Method of preparation of the tetradecapeptide

Somatostatin (also known as somatotropin release inhibitory factor) is a tetradecapeptide of the formula

L-Ala-Gly-L-Cys-L-Lys-L-Asn-L-Phe-L-Phe-L-Trp-L-Lys-L-Thr-Li-Phe-L-Thr-L-Ser- L-Cys-OH.

This tetradecapeptide was isolated from sheep hypothelamic extracts and was found to be effective in inhibiting growth hormone secretion, also known as somatotropin (see P. Brazeau, W. Vale, R. Burgus, N. Ling, M. Butcher, J. River. and R. Guillemin, Science, 179. 77 (1973)].

In addition, U.S. Patent No. 3,904,594 discloses natural somatostatin and a generic class of other compounds with a dodecapeptide sequence corresponding to positions 3-14 of the natural hormone.

A compound usefully referred to as D-Ala'-somatostatin has also been described by Ferlend et al., Molecular and Cellular Endocrinology, 4, 79-88 (1976). D-Ala'-somatostatin, although structurally stereoisomeric of native L-Ale'-somatostatin, is approximately half-effective compared to native somatostatin in inhibiting gastric acid secretion in vivo. The compound of the invention, D-Val'-somatostatin, differs from D-Ala'-somatostatin by substituting two hydrogen atoms with methyl groups. Considering the potential fermacological activity of D-Val'-somatostatin, it should be concluded that its activity should be similar to that of D-Ala'-somatostatin. Thus, in theory, D-Val'-somatostatin would be expected to be less potent than the natural hormone, such as an in vivo inhibitor of gastric acid secretion. However, D-Val'-somatostatin has an efficacy somewhat higher than that of natural hormone. This result shows that the efficacy of D-Val'-somatostatin could not be predicted by comparison with structurally similar known compounds.

The present invention provides a process for the preparation of a tetradecapeptide of formula I,

HD-Val-Gly-L-Cys-L-Lys-L-Asn-L-Phe-L-Phe-L-Trp-L-Lys-L-Thr-L-Phr-L-Thr-L-Ser- L-Cys-OH (I) which is carried out by treating a compound of formula 11,

RD-Val-Gly-L-Cys (R) -L-Lys (Rg) -L-Asn-L-Phe-L-Phe-L-Trp (R)₅) -L-Lys (Rg) -L-Thr (H) -L-Phe-L-Thr (R)₃) -L-Ser (R₄-L-Cys (R₁ ) -X _(AND; where it represents

R is an alpha-amino protecting group,

R₁ a thio protecting group,

Rg is a epsilon-amino protecting group, each of the symbols

Rj and R₄ a hydroxy protecting group,

R 1 is hydrogen or formyl;

The X group of the resin formula wherein the resin is polystyrene is treated with hydrogen fluoride.

An example of the starting material of formula (II) is: N- (BOC) -D-Vel-Gly-L- (PMB) Cys-L- (CBzOC) -Lys-L-Asn-L-Phe-L-Phe- L- (Forz) Trp-L- (CBzOC) -Lys-L- (Bzl) Thr-L-Phe-L- (Bzl) Thr-L- (Bzl) Ser-L- (PMB) Cys-0-CH₂212246 resin

Ύ7 where the resin is polystyrene.

The protecting groups contained in the compounds of formula (II) must possess properties satisfying two aspects. On the one hand, protecting groups must prevent the reactive grouping present in a particular molecule from undergoing a reaction during the exposure of the molecule to conditions that would otherwise result in the disappearance of that reactive grouping. The protecting group, on the other hand, must be of a nature such that it can be readily cleaved while simultaneously regenerating the original reactive group under conditions which would not adversely affect other parts of the molecule. Suitable groups for this purpose, i.e. to protect amino, hydroxy and thio groups, are well known to those skilled in the art. These volumes have been described in detail in their entirety. One of them is the work of Frotective Groups in Organic Chemistry, red.

JFW Mc Omie, Ed., Plenum Press, New York, 1973.

