CA1339631C - Process and intermediates for grf peptide - Google Patents

Abstract

Disclosed herein is an improved process for preparing the amidated fragment of growth hormone releasing factor containing the first 29 amino acids of the N-terminal portion. The process is based on a modified solid phase peptide synthesis using highly purified key intermediates represented by the formulae Tyr-Arg-Lys-Val-Leu-Gly and Gln-Leu-Ser-Ala-Arg-Lys-Leu.

Description

133~631 Process and Intermediates for GRF Peptide Field of the Invention This invention relates to an improved process for preparing an active fragment of human growth hormone releasing factor (hGRF). More specifically, this invention relates to an efficient process for preparing the amidated fragment of hGRF containing the first 29 amino acids of the N-terminal portion thereof, and to intermediates for use in the process.

Background of the Invention hGRF is a linear peptide of 44 amino acids, having an amidated C-terminus. Structure activity studies have shown that amino acids can be deleted for the C-terminal portion of hGRF without loss of intrinsic activity; see, for example. N. Ling et al., Biochem.
Biophys. Res. Commun., 123, 854 (1984). Such studies have led to the conclusion that the amidated fragment containing the first 29 amino acids of the N-terminal portion, which is the product of the process of this invention, is a preferred active fragment since it retains much of the in vitro and in vivo activity of hGRF. This fragment, which according to convention is designated as hGRF(1-29)NH2 has the following structure:
H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Yal-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH
_~ 2 1339~31 The importance and potential use of growth hormone releasing factors, and their active fragments (including human as well as those from other species), have been well documented; for example, see F.X. Coude et al., Trends in Biotechnology, 2, 83 (1984), and A.M. Felix et al., Annu. Rep. Med.
Chem., 20, 185 (1985). These peptides stimulate the release of growth hormone (GH). Thus the peptides are indicated for treating GH deficiencies and for augmenting the desirable effects of GH. More explicitly, the peptides, including hGRF(1-29)NH2, or their therapeutically acceptable salts, are useful for treating growth related disorders due to insufficient production of endogeneous GH in animals, for example prepubertal growth hormone deficiency in humans; for healing wounds; for improving milk production in dairy herds, such as cows and goats;
for improving the quality of meat in meat-producing animals (i.e. increasing the ratio of meat to fat);
for increasing wool growth; and for improving feed efficiency in meat-producing animals and dairy cows.
The peptides also can be used diagnostically to evaluate pituitary function.

Accordingly, there is a need for an efficient process to prepare hGRF(1-29)NH2.

A number of preparations of hGRF(1-29)NH2 has been reported; see, for example, Felix et al., supra. The previously reported syntheses can be classified either as solid phase synthesis or as solution phase synthesis. A typical example of solid phase synthesis of hGRF(1-29)NH2, wherein amino acids are coupled serially to a solid support is described by Ling et al., supra. A typical example of solution synthesis, wherein peptide fragments are coupled in 1~39~3 L

solution is described by K. Ono et al., European patent application 193,910, published September 10, 1986 . Sol id phase synthesis of hGRF(1-29) NH2 is practical only for preparing experimental quantities of the peptide. Processes based on the solution methods for preparing GRF, or its active analogs, see Ono et al., supra, or J. Diaz et al., US patent 4,707,541, November 17, 1987, are more amenable to large scale production; however, they require numerous operati ons and use l arge amounts of solvents.

The present process has the features of being simple, rapid and avoids the use of obnoxious chemicals. It efficiently and economically produces hGRF(1-29)NH2 on a commercial scale and with a purity of greater than 98%. By the particular choice of reaction conditions and highly pure intermediate fragments, the process yields the desired peptide free of significant racemization and troublesome by-products.

