DESC:
FIELD OF THE INVENTION
The present invention relates to a process of preparation of glucagon-like peptide-1 (GLP-1) analogues. Particularly, the invention is in relation to preparation of peptides of pharmaceutical significance. More particularly, the disclosure provides synthesis method of glucagon like peptides via fragment approach and sequential amino acids couplings. Additionally, economical and facile purification method for the synthesized peptide is provided.

BACKGROUND AND PRIOR ART
The synthesis strategies adopted in the prior art for the glucagon-like peptide-1 peptide includes different approaches such as uses of different/multiple resins, coupling reagents, fragments, different protecting group for amino acids.
Liraglutide is an analogue of glucagon-like peptide-1 containing a backbone of 31 amino acids, wherein the Lys-20 is condensed with Glu-Pal group. Liraglutide is produced by covalently linking glucagon-like peptide-1 to a fatty acid. It is a once-daily injectable and has the effects of lowering blood sugar level, reducing body weight, promoting islet cell regeneration, as well as protecting cardiovascular system.
Semaglutide is a next generation glucagon-like peptide-1 analogue as a once-weekly injection. Semaglutide shares a similar backbone to Liraglutide, with Ala2 being substituted by Aib, and Lys20 is side modification moiety derivatized with N-(17-carboxy-1oxoheptadecyl)-L-?-glutamyl-2-[2-(2-aminoethoxy] ethoxy] acetyl-2-[2-(2-aminoethoxy) ethoxy] acetyl.
Due to massive demand of these peptides in the market, it is very crucial to come up with a method that provides more efficient industrially scalable process with reduced time cycle, easy purification and process, which avoids the use of toxic or otherwise undesirable reagents.
US20100317057A1 describes semi-recombinant method for preparation of GLP-1 analogue and derivatives with non-proteogenic amino acids in the N-terminal part combining the use of recombinant expression techniques and chemical peptide synthesis.
CN103848910A describes sequential synthesis of backbone peptide which includes Fmoc-Lys(Mmt)-OH and Boc-His-OH for branching.
WO 2019/069274 Al provides uses of different fragments (1-19) with a fragment (20-31), or a fragment (1-18) with a fragment (19-31). Another preferred coupling of fragment (1-17) with a fragment (18-31). In addition, with carboxyl terminal amino acid of the first fragment is a Leu residue coupling a fragment (1-14) with a fragment (15-31).
ACS Combinatorial Science 2020 22(12), 821-825 provides synthesis of semaglutide (GLP-1 analogue) with solid hydrophobic support assisted liquid phase synthesis method using 2,4-didocosyloxybenzyl alcohol (TAG), where complete sequence divided in six fragments. However, use of more number of fragments for synthesis increases the manufacturing cost.
Angew. Chem. Int. Ed. 2017, 56, 7803– 7807 and Angew. Chem. 2017, 129, 7911– 7915 provides synthesis of peptide with solid support assisted liquid phase synthesis of peptide by using another TAG, 3, 4, 5-tri(2',3'-dihydrophytyloxy)benzyl alcohol.
There is a long felt need in the art to develop a process for synthesis of GLP-1 peptide analogues which is economical, commercially scalable, cost efficient, has better yield, less impurities, that can be readily purify by well-known techniques and uses safe starting materials. The present inventors have surprisingly developed a process for synthesis of GLP-1 peptide analogues which ameliorates the drawbacks of the prior art.

OBJECTS OF THE INVENTION
It is an object of the present invention to overcome the drawbacks of the prior art.
It is an object of the present invention to provide a process for preparing GLP-1 peptide analogues.
It is another object of the present invention to provide a process for preparing GLP-1 peptide analogues which is economical, industrially scalable, cost efficient process, has a better yield, less impurities, that can be readily purify by well-known techniques and uses safe starting materials.
It is yet another object of the present invention to provide a process for preparing GLP-1 peptide analogues which is environmentally benign, simple, straightforward, and efficient and provides the end product in high yield and purity.

SUMMARY OF INVENTION
According to an aspect of the present invention, there is provided a process for preparing GLP-1 peptide analogues using novel fragments approach with tagging approach.
According to another aspect of the present invention, there is provided a method for preparing novel fragments for use in synthesis of GLP-1 peptide analogues.

BRIEF DESCRIPTION OF ACCOMPANYING FIGURES
Figure 1: Fragment-IG synthesized on TAG Fmoc Liquid phase peptide strategy.
Figure 2: Fragment SG synthesized on TAG Fmoc Liquid phase peptide strategy.
Figure 3: Fragment BG synthesized on TAG Fmoc Liquid phase peptide strategy.
Figure 4: Synthesis of Side chain (W part) on Tag
Figure 5: Synthesis of Fmoc-Lys(W)-OH ( Lys with side chain)
Figure 6: Synthesis of Semaglutide

