WO2008098693A2

WO2008098693A2 - Convergent synthesis of glp-1

Info

Publication number: WO2008098693A2
Application number: PCT/EP2008/000846
Authority: WO
Inventors: Joachim Krüger; Hans-Christian Militzer; Konrad Siegel; Torsten Minuth
Original assignee: Bayer Healthcare Ag
Priority date: 2007-02-16
Filing date: 2008-02-02
Publication date: 2008-08-21
Also published as: WO2008098693A3

Abstract

The present invention relates to a process for the manufacture of the polypeptide GLP-1 (SEQ ID NO 1) and its PEGylated derivative, bei fragment condensation (convergent synthesis).

Description

Manufacturing Process

The present invention relates to a process for the manufacture of the polypeptide SEQ ID NO 1 and its PEGylated derivative.

The polypeptide SEQ ID NO 1 and its PEGylated derivative are described in WO 2003/040309. The PEGylated derivative consists of the polypeptide SEQ ID NO 1 comprising 31 amino acids and a PEG portion bound to the sulfhydryl group of CyS₃ , on the C-terminus. The polypeptide SEQ ID NO 1 is described as an dual GLP 1 agonist and glucagon antagonist and is effective in the treatment of diseases and conditions in connection with metabolic disorders such as diabetes, hyperglycemia, impaired fasting glucose, impaired glucose tolerance, prediabetic states and obesity.

Normally the synthesis of polypeptides e.g. by sequential solid phase synthesis is a well-known and standard process in peptide synthesis. However, the sequential peptide synthesis provides the polypeptide SEQ ID NOl only in low yields and makes an extensive purification e.g. by chromatography necessary. That is due to an aggregation of the growing peptide chains which leads to deteriorated swelling properties of the resin and ultimately to incomplete reactions and low yields.

In order to solve the problem, a convergent synthesis route is chosen, i.e. peptide fragments of the polypeptide can be first synthesized by a sequential synthesis and then combined to the final polypeptide. The crucial step in the synthesis of the polypeptides according to the invention is to choose the right peptide fragments for a retrosynthesis. The following aspects for selecting the optimal fragments have to be considered: the synthesis has to proceed with a low level of racemization, C-terminal amino acids which are known to undergo side reactions (e.g. Arg, Asp, Cys) during the coupling of the peptide fragments are to be avoided, sterically accessible amino acids at the disconnections are to be selected to ensure smooth coupling kinetics, the peptide fragments should be of equal size, and long sequences including Cys₃i is to be avoided due to possible side reactions of the sensitive Cys.

The present invention provides a convergent synthesis route of the polypeptide SEQ ID NO 1 saving significant resources and time and can be the basis for a production process on technical scale. The process according to the invention provides the polypeptides in good yields and high purity including only one chromatography at the very end of the synthesis.

The process according to the invention is a 4-fragment convergent synthesis of the polypeptide SEQ ID NO 1. The retro-synthetic strategy separates the polypeptide of SEQ ID NO 1 into three fragments of similar size which are the peptides SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4, and as fourth fragment the Arg_3O-Cys3i-dipeptide hereinafter referred to as peptide SEQ ID NO 5. The following shows the one-letter-code of the polypeptide SEQ ID NO 1 in order to demonstrate the retrosynthesis:

H-H₁S₂Q₃G₄T₅F₆T₇S₈D₉Y₁₀A₁₁R₁₂Y₁₃L₁₄D₁₅A₁₆R₁₇R₁₈A₁₉R₂₀E₂₁F₂₂I₂₃K₂₄W₂₅L₂₆V₂₇R₂₈G₂₉R₃₀C₃₁-OH, (SEQ ID NO l).

The first retrosynthetic cut is between Alan and Argπ providing the peptide SEQ ID NO 2 consisting of the amino acids 1 to 1 1, the second cut is between AIa)₉ and Arg₂o providing the peptide SEQ ID NO 3 consisting of the amino acids 12 to 19, and the third cut is between Gly₂₉ and Arg3o providing the peptide SEQ ID NO 4 consisting of the amino acids 20 to 29 and the peptide SEQ ID NO 5 consisting of the amino acids 30 to 31.

Figure 1 shows the peptide sequence of SEQ ID NO 1.

Figure 2 shows the peptide sequence of SEQ ID NO 2.

Figure 3 shows the peptide sequence of SEQ ID NO 3.

Figure 4 shows the peptide sequence of SEQ ID NO 4.

Figure 5 shows the peptide sequence of SEQ ID NO 5.

Figure 6 shows the peptide sequence of SEQ ID NO 6, which is the combination of the peptide sequences SEQ ID NO 5 and SEQ ID NO 4.

Figure 7 shows the peptide sequence of SEQ ID NO 7, which is the combination of the peptide sequences SEQ ID NO 5, SEQ ID NO 4 and SEQ ID NO 3.

Certain terms used throughout this specification are defined below, and others will be defined as introduced. The single letter abbreviation for a particular amino acid, its corresponding amino acid, and three letter abbreviation are as follows: A, alanine (Ala); C, cysteine (Cys); D, aspartic acid (Asp); E, glutamic acid (GIu); F, phenylalanine (Phe); G, glycine (GIy); H, histidine (His); I, isoleucine (He); K, lysine (Lys); L, leucine (Leu); M, methionine (Met); N, asparagine (Asn); P, praline (Pro); Q, glutamine (Gin); R, arginine (Arg); S, serine (Set); T, threonine (Thr); V, valine (VaI); W, tryptophan (Tip) ; and Y, tyrosine (Tyr). The present invention provides a process for the manufacture of the polypeptide SEQ ID NO 1 or a protected derivative thereof wherein a protected derivative of the peptide SEQ ID NO 5 reacts with a protected derivative of the peptide SEQ ID NO 4 in the presence of one or more coupling agents applicable for preparing amide bonds, and

wherein the protected derivative of the peptide SEQ ID NO 5 carries a suitable protecting group at the C-terminus, a free amino group at the N-terminus and one or more suitable protecting groups at the side chain functionalities, and

wherein the protected derivative of the peptide SEQ ID NO 4 carries a suitable protecting group at the N-terminus and one or more suitable protecting groups at the side chain functionalities.

The product of the reaction between the protected derivative of the peptide SEQ ID NO 5 and the protected derivative of the peptide SEQ ID NO 4 is the peptide SEQ ID NO 6 or a derivative thereof which optionally carries one or more protecting groups.

The present invention also provides a process for the manufacture of the polypeptide SEQ ID NO 1 or a protected derivative thereof wherein a protected derivative of the peptide SEQ ID NO 6 reacts with a protected derivative of the peptide SEQ ID NO 3 in the presence of one or more coupling agents applicable for preparing amide bonds, and

wherein the protected derivative of the peptide SEQ ID NO 6 carries a suitable protecting group at the C-terminus, a free amino group at the N-terminus and one or more suitable protecting groups at the side chain functionalities, and

wherein the protected derivative of the peptide SEQ ID NO 3 carries a suitable protecting group at the N-terminus and one or more suitable protecting groups at the side chain functionalities.

The product of the reaction between the protected derivative of the peptide SEQ ID NO 6 and the protected derivative of the peptide SEQ ID NO 3 is the peptide SEQ ID NO 7 or a derivative thereof which optionally carries one or more protecting groups.

Further the present invention provides a process for the manufacture of the polypeptide SEQ ID NO 1 or a protected derivative thereof wherein a protected derivative of the peptide SEQ ID NO 7 reacts with a protected derivative of the peptide SEQ ID NO 2 in the presence of one or more coupling agents applicable for preparing amide bonds, and wherein the protected derivative of the peptide SEQ ID NO 7 carries a suitable protecting group at the C-terminus, a free amino group at the N-terminus and one or more suitable protecting groups at the side chain functionalities, and

wherein the protected derivative of the peptide SEQ ID NO 2 carries a suitable protecting group at the N-terminus and one or more suitable protecting groups at the side chain functionalities.