Summary of the Invention The process of this invention is directed to the preparation of hGRF(1-29)NH2, represented by formula 1 :

H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH2 The process comprises:

(a) stepwise coupling the required amino acid residues to the amino acid-resin of formula H-Gly-S-P

4 133~31 wherein S is a photosensitive spacer and P is a resin to obtain the HGRF(10-15)-resin of formula 2 X-Tyr(W1)-Arg(W2)-Lys(W3)-Val-Leu-Gly-S-P 2 wherein X is an a-amino protective group, preferably t-butyloxycarbonyl, W1 is a protective group for the hydroxyl of Tyr, preferably benzyl or 2,6-dichloro-benzyl, W is a protective group for the guanidino group of Arg, preferably tosyl or nitro, W3is a protective group for the ~-amino group of Lys, preferably 2-chlorobenzyloxycarbonyl or tosyl, and S
and P are as described above; and cleaving the hGRF(10-15)-resin by photolysis to obtain the corresponding hGRF(10-15)0H fragment of formula 3 X-Tyr(W )-Arg(W )-Lys(W )-Val-Leu-Gly-OH 3 wherein X, W1, w2 and W3 are as described above;

(b) stepwise coupling the required amino acid residues to the amino acid-resin of formula H-Leu-S-P wherein S and P are as defined above to obtain the hGRF(16-22)-resin of formula 4 X-Gln-Leu-Ser(W4)-Ala-Arg(W2)-Lys(W3)-Leu-S-P 4 wherein X is an ~-amino protective group, W2, W3, S
and P are as defined above, and W4 is a protective group for the hydroxyl of Ser, preferably benzyl; and cleaving the hGRF(16-22)-resin by photolysis to obtain the corresponding hGRF(16-22)0H fragment of formula 5 X-Gln-Leu-Ser(W4)-Ala-Arg(W2)-Lys(W3)-Leu-OH 5 in which X is an ~-amino protective group and W2, W3, 1339~31 and W are as defined above;
(c) coupling stepwise a hGRF(23-29)-resin of formula H-Leu-Gln-Asp(W5)-Ile-Met-Ser(W4)-Arg(W2)-Q 6 wherei n w2 and W4 are protecti ve groups as defined above, W is a protective group for the ~-carboxyl of Asp, preferably benzyl, 2,6-dichlorobenzyl or cyclohexyl, and Q is a benzhydrylamine type resin, with the previously noted hGRF(16-22)0H fragment of formula 5 and the hGRF(10-15)0H fragment of formula 3, to obtain a hGRF(10-29)-resin of formula 7 X-Tyr(W1)-Arg(W2)-Lys (W3)-Val-Leu-Gly-Gln-Leu-Ser (W4)-Ala-Arg(W2)-Lys (W3)-Leu-Leu-Gln- 7 Asp(W5)-Ile-Met-Ser (W4)-Arg(W2)-Q

wherein X is an a-amino protective group and W1, W2, W3, W4, W5 and Q are as defined above;

(d) sel ectively removing the a-amino protective group of the hGRF-(10-29)-resin to obtain the corresponding hGRF(10-29)-resin of formula 7 wherein X is hydrogen;

(e) coupling stepwise the last-named hGRF(10-29)-res-in with the required amino acid residues to obtain t h e hGRF(1-29)-resin of formula 8 X-Tyr(Wl)-Ala-Asp(W5)-Ala-Ile-Phe-Thr(W6)-Asn-Ser (W4)-Tyr-(W1)-Arg(W2)-Lys (W3)-Val-Leu-Gly-Gln-Leu-Ser(W4)-Ala-Arg(W2)-Lys(W3)-Leu- 8 Leu-Gln-Asp(W5)-Ile-Met-Ser (W4)-Arg(W2)-Q

wherein X is an a-amino protective group, W1, W2, W3, W4, W5 and Q are as def i ned above, and W6 is a 1339~31 protective group for the hydroxyl of Thr, preferably benzyl; and f) deprotecting the hGRF(1-29)-resin of formula 8 to obtain hGRF(1-29)NH2.

The hGRF(10-15)-resin of formula 2 and the hGRF-(16-22)-resin of formula 4 also are included within the scope of this invention.

Details of the Invention The term "residue" with reference to an amino acid means a radical derived from the corresponding a-amino acid by eliminating the hydroxyl of the carboxyl group and one hydrogen of the a-amino group. The term "amino acid residue" can include radicals derived from side chain protected amino acids.

In general, the abbrevi ati ons used herei n for designating the amino acids and the protective groups are based on recommendations of the IUPAC-IUB
Commission on Biochemical Nomenclature, see Biochemistry, 11, 1726-1732 (1972). For instance, Gln, Ala, Gly, Ile, Arg, Asp, Phe, Ser, Leu, Asn, Thr, Lys, Val, Met and Tyr represent the "residues"
of L-glutamine, L-alanine, glycine, L-isol eucine, L-arginine, L-aspartic acid, L-phenylalanine, L-serine, L-leucine, L-asparagine, L-threonine, L-lysine, L-valine, L-methionine and L-tyrosine, respectively.

