DESC:TECHNICAL FIELD
The present disclosure in general is in relation to synthetic organic chemistry.

Particularly, the present invention provides an improved method for preparation of Guanylate Cyclase 2C Agonist. More particularly, the disclosure provides an improved method for the preparation of Guanylate Cyclase 2C Agonist, Linaclotide.

The method provides high yield of Linaclotide with commendable purity.

BACKGROUND AND PRIOR ART
Linaclotide, a synthetic peptide, is a guanylate cyclase-C (GC-C) agonist for the treatment of chronic constipation (CC) and irritable bowel syndrome with constipation (IBS-C). Structurally, it is a heterodetic peptide and consists of fourteen amino acids. The peptide sequence of Linaclotide is: CCEYCCNPACTGCY. There are three disulfide bonds which are located between Cys1 and Cys6; between Cys2 and Cys10; and between Cys5 and Cys13. The structure of Linaclotide is represented by Formula 1.

15
Formula 1

Albericio and coworkers (Biopolymers 2011;96 (1):69-80) synthesized Linaclotide by using a random disulfide bond formation strategy. A linear peptide containing six Cys residues is prepared in solid phase and cyclized in solution.

US 9580471B2 provides a process for the preparation of Linaclotide, which includes condensation of three fragments. Process is lengthy and purification protocol after tricyclization is time consuming to produce Linaclotide.

PCT/IN2017/050051 uses solid phase peptide synthesis for Linaclotide peptide backbone preparation on to a resin. The peptide backbone is cleaved, deprotected and cyclised using H2O2 oxidation of Cys containing side chain protecting group as Trt or Phacm.

CN105884864A provides method for synthesizing Linaclotide on a resin by the conventional solid-phase peptide synthesis method and then cyclization is carried out by random oxidation by adding hydroquinone or TCEP.

WO2019113872A1 describes a process in which stepwise coupling of amino acids having an N-terminal Fmoc protecting group is adopted to obtain a Linaclotide linear peptide resin. Cyclization of the linear peptide resin is done by oxidation using N-Chlorosuccinimide solution. The resin is then subjected to a cleavage reaction, purified and lyophilized to obtain Linaclotide.

CN107936094A describes method for synthesizing Linaclotide in solid-liquid phase combination. Two fragments (6+8) based synthesis is done using different protecting groups for all the Cys to be used for disulphide bond preparation for selective synthesis of three disulfide rings on the solid phase resin. Though the cyclization is 20 quick and accurate, the use of different protecting groups makes the invention costly. Also, sequential disulphide bond formation process is complex and lengthy.

Considering the importance of Linaclotide, there is necessity in the art for a new, low cost and high-yielding process to suffice the dire industrial need for its synthesis.

OBJECT OF THE INVENTION
It is an object of the present invention is to develop an economically beneficial, environmentally benign, high yielding synthesis method for Linaclotide.

It is another object of the present invention to provide a synthesis method for Linaclotide by use of cost effective reagents, simple reaction operation and easy workup process.

It is a further object of the present invention to provide a synthesis method for Linaclotide by using novel peptide fragments to reduce time of the synthesis process.

It is yet another object of the present invention to provide Linaclotide with good yield and high purity greater than 99 % on an industrial scale, with efficient turnaround time.

SUMMARY OF THE INVENTION
The following disclosure presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the present invention. It is not intended to identify the key/critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concept of the invention in a simplified form as a prelude to a more detailed description of the invention presented later.

In an aspect of the present invention, there is provided a method for synthesizing Guanylate Cyclase 2c Agonist (Linaclotide) by solid phase synthesis, the method comprising the steps of: a. synthesizing a peptide fragment A having the sequence Fmoc-Asn(Trt)-Pro-Ala-OH; b. synthesizing a peptide fragment B having the sequence Fmoc-Glu(Otbu)-Tyr(tbu)-OH; c. synthesizing of a peptide resin 1 having the sequence of NH2-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu) using 2-CTC resin; d. coupling peptide fragment A to peptide resin 1 to obtain Fmoc-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin; e. coupling of two moieties of Fmoc-Cys(Trt)-OH with Fmoc-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin to obtain Fmoc-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin; f. coupling peptide fragment B to the resin obtained in step (e) to obtain Fmoc-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin; g. coupling of Fmoc-Cys(Trt)-OH and Boc-Cys(Trt)-OH with the resin obtained in step (f) to obtain Boc-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin; h. cleaving the peptide from the resin of step (g) to obtain a crude peptide; i. oxidising the crude peptide in isopropanol and water to obtain crude Linaclotide; and j. purifying the crude Linaclotide by passing through a resin to obtain pure Linaclotide.

Other aspects, advantages, and salient features of the invention will become apparent to those skilled in the art from the following detailed description, which, taken in conjunction with the annexed drawings, discloses exemplary embodiments of the invention.

BRIEF DESCRIPTION OF ACCOMAPNAYING DRAWINGS
Figure 1 illustrates a flowchart depicting hybrid convergent synthesis approach via two novel fragments.

Figure 2 illustrates a flowchart depicting linear synthesis approach.

Figure 3 illustrates a flowchart depicting synthesis of fragment A-Fmoc-Asn(Trt)Pro-Ala-OH.

Figure 4 illustrates a flowchart depicting synthesis of Fragment B-Fmoc-Glu(OtBu)-Tyr(tBu)-OH.

DETAILED DESCRIPTION OF THE INVENTION
The invention provides a process for the synthesis of a Guanylate cyclase 2C agonist, Linaclotide with high yield and less impurities.

The following description with reference to the accompanying drawings is provided to assist in a comprehensive understanding of various embodiments of the present disclosure as defined by the claims and their equivalents. 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 various embodiments described herein can be made without departing from the scope and spirit of the present disclosure. In addition, descriptions of well-known functions and constructions may be 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 present disclosure. Accordingly, it should be apparent to those skilled in the art that the following description of various embodiments of the present disclosure is provided for illustration purpose only and not for the purpose of limiting the present disclosure 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. Thus, for example, reference to "a component surface" includes reference to one or more of such surfaces.

