DE2647188A1 - METHOD FOR PRODUCING A PEPTIDE - Google Patents

Description

where A and B are identical or different and each represent an amino acid residue or a peptide residue "and where X is an amino protecting group and where Y is one of the following carboxyl protecting groups: tert-alkoxy group, Benzyloxy, benzylamino and benzhydrylamino groups, which are optionally substituted with an inert substituent is prepared by adding an amino acid or a peptide with an N-terminal protecting group or a salt thereof of the formula

X-A-OH

with an amino acid or a peptide of a C-terminal Protecting group or a salt thereof of the formula

H-B-Y

converts, in the presence of a thiol proteinase enzyme or a serine proteinase enzyme in an aqueous solution with a pH at which the enzyme activity of thiol proteinase or the serine proteinase is maintained "

The invention relates to a method for producing a peptide. In particular, the invention relates to a method for the production of a peptide using a specific enzyme as a catalyst.

7 0 β 81771113

Conventionally, peptides are prepared by the azide method or by the mixed acid anhydride method, the carbodiimide method, the active ester method or the acid chloride method or the like. These conventional methods occur however, poses various industrial problems. This leads to a racemization of the carboxyl component of the C-terminal amino acid residue. Then there must be side reactions, problems of temperature control, problems of solvent choice and with difficulty in the properties of the amino protecting groups and the carboxyl protecting groups can be expected, as well as with special effects, which by the functional groups of the side chains of the amino acids be evoked.

The fragment condensation method for the preparation of peptides can be used with advantage in compounds which have glycine as the carboxyl end group (the only amino acid that is not racemized). With other connections however, with amino acids other than carboxyl end groups, racemization cannot be prevented. In the ! Eat represents the racemization in all previous peptide syntheses poses a difficult problem. When racemization occurs, the purity of the product is very low and it is necessary to separate the contaminating isomer from the product. This is extremely detrimental to the industrial Implementation of the procedure.

Of all the conventional methods of making peptide bonds, the azide method is unique largely free of racemization problems and this azide method is therefore the method of choice. The azide process however, requires complicated reaction steps and urea derivatives are formed by a side reaction. For these reasons, the azide method is unfavorable from the standpoint of the yield.

In addition to the various organic

709817/1113

chemical method using peptide synthesis method proposed the enzymes papain and chymotrypsin (J.S. Fruton "Advances in Protein Chemistry", 5, Academic Press Inc., New York, If.Y. 1949). The following reactions occur a:

Bz-Leu-OH + H-Leu-NH0 (D (ID

Bz-Leu-Leu-NH0 (III)

Bz-Leu-OH + H-Gly-NH0 (D (II)

“Bz-Leu-Gly-NH0

(III)

Bz-Tyr-OH + H-Gly-NH0
(D (II)
^ Bz-Tyr-Gly-NH0

(III)

Z-Phe-Gly-OH + H-TyT-NH ₂ (D (II)

f Z-Phe-Gly-Iyr-NH ₂

(III)

The phenylamino group of peptide (III) must be right drastic conditions are removed because the phenylamino group attached to the C-terminal group of the amine component (II) bound cannot be easily cleaved from the peptide. This disadvantageously leads to a split the peptide chain. For these reasons, this peptide synthesis method has hitherto been used practically not applied. On the other hand, in reaction (4), traneamidation and transpeptidation occur one, so that this reaction is not practically usable either. (R.B. Johnston et alj J. Biol. Chem., 185, 629 (1950) and J.S. Fruton et al; J. Biol. Chem., 204, 891 (1953))

7 C 9 β 1 7 / i 11 3

In reaction (4) the primary amino group of the acid amide is which is connected to the terminal group of the amine component, a promoter for the catalyzed by papain Amidase reaction. Therefore, these "known methods only approach theoretical interest in that they show that papain and Chymotrypsin as catalysts for the synthesis of peptide endings can be used in which phenylamino groups as protective groups of the terminal carboxyl group serve the amine component. There is therefore a significant need for efficient methods of peptide synthesis.

It is therefore the object of the present invention to provide a method for the synthesis of oligopeptides or polypeptides, which leads to high yields with a simple procedure.

This object is achieved according to the invention by a method for Preparation of a peptide of the formula

X-A-B-Y

dissolved, where A and B are the same or different and mean an amino acid residue or a peptide residue and where X is a Amino protecting group and where Y is a carboxyl protecting group, namely a substituted or unsubstituted one tert-alkoxy, benzyloxy, benzylamino and benzhydrylamino groups means, wherein an amino acid or a peptide having an N-terminal protecting group or a salt thereof of formula

X-A-OH

with an amino acid or a peptide with a C-terminal Protecting group or a salt thereof of the formula

H-B-Y

is reacted, in the presence of thiol proteinase or serine proteinase in an aqueous solution, the pH of which is chosen so that the enzyme activity of the thiol proteinase or the serine proteinase is maintained.

The thiol proteinase enzyme includes the enzymes papain, stembromelein, Ficin, cathepsin B, chymopapain, streptococcal protein

709817/1113

nase, Asclepain, Clostridium histolyticum protenase B and yeast protenase B. Thiolproteinaee is suitable for the hydrolysis of a large number of different proteins, as well as for promoting the cleavage of a large number of peptide bonds, amino bonds and ester bonds, as well as for promoting the decomposition of benzoylalginine amide, benzoylglycylleucylleucyl-amide, benzoylglycine. The thiol proteinase is thus effective both as an exopeptidase and as an endopeptidase (JR Kimmel and EL Smith, Advance Enzymology, 19, 267 (1957)). Serine proteinase includes the enzymes substilisin, Aspergillus alkaline proteinase, elastase, α-lytin proteinase, chymotrypsin, metridium proteinase A, trypsin, thrombin, tlasmin, kininogenin, enteropeptidase, acrosin, phaseolus protenacterase, and tenemaria endopeptidase a protenacterase, arthropeptidase, arthropeptidase. It has been reported that serine proteinase has considerable hydrolysis activity against the leu (15) - Tyr (16) sequence and against the Tyr (16) - Leu (17) sequence of the insolin B chain, and also has a large esterase activity against Acyl amino acid esters. The subtilisin obtained from B. subtilis or analogous microorganisms includes types from Carsbery, Novo, BPN ¹ and the like. Alkaline proteinase which can be isolated from ray fungi is a known material.

In the following, the term acid component is used to denote the amino acid or the peptide starting material of the formula

X-A-OH

denotes, where X is a protecting group of the terminal amino group and where A is an amino acid residue or a peptide residue. Group A denotes an amino acid residue or a peptide residue, where suitable amino acids are preferably aliphatic amino acids, such as monoaminomonocarboxylic acids, z. B. Glycine (GIy), Alanine (AIa), Valine (VaI), Forvaline (nor-val), leucine (leu, isoleucine (iso-leu), Norleucine (nor-Leu); Oxyamino acids, such as serine (Ser),

709617/11 13

Threonine (Thr), homoserine (homo-Ser); sulfur-containing amino acids, such as methionine (Met) or cystine (CysS) and Cysteine (CysH); Monoamino diarboxylic acids such as aspartic acid (Asp), glutamic acid (GIu), asparagine (Asn) and Glutamine (GIn); Diamino monocarboxylic acid, such as ornithine (Orn), Lysine (Lys), arginine (Arg); aromatic amino acids such as phenylalanine (Phe) and tyrosine (Tyr); and heterocyclic Amino acids such as histidine (His), tryptophan (Try). These amino acids are denoted by symbols "which appear on the Field of proteins are common. Peptides are identified by combinations of these symbols.

Suitable protective groups for the free terminal amino group (N-terminal protective group) of the acid component are tert-alkoxycarbonyl groups, such as t-butyloxycarbonyl (BOC-), t-amyloxycarbonyl (t-Aoc-); Benzyloxycarbonyl (Z-), p-methoxybenzyloxycarbonyl (PMZ), 3,5-dimethoxybenzyloxycarbonyl Jz (OMe) ₂ -J, 2,4,6-trimethylbenzyloxycarbonyl (TMZ-), p-phenylazobenzyloxycarbonyl (PZ-); p-toluenesulfonyl (Tos-); o-nitrophenylsulfenyl (Nps-); or the like. The other amino acid or peptide starting material has the formula

H-B-Y.

