CA1059938A - Process for producing a peptide - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A peptide having the formula X-A-B-Y
wherein A and B are the same or different and each represents an amino acid residue or a peptide residue, X represents an amino protective group, Y represents a carboxyl protective group selected from the group consisting of tertiary alkoxy, and benzyloxy, benzylamino and benzhydrylamino which can be substit-uted with an inert substituent is prepared by a process which comprises reacting an amino acid or peptide having an N-terminal protective group, or a salt thereof of the formula:
X-A-OH
with an an amino acid or peptide having a C-terminal protective group or a salt thereof of the formula:
H-B-Y
in the presence of thiol proteinase or serine proteinase enzyme in an aqueous solution having a pH sufficient to maintain the enzyme activity of said thiol proteinase or serine proteinase.

Description

The present invention relates to a process for producing a peptide. More particularly, it relates to a process for producing a peptide by using a specific enzyme as a catalyst.
Typical conventional processes for producing peptides include the azide method, the mixed acid anhydride method, the carbodiimide method, the active ester method and the acid chloride method. However, various industrial problems are encountered by these conventional processes, such as racemiza-tion of the carboxyl component of the C-terminal amino acid residue occurs. Other probIems include side reactions, temper-ature control, selection of solvent, the properties of amino protective groups and carboxyl protective groups and the effects of functional groups on the side chains of amino acids.
The fragment condensation method of preparing peptides can be applied to advantage for compounds which contain glycine (the ; only amino acid which cannot be racemized) at the carboxyl terminal group. However, for compounds containing any other amino acid at the carboxyl terminal group the racemization cannot be prevented. In actuality, on any peptide synthesis, the racemization problem is serious. When racemization occurs, the purity of the product is decreased, and is necessary to separate the impure isomer fromthe product. This is very detrimental for any industrial operation.
Among the conventioanl methods for forming peptide bonds, the azide method is the only method in which racemization is not much of a problem and itis for this reason that it is a desirable method. However, the azide method involves complicated operational procedures and a urea derivative is produced in a side reaction. Because of these features the azide method is undesirable from the viewpoint of yield. In addition to ~osssas, the various organic chemical processes, for preparing peptides, a particular peptide synthesis using the enzyme papain or chymotrypsin has been disclosed (See, for example, J.S. Fruton "Advances in Protein Chemistry", 5, Academic Press`Inc. New York, N.Y. 1949).
The reactions of the method are as follows:
(1) Bz-Leu-OH + H-Leu NH0 (I) (II) papain Bz-Leu-Leu-NH0 ~ (III)

(2) Bz-Leu-OH + H-Gly-NH0 (I) (II) papain Bz-Leu-Gly-NH0 ) (III)

(3) Bz-Tyr-OH + H-Gly-NH0 (I) (II) chymotrypsin Bz-Tyr-Gly-NH0 ~ (III)

(4) Z-Phe-Gly-OH + H-Tyr-NH2 (I) (II) papain Z-Phe-Gly-Tyr-NH2 > (III) to remove the phenylamino group from the peptide (III) under severe conditions because the phenylamino group which is bonded to the C-terminal group of the amine component (II) cannot be easily separated fromthe peptide, and thus cleavage of the peptide chain is disadvantageous. Because of this deficiency, this mode of peptide synthesis cannot be practically used for peptide synthesis. On the other hand, reaction (4) is accompanied by transamidation and transpeptidation side reactions and thus is not practically suitable. (See, for example, R.B. Johnston et al; J. Biol, Chem., 185, 629(1950) and J.S. Fruton et al; J. Biol.
Chem., 204,891(1953). In reaction (4), the primary amino group of the acid amide bonded to the terminal group of the amine component, promotes the papain catalyzed amidase reaction.

~059938 Accordingly, these processes provide only a theoretical interest in showing that papain and chymotrypsin act as catalysts for the synthesis of peptide bonds in which the phenylamino group is used as the protective group for the terminal carboxyl group of the amine component.
A need, therefore, continues to exist for a method of peptide synthesis in which the variety of difficulties encountered by the prior art procedures can be overcome.
Accordingly, the present invention provides a process for synthesizing a desired oligopeptide or polypeptide by a simple operation in high yield.
According to the present invention there is provided a process for producing a peptide having the formula:
X-A-B-Y
wherein A and B are the same or different and represent an amino acid residue or a peptide residue, X represents an amino protective group, Y represents a carboxyl protective group selected from the group consisting of substituted or unsubstituted tertiary alkoxy, and benzyloxy, benzylamino and benzhydrylamino by reacting an amino acid or peptide having an N-terminal protective group or a salt thereof of the formlula X-A-OH
with an amino acid or peptide having a C-terminal protective group or a salt thereof of the formula H-B-Y
in the presence of thiol proteinase or serine proteinase in an aqueous solution having a pH sufficient to maintain the enzyme activity of said thiol proteinase or serine proteinase.
The thiol proteinase enzyme includes enzymes of papain, Stembromelein, Ficin, Cathepsin B, Chymopapain, Streptococcal proteinase, Asclepain, Clostridium histolyticum protenase B and Yeast protenase B. Thiol proteinase is characterized by having :

