Green solvent-based mixed-solvent strategy for the synthesis of peptide

Abstract

The present invention relates to a mixed green solvent based process for the preparation of peptide. More specifically, the present invention relates to a process of the preparation of Octreotide using greener solvent. The process of present invention also focused on minimizing solvent consumption, especially DMF, trifluoroacetic acid (TFA) and ethers. The process of present invention also substitutes acetonitrile with methanol in the chromatographic purification steps. The process of present invention only addresses environmental concerns associated with DMF usage but also maintains the efficiency and effectiveness of the Octreotide synthesis process.

Description

GREEN SOLVENT-BASED MIXED-SOLVENT STRATEGY FOR THE SYNTHESIS OF PEPTIDETECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to process chemistry. Particularly, the present invention relates to a mixed green solvent based method for the preparation of peptide. More specifically, the present invention relates to a process of the preparation of Octreotide using greener solvent.BACKGROUND OF THE INVENTION

[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Peptide synthesis can be achieved through two main approaches: liquid phase peptide synthesis (LPPS) and solid phase peptide synthesis (SPPS) chemistry. While LPPS is preferable for synthesizing small peptide molecules with fewer than 10 amino acids, it involves multiple steps, with each step requiring isolation of the target product at the highest possible purity level. However, this method contributes significantly to waste generation due to solvent and reagent consumption in each step.

[0004] In contrast, SPPS, which involves carrying out all reactions in a single reactor until the completion of the target sequence, is the conventional method for peptide synthesis. Although SPPS consumes less solvent compared to LPPS, the amount is still substantial, especially considering its environmental impact. Common solvents used in SPPS include dimethyl formamide (DMF), N-methyl pyrrolidone (NMP), and dichloromethane (DCM or MDC).

[0005] The environmental footprint of SPPS-based peptide synthesis is negative due to the large amount of solvent used. Consequently, efforts have been made to make peptide synthesis more environmentally friendly, focusing on solvent substitution, recycling, reduction, and alternative synthetic methods. Current regulations, such as those outlined by REACH, are moving toward restricting the use of classic SPPS solvents like DMF, DCM, NMP, and DMAc.

[0006] To address this, alternative solvents for SPPS must be identified promptly to avoid disruptions in industrial production of therapeutic peptides. DMF, a commonly used solvent in SPPS, is highly toxic, prompting the search for other substitute solvents.

[0007] However, finding a single solvent that can efficiently fulfill all the necessary functions in SPPS, including swelling of the resins, coupling, deprotection, and washing steps, has proven challenging. Consequently, many green solvents have been excluded from consideration. Instead, the use of solvent mixtures comprising two different green solvents has emerged as a promising alternative.

[0008] A recent study has focused on replacing DMF in SPPS with mixtures of two green solvents (GM-SPPS), including cyrene (dihydrolevoglucosenone), sulfolane, or anisole with dimethyl or diethyl carbonate. These mixtures were evaluated for their efficiency in resin swelling, couplings, deprotections, and washing processes, with the aim of developing a more sustainable approach to peptide synthesis.

[0009] Fmoc-protected amino acids are mostly used for SPPS. These derivatives of amino acids are highly water insoluble. On the other hand, coupling reaction in peptide bond formation requires highly polar aprotic solvent.

[0010] Vincent Martin, et., al (RSC Adv., 10, 2020, 42457-42492) titled 'Greening the Synthesis of Peptide Therapeutics: An Industrial Perspective' have effectively explained the need for green techniques in their review article on green chemistry. It provides insights into solvent selection based on process requirements, along with a comparison of solvents for GSPPS.

[0011] In the research article titled "In situ Fmoc removal - a sustainable solid-phase peptide synthesis approach" (Ashish Kumar A. S., et. al., Green chemistry, 24, 2022, 4887- 4896), explained the process and discuss the results of in situ Fmoc removal during SPPS. They synthesized the tripeptide GFL-NH2 and the pentapeptide YGGFL-NH2 using various protocols. The purity of the product and the impurity profile of Des and Endo impurities were studied at various stages.

[0012] Ashish Kumar, et. al., studied the use of y-valerolactone (GVL) and N- formylmorpholine (NFM) as substitutes for DMF in the synthesis of pentapeptides and decapeptides (Microwave-Assisted Green Solid-Phase Peptide Synthesis Using y- Valerolactone (GVL), Tetrahedron Letters, 58(3 A). 2017, 2986-2988). They investigated the swelling properties of polystyrene (PS) and ChemMatrix-based resins in different solvents such as 2-MeTHF, CPME, and IPA. PS resin exhibited poor swelling in these solvents,whereas swelling was satisfactory in GVL and NFM. Additionally, the solubility of amino acids and coupling reagents was found to be good in GVL and NFM.

[0013] Lucia Ferrazzano et. al., have investigated green solvent mixtures for solid-phase peptide synthesis (SPPS) (ACS Sustainable Chemistry & Engineering, 7(15), 2019, 12867- 12877), aiming to achieve a highly efficient synthesis of pharmaceutical-grade peptides without the use of dimethylformamide (DMF). They prepared and studied different solvent mixtures to evaluate their swelling properties on various resins. The main solvents selected for combination were anisole, cyrene, diethyl carbonate, dimethyl carbonate, and sulfolane. After conducting experiments, the best mixture obtained was anisole: dimethyl carbonate in ratio 70:30, based on the swelling properties of the resins.

[0014] Jonathan M. Collins et. al., has presented process for SPPS which completely remove solvent washing step (Nature communications, 14, 2023, 8168-8179). Key step of this process is effective for removal of Fomc- deprotection reagents by evaporation. Both research and commercial scale show comparative results. Impact on process is saving of around 95% solvents and reduction in synthesis process time.

[0015] Tobias M. Postma and Fernando Albericio have demonstrated that N-Chlorosuccinimide (NCS) (Organic Letters, 15(3), 2013 616-619) can serve as an efficient reagent for on-resin disulfide formation in solid-phase peptide synthesis (SPPS). On-resin oxidation can be achieved using NCS in SPPS, and these reactions are particularly useful for forming multiple disulfide bonds using various protecting groups for cysteine residues such as Mmt, Trt, and Tmp. Additionally, NCS is compatible with peptides containing methionine and tryptophan residues.

[0016] In a study by Mahama Alhassan et al. (Green Chemistry, (4), 2020, 996-1018), a greener approach for cleaving protected peptides from 2-chlorotrityl chloride (CTC) resin was developed to eliminate the use of hazardous dichloromethane (DCM). Few efforts have focused on greening the cleavage step of peptide synthesis, so the researchers conducted several experiments to identify greener alternatives to DCM for this process.

[0017] Liquid chromatography is the preferred technique for purification, typically employing reversed-phase, ion-exchange, or hydrophilic interaction chromatography, depending on the peptide's hydrophobic / hydrophilic properties and charge. Mixed-mode stationary phases, which combine reversed-phase and ion-exchange characteristics, are also gaining attention fortheir versatility.

