DESC:FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See section 10, rule 13]

A PROCESS FOR THE PREPARATION OF SEMAGLUTIDE

PIRAMAL PHARMA LIMITED, a company incorporated under the Companies Act 2013, of Ground Floor, Piramal Ananta, Agastya Corporate Park, Kamani Junction, LBS Marg, Kurla West, Mumbai 400070, State of Maharashtra, India

The following specification particularly describes the invention and the manner in which it is to be perfomed
FIELD OF THE INVENTION
The present invention relates to a process for the preparation of Semaglutide of formula (1) by fragment condensation using solid phase peptide synthesis.

H-1His-Aib-3Glu-Gly-5Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-16Gly-17Gln-Ala-Ala-Lys(ODDA-?-Glu-PEG-PEG)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-31Gly-OH
Formula (1)

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.

Semaglutide is an antidiabetic medication developed by Novo Nordisk used for the treatment of type 2 diabetes and an anti-obesity medication used for long-term weight management. It is a peptide similar to the hormone glucagon-like peptide-1 (GLP-1), modified with a side chain. It can be administered by subcutaneous injection or taken orally. Semaglutide was approved in United States on 5 December 2017, in Europe on 12 February 2018. It is sold under the brand names Ozempic and Rybelsus for diabetes, and under the brand name Wegovy for weight loss.

Semaglutide is described in US 8129343. According to the disclosure of US 8129343, the amino acid backbone of Semaglutide is prepared by standard sequential Fmoc-solid phase peptide synthesis followed by deprotection and then by coupling of the side chain fragment to the Lys20 side chain. The disclosed route has several disadvantages. For example, the sequential synthesis disclosed results in low purity. Additionally, the coupling of the side chain moiety to Lys side chain in the presence of additional amine group at the N-terminus is not selective enough and results in impurities due to wrong or double attachment of the side chain moiety. This results in the need of additional purification cycles and loss of the yield. US 8129343 further discloses using N -[1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl] (Dde) as protecting group on the Lys. This strategy is disadvantageous because hydrazine, which is a toxic reagent, is required for the removal of the Dde group.

Various researchers and scientists reported the fragment based as well as sequential synthesis of Semaglutide in solid and liquid phase.

Semaglutide synthesized by a linear/sequential synthesis as described in the prior art documents possess lot of technical difficulties, expensive production costs and such synthesis is not viable for the preparation on an industrial scale. Hence, there is a significant need to develop an improved process for the preparation of Semaglutide.

The inventors of the present invention after analysing the literature, have taken substantial efforts to develop a process for the preparation of Semaglutide which involves simple operations and easy to carry out chemical conversions.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a process for the preparation of Semaglutide of Formula (1),
H-1His-Aib-3Glu-Gly-5Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-16Gly-17Gln-Ala-Ala-Lys(ODDA-?-Glu-PEG-PEG)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-31Gly-OH
Formula (1)
comprising the steps of:
step i) coupling X-Gly to a resin to obtain X-Gly-resin; and elongation of the peptide with sequential addition of protected amino acid(s) by solid phase synthesis to X-Gly-resin to obtain protected peptide bound to a support to obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin; wherein Fmoc-Lys(Alloc)-OH is used as a raw material of Lys20;
OR
step i)
a) condensing X-17Gln(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-31Gly-Resin (Fragment P) with 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) in presence of a coupling agent and in a solvent to obtain X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin; and
b) condensing the peptide obtained in step (b) with:
X-Glu(Y)-Gly-OH (Fragment R) followed by X-His(Y)-Aib-OH (Fragment M); OR
X-Glu(Y)-Gly-OH (Fragment R) followed by X-Aib-OH (Fragment H) followed by X-His(Y)-OH (Fragment T); OR
X-Gly-OH (Fragment A) followed by X-Glu(Y)-OH (Fragment L) followed by X-His(Y)-Aib-OH (Fragment M); OR
X-Gly-OH (Fragment A) followed by X-Glu(Y)-OH (Fragment L) followed by X-Aib-OH (Fragment H) followed by X-His(Y)-OH (Fragment T); OR
X-Gly-OH (Fragment A) followed by X-His(Y)-Aib-Glu(Y) (Fragment X); OR
X-Aib-Glu(Y)-Gly-OH (Fragment V) followed by X-His(Y)-OH (Fragment T); OR
X-Gly-OH (Fragment A) followed by X-Aib-Glu(Y)-OH (Fragment Y) followed by X-His(Y)-OH (Fragment T); OR
X-His(Y)-Aib-Glu(Y)-Gly-OH (Fragment Z);
each coupling in presence of a coupling agent and solvent to obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
wherein each of the fragment’s A, H, L, M, T, V, X, Y and Z may be prepared by sequential coupling of suitable protected peptides in presence of a coupling agent and solvent;

step ii) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y);
each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin; and

step iii) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide of Formula (1);
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

In another embodiment, the present invention provides a process for the preparation of Semaglutide, which comprises:
a) Synthesizing Fragments P, I, R and M;
X-17Gln(Y)-Ala-Ala-Lys(Alloc)-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-His(Y)-Aib-OH (Fragment M)
wherein Fragment I contain the protected dipeptide comprising a pseudoproline dipeptide;
b) condensing Fragment P with Fragment I in presence of a coupling agent and in a solvent to obtain X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 obtain 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(Y)-Ala-Ala-Lys(Alloc)-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 obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
e) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y);
each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
f) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide;
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