In the above formulas defining the intermediate compounds, R represents an alpha-amino protecting group. The amino protecting groups are well known to those skilled in the art of peptide chemistry, many of which are cited in Mc Omie, supra, in Chapter 2 by JW Barton. Illustrative examples of such protecting groups include benzyloxycarbonyl-, p-chlorobenzyloxycarbonyl-, β-bromobenzyloxycarbonyl-, o-chlorobenzyloxycarbonyl-, 2,6-dichlorobenzyloxycarbonyl-, 2,4-dichlorobenzyloxycarbonyl-, o-bromobenzyloxycarbonyl-, tert -benzyloxycarbonyl-, o-chlorobenzyloxycarbonyl-, o-chlorobenzyloxycarbonyl- BOC), tert-amyloxycarbonyl-, 2- (p-biphenylyl) iaopropyloxycarbonyl- (BpOC), adamantyloxycarbonyl-, cyclohexyloxycarbonyl-, cycloheptyloxycarbonyl-, triphenylmethyl- (trityl) and p-toluenesulfonyl. A preferred alpha-amino protecting group for R is t-butyloxycarbonyl.

R 1 represents a protecting group of the sulfhydryl substituent. Many of these protecting groups are described in Mc Omie's work cited above in Chapter 7 by RG Blckey, VR Rao and WG Rhodes. Illustrative examples of such protecting groups include β-methoxybenzyl-, benzyl-, ρ-tolyl-, benzhydryl-, acetamidomethyl-, tri-yl, p-ni-trobenzyl-, tert-butyl-, isothytyloxymethyl, and any of a large number of three Tyl derivatives. Further examples can be found, for example, in the encyclopedia Houben-Weyl, Methoden der Organiechen Chemie, Synthese von Peptlden, Vol. 15/1 β 15/2 (1974). Stuttgart, Germany. A preferred protecting group for the sulfhydryl group represented by R1 is p-methoxybenzyl.

R 8 represents a suitable epsilon-amino protecting group. Illustrative examples include those listed above as suitable for alpha-amino protection, typical groups for this include benzyloxycarbonyl, tert-butyloxycarbonyl, tert-amyloxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzyl oxycarbonyl- , ρ-chlorobenzyloxycarbonyl-, ρ-bromobenzyloxycarbonyl-, o-chlorobenzyloxycarbonyl-2,6-dichlorobenzyloxycarbonyl-, 2,4-dichlorobenzyloxycarbonyl-, o-bromobenzyloxycarbonyl-, p-nitrobenzyloxycarbonyl-, isopropyloxycarbonyl-, cyclohexyloxycarbonyl-, cycloheptyloxycarbonyl- and p-toluenesulfonyl.

As will be apparent from the description below, the process for preparing the tetradecapeptides of Formula I comprises periodically cleaving the alpha-amino protecting group from the terminal amino acid present in the peptide chain. The only restriction on the identity of the epsilon-amino protecting group of the lysine residue is therefore that the protecting group must be such that it does not cleave under the conditions used for selective removal of the alpha-amino group. Appropriate choice of alpha-amino and epsilon-amino protecting groups is readily appreciated by those of ordinary skill in peptide chemistry, but the relative ease with which each protecting group can be cleaved is to be considered. For example, groups such as 2- (p-biphenylyl) isopropyloxycarbonyl (BpOC) and trityl are very labile and can be cleaved even in the presence of a weak acid. Moderately strong acids such as hydrochloric acid, trifluoroacetic acid or boron trifluoride in acetic acid are required to cleave other groups, such as tert-butyloxycarbonyl, tert-amyloxycarbonyl, adamantyloxycarbonyl and p-methoxybenzyloxycarbonyl. Even stronger acids have to be used for the cleavage of other protecting groups, such as the cleavage of benzyloxycarbonyl, halobenzyloxycarbonyl, β-nitrobenzyloxycarbonyl, cycloalkyloxycarbonyl and isopropyloxycarbonyl groups requiring strongly acidic conditions, i.e. the use of hydrogen bromide, hydrogen bromide or hydrogen fluoride.

Of course, even more labile groups are also cleaved using stronger acids. Thus, when selecting amino protecting groups, it is necessary to ensure that the alpha-amino group is more labile than the epsilon-amino protecting group, and the decoupling conditions must be more selective so that only the alpha-amino protecting group is cleaved.

The advantages of the combination of protecting groups meeting these conditions is the combination of o-chlorobenzyloxycarbonyl- or cyclopentyloxycarbonyl in the meaning of R₂ and tert-butyloxycarbonyl groups as alpha-amino protecting groups used for each amino acid added to the polypeptide chain.

The groups R1 and R1 represent the protective groups of the alcoholic hydroxyl groups of threonine and serine. Many such protecting groups are described in Mc Omie, supra, cited in Chapter 3 by CB Reese. Typical examples of such protecting groups include O, -C ^ alkyl groups such as methyl, ethyl and tert-butyl, benzyl, substituted benzyl such as p-methoxybenzyl, β-nitrobenzyl, p-chlorobenzyl and o-. chlorobenzyl, C 1 -C 4 alkanoyl, such as formyl, acetyl and propionyl, triphenyl methyl (trityl). When α and R ^ are protecting groups, preferably both are benzyl groups.