The term "photosensitive spacer" or "photolabile spacer", designated by the symbol "S", as used herein, means a divalent organic linking unit which, when incorporated into the peptide-resin system, links the first amino acid building block to the resin by orthogonal covalent bonds, the unit or spacer being further characterized in that the bond between the spacer and the first amino acid residue can be cleaved by photolysis to afford the peptide (or the first amino acid residue) with a C-terminal carboxyl. For exampl es of such spacers, see D.H Rich and S.K. Gurwara, Canadian patent 1,108,348, September 1, 1981; J.P. Tam et al., J. Amer. Chem.
Soc., 102, 6117 (1980); F.S Tjoeng and G.A. Heavner, J. Org. Chem., 48, 355 (1983), and J. Gauthier, Canadian patent application, SN 547,394, filed September 21, 1987. When utilized herein, the spacer is first attached to the resin to give the solid support of formula Q-S-P wherein Q is bromo, chloro or iodo, and S and P are as defined herein.
Preferred spacers are represented by the formulae -CH(CH3)CO~OCH2CO- and -CH(CH3)CO~CH 2CO-, when the resin is one of the benzhydrylamine type, and -CH(CH3)CO- when the resin is one of the styrene-divinylbenzene type.

The term "benzhydrylamine type resin", as used herein means a benzhydrylamine resin of the type commonly employed in solid phase peptide synthesis (SPPS).
Such resins include benzhydrylamine resin (BHA) and 4-methylbenzhydrylamine resin.

8 1~3~31 Turning to the process of this invention, one feature is the protection of labile side chain groups of the various amino acid residues with suitable protective groups to prevent a chemical reaction from occurring at those sites until after the completion of the stepwise coupling to produce the hGRF(1-29)resin of formula 8. Another common feature is the protection of the ~-amino group of an amino acid while the free carboxyl group of that reactant is coupled with the free ~-amino group of the second reactant; the ~-amino protective group being one that can be selectively removed to allow the subsequent coupling step to take place at the amino group from which the protective group is removed.

Still another feature involves the preparation by SPPS of protected peptide-resins, i.e. hGRF(10-15)-resin of formula 2 and the hGRF(16-22)resin of formula 4, from which the resin can be cleaved by photolysis to give corresponding ~-amino protected fragments with a free C-terminal carboxyl. These protected fragments, i.e. the fragments of formulae 3 and 5, are generated in a form suitable for suc-cessive coupling by SPPS methodology to the hGRF(23-29)-resin of formula 6. This manner of generation of the individual peptide fragments enables one to purify important intermediate products before coupling, thus decreasing the chances of carrying undesirable impurities through to the final product.

Two types of photochemical resins are employed to generate the protected fragments. In one case, the commercially available copoly(styrene-divinylbenzene) resin is reacted with 2-chloropropionyl chloride in the presence of aluminum chloride under Friedel-Crafts conditions to obtain the photolabile resin 1:~39S31 2-chloropropionyl copoly(styrene-divinylbenzene). In another case, the commercially available benzhydryl-amine (BHA) resin or 4-methylbenzhydrylamine resin was modified by attaching a photosensitive spacer thereto. Preferred spacers have been noted previously. A preferred photochemical resin of the second type is 4-(2-chloropropionyl)phenoxyacetyl BHA
resin. The actual choice of one type of resin is based on the optimized preparation of the various fragments on respective resins.

To initate the preparation of the fragments of formulae 3 and 5, a first amino acid is coupled to the photolabile resin. The preparation of the amino acid-resin is exemplified as follows: An a-amino protected amino acid, e.g. Na-Boc-glycine, is coupled to a solid support of formula Q-S-P wherein Q is bromo, chloro or iodo and S and P are as defined herein in the presence of potassium fluoride or cesium chloride to give the corresponding soid support having an a-amino protected amino acid linked thereto. Thereafter, the a-amino protective group of the latter resin derivative is removed to give the desired amino acid-resin starting material with a free amino group. Thus, the later amino acid-resin serves as the solid component to elaborate the desired fragment-resin by SPPS.