This component is referred to below as the amine component. In this formula, B denotes an amino acid residue or a peptide residue which corresponds to the definition of Remainder A follows.

Suitable protective groups for the carboxyl group (C-terminal protective groups) of the amino component are tert-alkoxy groups, such as t-butoxy (-0Bu ¹ *), benzyloxy £ OBzl), p-nitrobenzyloxy i-OBzl (ρ-Ν0 ₂ ) | , Benzhydryloxy (-OBzh), benzylamino (-NHBzI), 2,4-dimethoxybenzylamino (-NHDMB) and benzhydrylamino (-NHBzh). These protective groups for the terminal carboxyl group of the amine component are resistant to esterase reactions and amidase reactions which can be caused by the thiol proteinase enzymes and the serine proteinase enzymes.

700817/1113

Amino acid residues and peptide residues can also be used as the acid component and as the amine component in the method according to the invention veru / ends with functional groups in the side chains u / earth. In these cases it is preferable to use these functional ones as well Protect groups with protection groups. Suitable protecting groups for the a> -amino group (N) are N ^ -Benzyloxycarbonyl (N -Z), t-butoxycarbonyl (N ^ -BOC) and tosyl (N -Tos). Suitable protecting groups for the N-guanidino group

(N ^G ) of Arg are Nitro (N ^G -NO "), N ^G -Benzyloxycarbonyl (N ^G -Z)

GG G

and N, N -dibenzyloxycarbonyl (N -ZZ). Suitable protecting groups for the imidazole ^{rings (N lm} ) of His are N ^lm -benzyl (N -Bzl) and tosyl (N -Tos). Suitable protective groups for the ω- carboxyl group are ^ -Benzyloxy (-OBzl). Suitable protective groups for the hydroxyl group of the aliphatic or aromatic oxyamino acids are aralkyl groups, such as benzyl (Bzl). Suitable S-protecting groups for the mercapto group of CysH include the benzyl group (Bzl). These protective groups should be stable under the conditions of the main reaction and, on the other hand, should be easy to split off from the end product without leading to side reactions. The acid component used as starting materials and the amine component can have protective groups or the N -amino group of the amine component can be free or in the form of an inorganic or organic salt, e.g. B. a hydrochloride, a hydrobromide, oxalate, p-toluenesulfonate or acetate. In the inventive method, the condensation reaction, which leads to the formation of peptides, can be carried out in an aqueous solution which has a pH value at which the enzyme activity is retained. In the case of thiol proteinase, this pH is about 4 to 7.5, and in the case of serine proteinase, about 6 to 9.

709817/1113

There are two methods of maintaining the correct pH to maintain enzyme activity. The first method is to carry out the condensation reaction in a buffer solution, e.g. B. a citric acid buffer solution, a Mcllvaine buffer solution, a Kolthoff buffer solution, a Tris-HCl buffer solution or a veronal buffer solution perform, with the acid component and the amine component are dissolved in the buffer solution and whereupon the enzyme is added. The other method is, depending to add the acid or base to the reaction mixture according to the measured pH value during the condensation reaction, so the pH of the reaction mixture in a range necessary for maintaining the enzyme activity to keep.

The starting materials can be used in a proportion of 0.8 to 2 moles and preferably 1 to 1.5 moles of the acid component can be used per 1 mole of the amine component. If the starting materials are not too soluble in the aqueous solution a solvent can be added to improve the solubility of the reactants, e.g. B. an alcohol like Methanol or ethanol; Dimethylformamide; Dioxane; Tetrahydrofuran; Dimethyl sulfoxide or the like. The amount of added Solvent should be limited so that the activity of the enzyme for the reaction of the invention is not impaired or is inhibited. If a solvent is used, it should be used in amounts of less than 1 part by weight and preferably 0.2 to 1.0 part by weight per 1 part by weight of water are used. The inventive reaction will carried out in an aqueous solvent. It is necessary, to lower the relative solubility of the reaction products, so that they are preferably sparingly soluble in the system or are insoluble.

The amount of the thiol proteinase enzyme or the serine proteinase enzyme is in the range from 10 to 500 mg, preferably 10 to 400 mg and especially 50 to 300 mg per 1 mmol of the amine component.

709017/1113

Furthermore, the solution can also contain an enzyme activator, such as CysH or a salt thereof or 2-mercaptoethanol or a salt thereof can be added. The reaction temperature is usually in the range from 20 to 55 ° C and in particular in the range from 30 to 40 C. Under these conditions, the enzyme activity is retained. The reaction proceeds smoothly for 1 to 24 hours under the conditions mentioned. The reaction product falls out of the Reaction system and can be isolated easily.

According to the invention, you can learn a dipeptide, an oligopeptide or a polypeptide of the following formula

X-A-B-Y

obtained in a simple manner by adding the groups A and B the starting materials of the formulas

Appropriately selects X-A-OH and H-B-Y.

The dipeptide derivative LysyJrlysin can according to the following reaction formula getting produced.

Z-Lys (Z) -OH + H-Lys (Z) -0Bu ^fc

Z-Lys (Z) -Lys (Z) -OBu ^t

Lysine, whose co- amino group is protected with the carbobenzoxyl group (Z-) (Z-Deriwat), serves as the acid component and a t-butyl ester serves as the amine component. Lysyl-lysine is obtained, the amino group of which is protected in the side chain.

The dipeptide derivative arginyl-leucine containing arginine is obtained by the following reaction Z-Arg (Z, Z) -OH + H-Leu-OBzh

^ Z-Arg (Z, Z) -Leu-OBzh.

The Tri-Z-arginine is obtained by protecting the guanyl

709817/1113

group and the amino group of arginine with Z. It serves as Acid component. The benzhydryl ester of leucine serves as the amine component. A dipeptide derivative is obtained in a simple manner by reaction in the presence of papain. If you have an amino acid with a functional group in the side chain is used, the amino acid whose functional group is protected or unprotected, as in the examples below to be implemented. Some reactions of histidine are explained below.

B0C-His (Bzl) -0H + H-Leu-OBzh

f BOC-His (Bzl) -Leu-OBzh ... (a)

BOC-His-OH + H-Leu-OBzh

$ ► BOC-His-Leu-OBzh ... (b)

Reaction (a) denotes the case of protection of the side chain while in reaction (b) the side chain is unprotected.

The relations of dipeptide production have been explained above. Another situation exists in the case of an acid component with a peptide bond. In this case, an acyl dipeptide is reacted with an amino acid amide derivative. The reaction proceeds smoothly in the presence of papain without any side reactions occurring. It can be illustrated by the formula below:
Z-Pro-Gly-OH + H-Leu-NHBzh

Z-Pro-Gly-Leu-NHBzh

Carbobenzoxy-prolyl-glycyl-leucine-benzhydrylamide is obtained.

Examples of the synthesis of tripeptides are shown in Table 3. The following explains the relationships which exist when the acid component is a dipeptide and when the amine component is also a dipeptide.

70Ö817 / 1113

The following reaction can lead to the synthesis of the fragment (3-6) tetrapeptide of UaI -angiotensin-II, which as a polypeptide hormone known to be used.

BOC-l / al-Tyr (Bzl) -OH + H-Val-His (BzI) -OBzI

■ * BOC-l / al-Tyr (BzI) -l / al-His (BzI) -OH

When the l / alylhistidine ester is used as the amine component of the reaction, and when the reaction is carried out in the manner of papain, a tetrapeptide having a benzyl ester group cannot be obtained. This is believed to be due to an esterase effect, which is one of the characteristics of papain. The above reaction is particularly advantageous when the tetrapeptide is used as the acid component in the next stage of polypeptide synthesis. When the carboxyl group of the amine component is the benzhydrylamide, a tetrapeptide benzhydrylamide derivative can be obtained in high yields. Examples in which a dipeptide derivative is used as an acid component or an amine component are listed in Table 4. The same reaction can be carried out using serine proteinase, e.g. B. Subtilisin BPN ¹ can be performed. As already mentioned, oligopeptides and polypeptides can be synthesized by selecting suitable protective groups for the carboxyl group of the amine component and by using thiol proteinase or serine proteinase, although these show an esterase effect and an amidase effect. The catalytic effectiveness of these enzymes for peptide synthesis is completely unexpected.