1o59g38 the ability to hydrolyze a wide range of proteins and to promote the cleavage of many peptide bonds, amido bonds and ester bonds as well as promoting the decomposition of benzoyl alginine amide, benzoyl glycylleucylglycine and benzoyl tyrosylglycine amide thereby having the activity of exo and endo peptidase (J.R. Kimmel and E.L. Smith, Advance Enzymology, 19, 267 (1957). Serine proteinase includes enzymes of Substilisin, Aspergillus alkaline proteinase, Elastase, ~-Lytic proteinase, Chymotrypsin, Metridium protenase A, Trypsin, Thrombin, Tlasmin, Kininogenin, Enteropeptidase, Acrosin, Phaseolus protenase, Altemaria endopeptidase, Arthrobacter serine protenase and Tenebrio ~-protenase. It has been reported that the serine proteinase has substa..tial hydrolysis activity to the Leu(15) - Tyr (16) and Tyr (16) - Leu (17) sequence of the insulin B chain and also exhibits high esterase activity to acylamino acid ester. The subtilisin produced by using B. Subtilis or analogous germs includes types of Carsbery, Novo, BPN' and the like~ The alkaline proteinase isolated from ray fungus or fungi is a known material. The amino acid or peptide starting materials having the formula X-A-OH
wherein ~ represents a protective group for the terminal amino group and A represents an amino acid residue or a peptide residue which is used in the process of the invention are referred to as the acid component. The radical A in the formula more defin-itively represents an amino acid residue or peptide residue where-in suitable amino acids include aliphatic amino acids such as monoamino monocarboxylic acids, e.g. glycine (Gly), alanine (Ala), valine ~val), norvaline (nor-Val), leucine (Leu), isoleucine(iso-Leu), norleucine(nor-Leu); oxyamino acids, e.g.
serine (Ser), theronine (Thr), homo-serine(homo-Ser); sulfur-containing amino acids, e.g. methionine (~et) or cystine (CysS) 10599~8 and cysteine (CysH); monoamino dicarboxylic acids, e.g.
aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn) and glutamine (Gln); diamino monocarboxyiic acids, e.g. ornithine (Orn), lysine (Lys), arginine (Arg); aromatic amino acids, e.g.
phenylalanine (Phe), and tyrosine (Tyr); and heterocyclic amino acids, e.g. histidien (His), tryptophan (Try). (These amino acids are designated by symbols which are commonly used in the field.) (The peptides are also designated by combinations of these symbols).
Suitable protective groups for the free terminal amino group (an N-terminal protective group) of the acid component include tertiary alkoxy-carbonyl groups such as t-butyloxycarbonyl (BOC-), t-amyloxycarbonyl (t-Aoc-); benzyloxycarbonyl (Z-), p-methoxybenzyloxycarbonyl (PMZ), 3,5-dimethoxybenzyloxycarbonyl {Z(OMe)2-}, 2,4,6-trimethylbenzyloxycarbonyl (TMZ-),p-phenylazo-benzyloxycarbonyl (PZ-); p-toluenesulfonyl (Tos-); o-nitrophenyl-sulfenyl (Nps-); and the like. The other amino acid or peptide starting material having the formula H-B-Y
which is used in the process of the invention is referred to as the amine component. In the formula B represents an amino acid `
residue or peptide residue which can be the same defined above as A. The protective groups for the carboxyl group (C-terminal protective groups) of the amino component include tertiary alkoxy groups suchàs t-butoxy (-OBut), benzyloxy (-OBzl), p-nitrobenzyl-oxy~{-OBzl (p-NO2)}, benzhydryloxy (-OBzh), benzylamino (-NHBzl), 2,4-dimethoxy benzylamino (-NHDMB), and benzhydrylamino (-NHBzh).
These protective groups for the terminal carboxyl group of the amine component are resistant to esterase and amidase reactions which are caused by the thiol proteinase and serine proteinase enzymes.
The acid component and the amine component used in the process of the invention include the amino acid residues and peptide residues which have a functional group on a side chain.
In most of these cases, it is prefexable to protect the function-al group with a protective group. Suitable protective groups for ~-amino group (N~) can be N~-benzyloxycarbonyl (N~-Z), t-butoxy-carbonyl (N~-BOC) and tosyl (N~-Tos). Suitable protective groups for the N-guanidino group (NG) of ARg include nitro (NG-NO2), NG-benzyloxycarbonyl (NG-Z) and NG. NG-dibenzyloxycarbonyl (N -Z-Z). Suitable protective groups for imidazole rings (Nim) of His include N m-benzyl (N m-Bzl) and tosyl (N m-Tos). Suitable protective groups for the ~-carboxyl group include ~-benzyloxy (-OBzl). Suitable protective groups for the hydroxyl group of a liphatic or aromatic oxyamino acids include aralkyl groups such as -benzyl (Bzl). Suitable S-protective groups for the mercapto group of CysH include the benzyl group (Bzl). The prot-ective groups should possess the characteristics of being stable in the main reaction and that they can be easily separated from the product without being involved in side reactions. The acid component and the amine component starting materials 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 such as a hydrochloride, hydrobromide, oxalate, p-toluenesulfonate or acetate. In the process of the invention, the condensation reaction in which the peptide bond is formed can be conducted in an aqueous solution having a pH which maintains enzyme activity which is about 4 to 7.5 for the thiol proteinase and 6 to 9 for the serine proteinase.
There are two methods which can be employed to achieve the proper pH to maintain enzyme activity. One method is to conduct the condensation reaction in a buffer solution such as a citric acid buffer solution, McIlvaine buffer solution, Kolthoff ~059938 buffer solution, tris-HCl buffer solution, or veronal buffer solution in which the acid component and the amine component are dissolved and the enzyme is added. The other method is to conduct the condensation reaction by maintaining the pH of the reaction mixture in the proper range to maintain enzyme activity by adding the acid or the base to the reaction mixture depending upon the pH detected.
The starting materials are usually used in a ratio of 0.8 to two moles, preferably one to 1.5 moles of the acid component per one mole of the amine component. If the starting materials are not too soluble in the aqueous medium, it is possible to improve the solubility of the reactants by adding a solvent such as an alcohol, e.g. methanol, or ethanol; dimethylformamide;
dioxane; tetrahydrofuran; dimethylsulfoxide, or the like to the aqueous solution. The amount of the added solvent should be limited so as not to inhibit the activity of the enzyme in the reaction of the invention. If a solvent is employed, it is usually used in an amount of less than 1 part by weight, preferably 0.2 to 1.0 part by weight per one part by weight of water. The reaction of the invention is performed in an aqueous medium, and it is necessary to decrease the relative solubility of the reaction product preferably to a sparingly soluble or insoluble state in the system.
The amount of thiol proteinase or serine proteinase enzyme employed is in a range of 10 to 500 mg, preferably 10 to 400 mg, especially 50 to 300 mg per 1 mmole of the amine component. An enzyme activator such as CysH, or a salt thereof or 2-mercaptoethanol or a salt the.reof can also be added to the solution. The reaction temperature employed is usually in a range of 20 to 55C, preferably 30 to 40C which is sufficient to maintain enzyme activity. The reaction proceeds smoothly under these conditions for 1 to 24 hours. The reaction product ~059~38 precipitates from the reaction system and the reaction product can be easily isolated.
In accordance with the process of the invention, a minimum size dipeptide, oligo-peptide or polypeptide having the formula X-A-B-Y
can be easily produced by appropriately selecting the desired A and B radicals in the starting materials having the formulas X-A-OH and H-B-Y.
When the dipeptide derivative of lysyl lysine which is produced by the following reaction Z-Lys(Z)-OH + H-Lys(Z)-OBut ) Z-Lys(Z)-Lys(Z)-OBut wherein the Z-derivative produced by lysine whose ~-amino group is protected with the carbobenzoxyl group (Z-), is used as the acid component and a t-butyl ester is used as the amine component the lysyl lysine whose amino group in the side chain is protected, can be obtained. When the dipeptide derivative containing arginine i.e. arginyl leucine which is produced by the following reaction Z-Arg(Z,Z)-OH + H-Leu-OBzh ~ Z-Arg(Z,Z)-Leu-OBzh, wherein the tri-Z-arginine is produced by protecting the guanyl group and the amino group of arginine with Z, is used as the acid component and the benzhydryl ester of leucine is used as the amine component, a dipeptide derivative can be easily obtained by the reaction in the presence of papain. When an amino acid having a functional group in th~ side chain is used, the amino acid can be reacted with the functional group protected or unprotected as in the examples. Various reactions of histidine will be shown by the following reaction sequences.