In another embodiment, the present invention provides a process for the preparation of Semaglutide, which comprises:
a) synthesizing Fragments P, I, R, H and T;
X-17Gln(Y)-Ala-Ala-Lys(Alloc)-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-Aib-OH (Fragment H)
X-His(Y)-OH (Fragment T)
wherein Fragment I contain the protected dipeptide comprising a pseudoproline dipeptide;
b) condensing Fragment P with Fragment I in presence of a coupling agent and in a solvent to obtain X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 obtain 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(Y)-Ala-Ala-Lys(Alloc)-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 H in presence of a coupling agent and in a solvent to obtain X-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-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 T in presence of a coupling agent and in a solvent to obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
f) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y);
each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
g) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide;
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

In another embodiment, the present invention provides a process for the preparation of Semaglutide, which comprises:
a) synthesizing Fragments P, I, A, L and M;
X-17Gln(Y)-Ala-Ala-Lys(Alloc)-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-Gly-OH (Fragment A)
X-Glu(Y)-OH (Fragment L)
X-His(Y)-Aib-OH (Fragment M)
wherein Fragment I contain the protected dipeptide comprising a pseudoproline dipeptide;
b) condensing Fragment P with Fragment I in presence of a coupling agent and in a solvent to obtain X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 Fragment A in presence of a coupling agent and in a solvent to obtain X-Gly-Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 L in presence of a coupling agent and in a solvent to obtain 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(Y)-Ala-Ala-Lys(Alloc)-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 dipeptide Fragment M in presence of a coupling agent and in a solvent to obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
f) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y);
each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
g) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide;
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

In another embodiment, the present invention provides a process for the preparation of Semaglutide, which comprises:
a) synthesizing Fragments P, I, A, L, H and T;
X-17Gln(Y)-Ala-Ala-Lys(Alloc)-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-Gly-OH (Fragment A)
X-Glu(Y)-OH (Fragment L)
X-Aib-OH (Fragment H)
X-His(Y)-OH (Fragment T)
wherein Fragment I contain the protected dipeptide comprising a pseudoproline dipeptide;
b) condensing Fragment P with Fragment I in presence of a coupling agent and in a solvent to obtain X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 Fragment A in presence of a coupling agent and in a solvent to obtain X-Gly-Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 L in presence of a coupling agent and in a solvent to obtain 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(Y)-Ala-Ala-Lys(Alloc)-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 H in presence of a coupling agent and in a solvent to obtain X-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
f) condensing the peptide obtained in step (e) with a Fragment T in presence of a coupling agent and in a solvent to obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
g) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y);
each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
h) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide;
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

In another embodiment, the present invention provides a process for the preparation of Semaglutide, which comprises:
a) synthesizing Fragments P, I, A and X;
X-17Gln(Y)-Ala-Ala-Lys(Alloc)-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-Gly-OH (Fragment A)
X-His(Y)-Aib-Glu(Y) (Fragment X)
wherein Fragment I contain the protected dipeptide comprising a pseudoproline dipeptide;
b) condensing Fragment P with Fragment I in presence of a coupling agent and in a solvent to obtain X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 Fragment A in presence of a coupling agent and in a solvent to obtain X-Gly-Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 X in presence of a coupling agent and in a solvent to obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
e) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y);
each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
f) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide;
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

In another embodiment, the present invention provides a process for the preparation of Semaglutide, which comprises:
a) synthesizing Fragments P, I, V and T;
X-17Gln(Y)-Ala-Ala-Lys(Alloc)-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-Aib-Glu(Y)-Gly-OH (Fragment V)
X-His(Y)-OH (Fragment T)
wherein Fragment I contain the protected dipeptide comprising a pseudoproline dipeptide;
b) condensing Fragment P with Fragment I in presence of a coupling agent and in a solvent to obtain X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 Fragment V in presence of a coupling agent and in a solvent to obtain X-Aib-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(Y)-Ala-Ala-Lys(Alloc)-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 T in presence of a coupling agent and in a solvent to obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
e) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y);
each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
f) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide;
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

In another embodiment, the present invention provides a process for the preparation of Semaglutide, which comprises:
a) synthesizing Fragments P, I, A, Y and T;
X-17Gln(Y)-Ala-Ala-Lys(Alloc)-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-Gly-OH (Fragment A)
X-Aib-Glu(Y)-OH (Fragment Y)
X-His(Y)-OH (Fragment T)
wherein Fragment I contain the protected dipeptide comprising a pseudoproline dipeptide;
b) condensing Fragment P with Fragment I in presence of a coupling agent and in a solvent to obtain X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 Fragment A in presence of a coupling agent and in a solvent to obtain X-Gly-Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 Y in presence of a coupling agent and in a solvent to obtain X-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-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 T in presence of a coupling agent and in a solvent to obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
f) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y);
each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
g) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide;
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

In another embodiment, the present invention provides a process for the preparation of Semaglutide, which comprises:
a) synthesizing Fragments P, I and Z;
X-17Gln(Y)-Ala-Ala-Lys(Alloc)-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-His(Y)-Aib-Glu(Y)-Gly-OH (Fragment Z)
wherein Fragment I contain the protected dipeptide comprising a pseudoproline dipeptide;
b) condensing Fragment P with Fragment I in presence of a coupling agent and in a solvent to obtain X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 Fragment Z in presence of a coupling agent and in a solvent to obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
d) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y);
each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
e) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide;
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