From the above description it is evident that the inventive Process for the production of peptides with desired Amino acid sequence succeeds by utilizing the endopeptidase characteristics. Even catalytic amounts of the enzyme are sufficient and the enzyme can be used repeatedly will. The reaction proceeds under mild conditions in a buffer solution or in a solution with the desired one pH smoothly. The yield is relatively high and the product shows a very high purity. The inventive

709817/1113

Procedure is suitable for both gradual extension the peptide chains as well as condensation wan peptide fragments. Both reactions are suitable for industrial purposes. In addition, there is no racemization of the peptides, a result that cannot be achieved with conventional peptide syntheses.

In the following the invention is illustrated by means of embodiments explained in more detail.

example 1

A mixture of 20 ml Mcllvain buffer solution of pH 6.2 and

3 ml of methanol are added to 497 mg (1.20 mmol) of Z-Lys (Z) -OH and 366 mg (1.09 mmol) of H-Lys (z) -0Bu. 130 mg of papain (titer 1200 CSU / g, manufactured by Green-cross KK) and 0.05 ml of 2-mercaptoethanol are then added to the mixture with stirring at 38 ° C., whereupon the reaction is carried out for 24 hours. The oily product obtained is extracted with ethyl acetate and the extract solution is washed in sequence with water, 0.5N HCl, 7% ammonia water and again water. The ethyl acetate solution is concentrated and petroleum ether is added. 385 mg (48 %) of crude crystals of Z-Lys (Z) -Lys (Z) -OBu ^{t are obtained} . Some of the crystals are recrystallized from ethyl acetate / petroleum ether. A pure product is obtained with a melting point of 62 to 66 ^{° C. and 5 =} ~ ¹⁴ ' ² ° ( ^{c =} °' ⁵ methanol).

Elemental analysis

C HN

calculated (%) 65.55 7.15 7.65 found (%) 65.55 7.22 7.67

Example 2

A mixture of 20 ml Mcllvaine buffer solution with pH 7.0 and

4 ml of methanol is added to 692 mg (1.20 mmol) of Z-Arg (Z, Z) -OH and

709817/1113

470 mg (1.00 mmole) of H-Leu-OBzh.TosOH were given. 150 mg of papain and 0.1 ml of 2-mercaptoethanol are then added to the mixture with stirring at 38 ° C. and the reaction is carried out for 10 hours. The colorless precipitate obtained is filtered off and washed in sequence with water, 7% ammonia water and again water, Z-Arg (Z, Z) -Leu-0Bzh

receives.

Yield 850 mg (99 %)

Melting point 161 to 168 ^° C

[&>] p ⁵ = -9.4 ⁰ C (C = 1.0 Ν, Ν-dimethylformamide)

Elemental analysis

C HN

calculated {%) 68.76 6.24 8.18 found (%) 68.43 6.18 8.38

Example 3

20 ml of McI IV / aine buffer solution at pH 6.6 become 691 mg (2.00 mmol) of BOC-His (BzI) -G.-; and added 599 mg (1.80 mmol) of H-Leu-NHBzh.HCl. Then 50 mg of papain and 100 mg of cysteine hydrochloride are added to the mixture with stirring at 38 ° C. and the reaction is carried out for 24 hours. The colorless precipitate obtained is filtered off and washed in sequence with water, 7% ammonia water and water. 750 mg of crude crystals are obtained. The product is dissolved in 50 ml of hot methanol and the hot solution is treated with activated charcoal to remove the protein. The solution is then concentrated and water is added to the residue and the product is recrystallized to give a crystalline product, namely BOC-His (Bzl) -Leu-NHBzh. Yield 686 mg (62 %)
Melting point 113 to 115 ^° C

Ip = -8.1 ° (C = 1.0 chloroform)

Elemental analysis C HN

calculated {%) 71.24 7.27 11.23 found {%) 71.15 7.30 11.31

709817/1113

Example 4 «* V jT *

20 ml of MeIlvaine buffer solution of pH 6.6 are added to 511 mg (2.00 mmol) of BOC-His-OH and 599 mg (1.80 mmol) of H-Leu-NHBzh.HCl. Then gives 250 mg papain and 100 mg of cysteine hydrochloride with stirring at 38 ⁰ C to the mixture and the reaction is carried out u / hile 24 h. The colorless precipitate obtained is filtered off and washed in sequence with water, 7% ammonia water and water, a crystalline crude product being obtained. This is dissolved in ethanol / water and the hot solution is treated with activated charcoal to remove the protein. The solution is then concentrated. This gives the crystalline product BOC-His-Leu-NHBzh.

Yield 336 mg (35 %) melting point 221 to 223 ^° C

^ ⁵ = -23.6 ° (C = 0.5 chloroform) Elemental analysis CH ti '

calculated {%) 67.52 7.37 13.12 found {%) 67.39 7.38 13.21

Example 5

40 ml McIlvaine buffer solution υοη pH 6.2 are added to 531 mg (2.00 mmol) BOC-Phe-OH and 935 mg (2.00 mmol) H-His- (Bzl) -NHDMB.2HCl, whereupon 4 ml of 1N NaOH are added to the solution. 480 mg of papain and 240 mg of cysteine hydrochloride are then added to the mixture at 38 ° C. with stirring. The reaction is then carried out for 24 hours. The colorless precipitate obtained is filtered off and washed in succession with water, 7% ammonia and then again with water. 960 mg of a crystalline crude product are obtained. This is dissolved in 100 ml of hot methanol and the hot solution is treated with activated charcoal for 30 minutes in order to remove the protein. The solution is then concentrated and water is added, the crystalline product BOC-Phe-His (BzI) -NHDMB.1 / 2H ₂ O appearing.

709817/1113

26A7188

Yield 650 mg (40 %) melting point 128 to 132 ⁰ C: = +1.6 (C = 1.0 chloroform)

Elemental analysis

C HN

calculated {%) 66.44 6.82 10.76 found {%) 66.34 6.66 10.78

Examples 6 to 23

The experience of Example 1 is repeated, but here the N * -acylamino acid as the acid component and the amine component the amino acid ester of H-l / al-0Bzh according to the table is used. The results are shown in Table 1. Example 1 is also repeated, but with this the N-acylamino acid of Z-AIa-OH is used as the acid component and u / obei as the amine component of the amino acid ester or the amide according to Table 2 can be used. The results are also compiled in Table 2 '.

709817/1113

acid
component Z-M et-OH Amine
component Table 1 the end
beu
te ^) - 16 - H
7.22
7.12 NS
5.28
4.74 Z-Leu-OH Z-Asn-OH H-Val-OBzh product 73 Enamel
Point
( ⁰ C) 6.71
6.34 4.66
4.80 E.g,
No, Z-Phe-OH £ -Glu-OH H-Val-OBzh Z-Leu-Val-OBzh 91 oily Φ
(2) Elemental analysis
(1) calculated (%)
(2) found (%) 6.39
6.44 5.55
4.66 6th Z-Ser-OH Z-GIu-OH H-Val-OBzh Z-Phe-Val-OBzh 34 100-106 Φ C.
72.43
72.54 6.61
6.59 5.40
5.26 7th _g e-Thr-OH Z-Arg (NO ₂ ) -OH H-Val-OBzh Z-Ser-Val-OBzh 66 oily Φ 69.98
71.21 6.61
6.60 5.12 5.84
5.16 5.79 θ 10 Z-Lys (Z) -OH H-Val-OBzh Z-Thr-Val-OBzh 81 89-95 Φ
Φ 69.03
71.11 6.26
6.27 7.90
7.41 11 H-Val-OBzh Z-Met-Val-OBzh 25th 95-98 © 69.48
69.19 6.27
6.31 5.12
4.84 12th H-Val-OBzh Z-Asn-Val-OBzh 61 116-125 Φ
Φ 67.86
67.83 6.47.
6.39 7.74
7.74 13th H-Val-OBzh Z-Glu-Val-OBzh 69 125-131 Φ
(2) 67.78
68.18 6.19
6.21 13.58
13.40 14th H-Val-OBzh Z-GIu-VaI-OB zh 83 178-183 ® 68.12
68.23 6.67
6.66 6.18
6.33 15th H-Val-OBzh Z-Arg (NO ₂ ) -Val-OBzh 70 139-142 Φ 68.24
67.94 Z-Lys (Z) -Val-OBzh 110-117 Φ 62.12
62.98 70.67
70.28