The product of the reaction between the derivative of the peptide SEQ ID NO 7 and the derivative of the peptide SEQ ID NO 2 is the peptide SEQ ID NO 1 or a derivative thereof which optionally carries one or more protecting groups.

Preference is given to a process for the manufacture of the polypeptide SEQ ID NO 1 or a protected derivative thereof comprising the following steps:

a) a protected derivative of the peptide SEQ ID NO 5 reacts with a protected derivative of the peptide SEQ ID NO 4 in the presence of one or more coupling agents applicable for preparing amide bonds, and

b) the product of step a), which is a protected derivative of the peptide SEQ ID NO 6, reacts with a protected derivative of the peptide SEQ ID NO 3 in the presence of one or more coupling agents applicable for preparing amide bonds, and

wherein the protected derivative of the peptide SEQ ID NO 3 carries a suitable protecting group at the N-terminus and one or more suitable protecting groups at the side chain functionalities. c) the product of step b), which is a protected derivative of the peptide SEQ ID NO 7, reacts with a protected derivative of the peptide SEQ ID NO 2 in the presence of one or more coupling agents applicable for preparing amide bonds, and

wherein the protected derivative of the peptide SEQ ID NO 7 carries a suitable protecting group at the C-terminus, a free amino group at the N-terminus and one or more suitable protecting groups at the side chain functionalities, and

The protected derivative of the polypeptide SEQ ID NO 1 can be deprotected for example by standard techniques known in the art.

The crude deprotected polypeptide SEQ ID NO 1 can be purified by standard purification techniques as for example chromatography, precipitation or crystallisation.

The peptides SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5 and/or their protected derivatives according to the invention may be synthesized by any of the chemical synthesis techniques known in the art, for example solution or solid phase, preferably solid phase synthesis techniques, for example, using commercially available automated peptide synthesizers (see, e.g., Merrifield, J. Amer. Chem. Soc. 85:2149-54, 1963; Carpino, Ace. Chem. Res. 6: 191 -98, 1973; Birr, Aspects of the Merrifield Peptide Synthesis, Springer- Verlag: Heidelberg, 1978; The Peptides: Analysis, Synthesis, Biology, VoIs. 1, 2, 3, and 5, (Gross & Meinhofer, eds.), Academic Press: New York, 1979; Stewart, et al., Solid Phase Peptide Synthesis, 2nd. ea., Pierce Chem. Co.: Rockford,l l l ., 1984; Kent, Ann. Rev. Biochem. 57:957-89, 1988; and Gregg, et al., Int. J. Peptide Protein Res. 55:161 -214, 1990, Sewald, N; Jakube, H.-D. Peptides: Chemistry and Biology, Wiley- VCH, Weinheim, 2002).

In particular solid phase methodology may be used. Briefly, an N-protected C-terminal amino acid residue is linked to an insoluble support such as divinylbenzene cross-linked polystyrene, polyacrylamide resin, Kieselguhr/polyamide (pepsyn K), controlled pore glass, cellulose, polypropylene membranes, acrylic acid-coated polyethylene rods, or the like. Cycles of deprotection, neutralization, and coupling of successive protected amino acid derivatives are used to link the amino acids from the C-terminus according to the amino acid sequence. For some synthetic peptides, an Fmoc strategy using an acid-sensitive resin may be used. Solid supports in this regard are divinylbenzene cross-linked polystyrene resins, which are commercially available in a variety of functionalized forms, including chloromethyl resin, hydroxymethyl resin, paraacetamidomethyl resin, benzhydrylamine (BHA) resin, 4-methylbenzhydrylamine (MBHA) resin, oxime resins, 4- alkoxybenzyl alcohol resin (Wang resin), 4-(2',4'-dimethoxyphenyl; aminomethyl)-phenoxymethyl resin, 2,4-dimethoxybenzhydryl-amine resin, 4-(2',4' dimethoxyphenyl-Fmoc-amino-methyl)- phenoxyacetamidonorleucyl-MBHA resin (Rink amide I MBHA resin), and chlorotrityl resin. Preference is given to the chlorotrityl resin. In addition, acid-sensitive resins also provide C-terminal acids, if desired. A protecting group for the amino function in alpha amino acids is the base-labile 9- fluorenylmethoxy-carbonyl (Fmoc) group or the acid-labile tert.-butyloxycarbonyl (Boc) group.

Suitable protecting groups for the side chain functionalities of amino acids chemically are well known in the art (Peter G. M. Wuts and Theodora W. Greene, Greene's Protective Groups in Organic Synthesis, 4th Edition, 2006, Willey&Sons).

Examples of protecting groups include, but are not limited to, tert.-butyloxycarbonyl (Boc), fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Aloe), tert.-butyloxy (OtBu), tert.-butyl (tBu), trityl or triphenylmethyl (Trt), pyridyldiphenylmethyl (Pdpm), 2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), 4-toluenesulfonyl (Ts), 4-methoxy-2,3,6-trimethylbenzene-solfonyl (Mtr), 4-methoxybenzenesulfonyl (Mbs), allyl (Al), benzyl (BzI), 4-methoxybenzyl (Mob), 2,4,6-trimethoxybenzyl (Tmb), acetamidomethyl (Acm).

The following protected amino acid derivatives are preferred as building block for the synthesis of the polypeptides and its protected derivatives according to the invention: Fmoc-Asn(Trt)-OH, Fmoc- Thr(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Boc)₂-OH, Fmoc-Pro-OH, Boc-His(Trt)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc- Asp(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc- GIu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tφ(Boc)-OH, Fmoc-Val-OH, Fmoc- Cys(Trt)-OH and H-Cys(Trt)-OH.

The protected derivative of the polypeptide SEQ ID NO 1 is preferably the compound A which is Boc-His(Trt)-Ser(tBu)-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ala- Arg(Pbf)-Tyr(tBu)-Leu-Asp(OtBu)-Ala-Arg(Pbf)-Arg(Pbf)-Ala-Arg(Pbf)-Glu(OtBu)-Phe-Ile- Lys(Boc)-Tφ(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Cys(Trt)-OtBu. The protected derivative of the polypeptide SEQ ID NO 2 is preferably the compound B which is

Boc-His(Trt)-Ser(tBu)-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ala-

OH.

The protected derivative of the polypeptide SEQ ID NO 3 is preferably the compound C which is Fmoc-Arg(Pbf)-Tyr(tBu)-Leu-Asp(OtBu)-Ala-Arg(Pbf)-Arg(Pbf)-Ala-OH.

The protected derivative of the polypeptide SEQ ID NO 4 is preferably the compound D which is Fmoc-Arg(Pbf)-Glu(OtBu)-Phe-Ile-Lys(Boc)-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-OH.

The protected derivative of the polypeptide SEQ ID NO 5 is preferably the compound E which is H- Arg(Pbf)-Cys(Trt)-OtBu.

The protected derivative of the polypeptide SEQ ID NO 6 is preferably the compound F which is ^' Fmoc-Arg(Pbf)-Glu(OtBu)-Phe-Ile-Lys(Boc)-Tφ(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Cys(Trt)- OtBu.

The protected derivative of the polypeptide SEQ ID NO 7 is preferably the compound G which is Fmoc-Arg(Pbf)-Tyr(tBu)-Leu-Asp(OtBu)-Ala-Arg(Pbf)-Arg(Pbf)-Ala-Arg(Pbf)-Glu(OtBu)-Phe-Ile- Lys(Boc)-Tφ(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Cys(Trt)-OtBu.