In another embodiment, the present invention provides a process for the preparation of Semaglutide, which comprises:
a) coupling X-Gly to a resin by solid phase synthesis to obtain X-Gly-resin; and
b) elongation of a peptide with sequential addition of protected amino acid(s) by solid phase synthesis to X-Gly-resin to obtain protected peptide bound to a support;
wherein Fmoc-Lys(Alloc)-OH is used as a raw material of Lys20;
c) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y); each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
d) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide;
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

OBJECTIVE OF INVENTION
An objective of the present invention is to provide a process for preparing Semaglutide, which is simple, industrially applicable, and robust.
BRIEF DESCRIPTION OF ABBREVIATIONS
ACN: Acetonitrile
Aib: 2-Aminoisobutyric acid
Ala: Alanine
Alloc: Allyloxycarbonyl
Arg: Arginine
Asp: Aspartic acid
Boc: t-Butyloxycarbonyl
Bpoc: 2-(4-Biphenyl)isopropoxycarbonyl
Cbz: Benzyloxycarbonyl
DCM: Dichloromethane
DIC: Diisoprpopylcarbodiimide
DIPEA: N,N-Diisopropylethylamine
DMAC: N,N-Dimethylacetamide
DMF: N,N-Dimethylformamide
DMT: dimethoxy trityl
DCC: N,N-Dicyclohexylcarbodiimide
DCU: Dicyclohexylurea
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
MTB Ether: Methyl tert-butyl ether
NMP: N-methyl Pyrrolidone
ODDA: Octadecanedioic acid
OtBu: t-butyl ester
Pbf: 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl
Pd(PPh3)4: Tetrakis(triphenylphosphine)palladium(0)
PEG: Polyethylene Glycol
PFP: Pentafluorophenol
Phe: Phenylalanine
Pmc: 2,2,5,7,8-pentamethylchroman-6-sulfonyl
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
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 in 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 Semaglutide by coupling of three or more suitably protected fragments on solid support.
It has surprisingly been found that the use of at least one Fmoc pseudoproline dipeptide advantageously suppresses peptide aggregation and hence the formation of by-products due to inefficient synthesis. Preferably, the pseudoproline dipeptide is introduced at a position corresponding to or identical with a position selected from Gly4-Thr5, Phe6-Thr7, Thr7-Ser8, Val10-Ser11 or Ser11-Ser12 of the peptide.
The term “pseudoproline dipeptides” as used herein refers to temporary proline mimics, which can be readily obtained from Ser and Thr by oxazolidine formation and from Cys by thiazolidine formation. These dipeptides are one possible option to mitigate on-resin aggregation during SPPS. The 2,2-dimethyloxazolidines (Psi(Me,Me)pro) are smoothly cleaved by TFA and thus particularly suitable for Fmoc-SPPS. Hence, in particular Fmoc-Gly-Thr(Psi(Me,Me)pro)-OH, Fmoc-Phe-Thr(Psi(Me,Me)pro)-OH, Fmoc-Thr(tBu)-Ser(Psi(Me,Me)pro)-OH, Fmoc-Val-Ser(Psi(Me,Me)pro)-OH, and/or Fmoc-Ser(tBu)-Ser(Psi(Me,Me)pro)-OH may be used. One or more pseudoproline dipeptides are introduced at a position selected from the group consisting of Gly4-Thr5, Phe6-Thr7, Thr7-Ser8, Val10-Ser11 or Ser11-Ser12 of the peptide of formula (1).
In one embodiment of the present invention is to provide a process for the preparation of Semaglutide, 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 yield Semaglutide.

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 obtain protected Semaglutide and further deprotected to obtain Semaglutide.

The suitable fragments selected for the preparation of Semaglutide are as follows:
• X-17Gln(Y)-Ala-Ala-Lys(Alloc)-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-His(Y)-Aib-OH (Fragment M)
• X-Gly-OH (Fragment A)
• X-Glu(Y)-OH (Fragment L)
• X-Aib-OH (Fragment H)
• X-His(Y)-OH (Fragment T)
• X-His(Y)-Aib-Glu (Y) (Fragment X)
• X-Aib-Glu(Y)-Gly-OH (Fragment V)
• X-Aib-Glu(Y)-OH (Fragment Y)
• X-His(Y)-Aib-Glu(Y)-Gly-OH (Fragment Z)

wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

In one embodiment, the present invention provides a process for the preparation of Semaglutide of Formula (1),
H-1His-Aib-3Glu-Gly-5Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-16Gly-17Gln-Ala-Ala-Lys(ODDA-?-Glu-PEG-PEG)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-31Gly-OH
Formula (1)
comprising the steps of:
step i) coupling X-Gly to a resin to obtain X-Gly-resin; and elongation of the peptide with sequential addition of protected amino acid(s) by solid phase synthesis to X-Gly-resin to obtain protected peptide bound to a support to obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin; wherein Fmoc-Lys(Alloc)-OH is used as a raw material of Lys20;
OR
step i)
a) condensing X-17Gln(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-31Gly-Resin (Fragment P) with 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) in presence of a coupling agent and in a solvent to obtain X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin; and
b) condensing the peptide obtained in step (b) with:
X-Glu(Y)-Gly-OH (Fragment R) followed by X-His(Y)-Aib-OH (Fragment M); OR
X-Glu(Y)-Gly-OH (Fragment R) followed by X-Aib-OH (Fragment H) followed by X-His(Y)-OH (Fragment T); OR
X-Gly-OH (Fragment A) followed by X-Glu(Y)-OH (Fragment L) followed by X-His(Y)-Aib-OH (Fragment M); OR
X-Gly-OH (Fragment A) followed by X-Glu(Y)-OH (Fragment L) followed by X-Aib-OH (Fragment H) followed by X-His(Y)-OH (Fragment T); OR
X-Gly-OH (Fragment A) followed by X-His(Y)-Aib-Glu(Y) (Fragment X); OR
X-Aib-Glu(Y)-Gly-OH (Fragment V) followed by X-His(Y)-OH (Fragment T); OR
X-Gly-OH (Fragment A) followed by X-Aib-Glu(Y)-OH (Fragment Y) followed by X-His(Y)-OH (Fragment T); OR
X-His(Y)-Aib-Glu(Y)-Gly-OH (Fragment Z);
each coupling in presence of a coupling agent and solvent to obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
wherein each of the fragments A, H, L, M, T, V, X, Y and Z may be prepared by sequential coupling of suitable protected peptides in presence of a coupling agent and solvent;