lOS9938 BOC-His(Bzl)-OH + H-Leu-OBzh ) BOC-His(Bzl)-Leu-OBzh ...(a) BOC-His-OH + H-Leu-OBzh - > BOC-His-Leu-OBzh --(b) Reaction (a) represents the situation in which the side chain is protected, and the reaction (b) represents the case in which the side chain is unprotected.
The dipeptide situation has been illustrated. Another situation is the case of the acid component which has one peptide bond and is represented by the reaction of an acyl dipeptide with an amino acid amide derivative. The reaction can be smoothly performed without the occurence of side reactions in the presence of papain. This situation is shown by the ~ollowing reaction .
Z-Pro-Gly-OH + H-Leu-NHBzh ) Z-Pro-Gly-Leu-NHBzh The above reaction proceeds smoothly to form carbobenzoxy-prolyl-glycyl-leucine-benzhydrylamide.
Examples of the synthesis of tripeptides are shown in Table 3. The situation in which the acid component is a dipeptide and the amine component is a dipeptide will now be illustrated. The following reaction can be applied for the synthesis of fragment (3-6) tetrapeptide of Val5-angio-tension-II
which is known as a polypeptide hormone BOC-Val-Tyr(Bzl)-OH + H-Val-His(Bzl)-OBzl ~ BOC-Val-Tyr(Bzl)-Val-His(Bzl)-OH

When valyl histidine ester is used as the amine component of the reaction and the reaction is performed in the presence of papain, a tetrapeptide containing a benzyl ester group cannot be obtained. This is believed to be the result of an esterase action which is one of the characteristics of papain. The above lOS9938 reaction is especially advantageous when the tetrapeptide is used as the acid component in the next step of a polypeptide synthesis. When the carboxyl group of the amine component is the benzhydryl amide, a tetrapeptide benzhydrylamide derivative can be obtained in high yield. Examples in which a dipeptide derivative is used as the acid or amine component of the reaction are shown in Table 4. The same reaction can be performed by using a serine proteinase such as Subtilisin BPN'. As stated above, the synthesis of oligopeptides and polypeptides can be achieved by selecting the appropriate protective group for the carboxyl group of the amino component by using the thiol proteinase or the serine proteinase though they exhibit esterase and amidase action. The catalytic effect of the present enzymes for peptide synthesis is completely unexpected. As it is clear from the foregoing description, in the process of the present invention, the synthesis of peptides having the desired amino acid sequence can be attained by utilizing the characteristics of an endopeptitase. Only a catalytic amount of the enzyme is sufficient and the enzyme can be repeatedly used. The reaction proceedssmoothly under mild conditions in a buffer solution or in a soluiton having a desired pH. The yields are relatively high and the purity of the products is substantially high. The process of the invention can be utilized both in stepwise elongation of peptide chains and in condensation of peptide fragments. Both reactions are effective for industrial purposes. Moreover, racemization of the peptides does not occur, which is a result which could not be attained by the conventional synthesis methods.
The present invention will be further illustrated by way of the following Examples.

_XAMPLE 1 A mixture of 20 ml of McIlvaine buffer solution having a pH of 6.2 and 3 ml of methanol was added to 497 mg (1.20 mmol) of Z-Lys(Z)-OH and 366 mg (1.09 mmol) of H-Lys(Z)-OBut. Then, 130 mg of papain (titre 1200 CSU/g manufactured by Green-cross K.K.) and 0.05 ml of 2-mercapto ethanol were added to the mixture with stirring at 38C for 24 hours, and the reaction was conducted. The resulting oily product was extracted with ethyl acetate and the extracted solution was sequentially washed with water, 0.5 N HCl, 7% ammonia water and water. The ethyl acetate solution was condensed and petroleum ether was added to the solution whereby 385 mg (48%) of crude crystals of Z-Lys(Z)-Lys(Z)-OBut were obtained. A portion of the crystals was recrystallized from ethyl acetate-petroleum ether whereby a pure product having a melting point of 62 to 66C and an [~]25= 14.2O (C=0.5 methanol) was obtained.

ELEMENTAL ANALYSIS
C H N
Calculated(%) 65.557.15 7.65 Found (%) 65.557.22 7.67 A mixture of 20 ml of McIlvaine buffer solution having a pH of 7.0 and 4 ml of methanol was added to 692 mg (1.20 mmol) - of Z-Arg(Z,Z)-OH and 470 mg (1.00 mmol) of H-Leu-OBzh TosOH.
Then, 150 mg of papain and 0.1 ml of 2-mercapto ethanol were added to the mixture with stirring at 38C for 10 hours and the reaction was conducted. The resulting colorless precipitate was filtered and was sequentially washed with water, 7~ ammonia water, and water whereby the product Z-Arg(Z,Z)-Leu-OBzh was obtained.
yield 850 mg (g9%) melting point 161 to 168C

