FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)
1. Title of the invention
PROCESS FOR THE SYNTHESIS OF PROTECTED UNNATURAL AMINO ACID
2. Applicant(s)

Name
Nationality

Indian company incorporated under Companies Act, 1956
Address
USV Limited
B.S.D. Marg, Station Road, GovandifE), Mumbai 400 088 Maharashtra
3. Preamble to the description
The following specification particularly describes the invention and the manner in which it is to be performed.

TECHNICAL FIELD
This invention relates to a cost effective process for synthesis of protected unnnatural amino acid which is used as an intermediate in the synthesis of peptides by solid phase peptide synthesis. More particularly, the invention relates to the synthesis of protected homoarginine and synthesis of peptides containing homoargmine by solid phase peptide synthesis.
BACKGROUND AND PRIOR ART
Solid phase peptide synthesis, the alternative to conventional synthesis in solution, is distinguished by its speed and convenience of operation. Successful applications to the synthesis of a large variety of biologically active peptides has been commercially explored. In the last decade, there has been a shift from classical solution phase peptide synthesis (CSPS) towards solid phase peptide synthesis (SPPS) for the manufacturing of peptides on a large scale. Two intrinsic properties of the SPPS approach contribute to its commercial competitiveness. First, no intermittent isolation occur during synthesis on a solid support. Second, SPPS follows a generic protocol and is therefore automatable. Both of these aspects increase the manufacturing efficiency and are not applicable to CSPS.
Despite these benefits of the SPPS approach, manufacturing efficiency is compromised somewhat by cost of starting materials. These are relatively high due to to the use of expensive non-reusable resins and amino-acid derivatives. Moreover, all functional side-chain must be protected in SPPS in order to make the hydrophobic resin accessible to the otherwise polar amino acids, and avoid the accumulation of sequences containing modified side-chains.
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SPPS of peptides in commercial quantities suffers major setback if the ready availability of the protected amino acids as precursors is not consistent with respect to the quality and quantity. Synthesis of protected amino acids at requisite cost and quality is of prime importance to the commercial competitiveness of the peptide process. The Fmoc-protected amino acids, either with or without side-chain protecting groups as required, that are used in loading the resin and in peptide synthesis are available commercially from inter alia, Genzyme Pharmaceuticals Inc., Cambridge, MA, Bachem Biosciences Inc. Torrance, CA; Senn Chemicals, Dielsdorf. Switzerland, and Orpegen Pharma, Heidelberg, Germany, or are readily synthesized using materials and methods well known in the art.
The solid phase synthesis of peptides containing one or several Arg residues in there chains inevitable confronts problems associated with the protection of the guanidine group (N6), because this group can be acylated during the coupling process and the removal of the JV° protecting groups might also cause some complications. It has been found that the protection of Ncc-Fmoc derivatives of Arg by the N° protecting urethane type groups (Boc or Adoc) does not prevent the acylation of the guanidine function and the formation of Ornithine. On the other hand, the deprotection of the peptide containing several Mtr protected Arg residues require rigorous conditions and prolonged treatment that can result in the formation of some by-products. Similarly use of unnatural amino acid like hydroxyproline, homoarginine, norleucine, citruUine, ornithine, tertiary butylglycine, tertiary butylalanine, homocysteine in the synthesis of peptides presents a considerable challenge while using SPPS for synthesis of such peptides as these amino acids have to be adequately protected at Na-NH2 and side chains by appropriate protecting agents. Readymade availability of the reagents, consistent supply, adequate purity and most importantly reasonable cost decide the
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overall cost-competitiveness of the commercial process for synthesis of peptides. Synthesis of high volume, high value peptides like Eptifibatide wherein usage of two unnatural amino acids hence remains a challenge for the manufacturers. Incorporation of homoarginine (HoArg) by guanylation of lysine though cost-effective has impurity profile which is precedent in the prior art. Hence the present invention presents a cost-effective and efficient process for synthesis of protected homoarginine.
OBJECT OF INVENTION:
One of the object of the present invention is to prepare a cost effective process for synthesis of protected unnatural amino acid which is used as an intermediate in the synthesis of peptides by Solid Phase Peptide Synthesis.
Another object of the present invention is synthesis of protected homoarginine and synthesis of peptides containing homoarginine by Solid Phase Peptide Synthesis.
Still another object of present invention is a process for synthesis of a protected unnatural amino acid by reacting the same with an a-amino protecting reagent dissolved in aqueous organic phase essentially in the presence of an organic base.
Still another object of the invention is a process for solid phase synthesis of Eptifibatide by using protected unnatural amino acid obtained by the present process .
SUMMARY OF THE INVENTION
One aspect of the present invention is a process for synthesis of a protected unnatural amino acid of the Formula 1,
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wherein Rl is any guanidino protecting group; and R2 is any a-amino Nitrogen protecting group; wherein the process comprises reacting unnatural amino acid of Formula 2,

