CA1177429A - Process for enzymatic production of peptides - Google Patents

Abstract

Abstract A peptide having the formula A-B
wherein A represents an N-terminal protected amino acid residue or an optionally N-terminal protected peptide residue and B represents an optionally C-terminal protected amino acid residue, is prepared by reacting a substrate component selected from the group consisting of optionally N-terminal protected peptides of the formula A-X
wherein A is as defined above and X represents an amino acid with an amine component selected from the group consis-ting of (a) amino acids of the formula H-B-OH, (b) optionally N-substituted amino acid amides of the formula wherein B is an amino acid residue and R3 represents hydrogen, hydroxy, amino or alkyl, aryl or aralkyl, and (c) amino acid esters of the formula H-B-OR4 or H-B-SR4 wherein B is an amino acid residue and R4 represents alkyl, aryl and aralkyl, in the presence of a carboxypeptidase enzyme in an aqueous solution or dispersion having a pH from 5 to 10.5, preferably at a temperature of from 20 to 50°C, to form a peptide, and subsequently cleaving a group H, -NR3R3', -OR4, -SR4 or-SeR4 or an N-terminal protective group, if desired.

Description

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A Process for Enzymatic Production of Peptldes ______________________________________________ Back~round of the Invention 1. Field of the Invention The present invention relates to a process for enzymatic production of peptides. More particularly, the invention relates to a process for producing peptides by using a specific group of enzymes as catalysts.

2. Description of the Prior Art It is known to carry out syntheses of peptides by more or less sophisticated coupling reactions, both in respect of ~lomogeneous synthesis and of heterogeneous solid phase synthesis. All these chemical methods, ho~ever, involve the risk o~ undesirable secondary reactions and raccmizat-ions, and it is therefore necessary to control the chemical reactions carefully to minimize or eliminate these problems.
Moreover, the amino acid side chains must often be pro-tected, requiring deblocking as the last chemical step to produce the desired peptides. Depending upon the size of the synthetized peptide, the yields may be low, and the secondary reactions frequently necessitate cumbersome purification procedures to obtain the pure peptide. All these inherent problems of chemical peptide syntheses plus the high price of several of the coupling reagents and the blocking amino acid derivatives mean that even small synthetic peptides, e.g. pentapeptides, are relatively expensive to produce.

~ince enzylrles are very specific catalysts, and proteolytic enzymes are thus able to~hydrolyze peptide bonds in proteins, studies have previously been made of the pos-., ~

, ~ 7~ ,g sibilities of reversing this hydrolysis reaction or, in other words,of utilizing enzymes as catalysts in the synthesis of peptide bonds. Bergmann and Fraenkel-Conrat (ref. 1) and sergmann and Fruton (ref. 2) made a detailed examination of this and showed in 1937 that papain and chymotrypson c~uld catalyze the coupling of certain acylamino acids and amino acid anilides. These studies have been continued and intensified on the basis of two fundament-ally different ap~roaches,viz. a thermodynamic and kinetic one.
Funda~ental features in the thermodynamic methods are the use of a suitable protease for catalyzing thé establishment of the thermodynamic equilibrium between the reactants, and a removal of the reaction product from the reaction mixture. Thus, Isowa et al (ref. 7-9) and U.S. patent No. 4,119,~93 and British patents Nos. 1,523,546 and 1,533,129, ~uisi et al (ref. 12, 18) and Morihara et al (ref. 14) found that se~eral serine, thiol and metalloendoproteases catalyze the synthesis of peptides from protected, but very easily soluble di-, tri- and tetrapeptides, provided the final products precipitate from the reaction mixture on account of their solubility being lower than the equi-librium concentration. Examples of such syntheses are illustrated in the follo\~ing reaction schemepapain 1) Z-leu-Phe-OH + Phe-ODPM _ ~ Z-leu-Phe-Phe-ODPM
ODPM = diphenyl methyl ester g( 2) OH + phe_val_OBut Thermolysin A-Arg(N02)Phe-Val-OBu

3) B05-Val-Tyr(Bz)OH + Val-His(Bz)-Pro-Phe-OEt BOC-Val-Tyr(Bz)-Val-His(Bz -Pro-Phe-OH
(Z = carbobenzoxy, Bz = benzoyl, BOC = tert. butylox~carbonyl, all amino acids being in the L-form).

However, this reaction principle can only be used as general method of synthesis if, like in the conventional chemical coupling procedure, all the amino side chains with potentially ionizable groups are blocked before the reaction and deblocked af-ter the reaction. The method is also de~icient in that -the high specificity of the various enzymes calls for the use of various enzymcs, depending upon the type of the peptide bond to be synthetized, or rather of the amino acids forming the peptide bond. Even though these complications might be overcome, the method involves several other problems because it re~uires a very high concentration of enzyrne (100 mg/mmole peptide), long reaction periods (1 to 3 days) and gives very varying yields ~typically 20 to ~O,o, 1~ cf.: theabove mentioned U.S. and British patents).

Klibanov et al (ref. 103 have proposed to ~ork in a system consisting of water and an organic solvent immiscible with water. This procedure, too, requircs a high concentration of enzyme and a ]ong reaction period of up to several days.

The other type of enzymatic syntheses rely on the kinetic approach of the reaction. It has been shown for most serine and thiol proteases that an acyl enzyme intermediate is formed in one of the catalytic steps during the hydro-lysis of peptides or pep-tide esters, which is then hydro-lyzed by water in the subsequent step or steps. If other nucleophiles than water are present during the hydrolysis, they too will accept the acyl group from the acyl enzyme, resulting in the formation of an amide bond. This has been studied e.g. by Fastrez and Fersht (re~. 3) who rc-port chymotrypsin-catalyzed hydrolysis of N-acetyl-L-phenyl-alanine ethyl ester (Ac-Phe-OEt) in the presence of various arnino acid amides. The reaction is sho~n in scheme (4):

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, - , Ac-Phe-OEt + CT;~ Ac-Phe-OH + CT
k2 (Ac-Phe-OEt)-CT~ Ac-Phe-CT~'' + EtOH ~ ~ ~
k~ Ac-Phe-NH-R + CT

Scheme (4) (CT = chymotrypsin) First an enzyme substrate complex is for~ed followed by the formation of the acyl enzyme intermediate (Ac-Phc-CT).
This is hydrolyzed by water to Ac-Phe-OH, but if a nucleophile (R-NH2) is also present, the acyl enzyrne intermediate will be subject to aminolysis in addition to hydrolysis. Assuming that k~ k 3 and kL~>~ k_4, the ratio of aminolysis to hydrolysis clearly depends on k4/k3 as well as on the concentration of thé nuclcophile which is in competition with 55 M water. Fastrez and Fersht found that e.g. 1 M alanine amide at pH 10 is a 44 times stronger nucleophile than 55 M water, which resulted in a predominant formation (larger -than 95%) of the shown N-acyldipeptide-amide. Morihara and Oka (ref.
13-16) have further exploited this principle for enzymatic peptide synthesis. Using chymotrypsin and trypsin they synthetized a plurality of pep-tides from N-acyiamino acid esters and amino acid derived nucleophiles and demonstrated that the reaction was purely kinetic since high yields could be obtained, independent of the solubility of the product.

~he studies mentioned above secm to indicate that kinetic approaches possess several advantages over the thermo-dynamic methods:

1. Quicker reaction (in certain cases completed within few minutes).

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2. Possibility of using low concentrations of enzyme.

3. Amino acid side chains must not necessarily be blocked.

4. Immobilized and insoluble enzymes may be used as the peptides are in solution, permit'cing automatization.

As, however, the reaction products may be soluble the synthesis must be quicker than the secondary hydrolysis of the product so that they can be separated in time.

Moreover, the known kinetic approaches are limited in their applicability because the specific properties of the enzymes examined call for the use of various enzymes for the synthesis of the various peptide bonds. Additionally, synthesis of large peptide molecules invariably causes the internal peptide bonds in the molecule to be hydro-lyzed, independent of the esterolytic activity of the cnzymes, due to the endopeptidase acti~i-ty of the en~yrnes used till now.

Sum ary of the Invention The object of the present invention is to provide an enzymatic peptide synthesis which eliminates the draw-backs mentioned in the foregoing, and more particularly asynthesis that is of a general nature so that it is not limited to specific amino acid components, and which ~oes not involve any risks of subsequent hydrolysis of the internal peptide bonds.

