DK163365B - Process for enzymically preparing dipeptide derivatives - Google Patents

Description

DK 163365 B

The present invention relates to a process for enzymatically preparing dipeptides and derivatives of dipeptides of the kind set forth in claim 1.

In recent years there has been a growing interest in dipeptides and dipeptide derivatives, possibly containing an amino acid residue of D configuration, for their potential pharmacological effects, such as antibiotics. -10 peptides in areas such as artificial nutrition - both human and veterinary, sweeteners and agrochemicals, such as insecticides.

Such dipeptides HABY can be prepared by 15 known chemical coupling reactions, but common to all these methods is that it is usually necessary to protect the amino acids involved - A and B - on the amino and carboxylic acid groups, as well as often on the side chains, if these carrying functional groups. Furthermore, due to the reagents and conditions used, there is an inherent risk of side reactions during the chemical coupling step. Racemization, preferably on the A component, is such a significant side reaction. By replacing the chemical coupling step with an enzymatic coupling step which runs under mild conditions, such side reactions and racemization can be avoided, thereby forming a stereochemically pure product.

The presence of amino or carboxy protecting groups is mandatory both by chemical coupling and by enzymatic coupling using endoproteases, and according to the prior art, also on the amino function of the substrate by enzymatic exoprotease-catalyzed dipeptide preparation.

Hereby, as explained below, these processes are added to several undesirable features that severely affect 35 2

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their process economics on an industrial scale, which is particularly evident in dipeptide production. The disadvantages arise from the introduction, removal and presence of the protecting groups during the process, which increases the overall process cost and time as well as the yield as such.

Typical examples of commonly used amino protecting groups are carbobenzoxy (Z-) and tert-butoxycarbonyl (Boc) -type groups having a molecular weight comparable to the amino acid residues. First, the protecting groups must be introduced into the starting materials by suitable, expensive agents in a separate reaction step followed by an isolation step. The presence of these hydrophobic groups often has a drastic effect on the solubility of the intermediates and the reaction products and can affect both the type and amount of solvents required for their treatment, and the ease with which they can be purified and de-protected. Furthermore, the de-protection must be done in a separate step followed by a purification step.

There are a number of reactions for this purpose, but with the exception of catalytic hydrogenation, which in itself causes 25 industrial problems, these reactions take place under severe and often highly acidic or basic conditions, often causing a number of side reactions. The result of this will be an unclean product, unless it is subject to demanding and elaborate purification.

30

Thus, the final steps of this relatively long series of synthesis steps can become quite extensive deb protection to obtain the desired dipeptides. In addition, due to the · almost inevitable secondary reactions, 35 fairly clean cleaning procedures are required to obtain a product of the desired high purity.

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Attempts to avoid amino terminal protection in the preparation of dipeptides have included microbial fermentation, e.g. the fermentation process described in EP-A1-074095 and EP-A2-102529 to form aspartame. This technique is fundamentally different from synthetic methods and relies on specific organisms for each peptide. It is therefore not generally applicable. Furthermore, the yields are often low and the recovery from the fermentation medium is cumbersome.

10 In view of the overall process economy, it is therefore an obvious advantage to be able to avoid protecting groups, also at the amino and carboxy terminals. It is the object of the present invention to enable this by dipeptide synthesis on the basis of serine and thiol carboxypeptidase catalysis.

It may be of interest to be able to produce a dipeptide having side chain protection but no terminal protection, and it will be seen that this is also possible in the process of the invention when starting from side chain protected but amino and carboxy protected 20 starting materials, respectively. Hereby the same benefits can be obtained! in terms of mild reaction conditions and overall process economics, as with side chain protected starting materials.

The C-terminal protecting group can be removed enzymatically immediately or with an enzyme other than that used for the synthesis. The presence of a protecting group, e.g. an amide, may have an effect on the biological effect of the compounds, so it is not always removed. If desired, the side chain protecting group can be removed chemically or enzymatically, usually under milder conditions than is possible for the amino protecting group.

Known are the enzyme catalyzed coupling reactions which allow the use of side chain unprotected amino acid derivatives and optionally a C-terminal unprotected B component (nucleophile). For example, reference may be made to Danish Patent Specification No. 155613 (App. No. 5202/80) and the corresponding 4

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No. EP-A-17 485 (EP-B1-17485).