The terminology used herein is for the purpose of describing particular various embodiments only and is not intended to be limiting of various embodiments. As used herein, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising" used herein specify the presence of stated features, integers, steps, operations, members, components, and/or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, operations, members, components, and/or groups thereof. Also, expressions such as "at least one of," when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

The present invention enables for the synthesis of Linaclotide comprising solid phase and/or solution phase synthesis of necessary unique fragments, subsequent condensation of fragments in solid phase or by linear approach to form pure linear crude peptide followed by sequential oxidative cyclization of cysteine amino acid of linear crude peptide to form crude Linaclotide. Typically, Cys 1, Cys 2, Cys 5, Cys 6, Cys 10 and Cys 13 are protected by Trt and peptides are synthesized by Fmoc / tBu solid-phase synthesis. After coupling of necessary fragments, global deprotection is carried out by Trifluoroacetic acid with suitable scavengers. First oxidation is carried out with charcoal to form three disulfide bond between Cys1 and Cys6, between Cys2 and Cys10, and between Cys5 and Cys13, and then the subsequent crude peptide is purified by reverse phase preparative HPLC and or using Biotage purification techniques to remove all unwanted impurities.

In another approach aforementioned oxidative cyclization and purification is followed after preparation of crude peptide by linear synthesis.

The present invention enables convenient synthesis of Linaclotide with good yield and high purity greater than 99 % on an industrial scale, with efficient turnaround time.

In an embodiment of present invention, linear peptide is prepared using either hybrid convergent synthesis approach via two novel fragments: Fragment A and Fragment B as depicted in Flowchart 1 (Figure 1) or by solid phase synthesis approach by linear sequential protected amino acid addition as depicted in Flowchart 2 (Figure 2) followed by oxidative cyclization using charcoal and purification.

The embodiments depicted in flow chart 1 and 2 include cost effective and environmentally benign reagents and simple reaction operations.

In a preferred embodiment, Linaclotide is prepared by the convergent synthesis approach using novel fragments A and B.

In the present invention the Fragment A is Fmoc-Asn(Trt)-Pro-Ala-OH and Fragment B is Fmoc-Glu(OtBu)-Tyr(tBu)-OH.

In an embodiment Fragment A-Fmoc-Asn(Trt)-Pro-Ala-OH is prepared by solid phase and Fragment B-Fmoc-Glu(OtBu)-Tyr(tBu)-OH is prepared by liquid as well as solid phase synthesis.

The significance of fragment approach is minimization of impurities and increased yield compared to the solid phase synthesis. The fragments A and B are prepared by solid and/or liquid phase and suitably combined to minimize the impurities and increase the yield.

The schematic of general method adopted for coupling of Fragment A-Fmoc-Asn(Trt)Pro-Ala-OH and Fragment B-Fmoc-Glu(OtBu)-Tyr(tBu)-OH on solid phase is given in Flowchart 3 (Figure 3) and Flowchart 4 (Figure 4) respectively.
In one embodiment of the present invention, resins for the solid phase synthesis are selected from a group comprising Chlorotrityl Chloride (CTC), Sasrin, Wang Resin, 4-methytrityl chloride, TentaGel S, TentaGel TGA and the like. In a preferred embodiment, the resin for solid phase synthesis is CTC.

In an embodiment of the present invention, the amino protecting groups of the peptides are selected from a group comprising fluorenylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc), Benzyl chloroformate (Cbz), 2-(p-biphenylyl)-2-propyloxycarbonyl (Bpoc), and the like. In a preferred embodiment, the amino protecting group is Fmoc. In another preferred embodiment, the amino protecting group is Boc.

In an embodiment of present invention, the side protecting group for Cys 1, Cys2, Cys 5, Cys 6, Cys 10 and Cys 13 is trityl (Trt). However, Fmoc-Cys(Mtt)-OH and Fmoc-Cys(Mmt)-OH can also be used.

In yet another embodiment of the present invention, coupling reagents are selected from a group comprising DIPC (Di-isopropyl carbodimide), TBTU(2-(1HBenzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate), HBTU (2-(1Hbenzotriazol-1-yl)-1,1,3,3-tetramethyluronium Hexafluorophosphate), HATU (1[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3- oxide hexafluorophosphate), HOBt (Hydroxybenzotriazole), 6-chloro HOBt, 1- DIC (3dicyclohexylcarbodiimide), Diisopropylcarbodiimide (DCC), Diisopropyl carbodiimide (DIC), BOP(Benzotriazol- l -yl-oxy-tris (dimethyl amino) phosphonium hexafluorophosphate), PyBOP (Benzotriazol-1-yloxy tri(pyrrolidino) phosphoniumhexafluorophosphate), PyBrOPBromotri (pyrrolidino) phosphonium hexafluorophosphate), PyClOP (Chlorotri (pyrrolidino) phosphonium hexafluorophosphate), Oxyma Pure (Oxyma - Ethyl-2-cyano-2-(hydroxyimino) acetate), TCTU (0-(6-Chlorobenzotriazol-l -yl)-l , l,3,3-tetramethyluronium tetrafluoroborate), EEDQ (Ethyl 1 ,2- dihydro-2-ethoxyquinoline-l -carboxylate ), COMU (1 -Cyano-2-ethoxy-2-oxoethy Hdenaminooxy) dimethyl aminomorpholino carbeniumhexafluoro phosphate), DEPBT (3-(Diethoxy-phosphoryloxy)-3H-benzo[d][l ,2,3] triazin-4- one) and the like; and mixtures thereof.

In yet another embodiment of the present invention, the solvents for coupling are selected from a group comprising Dimethyl formamide, N-Methyl-2-pyrrolidone, Dichloromethane, N,N-dimethylacetamide (DMA), Acetonitrile, tetrahydrofuran (THF), 2-Methyl tetrahydrofuran N-butylpyrrolidinone and mixtures thereof.

In another embodiment, loading of resin is preferably in the range of 0.5-0.7 mmol/g.