Briefly, this and other objects of the invcntion can be attained in a proccss for producing a pcptide of the gcneral formula A-B

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-- ~ ~ 7 7 ~ ~ 3 wherein A represents an N-terminal protected L-amino acid residue or an optionally N-terminal protected peptide residue having a C-terminal L-amino acid and B represents an optionally C-terminal protected L-amino acid residue, which comprises:
S reacting a substrate component selected from the group consisting of (a) amino acid esters, peptide esters and depsipep-tides of the formula A-OR , A-SRl or A-SeRl wherein A is as defined above and Rl represents alkyl, aryl, heteroaryl, aralkyl or an c~-des-amino fragment of an L-amino acid residue, (b) optionally N-substituted amino acid amides and peptide amides of the formula wherein A is as defined above and R2 and ~ each represent hydrogen, alkyl, aryl, heteroaryl or aralkyl, and (c) optionally N-terminal protected peptides of the formula A-X
wherein A is as defined above and X represents an L-amino acid residue with an amine component selected from the group consisting of (a) L-amino acids of the formula H-B-OH, wherein B is an L-amino acid residue, (b) optionally N-substituted amino acid amides Oc the formula ~7~

wherein B is an L-amino acid residue and R3 and R each represent hydrogen, hydroxy, amino or alkyl, aryl, heteroaryl or aralkyl, and (c) amino acid esters of the formula H-B-OR , H-B-SR- or H-B-SeR
wherein B is an L-amino acid residue and R4 represents alkyl, aryl, heteroaryl and aralkyl, in the presence of a L-specific serine or thiol of a carboxypeptidase ~10 enzyme from yeast or of animal, vegetable or microbial origin in an aqueous solution or dispersion having a pH from 5 to 10.5, preferably at a temperature of from 20 to 50C, to form a peptide, and subsequently cleaving a group H, -NR3R , -OR , -SR or SeR or an N-terminal protective group, if desired.

Detailed description of the preferred embodiments The invention is based on a fundame~tal change in relation to the prior art, viz. the use of exopeptidases instead of the enzymes employed till now which have all displayed predo-minant or at any rate significant endopeptidase activity.
It has been found that besides being exopeptidases the useful enzymes must display a peptidase activity of broad specific-ity to thereby allow a synthesis activity of corresponding broad specificity, which is not restricted to a single or a few types of peptide bonds. The enzymes must be capable of forming acyl enzyme intermediates of the type describded above under the kinetic approaches, and must especially possess such characteristics as will allow the aminolysis of the intermediate ur~er conditions . .

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where K 4 ~ K4, cf. scheme (4) above, to avoid immediate hydrolysis of the peptides produced. The ability to form acyl enzyme inter-mediates may also be expressed as the ability to cleave the C-terminal protective group in the su~strate component used, i.e.
in the present case an esterase or ami~ase activity. For a systhe-sis to take place, said activity must dominate the peptidase ac-tivity of the enzyme under the reaction conditions used.
This multiplicity of characteristics is found in the group of exopeptidases called carboxypeptidases, which are capable of hydrolyzing peptides with free carboxyl groups. It has been found that a plurality of such carboxypeptidases exhibit different enzymatic activities ~hich are very depenaent on pH so that e.g.
in a basic environment at a pH from 8 to 10.5 they display pre-dominantly esterase or amidase activity and at a pH from 9 to 10.5 no or only insignificant peptidase activity. These properties can be advantageously used in the process of the invention because they contribute to the achievement of good yields.
Further, it has been found that the ability to form said acyl enzyme intermediates are particularly pronounced in serine or thiol proteases. Accordingly the carboxypeptidases used in the process for the conversion are L-specific serine or thiol carboxypeptidases. Such enzymes can be produced by yeast fungi, or they may be of animal, vegetable or microbial origin.

A particularly expedient enzyme is carboxypeptidase from yeast fungi (CPD-Y). This enzyme is described by ~ayashi et al. (ref. 5) and by Johansen et al. (ref. 6) who developed a p~rticularly expedient purification method by affinity chromato-graphy on an affinity resin comprising a polymeric resin matrix with coupled benzylsuccinyl groups. CPD-Y, which is ~ serine ~ t~ , "
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enzyme, is characterized by having the above relation between the different enzymatic activities at pH~ 9 and by having no endopepti-dase activity. In addition to its specificity for C-terminal amino acids or esters with free ~-carboxyl groups, CPD-Y can also hydrolyze peptides in which the C-terminal ~-carboxyl group is blocked in the form of an ester group, e.g. an alkyl or aryl ester or as an amide group or an N-substituted amide, e.g. an anilide. Importantly, the enzyme hydrolyzes most substrates, inde-pendent of the type of the C-terminal amino acid residue. Another advantage of CPD-Y is that it is available in large amounts and displays relatively great stability.
In addition to CPD-Y, which is the preferred enzyme at present, the process of the invention is feasible with other L-specific serine or thiol carboxypeptidases, such as those listed in the following survey:
Enzyme Oriqin Penicillocarboxypeptidase S-l Penicillium janthinellum S-2 ~ "
Carboxypeptidase(s) from Aspergillus saitoi " Aspergillus oryzae Plants Carboxypeptidase(s) C Orange leaves Orange peels Carboxypeptidase C~ Citrus natsudaidai Hayata Phaseolin French bean leaves Carboxypeptidase(s) from Germinating barley Germinating cotton plants - (Plant Cont.) Tora~toes Watermelons Bromelain pineapple po~der Bovine carboxypeptidases A and B (CPD-A and CPD-B), S however, are not suitable because they are metallocarboxypeptidases.
As explained above, the synthesis is based on a reaction of a so-called substrate component also called acid component or donor, and containing the moiety A with a so-called amine component, also called nucleophile component or acceptor, and containing the moiety B thereby to form a peptide A-B.
The moiety A, which is an L-amino acid residue or a peptide residue having a C-terminal L-amino acid can, if desired, be N-terminal amino protected to avoid undesirable secondary reactions.
The need for amino protection diminishes with increasing chain length of the peptide residue and is essentially non existent when the peptide residue consists of three amino acids, depending, however, upon their type and sequence.
Examples,of useful amino acids are aliphatic amino acids, such as monoamino monocarboxylic acids, e g. Glycine (Gly), alanine (Ala), valine (Val), norvaline (Nva), leucine (Leu), isoleucine (iso-Leu), and norleucine (Nle), hydroxy amino acids, such as serine (Ser), threonine (~nr) and hornoserine (homo-Ser), sulfur-containing arnino acids, such as methionine (Met) or cystine (CysS) and Cysteine (CysH), monoamino dicarboxylic acids and amides thereof, such as aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn) and glutamine (Gln), diaminomonocarboxylic acids, such as ornithine (Orn) and lysine (Lys), ar~inine (Arg), aromatic amino acids, such as phenylalanine (Phe) and tyrosine (~yr), as well as heterocylic amino acids, such as histidine (His) and tryptophan (Trp).
As protective groups may be used the amino protective groups common within the peptide chemistry, such as benzoyl, (Bz), acetyl (Ac) or tertiary alkoxycarbonyl groups, e.g. t-butyloxycar-bonyl (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-nitrophenylsul-1~ fenyl(Nps-), or the like.
Preferred protective groups are benzyloxycarbonyl and t-butyloxycarbonyl since these derivatives are easy to produce, economic in use and easy to cleave again.
As stated above the substrate component may be selected from the group consisting of (a) amino acid esters, peptide esters and depsipeptides of the formula A-ORl, A-sRl or A-SeRl wherein A is as defined above and R represents alkyl, aryl, heteroaryl, aralkyl or an ~-des-amino fragment of an L-amino acid residue, and (b) optionally N-substituted amino acid amides and peptide amides of the formula A-NR R
wherein A is as defined above and R2 and R2 each represent hydrogen, alkyl, aryl, heteroaryl or aralkyl, and ~c) optionally N-terminal protected peptides of the formula , A-X
wherein A is as defined above and ~ represents an an amino acid residue.
In this context "alkyl" means straight chain or branched alkyl, preferably with 1 to 6 carbon atoms, e.g. methyl, ethyl, propyl, isopropyl, butyl, isobytyl, tert. butyl, amyl, hexyl, and the like.
"Aryl" means phenyl and the like.
"Aralkyl" means benzyl, phenethyl, and the like.
All of these groups may be substituted with substi-tuents which are inert with relation to the enzyme, e.g. halo (fluoro, chloro, bromo, iodo) nitro, alkoxy (methoxy, ethoxy, etc.), or alkyl (methyl, ethyl, etc.), provided the amino acid residue or the peptide derivative is a substrate for th~ carboxypeptidase.
Thus in case of esters the group ORl is preferably selected from among alkoxy groups, such as methoxy, ethoxy or t-butoxy, phenyloxy, and benzyloxy-group. m e groups may optionally be substituted with inert substituents, such as nitro groups (p-nitrobenzyloxy). Other groups may be used as well if the amino acid residue or the peptide derivative is a substrate for the carboxypeptidase. An example is the so-called depsipeptides ~here the C-terminal amino acid is linked via an ester bond instead of a peptide bond, e.g. benzoyl-glycine-phenyl lactate (Bz-Gly-O-des-NH -Phe).