Tabel

- 17 -

C3> CO OO

E.g.
No. acid
component Amine
component product the end
prey
{%) Enamel
Point
( ⁰ C) 2> Elemental Analysis
(1) calculated (#)
(2) found {%) H
6.60
6.54 N
5. 73
5.82 16 Z-AIa-OH H-Val-OBzh Z-Ala-Val-OBzh 80 92-96 (P
(D C.
71.29
71.28 8.22
8.15 7.14
7.14 17th Z-AIa-OH H-Leu-OBu * Z-Ala-Leu-OBu * 49 95.5-97.0 © 64.26
64.65 6.01
6.01 5. 52
5.11 18th Z-AIa-OH H-Phe-OBzh Z-Ala-Phe-OBzh 89 123-125 73.86
73.97 6.82
6.83 5. 58
5.44 19th Z-AIa-OH H-Ile-OBzh Z-Ala-Ile-OBzh 92 88-96 (D 71.69
71.72 6.36
6.10 9. 14
9.24 20th Z-AIa-OH H-Ala-NHBzh Z-Ala-Ala-NHBzh 61 230-232 Φ 70.57
69.48 6.02
6.11 11.15
10.92 21 Z-AIa-OH H-Asn-NHBzh Z-Ala-Asn-NHBzh 53 ! 252-254 UD
(D 66.91
66.70 6.06
6.10 16.38
16.42 22nd Z-AIa-OH H-Arg (NO ₂ ) -
NHB zh Z-Ala-Arg (NO ₂ ) -
NHBzh-1/2 H _? O 52 123-125 60.19
60.06 7.15
7.22 7.65
7.67 23 Z-AIa-OH H-LyS (Z) -OBu * Z-AIa-LyS (Z) -OBu * 48 62-66 65.55
65. 55

Example 24 V \ 4 ^s

1.5 ml of Ν, Ν-dimethylpormamide are added to 370 mg (1.20 mmol) of Z-Pro-Gly-OH and 305 mg (1.00 mmol) of H-Leu-NHBzh.i / 2H ₂ O with stirring. 15 ml of citric acid buffer solution having a pH of 5.5 is then added to the mixture. The mixture is then replaced with 200 mg of papain and 0.2 ml of 2-mercaptoethanol with stirring at 38 ° C. and the mixture is reacted for 24 hours. The colorless precipitate obtained is filtered off and washed in succession with water, a 5% aqueous citric acid solution, water and 7 % ammonia solution. This gives 310 mg (51%) of crude crystals having a melting point v / on 146 to 149 ⁰ C. The product ujird dissolved in methanol and a small amount of insoluble material by filtration luird separated. The solution is concentrated and water is added to the residue. The product obtained is Z-Pro-Gly-Leu-NHBzh.H ^ O having a melting point 148-150 ⁰ C, and (cc / ^ = -41.0 ° (C = 0.5, methanol).

Elemental analysis calculated {%) _6? ^ _75? ^ ₂ ^ ₃₀

found {%) 68.05 6.96 9.24 Examples 25-32

The experience of Example 24 is repeated if one is furniture however, the acid component is the N-acyl dipeptide from Z-Phe-Ala-OH and the amino acid or the amide according to Table 3 is used as the amine component. The results are shown in Table 3.

709817/1113

Tabel

- 19 -

E.g. acid no. composed

Amine component

Product yield

Schmel ;:
Point

( ⁰ C)

Elemental analysis

(1) calculated (%) (2) found (%)

25th

Z-Phe-Ala-OH

H-Gly-NHBzh

Z-Phe-Ala-Gly-NHBzh 218-219

70.92 71.17

6.12 6.15

9.45 9.46

26th

Z-Phe-Ala-OH

H-Ala-NHBzh Z-Phe-Alä-Ala-NHBzh

1 / 2H ₂ O 219-220

70.22 70.39

6.38 6.22

9.10 9.28

27

Z-Phe-Ala-OH

H-Val-NHBzh ] Z-Phe-Ala-Val-NHBzh 258-259

φ 71.90 (Zj 71.97

6.67 6.69

8.83 8.89

28

Z-Pro-Gly-OH

H-Ile-NHBzh ■ Z-Pro-Gle-NHBzh

211-213

69.84 69.63

6.90 6.86

9.58 9.41

29

Z-Pro-Gly-OH

H-Val-NHBzh

Z-Pro-Gly-VaI-NHB zh · 1 / 2H ₂ O

197-199

Φ 68.3 7 φ 68.77

6.78 6.61

9.67 9.65

30th

Z-Pro-Gly-OH

H-M et-NHB zh

Z -Pro-Gly-Met-NHBzh

190

φ 65.76 © 65.61

6.35 6.38

9.30 am 9.46

31

Z-Pro-Gly-OH

H-Arg (NO ₂ ) -NHB zh

Z-Pro-Gly-Arg (NO ₂ ) -NHB zh! 120-125

60.70 60.65

5.99 6.23

16. 16.

CO

11.96 OO 12.01

32

Z-Pro-Gly-OH

H-A sn-NHB zh

Z-Pro-Gly-Asn-NHBzh

239-240

U) 65.62 (D 65.62

6.02 6.00

Example 33

A mixture of 15 ml of McIlvaine buffer solution with a pH of 8.0 and 15 ml of methanol become 564 mg (1.2 mmol) of BOC-ual-Tyr- (Bzl) -OH and 516 mg (1.0 mmol) of H-Val-His (Bzl) -OBzI.2HCl given. 300 mg of papain and 150 mg of cysteine hydrochloride are then added to the mixture while stirring at 38 ° C. whereupon the reaction is carried out for 24 hours. The colorless precipitate obtained is filtered off and washed with Water washed. 450 mg of crude crystals are obtained. The product is dissolved in 40 ml of ethanol and the hot solution is treated with activated charcoal to remove the protein. The solution is then cooled and the precipitate obtained is filtered off and the filtrate is concentrated. The residue is added to the residue by adding ether recrystallized. BOC-Wal-Tyr (Bzl) - \ / al-His (Bzl) -OH is obtained.

Yield 100 mg (12 %) ninhydrin test: negative

Melting point: 159 to 165 ^° C

Va \ _D = -8.16 ° (C = 0.5 Ν, Ν-dimethylformamide)

Elemental analysis C HN

calculated (%) 63.44 7.26 10.00 found {%) 63.67 7.04 10.24

Example 34

A mixture of 20 ml Mcllvaine buffer solution with pH 6.1 and 10 ml methanol become 518 mg (1.1 mmol) BOC-Ual-Tyr (Bzl) -DH and 583 mg (1.0 mmol) H - \ / al-His (Bzl) -NHBzh.2HCl, whereupon 2 ml of 1N NaOH are added to the solution. 240 mg of papain and 120 mg of cysteine hydrochloride are then added to the solution with stirring at 38 ° C. The reaction is then carried out for 48 hours. The colorless precipitate obtained is filtered off and washed in succession with 0.5N HCl, 7% ammonia water and water. 950 mg of crude crystals are obtained. The product iuird dissolved in 300 ml of methanol and the hot

709817/1113

Solution is treated with activated charcoal to remove the protein. The solution is then concentrated and the residue becomes recrystallized from N, N-dimethylformamide / Wassar, ujobei man BOC-Val-Tyr (BzI) -Val-His (BzI) -NHBzh.

Yield 410 mg {% 2%) Melting point 237 to 239 ⁰ C ^ = -6.1 ° (C = 1.0 N, N-dimethylformamide)

Elemental analysis calculated {%) ^ ₈₄ ^ ₀ ^ _{Q> [] n}

found (%) 70.18 7.01 9.94 Examples 35 to 41

The procedure of Example 34 is repeated, if one as the acid component the N ^ -acyldipeptide according to Table 4 and as the amine component the jeu / hasty dipeptide amide of H-Phe-Ser-NHBzh according to Table 4 is used. The results are compiled in Table 4.