The resin is swelled with a solvent before use. Cleavage of resin is carried out by using mixed solution of TFA: DCM (1-2%:99-98%) to react with the resin for 3-4 times, each time for 10-20 minutes.

In another embodiment, the coupling reaction of each amino acid is varied ranging from about 2 to about 6 hours, preferably 2 to 4 hours at temperature ranging from about 20°C to about 35°C; or at suitable elevated temperature.

In another embodiment, the linear peptide is cleaved from the resin, deprotected globally and cyclized by using cost effective, simple, single step oxidation method.
In one embodiment of the present invention, the linear peptide is cleaved from resin using TFA (Trifluoro acetic acid) and DCM (Dichloromethane).

In another embodiment of the present invention, the linear protected peptide is de-protected with a mixture of reagents selected from the group comprising of TFA (Trifluoro acetic acid), TIS (Triisopropylsilane), DTT (Diothreitol), EDT (Ethanedithiol), ammonium iodide, 2,2'-(ethylenedioxy)diethane and acetyl cysteine, DMS, phenol, cresol and thiocresol.

In yet another embodiment of the present invention, the cyclization is carried out by means of intramolecular cysteine S-S bridge formation in presence of granular charcoal as catalyst. More particularly, disulfide cyclization reaction is carried with 1% w/w of activated charcoal in IPA: water (60:40) for about 16-18 hours at pH of about 9-9.3. The present method avoids use of other conventional oxidising reagents like hydrogen peroxide, DMSO, Cysteine.

An essential feature of the present invention is purification using HPLC or using Biotage purification techniques to remove all unwanted impurities. Post cyclization the reaction product is filtered and purified by reverse-phase high-pressure liquid chromatography which includes octadecylsilane as a stationary phase and 0.1% ammonium acetate aqueous solution/acetonitrile as a mobile phase and fractions are pooled for lyophilization to obtain pure Linaclotide.

The present method of synthesizing Linoclotide using the fragment based approach uses eco-friendly and cost-effective reagents. The method for synthesizing pure Linaclotide is a faster and cheaper method than the methods available in the art.

Particularly, the present invention provides a method for synthesizing Guanylate Cyclase 2c Agonist (Linaclotide) by solid phase synthesis, the method comprising the steps of:
a. synthesizing a peptide fragment A having the sequence Fmoc-Asn(Trt)-Pro-Ala-OH;
b. synthesizing a peptide fragment B having the sequence Fmoc-Glu(Otbu)-Tyr(tbu)-OH;
c. synthesizing of a peptide resin 1 having the sequence of NH2-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu) using 2-CTC resin;
d. coupling peptide fragment A to peptide resin 1 to obtain Fmoc-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin;
e. coupling of two moieties of Fmoc-Cys(Trt)-OH with Fmoc-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin to obtain Fmoc-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin;
f. coupling peptide fragment B to the resin obtained in step (e) to obtain Fmoc-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin;
g. coupling of Fmoc-Cys(Trt)-OH and Boc-Cys(Trt)-OH with the resin obtained in step (f) to obtain Boc-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin;
h. cleaving the peptide from the resin of step (g) to obtain a crude peptide;
i. oxidising the crude peptide in isopropanol and water to obtain crude Linaclotide; and
j. purifying the crude Linaclotide by passing through a resin to obtain pure Linaclotide.

The synthesis of peptide fragment A comprises the steps of (i) coupling each amino acid Fmoc-Ala-OH, Fmoc-Pro-OH and Fmoc-Asn(trt)-OH sequentially to 2-CTC resin followed by deprotection after each coupling step; and (ii) cleaving the synthesized peptide from the resin.

The synthesis of peptide fragment B comprises the steps of (i) coupling each amino acid Fmoc-Tyr(tbu)-OH and Fmoc-Glu(OtBu)-OH sequentially to 2-CTC resin followed by deprotection after each coupling step; and (ii) cleaving the synthesized peptide from the resin.

The synthesis of peptide resin 1 comprises the steps of (i) coupling each amino acid Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH sequentially to 2-CTC resin followed by deprotection after each coupling step.

In the method as described herein, the coupling of amino acids are carried out by coupling reagents selected from a group consisting of DIPC (Di-isopropyl carbodimide), TBTU(2-(1HBenzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate), HBTU (2-(1Hbenzotriazol-1-yl)-1,1,3,3-tetramethyluronium Hexafluorophosphate), HATU (1[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3- oxide hexafluorophosphate), HOBt (Hydroxybenzotriazole), 6-chloro HOBt, 1- DIC (3dicyclohexylcarbodiimide), Diisopropylcarbodiimide (DCC), Diisopropyl carbodiimide (DIC), BOP(Benzotriazol- l -yl-oxy-tris (dimethyl amino) phosphonium hexafluorophosphate), PyBOP (Benzotriazol- 1-yloxy tri(pyrrolidino) phosphoniumhexafluorophosphate), PyBrOPBromotri (pyrrolidino) phosphonium hexafluorophosphate), PyClOP (Chlorotri (pyrrolidino) phosphonium hexafluorophosphate), Oxyma Pure (Oxyma - Ethyl-2-cyano-2-(hydroxyimino) acetate), TCTU (0-(6-Chlorobenzotriazol-l -yl)-l , l,3,3-tetramethyluronium tetrafluoroborate), EEDQ (Ethyl 1 ,2- dihydro-2-ethoxyquinoline-l -carboxylate ), COMU (1 -Cyano-2-ethoxy-2-oxoethy Hdenaminooxy) dimethyl aminomorpholino carbeniumhexafluoro phosphate), DEPBT (3-(Diethoxy-phosphoryloxy)-3H-benzo[d][l ,2,3] triazin-4- one) and mixtures thereof.

In the method as described herein, the coupling takes place in solvents selected from a group consisting of Dimethyl formamide, N-Methyl-2-pyrrolidone, Dichloromethane, N,N-dimethylacetamide (DMA), Acetonitrile, tetrahydrofuran (THF), 2-Methyl tetrahydrofuran N-butylpyrrolidinone and mixtures thereof.

In the method as described herein, the coupling reaction of each amino acid is between 2 to 6 hours at temperature ranging from about 20°C to about 35°C.