R 8 is hydrogen or formyl and defines> nr₅ tryptophan residue. The formyl group serves as a protecting group. The use of this protecting group is not obligatory and therefore Rg may be either hydrogen (a group not protected by nitrogen) or formyl (a group protected by nitrogen).

The following abbreviations are used throughout the description, most of which are commonly introduced in the art:

Ala - alanine

Asn - asparagine

Cys - cysteine

Gly - glycine

Lys - lysine

Phe - phenylalanine

Ser-serine

Thr - třeonin

Trp - tryptophan

Val - valine

DCC-N, N'-dicyclohexylcarbodiimide

DMF-N, N-dimethylformamide

BOC - tert-butyloxycarbonyl

PMB - p-methoxybenzyl

CBzOC - o-chlorobenzyloxycarbonyl

CPOC - cyclopentyloxycarbonyl

Bzl - benzyl

Forms 1

BpOC-2- (p-biphenylyl) isopropyloxycarbonyl.

While the choice of particular protecting groups to be used in the preparation of compounds of Formula I is within the skill of the art in peptide synthesis,

The sequence of reactions that must be performed influences the choice of particular protecting groups.

In other words, the chosen protecting group must be stable both to the reactants used and to the conditions of the subsequent reaction steps. For example, as already stated to some extent, the particular protecting group used must remain intact under the conditions used to cleave the alpha-amino protecting group of the terminal amino acid residue of the peptide fragment in preparation for condensation of the next amino acid fragment to the peptide chain. It is also important to select as a protecting group a group that remains intact during the building of the peptide chain and which can be easily cleaved after completion of the synthesis of the desired tetradecapeptide product. However, all these factors are within the skill of the art.

As can be seen from the above discussion, the tetradecapeptides of formula II can be prepared by solid phase synthesis. This synthesis consists in the gradual build-up of the peptide chain, starting at the terminal carbon end of the peptide. In most cases, the cysteine ​​is first coupled to the resin via its carboxy function, by reacting the cysteine ​​with the protected amino group and the protected sulfhydryl group with a chloromethyl or hydroxymethyl resin. The preparation of a hydroxymethyl resin is described in Bodanszky et al., Chem. Indian. (London), £ 8, 1597-1598 (1966). Chloromethylated resin is commercially available.

When carrying out the coupling reaction of the carboxyl function of the cysteine ​​and the resin, the protected cysteine ​​is first converted to the cesium salt. This salt is then reacted with the resin as described by BF Gisin, Helv. Chim. Acta, £ 6. 1476 (1973). Alternatively, the cysteine ​​may bind to the resin by first activating its carboxyl functional group in a conventional manner. For example, cysteine ​​can be reacted with a resin in the presence of a carboxy-activating compound, such as Ν, Ν'-dlcyclohexylcarbodimide (DCC).

Upon binding of the free carboxy group of the cysteine ​​to the resin support, the peptide chain begins to build gradually, by progressively condensing the individual amino acids to the N-terminal portion of the peptide chain. Thus, it is necessary at each stage to cleave the alpha-amino protecting group from the amino acid in the terminal portion of the peptide fragment, and then join the following amino acid residue to the free and reactive N-terminus of the terminal amino acid.

The cleavage of the alpha-amino protecting group can be carried out in the presence of an acid such as hydrobromic, hydrochloric, trifluoroacetic, p-toluenesulfonic, benzenesulfonic, naphthalenesulfonic and acetic acid to give the corresponding acid addition salt of the product.

Another method of cleavage of the amino protecting group is by treatment with boron trifluoride. For example, by treatment with diethyl ether of boron trifluoride in glacial acetic acid, a fragment of an amino-protected peptide is converted to a complex with boron trifluoride, which is then converted to the free peptide fragment by treatment with a base such as aqueous potassium bicarbonate. Any of these methods can be used provided that it is achieved by cleavage of the N-terminal alpha-amino protecting group without disrupting any other protecting groups present in the peptide chain. In view of this requirement, it is preferable to cleave the N-terminal protecting group using trifluoroacetic acid. Typically, the cleavage is carried out at a temperature of about 0 ° C to room temperature.