EP-B1-17485 discloses a process for the preparation of peptides of general formula 5 A1 "B1" Z1 wherein A 1 represents an N-terminal protected L-amino acid residue or an optional N-terminal protected peptide residue with a 10 C-terminal L-amino acid residue and represents an L-amino acid residue, and is OH or a C-terminal protecting group, by reacting a substrate component with an amine component in the presence of an enzyme and, if desired, cleavage of any terminal protecting groups to form a peptide of the formula

Al "B1" Z1, which is characterized by reacting a substrate component selected from amino acid esters, peptide esters, depsipeptides, optionally N-substituted amino acid amides or peptide amides, and optionally N-terminal protected peptides of the formula

A1-0R1, A ^ NR ^ 'or Aj-Xj-OH

wherein A 1 is as defined above, R 2 represents alkyl, aryl, heteroaryl, aralkyl or an α-des amino fragment of an amino acid residue, R 2 and R 2 'each represent hydrogen, alkyl, aryl, heteroaryl or aralkyl, and represents an L-amino acid residue, with an amine component (nucleophile) selected from 35

DK 163365 B

optionally N-substituted L-amino acid amides, L-amino acids and L-amino acid esters, of the formula HB ^ NRgRg 'or HB ^ OR ^ wherein B ^ is as defined above Rg and Rg' each represents hydrogen, hydroxy, amino, alkyl, aryl, heteroaryl or aralkyl, and is hydrogen, alkyl, aryl, heteroaryl or aralkyl in the presence of an L-specific serine or thiol carboxypeptidase enzyme derived from yeast or of animal, vegetable or other microbial origin, in a aqueous solution or dispersion at pH 5-10.5, preferably at a temperature of 20-50 ° C. The preferred enzyme is carb oxypeptidase Y from yeast, hereinafter referred to as CPD-Y.

(It should be noted that the above symbol meanings are not identical to those used in EP-B1-17485 to avoid confusion with those used in the application).

Thus, if a dipeptide is to be prepared by the method of EP-B1-17485, an N-terminal protected amino acid derivative is used as the substrate component and the incoming amino acid is mandatory 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 essentially lapses when the peptide residue consists of 3 amino acids, however depending on their nature and sequence.

This is illustrated in Breddam et al. "Influence of the 35 substrate structure on carboxypeptidase Y catalyzed peptide bond formation", Carlsberg Res. Commun., Vol. 45, pp. 361-67, December 30, 1980, where Ac-Ala-Ala-Ala-OMe and 6

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H-Ala-Ala-Ala-OMe was 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 90, respectively. 80%.

Breddam et al. also investigated the importance of the amino acid configuration for the coupling yield in CPD-Y catalyzed peptide synthesis, cf. the following table wherein 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-NH 2 0 15 _

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 stopped after 20 min.

20

It is evident from the table that if the C-terminal is in D-form, as in H-Ala-Ala-ala-OMe, no peptide synthesis occurs because the ester is not reacted. With the D-amino acid adjacent to the C-terminal as in H-Ala-ala-Ala-OMe, 25 reactions occur, but the coupling yield is reduced to 40% compared with the 80% in the pure L-configuration H-Ala-Ala-Ala. OMe.

Furthermore, it has long been recognized that some endo-proteases can catalyze the oligomerization of certain N-unprotected amino acid esters with L configuration, but it has never been tried before to make dipeptides which are not simple dimers. The results of such observations have usually been a mixture of 35 occasionally oligomers, and only in the case of product precipitation has it been possible to isolate a single product.

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For this reason, the use of endoproteases for peptide preparation has been limited to the use of amino- and carboxy-terminal protected starting materials, as described in US-A-4,086,136.

5

The above amino and carboxy terminal protected starting materials are also mandatory if aspartate endoproteases as described in US-A-3,972,773 or metalloendoproteases as described in EP-A1-10 0099585 are used in the preparation of Z-AspPheOMe. PheOMe salt.

