GB1584669A - Process for synthesising peptides containing a sulphated tyrosine residue - Google Patents

Description

(54) PROCESS FOR SYNTHESISING PEPTIDES CONTAINING A SULPHATED TYROSINE RESIDUE (71) We, AKADEMIE DER WISSENSCHAFTEN der DDR, of 5, Rudower Chaussee, 1199 Berlin, German Democratic Republic, a Corporation organised under the laws of the German Democratic Republic, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: The present invention is concerned with a process for synthesising peptides containing a sulphated tyrosine residue.

More particularly, the present invention is concerned with a process for synthesising peptides containing 6 to 20 amino acid residues and a sulphated tyrosine residue necessary for biological activity, for example peptides of cholecystokinin-pancreozymin (PZ) and the derivatives and salts thereof, which are suitable for pharmaceutical use.

The gastrointestinal hormone cholecystokinin-pancreozymin, its C-terminal heptapeptide and octapeptide, as well as caerulein, a decapeptide with similar structure isolated from the skin of the frog Hyla caerula, are able to contract the gall bladder, relax the bile duct, stimulate the excretion of proteolytic enzymes in the pancreas and accelerate intestinal passage. They can be used, for example, as a remedy in intestinal diagnosis by X-rays and have a potential therapeutic use. They have the following foitnulae: PZ-octapeptide: H-Asp-Tyr(SO 3-)-Met-Gly-Trp-Met-Asp-Phe-NH 2 Caerulein: PCA-Gln-Asp-Tyr(SO 3)-Thr-Gly-Trp-Met-Asp-Phe-NH2 Human gastrin II. the hormone which stimulates gastric acid secretion, also contains a sulphated tyrosine although sulphate is here not necessary for biological activity. Analogues of C-terminal caerulein heptapeptide with an antigastrin activity are also known.

The preparation and biological activity of caerulein and of a threonine-O-acetyl derivative are described in Federal Republic of Germany Patent Specification No.1,643,504. For the introduction of an SO3H residue into the decapeptide, use has been made of N-sulphonylpyridinium but sulphation does not seem to be complete. After deacetylation by alkaline saponification and purification, the yield obtained is about 40% (see also British Patent Specification No.1,173,539).

The preparation of the C-terminal octapeptide of cholecys'tokinin-pancreozymin and the pharmaceutical use thereof are disclosed in U.S. Patent Specifications Nos. 3,723,406 and 3,734.946. In these cases. a sulphated tyrosine residue was introduced by means of concentrated sulphuric acid into the non-protected octapeptide. Due to side reactions. the yield. after purification. was not more than 25% (see J.A.C.S.. 92, 195/1970). The condensation of a protected dipeptide to a C-terminal sulphated cholecystokinin-pancreozymin decapeptide is there described. In the same manner, several biologically-active octapeptides have been obtained by an exchange of the aspartyl residue by other amino acid residues and by maleic and fumaric acid (see British Patent Specification No.1.304,074 and also J.Medicinal Chem.. 13. 439/1970). Using concentrated sulphuric acid, ring substitution in the 3'-position of tyrosine occurred. C-sulphonated octa-, deca- and dodecapeptides of cholecystokinin-pancreozymin with a high biological activity are described in U.S. Patent Specification No.3.579.494.

Additional by-products in the production of C-terminal cholecystokinin peptides are: a) the non-sulphated sequence from incomplete sulphation and from saponification by the acid medium during the isolation procedure, b) derivatives substituted in the indole ring of tryptophan originating from repeated acid deprotection steps, favoured by using trifluoroacetic acid (see Angew. Chem., 89, 330/1977).

Boc-Tyr(SO 3-)-Met-Gly-Trp-Met-Asp-Phe-NH 2 (see Experientia, 24, 771/1968) seems to be the simplest cholecystokinin derivative, from the synthetic point of view, which exhibits biological activity. The sulphate residue has hitherto only been introduced into the complete peptide sequence in the last step of the synthesis. It is known from other peptide hormones that the omission of the N-terminal amino group results in the maintenance of increase of the biological activity and that acylation of the terminal nitrogen may prevent degradation by aminopeptidases, which may be of some advantage for biological activity.