In the method as described herein, wherein the peptide is cleaved from resin using Trifluoro acetic acid and Dichloromethane.

In the method as described herein, wherein de-protection of the peptides is carried out with a mixture of reagents selected from the group comprising of Trifluoro acetic acid, Triisopropylsilane, Diothreitol, Ethanedithiol, ammonium iodide, 2,2'-(ethylenedioxy)diethane and acetyl cysteine, DMS, phenol, cresol and thiocresol, and mixtures thereof.

In the method as described herein, wherein the cyclization takes place in presence of granular charcoal as catalyst.

Thus, the present invention provides a potential method for the synthesis of Linaclotide with the characteristics of simple reaction operation, easy post-processing, low cost, high yield.

The present invention is now being illustrated by way of non-limiting examples. The examples are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way. Efforts have been made to ensure accuracy with respect to numbers used, but some experimental errors and deviations should be accounted for.

Examples
All amino acids are commercially purchased from Chem-Impex, CS Bio, GL Bio Advance ChemTech, and the like. The synthesis of peptides is carried out by manual or in CS Bio peptide synthesizer (CS536XT).

A. General procedure for fragments synthesis-
Fragment-A and Fragment-B are synthesized on 2-CTC resin. 2-CTC is swelled 2x with DCM (10 vol.) each time for 20 minutes then is filtered for further use.

1st amino acid coupling- Protected amino acid(3.0eq) with DIPEA(6.0eq) in DCM(10vol) is added to pre-swelled resin; bubbled for 3h and filtered. The resin is washed with DCM (2 x 10 vol.) each time for 2 minutes with bubbling and then filtered. Capping is carried out with solution comprising solvents in the ratio DCM: MeOH: DIPEA (85:10:5, 10 vol.), bubbled for 30min and then filtered. Further CTC resin is washed with DCM (2 x 10 vol.) each time 2 minutes with bubbling, filtered, washed with DMF (2 x10 vol.) each time for 2 minutes, bubbled with nitrogen for 2 minutes and then filtered.

Fmoc deprotection- Fmoc is cleaved from peptide resin by using 20% piperidine in DMF (2 x 10 vol.) for 10 min followed by 15 min each time, filtered, washed with DMF (3 x 10 vol.) for 2 minutes under bubbling and then filtered. Resin is washed with DCM (3 x 10 vol.) each time 2 minutes bubbling then filtered. After DCM washing, it is repeated with DMF solvent.

2nd amino acid coupling – Pre-activated amino acid (3.0eq) with HOBt(3.0eq), DIC (3.0eq) in DMF (10 vol.) are added to peptide resin and bubbled for 2h and then filtered. Resin is washed with DMF (3x10 vol.) followed by DCM (3 x 10 vol.), each time resin is bubbled 2 minutes and then filtered. After DCM washing, it is repeated with DMF.

For rest of couplings of amino acids followed 2nd amino acid coupling procedure and Fmoc is deprotected using above deprotection procedure. Finally, peptide resin is washed with 20% MeOH in DCM and MTBE (3 x10 vol.), each time resin is bubbled 2 minutes and then filtered.

Resin cleavage- Peptide is cleaved from the resin using 1%TFA in DCM (10 vol.) for 3 times, 3 minutes each and cleaved fractions are collected, neutralized with pyridine. The peptide resin washed with DCM (7.5 vol.); collected fractions are combined and concentrated. Finally, the peptide is precipitated out by addition of ethanol followed by water (5 vol.), solid is collected by vacuum filtration and washed with water; dried under reduced pressure.

B. Couplings of fragments
STEP 1
Synthesis of NH2-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin:
To a Pre-swelled 2-CTC resin, a solution of Fmoc-Tyr(tBu)-OH(3.0eq) with DIPEA(6.0eq) in DCM (10vol) is added; resin is bubbled for 3h and filtered. Resin is washed with DCM (2 x 10 vol.) each time for 2 min with bubbling and then filtered. Capping is carried out with solution of DCM: MeOH: DIPEA (85:10:5, 10 vol.). Resin is bubbled for 30min and then filtered, washed with DCM (2 x 10 vol.) each time for 2 min with bubbling, filtered and washed with DMF (2 x10 vol.) for 2 min.

Fmoc deprotection- Fmoc is cleaved from peptide resin by using 20% piperidine in DMF (2 x 10 vol.) for 5 min followed by 10 min, filtered, washed with DMF (3 x 10 vol.) for 2 min under nitrogen bubbling and then filtered. Resin is washed with DCM (3 x 10 vol.) each time for 2 min under bubbling and then filtered. After DCM washing, DMF washing is carried out twice.

2nd amino acid coupling- To above Fmoc deprotected resin, pre-activated Fmoc-Cys(Trt)-OH(3.0eq) with HOBt(3.0eq), DIC (3.0eq) in DMF (10 vol.) in DMF (10 vol.) is added and resin is bubbled for 2 h. Completion of coupling is monitored by Bromo phenol blue indicator. After completion of coupling, peptide resin is washed with DMF (3x10 vol.) followed by DCM (3 x 10 vol.). For each washing resin is bubbled for 2 min and then filtered. After DCM washing, DMF washing is carried out twice.

Remaining three amino acids Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH andFmoc-Cys(Trt)OHwere coupled in same manner as mentioned above.

After coupling of amino acids; Fmoc is deprotected from peptide resin as explained earlier using 20% piperidine in DMF.

STEP 2
Synthesis of Fmoc-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu) 2-CTC resin:
Fragment-1, Fmoc-Asn-Pro-Ala-OH(3.0eq) is pre-activated with COMU(3.0eq) and DIPEA(3.0eq) in DMF (10 vol.) and is added to peptide resin. Resin is bubbled with nitrogen for 4h. After 4h, resin is washed with DMF (3x10 vol.) followed by DCM (3 x 10 vol.), each time resin is bubbled 2 min and then filtered.

Completion of coupling is monitored by Bromophenol blue indicator.