DETAILED DESCRIPTION OF THE INVENTION
The following description is provided to assist in a comprehensive understanding of exemplary embodiments of the invention. It includes various specific details to assist in that understanding but these are to be regarded as merely exemplary.
Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope of the invention. In addition, descriptions of well-known functions and constructions are omitted for clarity and conciseness.
The terms and words used in the following description and claims are not limited to the bibliographical meanings, but, are merely used by the inventor to enable a clear and consistent understanding of the invention. Accordingly, it should be apparent to those skilled in the art that the following description of exemplary embodiments of the present invention are provided for illustration purpose only and not for the purpose of limiting the scope of the invention as defined by the appended claims and their equivalents.
It is to be understood that the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
Features that are described and/or illustrated with respect to one embodiment may be used in the same way or in a similar way in one or more other embodiments and/or in combination with or instead of the features of the other embodiments.
It should be emphasized that the term “comprises/comprising” when used in this specification is taken to specify the presence of stated features, steps or components but does not preclude the presence or addition of one or more other features, steps, components or groups thereof.
The present invention provides a novel method for preparation of glucagon-like peptide-1 using novel fragments approach with tagging approach. The preparation method provides commercially scalable cost efficient process with better yield with less impurities that can be readily purify by well-known techniques. The present invention also provides novel fragments and method of preparation of novel fragments using in-house prepared starting material.
The inventors of the present invention have developed a process for synthesizing GLP-1 peptide, synthesis of side chain with Tagging and Lys coupling over resin to get cost efficient process. The present invention allows introduction of the side chain part at Lys at an early stage of the synthesis by using fairly safe reagents.
The present invention provides a synthesis method for glucagon-like peptide-1. The peptide is selected from a group comprising Exenatide, Liraglutide, Lixisenatide, Albiglutide, Dulaglutide, Semaglutide and Taspoglutide. In an embodiment, there is provided a method of preparation of Semaglutide.
The invention provides a robust process for preparation of fragments which was tedious with complete solution phase peptide synthesis.
The invention provides a fragment based process followed by selectively sequential coupling of few amino acids for synthesis of glucagon-like peptide-1 comprising solid phase peptide synthesis, wherein process comprises lysine derivatives at Lys (20) with branching part (w).
In an embodiment, there is provided a process for preparing a GLP peptide analogue, comprising coupling on solid phase at least a first fragment with peptidyl resin, wherein the coupling comprises reacting the carboxy terminal amino acid of the first fragment with the amino terminal amino acid of peptidyl resin, and wherein the carboxy terminal amino acid of the first fragment is a Gly or Alanine or Phenyl alanine residue; preferably Glycine.
The present invention also provides a synthetic approach allowing introduction of the N-(1 7-carboxy-1 -oxoheptadecyl)-L-Y-glutamyl-2-[2-(2-aminoethoxy)ethoxy] acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl with Lys at an early stage of the synthesis of Semaglutide.
In one embodiment of the present invention, the amino protecting groups are selected from but not limited to a group comprising of Fmoc, Boc, Cbz, Bpoc, and the like.
In another embodiment of the present invention, the carboxyl, phenolic and alcoholic groups are protected with groups selected from but not limited to a group comprising of DMT, MMT, Trt, tert-butyl, t-butoxy carbonyl, and the like.
In yet another embodiment of the present invention, the coupling agents are selected from the group comprising of hydroxybenzotriazole (HOBt); O-(7-azabenzotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-N,N,N',N'tetramethyluronium tetrafluoroborate (TBTU), 1,3-dicyclohexylcarbodlimide (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-oxytris(dimethyl-amino)-phosphonium hexafluorophosphate (BOP), N,N-bis-(2-oxo-3-oxazolidinyl)phosphonic dichloride (BOP-C1), benzotriazo1yloxytri(pyrolidino)phosphonium hexafluorophosphate (PyBOP), bromotri(pyrrolidino)phosphonium hexafluorophosphate (PyBrOP), chlorotri(pynolidino)phosphonium hexafluorophosphate (PyClOP), ethyl-2-cyano-2(hydroxyimino) acetate (Oxyma Pure), O-(6-Chloro-1-hydrocibenzotriazol-1-yl)-1,1,3,3tetramethyluronium tetrafluoroborate (TCTU), 245-norbornen-2,3-dicarboximido)-1,1,3,3tetramethyluronium tetrafluoroborate (TNTU), propane phosphonic acid anhydride (PPAA), 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoro borate (TSTU), bromo-tris-pyrrolidinophosphonium hexafluorophosphate (PyBroP), iso-butylchloroformate (IBCF), Ethyl 1,2dihydro-2-ethoxyquinoline-1-carboxylate (EEDQ), 1-Cyano-2-ethoxy-2oxoethylidenaminooxy)dimethylamino-morpholino-carbeniu- m hexafluorophosphate (COMU), 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) or mixtures thereof.
In yet another embodiment of the present invention, the coupling takes place in a solvent. The solvent for coupling is selected from a group comprising of DMF, DCM, THF, NMP, DMAC methanol, ethanol, isopropanol, dichloroethane, 1,4-dioxane, 2-methyl tetrahydrofuran ethyl acetate, acetonitrile, acetone, and the like or a mixture of the listed solvents.
In still another embodiment of the present invention, the coupling reaction is carried out in presence of a base. The base is selected from a group comprising but not limiting to di-C1-C1O -alkyl sulphides, alkyl phenyl sulphides, piperidine, 1,2-aminothiol of cysteine, a 1,2-aminoethanol of serine, a 1,2-aminoethanol of threonine, an amino oxyacetyl functional group, a mono-hydrazine succinyl functional group, ammonia, hydrazine, an alkoxide, a 4-hydrazinobenzoyl functional group, diisopropylamine, N,N-diisopropylethylamine triethylamine, dimethylamine, trimethyl amine, isopropyl ethylamine, pyridine, N-methyl morpholine and mixtures thereof.
In yet another embodiment of the present invention, the synthesis of linear peptide backbone is carried out on TAG which is acid sensitive.
In an embodiment, the TAG is selected from 2,4-Bis-octadecyloxy-phenyl)-methanol; 3,4,5-tri (2’,3’-dihydrophytyloxy) benzyl alcohol and 2,4-didocosyloxybenzyl alcohol or combinations thereof. Preferably, the TAG is 2,4-Bis-octadecyloxy-phenyl)-methanol.
In yet another embodiment of the present invention, the protected peptide is de-protected with a mixture of reagents selected from the group comprising of TFA, TIS, TIPS, DTT, EDT, ammonium iodide, 2,2'-(ethylenedioxy)diethane and acetyl cystein, DMS, phenol, cresol and thiocresol.
In an embodiment, the acid sensitive polymeric resin is selected from 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.
In yet another embodiment of the present invention, the purification of peptides is carried out by reverse phase HPLC using solvents: TFA in water, acetic acid, acetonitrile, orthophosphoric acid in water, triethylamine in water, ammonium acetate in water and ammonium bicarbonate in water.
In yet another embodiment of the present invention, the final isolation of crude peptide is carried out by lyophilization.
In yet another embodiment of the present invention, the side modification is done either in early stage of synthesis or after complete peptide backbone synthesis.
In a preferred embodiment, the present synthetic approach allows introduction of the N-(1 7-carboxy-1-oxoheptadecyl)-L-Y-glutamyl-2-[2-(2-aminoethoxy) ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl with Lys at an early stage of the synthesis.
In an embodiment, synthesis of Semaglutide via different fragment approach and individual amino acid couplings are provided as below,
For Semaglutide, W is N-(17-carboxy-1-oxoheptadecyl)-L-Y-glutamyl-2-[2-(2-aminoethoxy] ethoxy] acetyl-2-[2(2-aminoethoxy) ethoxy] acetyl.
Fragment-IG [SEQ ID NO.1]:-Fmoc-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-OH;
Fragment SG[SEQ ID NO.2]: Fmoc-Ser (tBu) - Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH;
Fragment-BG: Fmoc-Aib-Glu (OtBu)-Gly-OH;

The present invention provides a method for preparing Semaglutide, wherein the said method is carried out by fragment coupling on a resin by solid phase synthesis, comprising the steps of:
a. Providing a first protected peptide fragment having amino acid sequence of Arg (Pbf)-Gly on a resin;
b. Deprotecting said first peptide fragment in presence of a deprotecting agent and coupling the deprotected first peptide fragment with a protected peptide fragment having amino acid sequence SEQ ID NO:1 [Fragment IG] to form a protected peptide fragment having amino acid sequence SEQ ID NO:3 in presence of a coupling agent, a base and a solvent;
c. Deprotecting said peptide fragment having amino acid sequence SEQ ID NO:3 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:3 with protected amino acids P-Phe-OH and P-Glu(OtBu)-OH to form protected peptide fragment having amino acid sequence SEQ ID NO:4 in presence of a coupling agent, a base and a solvent;
d. Deprotecting said peptide fragment having amino acid sequence SEQ ID NO:4 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:4 with a modified protected amino acid Lys(W) to form protected peptide fragment having amino acid sequence SEQ ID NO:5 in presence of a coupling agent, a base and a solvent,
Wherein W is N-(17-carboxy-1-oxoheptadecyl)-L-Y-glutamyl-2-[2-(2-aminoethoxy] ethoxy] acetyl-2-[2(2-aminoethoxy) ethoxy] acetyl;
e. Deprotecting said peptide fragment having amino acid sequence SEQ ID NO:5 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:5 with protected amino acids P-Ala-OH, P-Ala-OH and P-Gln(trt)-OH respectively to form protected peptide fragment having amino acid sequence SEQ ID NO:6 in presence of a coupling agent, a base and a solvent;
f. Deprotecting said peptide fragment having amino acid sequence SEQ ID NO:6 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:6 with protected peptide fragment having amino acid sequence SEQ ID NO:2 [Fragment SG] to form protected peptide fragment having amino acid sequence SEQ ID NO:7, in presence of a coupling agent, a base and a solvent;
g. Deprotecting said peptide fragment having amino acid sequence SEQ ID NO:7 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:7 with protected amino acids P-Val-OH, PAsp( OtBu)-OH, P-Ser(tBu)-OH, P-Thr(tBu)-OH, P-(Phe-OH, P-Thr(tBu)-OH respectively to form protected peptide fragment having amino acid sequence SEQ ID NO:8, in presence of a coupling agent, a base and a solvent;
h. Deprotecting said peptide fragment having amino acid sequence SEQ ID NO:8 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:8 with protected peptide fragment having sequence of Fmoc-Aib-Glu (OtBu)-Gly-OH [Fragment BG] to form a protected peptide fragment having amino acid sequence SEQ ID NO:9 in presence of a coupling agent, a base and a solvent;
i. Deprotecting said peptide fragment having amino acid sequence SEQ ID NO:9 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:9 with protected amino acid fragment Boc His(Boc)-OH to form a protected peptide fragment having amino acid sequence SEQ ID NO:10 in presence of a coupling agent, a base and a solvent;
j. Deprotecting followed by resin cleavage of the peptide sequence having amino acid sequence SEQ ID NO:10 followed by filtration to obtain crude GLP-1 peptide;
Wherein said P is a N-terminal protecting group;
Wherein said resin is an acid sensitive polymeric resin; and

The fragments of SEQ ID NO:1 [Fragment IG], SEQ ID No.2 [Fragment SG] and Fragment BG are individually prepared on a TAG support by liquid phase synthesis.