In practice, the desired fragments of formulae 3 and 5 are prepared by stepwise coupling in the desired order the appropriate a-amino protected amino acids to the growing peptide-resin using a modified form of solid phase synthesis. (For a recent review of solid phase synthesis, see J.M. Stewart and J.D. Young, "Solid Phase Peptide Synthesis", 2nd ed, Pierce Chemical Company, Rockford, Illinois, USA, 1984.) More explicitly, the coupling of the amino acid residues is achieved by using dicyclohexylcarbodii-mide (optionally adding 1-hydroxybenzotriazole, 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine or N-hydroxysuccinimide) as the coupling agent, or by employing the "mixed anhydride" activated form of the a-amino protected acids. Another useful agent is benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), described by B. Castro et al., Tetrahedron Letters, 14, 1219 (1975). Subse-quent coupling of the fragments is achieved using di-cyclohexylcarbodiimide, dicyclohexylcarbodiimide/-1-hydroxybenzotriazole or BOP. Each ~-amino protected amino acid or protected fragment is introduced into the reaction system in a relatively low excess (two molar equivalents). The success of the coupling reaction at each stage is monitored by the ninhydrin reaction as described by E. Kaiser et al., Anal. Biochem., 34, 595 (1970). Removal of the a-amino protective group completes the coupling cycle.
In the instance where the a-amino protective group is a t-butyloxycarbonyl, trifluoroacetic acid in methylene chloride is used to effect deprotection.

The cleavage of the protected peptide-resins of formulae 2 and 4 is achieved by photolysis. The photolysis is accomplished by dissolving or suspend-ing the protected peptide-resin in a photolytically stable liquid medium; for example, dioxane, dimeth-ylformamide, methanol, ethanol or N-methylpyrroli-dine; purging the solution or suspension of the pept-ide-resin with argon or nitrogen to remove any dis-solved oxygen; and then irradiating the suspension or solution with photolytically effective ultraviolet light. In practice, irradiation at a wavelength of 350 nm has been found to be very effective. In this manner, the fragments hGRF(10-15) of formula 3 and hGRF(16-22)0H of formula 5 are obtained with a high degree of purity. Noteworthy at this point is the fact that the aforementioned procedure, based on the - B

11 1339~31 use of photosensitive resins, yields highly pure (295% pure) fragments having a protected N-terminal amine and a free C-terminal carboxyl, rendering them as ideal intermediates for eventual preparation of high quality hGRF(l-Z9)NH2.

The two fragments of formulae 3 and 5 are now coupled successively and in proper order with a hGRF(23-29)-resin to yield the hGRF(10-29)-resin of formula 7.
Standard SPPS techniques are applied both for the preparation of the hGRF(23-29)-resin and the subsequent coupling of the two fragments to the hGRF(23-29)-resin. BHA resin serves as a very practical resin for the preparation of the hGRF-(23-29)-resin. The conditions, which were described previously for achieving in practice the coupling of the amino acid residues for the preparation of the fragments of formulae 3 and 5, apply likewise to the preparation of the hGRF(23-29)-resin and its subsequent coupling with the fragments to give the hGRF(10-29)-resin.

Thereafter, the latter peptide-resin is coupled stepwise and in the order of the amino acid sequence of hGRF with the remaining amino acid residues, using the coupling conditions described hereinbefore, to yield hGRF(1-29)-resin.

Subsequent deprotection of the latter peptide resin yields the desired hGRF(1-29)NH2. The deprotection is readily achieved with hydrogen fluoride which simultaneously removes the side chain protecting groups and cleaves the peptide residue form the resin.

The following examples illustrate further this invention. Abbreviations used in the examples 12 1~39~31 ...

include Boc: t-butyloxycarbonyl; TFA: trifluoroace-ti c aci d; CH2Cl 2: methyl ene chl oride; DIEA:
diisopropylethylamine; DMF: dimethylformamide; EtOH:
ethanol; DCC: N,N'-dicyclohexylcarbodiimide; HOBT:
1-hydroxybenzotri azol e; and MeOH: methanol .
Sol uti on percentages are cal cul ated on a vol ume/vol -ume basis unless stated otherwise. Temperatures refer to the centigrade scale. The following terms are trademarks : Pyrex , Sep Tech, and Vydac .