According to the present invention, there is provided a process for the preparation of a peptide containing 6 to 20 amino acid residues and containing tyrosine, the hydroxyl group of which is esterified with sulphuric acid, wherein the tyrosine hydroxyl group of an N-protected tyrosine-containing fragment, which consists of 2 to 6 amino acid residues, is esterified with sulphuric acid, whereafter this fragment is condensed with another peptide containing 2 to 18 amino acid residues, followed by N-deprotection and then by acylation.

According to one embodiment of the present invention, there is provided a process for preparing X-Tyr(SO3~)-Met-Gly-Trp-Met-Asp-Phe-NH2, X being an acyl residue, by esterification of the tyrosine hydroxyl group of an N-protected Tyr-Met-Gly-alkyl ester with sulphuric acid, alkaline saponification of the alkyl ester group, coupling to H Trp-Met-Asp-Phe-NH2 by the mixed anhydride method to give a heptapeptide which is then N-deprotected and finally acylated in the presence of a tertiary amine.

The process according to the present invention can be used, for example, for the prepara tion'of the heptapeptide Boc-Tyr(SO3~)-Met-Gly-Trp-Met-Asp-Phe-NH2 in good yield and high purity, using H-Trp-Met-Asp-Phe-NH2, a commercially available gastrin tetrapeptide, and to use this heptapeptide to obtain derivatives thereof containing minimum structural features with minimum expense and maximum biological effect.

In the following, a preferred method is described for the synthesis of peptides containing tyrosine-O-sulphate, using N-acylated C-terminal heptapeptides of cholecystokininpancreozymin as examples: X-Tyr(SO 3-)-Met-Gly-Trp-Met-Asp-Phe-NH2 X = HOOC-CH2CH2-CO-, H 3C-CH 2-O-CO-CH 2CH 2-CO- or 4-HO-C6H4CH2-CO The N-protected, tyrosine residue-containing peptide, which contains 2 to 6 amino acid residues, is sulphated with N-sulphonyl-pyridinium or a mixture of chlorosulphuric acid and pyridine in a mixture of chloroform-dimethylformamide. In the case of Boc-Tyr-Met-Gly OEt, using a 30 fold excess of CISO3H, sulphation is quantitative.

After introducing the sulphate residue with chlorosulphonic acid in a pyridinechloroform-dimethylformamide mixture, the reaction mixture containing the peptide alkyl ester is worked up with an aqueous alkaline buffer system and preferably with an aqueous ammonia/ammonium chloride buffer. It is possible to isolate the sulphated, N-protected tripcptide ester from the buffer with mixtures of ethyl acetate with C3-C8 alcohols, especially ti-butanol, the yields being about 70%.

It is an advantage of this process that by-products due to O-acylated tyrosine derivatives from preceding steps, for example from Boc-Tyr(Boc)-O-activated esters, can be removed quantitatively and an expcnsive purfication of Boc-tyrosine-activated ester is not necessary.

Boc-Tyr(SO3~)-Met-Gly-OEt is saponified with sodium hydroxide and extracted from weakly acidic solution in the above-mentioned manner. It is possible to couple the N-protected peptide containing Tyr(SO3~ Na+) to other peptides with a free a-amino group. using methods conventional in peptide synthesis. In the synthesis of Boc heptapcptide (see formula), the mixed anhydride method can be used to give 88% yields of a uniform product.

After this procedure, the overall yield is much higher and the peptide is purer than in the case of products obtained by direct sulphation of the Boc-heptapeptide or of other higher peptides in the last synthesis step.