~059938 [~]D5= -9.4C (C=1.0 N,N-dimethylformamide) Elemental Analysis C H N
Calculated (%) 68.76 6.24 8.18 Found (%) 68.43 6.18 8.38 A 20 ml amount of McIlvaine buffer solution having a pH of 6.6 was added to 691 mg (2.00 mmol) of soc-His(szl)-oH
and 599 mg (1.80 mmol) of H-Leu-NHbzh-HCl. Then, 250 mg of papain and 100 mg of cysteine hydrochloride were added to the mixture with stirring at 38C for 24 hours and the reaction was conducted. The resulting colorless precipitate was filtered and sequentially washed with water, 7% ammonia water and water whereby 750 mg of crude crystals were obtained. The product was dissolved in 50 ml of hot methanol and the hot solution was treated with activated c~rbon to remove protein.
The solution was concentrated, water was added to the residue and the product was recrystallized whereby the crystalline product, BOC-His(Bzl)-Leu-NHBzh was obtained.
yield 686 mg(62%) melting point 113 to 115C [~]25= -8.1(C-1.0 chloro-D form) Elemental Analysis C H N
Calculated (%) 71.24 7.27 11.23 Found (%) 71.15 7.30 11.31 A 20 ml amount of McIlvaine buffer solution having a pH of 6.6 was added to 511 mg (2.00 mmol) of BOC-His-OH and 599 mg (1.80 mmol) of H-Leu-NHBzh-HCl. Then, 250 mg of papain and 100 mg of cysteine hydrochloride were added to the mixture with stirring at 38C for 24 hours and the reaction was conducted. The resulting colorless precipitate was filtered and sequentially washed with water, 7% ammonia water and water whereby crude crystals of product were obtained. The product was dissolved in methanol-water and the hot solution was treated with activated carbon to remove protein. The solution was concentrated whereby the crystalline product, BOC-His-Leu-NHBzh was obtained.
yield 336 mg (35%) melting point 221 to 223C
[~]25= _ 23.6 (C=0.5 chloroform) Elemental Analysis C H N
Calculated (%) 67.52 7.37 13.12 Found (%) 67.39 7.38 13.21 EXAMPLE S
A 40 ml amount of McIlvainebuffer solution having a pH
of 6.2 was added to 531 mg (2.00 mm~l) of BOC-Phe-OH and 935 mg (2.00 mmol) of H-His-(Bzl)-NHDMB~2HCl and then 4 ml of lN NaOH
was added to the solution. Then, 480 mg of papain and 240 mg of cysteine hydrochloride were added to the mixture with stirring at 38C for 24 hours and the reaction was conducted. The resulting colorless precipitate was filtered and was sequentially washed with water, 7% ammonia and water, wherein 960 mg of crude crystals of product were obtained. The product was dissolved in 100 ml of hot methanol and the hot solution was treated with activated carbon for 30 minutes to remove protein.
The solution was condensed and then water was added to the solution whereby the crystalline product of BOC-Phe-His(Bzl)-NHDMB~1/2H2O
was obtained.
yield 650 mg (50%) melting point 128 to 132C
[~]D = +1.6 (C=1.0 chloroform) Elemental Analysis C H N
Calculated (%) 66.44 6.82 10.76 Found (%) 66.34 6.66 10.78 EXAMPLES 6 to 23 The process of Example 1 was repeated except that the acid component of N~-acylamino acid and the amine component of the amino acid ester of H-Val-OBzh were used as shown in Table 1. The results are shown in Table 1. The process of Example 1 was repeated except that the acid component of the N~-acylamino acid of Z-Ala-OH and the amino component of the amino acid ester or amide were used as shown in Table 2. The results are shown in Table 2.

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~1 ~ ~1 _ lOS9938 A 1.5 ml amount of N,N-dimethylformamide was added to 370 mg (1.20 mmol? of Z-Pro-Gly-OH and 305 mg (1.00 mmol) of H-Leu-NHBzh l/2H2O with stirring, and then 15 ml of citric acid buffer solution having a pH of 5.5 was added to the mixture. Then, 200 mg of papain and 0.2 ml of 2-mercapto ethanol were added to the mixture with stirring at 38C for 24 hours and the reaction was conducted. The resulting colorless precipitate was filtered and was sequentially washed with water, 5% citric acid aqueous solution, water and 7% ammonia solution whereby 310 mg (51~) of crude crystals having a melting point of 146 to 149C were obtained. The product was dissolved in methanol and a small amount of insoluble material was separated by filtration. The solution was concentrated and water was added to the residue whereby the product, Z-Pro-Gly-Leu-NHBzh H2O having a melting point of 148 to 150C and an ~]25= 41.0(C=0.5 methanol) was obtained.
Elemental Analysis C H N
Calculated (%)67.75 7.02 9.30 Found (%) 68.05 6.96 9.24 EXAMPLES 25 to 32 _. .
The process of Example 24 was repeated except that an acid component of the N~-acyldipeptide of Z-Phe-Ala-OH and an amine component of the amino acid or amide shown in Table 3 were~used. The results are shown in Table 3.

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A mixture of 15 ml of McIlvaine buffer solution having a pH of 8.0 and 15 ml of methanol was added to 564 mg (1.2 mmol) of BOC-Val-Tyr-(Bzl)-OH and 516 mg (1.0 mmol) of H-Val-His(Bzl)-OBz1-2HCl. Then, 300 mg of papain and 150 mg of cysteine hydro-chloride were added to the mixture with stirring at 38C for 24 hours and the reaction was conducted. The resulting colorless precipitate was filtered and washed with water whereby 450 mg of crude crystals were obtained. The product was dissolved in 40 ml of ethanol and the hot solution was treated with activated car~on to remove protein. The solution was cooled and the resulting precipitate was filtered and the filtrate was condensed.
The residue was recrystallized by adding ether to the residue whereby the product BOC-Val-Tyr(Bzl)-Val-His(Bzl)-OH was obtained.
yield 100 mg (12%) ninhydrin test negative melting point 159 to 165C
[~]25= -8.16 (C-0.5 N, N-dimethylformamide) Elemental Analysis C H N
Calculated (~) 63.44 7.26 10.00 Found (~) 63.67 7.04 10.24 A mixture of 20 ml of McIlvaine buffer solution having a pH of 6.1 and 10 ml of methanol were added to 518 mg (1.1 mmol) of BOC-Val-Tyr(Bzl)-OH and 583 mg (1.0 mmol) of H-Val-His(Bzl)-NHBzh 2HCl and then 2 ml of lN NaOH was added to the solution.
Then, 240 mg of papain and 120 mg of cysteine hydrochloride were added to the mixture with stirring at 38C for 48 hours and the reaction was conducted. The resulting colorless precipitate was filtered and was sequentially washed with 0.5 N HCl, 7% of ammonia water, and water whereby 950 mg of crude lOS9938 crystals were obtained. The product was dissolyed in 300 ml of methanol and the hot solution was treated with activated carbon to remove protein. The solution was concentrated and the residue was recrystallized from N,N-dimethylforamide-water whereby BOC-Val-Tyr(Bzl)-Val-His(Bzl)-NHBzh was obtained.
yield 410 mg (42%) - melting point 237 to 239C
[a]D =-6.1 (C=1.0 N,N-dimethylformamide) Elemental Analysis C H N
Calculated (%) 69.84 7.10 10.00 Found (~) 70.18 7.01 9.94 EXAMPLES 35 to 41 The process of Example 34 was repeated except that an acid component of N~ acyldipeptide and an amine component of a dipeptide amide of H-Phe-Ser-NHBzh as shown in Table 4 were used. The results are shown in Table 4.