with an a-amino protecting reagent dissolved in aqueous organic phase essentially in the presence of an organic base.
Another aspect of the present invention is a protected unnatural amino acid of Formula 8

synthesized by process claimed with a purity of atleast 95%.
Still another aspect of the present invention is a process for solid phase synthesis of Eptifibatide by using protected unnatural amino acid of Formula 1 obtained by the claimed process .
DETAILED DESCRIPTION OF DRAWINGS:
Figure 1: RP-HPLC Chromatogram of Z-HoArg-OH; (Retention time:32.445 minutes; Purity:94.05%)
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Figure 2.RP-HPLC Chromatogram of Z-HoArg (Pbf)-OH process by-product profile;
(Retention time of PbfO-Na+ byproduct: 16.665 minutes; Purity:87.659%) Figure 3:RP-HPLC Chromatogram of water washing layer of Z-HoArg(Pbf)-OH Figure 4:RP-HPLC Chromatogram of ethyl acetate layer of Z-HoArg(Pbf)-OH Figure 5:RP-HPLC Chromatogram of Z-HoArg(Pbf)-OH ; (Retention time:40.244
minutes; Purity:92.395%) Figure 6:RP-HPLC Chromatogram of H-HoArg(Pbf)-OH ; (Retention time:23.696
minutes; Purity:94.765%) Figure 7:RP-HPLC Chromatogram of Fmoc-HoArg (Pbf)-OH (Retention time:20.250
minutes; Purity:96.32%) Figure 8:RP-HPLC Chromatogram of crude Eptifibatide using Fmoc-HoArg (Pbf)-
OH from Suzhou Tianma Speciality Chemicals Limited, China;(Retention
time: 19.5 minutes; Purity:62.07%) Figure 9:RP-HPLC Chromatogram of crude Eptifibatide using Fmoc-HoArg (Pbf)-
OH prepared using the instant invention ;(Retention time: 19.45 minutes;
Purity:67.89%) DETAILED DESCRIPTION OF THE INVENTION: Glossary of terms used in the Specification:
DCM: Dichloromethane;DMF:Dimethylformamide;DTT: Dithiothreitol;
HoArg:Homoarginine; NMM: N-methyl morpholine; RP-HPLC: Reverse Phase High Performance Liquid Chromatography; TFA: Trifluoroacetic acid; TIS:Triisopropyl silane; DIPE: Diisopropyl ether; CH2CI2: methylene chloride.
Usage of HoArg in peptides as GpIIb-IIIa Platelet aggregate inhibitors is illustrated in US 5686570. The results indicated that replacement of Arg by Har may be a good
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avenue for the design of biologically active peptides with increased resistance to degradation by trypsin-like enzymes.
Eptifibatide is a highly specific cyclic heptapeptide antagonist of the platelet glycoprotein Ilb/IIIa. It is a short-acting parenteral antithrombotic agent that is used during percutaneous coronary interventions for the treatment of unstable angina and as an adjunct to thrombolytic agents for the treatment of acute myocardial infarction. Eptifibatide is marketed in the U.S. under the trademark INTEGRILIN®. Many reported synthetic approaches to eptifibatide have employed known techniques of solid-phase peptide synthesis as described, for example, in U.S. Patents 5,318,899; 5,686,570 and 5,747,447. A commercial-scale, liquid phase process was also reported at the 1999 IBC Conference on Peptide Technologies, "Peptisyntha's Method of Producing GMP Peptides on an Industrial Scale". The commercial process is a convergent synthesis involving the separate preparation of two fragments: Mpa-Har-Gly and Asp-Trp-Pro. The last residue attached is an S-trityl-protected cysteinamide as described, for example, in U.S. Patent 5,506,362. After removal of the S-trityl protecting groups (on the cysteinamide and mercaptopropionyl residues), ring closure is then achieved by disulfide bond formation. Crude eptifibatide obtained by the commercial process has a reported purity of about 80%. Two column chromatography steps improve the purity to greater than 99%. Liquid-phase synthesis has generally been viewed as more feasible than solid- phase synthesis for the large-scale manufacture of eptifibatide. However, solubility issues and the generation of complex reaction mixtures present challenges for large-scale liquid phase processes. Complex reaction mixtures, for example, make purification of the product more difficult. Ways exist to overcome these problems, such as the use of persilylated amino acids and phase transfer reagents, as described, for example, in U.S. Patent 4,954,616, and

