A process for enzymatic production of dipeptides

Abstract: Dipeptides having the general formula H-A-B-Y wherein A represents an optionally side-chain protected L- or D-α-amino acid or ω-amino acid and B represents an optionally side-chain protected L- or D-α-amino­ carboxylic acid which may be the same as or different from A, an L- or D-aminophosphonic acid or L- or D-­ aminosulfonic acid or the corresponding ω-amino acids or salts and hydrates thereof, and Y is OH or a C-terminal blocking group, are prepared by reacting a substrate component, which is an amino acid derivative having the formula wherein A is as defined above, R¹ represents hydrogen, alkyl, aryl or aralkyl optionally substituted with inert substituents or an α-des-amino fragment of an amino acid, and R² and R³ are the same or different and each represents hydrogen, alkyl, aryl or aralkyl optionally substituted with inert substituents, with a nucleophile component which when A = B may be formed in situ and is selected from (a) L-amino acids having the formula H-B-OH (b) L-amino acid amides having the formula wherein B is an L-amino acid, and R² and R³ have the above meaning, except that when R² represents hydrogen, R³ may also represent hydroxy or amino (c) L-amino acid esters having the formula H-B-OR⁴ wherein B is an L-amino acid, and R⁴ represents alkyl, aryl or aralkyl, and (d) optionally acid group protected straight chain or branched amino phosphonic acids or amino sulfonic acids having the formula NH₂C x H z PO₃H₂ or NH₂C x H z SO₃H wherein x is 1-6 and z is 2-12 in the presence of a serine or thiol carboxypeptidase from yeast or of animal, vegetable or other microbial origin, preferably CPD-Y from yeast in an aqueous solution or suspension having a pH value between 5 and 10.5 optionally containing an organic solvent and/or a salt, and then, if desired, cleaving a present side-­ chain protecting group or protective group Y and/or, if desired, converting the resulting dipeptide derivative to a salt or hydrate. The process allows for production of L,L-, LD-, DL- and DD-dipeptides without risk of racemization in a simple and economic manner.

Description: The present invention concerns a process for enzymatic production of dipeptides and derivatives of dipeptides and of the type stated in the introductory portion of claim 1. In recent years there has been an increasing interest in dipeptides and dipeptide derivatives optionally containing an amino-acid residue of D-configuration, with a view to their potential pharmacological effects, such as e.g. antibiotics. Likewise, there has been a great interest in dipeptides within fields such as artificial nutrition - human as well as veterinary, sweeteners and within agrochemistry, such as e.g. herbicides. Such dipeptides H-A-B-Y can be produced by means of known chemical coupling reactions, but all these methods share the feature that, generally, it is necessary to protect the amino acids involved - A and B - on the amino group and the carboxylic acid group, respectively, and frequently also on the side chains if these carry functional groups. Further, there is an inherent risk of side reactions during the chemical coupling step because of the reagents and conditions employed, a major side reaction being racemization, particularly of the A-component. By replacing the chemical coupling step with an enzymatic coupling step proceeding under mild conditions, such side reactions and racemization can be avoided, yielding a stereochemically pure product. The presence of amino- and carboxyl protective groups is mandatory in chemical coupling as well as in enzymatic coupling using endoproteases, and according to prior knowledge also on the amino function of the substrate in enzymatic exoprotease catalyzed formation of dipeptides. This adds several undesired features to these processes seriously afflicting their process economy on an industrial scale, particularly apparent in dipeptide synthesis. The disadvantages are concerned with the introduction of these groups, as well as their removal and presence during process operation, increasing overall process cost and time and affecting overall yield. Typical examples of amino protective groups commonly used are those of the carbobenzoxy (Z-) and tert-butoxycarbonyl (Boc-) type, which are of a molecular weight comparable to those of the amino acid residues. Firstly, the protective groups will have to be introduced in the starting materials by means of appropriate costly agents in a separate reaction step followed by an isolation step. While present, these hydrophobic groups often have a drastic effect upon the solubility of the intermediates and reaction products, and may afflict both the nature and the amount of solvents required in their processing as well as ease of purification and of deprotection. The deprotection will also take place in a separate step with a following purification step. For this purpose a series of reactions are available, but with the exception of catalytic hydrogenation, posing industrial problems of its own, these methods are occuring under violent often strongly acidic or basic conditions, frequently giving rise to a series of side reactions, resulting in an impure product or demanding laborious purification. The last steps in this relatively long series of synthesis steps may thus be a rather comprehensive deprotection to obtain the desired dipeptides, and, owing to the almost inevitable secondary reactions, rather laborious purification procedures are frequently required to provide a product with the desired high purity. Attempts to avoid amino terminal protection in the production of dipeptides have led to microbial fermentation approaches, like the fermentation process under formation of aspartame described in EP-A1-074095 and EP-A2-102529. This technique is fundamentally different from synthetic approaches and relies on specific organisms