In order to build up the polypeptides and/or their protected derivatives according to the invention the amino acid derivatives and/or the polypeptide fragments may be coupled by using a variety of coupling agents and chemistries known in the art, such as direct coupling with DIC (diisopropyl- carbodiimide), DCC (dicyclohexylcarbodiimide), BOP (benzotriazolyl-N-oxytrisdimethylam inophosphonium hexa-fluorophosphate), PyBOP (benzotriazole-1-yl-oxy-tris- pyrrolidinophosphonium hexafluoro-phosphate), PyBrOP (bromo-tris-pyrrolidinophosphonium hexafluorophosphate); via preformed symmetrical anhydrides; via active esters such as pentafluorophenyl esters; or via preformed HOBt (1-hydroxybenzotriazole) active esters or by using Fmoc-amino acid fluoride and chlorides or by using Fmoc-amino acid-N-carboxy anhydrides; via activation with HBTU (2-(lH-benzotriazole-l-yl),l ,l,3,3-tetramethyluronium hexafluorophosphate), HATU (2-(lH-7-aza-benzotriazole-l-yl),l,l,3,3-tetramethyluronium hexafluoro-phosphate) or TBTU (O-(Benzotriazol-l-yl)-N,N,N^N'-tetramethyluronium tetrafluoroborate) optionally in the presence of HOBt or HOAt (7-azahydroxybenztriazole). Preference is given to TBTU in the presence of HOBt.

The solid phase method may be carried out manually, although automated synthesis on a commercially available peptide synthesizer (e.g., Applied Biosystems 43 IA or the like; Applied Biosystems, Foster City, CA) may be used. In a typical synthesis, the first N-protected (C-terminal) amino acid, preferably Fmoc protected, is loaded on the resin, preferably the chlorotrityl resin using in inert solvent in the presence of an organic base. Successive deprotection, preferably for example with a 20% solution of piperidine in NMP (N-methylpyrrolidone) or DMF (dimethylformamide), and coupling cycles according to ABI FastMoc protocols (Applied Biosystems) may be used to generate the peptide sequence. Double and triple coupling, with capping by acetic anhydride, may also be used.

The protected peptide derivatives according to the invention may be cleaved from the resin by using a dilute solution of trifluoroacetic acid (TFA) in for example dichloromethane, mixtures of trifluoroethanol/dichloromethane, trifluoroethanol/dichloromethane/acetic acid, hexafluoro-iso- propanol/dichloromethane and hexafiuoro-iso-propanol/dichloromethane/actic acid. The peptide may be separated from the resin by filtration and isolated by precipitation from a less polar solvent such as ether. Further purification may be achieved by conventional methods, such as flash chromatography, gel filtration, reverse phase HPLC (high performance liquid chromatography) or lyophilisation.

In particular compound B can be synthesized preferably by solid phase synthesis. In the first step the resin, preferably a chlorotrityl resin, is loaded with Fmoc -AIa-OH in the presence of a base, preferably diisopropyl ethyl amine, and in an inert solvent, preferably dichloromethane. After 2 hours the resin can be filtered and optionally the loading procedure can be repeated. Subsequently, the resin is optionally capped using an excess of methanol in the presence of a base, preferably diisoproyl ethyl amine. Finally the resin is filtered, washed with inert solvents, for example dichloromethane and/or dimethylformamide and dried. The Fmoc-deprotection is accomplished with a base, preferably piperidine in a polar solvent preferably dimethylformamide. Optionally the Fmoc- deprotection can be repeated. The coupling reaction with the next amino acid building block of the peptide chain is accomplished by adding the side chain protected amino acid, which is Fmoc- protected at the N-terminus, to the resin in an inert solvent, preferably in dimethylformamide, in the presence of a coupling agent, for example preferably HOBt and TBTU, and a base, preferably diisopropyl ethyl amine. The resin is filtered and optionally the coupling can be repeated. After successful coupling and before the next coupling step the Fmoc-protecting group is to be removed and the coupling procedure is to be done with the next amino acid building block. After all couplings the resin is filtered, washed with an inert solvent, preferably dimethylformamide, and dried. Finally the product is cleaved from the resin in the presence of trifluoroethanol in for example dichloromethane as solvent. The compound B is isolated by drying the filtrate in vacuo and can be precipitated from for example diisopropyl ether. In particular compound C can be synthesized preferably by solid phase synthesis. In the first step the resin, preferably a chlorotrityl resin, is loaded with Fmoc -AIa-OH in the presence of a base, preferably diisopropyl ethyl amine, and in an inert solvent, preferably dichloromethane. After 2 hours the resin can be filtered and optionally the loading procedure can be repeated. Subsequently, the resin is optionally capped using an excess of methanol in the presence of a base, preferably diisoproyl ethyl amine. Finally the resin is filtered, washed with inert solvents, for example dichloromethane and/or dimethylformamide and dried. The Fmoc-deprotection is accomplished with a base, preferably piperidine in a polar solvent preferably dimethylformamide. Optionally the Fmoc- deprotection can be repeated. The coupling reaction with the next amino acid building block of the peptide chain is accomplished by adding the side chain protected amino acid, which is Fmoc- protected at the N -terminus, to the resin in an inert solvent, preferably in dimethylformamide, in the presence of a coupling agent, for example preferably HOBt and TBTU, and a base, preferably diisopropyl ethyl amine. The resin is filtered and optionally the coupling can be repeated. After successful coupling and before the next coupling step the Fmoc-protecting group is to be removed and the coupling procedure is to be done with the next amino acid building block. After all couplings the resin is filtered, washed with an inert solvent, preferably dimethylformamide, and dried. Finally the product is cleaved from the resin in the presence of trifluoroethanol in for example dichloromethane as solvent. The compound C is isolated by drying the filtrate in vacuo and can be precipitated from for example diisopropyl ether.

In particular compound D can be synthesized preferably by solid phase synthesis. In the first step the resin, preferably a chlorotrityl resin, is loaded with Fmoc-Gly-OH in the presence of a base, preferably diisopropyl ethyl amine, and in an inert solvent, preferably dichloromethane. After 2 hours the resin can be filtered and optionally the loading procedure can be repeated. Subsequently, the resin is optionally capped using an excess of methanol in the presence of a base, preferably diisoproyl ethyl amine. Finally the resin is filtered, washed with inert solvents, for example dichloromethane and/or dimethylformamide and dried. The Fmoc-deprotection is accomplished with a base, preferably piperidine in a polar solvent preferably dimethylformamide. Optionally the Fmoc- deprotection can be repeated. The coupling reaction with the next amino acid building block of the peptide chain is accomplished by adding the side chain protected amino acid, which is Fmoc- protected at the N-terminus, to the resin in an inert solvent, preferably in dimethylformamide, in the presence of a coupling agent, for example preferably HOBt and TBTU, and a base, preferably diisopropyl ethyl amine. The resin is filtered and optionally the coupling can be repeated. After successful coupling and before the next coupling step the Fmoc-protecting group is to be removed and the coupling procedure is to be done with the next amino acid building block. After all couplings the resin is filtered, washed with an inert solvent, preferably dimethylformamide, and dried. Finally the product is cleaved from the resin in the presence of trifluoroethanol in for example dichloromethane as solvent. The compound D is isolated by drying the filtrate in vacuo and can optionally be precipitated from for example diisopropyl ether.

In particular compound E can be synthesized preferably by solution phase synthesis. For example H- Cys(Trt)-OH is added to methyl sulfonic acid at room temperature under inert gas atmosphere in methyl tert. -butyl ether. Subsequently, trichloroacetimidate is added and the mixture is stirred for 5 to 20 h at room temperature. Afterwards the reaction mixture is worked-up and H-Cys(Trt)OtBu is isolated. For the coupling with the arginine building block, H-Cys(Trt)OtBu reacts with Fmoc- Arg(Pbf)-OH in the presence of a coupling agent, for example preferably HOBt and TBTU, and a base, preferably diisopropyl ethyl amine, in an inert solvent for example dimethylformamide at room temperature. After work-up of the reaction mixture Fmoc-Arg(Pbf)-Cys(Trt)-OtBu can be isolated. For Fmoc-deprotection piperidine is added in an inert solvent, for example dimethylformamide. After work-up of the reaction mixture compound E can be isolated.