extensive chromatographic purification, but such means add to the cost of the overall process. A need thus exists for alternative processes for the manufacture of eptifibatide by SPPS using cost competent starting materials.
Two SPPS strategies extensively used for synthesis employ the Fmoc based or Boc based strategies. Most of the Fmoc-amino acids derivatives are commercially available. However, a problem exists in the art for the preparation of peptides containing some amino acid analogs like homoarginine as well as cyclic peptide compounds based on disulfide links, because separate operations are required before purifying the end product, which increases expense and may affect final product purity and yield. Fmoc-homoarginine residue if purchased commercially for use in the assembly of the peptide chain becomes very expensive. Alternatively the peptide assembly can be built using lysine followed by guanylation of the lysine residue at the .alpha.-NH.sub.2 (Lindeberg et al., Int. J. Peptide Protein Res. 10, 1977, 240-244). Howsoever this strategy suffers from certain drawbacks. Hence synthesis and incorporation of direct Fmoc protected HoArg in Eptifibatide holds greater promise.
There is enough prior art which discloses the peptides containing HoArg with therapeutic value. Lindeberg et al. (Int. J. Peptide Protein Res. 1977, 10, 240) discloses the synthesis of l-deamino-4-L-valine-8-DL-homolysine-vasopressin and protected l-deamino-4-L-valine-8-D-lysine-vassopressin in which with non-natural amino acids were incorporated. The addition of a methylene group to lysine and arginine was made to generate the non-natural amino acids homo lysine and homoarginine, respectively. The study revealed that peptides with homolysine and homoarginine reduced the antidiuretic activity of the peptides.
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Nestor et al. (J. Med. Chem. 1988, 31, 65) disclose the synthesis of a new series of unnatural amino acids and their incorporation into antagonistic analogues of lutenizing hormone-releasing hormone (LH-RH). In particular, non-natural aminoacids of arginine exhibited high acute potency and very prolonged duration of action. Biological and clinical pharmacology studies revealed that these LH-RH antagonists cause mast cell degranulation, and were removed from consideration as candidates for full commercial development.
There is a need in the art for unnatural amino acids and for peptides incorporating such acids to achieve superior effects, such as, for example, improved diagnostic or disease fighting activity. The present invention accordingly describes unnatural amino acids, methods of making thereof, and utilization thereof in peptides.
According to the present invention, it is one preferred embodiment to couple Arg or Har? preferably when being used as FMOC-Arg and FMOC-HoArg respectively, with or without the use of side chain protecting groups.
Arginine is the most basic naturally occurring a- amino acid due to the resonance stabilised guanidinium cation (pKl 1.82, pK2 8.99 & pk3 13.20). There are four main approaches to the problem of arginine side chain protection of the guanidine group:
1) preferential protonation
2) nitration,
3) (di) urethane protection,
4) aryl sulfonyl protection.
Each of these options offers specific advantages & incurs disadvantages. The following criteria are essential while designing a N-a-NH2 group protected homoarginine or arginine derivatives:
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1) reagent availability
2) stability to basic conditions required for Fmoc deprotection
3) removal by TFA: H20 (95:5) & TFA:CH2Cl2(50:50) within 1 hour at room temperature.
PCT publication WO 89/07609 exemplifies the process for preparation of Boc-ON [(2-(tert-butyloxycarbonylimino)-2-phenylacetonitrile] protected homoarginine. In step 1 the homoarginine is treated with Boc-ON under basic pH conditions in an aqueous dioxane mixture until reaction is essentially complete, say for 20 to 24 hours. After workup of the mixture the Boc protected homoarginine can be obtained as an essentially white powder and processed further without further purification. In step 2, ter-Boc-HoArg can be treated to protect guanidino moiety and [H2N-((NH)-NH)-] of the tert-Boc-protected HoArg by deprotonating it at a basic pH of 11 to 12 in a suitable diluent such as aqueous acetone with an amino protecting reagent such as p-toulenesulfonyl chloride for time sufficient to react with essentially all of the amino nitrogen for about three hours. The resulting reaction mixture can be processed to separate the desired product, tert-Boc-HoArg-fA^-tOsyl), from small amounts of side reaction products such as (A^-TosjHoArg and trace amounts of unreacted starting material.
R. Ramage et al.,1991 discussed synthesis and application of FmocArg(Pmc).OH and Bnpeoc.Arg(Pmc).OH. The construction of 2,2,5,7,8-pentamethyl chroman (Pmc) was achieved in a single step from 2,3,5-trimethylphenol and isoprene, using zinc chloride as catalyst in HO Ac. Chlorosulfonation of Pmc afforded the required reagent Pmc.CI which was reacted with Na-Z.Arg.OH under the usual alkaline conditions to give Na-benzyloxycarbonyl-NG--(2J2,5,7,8-pentamethylchroman-6-sulphonyl)-Arg (Z.Arg(Pmc).OH). Purification of Z.Arg(Pmc).OH was achieved by crystallization of the cyclo-hexyl amine salt. Reconvertion of this salt to Z.Arg(Pmc).OH and