step ii) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y);
each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin; and

step iii) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide of Formula (1);
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

In an embodiment, X is an amino protecting group selected from Fmoc, Boc, Cbz or Bpoc.

In an embodiment, Y is a carboxyl, amide, phenolic and alcoholic protecting group selected from DMT, MMT, Trt, tert-butyl or t-butoxy carbonyl.

In an embodiment, Z is a guanidine protecting group 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 mixture thereof.

The coupling reaction is carried out in presence of a base. The base used may be an 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, lithium hydroxide, 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 or 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 include, most preferred super acid labile resin is 2-chlorotrityl resins.
In an embodiment of the present invention the solid support is a resin, wherein said resin is chosen from Wang resin or 2-CTC resin.

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

In the present invention, the protection group is preferably 9-Fluorenylmethyloxycarbonyl (Fmoc) group.

The organic base is selected from the group comprising piperidine, piperazine, N-methyl morpholine, diethyl amine, triethyl amine, 1,8-Diazabicyclo [5.4.0]undec-7-ene (DBU) or mixtures thereof.

The organic solvent is selected from the group comprising dimethyl formamide (DMF), N-Methyl-2-Pyrrolidone (NMP), dichloromethane (DCM), tetrahydrofuran (THF), N,N-dimethylacetamide (DMAC) or mixture thereof.

In an embodiment, the cleavage of the protected peptide from the resin and the deprotection of the groups on amino acids are simultaneous, to yield Semaglutide.
In an embodiment of the present invention in step i (a), 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 i (b), 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 (ii), the coupling agent used is a mixture of hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide (DIC) in DMF solvent.

In an embodiment, step (iii) of the present process 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 (tBu, Boc, Trt, Pseudoproline, Pbf) was carried out by the treatment of cocktail mixture/cleaving reagent.

In an embodiment, the “cocktail mixture/cleaving reagent” used in step (iii) is selected from HF, TFA (trifluoroacetic acid), TIS or TIPS (triisopropyl silane), Phenol, water, Anisole, Thioanisole, EDT (Ethane- 1,2-di thiol), 1 -dodecanethiol (DDT), Dithiothreitol (DTT), methanesulfonic acid or mixtures thereof.

In one embodiment, the “cocktail mixture/cleaving reagent” used in step (iii) is selected from TFA:TIS:Anisole:water:phenol (84:5:5:3:3), TFA:TIS:Anisole:water:phenol (85:5:5:4:1), TFA:TIS:Anisole:water:phenol (86:5:5:3:1), TFA:TIS:Anisole:water:phenol (87:5:5:2:1), or TFA:TIS:Anisole:water:phenol (88:5:5:1:1).
In another embodiment of the present invention in step (iii), the deprotection of all the protecting groups was carried out by the treatment of the cocktail cleavage mixture of TFA: TIS: Anisole: Water: Phenol (85:5:5:4:1).
In yet another embodiment of the present invention in step (iii), the deprotection of all the protecting groups was carried out by the treatment of the cocktail cleavage mixture of TFA: TIS: Anisole: Water: Phenol (86:5:5:3:1).
In a further embodiment of the present invention in step (iii), the deprotection of all the protecting groups was carried out by the treatment of the cocktail cleavage mixture of TFA: TIS: Anisole: Water: Phenol (87:5:5:2:1).

In another embodiment the present invention is to provide a process for the preparation of Semaglutide, which comprises:
a) synthesizing Fragments P, I, R and M;
X-17Gln(Y)-Ala-Ala-Lys(Alloc)-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-His(Y)-Aib-OH (Fragment M)
wherein Fragment I contain the protected dipeptide comprising a pseudoproline dipeptide;
b) condensing Fragment P with Fragment I in presence of a coupling agent and in a solvent to obtain X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 obtain 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(Y)-Ala-Ala-Lys(Alloc)-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 obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
e) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y); each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
f) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide;
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

In another embodiment, the present invention provides a process for the preparation of Semaglutide, which comprises:
a) synthesizing Fragments P, I, R, H and T;
X-17Gln(Y)-Ala-Ala-Lys(Alloc)-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-Aib-OH (Fragment H)
X-His(Y)-OH (Fragment T)
wherein Fragment I contain the protected dipeptide comprising a pseudoproline dipeptide;
b) condensing Fragment P with Fragment I in presence of a coupling agent and in a solvent to obtain X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 obtain 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(Y)-Ala-Ala-Lys(Alloc)-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 H in presence of a coupling agent and in a solvent to obtain X-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-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 T in presence of a coupling agent and in a solvent to obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
f) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y);
each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
g) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide;
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.
In another embodiment, the present invention provides a process for the preparation of Semaglutide, which comprises:
a) synthesizing Fragments P, I, A, L and M;
X-17Gln(Y)-Ala-Ala-Lys(Alloc)-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-Gly-OH (Fragment A)
X-Glu(Y)-OH (Fragment L)
X-His(Y)-Aib-OH (Fragment M)
wherein Fragment I contain the protected dipeptide comprising a pseudoproline dipeptide;
b) condensing Fragment P with Fragment I in presence of a coupling agent and in a solvent to obtain X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 Fragment A in presence of a coupling agent and in a solvent to obtain X-Gly-Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 L in presence of a coupling agent and in a solvent to obtain 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(Y)-Ala-Ala-Lys(Alloc)-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 dipeptide Fragment M in presence of a coupling agent and in a solvent to obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
f) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y);
each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
g) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide;
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