for each peptide, and is thus not generally applicable. In addition, yields are often low and recovery from the fermentation broth laborious. Thus, it is an obvious advantage in terms of overall process economy to be able to avoid protective groups, also on the amino and carboxy termini. It is the object of the present invention to make this possible in the synthesis of dipeptides mediated by serine- and thiol carboxypeptidase catalysis. In some cases, it may be of interest to be able to produce a dipeptide which may carry side-chain protection, but no terminal protection, and it will be shown that it is possible in the process according to this invention, starting from side-chain protected, but amino respectively carboxy unprotected starting materials. In this case, the same advantages of mild reaction conditions and overall process economy may be obtained. If desired, the side-chain protective group may be cleaved by chemical or enzymatic means. The enzyme catalyzed coupling reactions enabling the use of side-chain unprotected amino acid derivatives and an optionally C-terminal unprotected B-component (nucleophile) are known. See e.g. EP-A-0017485 or the granted equivalent (EP-B1-17485). EP-B1-17485 describes a process for producing peptides of the general formula A₁-B₁-Z₁ wherein A₁ represents an N-terminal protected L-amino acid residue or an optionally N-terminal protected peptide residue having a C-terminal L-amino acid residue, and B₁ represents an L-amino acid residue, and Z₁ is OH or a C-terminal protective group, by reaction of a substrate component with an amine component in the presence of an enzyme, and, if desired, cleavage of optional terminal protective groups to provide a peptide of the formula A₁-B₁-Z₁ which is characterized by reacting a substrate component selected from amino acid esters, peptide esters, depsipeptides, or optionally N-substituted amino acid amides or peptide amides, or optionally N-terminal protected peptides of the formulae A₁-OR₁, A₁-NR₂R₂' or A₁-X₁-OH wherein A₁ is as defined above, R₁ is alkyl, aryl, heteroaryl,aralkyl or an α-desamino fragment of an amino acid residue, R₂ and R₂' are each hydrogen, alkyl, aryl, heteroaryl or aralkyl, and X₁ is an L-amino acid residue, with an amine component (nucleophile) selected from optionally N-substituted L-amino acid amides, L-amino acids or L-amino acid esters, of the formula H-B₁-NR₃R₃' or H-B₁-OR₄ wherein B₁ is as defined above, R₃ and R₃' are each hydrogen, hydroxy, amino, alkyl, aryl, heteroaryl or aralkyl, and R₄ is hydrogen, alkyl, aryl, heteroaryl or aralkyl, in the presence of an L-specific serine or thiol carboxypeptidase enzyme originating from yeast or of animal, vegetable or other microbial origin, in an aqueous solution or dispersion at pH-5-10.5, preferably at a temperature of 20-50°C. The preferred enzyme is carboxypeptidase Y from yeast, called CPD-Y in the following. (It is noted that in order to avoid confusion with the present symbol meaning the above-mentioned symbol meanings are not identical with those used in EP-B-17485). Thus, if a dipeptide is to be produced by the process of EP-B1-17485, the substrate component is necessarily an N-terminal protected amino acid derivative, and the constituent amino acid is necessarily an L-amino acid. More generally, it is said that the need for N-terminal amino group protection of the substrate component decreases with increasing chain length of the peptide residue and is substantially absent when the peptide residue consists of 3 amino acids, depending, however, upon their type and sequence. This is illustrated by Breddam et al, "Influence of the substrate structure on carboxypeptidase Y catalyzed peptide bond formation", Carlsberg Res. Commun., vol. 45, p. 361-67, 30th December 1980, in which Ac-Ala-Ala-Ala-OMe and H-Ala-Ala-Ala-OMe were coupled with H-Leu-NH₂ in the presence of CPD-Y to form Ac-Ala-Ala-Ala-LeuNH₂ and H-Ala-Ala-Ala-LeuNH₂ in yields of 90 and 80%, respectively. Breddam et al also examined the importance of the amino acid configuration for the coupling yield in CPD-Y catalyzed peptide synthesis, cf. the following table, where Ala represents L-alanine and ala represents D-alanine. Substrate Product Yield (%) H-Ala-Ala-Ala-OMe H-Ala-Ala-Ala-Leu-NH₂ 80 H-Ala-ala-Ala-OMe H-Ala-ala-Ala-Leu-NH₂ 40 H-Ala-Ala-ala-OMe H-Ala-Ala-ala-Leu-NH₂ 0 Conditions: 25 mM substrate, 0.1 M KCl, 1 mM EDTA, pH 9.5, CPD-Y = 12 µm, 0.2 M Leu-NH-₂. The reaction was stopped after 20 min. It appears from the table that if the C-terminal is in D-form, like in H-Ala-Ala-ala-OMe, no peptide synthesis takes place because the ester is not reacted. With the D-amino acid juxtaposed to the C-terminal like in H-Ala-ala-Ala-OMe, reaction takes place, but the coupling yield is reduced to 40% compared with the 80% in the pure L-configuration H-Ala-Ala-Ala-OMe. Further, it has long been known that some endoproteases can catalyze oligomerization of certain N-unprotected amino acid esters with L-configuration, but it has never been attempted to use this for production of dipeptides which are not simple dimers. Generally, the results of such observations have been a mixture of a series of oligomers, sometimes long, and only in case of product precipitation has it been possible to isolate a single product. For this reason the use of endoproteases for peptide synthesis has been limited to the use of amino and carboxy terminal protected starting materials, as exemplified by US-A-4.086.136. The abovementioned amino and carboxy terminal protected starting materials are also mandatory if aspartate endoproteases are used as exemplified by US-A-3.972.773, and if metallo endoproteases are used, as exemplified by the synthesis of Z-AspPheOMe.PheOMe-salt in EP-A1-009585. Finally, the synthesis of the diastereomeric dipeptides of DL, LD and DD-configuration as well as peptides containing beta-amino acid residues from