The above-mentioned fragments are prepared by liquid phase easily by using Tagging technique on industrial scale without any difficulty with good purity and in high yield. The fragments were prepared on acid labile tagging technique to provide desired protected linear peptide and subsequently desired final Semaglutide with good yield and purity.
The present invention provides a preparation method for fragment synthesis on less expensive Tag instead of costly resins. This provides better yield and less impurity wherein the peptides can be easily purified using simple non-expensive techniques. The process disclosed herein can provide glucagon-like peptide-1 such as Liraglutide and Semaglutide with good yield and high purity on an industrial scale.

In an embodiment, there is provided a method for preparing a peptide fragment having amino acid sequence of SEQ ID NO:1 [Fragment IG] for synthesis of Semaglutide, wherein the said method is carried out on a TAG support by liquid phase synthesis, comprising the steps of:
i. Tagging a protected amino acid of P-Gly-OH with TAG to form P-Gly-TAG;
ii. Deprotecting the tagged amino acid fragment in presence of a deprotecting agent and successive coupling the deprotected tagged amino acid fragment with protected amino acid fragments of P-Arg (Pbf)-OH, P-Val-OH, P-Leu-OH, P-Trp(Boc)-OH, P-Ala-OH, P-Ile –OH to form a protected tagged peptide fragment having amino acid sequence of SEQ ID NO:1 in presence of a coupling agent, a base and a solvent;
iii. Cleaving the TAG by dissolving the peptide fragment of SEQ ID NO.1 in cleaving agents followed by neutralization and filtration to obtain the solid peptide fragment having SEQ ID NO.1;
Wherein said P is a N-terminal protecting group; and said TAG is acid sensitive.

In another embodiment, there is provided a method for preparing a peptide fragment having amino acid sequence SEQ ID NO:2 [Fragment BG] for synthesis of Semaglutide, wherein the said method is carried out on a TAG support by liquid phase synthesis, comprising the steps of:
i. Tagging a protected amino acid of P-Gly-OH with TAG to form P-Gly-TAG;
ii. Deprotecting the tagged amino acid fragment in presence of a deprotecting agent and successive coupling the deprotected tagged amino acid fragment with protected amino acid fragments of P-Glu(OtBu), P-Leu-OH, P-Tyr(tBu)-OH, P-Ser(tBu)-OH, P-Ser(tBu)-OH to form a protected tagged peptide fragment having amino acid sequence of SEQ ID NO:2 in presence of a coupling agent, a base and a solvent;
iii. Cleaving the TAG by dissolving the peptide fragment of SEQ ID NO.2 in cleaving agents followed by neutralization and filtration to obtain the solid peptide fragment having SEQ ID NO.2;
Wherein said P is a N-terminal protecting group; and said TAG is acid sensitive.

In yet another embodiment, there is provided a method for preparing a peptide fragment having amino acid sequence Fmoc-Aib-Glu (OtBu)-Gly-OH [Fragment BG] for synthesis of Semaglutide, wherein the said method is carried out on a TAG support by liquid phase synthesis, comprising the steps of:
i. Tagging a protected amino acid of P-Gly-OH with TAG to form P-Gly-TAG;
ii. Deprotecting the tagged amino acid fragment in presence of a deprotecting agent and successive coupling the deprotected tagged amino acid fragment with protected amino acid fragments of P-Glu(OtBu), P-Aib-OH, to form a protected tagged peptide fragment having amino acid sequence of Fragment BG in presence of a coupling agent, a base and a solvent;
iii. Cleaving the TAG by dissolving the peptide fragment of Fragment BG in cleaving agents followed by neutralization and filtration to obtain the solid peptide fragment;
Wherein said P is a N-terminal protecting group; and said TAG is acid sensitive.

In another embodiment, there is provided a method for preparing an amino acid fragment of Lysine with side chain (Lys (W)) for synthesis of Semaglutide, comprising the steps of:
i. Tagging a protected first compound of 8-amino 3,6 dioxa octanoic acid (P-PEG-OH) with TAG to form P-PEG-OTAG;
ii. Deprotecting the tagged fragment in presence of a deprotecting agent and coupling the deprotected tagged first fragment with protected compound fragment of P-PEG-OH and successive coupling with P-Glu (Otbu), 18-ter-butoxy-18-oxo-octadecanoic acid to form a protected tagged fragment of 18-ter-butoxy-18-oxo-octadecanoic acid-Glu-Otbu-PEG-PEG-OTAG (W-OTAG) in presence of a coupling agent, a base and a solvent,
iii. Cleaving the TAG from the fragment in presence of cleaving agents followed by filtration to obtain Fragment-W-OH;
iv. Providing a protected amino acid sequence of P-Lys (Alloc)-OH on a resin;
v. Deprotecting the Alloc fragment in presence of a deprotecting agent and coupling the deprotected fragment with Fragment-W-OH obtained in step (iii) in presence of a coupling agent, a base and a solvent,
vi. Cleaving of the resin from the peptide fragment in presence of cleaving agents followed by filtration to obtain the amino acid fragment of Lysine with side chain (Lys (W);
Wherein said P is a N-terminal protecting group; and said TAG is acid sensitive;
Wherein W is N-(17-carboxy-1-oxoheptadecyl)-L- ? -glutamyl-2-[2-(2-aminoethoxy] ethoxy] acetyl-2-[2(2-aminoethoxy) ethoxy] acetyl

In an embodiment, the TAG cleaving agents are selected from CH2Cl2, TFE, TFA or a mixture thereof.
In an embodiment, the neutralization is with DIPEA.
List of Abbreviations used in the specification are given in below Table 1:
Sr. No. Abbreviation Name
1 Fmoc 9-fluorenylmethyloxycarbonyl
2 TAG (2,4-Bis-octadecyloxy-phenyl)-methanol),
3,4,5-tri (2’,3’-dihydrophytyloxy) benzyl alcohol and 2,4-didocosyloxybenzyl alcohol
3 DMAP Dimethylaminopyridine
4 HBTU O-Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate
5 COMU 1-Cyano-2-ethoxy-2oxoethylidenaminooxy)dimethylamino-morpholino-carbeniu- m hexafluorophosphate
6 HATU O-(7-Aza-benzotriazole-1-oxy)-N,N,N',N'-tetramethyluronium hexafluorophosphate
7 DIC Diisopropylcarbodiimide
8 HOBt 1-hydroxybenzotrizole
9 HOAt 1-hydroxy-7-azabenzotriazole
10 DIEA N,N-diisopropylethylamine
11 Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl Trt Trityl
tBu Tert-butyl
12 DMF N,N-dimethyl formamide
13 DCM Dichloromethane
14 TFA Trifluoroacetic acid
15 NMP N-methyl pyrrolidone
16 DMSO Dimethyl sulfoxide
17 EDT Ethanedithiol

18 DODT 3,6-dioxa-1,8-octanedithiol
19 TIPS Triisopropyl silane

EXAMPLES
The following examples are meant to illustrate the present invention. The examples are presented to exemplify the invention and are not to be considered as limiting the scope of the invention.