According to the present invention, the quantitative deblocking of a Boc-heptapeptide, combined with a minimum hydrolysis of the sulphuric ester and almost without side reactions in the indole ring system of Trp. is possible at ambient temperature when using trifluoroacetic acid containing 15 to 25% by weight of water and an upper layer of methoxybenzene/2-mercaptoethanol (5:1 v/v) is present and an optimum reaction time. In this case, desulphation is less than 20% and the use of the Boc protecting group is preferable in comparison with other more expensive groups which are removed by weakly acidic, neutral or basic reagents. The identical sequence without sulphuric acid is easy to remove after the next step, i.e. the N-acylation, if necessary for the final use.

Acylation of the free heptapeptide H-Tyr(SO3-)-Met-Gly-Trp-Met-Asp-Phe-NH2 is carried out by reaction with activated esters of carboxylic acids, for example with 4-hydroxyphenylacetic acid and monoethylsuccinic acid 2,4,5-trichlorophenyl ester, as well as with succinic anhydride.

Neutralisation of trifluoroacetic acid and acylation can be carried out by the addition of a tertiary amine, for example N-ethylmorpholine.

After N-deprotection and acylation, gel filtration can be used for separating sulphated peptides from non-sulphated peptides.

The acylated heptapeptides of cholecystokinin exhibit gall bladder-contracting activities.

The succinyl-heptapeptide (i.e. C-terminal deaminopancreozymin octapeptide) has a very potent activity on the isolated gall bladder which is nearly of the same order of magnitude as caerulein. On the other hand, the 4-hydroxyphenylacetyl and ethoxysuccinyl derivatives show only about 1 % of the activity of caerulein.

The separation of the non-sulphated sequences, X-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2 can be successfully carried out by countercurrent distribution in a CHCl3/CH3OH/NH3/H2O system and by partition chromatography with 0.1 N ammonium acetate buffer solution on a "Sephadex" G25 column, despite the small difference in molecular weight of both products, i.e. the sulphated and the non-sulphated deaminooctapeptide. ("Sephadex" is a Registered Trade Mark).

The polypeptide succinyl-Tyr(SO3-)-Met-Gly-Trp-Met-Asp-Phe-NH2 and its salts are new and pharmaceutically useful. Consequently, the present invention also provides compositions containing this polypeptide and/or at least one non-toxic salt thereof, in admixture with a solid or liquid pharmaceutical diluent or carrier.

The following abbreviations are used herein: Asp = aspartic acid Gln = glutamine Gly = glycine Met = methionine Phe = phenylalanine Thr = threonine Trp = tryptophan Tyr = tyrosine PCA = pyrrolidone-2-carboxylic acid PZ = pancreozymin Boc = tert.-butoxycarbonyl OEt = ethyl ester OTcp = 2,4,5-trichlorophenyl ester NEM = N-ethylmorpholine TFA = trifluoroacetic acid DMF = dimethylformamide The following Examples are given for the purpose of illustrating the present invention: Example 1.

N-tert.-Butyloxycarbonyl- O-sulphate-L-tyrosyl-L-methionylglycine ethyl ester ammonium salt.

2.8 g. (0.036 mol) pyridine in 10 ml. chloroform are cooled to -10 C. While stirring, 2.4 ml. (0.037 mol) chlorosulphuric acid are added dropwise to this solution in the course of 30 to 40 minutes, a temperature of + 10 C. being maintained. When the addition is complete, stirring is continued for 30 minutes at 0 to 100C., whereafter the mixture of the pyridinesulphur trioxide complex and pyridine hydrochloride is brought into solution with 10 ml. dimethylformamide.

0.600 mg; (1.21 mMol) Boc-Tyr-Met-Gly-OEt are dissolved in 10 ml. dimethylformamide and 10 ml. pyridine and, while stirring, added to the solution of the pyridinesulphur trioxide complex. The reaction mixture is subsequently stirred for 5 hours at 35"C. on a water-bath, the solvent is removed at oil pump vacuum at 45"C. and the oily residue rapidly mixed at a temperature of -5 to 0 C., with vigorous stirring, with 200 ml. 0.1N ammonium chloride solution/0.lN ammonia buffer (pH 10.5). The pH is subsequently rapidly adjusted to 7.0 with this buffer. The solid material which precipitates out of the aqueous phase is brought into solution by the addition of 70 ml. of an n-butanol/ethyl acetate mixture (2:1 v/v). The aqueous phase is separated off, saturated with sodium chloride and extracted three times with 70 ml. amounts of the above-mentioned mixture.