After cleavage of the protecting group in the N-terminal position, the product obtained is usually in the form of an acid addition salt which has been used for the cleavage of the protecting group. This product can then be converted to a free amino compound by treatment with a weak base, typically a tertiary amine such as pyridine or triethylamine.

Now the peptide chain is prepared for reaction 8 with the following amino acid. The reaction with the amino acid can be carried out by any of several known techniques. For coupling the following amino acid to the N-terminus of the peptide chain, an amino acid having a free carboxy group but having a suitably protected alpha-amino group and any other reactive groups optionally present is used. The amino acid needs to be activated to react with the N-terminus of the peptide chain. One method of activation that can be used in the synthesis is to convert the amino acid to the mixed anhydride. Here, the free carboxylic acid function of the amino acid is activated by reaction with another acid, typically with an acid chloride derivative in the form of an acid chloride. Examples of acid chlorides which can be used to form mixed anhydrides include ethyl chloroformate, phenyl chloroformate, sec-butyl chloroformate, isobutyl chloroformate and pivaloyl chloride.

Another way of activating the carboxyl function of an amino acid for coupling with a peptide chain is by converting the amino acids into a reactive ester derivative. Examples of such reactive esters are 2,4,5-trichlorophenyl ester, pentachlorophenyl ester, p-nitrophenyl ester, 1-hydroxybenzotriazole ester and N-hydroxysuccinimide ester.

Another method of linking the C-terminal amino acid portion to a peptide fragment is a method wherein the condensation is carried out in the presence of at least an equimolar amount of N, N'-dlcyclohexylcarbodiimide (DCC). a group of resin formulas

After the peptide chain has been prepared with the desired amino acid sequence, the resulting peptides can be removed from the resin support. This is done by treating the tetradecapeptide with the reactive groups protected by hydrogen fluoride. The action of hydrogen fluoride cleaves the peptide from the resin, but in addition cleaves all other protecting groups present on the reactive groups of the peptide chain, as well as the alpha-amino protecting group of the terminal amino acid. When the peptide is cleaved from the resin while hydrogen fluoride is deprotected, the reaction is preferably carried out in the presence of anisole. The presence of anisole has been found to inhibit the potential alkylation of certain amino acid residues present in the peptide chain. In addition, the cleavage is preferably carried out in the presence of ethyl mercaptan. Ethyl mercaptan serves to protect the indole ring of the tryptophan residue and furthermore facilitates the conversion of protected cysteine ​​residues into the thiol form. Also, when R1 is a formyl group, the presence of ethyl mercaptan facilitates the cleavage of the formyl group by hydrogen fluoride.

For the above-described cleavage, a straight chain peptide containing 14 amino acid residues is obtained.

The following Examples illustrate the preparation of compounds of formula (II) and intermediates. The examples are only illustrative and do not limit the scope of the invention in any way.

Example 1

N-tert-butyloxycarbonyl-L-cysteinyl- (Sp-methomybenzyl) methylated polystyrene resin

To 500 ml of Ν, dl-dimethylformamide (DMF) containing the cesium salt of N-tert-butyloxycarbonyl- (Sp-methoxybenzyl) cysteine ​​[prepared from 9.06 g (26.5 mmol) of the selected acid] was added 51.0 g of chloromethylated polystyrene resin (0.75 mmol / g). The mixture was stirred at room temperature for 6 days. The resin was filtered off and washed successively three times with a mixture of 90% DMF and 10% water, three times with 95% ethanol and three times with DMF. To the resin suspended in 500 mL of DMF was added a solution of 10.5 g cesium acetate. The mixture was stirred at room temperature for 6 days. The resin was filtered off and washed successively once with aqueous DMF, three times with 90% DMF and 10% water, three times with 95% ethanol, three times with methylene chloride, three times with 95% ethanol and three times with chloroform. The dust particles were removed by suspending the resin four times in chloroform and separating the liquid each time. The resin was then dried under vacuum at 40 ° C overnight. 44.8 g of the title product are obtained. Analysis of the amino acids shows that the product contains 0.25 mmol of cysteine ​​per gram of resin. Cysteine ​​is determined as cysteine ​​acid in the hydrolysis product using a 1: 1 mixture of dioxane and concentrated hydrochloric acid to which a small amount of dimethyl sulfoxide has been added.