In another embodiment, the present invention provides a process for the preparation of Semaglutide, which comprises:
a) synthesizing Fragments P, I, A, L, H and T;
X-17Gln(Y)-Ala-Ala-Lys(Alloc)-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-Gly-OH (Fragment A)
X-Glu(Y)-OH (Fragment L)
X-Aib-OH (Fragment H)
X-His(Y)-OH (Fragment T)
wherein Fragment I contain the protected dipeptide comprising a pseudoproline dipeptide;
b) condensing Fragment P with Fragment I in presence of a coupling agent and in a solvent to obtain X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 Fragment A in presence of a coupling agent and in a solvent to obtain X-Gly-Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 L in presence of a coupling agent and in a solvent to obtain 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(Y)-Ala-Ala-Lys(Alloc)-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 H in presence of a coupling agent and in a solvent to obtain X-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
f) condensing the peptide obtained in step (e) with a Fragment T in presence of a coupling agent and in a solvent to obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
g) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y);
each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
h) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide;
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

In another embodiment, the present invention provides a process for the preparation of Semaglutide, which comprises:
a) synthesizing Fragments P, I, A and X;
X-17Gln(Y)-Ala-Ala-Lys(Alloc)-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-Gly-OH (Fragment A)
X-His(Y)-Aib-Glu(Y) (Fragment X)
wherein Fragment I contain the protected dipeptide comprising a pseudoproline dipeptide;
b) condensing Fragment P with Fragment I in presence of a coupling agent and in a solvent to obtain X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 Fragment A in presence of a coupling agent and in a solvent to obtain X-Gly-Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 X in presence of a coupling agent and in a solvent to obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
e) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y);
each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
f) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide;
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

In another embodiment, the present invention provides a process for the preparation of Semaglutide, which comprises:
a) synthesizing Fragments P, I, V and T;
X-17Gln(Y)-Ala-Ala-Lys(Alloc)-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-Aib-Glu(Y)-Gly-OH (Fragment V)
X-His(Y)-OH (Fragment T)
wherein Fragment I contain the protected dipeptide comprising a pseudoproline dipeptide;
b) condensing Fragment P with Fragment I in presence of a coupling agent and in a solvent to obtain X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 Fragment V in presence of a coupling agent and in a solvent to obtain X-Aib-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(Y)-Ala-Ala-Lys(Alloc)-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 T in presence of a coupling agent and in a solvent to obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
e) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y);
each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
f) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide;
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

In another embodiment, the present invention provides a process for the preparation of Semaglutide, which comprises:
a) synthesizing Fragments P, I, A, Y and T;
X-17Gln(Y)-Ala-Ala-Lys(Alloc)-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-Gly-OH (Fragment A)
X-Aib-Glu(Y)-OH (Fragment Y)
X-His(Y)-OH (Fragment T)
wherein Fragment I contain the protected dipeptide comprising a pseudoproline dipeptide;
b) condensing Fragment P with Fragment I in presence of a coupling agent and in a solvent to obtain X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 Fragment A in presence of a coupling agent and in a solvent to obtain X-Gly-Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 Y in presence of a coupling agent and in a solvent to obtain X-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-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 T in presence of a coupling agent and in a solvent to obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
f) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y);
each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
g) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide;
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

In another embodiment, the present invention provides a process for the preparation of Semaglutide, which comprises:
a) synthesizing Fragments P, I and Z;
X-17Gln(Y)-Ala-Ala-Lys(Alloc)-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-His(Y)-Aib-Glu(Y)-Gly-OH (Fragment Z)
wherein Fragment I contain the protected dipeptide comprising a pseudoproline dipeptide;
b) condensing Fragment P with Fragment I in presence of a coupling agent and in a solvent to obtain X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-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 Fragment Z in presence of a coupling agent and in a solvent to obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
d) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y);
each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
e) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide;
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

In another embodiment, the present invention provides a process for the preparation of Semaglutide, which comprises:
a) coupling X-Gly to a resin by solid phase synthesis to obtain X-Gly-resin; and
b) elongation of a peptide with sequential addition of protected amino acid(s) by solid phase synthesis to X-Gly-resin to obtain protected peptide bound to a support;
wherein Fmoc-Lys(Alloc)-OH is used as a raw material of Lys20;
c) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y); each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
d) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide;
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

In an embodiment, the present invention provides a process for the purification of crude Semaglutide comprising, subjecting crude Semaglutide to RP-HPLC (Reverse phase high performance liquid chromatography), which is a three stage purification with different mobile phase buffer composition (ammonium acetate/ ammonium bicarbonate/ trifluoroacetic acid/ acetic acid) and acetonitrile, methanol as organic solvents on C-8/C-4/phenyl silica column or daisogel silica.