Example 1 RESINS:

a) Preparation of 2-chloropropionyl copoly(styrene-1% divinylbenzene) resin 2-Chloropropionyl chloride (140ml, 183.12 9, 1.44 moles) was added to a suspension of aluminum chloride (230.0 y, 1.72 moles) in 1,2-dichloroethane (1.6 liters). The mixture was stirred until complete solution occurred. The solution was then added over a 5 min period to a mechanically stirred suspension of copoly(styrene-1% divinylbenzene) resin (200-400 mesh; 1 Kg). The reaction mixture was allowed to stir at room temperature (20-22~) for 4h. The resin was filtered, washed sequentially with MeOH (3x), CH2Cl2 (3x), and EtOH (3x), and then dried to constant weight in a vacuum oven. The dried material (1.20 Kg) was ready for use.

b) Preparation of 4-(2-chloropropionyl)phenoxyacetyl BHA-resin 4-(2-Chloropropionyl)phenoxyacetic acid (8.35 9, 34.5 mmoles) and HOBT (4.66 9, 34.5 mmoles) were dissolved separately in DMF (2 x 40 ml). The two solutions were mixed and the resulting mixture was cooled at 0~ for 20 min. A solution of DCC in CH2Cl2(27.5 ml, 1.256 mmoles/ml) was added to the solution. The mixture of activated acid was stirred for 30 min. at 0~. The free base of benzhydrylamine copoly (sty-rene-1% divinylbenzene) resin (200-400 mesh, 50.0 9, amine content = 0.46 mmole/g) was generated with DIEA

14 133g~31 in CH2Cl2. The resulting resin was stirred in CH2Cl2 (9OOml). The above noted mixture of activated acid was added in one portion to the stirred resin. The resulting mixture was stirred for 20h at room temper-ature. The resin was collected by filtration, washed with DMF (3X), MeOH (3X), CH2Cl2 (3X), EtOH (3X) and finally dried to constant weight in a vacuum oven to yield 54.3 g of resin. The Kaiser test, E. Kaiser et al., Anal. Biochem., 34, 595 (1970), was negative indicating no starting material.

Example 2 BOC-AMINO ACID RESINS:

a) Preparation of 2-(Boc-glycyl)propionyl copoly-(sty rene-1% divinylbenzene) 2-Chloropropionyl copoly(styrene-1% divinylbenzene) resin of Example la (20-400 mesh, 100g) was stirred in DMF at 60~ for lh. Boc-Gly-OH (56.09, 320 mmoles) and anhydrous potassium fluoride (46.00 g, 70 mmoles) were added in portions to the mixture. The resultant mixture was stirred at 60~ for 24 h. The resin was collected by filtration and washed three times each with DMF, DMF-water, water, DMF-water, EtOH, CH2Cl2 and EtOH. The resin was dried in a vacuum oven to yield 115.22 g of the title compound.

Titration with picric acid according to the method of B.F. Gisin, Anal. Chem. Acta, 58, 248 (1972) indicat-ed an amine content of 0.79 mmole/g resin for glycine.

b) Preparation of 2-(Boc-leucyl)propionyl copoly(sty-rene-1% divinylbenzene) By following the procedure of section (a) of this example but replacing Boc-Gly-OH with an equivalent amount of Boc-Leu-OH, 2-(Boc-leucyl)propionyl copoly(styrene-1% divinylbenzene) was obtained.

c) Preparation of 4-[2-(Boc-glycyl)propionyl]phe-noxyacetyl BHA-resin 4-[2-Chloropropionyl)phenoxyacetyl]-BHA resin of Ex-ample lb (15.0 g, 0.32 mmole/g resin was stirred in dry DMF (160 ml) for lh to allow the resin to swell;
then potassium fluoride (2.53 g, 43.2 mmoles) and Boc-Gly-OH (3.36 g, 19.2 mmoles) were added in por-tions to the mixture. The reaction mixture was stirred for 24h at 70~ and then filtered. The col-lected resin was washed three times each with DMF, DMF-H20, H20, dioxane-H20, dioxane, MeOH, CH2C12 and EtOH. The resin was dried to constant weight in a vacuum oven to afford free-flowing granules (14.90 9 ) -Titration with the picric acid indicated an aminocontent of 0.28 mmoles/g for glycine.