Example 2

N-tert-butyloxycarbonyl-L-valyl-glycyl-L- (Sp-methoxybenzyl) cysteinyl-L- (N-epsilon-o-chlorobenzyloxycarbonyl) -lysyl-L-aeparaginyl-L-phenylalanyl-L-phenylalahyl-L- ( formyl) tryptophyll-L- (N-epeilone-o-chlorobenzyloxycarbonyl) lyeyl-L- (O-benzyl) threonyl-L-phenylalanyl-L- (O-benzyl) threonyl-L- (O-benzyl) seryl-L- (Sp-methoxybenzyl) cysteinylmethylated polystyrene resin

The product of Example 1 (7.0 g) was placed in a reaction vessel of an automated peptide synthesizer (Beekman 990) and 12 of the remaining thirteen amino acids were added to the product in this apparatus. The resulting protected resin-bound tridecapeptide is divided into two equal portions and a terminal residue is attached to the product contained in one of these portions. The amino acids used and their order are:

, 2, 3 4.

7 8, 9.

11 , 12, 13.

N-tert.butyloxycarbonyl- (O-benzyl) -L-serine,

N-tert-butyloxycarbonyl- (O-benzyl) -L-threonine,

N-tert-butyloxycarbonyl-L-phenylalanine,

N-tert-butyloxycarbonyl- (O-benzyl) -L-threonine,

N-tert-butyloxycarbonyl-N-epsilon-o-chlorobenzyloxycarbonyl-L-lysine, N^and-tert-butyloxycarbonyl-N-formyl) -L-tryptophan,

N-tert. butyloxycarbonyl-L-phenylalanine,

N-tert-butyloxycarbonyl-L-phenylalanine, N-tert-butyloxycarbonyl-L-asparagine, N-tert-butyloxycarbonyl-N-epililone-o-chlorobenzyloxycarbonyl-L-lysine, N-tert-butyloxycarbonyl- ( Sp-methoxybenzyl) -L-cysteine,

N-tert-butyloxycarbonylglycine a

N-tert-butyloxycarbonyl-D-valine.

The sequence of operations to deprotect, neutralize, and attach an amino acid for each of the amino acids introduced into the peptide is as follows:

1. three washes with chloroform (10 ml / g resin each) for 3 minutes,

2. cleavage of the BOC group by treatment with 29% trifluoroacetic acid 48% chloroform and 6% triethylsilane at 20 ml for 20 minutes for 20 minutes, repeating the procedure twice,

3. two washes with chloroform (10 ml / g resin each) for 3 minutes,

4. one wash with methylene chloride (0 ml / g resin each) for 3 minutes,

5. Wash three times with a mixture of 90% tert-butyl alcohol and 10% tert-butyl alcohol (10 ml / g resin each) for 3 minutes,

6. three washes with methylene chloride (10 ml / g resin each) for 3 minutes,

7. neutralization with 3% triethylamine in methylene chloride (10 ml / g resin) for 3 minutes - repeated three times,

8. Wash with methylene chloride (10 ml / g resin) for 3 minutes - repeat three times,

9. Wash with 90% t-butyl alcohol and 10% t-butyl alcohol (10 ml / g resin) for 3 minutes - repeat three times,

10. Wash with methylene chloride (10 ml / g resin) for 3 minutes - repeat three times, i

11. adding 1.0 mmol / g of protected amino acid resin and 1.0 mmol / g of Ν, Ν'-dicyclohexylcarbodiimide resin (DCC) in 10 ml / g methylene chloride resin and mixing for 120 minutes,

12. Wash with methylene chloride (10 ml / g resin) for 3 minutes - repeat three times,

13. Wash with a mixture of 90% t-butyl alcohol and 10% t-butyl alcohol (10 ml / g resin) for 3 minutes - repeat three times,

14. Wash with methylene chloride (10 ml / g resin) for 3 minutes - repeat three times,

Neutralization by treatment with 10 ml / g of 3% triethylamine resin in methylene chloride for 3 minutes - repeated three times,

16. Wash with methylene chloride (10 ml / g resin) for 3 minutes - repeat three times,

17. Wash with a mixture of 90 56 tert - butyl alcohol and 10% tert - butyl alcohol (10 ml / g resin) for 3 minutes - repeat three times,.