STEP 3
Synthesis of Fmoc-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-GlyCys(Trt)-Tyr(tBu)-2-CTC resin:
Fmoc deprotection of Fmoc-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)- Tyr(tBu)-2-CTC resin is carried out as mentioned above.

After Fmoc deprotection, coupling of Fmoc-Cys(Trt)-OH is carried out by similar method as mentioned above. Upon coupling of Fmoc-Cys(Trt)-OH, again Fmoc is deprotected and another Fmoc-Cys(Trt)-OH is coupled to afford titled peptide resin.

STEP 4
Synthesis of Fmoc-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-AlaCys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin:
Fmoc deprotection of Fmoc-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin is carried out as mentioned above.
After Fmoc deprotection, Fragment-2, Fmoc-Glu(OtBu)-Tyr(tBu)-OH(3.0eq) is preactivated with COMU(3.0eq) and DIPEA(3.0eq) in DMF (10 vol.) and is added to peptide resin. Resin is bubbled with nitrogen for 4h.After 4h, resin is washed with DMF (3x10 vol.) followed by DCM (3 x 10 vol.), each time resin is bubbled 2 min and then filtered. Completion of coupling is monitored by Bromophenol blue indicator.

STEP 5
Synthesis of Boc-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin: Fmoc deprotection of Fmoc-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin is carried out as mentioned above.

After Fmoc deprotection, coupling of Fmoc-Cys(Trt)-OH is carried out by similar method as mentioned above. Upon coupling of Fmoc-Cys(Trt)-OH, again Fmoc is deprotected and another Boc-Cys(Trt)-OH is coupled to afford titled peptide resin.

Global deprotection: Peptide resin is treated with cocktail solution (TFA: TIPS: Thioanisol: H2O: Phenol: DTT-82.5:2.5:5:5:2.5:2.5%, 20vol.) and stirred for 4h. After 4hr, cleavage mixture is concentrated to minimum volume (~ 5 vol.) and then added to cold 10% DCM in MTBE under slow stirring. After 10 min., solid is precipitated out. Solid is filtered over Buchner funnel, washed with MTBE (3 x10 vol.) and well dried under reduced pressure.

Oxidation with Charcoal (Three Disulfide bridge formation): Peptide is dissolved in IPA and diluted with purified water (60:40, 2000 Vol.). The pH of solution is adjusted with aq. ammonia solution to 9-9.3 and then 1% w/w activated charcoal are added to reaction mixture. Reaction mixture is stirred for about 18 h. After completion of reaction; reaction mixture is filtered and concentrated to 30 vol. and lyophilized to afford white solid. Solid is purified by preparative HPLC to provide pure Linaclotide. Desired purity fractions are pooled and lyophilized to afford white solid (Purity > 99%).

In similar manner Linaclotide is synthesized by linear approach. For coupling of all amino acids, Fmoc deprotection, cleavage, disulfide bridge formation and purification above procedure are implemented.

C. Synthesis Protocol for the peptide using linear solid phase synthesis approach

Swelling of Resin: 10 g of 2-CTC resin is swelled with DCM (100mL) for 30 min and then filtered. This procedure is repeated for three times, each time for 30 min. with bubbling of nitrogen.

1st amino acid coupling- To pre-swelled resin in DCM (100 mL) is added FmocTyr(tBu)-OH (3.0eq) followed by DIPEA(6.0eq). Resin is bubbled for 4h and filtered.

Resin is washed with DCM (2 x 10 vol.) each time for 2 minutes with bubbling and then filtered. Capping is carried out with solution DCM: MeOH: DIPEA (85:10:5, 10 vol.), bubbled for 30min and then filtered. Resin is washed with DCM (2 x 10 vol.) each time 2 minutes with bubbling, followed by DMF (2 x10 vol.).

Fmoc deprotection- Fmoc is cleaved from peptide resin by using 20% piperidine in DMF (2 x 10 vol.) for 10 min followed by 15 min each time, filtered. Resin is washed with DMF (3 x 10 vol.) for 2 minutes under bubbling and then filtered followed by DCM (3 x 10 vol.).

2nd amino acid coupling – After deprotection of Fmoc, pre-activated Fmoc-Cys(Trt)-OH (3.0eq), DIC (3.0eq) and HOBt (3.0eq)in DMF (10 vol.) is added to peptide resin. Reaction is bubbled for 2h with nitrogen and then filtered.

Resin is washed with DMF (3x10 vol.) followed by DCM (3 x 10 vol.), each time resin is bubbled 2 minutes and then filtered. After DCM washing, and later washed with DMF twice repeatedly.

Following the procedure of 2nd amino acid coupling Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, FmocCys(Trt)-OH, Boc-Cys(Trt)-OH are coupled. Also, for Fmoc deprotection as described in the process above is followed.

After coupling of all amino acids sequentially, Peptide resin is washed with 20% MeOH in DCM and MTBE (3 x10 vol.), Each time resin is bubbled 2 minutes and then filtered.

Resin cleavage followed by global deprotection: Peptide resin is treated with cocktail solution (TFA: TIPS: Thioanisol: H2O: Phenol: DTT-82.5:2.5:5:5:2.5:2.5%, 20vol.) and stirred for 4h. After 4hr, cleavage mixture is concentrated to minimum volume (~ 5 vol.) and then added to cold 10% DCM in MTBE under slow stirring. After 10 min., solid is precipitated out. Solid is filtered over Buchner funnel, washed with MTBE (3 x10 vol.) and well dried under reduced pressure.

Oxidation with Charcoal (Three Disulfide bridge formation): Peptide is dissolved in IPA and diluted with purified water (60:40, 2000 Vol.). The pH of solution is adjusted with aq. ammonia solution to 9-9.3 and then 1% w/w activated charcoal are added to reaction mixture. Reaction mixture is stirred for about 18 h. After completion of reaction; reaction mixture is filtered and concentrated to 30 vol. and lyophilized to afford white solid. Solid is purified by preparative HPLC to provide pure Linaclotide. Desired purity fractions are pooled and lyophilized to afford white solid (Purity > 99%).