EXAMPLE 1:
Synthesis of Semaglutide according to an embodiment of present invention:
Synthesis of Fragments:-
Synthesis of Fragment-IG:-Fmoc-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-OH [SEQ ID NO.1]:-
Figure 1 illustrates Fragment-IG synthesized by a TAG supported Fmoc Liquid phase peptide strategy.
In an embodiment, the TAG is 2, 4-Bis-octadecyloxy-phenyl)-methanol.
1st amino acid coupling: In a clean and dry RBF charged THF (500 mL, 10V) and TAG-OH (50g, 1eq) at RT and stirred to dissolve RM cool to 0-5°C, Charged Fmoc-Gly-OH (34.56 g, 1.5 eq) Charge DIC (18.78 mL, 1.5 eq) and DMAP (4.72 g, 0.5 eq) at 0-5°C RM maintained at 5 to 10°C for 10 min. and at 25-30°C for 20Min TLC checked for complete conversion of TAG-OH (Mobile Phase: 20% EA in Hexane) found complies RM filtered through celite bed to remove solid urea so formed Filtrate ML added in to precooled Acetonitrile 600 ml at 5-10°C Solid filtered and washed with Acetonitrile 5V Solid suck dried and dried in VTD for 4 to 6 hr at 35 to 40°C
Dry wt: 70g (97.69%)
Fmoc deprotection: In a clean and dry RBF charged DCM(700ml, 10V) and Fmoc-Gly-OTAG (70g, 1.0 eq) at RT and stirred to dissolve RM cool to 5-10°C Charged Piperidine (16.1 mL, 1.5 eq) and DBU (17.1 mL, 1.5 eq) at 5-10°C RM maintained at 5 to 10°C for 5 min. and at 25-30°C for 10Min TLC checked for complete conversion of Fmoc-Gly-OTAG (Mobile Phase: 20% EA in Hexane) found complies. RM cool to 5 to 10°C and pH adjusted 7 to 8 with 1M HCl solution RM washed with purified water 5V x2 time Organic layer dried over sodium sulphate Organic layer as such taken for next coupling reaction (Note: yield considered as 100% and proceeded for next step)
2nd amino acid coupling: Charged the above solution (H-Gly-OTAG, 53 g, 1eq), Fmoc-Arg(Pbf)-OH (58.74 g, 1.2 eq) with HOBt monohydrate (17.3 g, 1.5 eq) and DIC (18.65 mL, 1.5 eq) in THF (265 mL, 5 Vol.) stirring continued for 2-3 hrs. After 3 hrs, TLC checked for complete conversion of H-Ala-OTAG, RM washed with 2.5% sodiumbicarbonate solution and brine solution and Organic layer dried over sodium sulphate Organic layer as such taken for next deprotection reaction
Fmoc deprotection and coupling procedure were followed for following amino acids as per sequence of amino acids, Fmoc-Val-OH ( 28 g, 1.2 eq), Fmoc-Leu-OH ( 31.37 g, 1.2 eq), Fmoc-Trp(Boc)-OH ( 46.76 g, 1.2 eq), Fmoc-Ala-OH ( 27.64 g, 1.2 eq), Fmoc-Ile-OH ( 31.37 g, 1.2 eq) were performed similar to the above procedure.
Dry wt: 106.05 g , 64%

Tag cleavage: Fmoc-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-OTAG
Tagged-fragment (106 g) was then dissolved in a 100:10:1 (v/v) mixture of CH2Cl2, TFE, and TFA ( 1060 mL). The reaction mixture was stirred at room temperature until the reaction was completed (30 min). After completion, the solution was filtered, and neutralized with DIPEA. Reaction mass were concentrated under reduced pressure at 30-35 oC. Solid Isolated In n-Hepatne (1060 mL, 10V) Solid filtered over Buchner funnel, washed with n-Hepatane (265 ML, 2 x 2.5 Vol.) Solid was dried for 4-6 hrs under reduced pressure at 35 - 40 oC to afford white solid. Desired fragment was confirmed by LCMS; m/z =1390 [M+H]+.
Yield: 46.40 g, 63 %

Synthesis of Fragment SG: Fmoc-Ser (tBu) - Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH [SEQ ID NO.2]:-
Figure 2 illustrates Fragment SG synthesized by a TAG supported Fmoc Liquid phase peptide strategy.
In an embodiment, the TAG is 2, 4-Bis-octadecyloxy-phenyl)-methanol.

1st amino acid coupling: In a clean and dry RBF charged THF (500 mL, 10V) and TAG-OH (50g, 1eq) at RT and stirred to dissolve RM cool to 0-5°C, Charged Fmoc-Gly-OH (34.56 g, 1.5 eq) Charge DIC (18.78 mL, 1.5 eq) and DMAP (4.72 g, 0.5 eq) at 0-5°C RM maintained at 5 to 10°C for 10 min. and at 25-30°C for 20Min TLC checked for complete conversion of TAG-OH (Mobile Phase: 20% EA in Hexane) found complies RM filtered through celite bed to remove solid urea so formed Filtrate ML added in to precooled Acetonitrile 600 ml at 5-10°C Solid filtered and washed with Acetonitrile 5V Solid suck dried and dried in VTD for 4 to 6 hr at 35 to 40°C
Dry wt: 70g (97.69%)
Fmoc deprotection: In a clean and dry RBF charged DCM (700mL, 10V) and Fmoc-Gly-OTAG (70g, 1.0 eq) at RT and stirred to dissolve RM cool to 5-10°C Charged Piperidine (16.1 mL, 1.5 eq) and DBU (17.1 mL, 1.5 eq) at 5-10°C RM maintained at 5 to 10°C for 5 min. and at 25-30°C for 10Min TLC checked for complete conversion of Fmoc-Gly-OTAG (Mobile Phase: 20% EA in Hexane) found complies. RM cool to 5 to 10°C and pH adjusted 7 to 8 with 1M HCl solution RM washed with purified water 5V x2 time Organic layer dried over sodium sulphate Organic layer as such taken for next coupling reaction (Note: yield considered as 100% and proceeded for next step)
2nd amino acid coupling: Charged the above solution (H-Gly-OTAG, 53g, 1eq), Fmoc-Glu(OtBu)-OH ( 29.28 g, 1.2 eq) with HOBt monohydrate (13.08 g, 1.5 eq) and DIC (13.65 mL, 1.5 eq) in THF (265 mL, 5 Vol.) stirring continued for 2-3 hrs. After 3 hrs, TLC checked for complete conversion of H-Gly-OTAG, RM washed with 2.5% sodiumbicarbonate solution and brine solution and Organic layer dried over sodium sulphate Organic layer as such taken for next deprotection reaction.
Fmoc deprotection and coupling procedure were followed for following amino acids as per sequence of amino acids, Fmoc-Leu-OH ( 32.00 g, 1.2 eq), Fmoc-Tyr(tBu)-OH ( 41.36 g, 1.2 eq), Fmoc-Ser(tBu)-OH ( 34.50 g, 1.2 eq), Fmoc-Ser(tBu)-OH ( 34.50 g, 1.2 eq) were performed similar to the above procedure.
Dry wt: 91.53 g, 64.5%

Tag cleavage: Fmoc-Ser (tBu) - Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OTAG
Tagged-fragment (90 g) was then dissolved in a 100:10:1 (v/v) mixture of CH2Cl2, TFE, and TFA ( 900 mL). The reaction mixture was stirred at room temperature until the reaction was completed (30 min). After completion, the solution was filtered, and neutralized with DIPEA. Reaction mass were concentrated under reduced pressure at 30-35 oC. Solid Isolated In n-Hepatne (900 mL, 10V) Solid filtered over Buchner funnel, washed with n-Hepatane (225 ML, 2 x 2.5 Vol.) Solid was dried for 4-6 hrs under reduced pressure at 35 - 40 oC to afford white solid. Desired fragment was confirmed by LCMS; m/z =1101[M+H]+.
Yield: 47.3 g, 82.3 %

Synthesis of Fragment-BG: Fmoc-Aib-Glu (OtBu)-Gly-OH:-
Figure 3 illustrates Fragment BG synthesized by a TAG supported Fmoc Liquid phase peptide strategy.
In an embodiment, the TAG is 2, 4-Bis-octadecyloxy-phenyl)-methanol.

1st amino acid coupling: In a clean and dry RBF charged THF (500 mL, 10V) and TAG-OH (50g, 1eq) at RT and stirred to dissolve RM cool to 0-5°C, Charged Fmoc-Gly-OH (34.56 g, 1.5 eq) Charge DIC (18.78 mL, 1.5 eq) and DMAP (4.72 g, 0.5 eq) at 0-5°C RM maintained at 5 to 10°C for 10 min. and at 25-30°C for 20Min TLC checked for complete conversion of TAG-OH (Mobile Phase: 20% EA in Hexane) found complies RM filtered through celite bed to remove solid urea so formed Filtrate ML added in to precooled Acetonitrile 600 ml at 5-10°C Solid filtered and washed with Acetonitrile 5V Solid suck dried and dried in VTD for 4 to 6 hr at 35 to 40°C
Dry wt: 70g (97.69%)
Fmoc deprotection: In a clean and dry RBF charged DCM(700mL, 10V) and Fmoc-Gly-OTAG (70g, 1.0 eq) at RT and stirred to dissolve RM cool to 5-10°C Charged Piperidine (16.1 mL, 1.5 eq) and DBU (17.1 mL, 1.5 eq) at 5-10°C RM maintained at 5 to 10°C for 5 min. and at 25-30°C for 10Min TLC checked for complete conversion of Fmoc-Gly-OTAG (Mobile Phase: 20% EA in Hexane) found complies. RM cool to 5 to 10°C and pH adjusted 7 to 8 with 1M HCl solution RM washed with purified water 5V x2 time Organic layer dried over sodium sulphate Organic layer as such taken for next coupling reaction (Note: yield considered as 100% and proceeded for next step)
2nd amino acid coupling: Charged the above solution (H-Gly-OTAG, 53g, 1eq), Fmoc-Glu(OtBu)-OH ( 29.28 g, 1.2 eq) with HOBt monohydrate (13.08 g, 1.5 eq) and DIC (13.65 mL, 1.5 eq) in THF (265 mL, 5 Vol.) stirring continued for 2-3 hrs. After 3 hrs, TLC checked for complete conversion of H-Gly-OTAG, RM washed with 2.5% sodiumbicarbonate solution and brine solution and Organic layer dried over sodium sulphate Organic layer as such taken for next deprotection reaction.
Fmoc deprotection and coupling procedure were followed for following amino acids as per sequence of amino acids, Fmoc-Aib-OH ( 29.46 g, 1.2 eq), were performed similar to the above procedure.
Dry wt: 69.04 g, 74.5%