Preferred carboxylic acid protective groups are alkoxy groups, in particular methoxy groups, or in other words: The substrate component preferably contains or consists of an amino acid methyl ester since these derivatives are easy to produce and good substrates at the same time. However, e.g. ethyl esters, ~ ' ~
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propyl esters, isopropyl esters, bu~yl esters, t-butyl esters, benzyl esters or tert. butyloxy esters may be used equally well.
It should be mentioned that ionizable groups which may be present in the individual amino acids, which are constituents of a peptide residue A, may, if desired7 be blocked in a manner kno~n per se, depending upon the type of the group. However, this is not always required, which is precisely one of the advantages of the present process. If it is desired to protect the function-al groups, suitable protective groups for the ~ -amino group N
are e.g. N~lJ-benzyloxycarbonyl (N ~-Z), t-butoxycarbonyl (N ~V-BOC) or tosyl (N~ -Tos). Suitable protective groups for the N-guanidino group (N ) in Arg are Nitro (N -NO2), N -benzyloxycarbonyl (N -Z) and N , N -dibenzyloxycarbonyl tN -Z-Z). Suitable protective groups for the imidazole ring (N ) in His are N -benzyl (N Bzl) and Tosyl (N m-Tos). Suitable protective groups for theuJ-carboxyl groups are ~ -benzyloxy (-OBzl). Suitable protective groups for the hydroxyl group in aliphatic or aromatic hydroxy amino acids are aralkyl groups, such as benzyl (Bzl). Suitable S-protective groups for the mercapto group in CysH are e.g. the benzyl group (Bzl). The protective groups must be stable during the primary reaction and be easy to cleave from the final product without causing any secondary reactions.
The process of the invention can in principle be carried out with any amino acid as substrate component. In fact, the preferred substrate component is an L-amino acid ester or peptide ester selected from benzyl or Cl-C~ alkyl esters or a p-nitroanilide.
The second participant in the reaction is the so-called amine component which is selected from the group consisting of (a) L-amino acids of the form~la H-B-OH

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wherein B is an L-amino acid residue, and (b) optionally N-substituted amino acid amides of the formula wherein B is an L-amino acid residue and R and R each represent hydrogen, hydroxy, amino or alkyl, aryl, heteroaryl or aralkyl, and (c) amino acid esters, thioesters or selenioesters of the form~la H-B-OR, H-B-SR or H-B-Se~ respec-tively wherein B is an L-amino acid residue and R4 represents alkyl, aryl, heteroaryl and aralkyl, The alkyl, aryl, heteroaryl and aralkyl groups may be substituted and are defined as explained in connection with the substrate component.
It is seen that when R3 = hydrogen, R3 = hydro~en, H-B-NR3R3 represents the free amide while when R3 = OH, H-B-NR3R3 is a hydroxamic acid when R3 = amino H-B-NR3R3 is a hydrazide, and when R3 = phenyl H-B-NR R3 represents an anilide.
m e preferred amine components are amides of the above formula, wherein B is an L-amino acid residue, R3 = H and R3 = H or Cl 3 alkyl. The preferred esters are H-B-OR~ wherein B is an L-amino acid residue and R4 is Cl 3 alkyl.
The process of the invention thus possesses the decisive advantage over e.g. Isowa et al and Morihara et al (op.
cit.) that it can be carried out both with free (not C-terrninal protected) amino acids, and with amino acids which are C-terminal protected e.g. by conversion into the corresponding arnides, anilides, hydrazides, esters or other specified carboxyl derivatives of amino acids.

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- 14a -As regards the amine component, the reac~ion sequence, yields, etc. depend very much upon whether the components are intro-duced as free acid or a.s C-protected derivative, e.g. as amide.
This will be illustrated in greater detail below in connection with the examples; the following general picture seems to emerge, however:
It applies to the free acids that the hydrophobic amino acids, such as alanine, leucine, valine and phenylalanine, as well as acids with a positively charged side chain, such as lysine and arginine, are relatively easy to incorporate in the chain of the substrate component, while amino acids whose side chains contain either carboxyl groups (aspartic acid or glutamic acid), hydroxyl groups (serine and threonine) or amide groups (asparagine and glutamine) are more difficult to incorporate.
Heterocyclic amino acids, such as proline and histidine, are extremely difficult to incorporate as free acids.
It is a different matter if C-terminal protected amino acids e.g. amino acid amides, or N-substituted amides, e.g. hydra-~ides or anilides are u~ed as amine component. This will be elaborated below, but it may be said quite generally that in this case the reaction is highly independent of the structure and allows even very high yields (up to 90%) to be obtained, however, here too, heterocyclix amino acids are more difficult to incor-porate than aliphatic ones.

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. 15 As stated above, the process of the ~invention is carried at pH 5.0 to 10.5, preferably at pH 8.0 to 10.5. I'he preferred pH-value, which is often within a very narrow range, depends upon the pH-optima and plH-minima, respectively, for the different enzyrnatic activities of the enzyme used, it being understood that the pH-value should be selected so that the activities are counter-balanced as explained in the foregoing.

Generally, the peptidase activity increases with dccreas-ing pH-values below about 9.0 thereby rendering the process less advantageous in terms of yield. However, in some cases, e.g. with Bz-Ala-Gly or Bz-Ala-Gly-NH2, the formed peptides or peptide amides are very poor substrates for the carboxypeptidase, so tha-t the esterase activity versus the amino acid ester or peptide cster preferably used as substrate component still dominates, thereby making a synthesis possible even at pH-values about or below 5Ø
Generally speaking, the optimum pH depends on the ac-tual starting materials, the formed peptides and the enzyme.

If CPD-Y is used as enzyme, the pH-value is preferably 8.0 to 10.5, particularly 9.0 to 10.5, as explained below~

This pH-value should be maintained throughout the coupling reaction, and may then be ch~nged for precipitation of the reaction product, cleavage of protective groups, etc. This may be provided for by incorporating a suitable buffer for the selected pH-range in the reaction medium, such as a bicarbonate buffer.

However, the selected bu~fer is not critical to the reaction, provided the proper pH is maintained.

The pH-value may also be maintained by adding an acid, such as HCl, or a baser such as ~aOH, during the reaction.

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.

The reaction is, as mentioned, carried out in an aqueous reaction medium which, if desired, may contain up to 50% by volume of an organic solvent. Preferred organic solvents are alkanols, e.g. methanol and ethanol, glycols, e.g. ethylene glycol or poly-ethylene glycols, dimethyl formamide, dimethyl sulfoxide, tetra-hydrofuran, dioxane and dimethoxyethane.
The selection of the composition of the reaction medium depends particularly upon the solubility, temperature and pH of the reaction components and the peptide product, and upon the stability of the enzyme.
The reaction medium may also comprise a component that renders the enzyme insoluble, but retains a considerable part of the enzyme activity, such as an ion exchanger resin. Alternatively the enxyme may be immobilized in known manner, cf. Methods in Enzymology, Vol. 44, 1976, e.g. by bondins to a matrix, such as a cross-linked dextran or agarose, or to a silica, polyamide or cellulose, or by encapsulating in polyacrylamide, alginates or fibres. Besides, the enzyme may be modified by chemical means to improve its stability or enzymatic properties.
~ne concentration of the two participants in the reaction may vary within wide limits, as explained below. A preferred starting concentration for the substrate component is 0.01 to 1 molar and for the amine component 0.05 to 3 molar.
The enzyme concentration may vary as well, but is prefer-ably 10 6 to 10 4 molar.
Accor~ing to the invention the reaction temperature is preferably 20 to 50C. The most appropriate reaction temperature for a given synthesis can be determined by '7'7~
. .

; -_ 17 .
experiments, but depends particularly upon the used amine component and enzyme. An appropriate temperature will usually be about ~5 to 45C, preferably about 35C. At temperatures lower than 20C the reaction time will usually be inappropriately ]ong~ while -temperatures above 50C often cause problems with the stabili-ty of the enzyme and/or reactants or o~ the reaction products.

Similar variations occur for the reaction time ~jhich depends very much upon the other reaction parameters, as explained below. The standard reac-tion time in the process o~ the invention is about 10 minutes, but may take up to a few hours.

It should be added that when using an amide or substituted amide as the amine component, it is often advantageous or even necessary to cleave the amide group specifically ~rom the formed peptide amide in order to continue the synthesis.
Also in this respect the carboxypeptidase, especially CPD-Y is very suitable since as described above CPD-Y
exhibits amidase activity at pH ~ 9 while the carboxy-peptidase activity is negligible.

By the same token the carboxypeptidase might generally beused to cleave the groups H-, NR3~3 , -OR , -SR or SeR
as defined from the formed peptide whether it is desired to continue the synthesis or just to obtain a final peptide which is not C-terminal protected.
As will be further illustxated below the process of the invention is applicable for the formation of an unlimited number of dipeptides, oligopeptides and polypeptides, based 3Q on the same inventive principle, viz. reacting a substrate cornponent with an amine component in the form of an amino acid or amino acid derivative in the presence of a carboxypeptidase.

Examples of well-known oligopeptides or polypeptides which may be produced accordingly are enkephalins, somatostatin 7 somatostatin analo~s and peptides with similar biological activities, and the so-called "sleep peptide" (Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu).

In certain cases, e.g. when dealing with peptides consist-ing of more than five amino acids, it might be advantageous to synthesize parts of the desired peptide in the form of oligopeptide fragments e.g. pentapeptides with the suitable amino acid sequences and subject the oligopeptides to fragment condensation in any manner known per se.

It might also be advantageous in order to improve e.g.
the solubility characteristics of the peptides formcd to use as the N-terminal amino acid arginine or another ionizable amino acid during the synthesis steps and then cleave the arginine from the pep-tide with a specific enzyme e.g. trypsin, when the dcsired amino acid sequence is otherwise in order.

These and other modifications also form part of the invention.

Before the process of the invention will be illustrated by examples, starting materials, methods of measurement, etc.
~rill be explained in general terms.

