preparation of Liraglutide comprising the following steps:
a. Acylation of linear peptide component of Liraglutide with Pal-Glu(ONSu)-OR in presence of base to form protected Liraglutide;
b. De-protection of protecting group under acidic conditions in presence of an amino acid to form Liraglutide.
Wherein R is C1-6 alkyl.

7. The process as claimed in claim 6, wherein amino acid of step b) is selected from Tryptophan, Methionine and Cysteine.

8. The process as claimed in claim 6, wherein base of step a) is selected from triethylamine, diisopropylethylamine, tetramethylpiperidine and N-methylmorpholine.
, Description:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to the improved process for the synthesis of Liraglutide represented by Formula-I.

BACKGROUND OF THE INVENTION
Liraglutide, is a long-acting analogue of the naturally occurring human glucagon like peptide-1 (GLP-1(7-37)) in which lysine at position 34 has been replaced with arginine and palmitoyl group has been attached via glutamoyl spacer to lysine at position 26.

Liraglutide (VICTOZA®), developed by Novo Nordisk got initial approval in United States in 2010 as subcutaneous injection.

US 6,268,343 discloses liraglutide and process for preparing it. Wherein recombinant technology is involved in preparing Arg34-GLP-1(7-37)-OH followed by reaction with Nα-hexadecanoyl-Glu(ONSu)-OtBu

Wherein Nα-hexadecanoyl-Glu(ONSu)-OtBu is prepared by reaction of H-Glu-OtBu and Pal-ONSu in presence of organic base in DMF, the obtained product is further treated with HOSu, in presence of coupling agent to get Nα-hexadecanoyl-Glu(ONSu)-OtBu

Further, Arg34-GLP-1(7-37)-OH is reacted with Nα-hexadecanoyl-Glu(ONSu)-OtBu in presence of base to form protected liraglutide. After quenching the reaction with Glycine, the t-Bu is deprotected by eluting onto a Varian 1 g C8 Mega Bond Elut® cartridge, using TFA. The eluate was concentrated in vacuum, and the residue purified by column chromatography using acetonitrile/TFA system.

US 7,273,921 B2 and US 6,451, 974 B1 discloses process for acylation of Arg34-GLP-1(7-37)-OH to obtain liraglutide. Wherein C1-12 alkyl or benzyl protected side or un-protected chains are used for acylation. De-protection of C1-12 alkyl ester is done by saponification under basic condition. Typically performed in a 0.01-4.0 M solution of an alkali metal hydroxide, e.g., sodium or potassium hydroxide. A benzyl protecting group may be removed by catalytic hydrogenation in an aprotic polar solvent, e.g., in acetone, at room temperature by using palladium-on-carbon and hydrogen. However, under basic condition the amino acids in the peptide degrades significantly and forms isomeric impurities which results in poor yields and tedious purification steps which yields the process uneconomical. The present invention overcomes the above-mentioned problems by employing the deprotection of alkyl ester under acidic condition which includes TFA, hydrochloric acid and the like.

US 20170283478 A1 discloses solid phase synthesis of Liraglutide, wherein the step of cleaving the 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 is a cocktail mixture of acid, scavengers, and solvents.

The acid utilized in the cleavage reagents may be selected from trifluoroacetic acid (TFA), difluoroacetic acid or monofluoroacetic acid. Scavengers is selected from EDT (1,2-Ethanedithiol), DDM (Dodecane mercaptan), TES (Triethylsilane), TIS (Triisopropylsilane), phenol, thioanisole and water or in any combination thereof.

CN 105294853 A discloses the liquid phase synthesis of Liraglutide, wherein after the side chain acylation, the reaction mixture is quenched by acid or alkali to terminate the side reaction of excess Palmitoyl-Glu(OSu)-OtBu. The removal of the side chain protecting group t-Bu can be carried out by using the deprotecting agent TFA and TIS or water commonly used in the art for removing the t-Bu on the carboxyl group.

The above discussed process leads to the formation of oxidative impurities and hence decrease the yield of the reaction during deprotection of alkyl ester and isolation of the peptide.

Further de-protection of protected side chain in the column is a tedious process, requires high reaction/cycle time and isolation of product requires additional work-up procedures.