Tag cleavage: Fmoc-Aib-Glu (OtBu)-Gly-OTAG
Tagged-fragment (65 g) was then dissolved in a 100:10:1 (v/v) mixture of CH2Cl2, TFE, and TFA (650 mL). The reaction mixture was stirred at room temperature until the reaction was completed (30 min). After completion, the solution was filtered, and neutralized with DIPEA. Reaction mass were concentrated under reduced pressure at 30-35 oC. Solid Isolated In n-Hepatne (650 mL, 10V) Solid filtered over Buchner funnel, washed with n-Hepatane (162.5 ML, 2 x 2.5 Vol.) Solid was dried for 4-6 hrs under reduced pressure at 35 - 40 oC to afford white solid. Desired fragment was confirmed by LCMS; m/z =569.6 [M+H]+.
Yield: 24.7 g, 80.2%

Synthesis of Side chain (W part) on Tag:
Figure 4 illustrates Synthesis of Side chain (W part) on Tag
1St Fmoc-8-amino 3,6 dioxa octanoic acid (Fmoc-PEG-OH)coupling: In a clean and dry RBF charged THF ( 100 mL, 10V) and TAG-OH (10 g, 1eq) at RT and stirred to dissolve RM cool to 0-5°C, Charged Fmoc-PEG-OH ( 8.96 g, 1.5 eq) Charge DIC (3.62 mL, 1.5 eq) and DMAP ( 1.89g, 0.5 eq) at 0-5°C RM maintained at 5 to 10°C for 10 min. and at 25-30°C for 20Min TLC checked for complete conversion of TAG-OH (Mobile Phase: 20% EA in Hexane) found complies RM filtered through celite bed to remove solid urea so formed Filtrate ML added in to precooled Acetonitrile ml at 5-10°C Solid filtered and washed with Acetonitrile 5V Solid suck dried and dried in VTD for 4 to 6 hr at 35 to 40°C
Dry wt: 14.98g (93.5%)
Fmoc deprotection: In a clean and dry RBF charged DCM(140 mL, 10V) and Fmoc-PEG-OTag (14.0g, 1.0 eq) at RT and stirred to dissolve RM cool to 5-10°C Charged Piperidine ( 2.04 mL, 1.5 eq) and DBU ( 3.1 mL, 1.5 eq) at 5-10°C RM maintained at 5 to 10°C for 5 min. and at 25-30°C for 10Min TLC checked for complete conversion of Fmoc-PEG-OTAG (Mobile Phase: 20% EA in Hexane) found complies. RM cool to 5 to 10°C and pH adjusted 7 to 8 with 1M HCl solution RM washed with purified water 70ml 5V x2 time Organic layer dried over sodium sulphate Organic layer as such taken for next coupling reaction (Note: yield considered as 100% and proceeded for next step)
2nd Fmoc-8-amino 3,6 dioxa octanoic acid (Fmoc-PEG-OH)coupling: Charged the above solution (H-PEG-OTAG, 10.92 g, 1eq), Fmoc-PEG-OH ( 6.39 g, 1.2 eq) with HOBt monohydrate (3.17 g, 1.5 eq) and DIC (3.26 mL, 1.5 eq) in THF (50 mL, 5 Vol.) stirring continued for 2-3 hrs. After 3 hrs, TLC checked for complete conversion of H-PEG-OTAG, RM washed with 2.5% sodiumbicarbonate solution and brine solution and Organic layer dried over sodium sulphate Organic layer as such taken for next deprotection reaction.
Fmoc deprotection and coupling procedure were followed for following amino acids as per sequence of Fmoc-Glu-Otbu (7.04 gm, 1.2eq.) and 18-ter-butoxy-18-oxo-octadecanoic acid (6.13g, 1.2 eq), were performed similar to the above procedure.
Wt.16.54 gm (80.2%)

Tag cleavage: :18-ter-butoxy-18-oxo-octadecanoic acid-Glu-Otbu-PEG-PEG-OTAG (Fragment W-OTAG)
Tagged-fragment (16 g) was then dissolved in a 100:10:1 (v/v) mixture of CH2Cl2, TFE, and TFA (160 mL). The reaction mixture was stirred at room temperature until the reaction was completed (30 min). After completion, the solution was filtered, and neutralized with DIPEA. Reaction mass were concentrated under reduced pressure at 30-35 oC. Solid Isolated In n-Hepatne (160 mL, 10V) Solid filtered over Buchner funnel, washed with n-Hepatane (40 ML, 2 x 2.5 Vol.) Solid was dried for 4-6 hrs under reduced pressure at 35 - 40 oC to afford oily product. Desired fragment was confirmed by LCMS; m/z =846s [M+H]+.
Yield: 9.7 g, 85.5%

Synthesis of Fmoc-Lys(W)-OH ( Lys with side chain):

Figure 5 illustrates Synthesis of Fmoc-Lys(W)-OH ( Lys with side chain)

2-CTC resin (5 g, loading capacity 1.2 mmol/g, 100 -200 mesh) charged in a clean dry peptide synthesizer vessel. Resin swelled in DCM (10Vol.) for 30 min, 2 times each.
1st amino acid coupling: To a pre-swelled resin, previously prepared solution of first amino acid Fmoc-Lys(Alloc)-OH ( 5.4 g, 2 eq) in DCM (50mL,10 Vol.) along with DIPEA ( mL, 10.0 eq) was added and agitated for 12 hrs at room temperature. After 12 hrs, solvent drained and resin was washed with DCM (2 x 10 Vol.). Capping solution mixture of DCM: MeOH: DIPEA (50mL,85:10:5, 10 Vol.) was added to resin and agitated for 30 min. Solvent drained and resin washed with DCM (50mL,2 x 10 Vol.) followed by DMF (50mL,2 x10 Vol.).
Alloc deprotection: Alloc deprotection performed in DCM (50mL 10V), phenyl silane 10eq. and palladium tetrakis 0.5eq. for 60 min. Drained solvent and resin was washed with DMF (50mL 2 x 10 Vol.), DCM (50ml, 2 x 10 Vol.) and DMF (50mL, 2 x 10 Vol.) respectively.
2nd amino acid coupling: The pre-activated Fragment-W-OH (7.6 g, 1.5 eq) with HOBt monohydrate (1.3 g, 1.5 eq) and DIC (1.4 mL, 1.5 eq) in DMF (10 Vol.) Coupling continued for 6 hrs. After 6 hrs, solvent drained and resin washed with DMF (50mL,2 x 10 Vol.), DCM (50mL 2 x 10 Vol.) and DMF (50mL 2 x10 Vol.) respectively.
After completion of synthesis, peptide resin was washed with 20% Methanol in DCM (1 x 10 Vol.), DCM (2 x 10 Vol.) and MTBE (2 x 10 Vol.) respectively.
Resin cleavage: Peptide Resin was washed with DCM (50mL 1 x 10 Vol.) for 2-3 mins. Peptide product was cleaved from resin using 1% TFA in DCM (50mL 3 x 10 Vol.), 5 minutes for each time. Cleaved fractions were collected and neutralized with pyridine (1:1 Volume ratio to TFA for each cleavage). Combined cleavage fractions were concentrated under reduced pressure at 30-35 oC. Resulting solution was co-evaporated with ethanol (2 x 5 Vol.) up to minimum volume and slurry was added to purified water (250mL,50 Vol.), stirred for 18 hrs. Solid was filtered over buchner funnel, washed with purified water (25 mL 2 x 5 Vol.), n-Heptane (25 mL 2 x 5 Vol) and well dried to afford white solid. Dried product was confirmed by LCMS, m/z = 1197[M+H]+.
Yield: 4.66 g, 65%

SYNTHESIS OF SEMAGLUTIDE:
Figure 6 illustrates synthesis of Semaglutide by coupling amino acids with Fragments IG, BG, SG and Lys with side chain prepared as above.