Finally, it has not been possible so far with carboxy peptidases and usually with no proteolytic enzyme (Class EC 3.4) to produce the diastereomeric dipeptides of DL, LD and DD configuration or peptides containing β-amino acid residues. from amino-unprotected starting compounds. A second class of enzymes, aminoacyl-t-RNA synthetase (Class EC 6.1), as exemplified in EP-A1-086053, has been tested. Here, a specific enzyme must be used for each type of amino acid residue, and furthermore expensive Co-factors such as ATP are needed. At the same time, the yields are very low, so although a certain amount of dipeptide ester or amide was isolated and identified, it typically required ten times as much co-factor-25 excess, several hundred times as much nucleophilic excess and several hundred times as much enzyme. excess weight, cf. Example 1, wherein 1 mg of Tyr is reacted with 4 g of L-Phe methyl ester in the presence of 0.6 g of tyrosyl-tRNA synthetase to give 0.4 mg of Tyr-Phe- OMe.

30

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 substrate component in controlled reactions for the synthesis of dipeptides and dipeptide derivatives and that it is possible to suppress a possible oligomerization of the substrate.

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Furthermore, it has surprisingly been found that N-unprotected amino acid derivatives of D configuration can also be used as substrates in these reactions, so that in addition to LL dipeptides, it is also possible to synthesize DL-5 dipeptides. However, the rate of turnover for D substrates is usually somewhat lower than for L substrates under similar conditions, however, as illustrated below, the difference in rate is far less than for the corresponding N-protected amino acid esters, where the D substrate is reacted at a rate. which is far less than the velocity of the L substrate. The yields are often as high or higher at the unprotected D substrates relative to the unprotected L substrates as shown by the following examples.

Even more surprisingly, it has also been found that with D-configuration substrates, D-configuration nucleophiles can be coupled by these enzymes, otherwise known to be L-specific at the amino side of the synthesis site. An amino acid ester incorporated in this manner is not further hydrolyzed.

The process according to the invention is thus characterized by the characterizing part of claim 1.

As examples of useful amino acids may be mentioned 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), hydroxyamino acids such 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 (Gin), diaminomonocarboxylic acids such as omin thin (Orn) and lysine (Lys), arginine (Arg), aromatic amino acids such as phenylalanine (Phe) and tyrosine (Tyr), and heterocyclic amino acids such as histidine 9

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(His), Prolin (Pro) and tryptophan (Trp). As examples of useful amino acids with more unusual ones. structure may be mentioned penicillamine (Pen), aminophosphonic acids such as alanine phosphonic acid (AlaP) amino sulfonic acids such as Taurine 5 (Tau), or'ω amino acids such as ε-alanine (BA1a). In the substrate component and in the nucleophilic component, as mentioned, the amino acid may also be in D form.

The advantages of the method according to the invention in relation to the known methods are minimal or no side chain protection, no N protection of the substrate component which can have both D and L configuration, no risk of racemization, few synthetic steps and a relatively pure end product, which together allows for a very simple and economical manufacturing method.

Preferred substrate components are esters of the formula HA-OR1, wherein A is as defined in claim 1, R is straight-chain or branched-chain alkyl of 1-6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl , amyl and hexyl, or aralkyl benzyl. Particularly suitable nucleophilic components are free L-amino acids or amino acid amides of the formula / Η2 Η-Β-Ν \ Η3 'wherein Β is as defined in claim 1, R 2 is H and R 3 is H or C 1 -C 6 alkyl, or amino acid esters of the formula HB-OR4, wherein B is as defined in claim 1 and wherein R4 represents straight or branched alkyl of 1-6 carbon atoms such as the above. As mentioned, R 1 may be alkyl, aryl or aralkyl optionally substituted by hydroxy, nitro or halogen.

In the process of the invention, depending on the nucleophile used, a peptide containing the group 10 may be formed intermediately

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R2 / -N or -OR * \ 5 * R 'wherein R2, R3 and R * have the meanings set forth in claim 1, whereupon this group is optionally cleaved to form a carboxylic acid group. This cleavage can be catalyzed by another or by the same enzyme used for the formation of the peptide.

Enzymes can also be used for cleavage of side chain protecting groups. Speaking as useful enzymes, depending on the type of protecting group, proteolytic enzymes, lipases and esterases, cf. "The Peptides, Analysis, Synthesis, Biology" Vol 9, Special Methods in Peptide Synthesis Part C. J.A. Glass, Enzymatic Manipulation of Protecting Groups in Peptide Synthesis, Academic Press 1987.