subsequent hydrogenolysis (10% Pd/C) gave NG-(2,2,5,7,8 pentamethylchroman-6-sulphonyl)-Arg (H.Arg(Pmc).OH).
Carpino et al.,I993 discusses that 2,2,4,6,7-pentamethyldihydrobenzofuran-5-suIfonyl residue (Pbf) is more easily deblocked by trifluoroacetic acid (TFA) than the corresponding 2,2,5J7,8-Pentamethylchroman-6-suIphonyl-L-arginine (Pmc) analog and can be used efficiently in the synthesis of arginine containing peptides.
Bodanzky et al., in Protocols in Peptide Synthesis, discusses the synthesis of Benzyloxy-carbanoyl-L-Arginine wherein arginine monohydrochloride is dissolved in ice-cold N NaOH with stirring. Both stirring and cooling to about 0°C are continued while benzyl chlorocarbonate and 2N NaOH are added in a few portions alternatingly. The pH of the mixture is kept between 9 and 10. After the addition of the reactants is completed stirring of the suspension is continued for 2 more hours. The pH drops during this time to 7.0 to 7.5. The precipitate is collected on the filter, washed with cold (ca. 10°C) water and recrystallized from boiling water. Crystallization in the cold (ice water bath). The product is collected, dried in air and then suspended in acetone filtered, washed with acetone and with ether. The purified material is dried in vacuo at 50°C.
The majority of a-amino acid sidechain protecting groups selected for Fmoc strategy of SPPS are easily cleaved by TFA:H20 (95:5) in ca 1 hour at room temperature although the time for t-butyl ether deprotection of Ser & threonine residues may vary significantly according to sequence. For some time the guanidino group of arginine (Arg) proved to be a problem, especially in sequences having many arginine residues, if the same mild acid conditions were set as constrains for guanadino protection or deprotection of arginine.

None of the cited prior art describes the synthesis of protected HoArg. When the protected Arg process was extended to HoArg using an inorganic base, the process could not progress to completion. There was a surprising finding that in the presence of organic base, the reaction progressed resulting in the formation of an intermediate with the N-CC-NH2 group being protected with benzyloxycarbonyl (Z) group. Another aspect of the present invention was the use of mixture of organic and aqueous phases for dissolution of starting material HoArg.HCl in the presence of an organic base.
One embodiment of the present invention is a process for synthesis of a protected unnatural amino acid of the Formula 1,

wherein Rl is any guanidino protecting group; and R2 is any a-amino Nitrogen protecting group; wherein the process comprises reacting unnatural amino acid of Formula 2,

with an a-amino protecting reagent dissolved in aqueous organic phase essentially in the presence of an organic base.
Another embodiment of the present invention is a process . wherein the unnatural amino acid of Formula 2 is reacted with any a-amino Nitrogen protecting group R2 in the presence of an organic base to yield protected a-amino acid of Formula 3


wherein the organic base is selected from group consisting of triethyl amine; cyclohexyl amine; 1,5-diazabicyclo (5,4,0) undec-5-ene; piperidene ethanolamine; pyrrolidine; diethylamine; morpholine; piperazine; dicyclohexylamine; hydroxylamine; N,N'-diisopropylethyl amine; N, N,N',N'-tetramethyl 1,8-napthalenediamine; tributylamine; and triethylenediamine.
Still another embodiment of the present invention is a process for the synthesis of Formula 3, wherein R2 is benzyloxycarbonyl succinimide (Z-OSu).