amino-unprotected starting compounds has so far not been possible with carboxypeptidases nor in general with any proteolytical enzymes (Class EC 3.4). Some efforts have been made with a different class of enzymes, aminoacyl-t-RNA-synthetase (Class EC 6.1) as exemplified by EP-A1-086053. In this case a specific enzyme must be used for each type of amino acid residue, and furthermore, expensive Co-factors like ATP are required. At the same time, yields are very poor, so even though some product was isolated and identified, typically a ten fold excess of Co-factor and a hundred fold excess of nucleophile and up to a thousand fold excess of enzyme by weight was required. It has now surprisingly been found that the serine and thiol carboxypeptidases used in EP-B1-17485 are capable of utilizing N-unprotected amino acid esters as a substrate component in controlled reactions for synthesis of dipeptides and dipeptide derivatives, and that it is possible to suppress a possible oligomerization of the substrate. It has moreover surprisingly been found that also N-unprotected amino acid derivatives of D-configuration can be used as substrates in these reactions, so that, in addition to LL-dipeptides, it is also possible to synthesize DL-dipeptides. The reaction rate for D-substrates, however, is generally somewhat lower than for L-substrates under uniform conditions, but, as illustrated below, the difference in rate is much smaller than for the corresponding N-protected amino acid esters, the D-substrate being reacted at a rate which is much smaller than the rate for the L-substrate. The yields are often just as high or higher with the unprotected D-substrates in relation to the unprotected L-substrates as shown by the following examples. Furthermore, it has even more surprisingly been found that with substrates of D-configuration, nucleophiles of D-configuration can be coupled by means of these enzymes, otherwise known to be L-specific on the amino side of the synthesis point. An amino acid ester incorporated in this manner is not hydrolyzed further. The process of the invention is thus characterized by the features defined in the characterizing portion of claim 1. Examples of useful amino acids include aliphatic amino acids, such as monoaminomonocarboxylic acids, e.g. glycine (Gly), alanine (Ala), valine (Val), norvaline (Nval), leucine (Leu), isoleucine (iso-Leu) and norleucine (Nleu), hydroxy amino acids, auch as serine (Ser), threonine (Thr) and homoserine (homo-Ser), sulfur-containing amino acids, such as methionine (Met) or cystine (CysS) and cysteine (CysH), monoaminodicarboxylic acids, such as aspartic acid (Asp), glutamic acid (Glu) and amides thereof, such as asparagine (Asn) and glutamine (Gln), diaminomonocarboxylic acids, such as ornithine (Orn) and lysine (Lys), arginine (Arg), aromatic amino-acids, such as phenylalanine (Phe) and tyrosine (Tyr), as well as heterocyclic amino-acids, such as histidine (His), proline (Pro) and tryptophan (Trp). Examples of useful amino-acids of a more unusual structure are penicillamine (Pen), aminophosphonic acids, such as alanine-phosphonic acid (Alap), aminosulfonic acids, such as taurine (Tau), and ω-amino-acids, such as β-alanine (BAla). As mentioned, they may be included in D-form in the substrate component and they may also be present in D-form in the nucleophile component. The advantages of the process of the invention over the given known methods are that minimum or no side-chain protection is required, that no N-protection of the substrate component (which may have both D- and L-configuration) is required, that there is little or no risk of racemization, that there are few synthesis steps, and that a relatively pure end product is obtained. The process therefore provides an extremely simple and economic method of production. Preferred substrate components are esters in which R¹ is a straight or branched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, amyl and hexyl, or the aralkyl group benzyl. Particularly preferred nucleophile components are free L-amino-acids or amino-acid amides, in which R² is H and R³ is H or C₁-C₆ alkyl, or amino-acid esters in which R⁴ is a straight or branched alkyl group having 1 to 6 carbon atoms such as any of those given above. More generally, as defined above, R¹ may be alkyl, aryl or aralkyl optionally substituted by inert substituents, e.g. hydroxy or nitro. R¹ will usually have no more than 12 carbon atoms, and substituents usually no more than 6. The invention also comprises the processes involving intermediate formation of a peptide containing the group -NR²R³ or -OR⁴, following which this group is cleaved to form a carboxylic acid group. This cleavage may be catalyzed by another or the same enzyme as was used to form the peptide. Enzymes may also be used to cleave side-chain protective groups. Suitable enzymes are proteolytic enzymes, lipases and esterases, according to the nature of the protective group, see "The Peptides, Analysis, Synthesis, Biology" Vol 9, Special Methods in Peptide Synthesis Part C, by J. A. Glaas, Enzymatic manipulation of Protecting Groups in Peptide Synthesis, Academic Press 1987. The process of the invention may be performed with CPD-Y, which is the presently preferred enzyme, and which is characterised more fully in EP-B1-17485, and also with other serine or thiol carboxypeptidases, such as those mentioned in the survey below, since these have a common mechanism of activity via acyl enzyme intermediates. The close relation between many of the above-mentioned carboxypeptidases has been discussed by Kubota et al , Carboxypeptidase C, J. Biochem., Vol. 74, No. (1973), p. 757-770. Carboxypeptidases from malt, wheat and other sources have been described by Breddam, Carlsberg Res. Comm. Vol. 51, p. 83-128, 1986. The carboxypeptidase used may also be chemically modified or be a biosynthetic mutant of the natural form. As