18. Wash with methylene chloride (10 ml / g resin) for 3 minutes - repeat three times,

19 Dec Wash 10 ral / g resin with dimethylformamide for 3 minutes - repeat three times,

20 May adding 1.0 mmol / g of protected amino acid resin and 1.0 mmol / g of Ν, Ν'-dicyclohexylcarbodiimide resin (DCC) in 10 ml / g resin 1: 1 DMF / methylene chloride and mixing for 120 minutes,

21. Wash with 10 ml / g dimethylformamide for 3 minutes - repeat three times,

22nd Wash with methylene chloride (10 ml / g resin) for 3 minutes - repeat three times,

23. Wash with a mixture of 90 56 tert-butyl alcohol and 1 0 56 tert-butyl alcohol (10 ml / g resin) for 3 minutes - repeat three times,

24. Wash with methylene chloride (10 ml / g resin) for 3 minutes - repeat three times,

25. neutralization by treatment with 10 ml / g of resin 356 triethylamine in methylene chloride for 3 minutes - repeated three times,

26. Wash with methylene chloride (10 ml / g resin) for 3 minutes - repeat three times,

27 Mar: Wash with a mixture of 90% t-butyl alcohol and 10% t-butyl alcohol (10 ml / g resin) for 3 minutes - repeat three times and

28. Wash with methylene chloride (10 ml / g resin) for 3 minutes - repeat three times.

The above sequence of operations is used to attach each of the amino acids except glycine and asparagine. The attachment of glycine is carried out using steps 1 to 18 only. The asparagine residue is introduced via the reactive p-nitrophenyl ester. In doing so, stage 11 is replaced by the following three stages:

a) washing of 10 ml / g resin with dimethylformamide for 3 minutes - repeated three times,

(b) addition of 1.0 mmol / g of K-tert-butyloxycarbonyl-L-asparagine p-nitrophenyl ester resin in 10 ml / g of a 1: 3 mixture of dimethylformamide and methylene chloride and stirring for 720 minutes; and

c) washing 10 ml / g resin with dimethylformamide for 3 minutes - repeated three times.

Also, step 20 was modified by adding p-nitrophenyl ester of N-tert-butoxycarbonyl-L-asparagine in a 3: 1 mixture of dimethylformamide and methylene chloride and stirring for 720 minutes.

The resulting resin bound peptide was dried under vacuum. The product is hydrolyzed at reflux in a mixture of concentrated hydrochloric acid and dioxane for 72 hours. Amino acid analysis of the resulting product gives the following results (lysine} is used as standard): Asn 1.04, 2Thr 2.68, Ser 1.08, Val 1.12, Gly 1.04, 3Phe 3.87, 2Lys 2.00 Trp 0.75.

Tryptophan is determined by 21 hours hydrolysis of a sample of the product in the presence of dimethylsulfoxide and thioglycolic acid. Cysteine ​​is not determined because it decomposes when analyzed by this method.

Příklaů 3

D-valyl-glycyl-L-cysteinyl-L-lysyl-L-asparaginyl-L-phenylalanyl-L-phenylalanyl-L-tryptophyll-L-lysyl-L-threonyl-L-phenylalanyl-L-threony.L-seryl- L-cysteine

To a mixture of 5 ml of anisole and 5 ml of ethyl mercaptan was added 2.828 g (at a substitution level of 0.150 mmol / g) of protected tetradecapeptide bound to the resin of Example 2. The mixture was cooled in liquid nitrogen and distilled with 56 ml of liquid hydrogen fluoride. The resulting mixture was allowed to warm to 0 ° C and stirred for 2 hours. The hydrogen fluoride is then distilled off. Ether was added to the remaining mixture and the resulting mixture was cooled to 0 ° C. The resulting solid was filtered and washed with ether. The product was dried and the deprotected tetradecapeptide was extracted from the resin mixture using 1M acetic acid and a small amount of glacial acetic acid.

The acetic acid solution is then lyophilized to dryness in the dark. The resulting white solid was suspended in a mixture of 10 mL degassed 0.2 M acetic acid and 4 mL glacial acetic acid. The suspension is heated, but all solids do not completely dissolve. The insoluble matter was filtered off and the opaque colorless filtrate was applied to a Sephadex G-25F column. Chromatography shall be carried out under the following conditions:

solvent: degassed 0.2 M acetic acid column dimensions: 75 x 1500 mm, temperature: 26 ° C, flow rate: 629 ml / h, fraction volume: 22.0 ml.

The absorbance at 280 nm of each fraction is plotted against the sizing number. A curve is obtained with a large wide maximum and an adjacent arm (inflection). UV-spectroscopy shows that the major reading of maxima is the product. Fractions 224-240 (i.e. eluate from 4.906 to 5.280 ml, peak corresponding to a total flow of 5.054 ml eluate) were pooled.

The pooled fractions do not contain a product corresponding to the curve arm. UV-spectroscopy shows that the combined fractions contained 175 mg of the product (yield 24.8 96). The content of sulfhydryl groups was found to be 93.6% of theory by the bllucan titration of an aliquot.