Still another embodiment of the present invention is converting the a-amino Nitrogen group protected unnatural amino acid of Formula 3, dissolving the same in C1-C4 alcohol and precipitating the same with cosolvent selected from diisopropyl ether or diethyl ether, or converting it in stable salt form by dissolving it in a mixture of an aqueous inorganic base and organic solvent, further mixing the mixture; removing the organic phase; and drying the aqueous phase or dissolving the stable salt in C1-C4 alcohol and precipitating the same with cosolvent selected from diisopropyl ether or diethyl ether.

Still another embodiment of the present invention is the stable sodium salt of Formula 4 with RP-HPLC purity of atleast 90%.
Still another embodiment of the present invention is a stable sodium salt of Formula 4
which is further lyophilized or is in the form of thick syrup or in the form of dried
powder.
Still another embodiment of the present invention is a process wherein the protected
a-amino acid of Formula 3 is further reacted with side chain protecting group Rl at
alkaline pH by continuous stirring at a temperature of about 2 to 10°C to yield
protected unnatural amino acid of Formula 5

Still another embodiment of the present invention is an intermediate of Formula 5 wherein side chain protecting group Rl is Pentamethyldihydrobenzofuran-5-sulfonyl chloride (Pbf-Cl) and R2 is benzyloxycarbonyl succinimide (Z-OSu) with a RP-HPLC purity of atleast 85%.
H
l\L .NH Pbf
T
Formula 6
Still another embodiment of the present invention is a protected unnatural amino acid of Formula 5 which undergoes hydrogenolysis in the presence of catalyst to yield a
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deprotected unnatural amino acid of Formula 7 and which is further protected with a-amino protecting reagent Rl to yield protected unnatural amino acid of Formula 1.
NH Formula 7
Still another embodiment of the present invention is an intermediate of Formula 1, wherin the a-amino protecting reagent Rl is 9-fiuorenylmethyloxycarbonyl (Fmoc) and R2 is Pentamethyldihydrobenzofuran-5-sulfonyl (Pbf).
Still another embodiment of the present invention is a protected unnatural amino acid of Formula 8
O H
H n^V^N^^^^N-s^NHPbf
HD ^Y T
Fmoc-NH NH
synthesized by process claimed with a purity of atleast 95%.
Still another embodiment of the present invention is a process for solid phase synthesis of Eptifibatide by using protected unnatural amino acid of Formula 1 obtained by the claimed process .
Still another embodiment of the present invention is a process for solid phase synthesis of Eptifibatide by using protected unnatural amino acid of Formula 8 obtained by the claimed process wherein the purity of crude Eptifibatide by RP-HPLC is atleast 55%.