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~r . _ _ ~OS9938 A mixture of 7.5 ml of McIlvaine buffer solution having a pH of 7.0 and 7.5 ml of methanol was added to 282 mg (0.6 mmol) of BOC-Val-Tyr-(Bzl)-OH and 345 mg (0.5 mmol) of H-Val-His(Bzl)-Pro-Phe-OEt-2HCl and then 1 ml of lN NaOH was added. Thereafter, 150 mg of papain and 70 mg of cysteine hydrochloride were added to the mixture with stirring at 38C for 24 hours and the reaction was conducted. The resulting colorless precipitate was filtered and was sequentially washed with water, 0.5 y HCl, 7% ammonia water and water whereby 400 mg of crude crystals of product were obtained. The product was dissolved in 50 ml of ethyl acetate and the hot solution was treated with activated carbon to remove protein. The solution was concentrated and the residue was recrystallized from methanol-water whereby Boc-Val-Tyr(Bzl)-Val-His(Bzl)-Pro-Phe-OEt was obtained.
yield 226 mg (42%) ninhydrin test negative melting point 167 to 173C
[~]D = ~34 0 (C=1.0 N,N-dimethylformamide) = -56.1 (C=l.Q methanol) Elemental Analysis C H N
Calculated (%) 66.82 7.19 10.39 Found (%) 66.72 7.16 10.55 The same product was produced by a solution method. The properties of the product are as follows:
melting point: 164 to 174C
comelting test: 161 to 167C
[~]D = ~35 3 (C=1.0 N,N-dimethylformamide) -59.2 (C=1.0 methanol) The racemization of the product produced by the enzyme method was not found in comparison with the product obtained by the solution method.

A mixture of 15 ml of McIlvaine buffer solution having a pH of 8.0 and 15 ml of methanol was added to 943 mg (1.20 mmol) of BOC-Asn-Arg(NO2)-Val-Tyr)Bzl)-OH and 690 mg (1.00 mmol) of H-Val-His(Bzl)-Prop-Phe-Oet 2HC1. Thereafter, 300 mg of papain and 150 mg of cysteine hydrochloride were added to the mixture with stirring at 38C for 4 hours and the reaction was conducted.
The resulting colorless precipitate was filtered and was sequentially washed with water, 0.5 N HCl, 7% ammonia and water whereby 1.20 g of crude crystals were obtained. The product was dissolved in 200 ml of ethanol and the solution was treated with activated carbon to remove protein. The solution was concentrated and the residue was recrystallized by adding ether whereby the crystalline product BOC-Asn-Arg(NO2)-Val-Tyr (Bzl)-Val-His(Bzl)-Pro-Phe-OEt was obtained.
Yield 530 mg (37%) melting point 185 to 197C
[~]D = ~47-3 (C = 1.0 methanol) [~]D5 = -25.3 (C = 1.0 N,N-dimethylformamide) Elemental Analysis C H N
Calculated (%) 59.17 6.88 14.79 Found (%) 59.11 6.62 15.02 The same product was produced by the solution method. The properties of the product are as follows.
melting point 185 to 198C
comelting point 185 to 190C
¦~]D = -48.4 (C = 1.0 methanol) -26.5 (C = 1.0 N,N-dimethylformamide) The racemization of the product produced by the enzyme method was not found in comparison with the product obtained by the solution method.

105~938 .
A 2 ml amount of N,N-dimethylformamide was added to 420 mg ~1.50 mmol) of Z-Gln-OH and 480 mg (1.25 mmol) of H-Leu-OBzh-(COOH)2 with stirring and then 20 ml of citric acid buffer solution having a pH of 5.5 was added to the mixture. Thereafter, 300 mg of Stembromelein and 0.2 ml of 2-mercapto ethanol were added to the mixture with stirring at 38C for 24 hours and the reaction was conducted. The resulting colorless precipitate was filtered and was sequentially washed with water, 5% citric acid, water, 7% ammonia water and water whereby 680 mg (97%) of the product, Z-Gln-Leu-OBzh having a melting point of 158 to 163C
were obtained. After drying, the product was dissolved in methanol and the hot solution was treated with active carbon. The solution was concentrated and the residue was crystallized by adding water to the concentrate whereby a pure product having a melting point of 160 to 163C and an [~]25 = -38.1 (C=1.0 methanol) was obtained.
Elemental Analysis C H N
Calculated (%) 68.67 6.66 7.51 Found (%) 68.65 6.65 7.61 A 15 ml amount of citric acid buffer solution having a pH of 5.5 was added to 403 mg (1.20 mmol) of Z-Leu-Ala-OH and 310 mg (1.00 mmol) of H-Phe-OBut (COOH)2. Thereafter, 200 mg of Stembromelein and 0.2 ml of 2-mercapto ethanol were~added to the mixture with stirring at 38C for 24 hours and the reaction was conducted. The resulting colorless precipitate was filtered and was sequentially washed with water, 5% citric acid, water 7% ammonia water and water whereby 310 mg of crude crystals of product were obtained. After drying, the product was dissolved with ethyl acetate and the solution was treated with activated carbon. The solution was concentrated and the residue was ~059938 recrystallized from petroleum ether whereby the product, Z-Leu-Ala-Phe-Osut was obtained.
yield 250 mg (4.6%) melting point 73 to 77C
~]25= _40 oo (C=0.5 methanol) Elemental Analysis C H N
Calculated (~) 66.77 7~66 7.79 Found (%) 66.76 7.71 7.90 A 2 ml amount of N,N-dimethylformamide was added 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 20 ml of citric acid buffer solution having a pH of 5.5 was added to the mixture. Thereafter, 300 mg of Stembromelein and 0.2 ml of 2-mercapto ethanol were added to the mixture with stirring at 38C for 24 hours and the reaction was conducted. The resulting colorless precipitate was filtered and was sequentially washed with water, 5% citric acid water, 7% ammonia solution and water whereby 610 mg (79%) of the product, Z-Leu-Ala-Ile-OBzh having a melting point of 167 to 170C were obtained. The product was dissolved in ethyl acetate and the solution was treated with activated carbon. The solution was concentrated and the residue was recrystallized b~ adding ether to the concentrate whereby a pure product having a melting point of 170 to 171C and an [~]25= -60.2 (C=1.0 methanol) was obtained.
Elemental Analysis C H N
Calculated (%) 70.22 7.37 6.82 Found (%) 69.95 7.40 6.91 EXAMPLES 47 to 56 The process of Example 46 was repeated except that the acid component of an N~-acyl dipeptide and an amine component of the amino acid ester or amide shown in Table 5 were used.