Example 2- Preparation of Linaclotide by Fragment approach
Synthesis of Fragment A: Fmoc-Asn(Trt)-Pro-Ala-OH
Fragment is synthesized using 2-CTC resin by Fmoc solid phase peptide strategy.
2-CTC resin (120 g, loading capacity 1.7 mmol/g, 100 -200 mesh) is charged in a clean dry peptide synthesizer vessel. Resin is 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-Ala-OH (190 g, 3.0 eq) in DCM (10 Vol.) along with DIPEA (213.12 mL, 6.0 eq) is added and agitated for 12 hr at room temperature. After 12 hr. solvent is drained, resin is washed with DCM (2 x 10 Vol.). Mixture of DCM: MeOH: DIPEA (85:10:5, 10 Vol.) is added to resin and agitated for 30 min. Solvent is drained and resin is washed with DCM (2 x 10 Vol.) followed by DMF (2 x10 Vol.).

Fmoc deprotection: Deprotection of Fmoc group is performed by adding 20 % piperidine in DMF (2 x 10 Vol.) for 10 min followed by 20 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.

2nd amino acid coupling: The pre-activated Fmoc-Pro-OH (206 g, 3.0 eq) with HOBt (82.6 g, 3.0 eq) and DIC (94.8 mL, 3.0 eq) in DMF (10 V) is added to above resin. Coupling is continued for 6hr. Solvent is drained and peptide resin is washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x10 Vol.) respectively.

Fmoc deprotection: Deprotection of Fmoc group is performed by adding 20 % piperidine in DMF (2 x 10 Vol.) for 10 min followed by 20 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.

3rd amino acid coupling: The pre-activated Fmoc-Asn(trt)-OH (365 g, 3.0 eq) with HOBt (82.6 g, 3.0 eq) and DIC (94.8 mL, 3.0 eq) in DMF (10 V) is added to above resin. Coupling is continued for 6 hr. Solvent is drained and peptide resin is washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x10 Vol.) respectively.

Each coupling and deprotection is monitored by Bromophenol blue test.

After completion of synthesis, peptide resin is 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 is cleaved from the resin using 1% TFA / DCM (3 x 10 Vol.), 5 minutes for each time. Cleavage fractions were collected and neutralized with pyridine (1:1 Volume ratio to TFA in each cleavage). Combined cleavage fractions were concentrated under reduced pressure at 30-35 oC. Resulting solution is co-evaporated with ethanol (2 x 5 Vol.) up to minimum volume and slurry is added to purified water(50 Vol), stirred for 18h. Solid is filtered over Buchner funnel washed with purified water. Kept for suck dry for 8h. Solid is collected and added to RB then n-Heptane is added to RB and stirred for 3h. Solid is filtered over Buchner funnel washed-Heptane Solid is dried in VTD under reduced pressure at 38-42 oC to afford white solid.
Yield: 118 g, 76%

Synthesis of Fragment B: Fmoc-Glu(Otbu)-Tyr(tbu)-OH
Fragment is synthesized using 2-CTC resin by Fmoc solid phase peptide strategy.
2-CTC resin (120 g, Loading capacity-1.7 mmol/g) is charged in a clean dry peptide synthesizer vessel. Resin is swelled with DCM (10V), 30 min, 2 times each.

1st amino acid coupling: First amino acid Fmoc-Tyr(tbu)-OH (281.2g, 3.0 eq) with DIPEA ( 213.12 mL, 6.0 eq) in DCM (10 Vol.) is added to pre-swelled resin; agitated for 12h. Drained the solvent, washed with DCM (2 x 10 Vol.). Capping solution of DCM: MeOH: DIPEA (85%:10%:5%, 10 Vol.) is charged to above resin, agitated for 30 min for capping the resin. Solvent is drained and resin is washed with DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

Fmoc deprotection: Deprotection of Fmoc group is performed by adding 20 % piperidine in DMF (2 x 10 Vol.) for 10 min followed by 20 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.

2nd amino acid coupling: The pre-activated Fmoc-Glu(OtBu)-OH (260.4 g, 3.0 eq) with HOBt (82.6 g, 3.0 eq) and DIC (96 mL, 3.0 eq) in DMF (10 Vol.) is added to resin, agitated for 6 h. Drained solvent and peptide resin is washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Each coupling and deprotection is monitored by Bromophenol blue test.

After completion of synthesis, peptide resin is washed with 20% Methanol in DCM (2 x 5 Vol.), DCM (2 x10 Vol.), MTBE (2 x 10 Vol.) and DCM (2 x10 Vol.) respectively.

Resin cleavage: Peptide is cleaved from the resin using 1% TFA / DCM (3 x 10 Vol.), 5 minutes for each time. Cleavage fractions were collected and neutralized with pyridine (1:1 Volume ratio to TFA in each cleavage). Combined cleavage fractions were concentrated under reduced pressure at 30-35 oC. Resulting solution is co-evaporated with ethanol (2 x 5 Vol.) up to minimum volume and slurry is added to purified water(50 Vol), stirred for 18h. Solid is filtered over Buchner funnel washed with purified water. Kept for suck dry for 8h. Solid is collected and added to RB then n-Heptane is added to RB and stirred for 3h. Solid is filtered over Buchner funnel washed-Heptane Solid is dried in VTD under reduced pressure at 38-42 oC to afford white solid.
Yield: 105.36 g, 80.2 %

Synthesis of NH2-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin: -
Fragment is synthesized using 2-CTC resin by Fmoc solid phase peptide strategy.
2-CTC resin (30.0g, Loading capacity- 0.5 mmol/g) is charged in a clean dry peptide synthesizer vessel. Resin is swell with DCM (10V), 30 min, 2 times each.

1st amino acid coupling: First amino acid Fmoc-Tyr(tBu)-OH (20.6g, 3.0 eq) with DIPEA (15.64 mL, 6.0 eq) in DCM (10 Vol.) is added to pre-swelled resin; agitated for 12h. Drained the solvent, washed the resin with DCM (2 x 10 Vol.), capping solution of DCM: MeOH: DIPEA (85%:10%:5%, 10 Vol.) is charged to above resin, agitated for 30 min for capping the resin. Drained the solvent and resin is washed with DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

Fmoc deprotection: Deprotection of Fmoc group is perform by adding 20% piperidine in DMF (2 x 10 Vol.) for 10 min followed by 20 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.