A protected unnatural amino acid of the Formula 1, wherein Rl is selected from 2,2,5,7;8-Pentamethylchroman-6-sulfonyl(Pmc);2,2J4,6,7-
Pentamethyldihydrobenzofuran-5-sulfonyl (Pbf); 2-(tert-butyloxy carbonylimino)-2-phenylacetonitrile Tos); 4-methoxy-2,3,6-trimethylbenzene -sulfonyl (Mtr); Bis(0-ter-butoxycarbonyl tetrachlorobenzoyl)-(Btb); 4-methoxy-benzenesulfonyl (mbs); 2,4,6-Trimethylbenzenesulfonyl (Mts) and R2 is selected from benzyloxycarbonyl succinimide (Z-OSu), trifluoroacetyl (Tfa), 9-fluorenylmethyloxycarbonyl (Fmoc), 4-toulenesulfonylethyloxycarbonyl (Tsoc), methyl sulphonyl ethyloxy carbonyl (Mesoc), 2-(triphenylphosphono)-ethyloxycarbonyl (Peoc), 2-cyano-t-butyloxycarbonyl (Cyoc), and pthaJyl (Pht) groups.
Additional advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
EXAMPLES:
Example l:Step-l: Synthesis of Z-Homoarginine-OH from Homoarginine. hydrochloride (Z.Harg.OH )
Homoarginine. monohydrochloride (100g) (l.Oeq) was dissolved in 50:50 Acetone: water (500ml) mixture. The solution was mixed at 0°C with constant stirring, while Na-(Benzyloxycarbonyloxy) Succinimide (1.3eq,, 145g) dissolved in acetone (500ml)
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was added gradually. To the above reaction mixture was then added triethyl amine as the base 2.1eq (94.6g) under constant stirring. The pH of the mixture was kept between 9 and 10. After the addition of the reactants is completed, stirring of the mixture was continued for 3 more hours.
After 3 hours, the reaction was quenched using citric acid and the reaction mixture was concentrated by evaporating acetone on a rotary evaporator. After the evaporation of acetone ceased, the reaction mixture was extracted with chloroform three times. The chloroform extracts were rejected and the aqueous layer was subjected to drying on a rotary evaporator. The drying was continued until a thick syrup was obtained. One alternative used was that the thick syrup was dissolved in methanol and the product was precipitated with DIPE. The product was further filtered and dried overnight. Other alternative was for further refinement of the product, the thick syrup was dissolved with 3.2N NaOH: acetone mixture (1:2 v/v). The mixture was thoroughly mixed with an overhead stirrer in a 20L separating funnel. The lower dark colored byproduct layer was rejected and the aqueous layer was subjected to drying on a rotary evaporator. The product on large scale was obtained as a thick syrup.The product was further dissolved in methanol and extracted by precipitation with DIPE. The progress of the reaction mixture was monitored by RP-HPLC, TLC. The Isolated yield ca 95% (146gm), RP-HPLC purity >90% (Figure 1), m/z 323.2 (sodium salt less).
Step-2: Synthesis of Z-Homoarginine(Pbf) -OH from Z-Homoarginine -OH Z.Harg.OH (146 g) was dissolved in acetone (760 ml). 3.2M sodium hydroxide solution (500ml) at 0°C. To this was added 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl chloride (Pbf-Cl), (215gm) in acetone (650ml) in a span of 10 minutes. The reaction mixture was stirred for 3 hrs in ice cold condition. After acidification to
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pH 4 with saturated citric acid, acetone was removed under reduced pressure. The remaining solution was further acidified with saturated citric acid to pH 3. The mixture was then diluted with ethyl acetate(lL). The insoluble PbfO-Na+ by-product was filtered on a Buchner funnel and the byproduct profile was analysed by RP-HPLC (Figure 2). The solution was then extracted with ethyl acetate (2 xl L). The combined organic extracts were then washed with water (5 x 500ml) (Figure 3), and Brine (5 x 500ml). The ethyl acetate layer was then dried over sodium sulfate (Figure 4). The solution was then concentrated in vacuum to a total volume of ca \L, cooJed in an ice bath and product was precipitated by n-hexane (5L). The thick syrup solidified on standing overnight at -20 °C. Washes were executed by 2L cold n-hexane. The syrup was then dried under reduced pressure to obtain a solid. The Isolated yield ca 55% (133gm), RP-HPLC purity >85% (Figure 5), m/z 575.3.
Step-3: Synthesis of H-Homoarginine(Pbf) -OH from Z-Homoarginine(Pbf) -OH (H.Harg(Pbf)-OH)
Z.Harg(Pbf).OH (133gm) was taken up in methanol (1.4Lml), 10% palladium on charcoal (30g) was added to it under a blanket of nitrogen and the resulting mixture was hydrogenated for 2-3hrs. The catalyst was removed by filtration over celite. The mixture was concentrated on a rotary evaporator. The desired product was then precipitated by the addition of ether to it and allowing it to stand at -20 °C overnight. After decanting the ether layer, through washes were given to the viscous syrup. This syrup was then dried to obtain a solid. The Isolated yield was ca 90% (90gm), RP-HPLC purity >85% (Figure 6), m/z 441.3.