illustrated more fully below, the process of the invention is rather simple; however, it is important to maintain a resonably or substantially constant pH value in the reaction mixture. This pH value is between 5 and 10.5, and preferably between 7 and 9.5, but also depends upon the particular starting materials, the peptide formed and the enzyme. The reaction is performed in an aqueous reaction medium, if desired containing up to 70% of an organic solvent which is miscible or immiscible with water, and compatible with the enzyme under the conditions specified. Preferred solvents are lower alcohols, dimethylformamide, dimethyl sulfoxide, dimethoxyethane, ethylene glycol and ethyl acetate. The reaction temperature is preferably room temperature or above, e.g. 20 to 50°C, but temperatures in the range 0 to 60°C may be used. The most advantageous temperature may be chosen according to the conditions otherwise given. The concentration of the two reaction components may vary within wide limits, but the nucleophile component is frequently in excess. In order to avoid oligomerization of the substrate component, the nucleophile component is often added in small portions at intervals during the entire reaction sequence. If an ethyl or higher ester is used, surprisingly no oligomerization is observed, and very high substrate concentrations may be used without side-reactions. In this case, a homo-dipeptide in which A = B may be formed without oligomerization from a single starting component, the nucleophile being generated in situ by hydrolysis of this ester. In some cases, this is a further technical advantage, cf. Fig. 2. Thus, the starting concentration of the substrate component may typically be 0.005 to 2 molar and for the nucleophile component, in cases where it is added separately, 0.005 to 3 molar. In most situations, it is possible to recover excess nucleophile component and the hydrolysis product from the substrate component for optional re-esterification and re-use. Recycling of the components is particularly easy because of their simple structure and the absence of side-reactions and deprotective losses. The enzyme concentration may likewise vary, but is frequently somewhat higher (5-50 µm) than the concentrations appropriate for the use of N-protected amino-acid ester substrates. However, as illustrated by the following Examples, the amount required for synthetic purposes may be reduced more than ten-fold by using a stable immobilized enzyme preparation, thereby enabling the enzyme to be used in a continuous process. The reaction medium may also contain salts, such as NaCl, which influence the binding of the enzyme to the substrate, as well as a complexing agent, such as EDTA, for metal ions present, which stabilises the enzyme. The reaction rate differences between D- and L-substrates mentioned above are illustrated in Fig. 1, which shows the reaction sequence for the CPD-Y hydrolysis of a protected and unprotected amino-acid ester (Tyr-OEt) in D- and L-form. It will be seen from the Figure that, while L-AcTyrOEt is hydrolyzed almost instantaneously, only insignificant hydrolysis (below 5%) of D-AcTyrOEt has taken place after 2 hours. In contrast, thre are only small differences in the hydrolysis sequence for the unprotected esters in L- and D-form. This surprising hydrolysis sequence is reflected in the following Examples, which illustrate the production of various dipeptides in the process of the invention using the enzymes CPD-Y and malt carboxypeptidase II, (CPD-MII) and wheat carboxypeptidase (CPA-W). General method for Examples 1-15 The reactions, performed on an analytical scale with a reaction volume of 1 ml, were carried out in a pH-stat, and the selected pH value was kept constant by automatic addition of 1 N NaOH. Reaction temperature was room temperature, unless otherwise stated. The table also includes reaction concentrations, content of organic solvent, product and yield. Reaction times are typically between 0.5 and 5 hours, and enzyme concentrations are typically 10-20 µm, unless otherwise stated. Product identification and determination of product yield were performed by means of reverse phase HPLC (Waters 6000 A pumps, 660 gradient blender, UK 6 injector) on a C₁₈NOVA PAK column (Waters, RCM) using suitable gradients of elution systems containing 50 mM triethylammonium phosphate, pH 3.0 from 0% to 80% acetonitrile with a flow of 2 ml/min. Elution was monitored by means of a UV detector (Waters 480) at 230 nm, 254 nm, 278 nm or 290 nm. The products were identified by amino-acid analysis of fractions from the HPLC analysis, which corresponded to the assumed product peak and/or by HPLC comparison with a chemically-synthesized reference product. These were produced according to known principles, usually via reaction between BOC-A-OSu - tertiary-butyloxycarbonyl - the succine imide ester derivative of the substrate amino-acid - and the used nucleophile component followed by deblocking of the N-terminal amino acid residue. In all cases, it was possible to separate LL-and DD-dipeptides from the diasteromeric DL-and LD-dipeptide products. For the products which can only be detected at 230 nm, the product yields were determined by means of the absorption/concentration curve of the chemically synthesized reference compound. For the other products, the yields were determined on the basis of the ratio between the integrated areas below the peaks in the elution chromatogram, corresponding to product respectively the reactant which absorbs at the wavelength concerned. The reaction conditions in the preparative Examples 16-21 are described in the individual Examples. The reactions were followed on analytical HPLC as described. The enzyme concentrations are generally lower and the reaction times longer than in the corresponding analytical Examples, but no attempt to optimize the reaction conditions has been made. Example 