[0018] Continuous chromatographic techniques, particularly those based on countercurrent chromatography concepts like MCSGP, are gaining traction due to improvements inproduct quality, increased productivity, and process simplification through automation. However, further theoretical studies focusing on process modeling are needed to optimize procedures efficiently.

[0019] Despite significant progress in purification processes, challenges remain, particularly regarding regulatory compliance to ensure adherence to Good Manufacturing Practice (GMP) standards. Overcoming these obstacles will be crucial for advancing purification techniques and ensuring the quality and safety of therapeutic peptides.

[0020] The solid-phase peptide synthesis (SPPS) method involves several steps. Initially, the process begins with attaching the first N-terminal protected amino acid, typically Fmoc- Amino acid, onto resin. The Fmoc group is then removed using 20% piperidine (pip.) in DMF. Subsequently, another Fmoc-Amino Acid is coupled using coupling reagents such as di-isopropyl carbodiimides (DIC) and N-hydroxy benzotriazole (HOBt) as additives in DMF solvent. To ensure efficient coupling, a large excess of molar concentration is utilized.

[0021] After each reaction, excess reagents are eliminated by filtration, as residual reagents can impact subsequent reactions. To prevent this, the peptidyl resin is washed with DMF. Typically, 5 to 10 volumes of solvent with respect to resin are required per operation. In a conventional synthesis protocol, the following steps are followed: coupling (5 to 10V), washings (3 X 10V), de-protections (2 X 10V), and washings after Fmoc- removal (5 X 10V). Each coupling cycle necessitates approximately 100 to 120 volumes of solvent. For instance, to synthesize a peptide consisting of 8 to 10 amino acids on a 10-gram scale, around 8000 to 12000 milliliters of solvent are required. Upon completion of the synthesis of the peptidyl resin, the crude peptide needs to be isolated from the resin using trifluoroacetic acid (TFA). Approximately 8 to 12 volumes of TFA are required for this process.

[0022] After the cleavage reactions are completed, the resin is removed by filtration, and the filtrate containing the crude peptide is transferred to ethers (100 to 120V) for precipitation. The crude peptide obtained is dried under vacuum at a moderately elevated temperature. Subsequently, the crude peptide is subjected to further reactions such as oxidation or other modifications.

[0023] The reaction mass is then subjected to chromatographic purification followed by lyophilization to obtain pure material. However, in conventional peptide synthesis using SPPS, large quantities of solvents are required: 1000 to 1200 volumes for synthesis and 120 to 200 volumes for the cleavage process. Additionally, the oxidation reaction and chromatographic purification further added to solvent utilization.

[0024] In purification, close-eluting impurities are removed using reverse-phase chromatography, where a large volume of acetonitrile is consumed. Overall, peptide synthesis using conventional SPPS has a significant environmental impact, contributing to negative carbon footprints.

[0025] In efforts to adopt more environmentally friendly practices for peptide production via solid-phase peptide synthesis (SPPS), recent research has primarily focused on a smaller scale, with limited implementation in commercial settings due to industry bottlenecks. Transitioning to greener methodologies poses challenges, particularly regarding potential changes in raw materials that could introduce new impurity profiles into the final product.

[0026] There is, therefore, a need in the art to provide an environmentally friendly i.e., greener method of preparation of peptides that consumes less hazardous solvents, has less carbon footprint and is industrially feasible.OBJECTIVES OF THE INVENTION

[0027] An objective of the present invention is to provide a process for preparation of peptide that can satisfy the existing need and can overcome one or more deficiencies found in the existing in the art.

[0028] Another objective of the present invention is to provide a process for preparing peptide that can be adapted as an economical and industrially scalable process.

[0029] Yet another objective of the present invention is to provide a process for preparing peptide that utilizes greener solvent and achieves substantially pure peptides.

[0030] Another object of the present disclosure is to provide a chemical synthetic process for preparing peptide.SUMMARY OF THE INVENTION

[0031] The present invention provides a process of preparation of peptide wherein the reaction conditions and processes are suitable for industrial-scale production without significant alterations to major raw materials. Accordingly, the present invention is all about replacement of hazardous solvents from synthesis, but complete removal of these solvents is not practically possible due to lack of commercial supply of green solvents. Use of green solvents will increase cost of raw materials. Rather than entirely eliminating N, N- dimethylformamide (DMF) from the synthesis, the process of present invention is conducted in greener solvents such as y-valerolactone (GVL) while using DMF for washings, thus introducing only GVL as an additional raw material. The process of present invention alsofocused on minimizing solvent consumption, especially trifluoroacetic acid (TFA) and ethers. In certain processes, ethers were completely phased out. The process of present invention substitutes acetonitrile with methanol in the chromatographic purification steps. This innovative approach was exemplified by the manufacturing process development for cyclic octapeptide, Octreotide. Octreotide serves as an ideal model due to its cyclic structure with a disulfide bond, alcohol at the C-terminal, and a composition of hydrophobic and D-amino acids, making it a paradigmatic example of a complex peptide.

[0032] In an aspect, the present invention relates to a process for preparation of linear or cyclic peptide with 1 to 40 amino acids comprising the steps of:(a) coupling Fmoc-N-protected amino acid to a resin capped amino acid using coupling reagent to obtain a dipeptide resin and avoiding washings by any solvents after coupling reactions;(b) selectively deprotecting Fmoc of N-protected amino acid at N terminal of the dipeptide resin using deprotecting reagent and greener solvent to obtain free amino dipeptide resin, and washing of resin using IPA, and DMF containing HOBt or Oxyma;(c) coupling a carboxyl terminus of Fmoc-N-protected amino acid to the free amino dipeptide resin of step (b) in the presence of a coupling reagent and greener solvent, and avoiding washings by any solvents after coupling reactions;(d) repeating step (b) & step (c) to form a peptidyl resin;(e) deprotecting Fmoc of N-protected amino acid at N-terminal of the peptidyl resin;(f) cleaving the peptide from the peptidyl resin to obtain linear crude peptide;(g) optionally oxidizing the linear crude peptide to obtain a crude cyclic peptide; and(h) optionally purifying the crude cyclic peptide to obtain pure cyclic peptide; wherein: the preparation process comprises a combination of a greener solvent as coupling solvent and conventional solvents as washing for each cycle of repeating of step (b) and (c) to form a peptidyl resin.

[0033] In another aspect of the present invention, the greener solvent used in the process of present invention is selected from the group consisting of selected from the group of solvents consisting of gamma-valerolactone (GVL), anisole, methanol, ethanol, isopropanol, 1,4-dioxane, 2-methyl tetrahydrofuran, N-methyl-2-pyrrolidinone (NMP), ethyl acetate, acetonitrile, acetone, and combination thereof.

[0034] In another aspect of the present invention, the process further comprises the step of lyophilizing the crude peptide or pure peptide to obtain solid peptide.