OBJECTS OF THE INVENTION
The objective of the present invention is to provide an improved process for the preparation of Liraglutide which minimizes the formation of oxidative impurities and hence improves the yield. The process involves conventional setup with significantly reduced cycle time.

SUMMARY OF THE INVENTION
One embodiment of the present invention provides a process for the preparation of side chain Pal-Glu (Y)-OR of Liraglutide comprising the following steps:
a) Activation of palmitic acid in presence of an activating agent to form Palmitic acid active ester Pal-Y;
b) Acylation of L-Glutamate- ester with Pal-Y in presence of an inorganic base to form Pal-Glu-OR;
c) Activation of Pal–Glu-OR in presence of an activating agent to form Pal-Glu (Y)-OR

Wherein R is C1-6 alkyl group and activating agent Y is selected from N-hydroxysuccinimide or 4-nitrophenol or 2,4,6-trichlorophenol or penta-fluorophenol and the like.

Second embodiment of the present invention provides a process for the preparation of Liraglutide comprising the following steps:
a) Acylation of linear peptide component of Liraglutide in a solvent using Pal-Glu(Y)-OR in presence of a base to form R-protected Liraglutide
b) De-protection of R group using an acid in presence of an amino acid to form Liraglutide

Wherein R is C1-6 alkyl and activating agent Y is selected from N-hydroxysuccinimide or 4-nitrophenol or 2,4,6-trichlorophenol or penta-fluorophenol and the like.

DETAILED DESCRIPTION OF THE INVENTION
One embodiment of the present invention provides a process for the preparation of side chain Pal-Glu(Y)-OR of Liraglutide comprising the following steps:
d) Activation of palmitic acid in presence of an activating agent to form Palmitic acid active ester Pal-Y;
e) Acylation of L-Glutamate- ester with Pal-Y in presence of an inorganic base to form Pal-Glu-OR;
f) Activation of Pal–Glu-OR in presence of an activating agent to form Pal-Glu (Y)-OR

Wherein R is C1-6 alkyl group and activating agent Y is selected from N-hydroxysuccinimide or 4-nitrophenol or 2,4,6-trichlorophenol or penta-fluorophenol and the like.

Second embodiment of the present invention provides a process for the preparation of Liraglutide comprising the following steps:
c) Acylation of linear peptide component of Liraglutide in a solvent using Pal-Glu(Y)-OR in presence of a base to form R-protected Liraglutide
d) De-protection of R group using an acid in presence of an amino acid to form Liraglutide

Wherein R is C1-6 alkyl and activating agent Y is selected from N-hydroxysuccinimide or 4-nitrophenol or 2,4,6-trichlorophenol or penta-fluorophenol and the like.

Another embodiment of the present invention provides a process for the preparation of side chain Pal-Glu(ONSu)-OR of Liraglutide comprising the following steps:
a) Coupling of palmitic acid, N-hydroxysuccinimide in presence of coupling agent to form Pal-ONSu
b) Acylation of L-Glutamate- ester with Pal-ONSu in presence of an inorganic base to form Pal-Glu-OR
c) Coupling of Pal–Glu-OR with N-hydroxysuccinimide in presence of a coupling agent to form Pal-Glu(ONSu)-OR
Wherein R is C1-6 alkyl group. Alkyl can be straight or branched chain selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, t-butyl, pentyl, hexyl etc.

Step a) Coupling of palmitic acid and N-hydroxysuccinimide in a solvent selected from MDC, THF, ethyl acetate; in presence of coupling agent selected from DIPC, DCC, EDCI.

Step b) Acylation of Glutamate -ester (H-Glu-OR) in a solvent using Pal-ONSu in presence of inorganic base is performed instead of organic base known in prior art. Also, this step employs less toxic solvents and milder reaction conditions.

Sodium bicarbonate, an inorganic base is used for the acylation of H-Glu-OR with palmitoyl succinate, instead of organic base.

Solvents like acetone, ethyl acetate, dichloromethane, and methanol are used instead of toxic dimethylformamide.