Startin~ materials Carboxypeptidase Y from baker's yeast was isolated by the affinity chromatography proccdure of Johansen ct al.
(ref. 6) and obtained as a lyophilized powder (16,~ enzy~c in sodium ~ trate). Before use the enzyme was desaltcd on Scphadex G-25 fine (1~5 x 25 cm) equilibrated and clutcd with distilled water. The conccntration of thc ~. ~

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enzyme was determined spectrophotome-trically using E18~o nm = 14.8 (ref. 6). A s-tock solution of 7 mg/ml (110 /uM) was prepared and stored in aliquots of 250-500 /ul at -21C. Benzoylalanine methyl ester (Bz-Ala-OMe) was purchased from Bachem, Liestal, Switzerland. Boron tri-fluoride etherate complex (for synthesis), solven-ts and reagents (all analytical grade) were from Merck, ~armstadt, West Germany. All amino acids and amino acid amides and their derivatives were from Sigma Chemical Company, St. Louis, USA. Carbobenzyloxy-phenylalanine methyl ester (Z-Phe-OMe) was prepared according to the procedure of Yamada et al. (ref. 20) and used as a syrup.
Phenylalanine hydrazide and alanine hydrazide hydro-chloride were prepared from the esters as described by Losse et al. (ref. 11). The uncorrected melting points were 86-88C (Lit.: 82-8~C (11)) and 182-185C (Iit.:
184-185C (21)), respectively. Asparagine amide dilly(lro-chloride was obtained from aspartic acid via the diethyl ester according to Fischer (ref. 4) by classical amino-lysis (m.p.: 210-214C, Lit.: 214-215C (19)). Other starting materials were provided from the above companies or produced in analogous manner.

Determination of product yields The purity was determined qualitatively by TLC on Silica Gel 60 F254 (Merck). The solvent system used was CHC13/
CH3(CI~)30H/CH3COOH/H20 (11:5:2:1) and the spots ~Jcre visualized by fluorescence quenching for estimation of the reactant composition.

The reactant compositions wcre determined quantitatively by reversc-phase l~PLC using an RP-8,10 /urn ~Merck) colu-nn and a Hewlett Packard 1084 Chromatogr~ph equipped with a variable wavelen~th W detector (Model 7~875 A). S~par-ation was achievcd using suitab]c spccific ~radicnts of .

~ ~L'7'~
-`~ 20 elution systems from 10/o CH3CN in 10 mM--NaAc, pH 4 to 100/o CH3CN or from 10 mM-NaAc, pH 4 to lOG~ CH3CN. The latter system was used for compounds such as Bz-Ala-Gly-OH, Bz-Ala-Ala-OH, Bz-Ala-Ser-OH and ~heir respective amides, cf. below. The flow rate was 3 ml/min, the column temperature 47C and the monitoring wavelength 260 nm. The yields were determined on the basis of the molar ratio of the reactants which was obtained from the integrated areas under the peaks in the elution profile.
Identification of products The spots were identified by thin-layer cochromatography with suitable standard compounds. Several products wére identified by a combination of HPLC and amino acid ana-lysis. For this purpose, 1 ml-aliquots were taken from the reaction mixture after 10 minutes, and the reaction was discontinued by adding 250 /ul 6 M-HCl: The pH was then adjusted to 4 with NaOH and the mixture separated by HPLC using Waters equipment including two pumps, a Model 660 Solvent Programmer, a Model U6K Injector, a Model 450 Variable Wavelength Detector combined with a Recorder (Radiometer REC 61) or a Hewlett Packard Recorder/
Integrator Model 3380A. Elution was monitored by scanning at a suitable wavelength between 255-280 nm. The chroma-tography was reverse-phase using a ~aters C-18 /u-Bondapak column with the elution system TEAP- 20% (v/v) TEAP
(triethylammoniumphosphate buffer) in methanol under suitable gradients and flow rates of 1.5-2.0 ml/min.
The TEAP-buffer was prepared according to Rivier (ref.
17). In many cases the system 0.1 M-HAc, pH 3 - 20/~
(v/v) 0.1 M-HAc, pH3 in methanol gave also sufficient resolution.

The effluent containing the N-acyldipeptides was collected manually and taken to ~ryness by lyophilization or on a Buchi Rotovap at 35-45C. Small samples of the residues were hydrolyzed in 6 M-HCl at 110C in vacuo for 36 h.

e~ C3 ,.
- - . 21 .
The evaporated hydrolyzates were then analysed on a Durrum D-500 amino acid analyzer.

Synthesis with free amino acids as amine component EX~MPLE 1 Asolution of 2 ml of 0.6 M Valinc-O.l M KCl-lmM EDTA, pH 9.8 was mixed with 100 /ul (0.1 mmole) of a 1 M
Bz-Ala-OMe (dissolved in 96~ ethanol) solution. The react--ion was carried out in a pH-stat at 35C and pH 9.8, the pH-value being kept constant by automa-tic addition of 0.5 M
NaOH~ The reaction was initiated by a~ding 0.7 mg of carboxypeptidase Y (150 U/mg, prepared by De ~orenede Bryggerier). After a reaction time of 30 minutes, the reaction was discontinued by adjusting pH to about pH = 1 with 6 M HCl. The reaction product was purified and isolated by means of high pressure chromatography. The yield of Bz-Ala-Val-OH was 40%. Quantitative amino acid analysis after hydrolysis of the product in 6 M HCl for 24 hours gave relatively 1.0 mole of Alanine and 1.0 mole of Valine.

The effect of pH, temperaturc and concentration of substrate cornponent, amine corrlponent and CPD-Y on -the yield in the above reaction:

Bz-Ala-OMe + H-Val-OH CPD ~ Bz-Ala-Val-OH + CH30H ~5 ~las studied in five separate series of experiments analo~ously with the above-mentioned procedure. One of the pararneters mentioned belo~ was varied in each e~peri-ment, while the four others ~Jere kept constant: t3.6 M
valine, 55 mM Bz-Ala-OMe, 4.5 mM CPD3-Y, pH 9.7, 35C. The results are shown in fig. 1. It ~li]l be scen Irom fig.
lA that the p}~-range for optirnal yield is rather n.3rro~, ~ '7~
.
; 22 :
extending over 0.5 pH units only. It will be seen from fig. lB that an increase in the reaction temperature caused an almost linear increase of synthesis on account of relatively less hydrolysis. At temperatures above 45C, enzyme inactivation and non-enzymatic ester hydrolysis became prohibitive. At lower tempera-tures and pH-values up to 10.0, the ester hydrolysis was negligible within the 10 minutes standard reaction time. It became significant, however, when the enzymatic reaction rate was inhibited and called for reaction times up to 2 to 5 hours.

While the yields of Bz-Ala-Val-OH increased with higher amine component concentration (fig. lD), the reverse was the case for the relationship between yield and concen~
tration of the substrate component Bz-Ala-OMe (fig. lC).
The latter observation, taken together with the depen-dency of yield on high enzyme concentration (fig. 1~), suggests an optimal ratio of substrate to enzyme concen-trations.

The time course of a typical reaction, illustrated by the above reaction under conditions little short of optimum is illustrated in fig. 2. In the presence of 0.5 M valine, the substrate Bz-Ala-OMe was rapidly converted (within 20 minutes) to 38% Bz-Ala-Val-OH and 62% Bz-Ala-OH. The figure also shows that the dipeptide was not hydrolyzed at pH 9.7 in the presence of excess valine. If, however, pH was adjusted to 5 -to 8, all Bz-Ala-Val-OH was hydrolyzed within seconds. This selective behaviour of CPD-Y at high pH is an important property for its usefulness in peptide syntheses.

EY~LE 2 A solution of 2 ml of 3M lysine - 0.1 M KCl - lmM EDTA, pH 9.8 ~las mixed ~lith 400 /ul of 100~' methanol and 7'~
~' 23 ~- -. ,; .............. .
100 /ùl (O.l mmole) of a IM Z-Phe-OMe (dissolved in 100/o methanol). The reac-tion was carried out as described in example 1 and initiated by adding 0.7 mg of carboxypep-ti-dase-Y. After a reaction -time of 30 minutes, pH was ad-justed to 1 with 6 M HCl. The reaction product waspurified and isolated by means of high pressure chromato-graphy. The yield of Z-Phe-Lys-OH-was 60/o. Quantitative amino acid analysis, as described in example 1, showed that the product relatively contained 1.0 mole of lysine and 1.0 mole of phenylalanine.

Analogously with examples 1 and 2, the peptides listed in table I were prepared from the starting materials stated.
The experiments were carried out in a Radiometer pH-stat and the yields were determined by HPLC ( cf. the foregoing).
The conditions were 4.5 /uM CPD-Y; pH 9.7 and 35C.

.~ ~

. .

~ >7~

~ _ . _ . . .
Table I
:
Carboxypeptidase-Y ca-talyzed synthesis of peptides with free amino acids as amine component.