[0035] In another aspect, the present invention relates to a process for preparing an octreotide comprising the steps of:(a) coupling Fmoc-Cys(Acm)-OH or Fmoc-Cys(Trt)-OH to a Thr(tBu)-OL-2-CTC resin using GVL as a coupling solvent to obtain a dipeptide resin, Fmoc-Cys(Acm)- Thr(tBu)-OL-CTC-Resin or Fmoc-Cys(Trt)-Thr(tBu)-OL-CTC-Resin;(b) selectively deprotecting Fmoc of Fmoc-Cys(Acm)-Thr(tBu)-OL-CTC-resin or Fmoc-Cys(Trt)-Thr(tBu)-OL-CTC-resin using deprotecting reagent, mixture of piperidine and DBU in greener solvent, gamma-valerolactone (GVL) to obtain dipeptide resin, H-Cys(Acm)-Thr(tBu)-OL-CTC-resin or H-Cys(Trt)-Thr(tBu)-OL- CTC-resin; washing of the dipeptide resin obtained using IPA, & DMF containing HOBt or Oxyma;(c) sequential coupling a carboxyl terminus of other Fmoc-N-protected amino acid of the octreotide to the free amino dipeptide resin of step (b) in the presence of a coupling reagent and gamma-valerolactone (GVL), as a greener solvent;(d) repeating step (b) & step (c) to form a peptidyl resin, Boc-D-Phe-Cys(Acm)-Phe-D- Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr(tBu)-OL-CTC-resin or Boc-D-Phe- Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-OL-CTC-resin;(e) cleaving the peptide from the peptidyl resin to obtain linear crude peptide using 30 to 70% TFA and DCM solution by directly quenching the reaction mixture in water, and subsequently performing an in-situ oxidation process to obtain a crude octreotide; and(f) optionally purifying the crude octreotide to obtain pure octreotide by reverse phase chromatography using methanol ranging from 10 to 60% in aqueous mobile phase containing 0.1 to 1.0% acetic acid. wherein: the process avoids washings by any solvents after coupling reactions in each cycle of repeating of steps (b) and (c) to form a peptidyl resin.

[0036] In another aspect of the present invention, the process of preparation of octreotide further comprises the step of lyophilizing the crude octreotide or pure octreotide to obtain solid octreotide.

[0037] Various objectives, features, aspects, and advantages of the inventive subject matter will become more apparent from the following detailed description of preferredembodiments.BRIEF DESCRIPTION OF THE FIGURES

[0038] Figure 1 is the flow diagram of process as exemplified in the example 1 of the present invention.

[0039] Figure 2 is the flow diagram of process as exemplified in the example 2 of the present invention.

[0040] Figure 3 is the flow diagram of process as exemplified in the example 3 of the present invention.

[0041] Figure 4 depicts route of synthesis of the process exemplified in the example 1 of the present invention.

[0042] Figure 5 depicts route of synthesis of the process exemplified in the example 3 of the present invention.DETAILED DESCRIPTION OF THE INVENTION

[0043] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0044] The description that follows, and the embodiments described herein, are provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification. All patents, published patent applications and publications cited herein are incorporated by reference as if set forth fully herein. It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0046] Wherever a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value inthat stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention.

[0047] The term “resin capped amino acid” as used herein refers to the amino acid capped with the resin. For example, H-Thr(tBu)-OL-2CTC.

[0048] The term “peptidyl resin” as used herein refers to the resin capped with linear amino acid sequence of the peptide of interest. For example, in case of octreotide then the peptidyl resin is Boc-D-Phe-Cys(Acm)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)- Thr(tBu)-OL-CTC-resin or Boc-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)- Cys(Trt)-Thr(tBu)-OL-CTC-resin.

[0049] The term “peptide of interest” refers to peptides that are formed by the process of the present invention.

[0050] Octreotide is the acetate salt of a cyclic octapeptide. It is a long -acting octapeptide with pharmacologic properties mimicking those of the natural hormone somatostatin. The chemical structure of Octreotide is given below.

[0051] In one embodiment, the present invention relates to a process for preparation of linear or cyclic peptide with 1 to 40 amino acids, wherein the process comprises the steps of:(a) coupling Fmoc-N-protected amino acid to a resin capped amino acid to obtain a dipeptide resin avoiding washings by any solvents after coupling reactions;(b) selectively deprotecting Fmoc of N-protected amino acid at N terminal of the dipeptide resin using deprotecting reagent and greener solvent to obtain free amino dipeptide resin, and washing of resin using IPA, and DMF containing HOBt orOxyma;(c) coupling a carboxyl terminus of Fmoc-N-protected amino acid to the free amino dipeptide resin of step (b) in the presence of a coupling reagent and greener solvent and avoiding washings by any solvents after coupling reactions;(d) repeating step (b) & step (c) to form a peptidyl resin;(e) deprotecting Fmoc of N-protected amino acid at N terminal of the peptidyl resin;(f) cleaving the peptide from the peptidyl resin to obtain linear crude peptide;(g) optionally oxidizing the linear crude peptide to obtain a crude cyclic peptide; and(h) optionally purifying the crude cyclic peptide to obtain pure cyclic peptide; wherein the preparation process comprises a combination of a greener solvent as coupling solvent and conventional solvents as washing solution for each cycle of step b & step c.

[0052] According to the present invention, the repeating step (b) & (c) includes the deprotection of Fmoc followed by the coupling of other amino acid as given in the route of synthesis scheme (Figures 4 and 5). The repeating cycle can be carried out using different protected amino acids up to the formation of peptidyl resin of peptide of interest.

[0053] In another embodiment of the present invention, the process further comprises the step of lyophilising the crude peptide or pure peptide to obtain solid peptide.

[0054] In yet another embodiment of the present invention, the linear or cyclic peptide with 1 to 40 amino acids is selected from octreotide, liraglutide, semaglutide, teriparatide, albiglutide, abaloparatide, dulaglutide, and exenatide.

[0055] In yet another embodiment of the present invention, the greener solvent is selected from the group of solvents consisting of gamma-valerolactone (GVL), anisole, methanol, ethanol, isopropanol, 1,4-dioxane, 2-methyl tetrahydrofuran, N-methyl-2-pyrrolidinone (NMP), ethyl acetate, acetonitrile, acetone, and combination thereof.

[0056] In another embodiment of the present invention, the coupling agent is selected from the group consisting of 1 -hydroxybenzotriazole (HOBt), N,N-diisopropylcarbodiimide (DIC), hexafluorophosphate benzotriazole tetramethyl uronium (HBTU), N,N- diisopropylethylamine (DIPEA), benzotriazol-l-yl-oxy- tris(dimethyl-amino)-phosphonium hexafluorophosphate (BOP), l-[Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5- b]pyridinium-3 -oxidehexa fluorophosphate (HCTU), hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU), 3-(Diethoxyphosphoryloxy)-l,2,3-benzotriazin-4(3H)-one (DEPBT), 2-chloro-4,6-dimethoxy-l,3,5-triazine (CDMT), 2-(lH-7-Azabenzotriazol-l-yl)- 1,1, 3, 3 -tetra methyluronium tetrafluoroborate (TBTU) and combination thereof.