1 Carboxypeptidase Y catalyzed synthesis of L-L-dipeptides with L-Tyrosine ethyl ester (50 mM) as substrate component and free amino-acids as nucleophiles Nucleophile (conc.) Solvent pH Product Yield % Alanine (1.9 M) Water 9.5 TyrAlaOH 10 Arginine (0.8 M) Water 9.5 TyrArgOH 31 Cysteine ( 1 M ) Water 8.0 TyrCysOH 30 LD-Cysteine ( 2 M ) Water 8.0 TyrCysOH(LL) 40 Leucine (0.2 M) Water 8.0 TyrLeuOH 1 Lysine ( 2 M ) Water 9.5 TyrLysOH 18 Methionine (0.3 M) Water 8.0 TyrMetOH 8 Methionine (0.3 M) Water 9.0 TyrMetOH 9 Methionine (0.3 M) 30% DMSO 9.0 TyrMetOH 15 Methionine (0.3 M) 15% EtOH 9.0 TyrMetOH 7 Glutamine (0.8 M) Water 9.5 TyrGlnOH 8 Pennicilamine (0.5 M) Water 8.0 TyrPenOH 7 Example 2 Carboxypeptidase Y catalyzed synthesis of L-L-dipeptides with L-Methionine (0.3 M) as nucleophile component in water at pH 9.0 Substrate (50 mM) Product Yield % Leucine methyl ester LeuMetOH 30 Leucine isopropyl ester LeuMetOH 36 Methionine ethyl ester MetMetOH 25 Phenylalanine methyl ester PheMetOH 16 Phenylalanine ethyl ester PheMetOH 19 Phenylalanine isopropyl ester PheMetOH 23 Serine isopropyl ester SerMetOH 21 Tryptophane methyl ester TrpMetOH 26 Tyrosine benzyl ester TyrMetOH 19 Example 3 Carboxypeptidase Y catalyzed synthesis of L-L-dipeptides with L-Tyrosine ethyl ester (50 mM) as substrate component and L-amino-acid amides or esters as nucleophiles Substrate Nucleophile (Conc.) Solvent pH Product Yield% TyrOEt Leucine amide (0.2 M) 30% DMSO 9.5 TyrLeuNH₂ 42 TyrLeuOH 4 TyrOEt Lysine amide (0.3 M) Water 9.5 TyrLysNH₂ 20 TyrLysOH 22 TyrOEt Arginine amide (0.2 M) Water 9.0 TyrArgNH₂ 50 TyrOEt Valine amide (0.3M) Water 9.0 TyrValNH₂ 77 TyrOEt Leucine methyl ester (0.2 M) 30% DMSO 9.0 TyrLeuOH 6 Example 4 Carboxypeptidase Y catalyzed synthesis of L-L-homodipeptides from a single starting compound in water at pH 8.5 Substrate (conc.) Product Yield % Methionine methyl ester (0.5 M) MetMetOH 14 Methionine ethyl ester (0.5 M) MetMetOH 16 Methionine isopropyl ester (0.5 M) MetMetOH 14 Tyrosine methyl ester (0.2 M) TyrTyrOH 1 Tyrosine ethyl ester (0.2 M) TyrTyrOH 1 Phenylalanine ethyl ester (0.2 M) PhePheOH 2 Alanine amide (0.2 M) AlaAlaNH₂ Example 5 Carboxypeptidase Y catalyzed synthesis of D,L-dipeptides with D-Tyrosine ethyl ester (50 mM) as substrate and free L-amino-acids as nucleophiles in water Nucleophile (conc.) pH Product Yield % Arginine ( 1 M ) 9.0 tyrArgOH 75 Cysteine ( 1 M ) 8.0 tyrCysOH 86 LD-cysteine ( 2 M ) 8.0 tyrCysOH(DL) 45 Leucine (0.2 M) 8.0 tyrLeuOH 22 Methionine ( 1 M ) 9.0 tyrMetOH 65 Pennicilamine ( 1 M ) 8.0 tyrPenOH 27 Example 6 Carboxypeptidase Y catalyzed synthesis of D,L-dipeptides with L-Methionine (0.3 M) as nucleophile component in water at pH 9.0 D-substrate (50 mM) Product Yield % leucine methyl ester leuMetOH 50 leucine isopropyl ester leuMetOH 71 methionine ethyl ester metMetOH 68 phenylalanine ethyl ester pheMetOH 71 phenylalanine isopropyl ester pheMetOH 72 serine isopropyl ester serMetOH 46 tryptophane ethyl ester trpMetOH 72 Example 7 Carboxypeptidase Y catalyzed synthesis of D,L-dipeptide amides with D-Tyrosine or D-Phenylalanine ethylester (50 mM) as substrate component and L-amino-acid amides as nucleophile components in water Substrate Nucleophile (conc.) pH Product Yield % tyrOEt Leucine amide (0.2 M) 9.0 tyrLeuNH₂ 82 tyrLeuOH 2 tyrOEt Valine amide (0.3 M) 9.0 tyrValNH₂ 94 tyrOEt Arginine amide (0.2 M) 9.0 tyrArgNH₂ 86 pheOEt Alanine amide (0.8 M) 9.0 pheAlaNH₂ 50 Example 8 Carboxypeptidase Y catalyzed synthesis of D,D-homodipeptide esters from D-substrate ester components in water at pH 9.0, also acting as nucleophile component D-substrate (conc.) Product Yield % tyrosin ethyl ester (0.05 M) tyrtyrOEt 9 phenylalanine ethyl ester (0.1 M) phepheOEt 10 tyrosine ethylene glycol ester (0.05 M) tyrtyrOEtOH 1 methionine methyl ester (0.1 M) metmetOMe 3 Example 9 Carboxypeptidase Y catalyzed synthesis of L-L-dipeptides with side-chain-protected carboxy-terminal amino-acids with L-TyrOEt (50 mM) as substrate component and side-chain-protected L-amino-acids and amides as Nucleophiles Nucleophile (Conc.) pH Solvent Product Yield% Acetamidomethyl cysteine (1 M) 8.5 Water TyrCys(-SAcm)OH 10 Acetamidomethyl cysteine amide (0.4 M) 8.5 Water TyrCys(-SAcm)NH₂ 12 TyrCys(-SAcm)OH 1 Beta-Benzylaspartic Acid (0.1 M) 9.0 30%DMSO TyrAsp(OBzl)OH 13 Epsilon Trifluoroacetyllysine (0.1 M) 8.5 30%DMSO TyrLys(Tfa)OH 12 Gamma-Tert-butylglutamic Acid Amide (0.1 M) 8.0 30%DMSO TyrGlu(OtBu)NH₂ 3 TyrGlu(OtBu)OH 12 Gamma-Methylglutamic Acid (0.3 M) 8.5 Water TyrGlu(OMe)OH 20 Gamma-Ethylglutamic Acid (0.3 M) 8.5 Water TyrGlu(OEt)OH 18 Example 10 Carboxypeptidase Y catalyzed synthesis of D-L-dipeptides with side-chain-protected carboxy-terminal amino-acids with D-TyrOEt (50 mM) as substrate component and side-chain-protected L-amino-acids and amides as Nucleophiles Nucleophile (Conc.) pH Solvent Product Yield% Acetamidomethylcysteine (1 M) 8.5 Water tyrCys(-SAcm)OH 75 Acetamidomethylcysteine amide (0.4 M) 8.5 Water tyrCys(-SAcm)NH₂ 71 tyrCys(-SAcm)OH 6 Beta-Benzylaspartic Acid (0.1 M) 9.0 30%DMSO tyrAsp(OBzl)OH 54 Epsilon-Trifluoroacetyllysine (0.1 M) 8.5 30%DMSO tyrLys(Tfa)OH 51 Gamma-Tert-butylglutamic Acid Amide (0.1 M) 8.0 30%DMSO tyrGlu(OtBu)NH₂ 42 tyrGlu(OtBu)OH 10 Gamma-Methylglutamic Acid (0.3 M) 8.5 Water tyrGlu(OMe)OH 75 Gamma-Ethylglutamic Acid (0.3 M) 8.5 Water tyrGlu(OEt)OH 71 Example 11 Carboxypeptidase Y catalyzed synthesis of L,L-dipeptides with side-chain-protected amino-terminal amino-acid residues from side-chain-protected substrate components (25 mM) and L-Methionine (0.3 M) as nucleophile component in 30% DMSO at pH 9.0 Substrate Product Yield % L-Aspartic Acid Dibenzyl ester Asp(OBzl)MetOH 65 L-Glutamic Acid Dibenzyl ester Glu(OBzl)MetOH 70 Example 12 Carboxypeptidase Y catalyzed synthesis of dipeptide esters and amides containing omega-amino-acids from L- and D-amino-acid ester substrates at 50 mM in