709817/11 13

Table 4

- 22 -

O (O OO

E.g. IMr./ Amine product The end- Enamel © Elemental analysis calculated N 30th S. 6th /Acid-
compose component beu-
• ip Point (D found 10. 36 6th te ( ⁰ C) H 10. 12th Example 35 Z-Ala-Gly-Phe-Ser- (2) C. 6.08 10. 26th Z-Ala-Gly-OH H-Phe-Ser-NHBzh NHB zh 85 254-256 © 67.17 5.94 10. 18th BOC-Val-Gly-Phe-Sert- <2> 67.02 7.14 10. 00 BOC-Val-Gly-OH H-Phe-Ser-NHBzh NHBzh-1 / 2H ₂ O 82 238-239 © 64.23 7.05 10. 27 Example "36 BOC-Leu-Gly-Phe-Ser- CS) 64.27 7.18 9. 46 BOC-Leu-Gly-OH H-Phe-Ser-NHBzh NHB zh 79 195-202 © 66.35 7.03 Q 92 Ex. 37 Z-Phe-Gly-Phe-Ser- (2) 66.17 6:00 am 9. 87 Z-Phe-Gly-OH H-Phe-Ser-NHBzh NHB zh 86 257-260 © 69.91 6.22 9 10 Ex. 38 Z-Pro-Gly-Phe-Ser- (D 69.97 6.14 9 04 4.1 Z-Pro-Gly-OH H-Phe-Ser-NHBzh NHB zh 61 228-230 Φ 68.07 6.16 9 57 4.1 Ex 39 PMZ-Met-Gly-Phe-Ser- (2) 67.70 6.15 10 .73 PMZ-Met-GIy-OH H-Phe-Ser-NHBzh NHB zh 88 200-210 63.96 6.23 10 Ex. 40 Z-Trp-Gly-Phe-Ser- 64.11 5.83 Z-Trp-Gly-OH H-Phe-Ser-NHBzh NHB zh 67 248-252 69.50 5.95 Example 41 69.44

Example 42

A mixture of 7.5 ml of McIluaine buffer solution at pH 7.0 and 7.5 ml of methanol lead to 282 mg (0.6 mmol) of BOC-l / al-Tyr- (Bzl) -OH and 345 mg (0.5 mmol) H-Val-His (Bzl) -Pro-Phe-0Et.2HCl and then the The mixture was replaced with 1 ml of 1N NaOH v /. 150 mg papain and 70 mg cysteine hydrochloride are then added the mixture is stirred at 38 ° C for 24 h. The received colorless precipitates are filtered off and sequentially with water, 0.5N HCl, 7 ^ ammonia water and again washed with water. 400 mg of a crystalline crude product are obtained. The product is dissolved in 50 ml of ethyl acetate and the hot solution is treated with charcoal to Remove protein. The solution is then concentrated and the residue is recrystallized from methanol / water. BOC-Val-Tyr (BzI) -l / al-His (Bzl) -Pro-Phe-OE t is obtained.

Yield 226 mg (42 %)

Ninhydrin test: negative / melting point 167 to 173 ^° C

fpq ² p = -34.0 (C = 1.0 Ν, Ν-dimethylformamide) = -56.1 (C = 1.0 methanol)

Elementary analysis C HN

calculated (%) 66.82 7.19 10.39

found (%) 66.72 7.16 10.55

The same product is obtained from a solution experience. This product has the following characteristics:

Melting point: 164-174 ° C mixed melting point: 161-167 ⁰ C.

&] ² q = -35.3 (C = 1.0 N, N-dimethylformamide) = -59.2 (C = 1.0 methanol)

In contrast to the solution method, no racemization is found with the enzyme method.

709817/1113

Example 43

A mixture of 15 ml Mcllvaine buffer solution with pH 8.0 and 15 ml methanol yielded 943 mg (1.20 mmol) BOC-Asn-Arg (NO ₂ ) -Ual-Tyr (Bzl) -0H and 690 mg (1 , 00 mmol) H-Ual-His (Bzl) -Pra-Phe-0Et.2HCl given. 300 mg of papain and 150 mg of cysteine hydrochloride are then added to the mixture with stirring at 3B C, and the reaction is then carried out for 4 hours. The colorless precipitate obtained is filtered off and washed successively with water, 0.5N HCl, 7 ^ ammonia water and then again with water. 1.20 g of crude crystals are obtained. The product is dissolved in 200 ml of ethanol and the solution is treated with charcoal to remove the protein. The solution is then concentrated and the residue is recrystallized by adding ether. The crystalline product BOC-Asn-Arg (NO ₂ ) -Wal-Tyr- (BzI) -Ual-His (BzI) -Pro-Phe-OEt is obtained.

Yield: 530 mg (37 %) melting point; 185 to 197 ⁰ C ² Q = -47.3 ° (C = 1.0 methane)

p = -25.3 ° (C = 1.0 N, N-dimethylformamide) Elemental analysis CHN

calculated (Ji) 59.17 6.88 14.79 found {%) 59.11 6.62 15.02

The same product is made by the solution method. It has the following characteristics:

Melting point: .185 to 198 ⁰ C mixed melting point: 185-190 ⁰ C.

M ² Q ⁼ ~ ⁴⁸ » ⁴ ° (C = 1.0 methanol)

= -26.5 ° (C = 1.0 N, N-dimethylformamide)

In contrast to the solution method, in the enzyme method no racemization took place.

709817/1113

Example 44

2 ml of N, N-dimethylformamide are added to 420 mg (1.50 mmol) of Z-GIn-OH and 480 mg (1.25 mmol) of H-Leu-OBzh. (COOH) "while stirring, whereupon another 20 ml Citric acid buffer solution with a pH of 5.5 is added to the mixture. 300 mg of stembromelein and 0.2 ml of 2-mercaptoethanol are then added to the mixture with stirring at 38 ° C., whereupon the reaction is carried out for 24 hours. The colorless precipitate obtained is filtered off and then washed in sequence with water, 5% citric acid, water, 7% ammonia water and again with water. This gives 680 mg (97%) of product Z-Gln-Leu-OBzh having a melting point 158-163 ⁰ C. After drying, the product is dissolved in methanol and the hot solution is treated with activated carbon. The solution is then concentrated and the residue is crystallized by adding water to the concentrate. A pure product with a melting point of 160 to 163 ° C. is obtained

and [K] ² D ^{= -38} » ¹ ° ( ^c = ¹ > ° methanol).

Elemental analysis C HN

calculated 00 68 , 67 6th , 66 7, 51 found ( :%) 68 , 65 6th , 65 7, 61 Example 45

15 ml of citric acid buffer solution with pH 5.5 becomes 403 mg

(1.20 mmol) Z-Leu-Ala-OH and 310 mg (1.00 mmol)

H-Phe-OBu. (COOH) ₂ given. 200 mg of strembromelein and 0.2 ml of 2-mercaptoethanol are then added to the mixture at 38 ° C. with stirring, whereupon the reaction is carried out for 24 hours

will. The colorless precipitate obtained is filtered off and one after the other with water, 5% citric acid,

Water, 7 ^ ammonia water and washed again with water, 310 mg of crude crystals are obtained. After drying it will

Product is dissolved with ethyl acetate and the solution is treated with activated charcoal. The solution is then concentrated and the residue is recrystallized from petroleum ether.