[0057] In another embodiment of the present invention, the resin is selected from the group consisting of 2-Chlorotrityl chloride (2-CTC), Sasrin™, TentaGel® S, TentaGel® TGA, Rink, Wang, and AmphiSpheres™

[0058] In another embodiment of the present invention, the resin deprotecting reagent in coupling reaction with greener solvent is a mixture piperidine with at least 1% concentration and DBU with at least 1% concentration in greener solvent.

[0059] In another embodiment of the present invention, the cleavage step of crude peptide from resin comprises the steps of: a) addition of a solvent mixture comprising dichloromethane (DCM) and trifluoroacetic acid (TFA) in a ratio ranging from at least 10% of TFA (v / v); b) precipitation of reaction mixture from step a) using ether; and c) optionally, direct quenching of the reaction mixture using water.

[0060] In another embodiment of the present invention, the oxidation step of the crude peptide containing two, four, six cysteine (Cys) residues is conducted by direct quenching with water and avoids use of ether for precipitation of crude peptide.

[0061] In another embodiment of the present invention, the purification step of crude peptide to obtain pure peptide is carried out using alcohol containing 1 to 4 carbon atoms in a mobile phase.

[0062] In another embodiment, the present invention relates to a process for preparing an octreotide, wherein the process comprises the step of:(a) coupling Fmoc-Cys(Acm)-OH or Fmoc-Cys(Trt)-OH to a Thr(tBu)-OL-2-CTC resin using GVL as a coupling solvent to obtain a dipeptide resin, Fmoc-Cys(Acm)- Thr(tBu)-OL-CTC-Resin or Fmoc-Cys(Trt)-Thr(tBu)-OL-CTC-Resin;(b) selectively deprotecting Fmoc of Fmoc-Cys(Acm)-Thr(tBu)-OL-CTC-resin or Fmoc-Cys(Trt)-Thr(tBu)-OL-CTC-resin using deprotecting reagent, mixture of piperidine and DBU in greener solvent, gamma-valerolactone (GVL) to obtain dipeptide resin, H-Cys(Acm)-Thr(tBu)-OL-CTC-resin or H-Cys(Trt)-Thr(tBu)-OL- CTC-resin; washing of the dipeptide resin obtained using IPA, & DMF containing HOBt or Oxyma;(c) sequential coupling a carboxyl terminus of other Fmoc-N-protected amino acid of the octreotide to the free amino dipeptide resin of step (b) in the presence of a coupling reagent and gamma-valerolactone (GVL), as a greener solvent;(d) repeating step (b) & step (c) to form a peptidyl resin, Boc-D-Phe-Cys(Acm)-Phe-D- Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr(tBu)-OL-CTC-resin or Boc-D-Phe- Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr(tBu)-OL-CTC-resin;(e) cleaving the peptide from the peptidyl resin to obtain linear crude peptide using 30 to 70% TFA and DCM solution by directly quenching the reaction mixture in water, and subsequently performing an in-situ oxidation process to obtain a crude octreotide; and(f) optionally purifying the crude octreotide to obtain pure octreotide by reverse phase chromatography using methanol ranging from 10 to 60% in aqueous mobile phase containing 0.1 to 1.0% acetic acid. wherein: the process avoids washings by any solvents after coupling reactions in each cycle, i.e., repeating step (b) and (c) to form a peptidyl resin.

[0063] In another embodiment of the present invention, further comprising the step of lyophilising the crude octreotide or pure octreotide to obtain solid octreotide.

[0064] In another embodiment, the peptide formed from the process of present invention can be cleaved from the resin using chemicals selected from but not limited to difluoroacetic acid, trifluoroacetic acid and the like.

[0065] In another embodiment, the processes well known in art can carry out the purification process of any given peptide. The purification process for the peptides can be selected from but not limited to preparative reverse phase HPLC, ion exchange chromatography, size exclusion chromatography affinity chromatography and the like.

[0066] In one embodiment, the present invention provides a process for synthesis of any given peptide thereof, wherein after completion of coupling reactions avoiding washing process, and in situ removal of N-terminal protecting group with piperidine-DMF solution or Piperidine-DBU-DMF solution or Piperidine-DBU-GVL solution, followed by washing with alcohols like methanol or ethanol or 1 -propanol or 2-propanol or any isomer of butanol or mixture of any 1 to 4 carbon containing alcohols. Washing of peptidyl resin with 1 to 10% composition of HOBt.H2O in DMF or GVL, or 1 to 10% composition of Oxyma in DMF or GVL or combination of any of the above.

[0067] In one embodiment, the present invention provides a process for cleavage of any given peptide thereof, wherein after completion of synthesis of peptidyl resin, crude peptide is isolated from resin using 1% to 70% Trifluoroacetic acid (TFA) in DCM, followed byprecipitation in ether including diethyl ether, di isopropyl ether, methyl tertiary butyl ether or quenching in water and subjected for purification by any chromatographic techniques.

[0068] In one embodiment, the present invention provides a process for purification of any given peptide thereof, wherein the chromatographic purification by reverse phase HPLC purification with an eluting mobile phase comprising 1 to 4 carbon containing alcohols in combination with water and acetonitrile as mobile phase and any salt of ionic form for buffer containing acetate, trifluoroacetate, formate, carbonate, phosphate, sulphate and halide anions at a linear gradient from 5% to 60%, or isocratic flow ranging from 1 % to 50% concentration of organic part in aqueous phase. Collecting and combining pooled fractions from HPLC purification, subjecting to concentration by HPLC or TFF or by nano filtration techniques. The concentrated mass was obtained and then subjected for lyophilization to provide the purified peptide.

[0069] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.EXAMPLES

[0070] The present invention is further explained in the form of following examples. However, it is to be understood that the following examples are merely illustrative and are not to be taken as limitations upon the scope of the invention.

[0071] Example 1: Synthesis of Octreotide peptidyl resin using Cys(ACM) and DIC- HOBT.H2O and GVL as solvent, De protection solution 2% Piperindine and 2% DBU in GVL.