1339~31 Example 3 PROTECTED FRAGMENTS:

a) Preparation of hGRH(16-22)-resin fragment, 2-(Boc-Gln-Leu-Ser(Bzl)-Ala-Arg(Tos)-Lys(2-ClZ)-Leu)propionyl copoly(styrene-1% divinylbenzene), and its subsequent cleavage to the corresponding C-terminal carboxyl fragment hGRF(16-22)OH

The Boc-amino acid resin of Example 2b (618 9, amine content: 0.805 mmole/g) was used to form the hGRF-(16-22)-resin fragment by a modification of the solid phase technique of R.B. Merrifield, J. Amer. Chem.
Soc., 85, 2149 (1963). The selected Boc-amino acids were added to the growing peptidyl-resin chain by the DCC-HOBT activated acid method (Lys, Arg, Ser, Gln) or by the symmetrical anhydride method (Ala, Leu).
The DCC-HOBT method comprised adding DCC (2 equiv.) in CH2Cl2 to a cold solution of HOBT (2 equiv.) and the selected Boc-amino acid (2 equiv.) in DMF, stirring the mixture at 0~ for 30 min. and adding the mixture to a suspension of the peptidyl resin in CH2Cl2. The symmetrical anhydride method comprised adding DCC (2 equiv.) in CH2Cl2 to the selected Boc-amino acid (4 equiv.) in CH2Cl2 at 0~, stirring the mixture at 0~ for 30 min, filtering the mixture and adding the filtrate to a suspension of the peptidyl resin in CH2Cl2. The coupling cycle consis-ted of i) deprotection with 30% TFA in CH2Cl2 (twice for 5 min, once for 25 min) (ii) neutralization with 5% DIEA in CH2Cl2 (twice for 3 min) and (iii) coupl-ing by addition of symmetrical anhydride or activated ester corresponding to the selected Boc-amino acid.
Intermediate washes were done successively with 17 1~396~1 CH2Cl2, 50~ isopropanol in CH2Cl2, isopropanol and CH2Cl2. The coupling reactions were monitored by the Kaiser tests and the fluorescamin test, see B.F.
Gisin, supra. The time required to complete the coupling reaction ranged from 4 to 24h . The final product was washed with DMF, CH2Cl2' isopropanol, CH2Cl 2 and EtOH, and then dried under vacuum to give 1217.4 g of the hGRF(16-22)-resin (100% yield, 96%
pure by HPLC).

The latter peptidyl resin was subjected to photolysis as follows: The peptidylresin (300g) was suspended in a mixture of DMF (7.5 l) and EtOH (3.9 l) in a Pyrex vessel. The suspension was purged with argon.
While being subjected to a continuous stream of argon, the suspension was stirred and irradiated at 0~ at a wavelength of 350 nm for 70h. The suspension was filtered. The filtrate was concentrated to dryness under reduced pressure at room temperature.
The residual oil was triturated with anhydrous Et20 to give a white solid. The solid was collected, washed with anhydrous Et20 and dried under vacuum over P205 to give 185 g of the corresponding pro-tected N-terminal, free C-terminal carboxyl segment, hGRF (16-22)OH, (100% yield, 95.4% pure by HPLC).

b) Preparation of hGRF(10-15)-resin fragment, 4-[2-(Boc-Tyr(2,6-diClBzl)-Arg(Tos)-Lys(2-ClZ)-Val-Leu-Gly)propionyl]phenoxyacetyl BHA resin, and its subsequent cleavage to the corresponding C-terminal carboxyl fragment hGRF(10-15)OH

The hGRF(10-15)-resin was assembled with the appropriate Boc-amino acids in the same manner as described for the preceding peptidylresin, using the symmetrical anhydride method (Leu ,Val ) and the DCC-HOBT method (Lys, Arg,Tyr) and the Boc-Amino acid ~ -~ ~ .

1~9~31 resin of Example 2c (574 g, amine content: 0.45 mmole/g) as a starting material. Thus, the hGRF(10-15)-resin (933 g) was obtained (100% yield).

The latter peptidylresin (466 g) was subjected to photolysis in the manner described for the preceding peptidylresin of Example 3a to give 161 g of the cor-responding protected N-terminal, free C-terminal carboxyl segment, hGRF(10-15)0H (87% yield, 97% pure by HPLC).

In the same manner but replacing the Boc-amino acid resin of Example 2c with an equivalent amount of 2-(Boc-glycyl)propionyl copoly(styrene-1% divinylben-zene) of Example 2a, h-GRF(10-15)0H also was obtained.