Alternatively compound E can be synthesized by reacting of Fmoc-Cys(Trt)-OH with isobutene in the presence of an acid, for example sulphuric acid in an inert solvent, for example dichlorormethane. After aqueous work-up the crude tert-butyl ester is used in the next step, optionally with purification. Fmoc-deprotection is accomplished by piperidine in an inert solvent, for example dichlorormethane. After aqueous work-up the crude mixture is submitted to the peptide coupling with Fmoc-Arg(Pbf)-OH in the presence of a coupling agent, for example preferably of HOBt and TBTU, and a base, preferably diisopropyl ethyl amine, in an inert solvent for example dichloromethane, dimethylformamide or a mixture thereof. After precipitation from water Fmoc is removed from the crude product by adding piperidine in the presence of an inert solvent, for example dichloromethane. The compound E can be isolated by precipitation from water and can be purified by crystallization and/or chromatography.

Another embodiment of the present invention is a process for preparing of compound F by reacting of compound D with compound E in the presence of a coupling agent, preferably of HOBt and TBTU, and a base, preferably diisopropyl ethyl amine, in an inert solvent, preferably dimethylformamide. After reaction for around 20 min. at room temperature compound F can be isolated by precipitation from water.

The concentration of compound D in the reaction mixture is from 10 to 200 mM, preferably from 20 to 100 mM. The concentration of compound E in the reaction mixture is from 10 to 200 mM, preferably from 20 to 100 mM. Another embodiment of the present invention is a process for preparing of compound G. Therefore the Fmoc-protecting group has to be removed from compound F. Fmoc-deprotection is accomplished by treating compound F with piperidine in dimethylformaide at room temperature. After adding of acetonitril the mixture is poured into diisopropyl ether, filtered and washed with diisopropyl ether. The Fmoc-deprotected product is isolated and dried. Alternatively, the Fmoc-deprotected product can also be isolated by flash-chromatography using dichloromethane/trifluoroethanol/methanol as eluent mixture. Afterwards the Fmoc-deprotected derivative of compound F reacts with compound C in the presence of a coupling agent, preferably of HOBt and TBTU, and a base, preferably diisopropyl ethyl amine, in an inert solvent, preferably dimethylformamide. After reaction for around 20 min. at room temperature compound G can be isolated by precipitation from water.

The concentration of compound F in the reaction mixture is from 10 to 200 mM, preferably from 20 to 100 mM. The concentration of compound C in the reaction mixture is from 10 to 200 mM, preferably from 20 to 100 mM.

Another embodiment of the present invention is a process for preparing of compound A. Therefore the Fmoc-protecting group has to be removed from compound G. Fmoc-deprotection is accomplished by treating compound G with piperidine in dioxane at room temperature. After adding of acetonitril the product is precipitated from a less polar solvent, preferably diisopropyl ether, filtered and washed with diisopropyl ether optionally under reduced pressure. The Fmoc-deprotected product is isolated and dried. Afterwards the Fmoc-deprotected derivative of compound G reacts with compound B in the presence of a coupling agent, preferably of HOBt and TBTU, and a base, preferably diisopropyl ethyl amine, in an inert solvent, preferably dimethylformamide. After reaction for around 20 min. at room temperature compound A can be isolated by precipitation from water.

The concentration of compound G in the reaction mixture is from 10 to 200 mM, preferably from 20 to 100 mM. The concentration of compound B in the reaction mixture is from 10 to 200 mM, preferably from 20 to 100 mM.

In order to remove traces of piperidine as an impurity stemming from the Fmoc-deprotection protocols, the crude products according to the invention can be precipitated from water or lipophilic solvents for example diisopropyl ether and then be filtered at reduced pressure. Alternatively, the crude product can be extracted with 1 M aqueous HCl optionally in the presence of a polar non- water soluble solvent preferably dichloromethane or a mixture of dichloromethane/trifluoroethanol. The excess of piperidine can also be removed by azeotropically destination using a carrier solvent, preferably dimethylformamide. Preferably the azeotropic distillation operates at a temperature of 35- 50⁰C, more preferably 40-45⁰C at reduced pressure. Another embodiment of the present invention is a process for preparing of peptide SEQ ID NO 1 wherein all protecting groups are removed from compound A. This can be accomplished by any means known to one skilled in the art. For instance by using a mixture comprising trifluoroactic acid, 1 ,4-dithioerythriole and triisopropylsilane in a solvent mixture of dichloromethane combined with trifluoroethanol, preferably a mixture of dichloromethane and trifluoroethanol in a ratio of from 10:1 up to 1 : 1, more preferably in a ratio from 5: 1 up to 3: 1. The reaction mixture can be stirred at room temperature for 2 to 6 h. Afterwards dichloromethane can be added and the mixture is poured into diisopropy ether at a temperature of 0 to 10⁰C, preferably 5°C. The crude peptide SEQ ID NO 1 can be isolated by precipitation, filtration and drying. The crude peptide SEQ ID NO 1 can be purified by chromatography.

The concentration in the reaction mixture of 1 ,4-dithioerythriole is from 0.5 up to 2 M, preferably around 1.5 M. The concentration in the reaction mixture of triisopropylsilane is from 0.5 up to 2 M, preferably around 1.2 M. In the reaction mixture the ratio of trifluoroacetic acid and the solvent mixture of dichloromethane and trifluoroethanol is from 1 :1 up to 3 : 1 , preferably around 2: 1.

Moreover, the present polypeptides can be fused with another compound, such as a compound to increase the half-life of the polypeptide and/or to reduce potential immunogenicity of the polypeptide (e.g., polyethylene glycol, "PEG" or fatty acid). In the case of PEGylation, the fusion of the polypeptide to PEG may be accomplished by any means known to one skilled in the art. For example, PEGylation may be accomplished by site-specific derivatization with PEG-maleimide at the Cys position. In addition to maleimide, numerous Cys reactive groups are known to those skilled in the art of protein crosslinking, such as the use of alkyl halides and vinyl sulfones (see, e.g., T. E. Creighton, Proteins, 2nd Ed., 1993).

The linker between the PEG and the peptide crosslinking group may be varied. For example, the commercially available Cys-reactive 41-45 kDa PEG (mPEG2-MAL; Nektar, San Carlos, CA) employs a maleimide group for conjugation to Cys, and the maleimide group is attached to the PEG via a linker that contains a Lys. As a second example, the commercially available Cys-reactive 43 kDa PEG (GL2-400MA; NOF, Tokyo, Japan) employs a maleimide group for conjugation to Cys, and the maleimide group is attached to the PEG via a bisubstituted alkane linker.

Numerous examples of suitable crosslinking agents are known to those skilled in the art (see, e.g., T. E. Creighton, Proteins, 2nd Ed., 1993). Such crosslinking agents may be linked to PEG as exemplified, but not limited to, by commercially available PEG derivatives containing amines, aldehydes, acetals, maleimide, succinimides, and thiols that are marketed, for example, by Nektar and NOF (e.g., Harris, et al., Clin. Pharmokinet. 40, 539-551, 2001). The present invention provides an improved process for preparation of the PEGylated derivative of polypeptide SEQ ID NOl, which is SEQ ID NO 1 PEG, carrying a PEG portion bound to the sulfhydryl group of Cys_3] on the C-terminus via a maleimide group,

wherein the polypeptide SEQ ID NO 1 is treated with the compound of the formula (I)

for example preferably at room temperature in a buffer system at a pH of 6.0 to 7.0, preferably 6.2 to 6.8, more preferably at a pH around 6.5,

wherein rnPEG is a polyethylenglycol chain carrying a terminal methyl group of a molecular weight of 41-45 kDa, preferably 43 kDa.

The concentration of the peptide SEQ ID NO 1 in the reaction mixture is from 0.1 to 10 %, preferably from 0.5 to 5 % by weight of the reaction mixture. The concentration of the compound of formula (I) in the reaction mixture is from 1 to 20 %, preferably from 5 to 15 % by weight of the reaction mixture.