[0072] Synthesis of octreotide was carried out on 2 gm scale of pre-loaded coupled Thr(tBu)-OL-2-CTC resin. The batch was assigned as P26. Initially resin was swelled using DMF, then first amino acid, Fmoc-Cys(Acm)-OH coupled using DIC- HOBt.H2O as coupling reagent in 5V GVL as coupling solvent. After completion of coupling, reaction solution was drained by applying vacuum. Deprotection solution solution 2% Piperindine and 2% DBU in GVL directly charged to reactor by avoiding washing to the resin after coupling process. Two deprotection wash were given, followed by 2 wash of 10V IPA, 2 wash of 10V 2 eq. HOBt.H2O in DMF washings. Same process was repeated for rest of sequential couplings ofamino acids. Peptidyl resin then subjected for cleavage was conducted using 6V DCM and 6 V TFA-cocktail for 3 hrs and the resin was fdtered off. TFA and DCM distilled out from filtrate under reduce pressure. The concentrated filtrate directly quenched in precooled water and subjected for oxidation followed by purification using aqueous Acetic acid buffer and Methanol. Figure 1 shows the flow diagram of the process of example 1. Figure 4 shows the route of synthesis of Octreotide.Table 1: Raw materialsProcess:1. Arranged clean and dry 100 mL SPPS reactor.2. Charged 2.0 gm H-Thr(tBu)-OL-CTC resin in reactor.3. Added 20 ml (10 V) DCM in reactor, allow to stir resin for 30 min and drain the solvent. Collect in spent.Coupling process for Fmoc-AA-OH4. In a clean beaker dissolve 1.32 mole (1.5 mole equivalent) Fmoc-AA-OH, 0.54 gm (2 mole equivalent) HOBt.H2O in 10.0 ml (5 V) GVL.5. Allow stirring until formation of a clear solution 2 to 5 min). Charge 0.55 ml (2 mole equivalent) DIPC in it.6. Transferred the coupling mixture in reactor containing resin.7. Allow to stir under nitrogen bubbling at 25 to 35 °C for 110 to 120 min.8. Checked Kaiser test, it showed colour-less bids of resin. Reaction was completed.9. Drain the reaction mass. Suck dry well.Removal of Fmoc10. Charged 20 ml (10 V)2% Piperindine and 2% DBU in GVL in reactor. Allow to stir under nitrogen bubbling at 25 to 35 °C for 10 to 12 min. Drain the de protecting solution.11. Repeat above operation.12. Charged 20 ml (10 V) IPA in reactor, allow to stir under nitrogen bubbling at 25 to 35 °C for 2 to 5 min. Drain the solution.13. Checked Kaiser test, it showed dark blue coloured bids of resin. Reaction was completed.14. Repeated above IPA operation.15. Charged 20 ml (10 V) of two eq. HOBt. H2O in DMF in reactor, allow to stir under nitrogen bubbling at 25 to 35 °C for 2 to 5 min. Drain the solution.16. Repeated above operation of DMF wash.After completion of Synthesis below process followed for drying ofpeptidyl resin17. Charged 20 ml (10 V) DCM in reactor, allow to stir under nitrogen bubbling at 25 to 35 °C for 2 to 5 min. Drain the solution.18. Repeat above operation of DCM wash.19. Charged 20 ml (10 V) IPA in reactor, allow to stir under nitrogen bubbling at 25 to 35 °C for 2 to 5 min. Drain the solution.20. Repeat above operation of IPA wash21. Charged 20 ml (10 V) MTBE in reactor, allow to stir under nitrogen bubbling at 25 to 35 °C for 2 to 5 min. Drain the solution.22. Repeat above operation of MTBE wash23. Suck dry well. Unload the material, transferred the peptidyl resin in Vacuum tray dryer for drying under vacuum at 45 to 50 °C for 6 to 8 hrs.Dry weight of peptidyl resin: 4.86 gmCleavage of Peptide from Resin24. Charged 4.86 gm Peptidyl resin in clean and dry 100 ml RBF25. Charged 24 ml (6 V) DCM in it and allowed to stir at 25 to 35 °C for 25 to 30 min.26. Prepared cocktail in another a clean RBF by adding add one by one TFA 28.8 ml (5.4 V), TIPS 1.44 ml (0.3 V), water 1.44 ml (0.3 V).27. Charged above cocktail in RBF containing Peptidyl resin. Allowed to stir at 25 to 35 °C for 180 to 200 min28. Filtered of resin and collected fdtrate in separate flask.29. Distilled of DCM and TFA under reduced pressure at below 40°C.30. Charged fdtrate in precooled (5 to 15 °C) 2000 ml (-500 V) water.Oxidation of linear peptide to get crude Octreotide31. Checked pH of linear solution, pH: 1.45.32. Removed before oxidation sample for Analysis.33. Charged 2 gm Iodine dissolved in 50 ml acetonitrile in reaction mass. A dark brown coloured solution was observed. Allowed to stir at 25 to 35 °C for 180 to 200 min. Taken out samples after every 60-minute interval.34. After completion of reaction, quenched the reaction mass using 0.1 M Ascorbic acid solution till the reaction mass transferred to colourless from dark brown.35. Adjusted pH of solution to 6 to 7 using 10 N NaOH water solution. pH- 6.2. (12.5 ml)36. Filtered the reaction mass thorough Ip, 0.8 p and 0.45 p in sequence. Transferred the filtered mass for purification.Results: Reaction monitored on HPLCTable 2 Result of crude Octreotide oxidation experiment P26Purification of crude OctreotideP26R1 Column Specification: - 21.2X250 mm Silica Specification: _YMC Triart, Cl 8, lOp, 120A U.V .= 220 &280nmTable 3 Gradient table for purification of crude Octreotide experiment P26R137. Charged 2.0 ml Acetic acid in glass bottle containing 2000 ml water, filter it through 0.45p filter. Labelled it as ‘Buffer A’. Charged 1000 ml Methanol in another glass bottle, labelled it as ‘Buffer B’.38. Purged all buffer lines with respective buffers. Column equilibrated with 5% buffer B with 95% Buffer A for 3 column volume (CV) at 20 ml / min flow.39. Crude octreotide solution obtained after oxidation process was loaded on column manually at flow rate 20 ml / min.40. After completion of loading, buffer A passed though column for 1 CV or up base line gets stable.41. Started gradient program as mentioned above table. Chromatographic elution was monitored at UV wavelength 220 and 280 nm. Material collected by observing UV in fractions.42. After completion of elution gradient program stopped. Column washed with 20%: 80% Buffer A: B for 3 CV.43. Samples from collected fractions were submitted for analysis.Table 4 Result of Purification fractions of P26R144. Fraction 3 to fraction 5 were mixed together, desired purity not meet so F3 to F5 taken for re-purification.P26R2In P26R1 desired purity not meet, to separate impurity re-purification done by following processTable 5 Raw materials for purification side fraction of Octreotide experiment P26R2Table 6 Gradient table for purification of crude Octreotide experiment P26R245. Charged 4 ml Trifluroacetic acid in glass bottle containing 2000 ml water, filter it through 0.45p filter. Labelled it as ‘Buffer A’. Charged 1000 ml Acetonitrile in another glass bottle, labelled it as ‘Buffer B’46. Purged all buffer lines with respective buffers. Column equilibrated with 5% buffer B with 95%Buffer A for 3 column volume (CV) at 15 ml / min flow.47. Pooled fraction of P26R1 F3-F5 mixed together and diluted with 1: 1 with water loaded on column manually at flow rate 14 ml / min.48. After completion of loading, buffer A passed though column for 1 CV or up base line gets stable.49. Started gradient program as mentioned above table. Chromatographic elution was monitored at UV wavelengths 230 and 280 nm. Material collected by observing UV in fractions.50. After completion of elution gradient program stopped. Column washed with 20%: 80% Buffer A: B for 3 CV.51. Samples from collected fractions were submitted for analysis.Table 7 Result of Purification fractions of P26R252. Fractions 3 to fractions 13 were mixed together, taken for de-salting.P26R3 Table 8 Raw materials for purification pure fraction of Octreotide experiment P26R3Table 9 Gradient table for purification of fraction of Octreotide experiment P26R353. Charged 2.0 ml Acetic acid in glass bottle containing 2000 ml water, filter it through 0.45p fdter. Labelled it as ‘Buffer A’. Charged 1000 ml Acetonitrile in another glass bottle, labelled it as ‘Buffer B’. Charged 3.85gm Ammonium acetate in glass bottle containing 500 ml fdter it through 0.45 i fdter. Labelled it as ‘Buffer C’. Charged 500 ml water in glass bottle, labelled it as ‘Buffer D’.54. Purged all buffer lines with respective buffers. Column equilibrated with 5% buffer B with 95% Buffer A for 3 column volume (CV) at 15 ml / min flow.55. Pooled fraction of P26R2 F3-F5 mixed together and diluted with 1: 1 with water loaded on column manually at flow rate 16 ml / min.56. After completion of loading, buffer D passed though column for 3 CV or up base line gets stable at flow rate 16 ml / min.57. For salt exchange buffer C passed though column for 3 CV at flow rate 16 ml / min.58. After that buffer D passed though column for 3 CV at flow rate 16 ml / min.59. Started gradient program as mentioned above table. Chromatographic elution was monitored at UV wavelength 220 and 280 nm. Material collected by observing UV in fractions.60. After completion of elution gradient program stopped. Column washed with 20%: 80% Buffer A: B for 3 CV.61. Samples from collected fractions were submitted for analysis.Table 10 Result of Purification fractions of P26R362. Collected single fraction, acetonitrile was removed by distillation under vacuum using rotary evaporator.63. The concentrated solution was transferred in lyophilisation tray.64. Kept tray in lyophilisation recipe as follows.