.
Substrate Amine component - Product Yield %
(conc.) (concentration) . ._ Bz-Ala-OMe Glycine (3.0 M) Bz-Ala-Gly-OH 62 Alanine (1.9 M~ Bz-Ala-Ala-OH 65 Valine (0.6 M) Bz-Ala-Val-OH 40 (Ex. 1) Leucine (0.17 M) Bz-Ala-Leu-OH 24 . Phenylalanine (0.16 M) Bz-Ala-Phe-OH 27 Serine ~3.2 M) Bz-Ala-Ser-OH 5o Threonine (0.7 M) Bz-Ala-Thr-OH 24 Methionine (0.6 M) Bz-A].a-Met-OH 46 Lysine ~1.5 M) Bz-Ala-Lys-OI~ 56 Arginine (0.8 M) Bz-Ala-Arg-OH 26 Aspartic acid (1.0 M) Bz-Ala-Asp-OH O
Asparagine (0.6 M) Bz-Ala-Asn-OH 5 Glutamic acid (1.2 M) Bz-Ala-Glu-OH O
Glutamic acid-r-methyl Bz-Ala-_ ester (1.0 M) Glu(OMe)-OH 3o Z-Phe-OMe Valine (0.6 M) Z-Phc-Val-OH 6 Lysine (3.0 M) Z-Phe-Lys-OH 60 Bz-Phe- . (Ex. 2) Cly-OMe Valine (0.6 M) Bz-Phe-Gly- 40 (30 mM) Val-OH
~ .. .
Z-Ala-OMe Glycine (2.0 M) Z-Ala-Gly-OH 3o (10 mM) Alanine (0.7 M) Z-Ala-Ala-OH 60 Leucine (0.15 M) Z-Ala-Leu-OH 21 Lysine (1.5 M). _ Z-Ala-Lys-OH 60 Bz-Gly-OJ~e Glycine (2.0 M) Bz-Gly-Gly-OH 63 (20 mM) Leucine (0.15 M) Bz-Gly-Leu-OH¦ ?l - . . . :
.

Table I (continued) . ~?--~ -I
Substrate I Amine component Product Yield %
(conc.) ¦ (concentration) _ _ Bz-Tyr-OEt Valine (0.6 M) . Bz-Tyr-Val-OH 3o ( 25 mM) Alanine (1.9 M) Bz-Tyr-Ala-OH 46 Ar~inine (0.8 M) Bz-Tyr-Arg-OH 35 _ -~ _ Ac-Phe-OEt Alanine (0.8 M) Ac-Phe-Ala-OH 85 (50 mM) ..__ Z-Ala-Ala- Glycine (2.0 M) Z-Ala-Ala- 40 OMe Gly-OH
(10 mM) Leucine (0.15 M) Z-Ala-Ala- O
Leu-OH
Phenylalanine (O.15 M) Z-Ala-Ala- - O
Phc-OH
Z-Ala-Phe- Glycine (2.0 M) Z-Ala-Phe- 35 OMe Gly-OH
(10 mM) Leucine (0.15 M) Z-Ala-~hc- O
Leu-OH
_......... .
Z-Ala-Ser- Leucine ~0.15 M) Z-Ala-Scr- ~ 40 OMe Leu-OH
~10,mM) ............ _ . .... _ Z-Ala-Val- Leucine (0.15 M) Z-Ala-Val- 25 OMe Leu-OH ..
(10 mM) Lysine (1.5 M) Z-Alu-Val- 60 __ Lys-OH
Z-A].a-NLeu- Glycine (2.0 M) Z-Ala-NLeu- 60 OMe Gly-OH
(10 mM) Leucine (0.15 M) Z-Ala-NLeu- 16 .. . -- --- _ Leu-OH
Z-Ala-Met- Glycine (2.0 M) Z-A]a-Met- 5o OMe Gly-OH
(10 mM) Leucine (O.15 M) Z-Ala-Met- 15 Lcu-OH
_ .. __ . . _ . _ Z-Ala-Trp- Glycine (2.0 M) Z-Ala-Trp- ~ 23 OJYe Cly-OH
(10 mM~ Lellcine (O.15 M) Z-Ala-Trp- 3 Leu-OH

:

.
;

. .

- . ~
~ 6 e .
Table I (continued) .. .
Substrate Amine component Product Yield (conc.) (concentrati~n) _ ; Z-Ala-Trp- Phenylalanine (0.15 M) Z-Ala-Trp- O
OMe Phe-OH
(10 mM) Lysine (1.5 M) Z-Ala-Trp- 36 Ly s- OH
Z-Ala-Ala- Leucine (0.15 M) Z-Ala-Ala- O
Tyr-OMe Tyr-Leu-OH
(10 mM) Lysine (1.5 M) Z-Ala-Ala- 23 Tyr-Lys-OH
Alanine (1.7 M) Z-Ala-Ala- 31 Tyr-Ala-OH

:
- : '` . `
`
.

.~. .
Synthesis wi-th amino acid amides as amine component EX~PLE 3 A solution of 2 ml of 0.6 M methionine amide (Met-NH2) -O.1 M KCl - 1 mM EDTA, pl~ 9.~, was r,lixed with 100/ul (0.1 mmole) of a 1 M Bz-Ala-OMe solution in 96S' methanol.
The reaction was carried out as described in e~ample 1 and initiated by adding O.7 mg of carboxypeptidase-Y.
After a reaction time of 30 minutes, pH was adjusted to 1 with 6 M HCl. The reaction product was purified and isolated by means of high prcssure chromato~raphy. The y~eld of Bz-Ala-Mct-NH2 was 95%. Quantitative amino acid analysis, as described in example 1, showed that the product relatively contained 1.0 mole of Ala, 1.0 mole of Met and 1.0 mole of NH3.

The course for the reaction is shown in fig. 3, from which it appears that within 20 minutes the substrate is converted almost completely to about 95% dipeptide, 5%
being hydrolyzed to Bz-Ala-OH.

.

A solution of 2 ml of 0.4 M valine amide (Val-NH2) -0.1 M KCl - 1 n~ ~DTA, pH 9.5, was rllixed with 100/ul (0.1 mmole) of a 1 M Bz-Ala-OMe solution in 96~ ethanol.
The reaction was carried out as dcscribed in exarnple 1.
~`he rcaction product Bz-Ala-Val-NH2 precipitated during the reaction. After 20 minutes' reaction pl~ was adjustcd to 1, and the precipitate was isolatcd by ccntri~u~ation.
The product ~.as dissolved in 1 ml o~ 96% ethanol and purified and iso]ated by hieh pressurc chromato~raphy.
The yield of Bz-Ala-Val-~1~2 was 95~', while, as shown in c~ample 1, it was only 4~' whcn the free acid was used .

. .

7~

as amine component. Quanti-tative amino acid analysis, ~-- as described in example 1, showed that the product relatively contained 1.0 mole of Ala, 1~0 mole of Val and 1.0 mole of NH3.

The dependency o~ the reaction sequence on pH and the valine amide concentration is S}lOWn in fig. 4. The react-ion was carried out analogously with the synthesis above, the constant parameters being: Valine amide 0.6 ~5, B~-Ala-OMe 55 mM, CPD-Y 4.5 /uM, pH 9.7, 35C.

It will be seen from ~ig. 4B that the yield is essen-tially insensitive to the variation in the valine amide concentration in contrast to fig. lD which shows the yield with varying valine concentration, at least a concentration e~uimolar to or higher than the substrate concentration (55mM).

It will be seen from fig. 4A tha-t the effective pH-range of valine amide extends down to 9.0, thcre being a sharp upper limit at pH 9.8 above which the yield decreases heavily.

Analogously with examples ~ and 4, the peptides listed in table II were prepared from -the starting materials s-tated. The experiments were carried out under the following conditions: 4.5 /uM CPD-Y; pl~ 9.6 and 35C, while the substrate concentration is stated in the table.

- ` ~
;~ 29 ~ Table ~I
:
Carboxypeptidase-Y catalyzed synthesi.s of peptides ~ith amino acid amides as amine component.