Step-4: Synthesis of Fmoc-Homoarginine(Pbf) -OH from H-Homoarginine(Pbf) -OH (Fmoc-Harg(Pbf)-OH)
H-Harg(Pbf)-OH (90gm) was dissolved in a mixture of 10% aqueous sodium carbonate (380ml) and dioxane (300ml) and the solution cooled to 0 °C. The solution of 9-fluorenylmethyl succinimidyl carbonate (71g) in dioxane (295 ml) was added drop wise and the reaction mixture was stirred for one hour on a ice bath, and then allowing the reaction to proceed further for 18hrs at ambient temperature. The solution was then diluted with cold water (1L) and washed with ether (5 x 500 ml) before acidification with 3N HC1 (pH 2.0). The solution was extracted with ethyl acetate (3 x 1L) and the combined extracts were washed with water (5 x 1L) and brine(5 x 1L), before drying over NaS04. This solution was concentrated in vacuum and the desired product precipitated by the addition of n-hexane. Washing were affected using cold n-hexane, followed by drying under vacuum to obtain a white to off white product. The Isolated yield ca 70% (95gm), RP-HPLC purity >96% (Figure 7),m/z663.6.
Example 2:
Process for Synthesis of Eptifibatide:
The heptapeptide Mpa-HoArg-Gly-Asp-Trp-Pro-Cys-NH2 was synthesized as peptide
amide by solid phase peptide synthesis technology on Rink amide resin using Fmoc
chemistry using CS936 Peptide Synthesizer. The side chain protecting groups used
were Asp-OBut; HoArg-Pbf, Cys-Trt, Mpa-Trt.The resin (17.24g) was transferred to
the reaction vessel of the CS936. The attachment of the first amino acid, viz cysteine
was carried out using combination of coupling solvent DMF/DCM. The preactivation
was carried out by dissolving the amino acid in the coupling solvent along with NMM
and HBTU. The remaining amino acids were coupled using DMF as the coupling
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solvent. Fmoc-HoArg(Pbf)-OH from Suzhou Tianma Speciality Chemicals Limited, China and prepared by process cited in Example 1 was used to prepare linear heptapeptide. A two time excess of the amino acids with respect to the mmole of resin was used for synthesis. Each coupling was carried out for 45-60 minutes. After the couplings were complete, the resin was washed with DMF/DCM under vacuum. The assembled peptide resin was treated with cleavage cocktail of TFA,TIS,DTT and water for 1.5-2 hours at room temperature. The reaction mixture was filtered and TFA was distilled out on Rotavap. Precipitation of the peptide was carried at -20°C by addition of cold DIPE with stirring. The crude peptide precipitate in the solvent was let to stand at -20°C overnight. The peptide was isolated by filtering followed by cold solvent wash. The crude SH Eptifibatide was dried under vacuum. The purity of crude Eptifibatide synthesized using Fmoc-HoArg(Pbf)-OH from Suzhou Tianma Speciality Chemicals Limited, China had the purity of 62.07% (Figure 8) whereas the purity of crude Eptifibatide using Fmoc-HoArg(Pbf)-OH prepared by the instant invention had the purity of 67.89% as determined by RP-HPLC (Figure 9).

We claim,
1) A process for synthesis of a protected unnatural amino acid of the Formula 1,
0 H
NHR2 NH
wherein Rl is any guanidino protecting group; and R2 is any a-amino Nitrogen
protecting group;
wherein the process comprises reacting unnatural amino acid of Formula 2,

with an a-amino protecting reagent dissolved in aqueous organic phase essentially in the presence of an organic base.
2) The process of claim 1, wherein Rl is selected from 2,2,5,7,8-Pentamethylchroman-6-sulfonyl (Pmc); 2,2,4,6,7-PentamethyIdihydrobenzofuran-5-sulfonyl (Pbf); 2-(tert-butyloxy carbonylimino)-2-phenylacetonitrile chloride(Tos); 4-methoxy-2,3,6-trimethylbenzene -sulfonyl (Mtr); Bis(0-ter-butoxycarbonyl tetrachlorobenzoyl)-(Btb); 4-methoxy-benzenesulfonyl (mbs); 2,4,6-Trimethylbenzenesulfonyl (Mts).
3) The process of claim 1, wherein R2 is selected from benzyloxycarbonyl succinimide (Z-OSu), trifluoroacetyl (Tfa), 9-fluorenylmethyloxycarbonyl (Fmoc), 4-toulenesulfonylethyloxycarbonyl (Tsoc), methylsulphonylethyloxy carbonyl (Mesoc), 2-(triphenylphosphono)-ethyloxycarbonyl (Peoc), 2-cyano-t-butyloxycarbonyl (Cyoc), and pthalyl (Pht) groups.
21