The results are shown in Table 5.

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N 14 N ~1 ~1 L~ 4~ Ul ~ Ul - 26a -~059938 A 2 ml amount of N,N-dimethylformamide was added to 380 mg (1.50 mmol) of Z-Thr-OH and 480 mg (1.25 mmol) of H-Leu-OBzh (COOH)2 with stirring and then 20 ml of citric acid buffer solution having a pH of 5.5 was added to the mixture. Thereafter, 300 mg of Ficin and 0.3 ml of 2-mercapto ethanol were added to the mixture with stirring at 38C for 24 hours and the reaction was conducted. The resulting colorless precipitate was filtered and was sequentially washed with water, 5% citric acid, water, 7%
ammonia water and water whereby 330 mg (50%) of the product, Z-Thr-Leu-OBzh having a melting point of 114 to 117C were obtained.
The product was dissolved in ethyl acetate and the solution was concentrated. The residue was recrystallized by adding ether to the concentrate whereby a pure product having a melting point of 120 to 122C and an [~]25= -41.4 (C=1.0 methanol) was obtained.
Elemental Analysis C H N
Calculated (%) 69.90 6.81 5.26 Found (%) 70.02 6.87 5.16 :
A 1.5 ml amount of N,N-dimethylformamide was added to 320 mg (1.20 mmol) of BOC-Met-OH and 254 mg (1.00 mmol) of H-Ala-NHBzh with stirring. Then, 15 ml of citric acid buffer solution having a pH of 5.5 was added to the mixture. Thereafter, 300 mg of Ficin and 0.3 ml of 2-mercapto ethanol were added to the mixture with stirring at 38C for 24 hours and the reaction was conducted.
The resulting colorless precipitate was filtered and was sequent-ially washed with water, 5% citric acid, water, 7% ammonia water and water, whereby 400 mg (82%) of the product BOC-Met-Ala-NHBzh having a melting point of 163 to 164C were obtained. The product was dissolved with ethyl ether and the solution was concentrated. The residue was recrystallized from ether-petroleum ether whereby a pure product having a melting point of 159 to 161C and an [~]D5=-29.6O (C=1.0 methanol) was obtained.
Elemental Analysis C H N
Calculated (%) 64.30 7.26 8.65 Found (%) 64.13 7.20 8.77 _AMPLE 59 A 2 ml amount of N,N-dimethylformamide was dissolved in 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 20 ml of citric acid buffer solution having a pH of 5.5 was added. Thereafter, 200 mg of Ficin and 0.2 ml of 2-mercapto ethanol were added to the mixture with stirring at 38C for 24 hours and the reaction was conducted. The resulting colorless precipitate was filtered and was sequentailly washed with water, 5~ citric acid, water, 7% ammonia water and water whereby 540 mg (67%) of crude crystals having a melting point of 156 to 158C were obtained. The product was dissolved in methanol and a small amount of insoluble material was removed. The solution was concentrated and the residue was recrystallized by adding water whereby the product, Z-Phe-Ala-Leu-OBzh having a melting point of 151 to 152C and [~]25 =_47 4O (C = 0.5 methanol) was obtained.
Elemental Analysis C H N
Calculated (%) 72.09 6.67 6.47 Found (%) 71.84 6.66 6.63 A mixture of 20 ml of McIlvaine buffer solution having a pH of 6.2 and 3 ml of methanol was added to 370 mg (1.26 mmol) of Z-Phe-OH and 360 mg (1.07 mmol) of H-Lys(Z)-OBut. Thereafter, 130 mg of papain and 0.05 ml of 2-mercapto ethanol were added to the mixture with stirring at 38C for 20 hours. The resulting colorless precipitate was filtered and was sequentially washed with water, 0.5 M citric acid, 7% ammonia water and water. The lOS9938 resulting crude crystals were dissolvedin ethyl acetate and the hot solution was treated with activated carbon. Thereafter, n-hexane was added to the filtrate whereby the product, Z-Phe-Lys(Z) -OBUt was obtained.
yield 531 mg (80%) melting point 118 to 121C
~]D = -13.5 (C = 1.0 methanol) Elemental Analysis C H N
Calculated (%) 68.057.02 6.80 Found (~ 68.196.98 6.85 -A mixture of 20 ml of McIlvaine having a pH of 6.2 and 3 ml of methanol was added to 359 mg (1.20 mmol)of Z-Phe-OH and 173 mg (1.00 mmol) of H-Val-OBut. Then, 130 mg of papain and 0.05 ml of 2-mercapto ethanol were added to the mixture at 38C for 20 hours and the reaction was conducted. The resulting oily product was dissolved in ethyl acetate and the solution was sequentially washed with water, 0.5 N HCl, 7% ammonia water and water. The solution was dried and treated with activated carbon to remove protein. The solution was concentrated and the petroleum ether was added to the concentrate whereby the crystalline product Z-Phe-Val-OBut was obtained.
yield 315 mg (69~) melting point 104 to 106C
~]D = -19.0C (C=1.0 methanol) The melting point and [~]25 were the same with the data disclosed in (Chem. Ber. 100,160 (1967) ).
The following are reference procedures for converting the peptides produced by the process of the invention to the corresponding free compounds.