709817/1113

Z-Leu-AJa-Phe-OBu is obtained. Yield: 250 mg (4.6 %) Melting point: 73 to 77 ⁰ C M ² D ⁼ -40.0 ° ( ^c = ° » ⁵ methanol)

E learning tariff analysis

C HN

calculated {%) 66.77 7.66 7.79 found (%) 66.76 7.71 7.90

Example 46

Add 2 ml of Ν, Ν-dimethylformamide to 470 mg (1.40 mmol) of Z-Leu-Ala-OH and 585 mg (1.25 mmol) of H-Ile-OBzh.TosOH with stirring, and then add 20 ml of citric acid buffer solution of pH 5.5 is added to the mixture. Thereafter u / ith the mixture with 300 mg Stembromelein and 0.2 ml 2-mercaptoethanol added u / orauf the mixture still at 38 ⁰ C for 24 h stirred and / ird. The colorless precipitate obtained is filtered off and washed in succession with water, 5% citric acid, water, 7% ammonia water and again with water. This gives 610 mg (79%) of product Z-Leu-Ala-Ile-OBzh having a melting point 167-170 ⁰ C. The product is dissolved in ethyl acetate and the solution is treated activated carbon. The solution is then concentrated and the residue is recrystallized by adding ether to the concentrate. Obtaining a pure product having a melting point of 170-171 ⁰ C and treated with ² M Q = -60.2 ° (C = 1.0 methanol)

Elementary analysis C HN

calculated (%) 70.22 7.37 6.82 found (%) 69.95 7.40 6.91

Examples 47 to 56

The procedure of Example 46 is repeated using however, the N ^ -acyldipeptide and as the acid component as the amine component, the amino acid ester or the amino acid amide according to table 5. The results are summarized in the table.

709817/1113

E.g.
No. acid
component Amine
component Table 5 the end
prey - 27 - 64
83 H
6.85
6.90 N
6.96
6.95 47 PMZ-A la-A la-OH H-Leu-OBzh product 93 Enamel
Point El
Ii 73
80 6.25
6.18 11.43
11.60 48 PMZ-AIa-AIa-OH H-Asn-NHBzn PMZ-Ala-Ala-Leu-OBzh 54 117-118 Φ
(D 22nd
15th 7.37
7.37 6.82
6.88 49 Z-Leu-Ala-OH H-Leu-OBzh PMZ-Ala-Ala-Asn-
NHBzn-1 / 2H ₂ O 90 270-273 (Γ
(D ementar analysis
) calculated (%)
) found {%) 20th
29 6.84
6.81 8.64
8.63 50 Z-Leu-Ala-OH H - Ph e-NHB zh Z-Leu-Ala-Leu-OBzh 63 145-146 (D C.
67.
67. 32
14th 6.71
6.72 11.38
11.33 0981 51 Z-Leu-Ala-OH H-Asn-NHBzh Z-Leu-Ala-Phe-NHBzh 68 227-230 φ 62.
62. 90
97 6.67
6.69 8.83
8.89 «4 52 Z-Phe-Ala-OH H-Val-NHBzh Z-Leu-Ala-Asn-NHBzh 47 255-256 φ
φ 70.
70. 77
.68 6.04
6.03 6. 15
6.37 113 53 Z-Phe-Ala-OH H-Phe-OBzh Z-Phe-Ala-Val-NHBzh 78 258-259 % 72.
72. .09
.17 6.85
7:00 7.33
7.45 54 Z-Phe-Ala-OH H-Phe-OBu * Z-Phe-Ala-Phe-OBzh 52 189-193 φ 66
66 .13
.88 7.11
7.04 9.63
9.78 55 A-Ala-Leu-OH H-AIa-NHB zh Z-Phe-Ala-Phe-OBuk 56 118-122 φ
φ 71
71 .22
.41 7.37
7.42 6.82
6.92 56 Z-Ala-Leu-OH H-Leu-OBzh Z-Ala-Leu-Ala-
NH zh-1 / 2H ₇ O 77 230-233 I. 73
73 Z-Ala-Leu-Leu-OBzh 110-111 φ
(D 69
69 68
67 70
70

2 S / f 718 8

Example 57

2 ml of N, N-dimethylformamide become 380 mg (1.50 mmol)
Z-Thr-OH and 480 mg (1.25 mmol) H-Leu-OBzh. (COOH) ₂ under
Stirring is added, whereupon 20 ml of citric acid buffer solution of pH 5.5 are added to the mixture. The whole thing is then replaced with 300 mg of ficine and 0.3 ml of 2-M8rcaptoethanol, and the mixture is stirred at 38 ° C. for 24 hours. The colorless precipitate obtained is filtered off and washed in sequence with water, 5% strength citric acid, water, 1% strength ammonia water and again with water. 330 mg (50 %) of the product Z-Thr-Leu-OBzh with a melting point of 114 to 117 ° C. are obtained. The product is dissolved in ethyl acetate and the solution is concentrated. The residue is made by adding
recrystallized from ether to the concentrate. A pure product with a melting point of 120 to 122 ° C. is obtained

and β *] ρ = -41.4 (C = 1.0 methanol).

Elemental analysis

C H N

calculated {%) 69.90 6.81 5.26 found (%) 70.02 6.87 5.16

Example 58

1.5 ml Ν, Ν-dimethylformamide become 320 mg (1.20 mmol)
BOC-Met-OH and 254 mg (1.00 mmol) H-Ala-NHBzh with stirring
given. 15 ml of citric acid buffer solution are then added
pH 5.5 to the mixture. Then you give another 300 mg of ficine and
Add 0.3 ml of 2-mercaptoethanol to the mixture with stirring at 38 ° C., the whole being reacted at 38 ° C. for 24 hours. The colorless precipitate obtained is filtered off
and washed in succession with water, 5% citric acid, water, 7 / ζ% ammonia water and again with water. 400 mg (82 %) of the product BOC-Met-Ala-NHBzh with a melting point of 163 ° to 164 ° C. are obtained. The product is dissolved with ethyl ether and the solution is concentrated. Then uiird
the residue recrystallized from ether / petroleum ether. Man
receives a pure product with a melting point of 159 to

709817/1113

o ". ., ι νΊ 25 or, ^ ο

161 C and JAl Q = -29.6 (C = 1.0 methanol).

Elemental analysis

CHN

calculated {%) 64.30 7.26 8.65 found {%) 64.13 7.20 8.77

Example 59

2 ml of IM, N-dimethylformamide are added to 516 mg (1.40 mmol) of Z-Phe-Ala-OH and 480 mg (1.25 mmol) of H-Leu-OBzh. (COOH) ₂ with stirring and then becomes the whole thing replaced with 20 ml citric acid buffer solution v / on pH 5.5 v /. Thereafter, 200 mg of ficin and 0.2 ml of 2-mercaptoethanol are added to the mixture with stirring at 38 ° C. and the whole is then reacted for a further 24 hours. The colorless precipitate obtained is filtered off and washed in sequence with water, 5% citric acid, water, 7% ammonia water and again with water. 540 mg (67 %) of crude crystals having a melting point of 156 to 158 ° C. are obtained. The product is dissolved in methanol and a small amount of an insoluble material is removed. The solution is concentrated and the residue is recrystallized by adding v / on water to the product. This gives Z-Phe-Ala-Leu-OBzh having a melting point v / on 151-152 ⁰ C and £ θζ] ² § = -47.4 ° (C = 0.5, methanol).

EIemeηtar analysis

C. , 09 H 67 N , 47 calculated (%) 72 , 84 6, 66 6th , 63 found {%) 71 6, 6th Example 60

A mixture of 20 ml of McIlv / ainB buffer solution of pH 6.2 and 3 ml of methanol is added to 370 mg (1.26 mmol) of Z-Phe-OH and mg (1.07 mmol) of H-Lys (Z) -0Bu. Then you give Add 130 mg of papain and 0.05 ml of 2-mercaptoethanol to the mixture with stirring at 38 ° C. for 20 h. The obtained colorless Precipitate is filtered off and sequentially with water,

709817/1113

0.5 M citric acid, 7 ^ ammonia water and washed again with water. The obtained crude crystals are dissolved in ethyl acetate and the solution is treated with activated charcoal. Then η-hexane is added to the fitrat. Z-Phe-Lys (Z) -0Bu ^{t is obtained} .