Examples of buffer systems include, but are not limited to, aqueous sodium dihydrogen phosphate solution. The pH can be adjusted by adding a sodium hydroxide solution, for example an aqueous 0.1 M sodium hydroxide solution.

After the PEGylation reaction, the reaction mixture is filtered and purified by chromatography preferably using commercially available cationic exchange resin such as Sepharose Fast Flow FF or Source 15S (source: GE Healthcare) followed by an ultrafiltration step.

The reactions are generally carried out at atmospheric pressure. However, it is also possible to work at elevated pressure or at reduced pressure (for example in a range of from 0.5 to 5 bar).

The present invention likewise includes all combinations of the areas of preference. The present invention will now be illustrated in detail with reference to non-limiting prefened examples. Unless stated otherwise, all amounts relate to percentages by weight.

Examples:

All solid phase reactions are performed using a custom made solid phase peptide synthesis (SPPS) devices with overhead stirring and porous filter plates at the bottom to allow for fast and efficient filtration.

HPLC method A:

column: Phenomenex Prodigy ODS3, column symmetry: 50x4.6 mm, 3 μm; T: 45°C; eluent: A = 1 mL formic acid in 1 L of water, B = 1 mL formic acid in 1 L of acetonitrile, gradient: 0-10 min from 95% A to 0% A, 10-20 min 100% B; flow rate: 1.2 ml/tnin; detection at 220 nm.

HPLC method B:

column: Chiral AD-H, column symmetry: 250x4.6 mm, 5 ?m; T: 45°C; eluent: A = n-heptane + 0.2% trifluoroacetic acid, B = ethanol + 0.2% trifluoroacetic acid, 0-20 min isocratic at 90% A; flow rate: 1 ml/min; detection at 210 nm.

HPLC method C:

column: ZORBAX 300SB-C3 (Agilent), column symmetry: 150x3.0 mm, 3.5 ?m; T: 45°C; eluent: A = water + 0.1% trifluoroacetic acid, B = tetrahydrofurane + 0.05% trifluoroacetic acid, gradient: 0-45 min from 90% A to 0% A, 45-50 min 100% B; flow rate: 0.5 ml/min; detection at 220 nm.

HPLC method D:

column: ZORBAX 3OOSB-C3 (Agilent), column symmetry: 150x3.0 mm, 3.5 ?m; T: 45°C; eluent: A = water + 0.1% trifluoroacetic acid, B = tetrahydrofurane + 0.05% trifluoroacetic acid, gradient: 0-15 min from 90% A to 100% B, 15-20 min 100% B; flow rate: 0.5 ml/min; detection at 220 nm.

LC-MS:

Is carried out using a Finnigan LCQ Deca XP MAX in combination with a HP 1 100 chromatography unit

Example 1 : Peptide SEO ID NO 1 PEG

Pegylation buffer: A solution of 7.9 g sodium dihydrogen phosphate in 5.0 L water for injection is adjusted to pH 6.5 using 11 mL of a 1 M solution of sodium hydroxide. Endotoxines are determined prior to use. Chromatopraphy buffer A: A solution of 150 g sodium acetate trihydrate in 55 L water for injection is adjusted to pH 4.5 using 750 mL of a 2 M solution of acetic acid. Endotoxines are determined prior to use.

Chromatopraphy buffer B: A solution of 133 g tris(hydroxymethyl)amino-methane and 321 g sodium chloride in 55 L water for injection is adjusted to pH 9.5 using 25 mL of a 1.2 M hydrochloric acid. Endotoxines are determined prior to use.

Washout buffer: A solution of 5.4 g sodium acetate trihydrate in 2 L water for injection is adjusted to pH 5.0 using 9 mL of a 2 M acetic acid. Endotoxines are determined prior to use.

All operations are carried out in a sterile environment.

To a clear solution of 40.0 g pegylation reagent (GL2-400 MA Sunbright from NOF, Japan) in 2 L of pegylation buffer a solution of 4.0 g of peptide SEQ ID NO 1 (69% peptide content according to UV-absorbance) in 2 L of pegylation buffer is added at 23°C. The pH of the resulting solution is adjusted to 6.5 using 4.5 mL of a 1 M aqueous sodium hydroxide solution. The mixture is stirred (250 rpm) for 30 min at 23⁰C before 0.96 g cysteine dissolved in 40 ml pegylation buffer is added. The reaction mixture is filtered through a Sartopore 2 filter (300 5441307H5-00-B) and the resulting solution (65 mL) is submitted to the subsequent chromatography in two batches, each of 2.02 L.

The column is packed with 2.9-3.0 L of SP Sepharose Fast Flow, which is equilibrated using ethanol (20% in water for injection). Prior to use, the column is flooded with 0.5 M sodium hydroxide and then washed with chromatopraphy buffer A. The column is charged with the first portion of the reaction mixture, it is washed with one column volume of chromatopraphy buffer A and then a gradient towards 100% chromatopraphy buffer B is initiated. The desired product elutes at a buffer A/ buffer B ratio of approx. 60:40. The pH of the product fraction (8.0 L) is adjusted to 5.0 using 1 1 ml of a 1.2 M hydrochloric acid. The solution is filtered through a Sartopore 2 filter (300 5441307H5-00-B) and stored at 2-8°C for two days.

The second batch of the reaction mixture is purified accordingly. Both product fractions are combined to give a total of 16.5 L of the purified product.

The ultrafiltration unit is equilibrated with 0.5 M sodium hydroxide and subsequently flushed with water for injection until the pH is neutral. The pooled product fractions from chromatography are concentrated to a volume of 1 L using a Slice Hydrosart 10 kD membrane. Eventually, the ultrafiltration unit is washed with 1 L of washout buffer to yield a combined product phase of 2 L. The level of endotoxines is determined (< 0.12 EE/mL), it is filtered through a Sartopore 2 filter (300 5441307H5-00-B) into sterile bottles and finally stored at -20⁰C.

The product concentration is determined by UV-absorbance (c= 1.18 g/L). This translates into a yield of 2.36 g based on peptide SEQ ID NO 1 (86%).

Example 2: H-Arg(Pbf)-Cvs(Trf)-OtBu, compound E

To a solution of 50.0 g (138 mmol) H-Cys(Trt)-OH in 500 mL THF, 33.2 g (345 mmol) methyl sulfonic acid is added in an inert gas atmosphere within 10 min. The temperature should not exceed 25°C during addition. Subsequently, at 20⁰C a solution of 151 g (690 mmol) tert. -butyl trichloroacetimidate in 1.0 L methyl tertbutyl ether is added and it is stirred for 18 hrs at 2O⁰C. For work-up, 500 mL water is added and the pH is adjusted to 9.0 employing 355 mL of a 1 molar aqueous NaOH. The phases are separated and the organic layer is washed with 500 mL water, dried over magnesium sulphate and concentrated in vacuo. The residue is suspended in 500 mL toluene, it is stirred at 2O⁰C for 30 min and then filtered. To the filtrate 250 mL water is added and the pH is adjusted to 11 using 1 molar aqueous NaOH. The layers are separated and the toluene layer is washed twice with each of 250 mL water. In an independent experiment, a portion of the toluene solution is evaporated to dryness and the residue is analyzed as outlined above (method B). Accordingly, the ester is formed with complete retention of the stereogenic centre (99.8 % ee).