REFERENCE EXAMæLE 1 A 2.00 g (3.20 mmol) amount of Z-Phe-Lys(Z)-OBut was dissolved in 15 ml of ethyl acetate and was admixed with 20 ml of 6.0 N HCl-ethyl acetate. The reaction was conducted at room temperature for 2 hours and the solution was concentrated. Dried ether was added to the residue whereby Z-Phe-Lys (Z)-OH was obtained.
yield 1.69 g (94%) melting point 94 to 96C
~]D = -6.2 (C = 0.5 methanol) Elemental Analysis C H N
Calculated (%) 66.29 6.28 7.48 Eound (%) 65.93 6.02 7.71 A 909 mg (2.00 mmol) amount of Z-Phe-Val-OBu was dissolved in a mixture of 8 ml of methanol and 0.12 ml of acetic acid and was admixed with 100 mg of 10% Pd-C. The reaction was carried out for 2 hours in a hydrogen atmosphere and then the catalyst was filtered. Methanol was removed by distillation and the residue was dissolved in ethyl acetate. The solution was washed with an aqueous solution of sodium bicarbonate and water. The ethyl acetate solution was dried and concentrated and petroleum ether was added to it whereby the crystalline product H-Phe-Val-OBut was obtained.
yield 612 mg (96%) melting point 65 to 67C
[~]25= _30.0o (C=1.0 methanol) The melting point and [~]25 were the same as the data disclosed in (Chem. Ber. 100, 160 (1967).

A 20 ml amount of Kolthoff buffer solution having a ph of 8.5 was added to 409 mg (1.10 mmol) of BOC-Tyr(Bzl)-OH and ~OS9938 303 mg (1.00 mmol) of H-Val-NHDMB HCl and then, 1.0 ml of lN NaOH
was added to the mixture. Thereafter, 100 mg of serine proteinase (titre 100 x 104 PU ~g sold by Nagase Sangyo K.K.) was added to the mixture with sitrring at 38C for 24 hours and the reaction was conducted. The gel precipitate was filtered and was sequentially washed with water, 0.5 N HCl, 7~ ammonia water and water, whereby 361 mg of crude crystals of product were obtained. The product was dissolvedinmethanol and the hot solution was treated with activ~ted carbon to remove protein. The solution was concentrated and the product was recrystallized by adding water whereby the pure product BOC-Tyr(Bzl)-Val-NHDMB was obtained.
yield 297 mg (48%) melting point 165 to 168C
-~ [~]25 = -0.8 (C=0.25 chloroform) Elemental Analysis C H N
Calculated (%) 67.83 7.32 6.78 Found (%) 67.71 7.35 6.60 A 20 ml amount of citric acid buffer solution having a pH of 7.5 was added to 518 mg (1.10 mmol) of BOC-Val-Tyr(Bzl)-OH and 517 mg (1.00 mmol) of H-Val-His(Bzl)-OBzl 2HCl and then 2.0 ml of lN NaOH was added to the solution. Then, 100 mg of the ; serine proteinase of Example 62 was added to the mixture with stirring at 38C for 24 hours and the reaction was conducted.
The resulting colorless precipitate was filtered and was washed with water whereby 620 mg of crude crystals were obtained. The product was dissolved in 300 ml of hot methanol and the hot solu-tion was treated with activated carbon for 1 hour to remove protein.
The solution was concentrated and water was added to the residue whereby the crystalline product BOC-Val-Tyr(Bzl)-Val-His(Bzl)-OH
H2O was obtained.

yield 450 mg (56%) melting point 176 to 180C
[~]25= -6.6-(C=0.5 methanol) Elemental Analysis C H N
Calculated (%) 64.84 7.17 10.31 Found (%) 64.84 7.16 10.90 A 7.5 ml amount of McIlvaine buffer solution having a pH of 8.97 was added to 282 mg (0.60 mmol) of BOC-Val-Tyr(Bzl)-OH and 345 mg (0.50 mmol) of H-Val-His(Bzl)-Pro-Phe-OEt-2HCl and then 1.2 ml of lN NaOH was added to the solution. Thereafter, 40 mg of serine proteinase was added to the mixture with stirring at 38C for 24 hours and the reaction was conducted. The result-ing gel precipitate was filtered and was sequentially washed with water, 0.5 N HCl and water whereby 400 mg of crude crystals of product were obtained. The product was dissolved in hot ethanol and the hot solution was treated with activated carbon to remove protein. The solution was concentrated and the residue was recrystallized from ethyl acetate whereby the product of BOC-Val-Tyr(Bzl)-Val-His(Bzl)-Pro-Phe-OH 2H2O was obtained.
yield 334 mg (62~) melting point 163 to 168C
[~]D = -26.9 (C=1.0 N,N-dimethylformamide) Elemental Analysis C H N
Calculated (%) 64.66 7.11 10.40 Found (%) 64.87 6.80 10.52 EXAMPLES 65 to 70 The process of Example 64 was repeated except that an acid component of an N~-acylamino acid or N~-acyl dipeptide and an amine component of the dipeptide t-butyl esters shown in Table 6 were used.

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In a flask, 280.2 mg (1 mmol) of Z-Gln-OH and 368.5 mg (1 mmol) of H-Phe-Phe-OBut were suspended in 10 ml of water. The glass electrode of a pH meter was inserted into the suspension.
A 150 mg amount of papain and 0.1 ml of 2-mercapto ethanol were added to the suspension with stirring while the pH of the mixture was adjusted to about 5.0 by adding 1/10 N NaO~ to dissolve the solid components. The mixture was stirred at 38C for 20 hours and the reaction was conducted. During the reaction, pH
of the reaction mixture was maintained at 5 to 6 by adding 1/10 N
NaOH while measuring the pH of the reaction mixture with a pH
meter. The resulting precipitate was filtered and was sequentially washed with water, lN HC1, water, 7~ ammonia water and water and dried. The product was recrystallized from ethyl acetate whereby 510 mg of the product of Z-Gln-Phe-Phe-OBut having a melting point of 197 to 200C were obtained.
yield 80.8~
[~]D5= -18.3 (C= 1.0 N,N-dimethylformamide) Elemental Analysis (C35H42O7N4) C H N
Calculated (~) 66.65 6.71 8.80 Found (~) 66.51 6.71 8.74 In a flask, 398.5 mg of Z-Phe-Val-OH and 320.4 mg of - H-Phe-Val-Osut were suspended in 10 ml of water. The glass elec-trode of a pH meter was inserted into the suspension. A 150 mg amount of papain and 0.1 ml of 2-mercapto ethanol were added to the suspension with stirring while adjusting the pH of the mixture to about 4.5 to 5.0 by adding a diluted HCl solution to dissolve the solid components. The mixture was stirred at 38C for 20 hours and the reaction was conducted. During the reaction, the pH of the reaction mixture was maintained at 4.5 to 5.0 by adding a diluted HCl solution to the reaction mixture.