[0073] After completion of drying, material was unloaded in HDPE container and sample was submitted for analysis. Dry weight: 620 mg HPLC Purity: 97.95% SMI: 1.72%.

[0074] Example 2: Synthesis of Octreotide using Cys(Acm) and DIC- HOBt.H2O and GVL as solvent on 5 gm scale

[0075] Synthesis of octreotide was planned on 5 gm scale of pre-loaded coupled Thr(tBu)-OL-2-CTC resin. The batch was assigned as P27. The synthesis process flowed same as mentioned for P26. Peptidyl resin then subjected for cleavage by two different ways. In first, cleavage was conducted using 6V DCM and 6 V TFA-cocktail for 3 hrs. Resin was filtered off and filtrate was concentrated under vacuum to remove DCM and TFA. Precooled diisopropyl ether was used for precipitation. Linear octreotide precipitate and dried under vacuum at elevated temperature. Same material then used for oxidation using Iodine. The crude octreotide obtained then purified by RP HPLC using 0.1% Acetic acid in water and methanol instead of acetonitrile. In second cleavage approach, filtrate was concentrated under vacuum to remove DCM and TFA then cocktail syrup quenched in precooled water and subjected for oxidation followed by purification. Purified fractions then freeze dried to get powder form of octreotide acetate. Figure 2 shows the flow diagram of the experimental steps of example 2.Process: Same as followed for P26 Op. no. 1 to 23 Dry weight of peptidyl resin: 12.5 gm Cleavage of Peptide from Resin: P27ATable 11 raw materials for Cleavage of Peptide from Resin experiment P27A1. Charged 2 gm Peptidyl resin in clean and dry 100 ml RBF2. Charged 12 ml DCM in it and allowed to stir at 25 to 35 °C for 25 to 30 min.3. Prepared cocktail in another a clean RBF by adding add one by one TFA 10.8 ml, TIPS 0.6 ml, water 0.6 ml.4. Charged above cocktail in RBF containing Peptidyl resin. Allowed to stir at 25 to 35 °C for 180 to 200 min5. Filtered of resin and collected filtrate in separate flask.6. Charged filtrate in precooled (5 to 15 °C) 1000ml water.P27BTable 12 raw materials for Cleavage of Peptide from Resin experiment P27B7. Charged 2.0 gm Peptidyl resin in clean and dry 100 ml RBF8. Charged 12 ml DCM in it and allowed to stir at 25 to 35 °C for 25 to 30 min.9. Prepared cocktail in another a clean RBF by adding add one by one TFA 10.8 ml, TIPS 0.6 ml, DODT 0.6 ml.10. Charged above cocktail in RBF containing Peptidyl resin. Allowed to stir at 25 to 35 °C for 180 to 200 min11. Filtered of resin and collected filtrate in separate flask.12. Distilled off TFA-DCM under vacuum using Rotary evaporator.13. Charged precooled (0 to 5 °C) 12 ml Diisopropyl ether in reaction mass.14. Allowed to stir for 10 to 20 mins at cooled temperature (below 10°C)15. Filter the precipitate using Buckner funnel, washed it with 2 washes of precooled (0 to 5 °C) 9 ml MTBE.16. Unload the material, and transferred it to vacuum tray dryer for drying. Allowed to dry it under vacuum at 35 to 40 °C for 5 to 6 hrs.17. Dry wt. 0.92gm sample submitted for analysis: HPLC Purity: 76,15%, Peptide content 61%,Oxidation of linear peptide to get crude Octreotide:P27ATable 13 Raw materials for Oxidation of linear peptide to get crude Octreotide experiment P27A18. Checked pH of linear solution, pH: 1.38. Removed before oxidation sample for Analysis.19. Charged 2 gm Iodine dissolved in 50 ml acetonitrile in reaction mass. Dark brown coloured solution observed. Allowed to stir at 25 to 35 °C for 180 to 200 min. Taken out samples after every 60min interval. 20. After completion of reaction, quenched the reaction mass using 0. Im Ascorbic acid solution till the reaction mass transferred to colourless from dark brown.21. Adjusted pH of solution to 6 to 7 using 10N NaOH water solution, pH- 6.5. (10 ml).22. Filtered the reaction mass thorough Ip, 0.8 p and 0.45 p in sequence. Transferred the filtered mass for purification.Results: Reaction monitored on HPLCTable 14 Result of crude Octreotide oxidation experiment P27AP27BTable 15 Raw materials for Oxidation of linear peptide to get crude Octreotide experiment P27B23. Charged 0.92 gm linear peptide in 1000 ml clean glass bottle. Added 900 ml water in it, allowed to stir at 25 to 35 °C for 25 to 30 min. till complete dissolution.24. Adjusted pH of solution to 2 to 3 using 20% TFA water solution. pH- 2.1. Removed before oxidation sample for Analysis.25. Charged 0.25 gm Iodine dissolved in 6 ml acetonitrile in reaction mass. Dark brown coloured solution observed. Allowed to stir at 25 to 35 °C for 180 to 200 min. Taken out samples after every 120 min interval.26. After completion of reaction, quenched the reaction mass using 0.1 M Ascorbic acid solution till the reaction mass transferred to colourless from dark brown.27. Filtered the reaction mass thorough Ip, 0.8 p and 0.45 p in sequence. Transferred the filtered mass for purification.Results: Reaction monitored on HPLCTable 16 Result of crude Octreotide oxidation experiment P27BPurification of crude Octreotide:P27R1Table 17 Raw materials for purification of crude Octreotide experiment P27R1Table 18 Gradient table for purification of crude Octreotide experiment P27R128. Charged 2.0 ml Acetic acid in glass bottle containing 2000 ml water, filter it through 0.45p filter. Labelled it as ‘Buffer A’. Charged 1000 ml Methanol in another glass bottle, labelled it as ‘Buffer B’.29. Purged all buffer lines with respective buffers. Column equilibrated with 5% buffer B with 95% Buffer A for 3 column volume (CV) at 10 ml / min flow.30. Crude octreotide solution obtained after oxidation process was loaded on column manually at flow rate 14 ml / min.31. After completion of loading, buffer A passed through column for 1 CV or up base line gets stable.32. Started gradient program as mentioned