Example 4 Preparation of protected hGRF(1-29)-resin By following the solid phase technique described in Example 3, the fragment hGRF(23-29) was assembled on a BHA resin. The appropriate Boc-amino acids were coupled by the DCC-HOBT method (Arg, Ser, Gln) or by the symmetrical anhydride method (Met, Ile, Asp, Leu). The t-butylcarbocation scavenger, DL-methion-ine (1% by volume of the scavanger in 30% TFA-CH2Cl2 solution), was used during Boc-deprotection following the coupling of Met, Ile, Asp and Gln, cf. D.
Le-Nguyen et al., J. Chem. Soc. Perkin Trans., 1, 1915 (1987). On completion of coupling the Leu residue to the growing peptide-resin, the resultant h G R F ( 2 3 - 2 9 ) - r e s i n , i . e .
Boc-Leu-Gln-Asp(Chxl)-Ile-Met-Ser(Bzl)-Arg(Tos)-BHA, was coupled serially (DCC-HOBT method) with the pro-1 9 1 ~ 3 '~

tected fragments of hGRF(16-22)OH and hGRF(10-15)OH
of Examples 3a and 3b, respectively. (For the depro-tection steps prior to the latter two couplings, and for all subsequent deprotection steps of this examp-le, DL-methionine was used as a scavenger.) There-after, the resultant hGRF(10-29)-resin was coupled stepwise with the appropriate Boc-amino acids, using the DCC-HOBT method for coupling Ser, Asn and Tyr, and the symmetrical anhydride method for Thr, Phe, Ile, Ala, Met and Asp. Following a final deprotect-ion (30% TFA in CH2Cl2 plus 1% by volume of DL-methi-onine), the protected hGRF(1-29)-resin, i.e. H-Tyr-(2,6-diClBzl)-Ala-Asp(Chxl)-Ala-Ile-Phe-Thr(Bzl)-Asn-Ser(Bzl)-Tyr(2,6-diClBzl)-Arg(Tos)-Lys(2-ClZ)-Val-Leu-Gly-Gln-Leu-Ser(Bzl)-Ala-Arg(Tos)-Lys(2-ClZ)-Leu-Leu-Gln-Asp(Chxl)-Ile-Met-Ser(Bzl)-Arg(Tos)-BHA
(124.45 g, 91% yield) was obtained.

Example 5 Preparation of hGRF(1-29)NH2 A mixture of the preceding protected hGRF(1-29)-resin (75.9g), DL-methionine (7.6 g) and anisole (distil-led, 75ml) was placed under nitrogen (positive pres-sure). The mixture was cooled at -80~ for 10 min (dry ice-isopropanol bath). Anhydrous hydrogen fluoride was distilled slowly into the cooled mixture over a period of about 70 min. The total amount of hydrogen fluoride added to the mixture was 750 ml.
The reaction mixture was stirred at 0~ for lh under nitrogen. Thereafter, a stream of nitrogen was passed immediately over the reaction vessel at 20-22~
until most of the hydrogen fluoride had evaporated.
The residue was dried under high vacuum for 100 min and then extracted with TFA (700 ml). The extract was concentrated to a small volume. Dilution of the concentrate with Et20 afforded a solid. The solid was collected, washed with Et20 and dried under high vacuum to give 44.7 9 of the crude title compound (TFA salt).

A portion of the latter crude product (4.0g) was purified further by HPLC (Sep Tech 1200, 5 x 35 cm;
Vydac ODS, 15-20~ particle size; 300 nm; linear gradient, 100ml/min, solvent A: 0.06% TFA in water, solvent B: 0.06% TFA in acetonitrile). The appropriate fractions were combined. Acetonitrile was removed under vacuum. The residue was lyophi-lized to give hGRF(1-29)NH2 (662 mg, 16.5% yield, 98.4% pure as indicated by HPLC).