20

In addition to CPD-Y, which p.t. is the preferred enzyme, and more specifically characterized in EP-B1-17485, the process of the invention is also carried out with other serine or thiol carboxypeptidases, such as those listed below, which serve a common mechanism of action via acylenzymmellemproduk-ter.

30 35 11

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Enzyme Origin

Mushrooms

Penicillocarboxypeptidase S-1 Penicillium janthinellum S-2,5 Carboxypeptidase (s) from Aspergillus saitoi

Aspergillus oryzae

plants

Carboxypeptidase (s) C Orange Leaves 10 Orange Shells

Carboxypeptidase C ^ Citrus natsudaidai

Hayata

Phased in Bean Leaves

Carboxypeptidase (s) from Germinating barley or ground Carboxypeptidase W from Wheat

Carboxypeptidase (s) from Germinating Cotton Plants

tomatoes

watermelons

Bromelain (pineapple) powder 20

The close relationship between a number of the above carboxypeptidases is discussed by Kubota et al., Carboxypeptidase C. J. Biochem., Vol. 74, no. (1973), pp. 757-770. Carboxypeptidase from malt, wheat and other sources is 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 a natural form.

As illustrated in the following, the process of the invention is quite simple, but it is important to maintain a fairly constant pH in the reaction mixture. This pH is between 5 and 10.5, but preferably between 7 and 9.5, and moreover depends on the specific starting materials, the dipeptide formed and the enzyme.

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The reaction is carried out in an aqueous reaction medium which, if desired, may contain an organic solvent which is miscible or immiscible with water and which is compatible with the enzyme used under the given conditions 5 in amounts up to 10% or more. Preferred solvents are lower alcohols, dimethylformamide, dimethylsulfoxide, dimethoxyethane, ethylene glycol and ethyl acetate.

The reaction temperature is preferably room temperature and above, 20-50 ° C, but temperatures in the range 0-60 ° C are applicable if advantageous under the other given conditions.

The concentration of the two reaction components can be varied within wide limits, but most often the nucleophile component will be in excess, and to avoid oligomerization of the substrate component, it is often added in smaller portions distributed over the entire reaction course. If an ester of formula H-A-OR1 is used, where R 1 is C 1 -C 10 alkyl, surprisingly no oligomerization is observed and very high substrate concentrations can be used without side reactions. Here, from a single starting compound, a homodipeptide can be formed, where A = 25 B, without oligomerization, the nucleophile being formed in situ by hydrolysis of this ester.

Thus, the initial concentration for the substrate component can typically be 0.005 - 2 molar and for the nucleophilic component in cases where it is added separately 0.005 - 3 molar. In most situations, it will be possible to recover excess of the nucleophilic component and the hydrolysis product from the substrate component for any residual refining and recycling. The components can easily be traced back to the process, due to their simple structure and the absence of side reactions and debarking losses.

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The enzyme concentrations can also be varied, but are often somewhat higher (5-50 μιη) than the concentrations useful using N-protected amino acid ester substrates, but as illustrated in the following examples, the amount of enzyme to carry however, the reaction is reduced more than tenfold by using a stable immobilized enzyme preparation, thereby utilizing the enzyme 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 complex binding agent for metal ions present, such as EDTA, which stabilize the enzyme.

The process according to the invention is further illustrated with reference to the drawing, wherein

FIG. Figure 1 shows the course of hydrolysis of L- and D-TyrOEt and L- and D-AcTyrOEt, respectively, at 50 mM concentration 20 in 20% DMS0 containing 1 mM EDTA at pH 9.0 ka talysed by 10 μΜ CPD-Y, and

Fig. 2 shows the course of the reaction by formation of the homodipep time L, L-MetMet from L-MetOEt at initial concentrations of 0.05 M and 0.5 M at pH 25 8.5 in the presence of 13 μΜ and 39 μΜ CPD, respectively. Y.

The differences mentioned initially in the rate of turnover of D and L substrates are illustrated in FIG. 1, showing the reaction course of the CPD-Y hydrolysis of a protected (Ac-TyrOEt) and unprotected amino acid ester (TyrOEt) in D and L form.