Step c) Coupling of Pal-Glu-OR and N-hydroxysuccinimide in a solvent selected from MDC, THF, ethyl acetate; in presence of coupling agent selected from DIPC, DCC, & EDCI.

Another embodiment of the present invention provides a process for the preparation of side chain Pal-Glu(ONSu)-OtBu of Liraglutide comprising the following steps:

a) Coupling palmitic acid with N-hydroxysuccinimide in presence of coupling agent to form Pal-ONSu
b) Acylation of L-Glutamate-tert-butyl ester with Pal-ONSu in presence of an inorganic base to form Pal-Glu-OtBu
c) Coupling of Pal–Glu-OtBu with N-hydroxysuccinimide in presence of a coupling agent to form Pal-Glu(ONSu)-OtBu,

Wherein,
In Step a) Coupling of palmitic acid and N-hydroxysuccinimide is done in a solvent selected from MDC THF, ethyl acetate; in presence of coupling agent selected from DIPC, DCC and EDCI.

Step b) Acylation of Glutamate-t-butyl ester (H-Glu-OtBu) in a solvent using Pal-ONSu in presence of inorganic base is performed instead of organic base known in prior art. Also, this step employs less toxic solvents and milder reaction conditions.

Sodium bicarbonate, an inorganic base is used for the acylation of H-Glu-OR with palmitoyl succinate, instead of organic base.

Solvents like acetone, ethyl acetate, dichloromethane, and methanol are used instead of toxic dimethylformamide.

Step c) Coupling of Pal-Glu-OtBu and N-hydroxysuccinimide in a solvent selected from MDC THF, ethyl acetate; in presence of coupling agent selected from DIPC, DCC and EDCI.

Second embodiment of the present invention provides a process for the preparation of Liraglutide comprising the following steps:
a) Acylation of linear peptide component of Liraglutide with Pal-Glu(ONSu)-OR in presence of base to form R-protected Liraglutide
b) De-protection of R group in acidic conditions in presence of an amino acid to form Liraglutide

Wherein R is C1-6 alkyl. Alkyl can be straight or branched chain selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, t-butyl, pentyl, hexyl etc.

Step a) Acylation is performed in a mixture of solvents selected from Acetonitrile: water, dimethylformamide: water and N-methylpyrolidone: water

In presence of base selected from triethylamine, diisopropylethylamine, tetramethylpiperidine and N-methylmorpholine and the like.

Step b) Deprotection is done in acidic conditions in presence of an amino acid

The acid used for de-protection is selected from the group consisting of trifluoro acetic acid, hydrochloric acid and the like.

Solvent used for de-protection is selected from MDC, 1,4-dioxane and the like. Amino acid used in the deprotection step is selected from Tryptophan, Methionine and Cysteine.

Another embodiment of the present invention provides a process for the preparation of Liraglutide comprising the following steps:
a) Acylation of linear peptide component of Liraglutide in a solvent using Pal-Glu(ONSu)-OtBu in presence of base to form t-Bu-protected Liraglutide
b) De-protection of t-Bu group in acidic conditions in presence of an amino acid to form Liraglutide

Wherein,
Step a) Acylation is performed in a mixture of solvents selected from Acetonitrile: water, dimethylformamide: water and N-methylpyrolidone: water

In presence of a base selected from triethylamine, diisopropylethylamine, tetramethylpiperidine and N-methylmorpholine.

Step b) De-protection of t-Bu group in acidic conditions in presence of an amino acid to form Liraglutide

The acid used in the acidic conditions in Step b) is selected from the group consisting of trifluoro acetic acid, hydrochloric acid and the like

Solvent used for de-protection is selected from MDC, 1,4-dioxane and the like

Amino acid used in the step b) is selected from Tryptophan, Methionine, Cysteine and the like.