water with beta-alanine and beta-alanine amide as nucleophiles Substrate Nucleophile (conc.) pH Product Yield % tyrOEt B-Alanine methyl ester (0.2 M) 8.5 tyrBAlaOMe 51 tyrOEt B-Alanine methyl ester (0.5 M) 8.5 tyrBAlaOMe 72 pheOEt B-Alanine methyl ester (0.5 M) 9.0 pheBAlaOMe 83 PheOEt B-Alanine methyl ester (0.5 M) 9.0 PheBAlaOMe 15 pheOEt B-Alanine amide (0.5 M) 9.0 pheBAlaNH₂ 70 PheOEt B-Alanine amide (0.5 M) 9.0 PheBAlaNH₂ 3 Example 13 Carboxypeptidase Y catalyzed synthesis of L,L and D,L dipeptides with hydroxyalkyl esters of L- and D-Tyrosine and L-Phenylalanine (50 mM) as substrate components and free L-Methionine (0.3 M) as nucleophile in water/ethylene glycol mixtures at pH 9.0 Substrate % glycol (vol/vol) Product Yield % TyrOEtOH 0 TyrMetOH 10 TyrOEtOH 40 TyrMetOH 8 TyrOEtOH 60 TyrMetOH 5 tyrOEtOH 0 tyrMetOH 56 PheOEtOH 0 PheMetOH 16 Example 14 Synthesis of L,L-dipeptides catalyzed by carboxypeptidases from barley and wheat at 50 mM initial L-substrate ester concentration at pH 8.0 in water and L-amino-acid and amides as nucleophile components Enzyme Substrate Nucleophile (conc.) Product Yield% CPD-MII PheOEt Methionine (0.4 M) PheMetOH 10 CPD-W PheOEt Arginine (0.8 M) PheArgOH 8 Example 15 Synthesis of D,L-dipeptides catalyzed by carboxypeptidases from barley and wheat at 50 mM initial D-Phenylalanine ethyl ester concentration at pH 8.0 in water and free L-amino-acids as nucleophile components Enzyme Substrate Nucleophile (conc.) Product Yield% CPD-MII pheOEt Methionine (0.4 M) pheMetOH 15 CPD-W pheOEt Arginine (0.8 M) pheArgOH 13 CPD-W pheOEt Methionine (0.4 M) pheMetOH 40 Example 16 Preparative synthesis of L,L-tyrosylcysteine, TyrCysOH Procedure L-tyrosine ethyl ester hydrochloride (1.5 g, 6 mmol) and L-cysteine (15.2 g, 125 mmol) were dissolved in 110 ml of-0,1 M KCl solution containing 1 mM EDTA. pH was adjusted to 8.0 with triethylamine. The reaction was initiated by addition of 7.5 ml of carboxypeptidase Y-solution (16 mg/ml), and was kept at pH 8.0 for the duration of the reaction by the addition of triethylamine under continuous stirring at room temperature. The remainder of the substrate (13.5 g, 54 mmol) was added in portions of 1,5 g during one hour. After 0,5 hour tyrosine precipitation started, and after 3.5 hours the reaction was stopped by heating to 45°C for 20 minutes. The formed tyrosine was filtered off, and the filtrate was purified by R-preparative HPLC (Waters Prep. L C/system 500A) using two columns (5.7 x 30 cm) packed with 60 um C₁₈-particles and 50 mM acetic acid as eluent. Collected fractions containing pure product were evaporated to dryness in vacuo under repeated additions of absolute ethanol. The remnant was stirred with diethyl ether. Following this, 3.88 g of L,L-tyrosine cysteine (14 mmol, 22%) was isolated by filtration and drying. Identification Choride and acetate were not detected, the product being present as a zwitter-ion. Amino-acid analysis following acid hydrolysis and derivation of Cys by acrylonitrile gave the result: Tyr (1.00) Cys (1.08) Purity Following derivation of the cysteine sidechain with acrylonitrile, only one spot was detected on TLC on Kiselgel 60-F using ninhydrine detection (Rf 0.73, eluent : ethyl acetate, butanol, acetic acid and water (1:1:1:1)). HPLC-purity: 99.5% (nucleosile 7 C₁₈, 0.1 M ammonium phosphate, pH 3.0/acetonitrile, 220 nm). UV-quantization: 98.5% (293 nm, tyrosine absorbance in 0,1 M NaOH). Example 17 Preparative synthesis of L,L-methionyl-methionine, MetMet-OH Procedure L-methionine ethyl ester hydrochloride (24.6 g, 115 mmol) and L-methionine (20.6 g, 138 mmol) were dissolved in 190 ml of 0.1 M KCl solution containing 1 mM EDTA. pH was adjusted to 9.0 with sodium hydroxide solution, and the reaction was initiated by addition of 14.2 ml of carboxypeptidase Y solution (20 mg/ml). The reaction was stirred overnight at room temperature, and pH was kept at 9.0 through the addition of sodium hydroxide solution by a pH-stat. pH was adjusted to 3.0 with HCl-solution at the end of reaction. Precipitated methionine was filtered off, and the filtrate was purified by R-preparative HPLC as described in example 16. Collected fractions containing pure product were concentrated by evaporation and finally freeze-dried. This procedure gave 10.6 g (37.8 mmol, 33%) of L-methionyl-L-methionine as a white amorphous powder. Indentification No chloride was detected, and only 1.9% (w/w) of acetate was determined, the product being predominantly in zwitter-ionic form. Amino-acid analysis showed methionine following acid hydrolysis and the absence of free methionine in unhydrolysed samples. Purity Purity by HPLC: 99.8% (nucleosile 7 C₁₈, 0.1 M ammonium phosphate, pH 3.0/acetonitrile, 220 nm). Quantization by reaction with trinitrobenzenesulfonic acid (TNBS) and UV-detection: 92.5%. Water content by Karl Fisher: 1.5%. Example 18 Preparative synthesis of L,L-tyrosylvalineamide, TyrValNH₂ Procedure L-tyrosine ethyl ester hydrochloride (16.0 g, 65 mmol) and L-valine amide hydrochloride (60 g, 390 mmol) were dissolved in 1150 ml of water and 65 ml of 20 mM EDTA was added. pH was adjusted to 9.0 with sodium hydroxide solution and the reaction was initiated by addition of 12 ml of a carboxypeptidase Y solution (20 mg/ml). The reaction was stirred at room temperature for four hours, and pH was maintained at 9.0 by the addition of sodium hydroxide solution. Following complete conversion the reaction was stopped by raising pH to 11. The denatured enzyme was filtered off, and the reaction mixture was diluted and purified by successive anion exchange on a Dowex A61x4 column and cation exchange on a Dowex A650Wx4 column using soidum and ammonium acetate salt gradients, respectively, and was finally desalted. Product fractions were combined and evaporated to dryness in vacuo under repeated additions of absolute ethanol, giving 11.2 g L,L-tyrosylvaline amide (40 mmol, 62%) as