Substrate Amine component Product IYield ~0 (conc.) (concentration) .~ __ . .. ___ Bz-.Ala-OMe Glycine amide (0.3 M) Bz-Ala-Gly-NH2 90 (55 mM) Serine amide (0.3 M~ Bz-Ala-Ser-NH290 (Ex. 4) Bz-Ala-Val-NH2 95b) Leucine amide (0.3 M) Bz-Ala-Lcu-NH285 . Methionine amide(O.6 M) Bz-Ala-Met-NH2 95 Phenylalanine amide Bz-Ala-Phe-NH2gob) Tyrosine amide (0.6 M) Bz-Ala Tyr-NH2 9o Asparagine amide(O.3M) Bz-Ala-Asn-NH2 80 Proline amide (0.3 M) Bz-Ala-Pro-NH2O
Glutamic acid amide Bz-Ala-Glu-NH O
(0.25 M) 2 Histidine amide (0.2 M) Bz-Ala-His-NH2 89 Threonine amide (0.2 M) Bz-Ala-Thr-NH2 ~8 ..
._ _ .......... . . _. ._ ._ . .
Z-Phe-OMe Valine amide (0.5 M) Z-Phe-Val-NH2 9,7b) ; (55 mM) Serine amide (0.4 M) Z-Phe-Ser-NH2 60 Tyrosine amide (0.4 M) Z-Phe-Tyr-NH2 64 . . ._. ... _ Bz-Phe- Valine amide (0.4 M) Bz-Phe-Gly-Val- 90 Gly-OMe NH
(30 mM) 2 __ _ __ .... __ ...................... ..
Z-Phe-Ala_ Valine amide (0.4 M) Z-Phe-Ala-Val- 90b) (20 ~1~ Glycine amide (0.4 M) Z-Phe-Ala-Gly- 95 ~H2 Histidine amide (0.4 M) Z-Phe-Ala-Hi~- 50 .. . . ._ - -- .. .... ._ b) Product precipitated ~ ~ 7t~c3 ~o .;; .
Table II (continued) Substrate Amine component Product ¦Yield %
(conc.) (Concentrati~n) . .
Z-Leu-Gly- Valine amide (0.4 M) - Z-Leu-Gly-Gly- 80 Gly-OEt Val-NH2 (10 mM) .
Bz-Tyr-OEt Valine amide (0.25 M) Bz-Tyr-Val-NH2 94 (50 mM) Glycine amide (0.55 M) Bz-Tyr-Gly-NH282 Z-Ala-OMe Leucine amide (0.15 M) Z-Ala-Leu-NH2 90 (10 mM) Glycine amide (0.15 M) Z-Ala-Gly-NH2 20 (75 mM) Tyrosine amide (0.2 M) Bz-Arg-Tyr-NH252 Bz-Tyr-OEt Valine amide (0.25 M) Bz-Tyr-Val-NH2 94 (50 mM) Glycine amide (O.55 M) Bz-Tyr-Gly-NH282 ~-Pro-OMe Leucine amide (0.25 M) Z-Pro--Leu-NH2 O
(50 mM) .. .
Bz-Gly-OMe Histidine amide (0.2 M) Bz-Gly-His-NH2 20 (100 mM) Glycine amide (0.55 M) Bz-Gly-Gly-NH2 95 Leucine amide (0.25 M) Bz-Gly-Leu-NH2 _ Z-Ala-Phe- Leucine amide (0.15 M) Z-Ala-Phe-Leu- 95 OMe NH2 (10 mM) Glycine amide (0.15 M) Z-Ala-Phe-Gly- 84 .. . ~ ..
Z-Ala-Ala- Glycine amide (0.15 M) Z-Ala-Ala-Gly- 100 OMe NH
(10 mM) Leucine amide (0.15 M) Z-Ala-Ala-Leu- 100 ._ _ ._ NH2 Z-Ala-Ser- Leucine amide (0.15 M) Z-Ala-Ser-Leu- 95 OMe NH
(10 mM) Glycine amide (0.15 M) Z-Ala-Ser-Gly- 70 . . ___ _ 2 .
Z-Ala-Val- Leucine amide (0.15 M) Z-Ala-Val-Leu- 84 OMe NH
(10 mM) Glycine amide (0.15 M) Z-Ala-Val-Gly- 100 ._ _...... _ ._ . .. _ NH2 _ _ 7 -, ; 31 : Table II (continued . ~ .

Substrate ¦ Amice component Product Yield %
(conc.) (concen~ration) Z-Ala-NLeu- Leucine amide (0.15 M) Z-Ala-NLeu- 100 OMe . ~Jeu-NH
(10 mM) Glycine amide (0.15 M) Z-Ala-NLeu- 100 Gly-NH2 _ Z-Ala-Met- Glycine amide (0.15 M3 Z-Ala-Met-Gly- 95 OMe NH
(10 mM) . 2 . ..
Z-Ala-Trp- Glycine amide (0.15 M) Z-Ala-Trp-Gly- 84 OMe NH
(10 mM) Leucine amide ~0.15 M) Z-Ala-Trp-Leu- 89 . NH2 Z-Thr-Pro- ~aline amide (0.2 M) Z-Thr-Pro-Val- 95 OMe NH
(25 rnM) .
Z-Ala-Ala- Glycine amide (0.15 M) Z-Ala-Ala-Tyr- .1.00 Tyr-OMe ~ly-NH2 (10 mM) Leucine amide (0.15 M) Z-Ala-Ala-Tyr- 100 L~U-NH2 __ .__ ._.__ .......... . __ _ Z-Tyr-Gly- Phenylalanine amide Z-Tyr-Gly-Gly- 40 r20 mM) (~-2 M) ,. _ - __ _ 7'7~
.
_ 32 EX~MPLE 5 Synthesis wi-th amino acid hydrazides as amine com~onent Analo~ously with examples 3 and 4 the peptides listed in Table III were prepared from the s~arting ma-terials stated. The experiments were carried out under the follow-ing conditions: 4.5 /uM CPD-Y, pH 9.6 and 35C.

Table III

Carboxypeptidase-Y catalyzed synthesis of peptides with amino acid hydrazides as amine component -- .
...
Substrate Amine component Product Yield ~' (conc.) (concentration) . , ~z-Ala-OMe Alanine-hydra~ide . Bz-Ala-Ala-NH- 37 (55 ml~) (0.6 M) NH2 Phenylalanine Bz-Ala-Phe-NH- ~0 _ hydra~ide (0.3 M) N~2 ...._ .

~MPLE 6 Synthesis with amino ~cid esters as amine com~onent The experiments with amino acid esters were carried out analo~ously with examples 1, 2, 3 and 4, in a Radiometer pH-stat at pH 9.0 ~ 9.7 and 23-35C. Products and yields were determined by HPLC. The CPD-Y eoncentration was froM
4.5 to 11 /uM.

Table IV states the peptides produced from the mcntioned starting materials.

i ., . -:
It is seen that in some cases a certain oligomerizationis obtained. Since the yields ~enerally are very hi~h, amino acid esters appear to be extremely useful if oligomerization can be further limited.
.
Table IV

Carboxypeptidase-Y catalyzed syn-thesis of peptides with amino acid esters as amine cornponent.

Substrate Amine component ProductIYield %
(conc.) (concentration) ._ Bz-Ala-OMe Glycine ethyl ester ~ Bz-Ala-Gly-OH~
(50 mM) (0.5 M) ~ Bz-Ala-Gly- ~ 100 ~Gly-OH J
Glycine propyl ester Bz-Alu-~ly-OH 85 Glycine isopropyl Bz-Ala-Gly-OH 90 ester (0.5 M) (0-5 M) Bz-Ala-Gly-OH 80 . fBz~Ala-Leu-OH ~
¦ Bz-Ala-Leu-Leu- ..
(O 25 M) ~ OH 85 . IBz-Ala-Leu-Leu-Leu-OH
Bz-Ala-Leu-Leu-Leu-Leu-OH
I.eucine ethyl ester ~ Bz-Ala-Lcu-OH ~
(0-~5 M) ~Bz-Ala-Lcu-Leu~ 5o Leucine-t-butyl Bz-Ala-Leu-OH O
ester (0.25 M) rBz-A] a-pnc-oH
Phenylalani~Je methyl ) Bz-Ala-Phe-Phe-cster (0.25 M) OH ~0 Bz-Ala-Phe-Phc-r),c-oll , 77~

.
- Table IV (continued) Substrate Amine component Product Yield %
(conc.) ~concentration) _ _ ,_ Bz-Ala-OMe Phenylalanine ethyl -(Bz-Ala-Phe-OH ~
(50 mM) cster (0 15 M) ~ Bz-Ala-Phe- ~ 70 LPhC-OH - J
Glutamic acid Bz-Ala-Glu (~--t-Bu~ methyl (~-t-Bu)-OH 35 ester (0.5 M) Glutamic acid (y-t-Bu~ Bz-Ala-Glu O
t-butyl ester (0.25 M) (~-t-Bu)-OH
. ~Bz-Ala-Met-OH
¦Bz-Ala-Met-Methionine methyl J M~t-OI~ ¦
ester (0.2 M) ~ Bz-Ala-Met- ' ~6 Mct-Mct-OH
~z-Ala-Mct-.
Met-Mct-Met-OH¦
Bz.-Ala-Met- ¦ .
Met-Met-Met-Met-OH -J
Methionine ethyl Bz-Ala-Met-OH 40 ester (0.2 M) Methionine isopropyl Bz-Ala-Met-OH 8 ester (0.2 M) Valinc methyl ester ~Bz-Ala-Val-OH ~
(0.7 M) ~ Bz-Ala-Val- ~ ~5 ~Val-OH J
(0.5 M) Bz-Ala-Ser-OH 50 Tyrosine methyl Bz-Ala-Tyr-OH~5 e~ter (0.5 M) Arginine methyl Bz-Ala-Arg-OH O
ester (0.5 M) ArGinine (NO ) methyl Bz-Ala-Ar~(N02)- ~0 cster (0.5 ~ OH

l~istidine methyl Bz-Ala-His-OH 2 cster (0.5 M) Threonine rncthyl Bz-Ala-Thr-OH
cstcr tO.5 l~) . , _ ~ - -- 35 s ~

-- - ~- Table IV (continued) . . _ .

Substrate Amine component Product Yield %
(conc.) (concentration) ___ Ac-Phe-OEt Alanine me-thyl ester Ac-Phe-Ala-OH
(50 mM) (0.5 M) Ac-Phe-Ala- 60 Ala-OH
Bz-Gly-OMe Histidine methyl Bz-Gly-His-OH 40 (50 mM) ester (0.5 M) EXA~LE 7 L- and D-stcreoisomers of the a_ _o comPonent Analogously with cxamples 1, 2, 3, 4, and 6, the pep~idcs stated in Table V wcre pro~uccd from the listed starting materials.

It is seen that only the L-isomers are incorporated. This is a very interesting fcature in tcrrns of process economy since it is consequently not necessary to purify the starting amino acids with a view to obtain the pure L,isomer.

. , ' _.

-. Table V
~ . . . .
Carboxypeptidasc-Y catalyzcd ~ynthcsis of peptides with L- and D-isomers of the amine component.