CAN
DCC
DDM
DIPEA
DIPC
DMF
EDCI
EDT
eq
g
GLP
h
HCl
HPLC
L
M
MDC
min.
mL
Mol.Wt.
MTBE
Nsu
Np
RP
tBu
Tcp
Pfp
TES
TFA
THF
TIS
VTD Acetonitrile
dicyclohexylcarbodiimide
dodecane mercaptan
N,N-Diisopropylethylamine
N,N'-diisopropylcarbodiimide
dimethylformamide
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
1,2-Ethanedithiol
equivalence
gram
glucagon like peptide
hours
hydrochloric acid
high pressure liquid chromatography
litre
molar
methylene dichloride
minutes
milliliter
molecular weight
methyl tert-butyl ether
N-hydroxysuccinimide
4 – Nitrophenol
reverse phase
tert-butyl
Trichlorophenol
Pentafluorophenol
triethylsilane
trifluoroacetic acid
tetrahydrofuran
triisopropylsilane
vacuum tray dryer

ADVANTAGES OF THE INVENTION
a. Use of inorganic base during the preparation of Pal-Glu-OtBu
Sodium bicarbonate, a mild inorganic base dissolved in water is used for the acylation of H-Glu-OtBu with palmitoyl succinate. Water, being an environment friendly solvent, which is more compatible for the reaction where inorganic base is involved. The process offers superior quality product and results with higher yield.

b. Use of less toxic and low-boiling solvents:
Less toxic and volatile solvents like acetone, ethyl acetate, dichloromethane, and methanol are used instead of toxic dimethyl formamide. The volatile solvents can be easily evaporated, and product can be isolated without any yield loss. Whereas dimethylformamide is miscible in water and hence yield loss is observed during the work-up process.

Further the process requires milder conditions to avoid isomeric impurities. The unit operations are simple and cost effective. The process offers superior yield compared to prior art processes.

c. Use of amino acid during t-Bu deprotection:
Use of amino acids like Tryptophan during t-Bu de-protection has considerable advantages. Firstly, it quenches the excess reagent i.e. Pal-Glu(ONSu)-OtBu used in acylation of linear peptide component of liraglutide. Secondly, peptides containing tryptophan are known to form oxidative degradants during cleavage and isolation.

Use of free tryptophan amino acid can help both as scavenger and to minimize kynurenine and other oxidative impurities. This renders critical impurities and helps in RP-HPLC purification to achieve the higher yield. Further, the employed process is simple, and all the unit operations are executed with conventional setup with significantly reduced cycle time.

The embodiments of the present invention are further described using specific examples herein after.

Example 1:
Charged palmitic acid (100 g) into dry MDC (800 mL) in RBF under nitrogen atmosphere. Charged DIPC (58.8 g). Stirred the reaction mass at 20 -25 °C for 5 min. Charged H-OSu (49.36 g) at 20 -25 °C and stirred for 30 min. - 1 h. Reaction progress has been monitored by TLC and upon completion, filtered the urea formed during reaction and washed the bed with THF (100mL). Concentrated the filtrate at 30 - 32 °C. Charged purified water (1.0 L) to the solid, stirred for 2 min and filtered. Repeated the slurry wash with water (1.0 L * 2). Dissolved the filtered solid with DCM (1.0 L), dried over sodium sulphate, filtered and concentrated which resulted in white solid. Dissolved the solid with 1:1 ratio of acetone/methanol mixture (1.8 L) at 33-35 °C. Stirred the clear solution obtained for 1 h at 5-10 °C and filtered. Washed the bed with pre-chilled methanol (100 mL*2) at 0 -5 °C. Dried the solid under vacuum at 27-30 °C for 10-12 h in desiccator. Yield ≥ 80.0 %, Purity ≥ 95.0%.

Example 2:
Charged H-Glu-OtBu (70 g) into sodium bicarbonate solution (31.78 g in 350 mL of purified water) in RBF under nitrogen atmosphere. Stir for 5 min. to obtain clear solution. Dissolved Pal-ONSu (109.5 g) with Acetone (700 mL) at 25-30 °C and charged to above reaction at 10-15 °C for a period of 5 min. Charged Acetone (700 mL) and stirred the mass at 20-25 °C. Monitored the progress of the reaction by TLC and upon completion concentrated the reaction mass completely and added purified water (300 mL). Chilled the mass and adjusted the pH of the reaction mass to 2-3 using 1.5 N HCl solution (583 mL). Extracted the product from aqueous layer using ethyl acetate (2.0 L). Washed the organic layer with purified water (700 mL*8) and saturated brine solution (700 mL). Dried the organic layer over sodium sulphate (70 g) and concentrated under vacuum till the weight of the concentrated mass reaches 7.5 - 8.0 times the weight of H-Glu-OtBu. Added EtOAc (70 mL) and n-hexane (1050 mL). Stirred at 25-30 °C for 30 min. and then stirred at 0-5 °C for 15 min. Filtered the solid and washed the bed with n-hexane (350 mL). Dried the solid under vacuum at 27-30 °C for 10-12 h in desiccator. Yield ≥ 90.0 %, Purity ≥ 95.0%.