a white powder. Identification 1.8% (w/w) of acetate was determined, the product being predominantly in zwitter-ionic form. Amino-acid analysis following acid hydrolysis gave the following results: Tyr (1.01) Val (0.99) Purity HPLC-purity: 99.5% (Novapak C₁₈, 0.1 M ammonium phosphate containing alkylsulfonate, pH 4.5/acetonitrile, 220 nm). UV-quantization: 97.8% (293 nm, tyrosine absorbance in 0.1 M NaOH). Example 19 Preparative synthesis of D,L-tyrosyl-arginine, D,L-tyrArg-OH Procedure 105 g of L-arginine (105 g, 603 mmol) was dissolved in 400 ml of water, and pH was adjusted to 9.0 with HCl-solution. D-tyrosine ethyl ester hydrochloride (6.1 g, 25 mmol) and 5 ml of 0.1 M EDTA were added, and pH readjusted to 9.0. The reaction was initiated by addition of 7 ml of a carboxypeptidase Y solution (20 mg/ml) and pH was kept at 9.0 by addition of NaOH-solution. After 4 hours the reaction was stopped by adjusting pH to 3 with HCl-solution. The reaction mixture was diluted and purified by cation exchange on a Dowex A650Wx4 column using an ammonium acetate salt gradient, and finally desalted. Collected product fractions were concentrated by evaporation and finally freeze-dried, giving 6.45 g of D,L-tyrosyl-arginine (19 mmol, 77%) as a white powder. Identification Acetate and chloride could not be detected, the product being present as a zwitter-ion. Amino-acid analysis following acid hydrolysis gave the following result: Arg (1.03) Tyr (0.97) Purity HPLC-purity: 99.5% (Novapak C₁₈, 0.1 M ammonium phosphate with alkylsulfonic acid, pH 4.5/acetonitrile, 220 nm). Water content by Karl Fisher: 3.4%. Example 20 Preparative synthesis of D,L-phenylalanylmethionine, D,L-pheMetOH by the use of immobilized carboxypeptidase Y Immobilization procedure Carboxypeptidase Y was immobilized on Eupergit C following the recommended procedure of the manufacturer. The immobilization was carried out in the phosphate buffer at pH 7.5, and residual active gel-groups were blocked with ethanolamine at pH 8.0 with subsequent washing. 93% of the enzyme was bound to the gel containing 2.5 mg protein/ml. The Eupergit C coupled enzyme was stored in 10 mM PIPES, 1 mM EDTA, 0.05% hydroxybenzoic acid ethyl ester, pH 7.0 at 4°C. Synthetic procedure D-phenylalanine ethyl ester hydrochloride (5.7 g, 25 mmol) and L-methionine (29.8, 200 mmol) were dissolved in 400 ml of H₂O, 5 ml of 0.1 N EDTA was added, and pH adjusted to 9.0 with sodium hydroxide solution, giving a reaction volume of 500 ml. The reaction mixture was continuously stirred and pH kept constant at 9.0 with sodium hydroxide solution, while the solution was being circulated over a column of immobilized CPD-Y at a flowrate of 3 ml/min. The coloumn contained immobilized CPD-Y on Eupergit C prepared as described above and was 2.5 cm x 5.5 cm with a volume of 27 ml, containing a total of 67 mg of CPD-Y. Circulation was continued for 10 hours. pH was then adjusted to 7 with HCl-solution and the product was purified on R-preparative HPLC as described in Example 16. Collected fractions containing pure product were combined, evaporated in vacuo and finally freeze-dried to give 3.5 g (12 mmol, 48%) of D,L-phenylalanylMethionine as a white, armorphous powder. Stability of the enzyme preparation The experiment could be repeated with the same enzyme gel preparation several times without noticable loss in conversion rate and comparable results. Thus, the enzyme is fairly stable at the reaction conditions, further improving process economy. Indentity of the product The product was free of chloride, but contained 7.0% (w/w) of acetate and so was partially on acetate form. Amino-acid analysis showed the absence of free amino-acids, and following acid hydrolysis a ratio of Met (0.98) Phe (1.03) Specific optical rotation using sodium D-line at 25°C and c=0.25 in water was -128.9° Purity of product HPLC: 99.6% (nucleosile 7 C₁₈, 0.1 M ammonium phosphate, pH 3/acetonitrile, 220 nm). Water content by Karl Fisher: 2.8% Example 21 Preparative synthesis of D,D-phenylalanylphenylalanine ethyl ester hydrochloride, D,D-phepheOEt.HCl Procedure D-phenylalanine ethyl ester hydrochloride (2.5 g, 11 mmol) was dissolved in 45 ml of water, and 0.5 ml of 0.1 N EDTA was added. pH was adjusted to 9.0 with sodium hydroxide solution, the substrate being present as a partially oily suspension at the beginning. The reaction was initiated by addition of 3.4 ml of a carboxypeptidase Y solution (20 mg/ml) and was stirred for 2.5 hours at room temperature, pH being kept at 9.0 with sodium hydroxide solution. The reaction was stopped by adjusting pH to 3 with HCl-solution. The reaction mixture was filtered and purified by R-preparative HPLC as described in example 16. Collected product fractions were concentrated by evaporation in vacuo and freeze-dried with added HCl-solution, giving 0.49 g (1.3 mmol, 12%) as a white, amorphous powder. Identification The product contained 9.2% (w/w) of chloride and no acetate, the product being present as hydrochloride. Amino-acid analysis showed phenylalanine following acid hydrolysis and no phenylalanine prior to acid hydrolysis. The product could be further hydrolysed with base, yielding a product chromatographically different from D,L-Phe-PheOH, and the product itself co-eluted with an L,L-PhePhe-OEt-standard in the used HPLC-system. Finally, specific optical rotation using sodium D-line at 25°C and c=0.5 in acetic acid was found to be -42.7°. This compared with a pure standard of L,L-PhePheOEt, which gave +52.1° under similar conditions. In this case, the discrepancy is believed to be due to the lesser purity of the synthesized peptide. Purity HPLC-purity: 82.3% (nucleosile 7 C₁₈, 0.1 M ammonium phosphate, pH 3/acetonitrile, 220 nm). The impurities detected were chromatographically different from substrate, substrate hydrolysis, product hydrolysis or diastereomers thereof. Water content by Karl Fisher: 7.5%.