The following steps are followed:

A] Synthesis of Fmoc-Arg (Pbf)-Gly-Wang resin: -
Synthesis carried out using Wang resin by Fmoc solid phase peptide strategy.
Fmoc-Gly-Wang resin (5.0 g, Loading capacity - 0.3 mmol/g) was charged in a clean dry peptide synthesizer vessel. Resin swelled with DMF (10 Vol.) for 30 min, 2 times each.
Fmoc deprotection: Fmoc deprotection performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 min followed by 15 min respectively. Drained the solvent, washed resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
1st amino acid coupling: The pre-activated Fmoc-Arg(Pbf)-OH (2.91 g, 3.0 eq) with HOBt monohydrate (0.69 g, 3.0 eq) and DIC (0.70 mL, 3.0 eq) in DMF (10 Vol.) added to above resin, agitated for 3hrs. Drained solvent and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

B] Synthesis of Fmoc-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin [SEQ ID NO. 3]:-
Fmoc deprotection: Fmoc deprotection was performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 min followed by 15 min respectively. Drained the solvent, washed resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
Coupling of Fmoc-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-OH with NH2-Arg(Pbf)-Gly-Wang resin:- Pre-activated Fmoc-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-OH (6.25 g, 3.0 eq) with HBTU (1.7g, 3.0 eq), HOBt monohydrate (0.69g, 3.0 eq) and DIPEA (0.78 mL, 3.0 eq) in 20 % DMSO/ DMF (10 Vol.) added to above Fmoc deprotected resin, agitated for 5 hrs. After 5 hrs. Drained solvent and peptide resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

C] Synthesis of Fmoc-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin [SEQ ID NO.4]
Fmoc-Phe-OH coupling :- The pre-activated Fmoc-Phe-OH (1.74 g, 3.0 eq) with HOBt monohydrate (0.69 g, 3.0 eq) and DIC (0.70 mL, 3.0 eq) in DMF (10 Vol.) added to above resin. Coupling was continued for 6 hrs. Solvent drained and peptide resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x10 Vol.) respectively.
Fmoc deprotection and coupling procedure were followed for following amino acid as per sequential. which are Fmoc-Glu (OtBu)-OH (1.91 g, 3.0 eq), were performed similar to the above procedure.

D]Synthesis of Fmoc-Lys(AEEAc-AEEAc-?-Glu17-Carboxyheptadecanoyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin [SEQ ID NO.5]
Fmoc deprotection: Fmoc deprotection performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 min followed by 15 min respectively. Drained the solvent, washed resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
Coupling of Fmoc-Lys(W)-OH:-The pre-activated Fmoc- Lys(AEEAc-AEEAc-?-Glu17-Carboxyheptadecanoyl)-OH (3.58 g, 2.0 eq), with HBTU (1.13 g, 2.0 eq) and HOBt monohydrate (0.46 g, 2.0 eq) and DIPEA (0.52 mL , 2.0 eq) in 20% DMSO/DMF (10 Vol.) added to above peptide resin, agitated for 5hrs. After 5 hrs., drained solvent and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

E]Synthesis of Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEAc-AEEAc-?-Glu17-Carboxyheptadecanoyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resin [SEQ ID NO.6]:-
Fmoc deprotection and coupling procedure were followed for following amino acid as per sequential. which are, Fmoc-Ala-OH (1.40 g, 3.0 eq), Fmoc-Ala-OH (1.40 g, 3.0 eq), Fmoc-Gln(Trt)-OH (2.77 g, 3.0 eq) were performed similar to the above Fmoc-Phe-OH coupling procedure.

F]Synthesis of Fmoc-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(AEEAc-AEEAc-?-Glu17-Carboxyheptadecanoyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resin [SEQ ID NO.7]:-

Coupling of Fragment-SG [SEQ ID NO.2]:-The pre-activated solution of Fmoc-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly -OH (4.95 g, 3.0 eq) with HBTU (1.7 g, 3.0 eq) and HOBt monohydrate (0.69 g, 3.0 eq) and DIPEA (0.78, 3.0 eq) in 20% DMSO/DMF (10 Vol.) added to above peptide resin, agitated for 5hrs. After 5 hrs. Drained solvent and resin was washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

G]Synthesis of Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(AEEAc-AEEAc-?-Glu17-Carboxyheptadecanoyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin[SEQ ID NO.8]
Fmoc deprotection: Fmoc deprotection performed by adding 20% Piperidine in DMF (2 x 10 Vol.) for 10 min. followed by 15 min respectively. Drained the solvent, washed resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
Coupling of Fmoc-Val-OH: The pre-activated solution of Fmoc-Val-OH (1.52 g, 3.0 eq) with HOBt monohydrate (0.69 g, 3.0 eq) and DIC (0.70 mL, 3.0 eq) in DMF (10 Vol.) added to above resin. Coupling was continued for 3 hrs. Solvent drained and peptide resin washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
Fmoc deprotection and coupling procedure were followed for following amino acids as per sequential. which are Fmoc-Asp (OtBu)-OH (1.85 g, 3.0 eq), Fmoc-Ser(tBu)-OH (1.72 g, 3.0 eq), Fmoc- Thr(tBu)-OH (1.78 g, 3.0 eq), Fmoc-Phe-OH (1.74 g, 3.0 eq), Fmoc-Thr(tBu)-OH (1.78 g, 3.0 eq), were performed similar to the above procedure.

H]Synthesis of Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(AEEAc-AEEAc-?-Glu17-Carboxyheptadecanoyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin[SEQ ID NO.9]:-
Fmoc deprotection: Fmoc deprotection performed by adding 20 % Piperidine in DMF (2 x 10 Vol.) for 10 min followed by 15 min respectively. Drained the solvent, washed resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
Fragment-BG coupling:- The pre-activated solution of Fmoc-Aib-Glu(OtBu)-Gly-OH (2.55 g, 3.0 eq) with HBTU (1.7 g, 3.0 eq) and HOBt monohydrate (0.69 g, 3.0 eq) and DIPEA (0.78, 3.0 eq) in 20% DMSO/DMF (10 Vol.) added to above peptide resin, agitated for 5h. Drained solvent and resin washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

I]Synthesis of Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(AEEAc-AEEAc-?-Glu17-Carboxyheptadecanoyl)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resin:-
Fmoc deprotection: Fmoc deprotection performed by adding 20% Piperidine in DMF (2 x 10 Vol.) for 10 min. followed by 15 min respectively. Drained the solvent, washed resin with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.
Boc-His(Boc)-OH coupling:- The pre-activated solution of Boc-His(Boc)-OH (2.23 g, 3.0 eq) with HBTU(1.7 g, 3.0 eq), HOBt monohydrate (0.69 g, 3.0 eq) and DIPEA (0.78 mL, 3.0 eq) in DMF (10 Vol.) added to above peptide resin. Resin was agitated for 4h. Drained solvent and resin washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.
Each coupling and deprotection monitored by Bromophenol blue test.
After completion of synthesis, peptide resin was washed with 20% Methanol in DCM (2 x 10 Vol.), MTBE (1 x 10 Vol) and well dried to afford 12.0 g of peptide resin.