Example 3:
Charged Pal-Glu-OtBu (110 g) into dry DCM (1.1 L) in a round bottom flask under nitrogen atmosphere. Charged H-OSu (34.44 g) at 20 -25 °C. Stirred the reaction mass for 5 min. Charged DIPC (46.5 mL), stirred for 5 min. Monitored by TLC for completion of the reaction. Upon completion, filtered the urea precipitated during reaction and washed the bed with DCM (110 mL*3). Concentrated the filtrate completely at 30-32 °C to get white solid. Dissolved the crude solid in 330 mL of acetone. Filtered the undissolved solid and added the clear solution to pre-chilled purified water at 10- 15 °C over a period of 10 min. Rinsed with 110 mL of acetone. Stirred the mass for 10 min and filtered. Washed the bed with water (110 mL*3). Dried the solid completely under vacuum in nitrogen atmosphere for 14-16 h. Dissolved the solid obtained from acetone/water crystallization in dry acetone (5 V) at 30-35 °C. Charged dry n-hexane (25 V) and chilled the mass to 5-10 °C. Charged remaining dry n-hexane (25 V) and stirred the mass at 5-10 °C for 1 h under nitrogen atmosphere. Filtered the solid, washed the bed with n-hexane (2V*2 times) and dried the solid under vacuum in nitrogen atmosphere for 4 h. Yield ≥ 80.0 %, Purity ≥ 95.0%.

Example 4:
Charged linear peptide component of liraglutide (12.0 g) to purified water (110.4 mL) at 25 °C. Stirred the mass to get clear solution. Charged ACN (24 mL). Stirred for 5 min. Charged triethylamine (7.08 mL, 15 eq.) at 25°C. Stirred the mass for 5 min at same temperature. Dissolved Pal-Glu(ONSu)-OtBu (1.326 g) in ACN (72 mL) and charged to above reaction mass at 25 °C. Rinsed with ACN (33.6 mL). Stirred for 30 min. and quenched the reaction with glycine (5.58 g) in 558 mL of 50 % aqueous ethanol solution. Adjusted the pH to 5.9 - 6.0 using neat TFA. Concentrated the mass to remove ACN at 30 °C. Centrifuged the solid and wash the solid. Yield ≥ 60.0 %, Crude Purity ≥ 80.0%.

Example 5:
Charged linear peptide component of liraglutide (12.0 g) to purified water (110.4 mL) at 25 °C. Stirred the mass to get clear solution. Charged ACN (24 mL). Stirred for 5 min. Charged DIPEA (15.54 mL, 15 eq.) at 25°C. Stirred the mass for 5 min at same temperature. Dissolved Pal-Glu(ONSu)-OtBu (1.326 g) in ACN (72 mL) and charged to above reaction mass at 25 °C. Rinsed with ACN (33.6 mL). Stirred for 30 min. and quenched the reaction with glycine (5.58 g) in 558 mL of 50 % aqueous ethanol solution. pH of the mass will be 9.0-10. Adjusted the pH to 5.9-6.0 using neat TFA. Concentrated the mass to remove ACN at 30 °C. Centrifuged the solid and wash the solid. Yield ≥ 60.0 %, Crude Purity ≥ 80.0%.

Example 6:
Charged tert-butyl protected Liraglutide (2.0 g) to methylene dichloride (6 mL) at 20 °C. Charged mixture of TFA (12 mL): Phenol (1 mL): TIS (1 mL) at 20 °C. Stirred the mass for 2- 3 h at 20-25 °C. Concentrated the mass to 40 % of the total volume and added to chilled MTBE (100 mL) at 5-10 °C. Stirred for 10 min and filtered the solid under nitrogen atmosphere. Washed the bed with MTBE (20 mL*4). Dried the solid in desiccator for 12 h at 25-30 °C and purified the crude by RP-HPLC.