It can be seen from the figure that while the L-AcTyrOEt is hydrolyzed almost instantaneously, after 2 hours only a negligible hydrolysis (less than 5%) of the D-AcTyrOEt has occurred. In contrast, there are only small differences in hydro- 14

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the course of light for the unprotected esters in the L and D form.

This surprising course of hydrolysis is reflected in the following examples which illustrate the preparation of 5 different dipeptides by the process of the invention using the enzymes CPD-Y and maltcarboxypeptidase II (CPD-MII) and wheat carboxypeptidase (CPD-W).

10 As can be seen from FIG. 2, no oligomerization is observed by CPD-Y catalyzed homodipeptide synthesis of L, L-MetMet from MetOEt.

General method for example 1-15 exemplified below 15

The reactions, performed on an analytical scale with a reaction volume of 1 ml, were carried out in a pH state, keeping the chosen pH value constant by the automatic addition of 1 N NaOH. Reaction temperature was room temperature, where not otherwise stated. The table also lists reactant concentrations, organic solvent content, product and yield. Reaction times are typically between 0.5 and 5 hours, and the enzyme concentrations are typically 10-20 μπι, where nothing else is stated.

25

Product identification and product yield determination were performed by reverse-phase HPLC (Waters 6000 A pumps, 660 gradient mixer, UK 6 injector) on a C ^ g NOVA PAK column (Waters, RCM) using appropriate gradients of elution systems containing 50 mM triethylammonium phosphate, pH 3.0 from 0% to 80% acetonitrile at a flow rate of 2 ml / min. The elution was followed by 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 assay corresponding to the putative 35

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15 and / or by HPLC comparison with a chemically synthesized reference product. These were prepared according to known principles, usually via reaction between BOC-A-OSu - tert-butyloxycarbonyl - succinimide ester derivative 5 of the substrate amino acid - and the nucleophilic component used followed by unblocking of the N-terminal amino acid residue. In all cases, it was possible to separate LL and DD dipeptides from the diastereomeric DL and LD dipeptide products.

10

The product yields for the products which can only be detected at 230 nm were determined by the absorption / concentration curve of the chemically synthesized reference compound. For the other products, the yields 15 were determined on the basis of the ratio of the integrated areas below the peaks of the elution chromatogram corresponding to the product and the reactant absorbing at the respective wavelength, respectively.

As an example of this method, preparation of D, L-PheØAlaOMe according to Example 12 is reproduced below.

Preparation of D, L-phenylalanine-beta-anatine methyl ester (D, L-phegAlaOMe) according to Example 12 In a pH state, D-phenylalanine-ethyl ester hydrochloride (11.4 mg; 50 moles) and beta-ananine methyl ester hydrochloride (70 mg; 0.5 mmol). This was dissolved in 800 μΐ of demineralized water and 10 L of a 100 mM EDTA solution was added, after which the pH was adjusted to 9.0 by ca. 40 μΐ 6N NaOH. 25 µl was taken for control (stored at room temperature) and 5 µl 0 sample for HPLC run. The reaction was started by adding 60 µl of carboxypeptidase-Y solution (333 µM) to a final concentration of ca.

35 20 μΜ. The pH was kept constant at 9.0 with 1 M NaOH, through the reaction a total of approx. 100 µl. The reaction temperature was room temperature. The reaction time was approx. 30 min, then 16

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ca. 90% of the substrate was consumed. 83% of these were converted to the product. Amino acid analysis of fractions from HPLC analysis corresponding to the putative product peak showed the expected composition.

5

The reaction conditions in Preparation Examples 16-25 are described in the individual Examples. The reactions were followed by analytical HPLC as described. The enzyme concentrations are generally lower and the reaction times longer than in the 10 corresponding analytical examples, but no attempt was made to optimize the reaction conditions.