Yield: 531 mg (80 %) Melting point: 181 to 121 ° C jXl ² p = -13.5 ° (C = 1.0 methanol)

Elemental analysis C HN

calculated (%) 68.05 7.02 6.80 found (%) 68.19 6.98 6.85

Example 61

A mixture of 20 ml Mclluaine buffer solution υοη pH 6.2 and 3 ml of methanol becomes 359 mg (1.20 mmol) of Z-Phe-OH and 173 mg (1.00 mmol) H-UaI-OBu given. Then another 130 mg are given Papain and 0.05 ml of 2-mercaptoethanol at 38 ° C. to the mixture and the whole is reacted for 20 hours. The oily product obtained is dissolved in ethyl acetate and the solution is washed in sequence with water, 0.5N HCl, 7 ^ ammonia water and again with water. The solution is dried and treated with activated charcoal to remove the protein. The solution is then concentrated and washed with petroleum

he added, with Z-Ph yield: 315 mg (69 %)

1 = -19.0 ⁰ C (C = 1.0 methanol)

ether added, whereby Z-Phe-Ual-OBu crystallizes out

Melting point: 104 to 106 ^° C

The melting point and / jKJ _D agree with the data according to Chem. Ber. JK] O, 160 (1967).

The following reference examples relate to the conversion that obtained with the process according to the invention Peptides into the free links.

709817/1113

Reference example 1

2.00 g (3.20 mmol) of Z-Phe-Lys (Z) -OBu are dissolved in 15 ml of ethyl acetate dissolved and mixed with 20 ml of 6, On HCl-ethyl acetate. The reaction is carried out at room temperature for 2 hours, whereupon the solution is concentrated. Dry ether is added to the residue. Z-Phe-Lys- (Z) -OH is obtained.

Yield: 1.69 g (94 %) Melting point: 94 to 96 ⁰ C • jj = -6.2 (C = 0.5 methanol)

Elementary analysis

C HN

calculated {%) 66.29 6.28 7.48 found {%) 65.93 6.02 7.71

Reference example 2

909 mg (2.00 mmol) Z-Phe-Wal-OBu are in a mixture of 8 ml of methanol and 0.12 ml of acetic acid dissolved and mixed with 100 mg of 10 / £ Pd-C. The reaction is for 2 h under a Carried out hydrogen atmosphere and then the catalyst is filtered off. The methanol is removed by distillation and the residue is dissolved in ethyl acetate. The solution is then mixed with an aqueous solution of sodium bicarbonate washed in water and then with water. The ethyl acetate solution is dried and concentrated and then with Petroleum ether was added, with H-Phe - \ / al-0Bu crystallizing out.

Yield: 612 mg (96 %) Melting point: 65 to 67 ⁰ C ^ = -30.0 (C s 1.0 methanol)

r -τ25
The melting point and L-SJ η ^agree with the data in FIG

Chem. Ber. 100, 160 (1967).

Example 62

20 ml Kolthoff buffer solution of pH 8.5 become 409 mg (1.10 mmol) BOC-Tyr (Bzl) -0H and 303 mg (1.00 mmol) H-t / al-NHDMB.HCl

709817/1113

and then another 1.0 ml of 1N NaOH is added to the mixture. Then 100 mg of serine proteinase (titer 100 10 PUN / g; sold by Nagase Sangyo KK) are added to the mixture with stirring at 3B C, and the whole is reacted for 2k h. The gel precipitate is filtered off and washed in sequence with water, 0.5N HCl, 7 ^ ammonia water and again with water. 361 mg of a crystalline crude product are obtained. This is dissolved in methanol and the hot solution is treated with activated charcoal to remove the protein. The solution is concentrated and recrystallized by adding water. Pure BOC-Tyr (BzI) -UaI-NHDMB is obtained.

Yield: 297 mg (48 %) Melting point: 165 to 168 ^° C

^ = -0.8 ° (C = 0.25 chloroform) elemental analysis C HN

calculated (%) 67.83 7.32 6.78 found (%) 67.71 7.35 6.60

Example 63

20 ml citric acid buffer solution obtained pH 7.5 u / earth to 518 mg (1.10 mmol) BOC-Val-Tyr (BzI) -0H and 517 mg (1.0 mmol) H-Val-His (Bzl) -0Bzl. 2HCl added, whereupon 2.0 ml of 1N NaOH are added to the solution. Then, the whole with 100 mg of serine in Example 62 was added with stirring at 38 ⁰ C and the whole iuird still mährend 2M h reacted. The colorless precipitate obtained is filtered off and washed with water. 620 mg of crude crystals are obtained. The product is dissolved in 300 ml of hot methanol and the hot solution is treated with activated charcoal for 1 hour to remove the protein. The solution is then concentrated and water is added to the residue, crystalline BOC-Wal-Tyr (Bzl) -Val-His (Bzl) -0H.H ₂ O being obtained.

Yield: 450 mg (56 %)

Melting point: 176 to 180 ^° C

709817/1113

l = -6.6 ° (C = 0.5 methanol)

Elemental analysis

CHN

calculated {%) 64.84 7.17 10.31 found {%) 64.84 7.16 10.90

Example 64

7.5 ml Mclluaine buffer solution of pH 8.97 and ground to 282 mg (0.60 mmol) BOC-Val-Tyr (BzI) -0H and 345 mg (0.50 mmol) H-Val-His (Bzl ) -Pro-Phe-0Et.2HCl, whereupon 1.2 ml of 1N NaOH are added to the solution. 40 mg of serine proteinase are then added to the mixture with stirring at 38 ° C. and the whole is reacted for 24 hours with stirring. The money precipitate obtained is filtered off and washed in sequence with water, 0.5N HCl and again with water. 400 mg of crude crystals are obtained. The product is dissolved in hot ethanol and the solution is treated with activated charcoal to remove the protein. The solution is then concentrated and the residue is recrystallized from ethyl acetate, BOC-Ual-Tyr (BzI) - \ / al-His (Bzl) -Pro-Phe-OH.2H ₂ O being obtained.

Yield: 334 <ng (62 %) Melting point: 163 to 168 ⁰ C P = -26.9 (C = 1.0 Ν, Ν-dimethylformamide)

Elementary analysis

C HN

calculated (%) 64.66 7.11 10.40 found {%) 64.87 6.80 10.52

Examples 65 to 70

The procedure of Example 64 is repeated using as The acid component is the N ^ -acylamino acid or the N -acyldipeptide and the dipeptide t-butyl ester as the amine component according to table 6. The results are also summarized in Table 6.

709817/1113

Tabel

E.g. acid Amine product the end Schmel; : - Elemental AnaJ calculated H Lyse N 40 No. component component prey Point (D found ι 7.99 (%) 7th 39 (%) ( ⁰ C) (2) 7.99 %) 7th 84 C. 70 7.71 7th 98 65 Z-Leu-OH H-Phe-Val-OBu * Z-Leu-Phe-Val-OBu * 46 106-108 φ 67. 87. 7.66 t, 24 © 67. 88 6.90 7th 04 66 Z-Leu-Phe-OH H-Phe-Val-OBu ^t Z-Leu-Phe-Phe-Val-OBu * 82 180-184 Φ 68. 75 6.81 7th 99 (D 68. 29 7.48 7th 00 67 Z-Phe-Tyr-OH H-Phe-Val-OBu ¹ Z-Phe-Tyr-Phe-Val- 35 158-166 φ 68. 43 7.50 8th. 96 OBu-t-1 / 2H ₂ O (D 68. 55 6.78 13th 14th 68 Z-Phe-Val-OH H-Phe-Val-OBu ⁴ Z-Phe-Val-Phe - \ ^ al-OBu ^t 22nd 181-186 φ 68. 33 6.79 13th 88 (D 68. 33 6.68 7th 74 69 Z-Phe-Arg (NO ₂ ) - H-Phe-Val-OBut Z-Phe-Arg (NO ₂ ) -Phe-Val- 21 105-116 ; φ 61. 42 6.60 7th OH OBuft ; φ 6ΐ. 08 70 Z-Phe-Ser-OH H-Lys (TOS) -Phe- Z-Phe-Ser-Lys (TOS) -Phe -59 168-172 ¹ Φ 62. 82 OB u * OBu ¹ φ 61.