The organic extracts are combined, washed with a saturated aqueous solution of sodium chloride until sulphate ions are no longer detectable and the organic phase then dried over anhydrous sodium sulphate. The solution is thereafter concentrated to one half of its volume. For the removal of the Boc-Tyr(Boc)-Met-Gly-OEt. the n-butanol containing solution is allowed to run slowly, while stirring. into the 2.5 fold amount of cold diethyl ether. The precipitated product is filtered off with suction. thoroughly washed with diethyl ether and petroleum ether and dried in a vacuum over phosphorus pentoxide. The yield is 0.50 g. (70% of theory); m.p. 139-141 C.

[α]D27 = -9.8 (c. = 1.0 in dimethylformamide) Amino acid analysis after alkaline hydrolysis: Tyr SO4 0.87; Tyr 0.05; Met 0.89; Gly 1.0 The thin layer chromatographic purity was tested on Silufol plates (produced by Kavalier.

Czechoslovakia) in system (A) - BuOH/glacial acetic acid/water (4:1:1 v/v/v) and (B) pyridine/n-butanol/glacial acetic acid/water (20:30:6:25 v/v/v/v). RfA = 0.66; RfB = 0.64.

Example 2.

N-tert. - Butyloxycarbonyl- O-sulphate-L-tyrosyl- L-nielliionyl-glycine sodium salt.

0.500 g. (0.84 mMol) Boc-Tyr(SO3NH4+)-Met-Gly-OEt are dissolved in 5 ml. water/ethanol (1:1 v/v) and stirred for 45 minutes at ambient temperature with 3 ml. IN aqueous sodium hydroxide solution. The solution is evaporated on a rotary evaporator at 40 C. to half of its volume and subsequently diluted with about 5 ml. water. The reaction solution is adjusted to pH 3.0 at 5 C. with 1N hydrochloric acid. The aqueous phase is extracted with 50 ml. of an n-butanol/ethyl acetate mixture (2:1 v/v). subsequently saturated with sodium chloride and thoroughly shaken out with two 50 ml. amounts of the above-mentioned solvent mixture. The organic phases are combined. washed with a saturated aqueous solution of sodium chloride and dried over anhydrous sodium sulphate. The solvents are removed in a vacuum at 40 C. and the residue evaporated to dryness several times with the addition of chloroform. The glassy residue is triturated with diethyl ether/petroleum ether. the solid material is thoroughly washed with diethyl ether and petroleum ether and dried in a vacuum over phosphorus pentoxide. Yield 0.360 g. (75% of theory); m.p. 196-198 C.

[α]D27 = -6.8 (C. = 0.5 in dimethylformamide) RfA = 0.64; RfB = 0.57.

Example 3.

N-tert.-Butyloxycarbonyl-O-sulphate-L-tyrosyl-L-methionyl-glycyl-L-tryptophyl-L-methionyl L-aspartyl-L-phenyl-alaninamide sodium salt.

0.360 g. (0.631 mMol) Boc-Tyr-(SO3Na)-Met-Gly-OH are dissolved in 20 ml. dimethylformamide, 0.079 ml. (0.631 mMol) N-ethylmorpholine is added thereto and the reaction solution is cooled to -25 C. 0.086 ml. (0.631 mMol) isobutyl chloroformate is added thereto and the mixture left for activation for 10 minutes at -25 C.

0.400 g. (0.631 mMol) H-Trp-Met-Asp-Phe-NH2 are separately dissolved in 20 ml. dimethylformamide. mixed with 0.079 ml. (0.631 mMol) N-ethylmorpholine and the reaction solution cooled to -25 C. This solution is added to the solution of the mixed anhydride of Boc-Tyr-(SO3Na)-Met-Gly-OH. The mixture is subsequently stirred for 30 minutes at -25 C. and then for 4 hours at ambient temperature. The amine hydrochloride which precipitates out is filtered off with suction, the solvent is removed at oil pump vacuum at 45 C. and the viscous residue is triturated in the cold with a mixture of n-butanol/diethyl ether (1:1 v/v). The solid material is filtered off with suction, thoroughly washed with chloroform and dried in a vacuum over phosphorus pentoxide. Yield 0.650 g. (88% of theory): m.p. 184- 186 C.