2nd amino acid coupling: The pre-activated Fmoc-Cys(Trt)-OH (26.3 g, 3.0 eq) with HOBt (6.0 g, 3.0 eq) and DIC (7.1 mL, 3.0 eq) in DMF (10 Vol.) is added to above resin, agitated for 3h. Drained solvent and peptide resin is washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Sequential coupling of next three amino acids Fmoc-Gly-OH (13.33 g, 3.0 eq), Fmoc-Thr(tBu)-OH (17.87 g, 3.0 eq), Fmoc-Cys(Trt)-OH (26.3 g, 3.0 eq) were performed similar to the above procedure.

After each coupling and deprotection the peptide resin is washed DMF (2 x10 Vol.), DCM (2 x 10 Vol.) and DMF (2 x 10 Vol.) respectively.

Each coupling and deprotection is monitored by Bromophenol blue test.

Synthesis of Fmoc-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin: -
Fmoc deprotection: Deprotection of Fmoc group is 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.

Fmoc-Asn(Trt)-Pro-Ala-OH (34.38g, 3.0 eq) with COMU(19.27g, 3.0eq) DIPEA(7.8ml, 3.0eq) in DMF (10V) is added to peptide resin, agitated for 16h. Drained solvent and peptide resin is washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Synthesis of Fmoc-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin: -
Fmoc deprotection: Deprotection of Fmoc group is 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-Cys(Trt)-OH: The pre-activated Fmoc-Cys(Trt)-OH (26.34 g, 3.0 eq) with HOBt (6.0g, 3.0 eq) and DIC (6.95mL, 3.0 eq) in DMF (10V ) is added to loaded resin, agitated for 3h. Drained solvent and peptide resin is washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Fmoc deprotection: Deprotection of Fmoc group is 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-Cys(Trt)-OH: The pre-activated Fmoc-Cys(Trt)-OH (26.34 g, 3.0 eq) with HOBt (6.0g, 3.0 eq) and DIC (6.95mL, 3.0 eq) in DMF (10V ) is added to loaded resin, agitated for 3h. Drained solvent and peptide resin is washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), and DMF (2 x 10 Vol.) respectively.

Synthesis of Fmoc-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin: -
Fmoc deprotection: Deprotection of Fmoc group is performed by adding 20% piperidine in DMF (2 x 10 Vol.) for 10 min followed by 20 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-Glu(OtBu)-Tyr(tBu)-OH: The pre-activated Fmoc- Fmoc-Glu(OtBu)-Tyr(tBu)-OH (29.0 g, 3.0 eq) with COMU (19.27g, 3.0 eq) and DIPEA (7.8 mL, 3.0 eq) in DMF (10V ) is added to loaded resin, agitated for 16h. Drained solvent and peptide resin is washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), DMF (2 x 10 Vol.) respectively.

Synthesis of Boc-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin: -
Fmoc deprotection: Deprotection of Fmoc group is 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-Cys(Trt)-OH:. The pre-activated Fmoc-Cys(Trt)-OH (26.34 g, 3.0 eq) with HOBt (6.0 g, 3.0 eq) and DIC (6.95 mL, 3.0 eq) in DMF (10Vol.) is added to loaded resin, agitated for 3h. Drained solvent and peptide resin is washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), and DMF (2 x 10 Vol.) respectively.
Fmoc deprotection: Deprotection of Fmoc group is 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 Boc-Cys(Trt)-OH: The pre-activated Boc-Cys(Trt)-OH (20.86 g, 3.0 eq) with HOBt (6.0 g, 3.0 eq) and DIC (6.95 mL, 3.0 eq) in DMF (10 Vol.) is added to loaded resin, agitated for 4h. Drained solvent and peptide resin is washed with DMF (2 x 10 Vol.), DCM (2 x 10 Vol.), and DMF (2 x 10 Vol.) respectively.
Each coupling and deprotection is monitored by Bromophenol blue test.

After completion of synthesis, peptide resin is washed with 20% Methanol in DCM (1 x 10 Vol.) and MTBE (1 x 10 Vol). Resin is dried under vacuum to provide well dried peptide resin (64.0 g).

Global deprotection: To a pre-cooled cleavage cocktail TFA: TIPS: Phenol: DTT: Thioanisole: H2O (82.5%:2.5%:2.5%:2.5%:5%:5%, 20 Vol.) is added protected peptide resin (64.0 g) at 5-10 oC. Resulting mixture is agitated for 5h at 20-25 oC. After completion of cleavage, resin is filtered and filtrate is concentrated up to 30 % and poured into pre-cooled (10 oC) 10 % DCM in MTBE (100 Vol.), solid is stirred for 15-30 minutes. Solid is filtered over Buchner funnel, washed with MTBE (2 x 5 Vol.). Crude peptide is dissolved in ACN/H2O (100 Vol.) and lyophilized to afford off white solid. Yield: 13.86 g, 60.3%

Oxidation with Charcoal (Three Disulfide bridge formation):
Crude peptide is dissolved in IPA and purified water (60: 40 %, 2000 Vol.), pH (9- 9.3) of solution is adjusted with aq. ammonia solution and added activated charcoal (w/w) at 25 – 30 oC. Reaction mixture is stirred for 24 h. Reaction is monitored by HPLC, after completion of reaction, reaction mixture is filtered and concentrated up to 40 vol. and lyophilized to afford white solid, Yield-12.37 g, 90 %.

HP 20 resin treatment: HP20 resin (198 g) is swelled with Methanol (14 Vol.) for 10 min then filtered. Resin is washed with purified water (3 x 14 Vol.), is agitated for 16 h in purified water (14 Vol.). Crude Linaclotide (12.3 g, assay 19.96%) is dissolved in 0.25M Ammonium bicarbonate (200 Vol.) and loaded to above resin, agitated for 2h. After 2 h, resin is loaded in glass column and washed with H2O (3 x 14 Vol.) followed by 2.5%, 5%, 7.5%, 9.0% and 10.0% of ACN/H2O. Fractions were collected and lyophilized to afford off white solid (6.23 g, assay 52%, HPLC purity 63.4 %). To achieve desired purity, impure Linaclotide is further purified by preparative HPLC. Desired purity fractions were pooled and lyophilized to afford white solid (1.34 g, HPLC Purity 99.12 %).