Example 71

280.2 mg (1 mmal) of Z-GIn-OH and 368.5 mg (1 mmol) of H-Phe-Phe-QBu are added to the suspension in 10 ml of water in a flask. A glass electrode of a pH meter is then immersed in the suspension. 150 mg papain and 0.1 ml 2-mercaptoethanol are added to the suspension with stirring, and the pH of the mixture is adjusted to about 5.0 by adding 1 / 10N NaOH in order to dissolve the solid components. The mixture is stirred at 38 ° C. for 20 hours and reacted in the process. During the reaction, the pH of the reaction mixture is kept at 5 to 6 by adding 1 / 10N NaOH, for which purpose the pH of the reaction mixture is continuously measured with the pH meter. The precipitate obtained is filtered off and washed in sequence with water, 1N HCl, water, 7 ^ ammonia water and again with water and then dried. The product is recrystallized from ethyl acetate. This gives 510 mg of the product Z-Gln-Phe-Phe-OBu having a melting point v / on 197-200 ⁰ C.

Yield: 80.8 %

(uCj _D = -18.3 ° (C = 1.0 Ν, Ν-dimethylformamide)

Elemental analysis (^ 35 ^ λ2 ° 7 ^Ν 4 ^

C HN

calculated {%) 66.65 6.71 8.80 found {%) 66.51 6.71 8.74

Example 72

398.5 mg of Z-Phe-1 / al-OH and 320.4 mg of H-Phe-1 / al-0Bu are placed in a flask for suspension in 10 ml of water. A glass electrode of a pH meter is immersed in the suspension. 150 mg papain and 0.1 ml 2-mercaptoethanol are added to the suspension with stirring, the pH of the mixture being adjusted to about 4.5 to 5.0 by adding dilute hydrochloric acid solution and the solid components being dissolved. The mixture obtained is ^{stirred at 38 °} C. for 20 h. During the reaction, the pH of the reaction mixture becomes

709817/1113

kept at 4.5 to 5.0 by adding dilute HCl solution to the reaction mixture. The precipitate obtained is filtered off and washed in sequence with water, 1N HCl, water, 7 ^ ammonia water and again with water, and then dried. The product is recrystallized from ethyl acetate / petroleum ether. This gives 30 mg (4.3% yield) of Z-Phe-Phe-Ual-Wal-OBu having a melting point of 130-145 ⁰ C.

Example 73

398.5 mg (1 mmol) of Z-Phe-Val-OH and 320.4 mg (1 mmol) of H-Phe-Ual-OBu are placed in a flask for suspension in 10 ml of water. The glass electrode of a pH meter is immersed in the suspension. 150 mg of serine proteinase are then added to the suspension with stirring while the pH of the mixture is maintained at 7.5 to 8.0 by adding dropwise i / 10N NaOH solution to the solution. The solid components are thereby dissolved. The mixture is ^{stirred at 38 °} C. for 20 h. During the reaction, the pH of the mixture is kept at 7.5 to 8 by adding 1 / Ιθη NaOH. For this purpose, the pH value of the mixture is continuously monitored with the pH meter. The precipitate obtained is filtered off and washed in sequence with water, 1N HCl, water, 7 ^ ammonia water and again with water and then dried. The product is recrystallized from ethyl acetate / petroleum ether. 190 mg (yield 27.1 % ) of the product Z-Phe-V / al-Phe-Val-OBu with a melting point of 130 to 145 ° C. are obtained.

Example 74

471.5 mg (1 mmol) of Z-Phe-Tyr-OH and 320.4 mg of H-Phe-Val-OBu for suspension in 10 ml of water. A glass electrode of a pH meter is immersed in the suspension. 150 mg serine proteinase and ground with stirring to the Suspension given while the pH of the mixture to 7.5 to

709817/1113

- 37 -

8.0 is adjusted by adding 1 / 10N NaQH to the mixture. The solid components are dissolved. The mixture is stirred at 38 ° C. for 20 h. During the reaction, the pH of the reaction mixture is maintained at 7.5 to 8.0 by adding 1/10 N NaOH to the mixture. The precipitate obtained is filtered off and washed in sequence with water, 1 η HCl, water, 7 ^ ammonia water and again with water and then dried. The product is then recrystallized from ethyl acetate / petroleum ether. 230 mg (yield 30 %) of the product Z-Phe-Tyr-Phe-VaI-OBu with a melting point of 155 ° C. are obtained.

- patent claims -

ORIGINAL INSPECTED

709817/1113

Claims (10)

PATENT CLAIMS luobei A and B are the same or different and one Mean amino acid residue or a peptide residue, ujobei X means an amino protective group and u / obei Y is one of the following carboxyl protecting groups: tert-alkoxy, benz yloxy, benzylamino and benzhydrylamino groups, which may optionally be substituted with an inert substituent, characterized in that the acid component is in the form of an amino acid or a peptide with an N-terminal Protecting group or a salt thereof of the formula X-A-OH with an amine component in the form of an amino acid or of a peptide with a C-terminal protecting group or a salt thereof of the formula H-B-Y converts, in the presence of a thiol proteinase or a serine proteinase in an aqueous solution, its pH value sufficient to maintain the enzyme activity of thiol proteinase or serine proteinase. 2. The method according to claim 1, characterized in that thiol proteinase is papain, stembromelein, ficin, cathepsin B, chymopapain, or streptococcal proteinase and that the serine proteinase is substilisin, Aspergillus alkaline proteinase, elastase, (K. lytin proteinase or using chymotrypsin. 3. V / experience according to one of claims 1 or 2, characterized in that that one keeps the pH at the geuiünschdes value by the amino acid or peptide reactants in a Buffer solution from pH 4 to 7.5 for the thiol proteinase and 7 0 9817/1113 originally inspected 6 to 9 for the serine proteinase. 4. l / experience according to one of claims 1 or 2, characterized characterized in that the pH is kept at the desired value by measuring the pH of the reaction mixture and an acid or a base to the aqueous solution depending on the measured pH of the reaction mixture. 5. The method according to any one of claims 1 to 4, characterized in that that the reaction is carried out at a reactant / ratio from 0.8 to 2 moles of the acid component per 1 mole of the amine component. 6. The method according to any one of claims 1 to 5, characterized in that that the reaction with the addition of 10 to 500 mg of thiol proteinase or serine proteinase of the solution per mmol of the amine component. 7. The method according to any one of claims 1 to 6, characterized in that the N-terminal protective group of the acid component is a tert-alkoxycarbonyl group or a benzyloxycarbonyl group which can be substituted with an inert substituent or p-toluenesulfonyl or o-nitrophenylsulfenyl and that the The C-terminal protective group of the amine component is a tert-alkoxy, benzyloxy, p-nitrobenzyloxy, benzhydryloxy, benzylamino, 2,4-dimethoxybenzylamino or benzhydrylamino group, and which can be substituted with an inert substituent _{or the like} 8. The method according to claim 7, characterized in that the tert-alkoxycarbonyl group a t-butyloxycarbonyl group or a t-amyloxycarbonyl group and that the substituted Benzyloxycarbonyl group the p-methoxybenzyloxycarbonyl group, the 3,5-dimethoxybenzyloxycarbonyl group, p-Phenyla zobenzyloxycarbonylgruppe, or the 2,4,6-trimethylbenz yloxycarbonyl group and that the 709817/1113 tert-alkoxy group is the t-butoxy group and that the inert substituent of the benzhydrylamino group 2,4-dimethoxyoxybenzylamino or benzhydrylamino. 9. The method according to any one of claims 1 to 8, characterized in that that A and B are identical or different and represent an amino acid residue or a peptide residue, in which the amino acid is an aliphatic amino acid, an oxyamino acid, a Schmefel-containing amino acid, a monoaminodicarboxylic acid, a diamino monocarboxylic acid or a is an aromatic amino acid or a heterocyclic amino acid. 10. The method according to claim 9, characterized in that the aliphatic amino acid is a monoaminomonocarboxylic acid and in particular glycine, alanine, \ / aline, norvaline, Leucine, isoleucine and norleucine are used and that serine, threonine or homoserine are used as oxyamino acids and that methionine, cystine or cysteine is used as the sulfur-containing amino acid and the monoaminodicarboxylic acid Aspartic acid or glutamic acid and, as diaminomonocarboxylic acid, ornithine, lysine or arginine, as aromatic Amino acid phenylalanine or tyrosine and as heterocyclic amino acid histidine or tryptophan. 709817/1113