In an embodiment, the present invention provides a process for preparation of Fragment P, comprising:
anchoring Fmoc-Gly-OH to a resin in presence of a coupling agent and solvent;
selective deprotection of amino acid using a base;
sequential coupling of Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH to the obtained resin in presence of a coupling agent and solvent; and
cleaving of protected peptide from solid support to get Fragment P.
Within the context of the present invention, Fragment P is prepared by solid phase synthesis as the process described in the Scheme 1:
Synthesis of Fragment P
Fmoc-Gly-Wang resin
20 % Piperidine/0.25 % HOBt.H2O in DMF
Fmoc-Arg(Pbf)-OH

Fmoc-Arg(Pbf)-31Gly-Wang resin
20 % Piperidine/0.25 % HOBt.H2O in 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/0.25 % HOBt.H2O in DMF
Fmoc-Lys(Alloc)-OH

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

20 % Piperidine/0.25 % HOBt.H2O in DMF
Fmoc-Ala-OH, Fmoc-Ala-OH
Fmoc-Gln(Trt)-OH,
Fmoc-Thr(tBu)-OH

Fmoc-17Gln(Trt)-Ala-Ala-Lys(Alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-31Gly-Wang resin (Fragment P)
Scheme 1

In an embodiment, the present invention provides a process for preparation of Fragment I, comprising:
anchoring Fmoc-Gly-OH to a resin in presence of a coupling agent and solvent;
selective deprotection of amino acid using a base;
sequential coupling of Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-Ser(PsiMe, MePro)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH to the obtained resin in presence of a coupling agent and solvent; and
cleaving of protected peptide from solid support to get Fragment I.
Within the context of the present invention, Fragment I is prepared by solid phase synthesis as the process described in the Scheme 2:
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 MTB% 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 (Fragment I)
Scheme 2
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 0.25 M 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(Trt)-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 ~0.37 mmol/gram (about 27.03 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 (0.25 M) 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 min, 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 (0.25 M) in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Gly-OH (20 mmol, 2.0 eq) was coupled using HOBt (20 mmol, 2.0 eq) and DIC (20 mmol, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 min 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 (0.25 M) in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Arg(Pbf)-OH (20 mmol, 2.0 eq) was coupled using HOBt (20 mmol, 2.0 eq) and DIC (20 mmol, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 min 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 (0.25 M) in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Val-OH (20 mmol, 2.0 eq) was coupled using HOBt (20 mmol, 2.0 eq) and DIC (20 mmol, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 min 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 (0.25 M) in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Leu-OH (20 mmol, 2.0 eq) was coupled using HOBt (20 mmol, 2.0 eq) and DIC (20 mmol, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 min 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 (0.25 M) in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Trp(Boc)-OH (20 mmol, 2.0 eq) was coupled using HOBt (20 mmol, 2.0 eq) and DIC (20 mmol, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 min 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 (0.25 M) in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Ala-OH (20 mmol, 2.0 eq) was coupled using HOBt (20 mmol, 2.0 eq) and DIC (20 mmol, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 min 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 (0.25 M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt (0.25 M) in DMF. The Fmoc-Ile-OH (20 mmol, 2.0 eq) was coupled using HOBt (20 mmol, 2.0 eq) and DIC (20 mmol, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 min 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 (0.25 M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt (0.25 M) in DMF. The Fmoc-Phe-OH (20 mmol, 2.0 eq) was coupled using HOBt (20 mmol, 2.0 eq) and DIC (20 mmol, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 min 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 (0.25 M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt (0.25 M) in DMF. The Fmoc-Glu(OtBu)-OH (20 mmol, 2.0 eq) was coupled using HOBt (20 mmol, 2.0 eq) and DIC (20 mmol, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 min 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 (0.25 M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt 0.25 M) in DMF. The Fmoc-Lys(Pal-Glu-OtBu)-OH (30 mmol, 3.0 eq) was coupled using HOBt (30 mmol, 3.0 eq) and DIC (30 mmol, 3.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 180 min 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 (0.25 M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt (0.25 M) in DMF. The Fmoc-Ala-OH (20 mmol, 2.0 eq) was coupled using HOBt (20 mmol, 2.0 eq) and DIC (20 mmol, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 min 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 (0.25 M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt (0.25 M) in DMF. The Fmoc-Ala-OH (20 mmol, 2.0 eq) was coupled using HOBt (20 mmol, 2.0 eq) and DIC (20 mmol, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 min 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(Trt)-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 (0.25 M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt (0.25 M) in DMF. The Fmoc-Gln(Trt)-OH (20 mmol, 2.0 eq) was coupled using HOBt (20 mmol, 2.0 eq) and DIC (20 mmol, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 min 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 the functionality 1.6 mmol/g in PP Bottle, add the activated amino acid solution to PP bottle having resin, shacked the bottle manually for next 5 minutes and then added 4 equivalents of DIEA/DCM mixture in PP bottle, allow the PP bottle 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 deprotected 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 s. 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 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 48.5 g 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(Trt)-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 (Fragment P; Example 1) was carried out by washing the resin using 20 % Piperidine, HOBt (0.25 M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt (0.25 M) in DMF. The Protected fragment from example 2 (Fragment I; 12 mmol,1.2 eq) was coupled using HOBt (24 mmol,2.4 eq) and DIC (24 mmol, 2.4 eq) in NMP solvent. The mixture was stirred via Nitrogen for 180 min at 40 °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:

Fmoc-Glu(OtBu)-OH (150 g,1 eq) dissolved in 1500 ml (10 ml/g) Ethyl acetate in 3L RB flask, after complete dissolution added solution of 71.4 g (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 g (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 g (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 g, 1 eq) in 2240 ml (40 ml/g) 1,4-dioxane stirred it to clear and cooled to 5 °C using ordinary ice bath. In another beaker, 1120 ml (20 ml/g) water, dissolved 7.95 g LiOH.H2O (2.0 eq) and 5.24 g 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 h, monitored reaction progress on HPLC. Reaction mixture taken for workup after 14 h, 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 h of aging at cooling washed with water and dried in hot air oven.
Yield: 32 g (70.06 %); Purity (by HPLC): 98.63 %.