Claims: A process for producing dipeptides having the general formula H-A-B-Y wherein A represents an optionally side-chain-protected L- or D-α-amino-acid residue or ω-amino-acid residue, and B represents an optionally side-chain-protected L- or D-α-amino-carboxylic acid residue which may be the same as or different from A, an L- or D-aminophosphonic acid residue or an L- or D-aminosulfonic acid residue or the corresponding ω-amino-acid or a salt or hydrate thereof, and Y is OH or a C-terminal blocking group, characterized by reacting a substrate component, which is an amino-acid derivative having the formula wherein A is as defined above, R¹ represents hydrogen, alkyl, aryl or aralkyl optionally substituted with inert substituents or an α-des-amino fragment of an amino-acid, and R² and R³ are the same or different and each represent hydrogen, alkyl, aryl or aralkyl optionally substituted with inert substituents, with a nucleophile component (which, when A = B may be formed in situ ) selected from (a) amino-acids having the formula H-B-OH wherein B is an aminocarboxylic acid residue as defined above, (b) amino-acid amides having the formula wherein B is an aminocarboxylic acid residue as defined above, and R² and R³ have the above meaning, except that when R² represents hydrogen, R³ may also represent hydroxy or amino, (c) L-amino-acid esters having the formula H-B-OR⁴ wherein B is an aminocarboxylic acid residue as defined above, and R⁴ represents alkyl, aryl or aralkyl, and (d) straight-chain or branched aminophosphonic acids or aminosulfonic acids having the formula NH₂C x H z PO₃H₂ or NH₂C x H z SO₃H wherein x is 1-6 and z is 2-12, in the presence of a serine or thiol carboxypeptidase from yeast or of animal, vegetable or other microbial origin, in an aqueous solution or suspension having a pH value between 5 and 10.5 and optionally containing an organic solvent and/or a salt, and then, if desired, cleaving any side-chain-protecting group or protective group Y and/or, if desired, converting the resulting dipeptide derivative to a salt or hydrate. A process according to claim 1, characterized by using carboxypeptidase Y from yeast as the carboxypeptidase. A process according to claim 2, characterized in that the carboxypeptidase used has been purified by affinity chromatography over an affinity resin consisting of a polymeric resin skeleton with a plurality of coupled benzyl succinyl groups. A process according to claim 1, characterized in that the carboxypeptidase used is penicillocarboxypeptidase S-1 or S-2 from Penicillium janthinellum, a carboxypeptidase from Aspergillus saitoi or Aspergillus oryzae, a carboxypeptidase C from orange leaves or orange peels, carboxypeptidase C N from Citrus natsudaidai Hayata, phaseoline from bean leaves or a carboxypeptidase from sprouted barley, malt, sprouted cotton plants, tomatoes, watermelons or Bromelein (pineapple) powder. A process according to claims 1-4, characterized in that the carboxypeptidase used has been chemically modified or is a biosynthetic mutant of a natural form. A process according to any of the preceding claims, characterized in that the carboxypeptidase enzyme used has been immobilized. A process according to any of the preceding claims, characterized by using an aqueous reaction solution or reaction dispersion containing from 0 to 70% of an organic solvent. A process according to claim 7, characterized in that the organic solvent is selected from alkanols, dimethyl sulfoxide, dimethylformamide, dimethoxyethane, ethylene glycol or ethyl acetate. A process according to any of the preceding claims, characterized by using as substrate component a D- or L-amino-acid ester selected from benzyl esters or straight or branched C₁-C₆ alkyl esters optionally substituted with inert substituents. A process according to any of the preceding claims, characterized by using as nucleophile component an amino-acid or amino-acid amide having the formula H-B-OH or H-B-NHR³ wherein R³ is hydrogen or C₁-C₃ alkyl and B is an L-amino-acid residue. A process according to claim 1, characterized by using as nucleophile component an ester having the formula H-B-OR⁴ wherein B is an aminocarboxylic acid residue and R⁴ is C₁-C₃ alkyl. A process according to any of the preceding claims, characterized in that the resulting dipeptide includes one or more C-terminal protective groups Y, and that the group or groups are cleaved enzymatically, preferably by means of the same carboxypeptidase enzyme as was used in the preceding reaction. A process according to any of the preceding claims, characterized in that the resulting dipeptide includes one or more side-chain protective groups and that the group or groups are cleaved enzymatically, preferably by means of an esterase or lipase or proteolytic enzyme.

Inventors: AASMUL-OLSEN STIG, THORBEK PIA, WIDMER FRED, AASMUL-OLSEN, STIG, THORBEK, PIA, WIDMER, FRED
Applicants: CARLBIOTECH LTD AS, CARLBIOTECH LTD. A/S
Application year: 1988
Main classification: C12P21
IPC codes: C12P21