4) The process of claim 1, wherein the unnatural amino acid of Formula 2 is reacted with any a-amino Nitrogen protecting group R2 in the presence of an organic base to yield protected a-amino acid of Formula 3

wherein the organic base is selected from group consisting of triethyl amine; cyclohexyl amine; 1,5-diazabicyclo (5,4,0) undec-5-ene; piperidene ethanolamine; pyrrolidine; diethylamine; morpholine; piperazine; dicyclohexylamine; hydroxylamine; N,N'-diisopropylethyl amine; N, tyN^N'-tetramethyl 1,8-napthalenediamine; tributylamine; and triethylenedi amine,
5) The process of claim 4, wherein the a-amino protecting reagent R2 is dissolved in an aqueous organic phase selected from chloroform/water, DMSO/water, acetone/water, methanol/water, DCM/water, DMF/water, ether/water and toluene/water.
6) The process of claim 4, wherein the reaction is carried out at an alkaline pH in the range from 8.0 to 12.0.
7) The process of claim 4, wherein R2 in Formula 4 is benzyloxycarbonyl succinimide (Z-OSu).

Z-
Formula 4 8) The process of claim 4, wherein the organic base is triethyl amine.
22
31 DEcaoie

9) The process of claim 8, wherein the triethyl amine is added as base in the range of 1.5 to 5.0 equivalents.
10) The process of claim 6. wherein the pH is about 9.0 to about 10.0.
11) An a-amino Nitrogen group protected unnatural amino acid of Formula 3 formed by process claimed in claim 1, further dissolving the same in C1-C4 alcohol and precipitating the same with cosolvent selected from diisopropyl ether or diethyl ether or further converting the same in stable salt form by dissolving in a mixture of an aqueous inorganic base and organic solvent, further mixing the mixture; removing the organic phase; and drying the aqueous phase or further dissolving the stable salt in C1-C4 alcohol and precipitating the same with cosolvent selected from diisopropyl ether or diethyl ether.
12) The process of claim 11, wherein the aqueous inorganic base is sodium hydroxide and organic solvent is acetone.
13) The process of claim 11, wherein the stable salt of Formula 4 is a sodium salt with RP-HPLC purity of atleast 90%.
14) The process of claim 11, wherein the stable salt of Formula 4 is further lyophilized or is in the form of thick syrup or in the form of dried powder.
15) The process of claim 4, wherein the protected a-amino acid of Formula 3 is further reacted with side chain protecting group Rl at alkaline pH by continuous stirring at a temperature of about 2 to 10°C to yield protected unnatural amino acid of Formula 5


T NH

23
31DEC2

16) The process of claim 15, wherein side chain protecting group Rl is
Pentamethyldihydrobenzofuran-5-sulfonyl chloride (Pbf-Cl) and R2 is
benzyloxycarbonyl succinimide (Z-OSu).
H N~,NHPbf
T
NH
Formula 6
17) The process of claim 16 wherein the side chain protected unnatural amino acid of Formula 6 has a RP-HPLC purity of atleast 85%.
18) The process of claim 15, wherein the protected unnatural amino acid of Formula 5 undergoes hydrogenolysis in the presence of catalyst to yield a deprotected unnatural amino acid of Formula 7 and further protected with a-amino protecting reagent Rl to yield protected unnatural amino acid of Formula I.

H
ML MHRl
NH

Y
2 NH

Formula 7
19) The process of claim 18, wherein the catalyst is 10% palladium on charcoal.
20) The process of claim 18, wherein the oc-amino protecting reagent Rl in Formula I is 9-fIuorenyImethyloxycarbonyl (Fmoc) and R2 in Formula I is Pentamethyldihydrobenzofuran-5-suIfonyl (Pbf)-
21) A protected unnatural amino acid of Formula 8

M H
"J T I
Fmoc-NH NH
synthesized by process claimed in claim 1 to claim 20 with a purity of atleast 95%.
22) A process for solid phase synthesis of Eptifibatide by using protected unnatural amino acid of Formula ] obtained by process of claim 1.
23) A process for solid phase synthesis of Eptifibatide by using protected unnatural amino acid of Formula 8 obtained by process of claims 1 to 21.
24) The process of c/aim 23, wherein the purity of crude Eptifibatide by RP-HPLC is atleast 55%.
25) Eptifibatide and process for preparation thereof substantially as described herein with reference to the foregoing examples and/or accompanying drawings.
Dated this the 31st day of December, 2010