[α]D25 = 7.1 (c. = 0.25 in dimethylformamide); [α]D25 = -3.7 (c. = 0.25 in water); Amino acid analysis after alkaline hydrolysis: Tyr(SO3H) 0.88; Tyr 0.03; Phe 0.9; Asp 1.0. RfA = 0.64; RfB = 0.67.

Exal7lple d.

O-Sulphate-L-tyrosyl-L-methionyl-glycyl-L-tryptophyl-L-methionyl-L-aspartyl-L-phenylalaninamide trifluoroacetate.

0.650 g. (0.565 mMol) Boc-Tyr(SO3Na)-Met-Gly-Trp-Met-Asp-Phe-NH2 are covered with 0.5 ml. methoxybenzene and 0.1 ml. 2-mercaptoethanol and subsequently stirred for 20 minutes at 18 - 200C. with 125 ml. 74% by weight trifluoroacetic acid.

The reaction solution is introduced into a two fold amount of-a diethyl ether/petroleum ether mixture (1:1 v/v) and the voluminous precipitate removed by suction filtration. This is thoroughly washed with petroleum ether and dried in a vacuum over phosphorus pentoxide/potassium hydroxide. Yield 0.550 g. (85 % of theory) .

RfA 0.52 and 0.62 (about 20%; RfB 0.57 and 0.77 (about 20%).

Amino acid analysis after alkaline hydrolysis: Tyr(SO3H) 0.68, Tyr 0.23, Asp 0.74, Gly 1.0, Met 1.6, Phe 0.9, Trp 1.1.

Example 5.

N-Succinoyl-O-sulphate-L-tyrosyl-L-methionyl-glycylp L-tryptophyl-L-methionyl-L-aspartyl L-phenylalaninamide-di-N-ethylmorpholinium salt.

0.550 g. (0.00048 mol) of the product of Example 4 is dissolved in 10 ml. dimethyl formamide, mixed, while stirring, with 181 ml. (0.00144 mol) N-ethylmorpholine and the reaction solution cooled to OOC. Then, within the course of 40 minutes, while stirring, there is introduced 0.062 g. (0.000624 mol) succinic anhydride and, at the same time, 0.079 ml.

N-ethylmorpholine is added dropwise. The reaction solution is then further stirred for 3 hours at 0 C. and subsequently left to stand overnight at 40C.

The solvent is removed at oil pump vacuum at a temperature of 40 C. and the viscous residue triturated with a cold mixture of n-butanol/diethyl ether. The solid material is filtered off with suction, thoroughly washed with tetrachloroethane, diethyl ether and petroleum ether and dried in a vacuum over phosphorus pentoxide. Yield 0.588 g. (91% of theory); m.p. 172 - 175 C. Amino acid analysis after alkanline hydrolysis: TyrSO O 72; Tyr 0.18; Asp 1.0; Gly 1.1; Met 1.6; Phe 0.93; Trp 0.75.

RfA 0.53 and 0.70 (about 20No); RfB 0.64 and 0.68 (about 20%).

Purification by gel filtration on Sephadex G-25.

Sephadex G-25 which has been swollen for several hours in 0.1N aqueous ammonium acetate solution (1N acetic acid adjusted to pH 6.5 by the addition of ammonia) is introduced into a column of 120 cm. length and 4 cm. diameter. There is applied thereto a solution of 200 mg. "crude desaminooctapeptide" in 8 ml. 0.1N ammonium acetate solution and 2 ml. dimethyl formamide and elution carried out with 0.1N aqueous ammonium acetate solution. At a flowthrough rate of 15 ml./hour, the concentration of peptide is determined by measurement of the extinction at 280 nm (Uvicord) and the eluate is collected in 8 ml. fractions. Fractions 9 - 31 contain the desamine-PZ octapeptide and fractions 32 - 34 desulphated by-products. Yield 0.116 g. (60% of theory) and 0.055 g.