above table. Chromatographic elution was monitored at UV wavelength 220 and 280 nm. Material collected by observing UV in fractions.33. After completion of elution gradient program stopped. Column washed with 20%: 80% Buffer A: B for 3 CV.34. Samples from collected fractions were submitted for analysis.Table 19 Result of Purification fractions of P27R135. Fraction 7 to fraction 9 were mixed together, desired purity not meet so F7 to F9 taken for re-purification.P27R236. Raw materials same as mentioned in Table 17. column and silica specification same as mentioned in P27R1, gradient program same as in Table 18.37. Buffers Preparation same as in P27R1.38. Purged all buffer lines with respective buffers. Column equilibrated with 5% buffer B with 95% Buffer A for 3 column volume (CV) at 10 ml / min flow.39. Crude octreotide solution obtained after oxidation process was loaded on column manually at flow rate 14 ml / min.40. After completion of loading, buffer A passed through column for 1 CV or up base line gets stable.41. Started gradient program as mentioned above table. Chromatographic elution was monitored at UV wavelength 220 and 280 nm. Material collected by observing UV in fractions.42. After completion of elution gradient program stopped. Column washed with 20%: 80% Buffer A: B for 3 CV.43. Samples from collected fractions were submitted for analysis.Table 20 Result of Purification fractions of P27R244. Fraction 6 to fraction 9 were mixed together, desired purity not meet so F6 to F9 taken for re-purification. P27R3In P27R1 and P27R2 desired purity not meet to separate impurity re-purification done by following process1. Table 21 Raw materials for purification side fraction of Octreotide experimentP27R3Table 22 Gradient table for purification of crude Octreotide experiment P27R245. Charged 4 ml trifluoroacetic acid in glass bottle containing 2000 ml water, filter it through 0.45p filter. Labelled it as ‘Buffer A’. Charged 1000 ml Acetonitrile in another glass bottle, labelled it as ‘Buffer B’ 46. Purged all buffer lines with respective buffers. Column equilibrated with 5% buffer B with 95% Buffer A for 3 column volume (CV) at 15 ml / min flow.47. Pooled fraction of P27R1 F7-F9 and P27R2 F6-F9 mixed together and diluted with 1: 1 with water loaded on column manually at flow rate 14 ml / min.48. After completion of loading, buffer A passed though column for 1 CV or up base line gets stable.49. Started gradient program as mentioned above table. Chromatographic elution was monitored at UV wavelength 230 and 280 nm. Material collected by observing UV in fractions.50. After completion of elution gradient program stopped. Column washed with 20%: 80% Buffer A: B for 3 CV.51. Samples from collected fractions were submitted for analysis.Table 23 Result of Purification fractions of P27R352. Fractions 5 to fractions 12 were mixed together, taken for de-salting.P27R4Table 24 Raw materials for purification pure fraction of Octreotide experiment P27R4Table 25 Gradient table for purification of fraction of Octreotide experiment P27R453. Charged 2.0 ml Acetic acid in glass bottle containing 2000 ml water, fdter it through 0.45p fdter. Labelled it as ‘Buffer A’. Charged 1000 ml Acetonitrile in another glass bottle, labelled it as ‘Buffer B’. Charged 3.85gm Ammonium acetate in glass bottle containing 500 ml fdter it through 0.45 p fdter. Labelled it as ‘Buffer C’. Charged 500 ml water in glass bottle, labelled it as ‘Buffer D’.54. Purged all buffer lines with respective buffers. Column equilibrated with 5% buffer B with 95% Buffer A for 3 column volume (CV) at 15 ml / min flow. 55. Pooled fraction of P27R3 F5-F12 mixed together and diluted with 1: 1 with water loaded on column manually at flow rate 16 ml / min.56. After completion of loading, buffer D passed though column for 3 CV or up base line gets stable at flow rate 16 ml / min.57. For salt exchange buffer C passed though column for 3 CV at flow rate 16 ml / min.58. After that buffer D passed though column for 3 CV at flow rate 16 ml / min.59. Started gradient program as mentioned above table. Chromatographic elution was monitored at UV wavelength 220 and 280 nm. Material collected by observing UV in fractions.60. After completion of elution gradient program stopped. Column washed with 20%: 80% Buffer A: B for 3 CV.61. Samples from collected fractions were submitted for analysis.Table 26 Result of Purification fractions of P27R462. Collected single fraction, acetonitrile was removed by distillation under vacuum using rotary evaporator.63. The concentrated solution was transfer in lyophilisation tray.64. Kept tray in lyophilisation recipe .

[0076] After completion of drying, material unloaded in HDPE container and sample submitted for analysis. Dry weight: 530 mg, HPLC Purity: 98.36% SMI 0.96%.

[0077] Example 3: Synthesis of Octreotide peptidyl resin using Cys(Trt) and DIC- HOBT.H2O and GVL as solvent on 5 gm scale

[0078] Synthesis of octreotide was planned on 5 gm scale of pre-loaded coupled Thr(tBu)-OL-2-CTC resin. Batch number assigned as P28. The synthesis process flowed same as mentioned for P26. Peptidyl resin was then subjected to cleavage, which was conducted using 6V DCM and 6 V TFA-cocktail for 3 hrs. The resin was filtered off. TFA and DCM distilled off under reduce pressure. The concentrated mass directly quenched in precooled water and subjected for oxidation followed by purification. Purified fractions then freeze dried to get powder form of octreotide acetate. Figure 3 shows the flow diagram of the experimental steps of example 3. Figure 5 depicts the route of synthesis of this process.

[0079] After completion of drying, material unloaded in HDPE container and sample submitted for analysis. Dry weight: 2700mg.