The resulting toluene solution of H-Cys(Trt)-OtBu (86 area%, method A) is diluted with 125 mL diemthylformamide. Subsequently, 9.3 g (69 mmol) HOBt monohydrate, 53.5 g (414 mmo) N,N- diisoproyl ethyl amine, 53.8 g (83 mmol) Fmoc-Arg(Pbf)-OH and 66.5 g (207 mmol) TBTU is added at 20⁰C and it is stirred for 30 min. HPLC analysis indicated a conversion of 90%. Therefore, additional 9.1 g (14 mmol) Fmoc-Arg(Pbf)-OH are added and the mixture is allowed to stir at 2O⁰C for further 30 min after which the conversion rises to 97%. For work-up, 500 mL water is added, the layers are separated and the organic layer is washed with 500 mL water. Then, the product solution is dropwise added to 2.5 L heptane and the resulting suspension is stirred for 1 h at 20⁰C prior to isolation. It is filtered, washed twice with each of 100 ml heptane and dried in vacuo to yield 98 g of

Fmoc-Arg(Pbf)-Cys(Trt)-OtBu in a quality of 85 area%. For further purification, the crude product is redissolved in 490 mL toluene and the resulting solution is added dropwise to 2.45 L diisopropyl ether. The resulting suspension is stirred at 20⁰C for 1 h, it is filtered and washed twice with each of

100 mL diisopropyl ether. Finally, it is dried in vacuo at 20⁰C to yield 84 g of Fmoc-Arg(Pbf)- Cys(Trt)-OtBu in a purity of 92 area% (method A). This corresponds to yield of 58% for two steps commencing from H-Cys(Trt)-OH.

The resulting 84 g (80 mmol) Fmoc-Arg(Pbf)-Cys(Trt)-OtBu are dissolved in 840 mL dimethylformamide / piperidine (3:1) at 0⁰C. After 10 min the reaction mixture is slowly added to 4.2 L water over a period of 3.75 hrs and it is stirred at O⁰C for additional 30 min. It is filtered and washed twice with each of 200 mL water. Subsequently, it is redissolved in methyl tertbutyl ether and the aqueous layer is separated. The organic layer is added to 2.1 L diisopropyl ether over a period of 1.5 hrs, and, subsequently, it is stirred for 30 min at 20⁰C. Eventually, it is filtered and washed twice with each of 100 mL diisopropyl ether. It is dried in vacuo for 18 hrs to yield 51 g (77%) of compound E.

HPLC: 93% purity (method C)

¹HNMR (500 MHz, D⁶-DMSO): d = 1.30-1.65 (m, 4H), 1.31 (s, 9H), 1.40 (s, 6H), 1.85 (m, broad, 2H), 1.98 (s, 3H), 2.35-2.50 (m, 2H), 2.42 (s, 3H), 2.50 (s, 3H), 2.95 (s, 2H), 3.03 (m, 2H), 3.11 (m, IH), 4.12 (m, IH), 6.40 (s, broad, IH), 6.58 (s, broad, IH), 7.22-7.35 (m, 15H), 8.27 (s, broad, I H).

MS (ES⁺): [M+H] = 829.

Example 3 : Fmoc-ArgfPbiyGlufOtBuVPhe-Ile-LvsfBocVTrpfBocVLeu- Val-Arg(Pbf)-Glv-OH. compound D.

Resin loading:

At 20⁰C, 100 g of the chlorotrityl resin (loading: 1.56 mmol/g, source: Iris Biotech) are allowed to swell for 30 min in 800 ml of dichloromethane. It is filtered and 800 ml of fresh dichloromethane, 14.8 g (49.8 mmol, source: Bachem) Fmoc-Gly-OH and 20.2 g (156.3 mmol) diisopropyl ethyl amine (DIPEA) are added. It is allowed to stir for 2 hours at 20⁰C. It is filtered and rinsed with 800 ml of dichloromethane. Subsequently, it is stirred with 500 ml of a solution of 5% diisopropyl ethyl almine and 10% methanol in dichloromethane for 10 min. After filtration, this process is repeated allowing a reaction time of 20 min. Subsequently, the resin is washed with 500 ml dimethylformamide (Ix) and 500 ml dichloromethane (3x) and dried in vacuo at 2O⁰C to yield 1 10 g of the loaded resin. The loading is determined as follows: a sample of ca. 10-20 mg of the resin is incubated with 50 mL of a solution of 20% piperidine in DMF for at least 4 hours. The absorbance of the supernatant is measured at 301 nm and the loading is calculated according to Lambert-Beer with. In this example the loading is 0.38 mmol/g.

Fmoc-deprotection:

After resin loading and prior to the first deprotection, the resin is allowed to swell in 500 ml dimethylformamide for 1 hour. For all following Fmoc-deprotections this measure is not necessary since the resin is sufficiently swollen prior to the Fmoc-removal step.

In general, Fmoc-deprotection is accomplished upon stirring of the resin in 1 L of a 20% piperidine solution in dimethylformamide for 10 min at 2O⁰C. It is filtered and the process is repeated with a fresh portion of 1 L of the piperidin solution for another 10 min. Finally, it is filtered and rinsed five times with each of 1 L of dimethylformamide. On a routine basis, the filtrates are tested on traces of piperdine using the chloranil test. Accordingly, three drops of the filtrate are added to a solution of 3 mL of acetone containing three drops of a concentrated solution of chloranil (tetrachloro- benzochinone) in toluene. The blue colour indicates the presence of an excess of amine in the filtrate.

Coupling reactions:

To a suspension of the resin in 1 L of dimethylformamide, 83 mmol (2 eq) of the amino acid, 2.8 g (21 mmol, 0.5 eq) HOBt, 10.8 g (83 mmol, 2 eq.) diisopropyl ethyl amine and 26.7 g (83 mmol, 2 eq.) TBTU are added and it is stirred at 20⁰C for 1 hour. The reaction mixture is drained off and the resin is washed three times with each of 1 L dimethylformamide.

Progress of the reaction can be monitored by using the "Kaiser test": a small aliquot of the resin is thoroughly rinsed with ethanol. Subsequently, 2 drops of each of the following solution are added: 80% phenol in ethanol, 5% ninhydrin in ethanol and 2 ml of an aqueous 0.001 M KCN solution in 98 ml pyridine. It is heated to 100⁰C for 5 min and the resin is washed with ethanol. A blue coloured resin indicates presence of amine, which corresponded to an insufficient conversion.

The following quantities of amino acids are used: 2 x 54.0 g Fmoc-Arg(Pbf)-OH, 28.2 g Fmoc- VaI-OH, 29.4 g Fmoc-Leu-OH, 43.8 g Fmoc- Trp(Boc)-OH, 39.0 g Fmoc-Lys(Boc)-OH, 29.4 g Fmoc-Ile-OH, 32.2 g Fmoc-Phe-OH, 35.4 g Fmoc-Glu(OtBu)-OH.

Cleavage from the resin

The resin is rinsed with 1 L of dichloromethane. Subsequently, it is stirred in 1 L of a 4: 1 mixture of dichloromethane/trifluoroethanol for 1 hour at 20°C. This step is repeated allowing for an extended reaction time of 1.5 hours. The resin is rinsed with 500 mL of dichloromethane and the filtrates are combined. The majority of the solvent is distilled off, leaving 285 g of a concentrated solution, which is slowly added to 2 L of diisopropyl ether at 20°C. It is filtered off and dried in vacuo at 20⁰C for 16 hours to yield 64 g of compound D in 93 area% (HPLC; method A). This corresponds to an overall yield of 67%.

The molecular mass is determined by LC-MS (LC: method C, MS: ion trap ESI⁺): C₁17H164N18O2₅S2: [M+H]⁺=2286.

Example 4: Fmoc-Arg(Pbf)-Tyr(tBu)-Leu-Asp(OtBu)-Ala-Arg(Pbf)-Arg(Pbf)-Ala-OH. compound

Resin loading

The loading of 100 g chlorotrityl resin (Iris Biotech) is accomplished as outlined under example 3 using 24.9 (80.0 mmol) of Fmoc- AIa-OH.

The loading is determined as outlined in example 3: 0.56 mmol/g.

Solid phase synthesis:

Fmoc-deptrotection, peptide coupling and cleavage from the resin is accomplished in analogy to the method described under example 3. The following quantities of amino acids are used:

3 x 81.7 g Fmoc-Arg(Pbf)-OH, 39.2 g Fmoc-Ala-OH, 51.8 gFmoc-Asp(OtBu)-OH, 44.5 g Fmoc- Leu-OH, 57.8 g Fmoc-Tyr(tBu)-OH. After cleavage from the resin and precipitation from diisopropyl ether, 109 g of compound C (51.6 mmol) are isolated with 93 area% (method A) according to HPLC analysis. This corresponds to a yield of 82%.