Reverse purification method- For the purification of Linaclotide the following mentioned column and purification method is used
COLUMN DETAILS
Column type Prepacked column
Dimension 250mm X 21.2mm
Resin C8 Welch Ulticil
Particle size 10µm
Pore size 100 Å
Column Volume 88mL
Column make Welch

Gradient details:-

Gradient Time (min) Flow rate (ml/ min) Mobile phase A % Mobile phase B %
0 15 90 10
12 15 85 15
25 15 80 20
30 15 78 22
50 15 78 22

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. The invention is, therefore, to be limited only by the terms of the appended claims along with the full scope of equivalents to which the claims are entitled.
,CLAIMS:
1. A method for synthesizing Guanylate Cyclase 2c Agonist (Linaclotide) by solid phase synthesis, the method comprising the steps of:
a. synthesizing a peptide fragment A having the sequence Fmoc-Asn(Trt)-Pro-Ala-OH;
b. synthesizing a peptide fragment B having the sequence Fmoc-Glu(Otbu)-Tyr(tbu)-OH;
c. synthesizing of a peptide resin 1 having the sequence of NH2-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu) using 2-CTC resin;
d. coupling peptide fragment A to peptide resin 1 to obtain Fmoc-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin;
e. coupling of two moieties of Fmoc-Cys(Trt)-OH with Fmoc-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin to obtain Fmoc-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin;
f. coupling peptide fragment B to the resin obtained in step (e) to obtain Fmoc-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin;
g. coupling of Fmoc-Cys(Trt)-OH and Boc-Cys(Trt)-OH with the resin obtained in step (f) to obtain Boc-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-2-CTC resin;
h. cleaving the peptide from the resin of step (g) to obtain a crude peptide;
i. oxidising the crude peptide in isopropanol and water to obtain crude Linaclotide; and
j. purifying the crude Linaclotide by passing through a resin to obtain pure Linaclotide.

2. The method as claimed in claim 1, wherein synthesis of peptide fragment A comprises the steps of (i) coupling each amino acid Fmoc-Ala-OH, Fmoc-Pro-OH and Fmoc-Asn(trt)-OH sequentially to 2-CTC resin followed by deprotection after each coupling step; and
(ii) cleaving the synthesized peptide from the resin.

3. The method as claimed in claim 1, wherein synthesis of peptide fragment B comprises the steps of (i) coupling each amino acid Fmoc-Tyr(tbu)-OH and Fmoc-Glu(OtBu)-OH sequentially to 2-CTC resin followed by deprotection after each coupling step; and
(ii) cleaving the synthesized peptide from the resin.

4. The method as claimed in claim 1, wherein synthesis of peptide resin 1 comprises the steps of (i) coupling each amino acid Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH sequentially to 2-CTC resin followed by deprotection after each coupling step.

5. The method as claimed in claims 1 to 4, wherein the coupling of amino acids are carried out by coupling reagents selected from a group consisting of DIPC (Di-isopropyl carbodimide), TBTU(2-(1HBenzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate), HBTU (2-(1Hbenzotriazol-1-yl)-1,1,3,3-tetramethyluronium Hexafluorophosphate), HATU (1[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3- oxide hexafluorophosphate), HOBt (Hydroxybenzotriazole), 6-chloro HOBt, 1- DIC (3dicyclohexylcarbodiimide), Diisopropylcarbodiimide (DCC), Diisopropyl carbodiimide (DIC), BOP(Benzotriazol- l -yl-oxy-tris (dimethyl amino) phosphonium hexafluorophosphate), PyBOP (Benzotriazol- 1-yloxy tri(pyrrolidino) phosphoniumhexafluorophosphate), PyBrOPBromotri (pyrrolidino) phosphonium hexafluorophosphate), PyClOP (Chlorotri (pyrrolidino) phosphonium hexafluorophosphate), Oxyma Pure (Oxyma - Ethyl-2-cyano-2-(hydroxyimino) acetate), TCTU (0-(6-Chlorobenzotriazol-l -yl)-l , l,3,3-tetramethyluronium tetrafluoroborate), EEDQ (Ethyl 1 ,2- dihydro-2-ethoxyquinoline-l -carboxylate ), COMU (1 -Cyano-2-ethoxy-2-oxoethy Hdenaminooxy) dimethyl aminomorpholino carbeniumhexafluoro phosphate), DEPBT (3-(Diethoxy-phosphoryloxy)-3H-benzo[d][l ,2,3] triazin-4- one) and mixtures thereof.

6. The method as claimed in claims 1 to 4, wherein the coupling takes place in solvents selected from a group consisting of Dimethyl formamide, N-Methyl-2-pyrrolidone, Dichloromethane, N,N-dimethylacetamide (DMA), Acetonitrile, tetrahydrofuran (THF), 2-Methyl tetrahydrofuran N-butylpyrrolidinone and mixtures thereof.

7. The method as claimed in claims 1 to 4, wherein the coupling reaction of each amino acid is between 2 to 6 hours at temperature ranging from about 20°C to about 35°C.

8. The method as claimed in claims 1 to 3, wherein the peptide is cleaved from resin using Trifluoro acetic acid and Dichloromethane.

9. The method as claimed in claims 1 to 4, wherein de-protection of the peptides is carried out with a mixture of reagents selected from the group comprising of Trifluoro acetic acid, Triisopropylsilane, Diothreitol, Ethanedithiol, ammonium iodide, 2,2'-(ethylenedioxy)diethane and acetyl cysteine, DMS, phenol, cresol and thiocresol, and mixtures thereof.

10. The method as claimed in claim 1, wherein the cyclization takes place in presence of granular charcoal as catalyst.