(11.6 % of theory) desulphated heptapeptide.

C49H60N9O16Sa (M.W. 1357.75) 2C6H13NO calc.: C53.96%; H6.38%; N 11.34%; S7.17% found: 54.15%; 6.38%; 11.70%; 6.90% a23 = -3.4 t 0.5 = 0.25 in water [α]D25 = -9.4 # 1 = 0.25 in dimethylformamide.

Amino acid analysis after alkaline hydrolysis Tyr(SO3@) Tyr Asp Gly Met Phe Trp 0.80 0,08 1.0 1.0 1.64 0.90 1.0 Example 6.

N-Ethoxysuccinyl-O-sulphate-L-tyrosyl-L-methionyl-glycyl-L-tryptophyl-L-methionyl-L aspartyl- L-phenylalaninamide, 0.190 g. (0.167 mMol) Tyr(OSO3-)-Met-Gly-Trp-Met-Asp-PheNH2 (see Example 4) are dissolved in 10 ml. dimethyl formamide and 0.0182 ml. (0.146 mMol) N-ethylmorpholine added thereto. The reaction mixture is cooled to 0 C. and 0.059 g. (0.182 mMol) ethoxysuccinic acid 2,4.5-trichlorophenyl ester added thereto. The reaction mixture is stirred for 30 minutes at 0 C. and then left to stand for 2 days at ambient temperature.

The solvent is removed in a vacuum at 45 C., the oily residue is digested with diethyl ether/petroleum ether and the solid material is filtered off with suction and washed with chloroform, diethyl ether and petroleum ether. Yield 0.146 g. (75% of theory); RfB = 0.61; Rfs = 0.70 about 15 % desulphated, acylated heptapeptide.

Purification by countercurrent partitioning: Chloroform/methanol/0.lN ammonium acetate solution (11.1:8:10 v/v/v) is used as partitioning system. After 32 partitioning steps and lyophilisation, the yield is 84% of theory (fractions 24 - 31), in addition to 15%desulphated, acylated heptapeptide (fractions 20 - 22). Acid-base titration and sulphate determination (BaSO4) gave 90% and 92%, respectively, of the calculated values.

Example 7. p-Hydroxyphenylacetyl- O-sulphate-L-tyrosyl-L-methionyl-L-tryptophyl- L-methionyl-L aspartyl- L-phenyThThninamide.

0.190 g. (0.167 mMol) of the heptapeptide sulphonate obtained according to Example 4 are dissolved in 10 ml. dimethylformamide, 0.0182 ml. (0.146 mMol) N-ethyl-morpholine is added thereto and the reaction mixture is cooled to 0 C. 56 mg. (0.182 mMol) p-hydroxyphenylacetic acid 2,4,5-trichlorophenyl ester are added thereto and the reaction mixture is stirred for 30 minutes at OOC. and for 2 days at ambient temperature. The solvent is removed in a vacuum at 450C. and the oily residue digested with diethyl ether/petroleum ether (1:1 v/v). The solid material is filtered off with suction and washed with chloroform, diethyl ether and petroleum ether. Yield 0.150g. (80%oftheory); Rfs = 0.59.

At Rfn = 0.69 about 15% desulphated, acylated heptapeptide.

Purification is carried out by countercurrent partitioning (cf. Example 6). Yield 80% of theory (fractions 24 - 31), in addition to 15% desulphated heptapeptide (fractions 11 - 23).

Acid-base titration and sulphate determination (BaSO4) gave, respectively, 95% and 93 % of the calculated values.