The molecular mass is determined by LC-MS (LC: method C, MS: ion trap ESI⁺): C₁₀₅H₁₄₇N₁₇O₂₃S₃: [(M+2H)/2]⁺= 1056.

Example 5 : Boc-His(TrO-Ser(tBuVGln(Trt)-Gly-Thr(tBu^')-Phe-Thr(tBu)-Ser(tBu^")-Asp(QtBu^')- Tyr(tBu)-Ala-OH. compound B.

Resin loading:

The loading of 200 g chlorotrityl resin (Iris Biotech) is accomplished as outlined under example 3 using 50 g (160 mmol) of Fmoc-Ala-OH. The loading is determined as outlined in example 3UV: 0.51 mmol/g.

Solid phase synthesis:

119.3 g Fmoc-Tyr(tBu)-OH, 99.4 g Fmoc-Asp(OtBu)-OH, 2 x 92.7 g Fmoc-Ser(tBu)-OH), 2 x 96.2 g Fmoc-Thr(tBu)-OH, 93.6 g Fmoc-Phe-OH, 71.9 g Fmoc-Gly-OH, 147.6 g Fmoc-Gln(Trt)-OH,

120.4 g Boc-His(Trt)-OH.

After cleavage from the resin and precipitation from diisopropyl ether, 135 g (63.3 mmol) of compound B are isolated with 93 area% (method A) according to HPLC analysis. This corresponds to a yield of 52%.

The molecular mass is determined by LC-MS (LC: method C, MS: ion trap APES): C119H1₅₆N₁4O22: [M+H]⁺= 2132, [(M+2H)/2]⁺= 1068, [(M-Trt)+H)]⁺= 1891.

Example 6: Fmoc-ArgrPbfi-GlurøtBuVPhe-Ile-LvsCBocVTrpCBoci-Leu-Val-ArgCPbfi-Glv- Arg(Pbf)-Cys(Trt)-OtBu, compound F. From a solution of 29.9 g (32.8 mmol, 95 area%) of example 2 (compound E) 1 L dimethylformamide approx. 500 mL dimethylformamide is distilled off at 40-45°C and 15 mbar. Fresh dimethylformamide (500 mL) is added and the distillation protocol is repeated twice to ensure complete removal of traces of piperidine.

Subsequently, 75.0 g (32.9 mmol, 93 area%) of example 3 (compound D), 2.22 g (16.4 mmol) HOBt monohydrate, 12.7 g (98.4 mmol) DIPEA and 21.1 g (65.6 mmol) TBTU is added and it is stirred at 20⁰C for 30 min. The reaction mixture is added to 3.75 L water and it is stirred for 30 min at 20⁰C. It is filtered and, washed with 1 L water, and, finally dried at 25⁰C in vacuo to give 98.4 g of compound F which corresponds to a yield of 97%.

The molecular mass is determined by LC-MS (LC: method C, MS: ion trap ESI⁺): C16₂H₂₁₉N₂₃O₃₀: [(M+2H)/2]⁺= 1549.

Example 7 : Fmoc- Arg(Pbf)-Tyr(tBuVLeu- AspfOtBuV Ala- Arg(Pbf)- Ar efPbfl- Ala- Arg(Pbf)- Glu(OtBu)-Phe-Ile-Lvs(Boc^")-Trp(Boc)-Leu-Val-Arg(Pbf)-Glv-Arg(Pbf)-Cys(Tr0-OtBu, compound α

a) Fmoc-deprotection:

A solution of 98.0 g (31.65 mmol) example 6 (compound F) in 490 mL dimethylformamide- piperidine (3: 1) is stirred at 20⁰C for 1 h. To the reaction mixture 490 mL acetonitrile is added and the resulting solution is poured within 10 min into 1.96 L diisopropyl ether. It is stirred at 20⁰C for 1 h, filtered and washed three times with each of 250 mL diisopropyl ether. Finally, it is dried at 20⁰C in vacuo to yield 78.2 g (86 % yield) of the Fmoc-deprotected example 6. HPLC (method D): 83 area%.

The molecular mass is determined by LC-MS (LC: method C, MS: ion trap ESI⁺): Ci₄₇H₂(_WN₂₃O₂₈S₄: [(M+2H)/2]⁺= 1437.

b) Coupling:

From a solution of 73.3 g (25.5 mmol, 83 area%) Fmoc-deprotected example 6 in 1.1 L dimethylformamide approx. 550 mL dimethylformamide is distilled off at 40-45⁰C and 15 mbar. Fresh dimethylformamide (550 mL) is added and the distillation protocol is repeated twice to ensure complete removal of piperidine. Subsequently, 56.5 g (26.8 mmol, 92 area%) of example 4 (compound C), 1.72 g (12.8 mmol) HOBt monohydrate, 9.9 g (76.5 mmol) DIPEA and 16.4 g (51 mmol) TBTU is added and it is stirred at 20⁰C for 30 min. The reaction mixture is poured into 1.83 L water and it is stirred for 30 min at 20⁰C. It is filtered and, washed twice with each of 250 mL water, and, finally dried at 3O⁰C in ) vacuo to give 126.0 g of compound G which corresponds to a yield of 99%. HPLC (method D): 79 area%.

The molecular mass is determined by LC-MS (LC: method C, MS: ion trap ESI⁺): C₂₅₂H₃₅₄N₁₄O₅₀S₇: [(M+3H)/3]⁺= 1656.

Example 8: Boc-His(Trt)-SerftBu)-Gln(Trt)-Glv-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- Tyr(tBu)-Ala-Arg(Pbf)-Tyr(tBuVLeu-Asp(OtBuVAla-Arg(Pbf)-ArgrPbf)-Ala-ArgrPbf)-Glu(QtBuV Phe-Ile-Lvs(Boc)-Trp(Boc)-Leu-Val-Arg(Pbf)-Glv-Arg(Pbf)-Cvs(TrO-OtBu. compound A.

a) Fmoc-deprotection:

To a suspension of 125.7 g (25.3 mmol, 79 area%) of example 7 (compound G) in 629 mL dioxane- piperidine (3: 1) is added 629 mL acetonitrile to give a clear solution after 5 min stirring at 20⁰C. It is maintained at 2O⁰C for additional 40 min. Then, the reaction mixture is poured into 4.71 L diisopropyl ether within 5 min and it is stirred at 20⁰C for 30 min and at O⁰C for additional 30 min. The Fmoc-deprotected intermediate is isolated by filtration using a vacuum not below 100 mbar to avoid blockage of the frit. It is rinsed three times with each of 330 mL diisopropyl ether.

Finally, it is dried at 30⁰C in vacuo to obtain 120.3 g (100% yield) of Fmoc-deprotected example 7. HPLC (method D): 77 area%

The molecular mass is determined by MS (ion trap ESI⁺): C₂₃₇H₃₄₄N₄₀O₄₈S₇: [(M+2H)/2]⁺= 2372.

b) Coupling:

From a solution of 1 12.1 g (23.6 mmol, 77 area%) of Fmoc-deprotected example 7 in 1.68 L dimethylformamide approx. 560 mL dimethylformamide is distilled off at 40-45⁰C and 15 mbar. Fresh dimethylformamide (560 mL) is added and the distillation protocol is repeated twice.

Subsequently, 55.4 g (25.96 mmol, 93 area%) of example 5 (compound B), 1.60 g (1 1.8 mmol) HOBt monohydrate, 9.15 g (70.8 mmol) DIPEA and 15.2 g (47.2 mmol) TBTU is added and it is stirred at 20⁰C for 40 min. The reaction mixture is poured into 3.73 L water and it is stirred for 30 min at 2O⁰C. It is filtered and, washed three times with each of 200 mL water, and, finally dried at 30⁰C in vacuo to give 160.5 g of compound A which corresponds to a yield of 99%. HPLC (method D): 79 area%

The molecular mass is determined by MS (ion trap ESI⁺): C₃₅₆H₄₉₈N₅₄O₆₉S₇ [(M+3H)/3]⁺= 1595, [(M+4H)/4]⁺= 1715, [(M+5H)/5]⁺= 1372.