[0080] Example 4: Synthesis of Octreotide peptidyl resin using Cys(Trt) and DIC- HOBT.H2O and GVL as solvent on 50 gm scale

[0081] Synthesis of octreotide was planned on 50 gm scale of pre-loaded coupled Thr(tBu)-OL-2-CTC resin. Batch number assigned as P30. The synthesis process flowed same as mentioned for P26. Peptidyl resin then subjected for cleavage, it was conducted using 6V DCM and 6 V TFA-cocktail for 3 hrs. The resin was filtered off. TFA and DCM distilled off under reduced pressure. The concentrated mass directly quenched in precooled water and subjected for oxidation followed by purification. Purified fractions then freeze dried to get powder form of octreotide acetate.

[0082] After completion of drying, material unloaded in HDPE container and sample submitted for analysis. Dry weight: 27.6 gm

[0083] The foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.ADVANTAGES OF THE INVENTION

[0084] The present invention provides a process of preparation of peptides that utilizes less volume of DMF.

[0085] The present invention provides a process of preparation of peptides that is industrially scalable and has reduced carbon footprint.

Claims

We Claim:

1. A process for preparation of linear or cyclic peptide with 1 to 40 amino acids, wherein the process comprises the steps of:(a) coupling Fmoc-N-protected amino acid to a resin capped amino acid using coupling reagent to obtain a dipeptide resin and avoiding washings by any solvents after coupling reactions;(b) selectively deprotecting Fmoc of N-protected amino acid at N terminal of the dipeptide resin using deprotecting reagent and greener solvent to obtain free amino dipeptide resin, and washing of resin using IPA, and DMF containing HOBt or Oxyma;(c) coupling a carboxyl terminus of Fmoc-N-protected amino acid to the free amino dipeptide resin of step (b) in the presence of a coupling reagent and greener solvent and avoiding washings by any solvents after coupling reactions;(d) repeating step (b) & step (c) to form a peptidyl resin;(e) deprotecting Fmoc of N-protected amino acid at N terminal of the peptidyl resin;(f) cleaving the peptide from the peptidyl resin to obtain linear crude peptide;(g) optionally oxidizing the linear crude peptide to obtain a crude cyclic peptide; and(h) optionally purifying the crude cyclic peptide to obtain pure cyclic peptide; wherein the preparation process comprises a combination of a greener solvent as coupling solvent and conventional solvents as washing solution for each cycle of step (b) & step (c).

2. The process as claimed in claim 1 further comprising the step of lyophilising the crude peptide or pure peptide to obtain solid peptide.

3. The process as claimed in claim 1, wherein the peptide is selected from the group consisting of octreotide, liraglutide, semaglutide, teriparatide, albiglutide, abaloparatide, dulaglutide, and exenatide.

4. The process as claimed in claim 1, wherein the greener solvent is selected from the group of solvents consisting of gamma-valerolactone (GVL), anisole, methanol, ethanol, isopropanol, 1,4-dioxane, 2-methyl tetrahydrofuran, N-methyl-2-pyrrolidinone (NMP), ethyl acetate, acetonitrile, acetone, and combination thereof.

5. The process as claimed in claim 1, wherein the coupling agent is selected from the group consisting of 1 -hydroxybenzotriazole (HOBt), N,N-diisopropylcarbodiimide (DIC), hexafluorophosphate benzotriazole tetramethyl uronium (HBTU), / V. / V-diisopropylcthylaminc (DIPEA), benzotriazol-l-yl-oxy- tris(dimethyl-amino)-phosphonium hexafluorophosphate (BOP), l-[Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium-3-oxidehexa fluorophosphate (HCTU), hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU), 3-(Diethoxyphosphoryloxy)-l,2,3-benzotriazin-4(3H)-one (DEPBT), 2-chloro-4,6- dimethoxy-l,3,5-triazine (CDMT), 2-(lH-7-Azabenzotriazol-l-yl)-l,l,3,3-tetra methyluronium tetrafluoroborate (TBTU) and combination thereof.

6. The process as claimed in claim 1, wherein the resin is selected from the group consisting of 2-chlorotrityl chloride (2-CTC), Sasrin™, TentaGel® S, TentaGel® TGA, Rink, Wang, and AmphiSpheres™7. The process as claimed in claim 1, wherein the resin deprotecting reagent in coupling reaction with greener solvent is a mixture of piperidine with at least 1% concentration and DBU with at least 1% concentration in greener solvent.

8. The process as claimed in claim 1, wherein the cleavage step of crude peptide from resin comprising the steps of: a) addition of a solvent mixture comprising dichloromethane (DCM) and trifluoroacetic acid (TFA) in a ratio ranging from at least 10% of TFA (v / v); b) precipitation of reaction mixture from step a) using ether; and c) optionally, direct quenching of the reaction mixture using water.

9. The process as claimed in claim 1, wherein the oxidizing step of the linear crude peptide containing two, four, six cysteine (Cys) residues is conducted by direct quenching with water and avoid use of ether for precipitation of crude peptide.

10. The process as claimed in claim 1, wherein purification step of crude peptide to obtain pure peptide is carried out using alcohol containing 1 to 4 carbon atoms in a mobile phase.

11. A process for preparing an octreotide, wherein the process comprising the step of:(a) coupling Fmoc-Cys(Acm)-OH or Fmoc-Cys(Trt)-OH to a Thr(tBu)-OL-2-CTC resin using GVL as a coupling solvent to obtain a dipeptide resin, Fmoc-Cys(Acm)- Thr(tBu)-OL-CTC-Resin or Fmoc-Cys(Trt)-Thr(tBu)-OL-CTC-Resin;(b) selectively deprotecting Fmoc of Fmoc-Cys(Acm)-Thr(tBu)-OL-CTC-resin or Fmoc-Cys(Trt)-Thr(tBu)-OL-CTC-resin using deprotecting reagent, mixture of piperidine and DBU in greener solvent, gamma-valerolactone (GVL) to obtain dipeptide resin, H-Cys(Acm)-Thr(tBu)-OL-CTC-resin or H-Cys(Trt)-Thr(tBu)-OL- CTC-resin; washing of the dipeptide resin obtained using IPA, & DMF containing HOBt or Oxyma;(c) sequential coupling a carboxyl terminus of other Fmoc-N-protected amino acid of the octreotide to the free amino dipeptide resin of step (b) in the presence of a coupling reagent and gamma-valerolactone (GVL), as a greener solvent;(d) repeating step (b) & step (c) to form a peptidyl resin, Boc-D-Phe-Cys(Acm)-Phe-D- Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr(tBu)-OL-CTC-resin or Boc-D-Phe- Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr(tBu)-OL-CTC-resin;(e) cleaving the peptide from the peptidyl resin to obtain linear crude peptide using 30 to 70% TFA and DCM solution by directly quenching the reaction mixture in water, and subsequently performing an in-situ oxidation process to obtain a crude octreotide; and(f) optionally purifying the crude octreotide to obtain pure octreotide by reverse phase chromatography using methanol ranging from 10% to 60% in aqueous mobile phase containing 0.1% to 1.0% acetic acid. wherein: the process avoids washings by any solvents after coupling reactions in each cycle of repeating of step (b) and (c) to form a peptidyl resin.

12. The process as claimed in claim 12, further comprising the step of lyophilising the crude octreotide or pure octreotide to obtain solid octreotide.

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