~059938 The resulting precipitate was filtered and was sequentially washed with water, lN HCl, water, 7% ammonia water, and water and was dried. The product was recrystallized from ethyl acetate-petroleum ether whereby 30 mg ~yield 4.3~) of the product Z-Phe-Val-Phe-val-Osut having a melting point of 130 to 145C
were obtained.

In a flask, 398.5 mg (1 mmol) of Z-Phe-Val-OH and 320.4 mg (1 mmol) of H-Phe-Val-OBut were suspended in 10 ml of water.
The glass electrode of a pH meter was inserted into the suspension.
- A 150 mg amount of serine proteinase was added to the suspension with stirring while adjusting the pH of the mixture to 7.5 to 8.0 by adding a 1~0 N NaOH solution dropwise to the solution to dissolve the solid components. The mixture was stirred at 38C
for 20 hours and the reaction was conducted. During the reaction, the pH of the mixture was maintained at 7. 5 to 8 by adding 1/10 N NaOH to the mixture while measuring the pH of the reaction mixture with a pH meter. The resulting precipitate was filtered and was sequentially washed with water, lN HCl, water, 7% ammonia water and water and was dried. The product was recrystallized from ethyl acetate-petroleum ether whereby 190 mg (yield of 27.1%) of the product of Z-Phe-Val-Phe-Val-OBut having a melting point of 130 to 145C were obtained.

In a flask, 471.5 mg (1 mmol) of Z-Phe-Tyr-OH and 320.4 mg of H-Phe-Val-OBut were suspended in 10 ml of water.
The glass electrode of a pH meter was inserted into the suspension.
A 150 mg amount of serine proteinase was added to the suspension with stirring while adjusting the pH of the mixture to 7.5 to 8.0 by adding l/10 N NaOH to the mixture to dissolve the solid components. The mixture was stirred at 38C for 20 hours and the reaction was conducted. During the reaction, the pH of the reaction mixture was maintained at 7.5 to 8.0 by adding 1/10 N NaOH to the mixture. The resulting precipitate was filtered and sequentially washed with water, lN HCl, water, 7% ammonia water and water and was dried. The product was recrystallized from ethyl acetate-petroleum ether whereby 230 mg (yield of 30%) of the product, Z-Phe-Tyr-Phe-Val-OBut having a melting point of 155C was obtained.

Claims (10)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for producing a peptide having the formula X-A-B-Y
wherein A and B are the same or different and represent an amino acid residue or a peptide residue;
X represents an amino protective group; and Y represents a carboxyl protective group selected from the group consisting of tertiary alkoxy, and benzyloxy, benzyl-amino and benzhydrylamino which can be substituted with an inert substituent, which comprises:
reacting an acid component of an amino acid or peptide having N-terminal protective group or a salt thereof of the formula X-A-OH with an amino component of an amino acid or peptide having a C-terminal protective group or a salt thereof the formula H-B-Y
in the presence of a thiol proteinase or serine proteinase in an aqueous solution having a pH sufficient to maintain the enzyme activity of said thiol proteinase or serine proteinase.

2. The process of Claim 1, wherein said thiol proteinase is papain, Stembromelein, Ficin, Cathepsin B, Chymopapain, or Streptococcal proteinase and said serine proteinase is Substilisin, Aspergillus alkaline proteinase, Elastase, .alpha.-Lytic proteinase or Chymotrypsin.

3. The process of Claim 1, wherein the pH is maintained at a sufficient level by reacting the amino acid or peptide reactants in a buffer solution at a pH of 4 to 7.5 for thiol proteinase and 6 to 9 for the serine proteinase.

4. The process of Claim 1, wherein the pH is maintained at the desired level by detecting the pH of the reaction mixture and adding an acid or a base to said aqueous solution depending upon the measured pH of the reaction mixture.

5. The process of Claim 1, wherein the reaction is conducted with a reactant ratio of 0.8 to 2 moles of the acid component per mole of the amine component.

6. The process of Claim 1, wherein the reaction is conducted by adding 10 to 500 mg of said thiol proteinase or serine proteinase to said solution per mmole of said amine component.

7. The process of Claim 1, wherein the N-terminal protective group of the acid component is a tertiary alkoxycarbonyl group or a benzyloxycarbonyl group which can be substituted with an inert substituent, p-toluenesulfonyl or o-nitrophenyl sulfenyl, and the C-terminal protective group of the amine component is tertiary alkoxy, benzyloxy (-OBzl), p-nitrobenzyloxy [-OBz] (p-NO2)], benzhydryloxy, (-OBzh), benzylamino (-NHBzl), 2,4-dimethoxy-benzylamino (-NHDMB), benzhydrylamino (-NHBzh) which can be substituted with an inert substituent.

8. The process of Claim 7, wherein said tert-alkoxy-carbonyl group is t-butyloxycarbonyl, or t-amyloxycarbonyl; said substituted benzyloxycarbonyl is p-methoxybenzyloxycarbonyl, 3-5-dimethoxybenzyloxycarbonyl, p-phenyl-azobenzyloxycarbonyl, or 2,4,6-trimethylbenzyloxycarbonyl, said tertiary alkoxy group is t-butoxy, and said inert substituent of said benzhydrylamino group is 2,4-dimethoxyoxybenzylamino or benzyhydrylamino.

9. The process of Claim 1, wherein A and B are the same or different and represent an amino acid residue or peptide residue wherein the amino acid is an aliphatic amino acid, an oxyamino acid, a sulfur-containing amino acid, a monoamino dicarboxylic acid, a diamino monocarboxylic acid, an aromatic amino acid or a heterocyclic amino acid.

10. The process of Claim 9, wherein said aliphatic amino acid is a monoaminomonocarboxylic selected from the group consis-ting of glycine, alanine, valine, norvaline, leucine, isoleucine, and norleucine, said oxyamino acid is serine, threonine or homoserine; said sulfur-containing amino acid is methionine, cystine or cysteine; said monoamino dicarboxylic acid is aspartic acid or glutamic acid; said diamino monocarboxylic acid is ornithine, lysine, or arginine; said aromatic amino acid is phenylalanine or tyrosine, and said heterocyclic amino acid is histidien or tryptophan.