Claims (8)

1. A process for preparing hGRF(1-29)NH2 which comprises:
(a) stepwise coupling the required amino acid residues to the amino acid-resin of formula H-Gly-S-P

wherein S is and P is benzhydrylamine resin or S is -CH(CH3CO- and P is a styrene-divinylbenzene resin, to obtain the hGRF(10-15)- resin of formula 2 X-Tyr(W1)-Arg(W2)-Lys(W3)-Val-Leu-Gly-S-P 2 wherein X is an .alpha.-amino protective group, W1, W2 and W3 are protective groups, and S and P are as described hereinabove; and cleaving the hGRF(10-15)-resin by photolysis to obtain the corresponding hGRF(10-15)OH
fragment of formula 3 X-Tyr(W1)-Arg(W2)-Lys(W3)-Val-Leu-Gly-OH 3 wherein X, W1, W2 and W3 are as described hereinabove;
(b) stepwise coupling the required amino acid residues to the amino acid-resin of formula H-Leu-S-P wherein S
and P are as defined hereinabove to obtain the hGRF((16-22)-resin of formula 4 X-Gln-Leu-Ser(W4)-Ala-Arg(W2)-Lys(W3)-Leu-S-P 4 wherein X is an a-amino protective group, W2, W3, S and P are as defined hereinabove, and W4 is a protective group; and cleaving the hGRF(16-22)-resin by photolysis to obtain the corresponding hGRF(16-22)OH fragment of formula 5 X-Gln-Leu-Ser(W4)-Ala-Arg(W2)-Lys(W3)-Leu-OH 5 in which X is an .alpha.-amino protective group and W2, W3, and W are as defined hereinabove;

(c) coupling stepwise a hGRF (23-29)-resin of formula H-Leu-Gln-Asp(W5)-Ile-Met-Ser(W4)-Arg(W2)-Q 6 wherei n W2, W4 and W5 are protective groups and Q is a benzhydrylamine type resin, with the previously noted hGRF(16-22)OH fragment of formula 5 and the hGRF(10-15)OH fragment of formula 3 to obtain a hGRF(10-29)-resin of formula 7 X-Tyr(W1)-Arg(W2)-Lys(W3)-val-Leu-Gly-Gln-Leu-Ser(W4)-Ala-Arg(W2)-Lys(W3)-Leu-Leu-Gln- 7 Asp(W5)-Ile-Met-Ser(W4)-Arg(W2)-Q

wherein X, W1, W2, W3, W4, W5 and Q are as defined hereinabove;

(d) selectiviely removing the .alpha.-amino protective group of the hGRF(10-29)-resi n to obtai n the corresponding hGRF(10-29)-resin of formula 7 wherein X is hydrogen;

(e) coupling stepwise the last-named hGRF(10-29)-resin with the required amino acid residues to obtain the hGRF(1-29)-resin of formula 8 X-Tyr(W1)-Ala-Asp(W5)-Ala-Ile-Phe-Thr(W6)-Asn-Ser(W4)-Tyr(W1)-Arg(W2)-Lys(W3)-Val-Leu- 8 Gly-Gln-Leu-Ser(W4)-Ala-Arg(W2)-Lys(W3)-Leu-Leu-Gln-Asp(W5)-Ile-Met-Ser(W4)-Arg(W2)-Q

wherein X is an .alpha.-amino protective group, W1, W2, W3, W4 and W5 and Q are as defined hereinabove, and W6 is a protective group; and (f) deprotecting the hGRF(1-29)-resin of formula 8 to obtain hGRF(1-29)NH2.

2. A process of claim 1 wherein W1 is 2,6-dichloro-benzyl, W2 is tosyl, W3 is 2-chlorobenzyloxycarbonyl, W4 is benzyl, W5 is cyclohexyl and W6 is benzyl.

3. A process of claim 1 wherein X is benzyloxycarbonyl.

4. A process of claim 1 wherein S and P of the amino acid-resin of formula H-Gly-S-P and the h-GRF(10-15)- resin of formula 2 is and benzylhydrylamine resin, respectively; S and P of the amino acid-resin of formula H-Leu-S-P and the hGRF(16-22)-resin of formula 4 is -CH(CH3)CO- and a styrene-divinylbenzene resin, respectively; and X is benzyloxycarbonyl.

5. A hGRF(10-15)-resin of formula 2 of claim 1.

6. A hGRF(10-15)-resin of formula 2 of claim wherein S is and P is benzylhydrylamine resin.

7. A hGRF(16-22)-resin of formula 4 of claim 1.

8. A hGRF(16-22)-resin of formula 4 of claim 1 wherein S is -CH(CH3)CO- and P is styrene-divinylbenzene resin.