Example 5: Coupling of dipeptide 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(Trt)-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 (0.25 M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt (0.25 M) in DMF. The Fmoc-Glu(OtBu)-Gly-OH (20 mmol, 2.0 eq) was coupled using HOBt (20 mmol, 2.0 eq) and DIC (20 mmol, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 min 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 dipeptide Fragment M with peptidyl resin of example 5.

Synthesis of Boc-(1)His(Trt)-(2)Aib-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(Trt)-Ala-Ala-Lys(Alloc)-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 (0.25- M) in DMF two times for 2 and 10 min, followed by washing the resin four times with HOBt (0.25 M) in DMF. The Boc-His(Trt)-Aib-OH (20 mmol, 2.0 eq) was coupled using HOBt (20 mmol, 2.0 eq) and DIC (20 mmol, 2.0 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 min 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 side chain group PEG with peptidyl resin of example 6.

Synthesis of Boc-(1)His(Trt)-Aib-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(Trt)-Ala-Ala-Lys(Fmoc-PEG)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-(31)Gly-Wang resin:

Alloc-deprotection of the 20th Lys(Alloc) from protected fragment (1-31) resin of example 6 was carried out by washing the resin using Pd(PPh3)4 (0.2 eq) /Phenyl silane (10 eq) in DCM three times for 30 min each, followed by washing the resin three times with DCM and three times with DMF followed by 0.5 % DIEA/DCM washings and three washings of 0.02 M to 0.05 M Sodium dithiodiethylcarbamate/DMF washings lastly wash the resin four times with five DMF wash. The Fmoc-PEG-OH (20 mmol, 2 eq) was coupled using HOBt (20 mmol, 2 eq) and DIC (20 mmol, 2 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 min 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 8: Coupling of side chain group PEG with peptidyl resin of example 7.

Synthesis of Boc-(1)His(Trt)-Aib-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(Trt)-Ala-Ala-Lys(Fmoc-PEG-PEG)-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 in 0.25 M HOBt/DMF two times for 2 and 10 min, followed by washing the resin four times with 0.25 M HOBt/DMF. The Fmoc-PEG-OH (20 mmol, 2 eq) was coupled using HOBt (20 mmol, 2 eq) and DIC (20 mmol, 2 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 min 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 9: Coupling of Glu with side chain group of peptidyl resin of example 8.

Synthesis of Boc-(1)His(Trt)-Aib-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(Trt)-Ala-Ala-Lys(Fmoc-Glu(PEG-PEG)-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 in 0.25 M HOBt/DMF two times for 2 and 10 min, followed by washing the resin four times with 0.25 M HOBt/DMF. The Fmoc-Glu(OH)-OtBu (20 mmol, 2 eq) was coupled using HOBt (20 mmol, 2 eq) and DIC (20 mmol, 2 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 min 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 10: Coupling of ODDA with side chain group of peptidyl resin of example 9.

Synthesis of Boc-(1)His(Trt)-Aib-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(Trt)-Ala-Ala-Lys(OtBu-ODDA-Glu(PEG-PEG)-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 in 0.25 M HOBt/DMF two times for 2 and 10 min, followed by washing the resin four times with 0.25 M HOBt/DMF. The Octadecanedioic Acid Mono-tert-butyl Ester (ODDA ester, 20 mmol, 2 eq) was coupled using HOBt (20 mmol, 2 eq) and DIC (20 mmol, 2 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 min 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: 85.0 g.

Example 11: Preparation of Semaglutide:
H-His1-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(ODDA-?-Glu-(PEG-PEG)-OH)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Ala27-Arg28-Gly29-Arg30-Gly31-OH:

Simultaneous deprotection of all the protecting groups was carried out by using cocktail cleavage mixture of TFA:TIS:Anisole:Water:Phenol (86:5:5:3:1). The cleavage was carried out using cleavage cocktail 10 ml/g of peptidyl resin at -10 °C for initial 15 min followed by the stirring of the peptidyl resin for 3 h 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 l of MTB ether were done to the product. Product was dried under vacuum for 16 h.
Yield: 36.50 g (88.74 % process yield)

Example 12: Coupling of side chain linker [OtBu-ODDA-Glu(PEG-PEG)-OtBu] with peptidyl resin of example 6.
Alloc-deprotection of the 20th Lys(Alloc) from Protected frag (1-31)-resin was carried out by washing the resin using Pd(pph3)4 (0.1 to 0.2 eq Preferably 0.2 eq) /Phenyl silane (5-10 eq preferably 10 eq) in DCM three times for 30 min each, followed by washing the resin three times with DCM & three times with DMF followed by 0.5 % DIEA/DCM washings & three washings of 0.02 M to 0.05 M Sodium dithiodiethylcarbamate/DMF washings lastly wash the resin four times with five DMF wash. The Semaglutide side chain linker i.e., OtBu-ODDA-Glu(PEG-PEG)-OtBu (3 mmol to 4 mmol preferably 4 mmol, 2 eq) was coupled using HOBt (3 mmol to 4 mmol preferably 4 mmol, 2 eq) and DIC (3 mmol to 4 mmol preferably 4 mmol, 2 eq) in DMF solvent. The mixture was stirred via Nitrogen for 120 min 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 MTB ether and drying of resin in desiccator. Weight of the peptidyl resin: 13.37 g.
,CLAIMS:We Claim