K]Synthesis of His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEAc-AEEAc-?-Glu17-Carboxyheptadecanoyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH, Semaglutide, crude [SEQ ID NO.10]
Global deprotection: To a pre-cooled cleavage cocktail of TFA: Phenol: DODT: Thioanisol: H2O (82.5 : 5.0 : 2.5 : 5.0 : 5.0, 20 Vol.) was added to protected peptide resin (12.0 g) at 5-10oC. Resulting mixture was agitated for 5 hrs at 20-25oC. After completion of cleavage, resin was filtered and filtrate was concentrated up to 30 % and poured into pre-cooled MTBE (20 Vol.), solid was stirred for 15-30 minutes and filtered over Buchner funnel, washed with MTBE (2 x 5 Vol.). Crude peptide was dissolved in H2O (10 Vol.) and lyophilized to afford off white solid. Yield: 5.82 g, 61.2 % HPLC purity.
The present invention provides process for manufacturing GLP-1 analogue with high yield and purity by fragment synthesis on Tagging technique and fragment coupling on resin. The invention also provides a method for preparation of peptides backbone fragments with cost efficient and industrially scalable process using different fragments combinations. The process of synthesis is environmentally benign, simple, straightforward, and efficient and provides the end product in high yield and purity
It is to be understood that the present invention is susceptible to modifications, changes and adaptations by those skilled in the art. Such modifications, changes, adaptations are intended to be within the scope of the present invention.
,CLAIMS:
1. A method for preparing Semaglutide, said method involving fragment coupling on a resin by solid phase synthesis, comprising the steps of:
a. Providing a first protected peptide fragment having amino acid sequence of Arg (Pbf)-Gly on a resin;
b. Deprotecting said first peptide fragment in presence of a deprotecting agent and coupling the deprotected first peptide fragment with a protected peptide fragment having amino acid sequence SEQ ID NO:1 [Fragment IG] to form a protected peptide fragment having amino acid sequence SEQ ID NO:3 in presence of a coupling agent, a base and a solvent;
c. Deprotecting said peptide fragment having amino acid sequence SEQ ID NO:3 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:3 with protected amino acids P-Phe-OH and P-Glu(OtBu)-OH to form protected peptide fragment having amino acid sequence SEQ ID NO:4 in presence of a coupling agent, a base and a solvent;
d. Deprotecting said peptide fragment having amino acid sequence SEQ ID NO:4 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:4 with a modified protected amino acid Lys(W) to form protected peptide fragment having amino acid sequence SEQ ID NO:5 in presence of a coupling agent, a base and a solvent,
Wherein W is N-(17-carboxy-1-oxoheptadecyl)-L-Y-glutamyl-2-[2-(2-aminoethoxy] ethoxy] acetyl-2-[2(2-aminoethoxy) ethoxy] acetyl;
e. Deprotecting said peptide fragment having amino acid sequence SEQ ID NO:5 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:5 with protected amino acids P-Ala-OH, P-Ala-OH and P-Gln(trt)-OH respectively to form protected peptide fragment having amino acid sequence SEQ ID NO:6 in presence of a coupling agent, a base and a solvent;
f. Deprotecting said peptide fragment having amino acid sequence SEQ ID NO:6 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:6 with protected peptide fragment having amino acid sequence SEQ ID NO:2 [Fragment SG] to form protected peptide fragment having amino acid sequence SEQ ID NO:7, in presence of a coupling agent, a base and a solvent;
g. Deprotecting said peptide fragment having amino acid sequence SEQ ID NO:7 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:7 with protected amino acids P-Val-OH, PAsp( OtBu)-OH, P-Ser(tBu)-OH, P-Thr(tBu)-OH, P-(Phe-OH, P-Thr(tBu)-OH respectively to form protected peptide fragment having amino acid sequence SEQ ID NO:8, in presence of a coupling agent, a base and a solvent;
h. Deprotecting said peptide fragment having amino acid sequence SEQ ID NO:8 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:8 with protected peptide fragment having sequence of Fmoc-Aib-Glu (OtBu)-Gly-OH [Fragment BG] to form a protected peptide fragment having amino acid sequence SEQ ID NO:9 in presence of a coupling agent, a base and a solvent;
i. Deprotecting said peptide fragment having amino acid sequence SEQ ID NO:9 in presence of a deprotecting agent and coupling the deprotected peptide fragment having amino acid sequence SEQ ID NO:9 with protected amino acid fragment Boc His(Boc)-OH to form a protected peptide fragment having amino acid sequence SEQ ID NO:10 in presence of a coupling agent, a base and a solvent;
j. Deprotecting followed by resin cleavage of the peptide sequence having amino acid sequence SEQ ID NO:10 followed by filtration to obtain crude GLP-1 peptide;
Wherein said P is a N-terminal protecting group;
Wherein said resin is an acid sensitive polymeric resin; and
Wherein said fragments of SEQ ID NO:1 [Fragment IG], SEQ ID No.2 [Fragment SG] and Fragment BG are individually prepared on a TAG support by liquid phase synthesis.

2. The method as claimed in claim 1, wherein the protecting group (P) is selected from Fmoc, Boc, Cbz and Bpoc.

3. The method as claimed in claim 1, wherein the coupling agents are selected from hydroxybenzotriazole (HOBt); O-(7-azabenzotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-N,N,N',N'tetramethyluronium tetrafluoroborate (TBTU), 1,3-dicyclohexylcarbodlimide (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-oxytris(dimethyl-amino)-phosphonium hexafluorophosphate (BOP), N,N-bis-(2-oxo-3-oxazolidinyl)phosphonic dichloride (BOP-C1), benzotriazo1yloxytri(pyrolidino)phosphonium hexafluorophosphate (PyBOP), bromotri(pyrrolidino)phosphonium hexafluorophosphate (PyBrOP), chlorotri(pynolidino)phosphonium hexafluorophosphate (PyClOP), ethyl-2-cyano-2(hydroxyimino) acetate (Oxyma Pure), O-(6-Chloro-1-hydrocibenzotriazol-1-yl)-1,1,3,3tetramethyluronium tetrafluoroborate (TCTU), 245-norbornen-2,3-dicarboximido)-1,1,3,3tetramethyluronium tetrafluoroborate (TNTU), propane phosphonic acid anhydride (PPAA), 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoro borate (TSTU), bromo-tris-pyrrolidinophosphonium hexafluorophosphate (PyBroP), iso-butylchloroformate (IBCF), Ethyl 1,2dihydro-2-ethoxyquinoline-1-carboxylate (EEDQ), 1-Cyano-2-ethoxy-2oxoethylidenaminooxy)dimethylamino-morpholino-carbeniu- m hexafluorophosphate (COMU), 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) or mixtures thereof.

4. The method as claimed in claim 1, wherein the coupling takes place in a solvent selected from DMF, DCM, THF, NMP, DMAC methanol, ethanol, isopropanol, dichloroethane, 1,4-dioxane, 2-methyl tetrahydrofuran ethyl acetate, acetonitrile, acetone, and the like or a mixture thereof.

5. The method as claimed in claim 1, wherein the coupling reaction is carried out in presence of a base selected from di-C1-C1O -alkyl sulphides, alkyl phenyl sulphides, piperidine, 1,2-aminothiol of cysteine, a 1,2-aminoethanol of serine, a 1,2-aminoethanol of threonine, an amino oxyacetyl functional group, a mono-hydrazine succinyl functional group, ammonia, hydrazine, an alkoxide, a 4-hydrazinobenzoyl functional group, diisopropylamine, N,N-diisopropylethylamine triethylamine, dimethylamine, trimethyl amine, isopropyl ethylamine, pyridine, N-methyl morpholine and mixtures thereof.

6. The method as claimed in claim 1, wherein the protected peptide is de-protected with a mixture of reagents selected from TFA, TIS, TIPS, DTT, EDT, ammonium iodide, 2,2'-(ethylenedioxy)diethane and acetyl cystein, DMS, phenol, cresol and thiocresol.

7. The method as claimed in claim 1, wherein said acid sensitive polymeric resin is selected from 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.

8. The method as claimed in claim 1, wherein the TAG is selected from 2,4-Bis-octadecyloxy-phenyl)-methanol; 3,4,5-tri (2’,3’-dihydrophytyloxy) benzyl alcohol and 2,4-didocosyloxybenzyl alcohol or combinations thereof.

9. A peptide fragment for preparing Semaglutide having amino acids sequence selected from sequences SEQ ID NO:1 (Fragment IG), SEQ ID NO:2 (Fragment SG) and Fragment BG.