Example 7:
Charged tert-butyl protected Liraglutide (2.0 g) to 1,4-dioxane- 4 N HCl (40 mL) at 20 °C. Stirred the mass for 2- 3 h at 25 °C. Concentrated the mass and add to chilled MTBE (100 mL) at 5-10 °C. Stirred for 10 min and filtered the solid under nitrogen atmosphere. Washed the bed with MTBE (20 mL*4). Dried the solid in VTD for 12 h at 25-30 °C and purified the crude by RP-HPLC.

Example 8:
Charged linear peptide component of liraglutide (12.0 g) to purified water (110.4 mL) at 25 °C. Stirred the mass to get clear solution. Charged ACN (24 mL). Stirred for 5 min. Charged triethylamine (7.08 mL, 15 eq.) at 25°C. Stirred the mass for 5 min at same temperature. Dissolved Pal-Glu(ONSu)-OtBu (1.326 g) in ACN (72 mL) and charged to above reaction mass at 25 °C. Rinsed with ACN (33.6 mL) and transferred to reaction mixture. Stirred for around 30 min. Upon completion of acylation added TFA over a period of time. TFA concentration in the mixture is around 60-80 %. Stirred the mixture for around 3 h. Upon of completion OtBu deprotection added the reaction mixture to chilled ether. The crude peptide will be precipitated out. The precipitated peptide is centrifuged and washed the pellet with MTBE. Dry the solid in VTD for 12 h. The crude yield ≥ 60.0 % and purity ≥ 80.0%.

Example 9:
Charged linear peptide component of liraglutide (12.0 g) to purified water (110.4 mL) at 25 °C. Stirred the mass to get clear solution. Charged ACN (24 mL). Stirred for 5 min. Charged triethylamine (7.08 mL, 15 eq.) at 25 °C. Stirred the mass for 5 min at same temperature. Dissolved Pal-Glu(ONSu)-OtBu (1.326 g) in ACN (72 mL) and charged to above reaction mass at 25 °C. Rinsed with ACN (33.6 mL) and transferred to reaction mixture. Stirred for around 30 min. Upon completion of acylation, added 5-10 eq. of tryptophan and TFA over a period of time. TFA concentration in the mixture is around 60-80 %. Stirred the mixture for around 3 h. Upon completion of OtBu deprotection added the reaction mixture to chilled ether. The crude peptide will be precipitated out. Filtered the precipitated peptide and give water slurry. Filtered, suck dried the solid and washed the bed with acetonitrile and MTBE. Dried the solid in VTD for 12 h. The crude yield is ≥ 60.0 % and purity ≥ 80.0%.

Example 10:
Dissolved the linear wet pellet (with 12 g equivalent of linear peptide component of liraglutide) in 5% TEA in water (120 mL) at 20 °C. Stirred the mass to get clear solution. Estimated the linear peptide component of liraglutide content by UV spectrophotometer. Charged ACN (24 mL) and stirred for 5 min. Dissolved Pal-Glu(ONSu)-OtBu (0.9-1.5 eq.) in ACN (72 mL) and charged to above reaction mass at 25 °C. Rinsed with ACN (24 mL) and transferred to reaction mixture. Stirred for around 30 min. Upon completion of acylation, added 5-10 eq. of tryptophan. Adjusted the pH of the reaction mass to 4 -5 using TFA. Concentrated the reaction mass to remove ACN. Added TFA (30-40 v) at 10-15 °C. Stirred the mixture for around 3-5 h. Upon completion of OtBu deprotection, concentrated the mass to 50 % of its initial volume and added the concentrated syrup to chilled ether. The crude peptide will be precipitated out. Filtered the precipitated peptide and gave water slurry. Filtered, suck dried the solid and washed the bed with acetonitrile and MTBE. Dried the solid in VTD for 12 h. The crude yield is ≥ 60.0 % and purity ≥ 80.0%.