Example 9: Peptide SEO ID NO 1

a) synthesis:

To a solution of 159 g (23.2 mmol) example 8 (compound A) in 636 mL of a mixture of dichloromethane / trifluoroethanol (4: 1 ) 239 g (1549 mmol) 1 ,4-dithioerythriole, 239 mL (1 166 mmol) triisopropylsilane and 1.35 L trifluoroacetic acid is added and it is stirred at 2O⁰C for 4 hrs.

To the reaction mixture 636 mL dichloromethane is added and the resulting solution is poured into

5.4 L cold diisopropyl ether within 2 min. It is stirred at 5°C for 1 h, filtered and rinsed three times with each of 500 mL diisopropyl ether. Finally, it is dried for three days at 20°C in vacuo to yield 126 g of the crude peptide SEQ ID NO 1. This formally corresponds to a yield of 145%. HPLC

(method D, scheme 45): 55 area%

b) purification

A portion of 1 1.9 g of the crude peptide SEQ ID NOl is dissolved in 1 125 mL of a mixture of acetonitrile / water+0.1 %TF A (30:70). The resulting solution is purified employing a Prodigy 10 μ ODSIII I OOA (Phenomenex), particle size 10 μm, column dimension: 250x50 mm. A total of 45 injections with each of 25 mL (264 mg crude peptide) are used. The eluent consisted of A: water+0.1% TFA and B: acetonitrile and a flow of 140 mL/min is applied. The following gradient is used: 0 min injection, 0.5-23.0 min A from 75 to 20%, 23.0 to 29.0 min A at 75%. The fractions are analyzed using HPLC method D. Fractions with >95 area% are pooled, acetonitrile is removed in vacuo and it is freeze dried to yield 2.05 g of the pure peptide SEQ ID NO 1.

HPLC (method A): 94% purity

MS: found 3757,1 Da; calculated monoisotopic mass: 3756,9 Da.

The product is submitted to an amino acid sequencing. The amino acid sequence is identical with the designated structure SEQ ID NOl . His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ala-Arg-Tyr-Leu-Asp-Ala-Arg-Arg-Alal9-Arg20-Glu- Phe-Ile-Lys-Trp-Leu-Val-Arg-Gly-Arg-(Cys)

The amino acid analysis matches well with the proposed amino acid sequence SEQ ID NOl (cf. table 8). The low values found for Thr and Ser can be explained by decomposition during hydrolysis:

^* determined by C.A. T., Tubingen, Germany.

Claims

Claims:

1. A process for the manufacture of the polypeptide SEQ ID NO 1 or a protected derivative thereof wherein a protected derivative of the peptide SEQ ID NO 5 reacts with a protected derivative of the peptide SEQ ID NO 4 in the presence of one or more coupling agents applicable for preparing amide bonds, and

2. A process for the manufacture of the polypeptide SEQ ID NO 1 or a protected derivative thereof wherein a protected derivative of the peptide SEQ ID NO 6 reacts with a protected derivative of the peptide SEQ ID NO 3 in the presence of one or more coupling agents applicable for preparing amide bonds, and

3. A process for the manufacture of the polypeptide SEQ ID NO 1 or a protected derivative thereof wherein a protected derivative of the peptide SEQ ID NO 7 reacts with a protected derivative of the peptide SEQ ID NO 2 in the presence of one or more coupling agents applicable for preparing amide bonds, and wherein the protected derivative of the peptide SEQ ID NO 7 carries a suitable protecting group at the C-terminus, a free amino group at the N-terminus and one or more suitable protecting groups at the side chain functionalities, and

4. The process of any of claims 1 to 3 for the manufacture of the polypeptide SEQ ID NO 1 or a protected derivative thereof comprising the following steps:

5. The process of any of claims 1 to 4 wherein the protected derivative of the polypeptide SEQ

ID NO 1 is compound A which is Boc-His(Trt)-Ser(tBu)-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)- Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ala-Arg(Pbf)-Tyr(tBu)-Leu-Asp(OtBu)-Ala-Arg(Pbf)-Arg(Pbf)-Ala- Arg(Pbf)-Glu(OtBu)-Phe-Ile-Lys(Boc)-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Cys(Trt)-OtBu.

6. The process of any of claims 1 to 4 wherein the protected derivative of the polypeptide SEQ

ID NO 2 is compound B which is Boc-His(Trt)-Ser(tBu)-Gm(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)- Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ala-OH.

7. The process of any of claims 1 to 4 wherein the protected derivative of the polypeptide SEQ

ID NO 3 is compound C which is Fmoc-Arg(Pbf)-Tyr(tBu)-Leu-Asp(OtBu)-Ala-Arg(Pbf)- Arg(Pbf)-Ala-OH.

8. The process of any of claims 1 to 4 wherein the protected derivative of the polypeptide SEQ ID NO 4 is compound D which is Fmoc-Arg(Pbf)-Glu(OtBu)-Phe-Ile-Lys(Boc)-Trp(Boc)-Leu-Val- Arg(Pbf)-Gly-OH.

9. The process of any of claims 1 to 4 wherein the protected derivative of the polypeptide SEQ ID NO 5 is preferably the compound E which is H-Arg(Pbf)-Cys(Trt)-OtBu.

10. The process of any of claims 1 to 4 wherein the protected derivative of the polypeptide SEQ ID NO 6 is preferably the compound F which is Fmoc-Arg(Pbf)-Glu(OtBu)-Phe-Ile-Lys(Boc)- Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Cys(Trt)-OtBu.

11. The process of any of claims 1 to 4 wherein the protected derivative of the polypeptide SEQ ID NO 7 is preferably the compound G which is Fmoc-Arg(Pbf)-Tyr(tBu)-Leu-Asp(OtBu)-Ala- Arg(Pbf)-Arg(Pbf)-Ala-Arg(Pbf)-Glu(OtBu)-Phe-Ile-Lys(Boc)-Tφ(Boc)-Leu-Val-Arg(Pbf)-Gly- Arg(Pbf)-Cys(Trt)-OtBu.

12. The process of any of claims 1 to 11 wherein the peptides SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4 and/or their protected derivatives are synthesized by solid phase synthesis.

13. The process of claim 12 wherein chlorotrityl resin is used as solid support.

14. The process of any of claims 1 to 13 wherein the coupling reagent is O-(Benzotriazol-l -yl)- N,N,N',N'-tetramethyluronium tetrafluoroborate.

15. The process of any of claims 1 to 13 wherein the coupling reagent is O-(Benzotriazol- 1 -yl)- N,N,N',N'-tetramethyluronium tetrafluoroborate in the presence of 1 -hydroxybenzotriazole and N,N- diisopropyl ethyl amine .

16. The process of any of claims 1 to 15 for preparing of peptide SEQ ID NO 1 wherein the protected derivative of peptide SEQ ID NO 1 is deprotected by trifluoroactic acid, 1,4- dithiocrythriolc and triisopropylsilane in a solvent mixture of dichloromethane combined with trifluoroethanol.

17. A process for preparation of the PEGylated derivative of polypeptide SEQ ID NO 1 , which is SEQ ID NO 1 PEG, carrying a PEG portion bound to the sulfhydryl group of Cys₃| on the C- terminus via a maleimide group,

at room temperature in a buffer system at a pH of 6.0 to 7.0,

wherein mPEG is a polyethylenglycol chain carrying a terminal methyl group of a molecular weight of 41 -45 kDa.

18. A compound which is selected from the group consisting of the compounds A, B, C, D, E, F and G.

19. Use of a compound of claim 18 for preparing the polypeptide SEQ ID NO 1 or its PEGylated derivative.