10. A method for preparing a peptide fragment having amino acid sequence of SEQ ID NO:1 [Fragment IG] for synthesis of Semaglutide, said method being carried out on a TAG support by liquid phase synthesis, comprising the steps of:
i. Tagging a protected amino acid of P-Gly-OH with TAG to form P-Gly-TAG;
ii. Deprotecting the tagged amino acid fragment in presence of a deprotecting agent and successive coupling the deprotected tagged amino acid fragment with protected amino acid fragments of P-Arg (Pbf)-OH, P-Val-OH, P-Leu-OH, P-Trp(Boc)-OH, P-Ala-OH, P-Ile –OH to form a protected tagged peptide fragment having amino acid sequence of SEQ ID NO:1 in presence of a coupling agent, a base and a solvent;
iii. Cleaving the TAG by dissolving the peptide fragment of SEQ ID NO.1 in cleaving agents followed by neutralization and filtration to obtain the solid peptide fragment having SEQ ID NO.1;
Wherein said P is a N-terminal protecting group; and said TAG is acid sensitive.

11. A method for preparing a peptide fragment having amino acid sequence SEQ ID NO:2 [Fragment BG] for synthesis of Semaglutide, said method being carried out on a TAG support by liquid phase synthesis, comprising the steps of:
i. Tagging a protected amino acid of P-Gly-OH with TAG to form P-Gly-TAG;
ii. Deprotecting the tagged amino acid fragment in presence of a deprotecting agent and successive coupling the deprotected tagged amino acid fragment with protected amino acid fragments of P-Glu(OtBu), P-Leu-OH, P-Tyr(tBu)-OH, P-Ser(tBu)-OH, P-Ser(tBu)-OH to form a protected tagged peptide fragment having amino acid sequence of SEQ ID NO:2 in presence of a coupling agent, a base and a solvent;
iii. Cleaving the TAG by dissolving the peptide fragment of SEQ ID NO.2 in cleaving agents followed by neutralization and filtration to obtain the solid peptide fragment having SEQ ID NO.2;
Wherein said P is a N-terminal protecting group; and said TAG is acid sensitive.

12. A method for preparing a peptide fragment having amino acid sequence Fmoc-Aib-Glu (OtBu)-Gly-OH [Fragment BG] for synthesis of Semaglutide, said method being carried out on a TAG support by liquid phase synthesis, comprising the steps of:
i. Tagging a protected amino acid of P-Gly-OH with TAG to form P-Gly-TAG;
ii. Deprotecting the tagged amino acid fragment in presence of a deprotecting agent and successive coupling the deprotected tagged amino acid fragment with protected amino acid fragments of P-Glu(OtBu), P-Aib-OH, to form a protected tagged peptide fragment having amino acid sequence of Fragment BG in presence of a coupling agent, a base and a solvent;
iii. Cleaving the TAG by dissolving the peptide fragment of Fragment BG in cleaving agents followed by neutralization and filtration to obtain the solid peptide fragment;
Wherein said P is a N-terminal protecting group; and said TAG is acid sensitive.

13. A method for preparing an amino acid fragment of Lysine with side chain (Lys (W)) for synthesis of Semaglutide, comprising the steps of:
i. Tagging a protected first compound of 8-amino 3,6 dioxa octanoic acid (P-PEG-OH) with TAG to form P-PEG-OTAG;
ii. Deprotecting the tagged fragment in presence of a deprotecting agent and coupling the deprotected tagged first fragment with protected compound fragment of P-PEG-OH and successive coupling with P-Glu (Otbu), 18-ter-butoxy-18-oxo-octadecanoic acid to form a protected tagged fragment of 18-ter-butoxy-18-oxo-octadecanoic acid-Glu-Otbu-PEG-PEG-OTAG (W-OTAG) in presence of a coupling agent, a base and a solvent;
iii. Cleaving the TAG from the fragment in presence of cleaving agents followed by filtration to obtain Fragment-W-OH;
iv. Providing a protected amino acid sequence of P-Lys (Alloc)-OH on a resin;
v. Deprotecting the Alloc fragment in presence of a deprotecting agent and coupling the deprotected fragment with Fragment-W-OH obtained in step (iii) in presence of a coupling agent, a base and a solvent;
vi. Cleaving of the resin from the peptide fragment in presence of cleaving agents followed by filtration to obtain the amino acid fragment of Lysine with side chain (Lys (W);
Wherein said P is a N-terminal protecting group; and said TAG is acid sensitive;
Wherein W is N-(17-carboxy-1-oxoheptadecyl)-L- ? -glutamyl-2-[2-(2-aminoethoxy] ethoxy] acetyl-2-[2(2-aminoethoxy) ethoxy] acetyl.

14. The method as claimed in any one of claims 10 to 13, wherein the TAG is selected from 2,4-Bis-octadecyloxy-phenyl)-methanol; 3,4,5-tri (2’,3’-dihydrophytyloxy) benzyl alcohol and 2,4-didocosyloxybenzyl alcohol or combinations thereof.

15. The method as claimed in any one of claims 10 to 13, wherein the protecting group (P) is selected from Fmoc, Boc, Cbz and Bpoc.

16. The method as claimed in any one of claims 10 to 13, wherein the coupling agents are selected from hydroxybenzotriazole (HOBt); O-(7-azabenzotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-N,N,N',N'tetramethyluronium tetrafluoroborate (TBTU), 1,3-dicyclohexylcarbodlimide (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-oxytris(dimethyl-amino)-phosphonium hexafluorophosphate (BOP), N,N-bis-(2-oxo-3-oxazolidinyl)phosphonic dichloride (BOP-C1), benzotriazo1yloxytri(pyrolidino)phosphonium hexafluorophosphate (PyBOP), bromotri(pyrrolidino)phosphonium hexafluorophosphate (PyBrOP), chlorotri(pynolidino)phosphonium hexafluorophosphate (PyClOP), ethyl-2-cyano-2(hydroxyimino) acetate (Oxyma Pure), O-(6-Chloro-1-hydrocibenzotriazol-1-yl)-1,1,3,3tetramethyluronium tetrafluoroborate (TCTU), 245-norbornen-2,3-dicarboximido)-1,1,3,3tetramethyluronium tetrafluoroborate (TNTU), propane phosphonic acid anhydride (PPAA), 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoro borate (TSTU), bromo-tris-pyrrolidinophosphonium hexafluorophosphate (PyBroP), iso-butylchloroformate (IBCF), Ethyl 1,2dihydro-2-ethoxyquinoline-1-carboxylate (EEDQ), 1-Cyano-2-ethoxy-2oxoethylidenaminooxy)dimethylamino-morpholino-carbeniu- m hexafluorophosphate (COMU), 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) or mixtures thereof.

17. The method as claimed in any one of claims 10 to 13, wherein the coupling takes place in a solvent selected from DMF, DCM, THF, NMP, DMAC methanol, ethanol, isopropanol, dichloroethane, 1,4-dioxane, 2-methyl tetrahydrofuran ethyl acetate, acetonitrile, acetone, and the like or a mixture thereof.

18. The method as claimed in any one of claims 10 to 13, wherein the coupling reaction is carried out in presence of a base selected from di-C1-C1O -alkyl sulphides, alkyl phenyl sulphides, piperidine, 1,2-aminothiol of cysteine, a 1,2-aminoethanol of serine, a 1,2-aminoethanol of threonine, an amino oxyacetyl functional group, a mono-hydrazine succinyl functional group, ammonia, hydrazine, an alkoxide, a 4-hydrazinobenzoyl functional group, diisopropylamine, N,N-diisopropylethylamine triethylamine, dimethylamine, trimethyl amine, isopropyl ethylamine, pyridine, N-methyl morpholine and mixtures thereof.

19. The method as claimed in any one of claims 10 to 13, wherein the protected peptide is de-protected with a mixture of reagents selected from TFA, TIS, TIPS, DTT, EDT, ammonium iodide, 2,2'-(ethylenedioxy)diethane and acetyl cystein, DMS, phenol, cresol and thiocresol.

20. The method as claimed in any one of claims 10 to 13, wherein said acid sensitive polymeric resin is selected from 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.

21. The method as claimed in any one of the claims 10 to 13, wherein the TAG cleaving agents are selected from CH2Cl2, TFE, TFA or a mixture thereof.

22. The method as claimed in any one of the claims 10 to 13, wherein the neutralization is with DIPEA.