_ Substrate Amine component ProductYield ~,o ( conc . ) ( concentration) L-isomcrlD-isomer . ._ ___ Bz-Ala-OMe Valine (0.6 M) Bz-Ala-Val- 42 O
(50 mM) OH
Alanine (1.8 M) Bz-Ala-Ala- 65 O
OH

Bz-Tyr-OEt Valine (o.6 M) Bz-Tyr-Val- 30 O
(30 mM) OH .
Alanine (1.8 M) Bz-Tyr-Ala- 46 O
01~ , Bz-Ala-OMe Val-NH2 (0.25 M) Bz-Ala-Val- 78 O
(50 m~l) NH2 Bz-Tyr-OEt Val-NH2 (0.25 M) Bz-Tyr-Val- 95 O
(30 mM) NH2 ..

Ac-Phc-OEt Ala-OMe (0.5 M) ~Ac-Phe-Ala-(50 1~) 01~ 1 60 O
~c-Phe- I
... (Ala)2-OH J

. . ~
.

3~ ~1 '7'7~Z~
.
~- ~7 ~ .

. . ._ Variation of the substrate ester ~roup _ Analogously with examples 1, 2~ 3, and 4 the peptides stated in Table VI were produced from -the ]isted starting materials.

The results prove tl~e f]exibility of the process of the invention as re~ards applicable substrates.

Table VI

Yield (%3 of carbox~peptidase-Y catalyzed synthesis of peptides with different ester groups on the substrate component.

Substrate I I
A ~ Bz Gly-OMe Bz-Gly-OEt Bz-Gly-OiPr Bz-Gly--OBz component ~ (20 mM) (20 mM) (20 mM) (20 TT~) . . . . _ _ .

Glycine _. . 45 47 Glycine amide 59 ! 95 ~ 88 ¦ 91 Leucine 21 59 74 80 (O 15 M) _ _ 100 95 ~ 95 .

.. . ~
. .

Depsipeptides as substrates Analogously with examples 3 and 4 the peptides stated in Table VII were produced from the listed starting materials.
The conditlons were 4.5 /uM CPD-Y, pH = 7.6 and 25C.

It is seen that very high yields are obtained.

Table VII

Carboxypeptidase-Y catalyzed synthesis of peptides with depsipeptides as substrate components.

Substrate Amine component Product Yield %
(conc.) (concentration) .

Bz-Gly- Glycine amide (0.25 M) Bz-Gly-Gly-NH2 90 Odes-NH2- Leucine amide (0.25 M) Bz-Gly-Leu-NH2 95 ( 20 mM) Phenylalanine amide Bz-Gly-Phe-NH2 95 ) _ . . .
Bz-Gly- Glycine amide (0.25 M) Bz-Gly-Gly-NH2 95 Odes-NH2- Leucine amide ~0.25 M) Bz-Gly-Leu-NH2 95 ( 20 m~S) ( 0. 25 M) --- Bz-Gly-Phe-NH2 95 Bz-Phe- Glycine amide (0.25 M) Bz-phe-Gly-NH2 90 Odes-NH2- Leucine amide (0.25 M) Bz-Phe-Leu-NH2 80 20 mM) Phenylalanine amide Bz-Phe-Phe-NH2 80 (0.25 M~ _ _ ~XAMPLE 10 . , Peptides as su~strate comp~nen-ts Analogously with examples 1, 2, 3, and 4 the peptides stated in Table VIII were pl~oduced. The experimen`ts were carried out in a Ra~iometer pH-stat and -the yields dc-termined by IIPLC. The condi~ions were 5 /uM CPD-Y, pH = 7.6 and 25C.

Table VIII

Carboxypeptidase-Y catalyzed synthesis of peptidcs ~Jith peptides as substra-tes.

Substrate Amine component Product Yleld %
(conc.) (concentration) .. _, (20_m ~ Leucine amide (0.25 M) Bz-Phe-Leu-NH2 90 Bz-Gly-Phe Leucine amide (0.25 M) Bz-Gly-Leu-NH2 10 .

(20 mM Leucine amide (0.25 M) Z-Ala-Leu-NH2 74 _ ~ __ _ Z-Phe-Ala Leucine amide (0.25 M3 Z-Phc-I.eu-NH2 60 (20 mM) . _ _ _ Z-Phe-Ser ¦Leucine amide (0.25 M) Z-Phc-Leu-NH2 7o (20 mM3 l _ _ .
. . .
.

~)7~

.
= . . . . 40 Pcptide synthesis as a function of pH .. .

In cases where the synthesized peptide is a poor substrate for CPD-Y the synthesis may be carried out at pH-v~lues bclow the preferred 9 - 10.5.

Analogously with e~arnples 1 and 3 the peptides statcd in Table IX were produced in a pl~-scat at the listed pH-values.

The conditions wcre 15 /uM CPD-Y and 25C.

Table IY

Carboxypcptidase-Y catalyzed synth~sis of peptidcs as function o~ pH.

..
Substrate Amine componcnt Product pH Yicld %
(conc.) (concentration) . . .
Bz-~la-OMe Glycine (1.3 M) Bz-Ala-Gly 99 05 57 _ .. 6 o 60 Glycine amide Bz-Ala-Cly- 9.5 77 (1-3 M) NH2 9.0 95 8 0 1~7 _ . 6.0 _ 4/~

- ~7~
_ .
.
.

~ . . . .
Various other amine components --. .
_ AMPLE 1 Analogously with examples 3 and 4 the peptides stated in Table X were produced. The experiments wcre carried out in a pl~-stat and the yields determined by ]~PJ,C. The conditions were 4.5 /uM CPD-Y, pH 9.7 and 25C.

Table X

Carboxypeptidase-Y catalyzed synthesis of peptides wi.th diflerent amino acid derivatives as amine components.

Substrate ¦ Amine component ¦ Product ¦Yield (conc.) ¦ (concentration) I l ._.............. _.__ _ ___ _ Bz-Al~-OMe ~-alanine amide (0.2 M) Bz-Ala-~Ala- 80 (50 mM) NH2 .
Glycine hydroxamic Bz-Ala-Gly- . 45 acid (0.2 M) NH-OH
D,L-Alanine hydroxamic Bz-A].a-Ala- 40 acid (O.2 M) NH-OH
. .

.1 - . -;~c~ ~ . . 42 ~ Y~PLE 13 ~~
. . .
Synth sis of ~ ides w th c~rboxypeptidases from bar].~y Gerlninating barley, e.~. in the form of rnalt, contains two dif~erent peptidascs denominated CP-l-l and CP-2-1 (see ~ 5 Lee E. Ray, Carlsberg Res. Cornm. 41, 169-182 (1976)).

CP-l-l and CP-2-1 ~ere isola-ted as described by L.E. Ray and the peptides listed in Table XI were produced analo{~ously with e~amples 1, 2~ 3, and 4 from the listed startjng materials. ~he conditions were 6 /uM CP-l-l or CP-2-1, pl1 8.0 and 25C.

Table XI

Synthesis of peptides usin~ carboxypcptida.ses from ~erillinating barley CP-l-l and CP-2-].

. . .
S~bstrate Amine component Product Yield i/o ~nzyme (conc.) (concentration) . ._ _ _ . ._ , Bz-Ala-OMe Valine amide Bz-Ala-Val- 43 CP-l-l (50 m~) (0.25 M) NH2 Alanine amide Bz-Ala-Ala- 58 CP-l-l (0.25 M) NH2 Lysine (1.7 M) Bz-Ala-Lys 11 CP-l-l Valine amide Bz-Ala-Val- 57 CP-2-1 (0.25 M) '~H2 Alanine amide Bz-~la-Ala- 64 CP-2-1 ~0.25 M) NH2 Lysine (1.7 M) Bz-Ala-Bys 11 P-2-1 ~7'7~

_3 ~ ~ L~ 3 EXAI~PLE 14 Carboxypeptidase-Y catalyzed deamidation o~ peptide amides To a solution of 2 ml 15 rnM Bz-Ala-Leu NH2 in 0.1 M KCl -

5 1 mM EDTA, pH 9.7, 25C and 10/o dimethyl formamide Jas added 2 mg CPD-Y. The reaction course as shown on fig. 5 was followed by HPLC as described in example 1. It is scen that Bz-Ala-Leu-NH2 after 20 minutes was completely converted to about 68% Bz-Ala-Leu~OH and 32% Bz-Ala-OH.
Amino acid analysis on the reaction mixture showed that the Bz-Ala-OH was mainly formed by cleavage of Leu-NH2 from Bz-Ala-Leu-NH2.

Com~arison of ~e~tide ester substrates with and without N-terminal ~rotective ~roups Analogously with examples ~ and 4 the peptides stated in Table XII were produced under the following conditions:
50 mM substrate, 5 ~uM CPD-Y 9 pH 9.5 and 25C.

Table XII

Comparison of carboxypeptidase-Y catalyzed syn-thesis of peptides using peptide esters with and without N-terminal protective groups as substrate.
. __ __ .. _ __ Substrate Amine component P Product Yield %
(conc.) (concentration~
, __ . _ . _. .. _ .
Ac-Al~-Ala- Leucine amide (0.15 M) Ac-Ala-Ala 90 Ala-OI-ie Ala-Leu-NH
(50-~'~) 2 Ala-Ala- Lcucine amide (0.15 M) Ala-Ala-Ala- 75 Ala-Oi;e Leu-NH2 (50 ~1) _ a. Y ~ ~,'d~J
,, . . __ . , .
_ 44 _. ~ __ . ... .