1. A process for preparation of Semaglutide of Formula (1),
H-1His-Aib-3Glu-Gly-5Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-16Gly-17Gln-Ala-Ala-Lys(ODDA-?-Glu-PEG-PEG)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-31Gly-OH
Formula (1)
comprising the steps of:
step i) coupling X-Gly to a resin to obtain X-Gly-resin; and elongation of the peptide with sequential addition of protected amino acid(s) by solid phase synthesis to X-Gly-resin to obtain protected peptide bound to a support to obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin; wherein Fmoc-Lys(Alloc)-OH is used as a raw material of Lys20;
OR
step i)
c) condensing X-17Gln(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-31Gly-Resin (Fragment P) with 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) in presence of a coupling agent and in a solvent to obtain X-(5)Thr(Y)-Phe-Thr(Y)-Ser(Y)-Asp(Y)-Val-Ser(PsiMe,MePro)-Ser(Y)-Tyr(Y)-Leu-Glu(Y)-Gly-Gln(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin; and
d) condensing the peptide obtained in step (b) with:
X-Glu(Y)-Gly-OH (Fragment R) followed by X-His(Y)-Aib-OH (Fragment M); OR
X-Glu(Y)-Gly-OH (Fragment R) followed by X-Aib-OH (Fragment H) followed by X-His(Y)-OH (Fragment T); OR
X-Gly-OH (Fragment A) followed by X-Glu(Y)-OH (Fragment L) followed by X-His(Y)-Aib-OH (Fragment M); OR
X-Gly-OH (Fragment A) followed by X-Glu(Y)-OH (Fragment L) followed by X-Aib-OH (Fragment H) followed by X-His(Y)-OH (Fragment T); OR
X-Gly-OH (Fragment A) followed by X-Glu(Y)-Aib-His(Y)-OH (Fragment X); OR
X-Gly-X-Glu(Y)-Aib-OH (Fragment V) followed by X-His(Y)-OH (Fragment T); OR
X-Gly-OH (Fragment A) followed by X-Aib-Glu(Y)-OH (Fragment Y) followed by X-His(Y)-OH (Fragment T); OR
X-His(Y)-Aib-Glu(Y)-Gly-OH (Fragment Z);
each coupling in presence of a coupling agent and solvent to obtain X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Alloc)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin;
wherein each of the fragments A, H, L, M, T, V, X, Y and Z may be prepared by sequential coupling of suitable protected peptides in presence of a coupling agent and solvent;

step ii) selectively deprotecting Alloc group on 20Lys by using a mixture of Pd(PPh3)4 and phenyl silane followed by sequential coupling using Fmoc-PEG-OH, Fmoc-PEG-OH, Fmoc-Glu(OH)-OtBu and Octadecanedioic acid mono-tert-butyl ester
OR
by coupling with Semaglutide sidechain linker i.e., (Y)-ODDA-Glu(PEG-PEG)-(Y);
each coupling in presence of a coupling agent and in a solvent to obtain resin bound protected Semaglutide;
X-(1)His(Y)-(2)Aib-(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(Y)-Ala-Ala-Lys(Y-ODDA-?-Glu(PEG-PEG)-Y)-Glu(Y)-Phe-Ile-Ala-Trp(Y)Leu-Val-Arg(Z)-Gly-Arg(Z)-(31)Gly-Resin; and

step iii) cleaving of peptide from resin and simultaneous side chain deprotection using “cocktail mixture/cleaving reagent” to obtain Semaglutide of Formula (1);
wherein, X represents amino protecting group, Y represents amide, carboxyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and Val-Ser(PsiMe,MePro) represents pseudoproline dipeptides.

2. The process as claimed in claim 1, wherein the coupling agent(s) is 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 a mixture thereof.

3. The process as claimed in claim 1, wherein the coupling takes place in presence of a solvent selected from the group comprising of DMF, DCM, THF, NMP, methanol, ethanol, isopropanol, dichloroethane, 1,4-dioxane, 2-methyl tetrahydrofuran ethyl acetate, acetonitrile, acetone or a mixture thereof.

4. The process as claimed in claim 1, wherein N-terminal protecting groups are deprotected using organic base prepared in organic solvent.

5. The process as claimed in claim 4, wherein the organic base is selected from piperidine, piperazine, N-methyl morpholine, diethyl amine, triethyl amine, 1,8-Diazabicyclo [5.4.0]undec-7-ene (DBU) or a mixture thereof.

6. The process as claimed in claim 4, wherein the organic solvent is selected from dimethylformamide (DMF), N-Methyl-2-Pyrrolidone (NMP), dichloromethane (DCM), tetrahydrofuran (THF), N,N-dimethylacetamide (DMAC) or a mixture thereof.

7. The process as claimed in claim 1, wherein the deprotection of the peptide from the resin is carried out in the presence of combination of Trifluoroacetic acid (TFA) and radical scavenger(s) selected from the group comprising of triisopropylsilane (TIS), dithiothreitol (DTT), 1,2-ethanedithiol (EDT), Phenol, cresol, anisole, thioanisole, ammonium iodide, DMS and water.