The following equations illustrate the reactions involved:

Boc-Tyr-Met-Gly-OEt 70% KJ, Pyridine-SO3 eomplex Boc-Tyr-(So3~NH4+)-Met-Gly-OEt (I) 75% L NaOH Boc-Tyr-(SO 3Na+)-Met-Gly-OH (2) + H-Trp-Met-Asp-Phe-NH2 88% J mixed anhydride synthesis Boc-Tyr(SO 3Na+)-Met-Gly-Trp-Met-Asp-Phe-N1l2 (3) 85 % + 75% bywt. TFA/C6H5OCH3/2-mercaptoethanol H-Tyr(So3+)-Met-GlypTrp-Met-Asp-Phe-NH2. TFA (4) 91 tCH2-CO + | O / + N-ethylmorpholine (NEM) CH2- CO/ 600/cm "Sephadex" G 25 HOOC-CH2CH2-CCO-Tyr(SO3)-Met-Gly-Trp-Met-Asp-Phe-NH2. 2 NEMH+ (5) and 12 % HOOC-CH2CH2-CO-Tyr-Met....NH 2 4 75% OXG 1 H ,C20-CO-CH 2CH2-CO-O-C6H2Cl3 H C2O-CC)-CH 2CH -CO-Tyr(SO 3)-MetpGly-Trp-Met-Asp-Phe-NH2 .2 NEMH- (6) 4 80% f HO-C6H4CH2-CO-O-C6H2Cl3 HO-C6H 4CH 2-CO-Tyr(SO 3)-Met-Gly-Trp-Met-Asp-Phe-NH2. 2 NEMH+ (7)

Claims (12)

WHAT WE CLAIM IS:

1. A process for the preparation of a peptide containing 6 to 20 amino acid residues and containing tyrosine, the hydroxyl group of which is esterified with sulphuric acid. wherein the tyrosine hydroxyl group of an N-protected tyrosine-containing fragment, which consists of 2 to 6 amino acid residues, is esterified with sulphuric acid, whereafter this fragment is condensed with another peptide containing 2 to 18 amino acid residues,followed by N-deprotection and then by acylation.

2. A process for preparing X-Tyr(SO3~)-Met-Gly-Trp-Met-Asp-Phe-NH2, X being an acyl residue, by esterification of the tyrosine hydroxyl group of an N-protected Tyr-Met Gly-alkyl ester with sulphuric acid, alkaline saponification of the alkyl ester group, coupling to H-Trp-Met-Asp-Phe-NH2 by the mixed anhydride method to give a heptapeptide which is then N-deprotected and finally acylated in the presence of a tertiary amine.

3. A process according to claim 2, wherein the tertiary amine is N-ethylmorpholine.

4. A process according to any of the preceding claims, wherein the tyrosine hydroxyl group is sulphated with chlorosulphonic acid in a pyridine-chloroform-dimethylformamide mixture, whereafter the reaction mixture is worked up with an aqueous alkaline buffer solution.

5. A process according to claim 4, wherein the aqueous alkaline buffer solution used is an aqueous ammonia/ammonium chloride buffer.

6. A process according to claim 2, wherein a mixture of ethyl acetate with a C3-C8 alcohol is used to isolate the sulphated, N-protected tripeptide ester.

7. A process according to any of claims 2 to 6, wherein trifluoroacetic acid containing 15 to 25% by weight of water and an upper layer of methoxybenzene/2-mercaptoethanol (5:1 v/v) is used at ambient temperature for the N-deprotection when the N-protecting group is the tert.-botoxycarbonyl radical.

8. A process according to any of claims 1 to 3 and 7. wherein gel filtration is used for separating sulphated peptides from non-sulphated peptides after N-deprotection and acylation.

9. A process according to claim 1 for preparing peptides. substantially as hereinbefore described and exemplified.

10. Peptides, whenever prepared by the process according to any of claims 1 to 9.

11. The polypeptide succinyl-Tyr(SO3~)-Met-Gly-Trp-Met-Asp-Phe-NH2 and the non-toxic salts thereof.

12. Pharmaceutical compositions. comprising the polypeptide according to claim 11 and,' or at least one non-toxic salt thereof. in admixture with a solid or liquid pharmaceutical diluent or carrier.