EX~MPLE 16 --~ . _ Svnthesis in the resence of or anic solvents P

Analogously with examples 1, 2, 3, 4, and 6 -the peptides stated in the below Tables XIII, XIV and XV were produced from the listed starting materials and with the listed concentrations of organic solvents.

The conditions were: 50 mM substrate, 5 /uM CPD-Y, pH 9.6 and 30 C.

Table XIII

Carboxypeptidase-Y catalyzed synthesis of peptides with 50 mM Bz-Ala-OMe as substrate and free amino acids as amine component with and without 20% methanol (MeOH) in 0.1 M KCl, 1 mM EDTA. ,.

_ . _ ...
Amine component Product _ Yield %
(concentration) without with MeOH MeOH
,, . _ _. _ _ .. _ Valine (0.6 M) Bz-Ala-Val-OH 42 53 Threonine (1.2 M) Bz-Ala-Thr--OH 52 61 Phenylalanine Bz-Ala-Phe-OH 16 18 (0.16 M) .
Leucine (0.16 M) Bz-Ala-Leu-OH 33 39 Methionine (0.6 M~ Bz-Ala-Met-OH 42 64 Glycine (3.0 M) Bz-Ala-Gly-OH 55 50 Serine (3.2 M) Bz-Ala-Ser-OH 50 52 Lysine (1.5 M) Bz-Ala-Lys-OH 53 41 Clutamic acid ~-t- Bz-Ala-Glu 54 55 butylester (1,0 M) (~-OBut)-OH
Asparagin (0.6 M) Bz-Ala-Asn-OH 18 .
r _ 4 5 __ = _~ ~
. Ta_le XIV
,_ . _ ...
Carboxypeptj.dasc-Y catalyzed synthesis of Bz-Ala-Thr-OH
from Bz-Ala-OMe (50 mM) and th~eoni.ne with varying conccntrati.ons of polyethylene ~lycol-~OO (PEG-300).
- Conditions: CPD-Y 4 /uM, pH 9.6 and 2~C.

.~
So PEG-300 in Threonine % Yield of 0.1 M KOl concentration Bz-Ala-Thr-OH

0 0~7 M 36 0.7 M 34 0.7 M 31 ~~ 30 0.7 M 29 0.35 M 28 .. . ..

Table XV

Carboxypeptidase-Y catalyzed synthesis of pcptides Irorn 50 mM Bz-Ala-OMe and the listed amino acid csters as arnine component in 30~0 polyethylene glycol-O.l M KCl, 1 mM ~DTA.
The other conditions were: CPD-Y = 8 /uM, pH 9.6 and 25C. ..

__. . . .. ... _ , Substrate Aminc component Product Yield ~0 (conc.) (concentration) . _ _ __ .. _ Bz-Ala-OMe Vali.nc-OMe (0.5 M~ Bz-Ala-Val_ 3 V~l-O~
Serinc-O~c (0,5 M) Bz-A].a~Scr OH 3o Clutarnic ~cid (y-OBut) B~-Al.a-Glu- ~8 -OM~ (0.5 M) (Y-OBut)_o~ ~
Bz-Ala-Phc-OlI
Phcnylal.1ninc-OMe ~ Bz-Ala-P}le- ~2 . ~ Table XV (continued) ,. . ~ . . .

Substrate Amine cornponent Product Yield %
(conc.) (concentration) Bz-Ala-OMe ~Bz-Ala-Met-OH ~
(50 m~) L-Methionine-OMe ~ Bz-Ala-Met- ~ 85 Bz-Ala-Met-Me-t-Met-OH J
D-Methionine-OMe Bz-Ala-D-Met-OH O
(0.5 M) _ Insoluble (immobilized) carboxy~e~tidase-Y

CPD-Y was bonded to Concanavalin A-Sepharose 4B (Pharmacia Fine Chemicals) followed by cross-lirking between CPD-Y and.
Concanavalin A with glutaraldehyde as described by Hsiao and Royer (Archives of Biochemistry and Biophysics, Vol.
198, 379-385 (1979). The CPD-Y concentration was 3 mg per ml packed Sepharose. ..

Analogously with examples 1 and 3 the peptides stated in Table XVI were produced under the following condi-tions:
50 mM substrate, 0.25 ml CPD-Y gel (5 /uM CPD-Y), pH 9.7 and 35C. After removal of the enzyme by fil-tration the products and yields were determined by HPLC.

. : .

7~7g,~9 .
_ ~ _ . 47 _ _ . Table XVI

Insoluble car~oxypeptidasc-Y c~talyzed synthesis of peptides.

__ . __ _ _ __ _ Substrate Amine component Product Yield ~' (conc.) (concentration) _ ___ _ .__ _ ____ Bz-Ala-OMe Phcnylalanine (0.15 M) Bz-Ala-Phe ~1 (50 !nM) --cin~ n z 11) gZ-Al L-Lcu-NH2 91 _ _

Claims (16)

The embodiments of the invention in which an exclusive property or privilege is clairned are defined as follows:

1. A process for producing a peptide of the general formula A-B
wherein A represents an N-terminal protected L-amino acid residue or an optionally N-terminal protected peptide residue having a C-terminal L-amino acid and B represents an optionally C-terminal protected L-amino acid residue, by reacting a substrate component with an amine component in the presence of an enzyme, characterized by reacting a substrate component selected from the group consisting of optionally N-terminal protected peptides of the formula A-X
wherein A is as defined above and X represents an L-amino acid residue with an amine component selected from the group consisting (a) L-amino acids of the formula H-B-OH
wherein B is an L-amino acid residue, and (b) optionally N-substituted amino acid amides of the formula H-B-NR3R3' wherein B is an L-amino acid residue and R3 and R3' each represents hydrogen, hydroxy, amino, or alkyl, aryl, heteroaryl or aralkyl, and (c) amino acid esters of the formula H-B-OR4, H-B-SR4 or H-B-SeR4 wherein B is an L-amino acid residile and R4 represents alkyl, aryl, heteroaryl and aralkyl, in the presence of an L-specific serine or thiol carboxypeptidase enzyme from yeast or of animal, vegetable or microbial origin in an aqueous solution or dispersion having a pH from 5 to 10.5, to form a peptide, and optionally cleaving any H, -NR3R3', -OR4, -SR4 or -SeR4, or N-terminal protective group.

2. The process according to claim 1, characterized by using carboxypeptidase Y from yeast as the carboxy-peptidase enzyme.

3. The process according to claim 2, characterized by using a carboxypeptidase Y which has been purified by affinity chromatography on an affinity resin comprising a polymeric resin matrix with a plurality of coupled benzylsuccinyl groups.

4. The process according to claim 1, characterized by using a carboxypeptidase enzyme selected from the group consisting of penicillocarboxypeptidase S-1 and S-2 from Pennicillium janthinellum, carboxypeptidases from Aspergillus saitoi or Aspergillus oryzae, carboxypeptidases C from orange leaves or orange peels, carboxypeptidase CN
from Citrus natsudaidai Hayata, phaseolain from bean leaves, carboxypeptidases from germinating barley, germinating cotton plants, tomatoes, watermelons and Bromelein (pineapple) powder.

5. The process according to claim 1, characterized by using an immobilized carboxypeptidase enzyme.

6. The process according to claim 1, characterized by maintaining the pH in the solution or dispersion at 8 to 10.5

7. The process according to claim 6, characterized by carrying out the reaction in a buffer solution with pH
8.0 to 10.5 or at a desired pH in the range of 8.0 to 10.5, which is kept constant by adding acid or base to the aqueous solution according to the measured pH-value in the reaction mixture.

8. The process according to claim 1, characterized by using an aqueous reaction solution containing from 0 to 50% by volume of organic solvent.

9. The process according to claim 8, characterized by using an organic solvent selected from the group consisting of alkanols, dimethyl sulfoxide, dimethyl formamide, dioxane, tetrahydrofuran, dimethoxy ethane, ethylene glycol and polyethylene glycols.

10. The process according to claim 8, characterized by carrying out the reaction with a starting concentration of the substrate component of 0.01 to 1 molar and 0.05 to 3 molar for the amine component.

11. The process according to claim 10, characterized by carrying out the reaction with a carboxypeptidase enzyme concentration of 10-6 to 10-4 molar.

12. The process according to claims 1, 2 or 3, characterized by using as the substrate component an L-amino acid ester or peptide ester selected from benzyl esters and straight chain or branched C1-4 alkyl esters, optionally substituted with inert substituents.

13. The process according to claims 1, 2 or 3, characterized by using a p-nitroanilide as the substrate component.

14. The process according to claims 1, 2 or 3, characterized by using as the amine component an amide of the formula where R3 is hydrogen or C1-3 alkyl and B is an L-amino acid residue.

15. The process according to claims 1, 2 or 3 characterized by using as the amine component an ester of the formula wherein R4 is C1-3 alkyl and B is an L-amino acid residue.

16. A process according to claims 1, 2 or 3, characterized by performing the cleaving of any group -NR3R3', -OR4, -SR4 or -SeR4 by means of the same L-specific serine or thiol carboxypeptidase enzyme as was used in the formation of the peptide.