15 20 25 30 35

Carboxypeptidase Y) catalyzed synthesis of LL dipeptides with L-tyrosine ethyl ester (50 mM) as substrate component 5 and free amino acids as nucleophiles

Example 1 17

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a

Nucleophil (Wife) Medium 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 Penicilamine (0.5 M) Water 8.0 TyrPenOH 7

a) 10 µm, 1 mM EDTA

25 30 35

Carboxypeptidase Y) catalyzed synthesis of LL dipeptides with L-methionine (0.3 M) as nucleophilic component in 5 water at pH 9.0

Example 2 18

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a

Substrate (50 mM) Product Yield%

Leucine methyl ester LeuMetOH 30 10 Leucine isoproyl ester LeuMetOH 36

Methionine ethyl ester b) MetMetOH 25

Phenylalanine methyl ester PheMetOH 16

Phenylalanine ethyl ester PheMetOH 19

Phenylalanine isopropyl ester PheMetOH 23 Serine isopropyl ester O) SerMetOH 21

Tryptophan methyl ester TrpMetOH 26

Tyrosine Benzyl Ester) TyrMetOH 19

A) 10 µm, 1 mM EDTA

b) 5 mM

°) Reaction time> 20 hours

d) 30% DMSO

25 30 35

Carboxypeptidase Y) catalyzed synthesis of LL dipeptides with L-tyrosine ethyl ester (50 mM) as substrate component 5 and L-amino acid amides or esters as nucleophiles

Example 3 19

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Sub-Nucleophil (Cone.) Medium pH Product Yield% 10% TyrOEt Leucinamide (0.2M) 30% DMSO 9.5 TyrLeuNH2b) 42

TyrLeuOH 4

TyrOEt Lysinamide (0.3 M) Water 9.5 TyrLysNH2 20

TyrLysOH 22

TyrOEt Argininamide (0.2 M) Water 9.0 TyrArgNH2 50 TyrOEt Valinamide (0.3M) Water 9.0 TyrValNH2 77

TyrOEt Leucine methyl ester (0.2 M) 30% DMSO 9.0 TyrLeuOH 6

20 a) 20 am, 1 mM EDTA

u °) Reaction time> 20 hours, 50% substrate reacted 35

Example 4 20

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Carboxypeptidase Y a) Catalyzed synthesis of LL homodi peptides from a single starting compound in water at pH 8.5

Substrate (Wife) Product Yield%

Methionine methyl ester (0.5 M) b) c) 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 c) 1

Tyrosine ethyl ester (0.2 M) TyrTyrOH c) 1

Phenylalanine ethyl ester (0.2 M 2) e) PhePheOH 2 Alanine amide (0.2 M) f) ^) AlaAlaN ^ a) 10 µm CPD-Y, 1 mM EDTA ^) Polymerization 20 C) Precipitation d) pH 9, 0

e) 30% DMSO

f) 50 µm CPD-Y, 1 mM EDTA 35

Carboxypeptidase Y a) catalyzed synthesis of DL dipeptides with D-tyrosine ethyl ester (50 mM) as substrate and free 5 L amino acids as nucleophiles in water

Example 5 21

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Nucleophil (Cone.) PH Product Yield% 10 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 Penicilamine (1 M) 13) 8.0 TyrPenOH 27

a) 10 µm, 1 mM EDTA

k) Reaction time> 20 h 20 25 30 35

Carboxypeptidase Y a) catalyzed synthesis of DL dipeptides with L-methionine (0.3 M) as nucleophilic component in water at pH 9.0

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Example 6 22 D substrate (50 mM) Product Yield% 10 leucine methyl ester leuMetOH 50 leucine isopropyl ester leuMetOH 71 methionine ethyl ester withMetOH 68 phenylalanine ethyl ester pheMetOH 71 phenylalanine isopropyl ester pheMetOH 72 (trine 1 mM EDTA 20 D) Reaction time 5 days 25 30 35

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Example 7 23

Carboxypeptidase Y a) Catalyzed synthesis of D, L-dipeptidamides with D-tyrosine or D-phenylalanine ethyl ester 5 (50 mM) as a substrate component and L-amino acid amides as nucleophilic components in water

Substrate Nucleophil (Con.) PH Product Yield% 10-tyrOEt Leucinamide (0.2 M) 9.0 tyrLeuNH ^ 82 tyrLeuOH 2 tyrOEt Valinamide (0.3 M) 9.0 tyrValN ^ 94 tyrOEt Argininamide (0.2 M) 9.0 tyrArgNH- 86 pheOt Alanine amide (0.8 M) 9.0 pheAlAn ^ J 50

a) 15 μm, 1 mM EDTA

b) Some polymerization

Example 8 24

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Carboxypeptidase Y a) Catalyzed synthesis of D, D homodi peptide esters from D-substrate ester components in water at pH 5 9.0, which also acts as nucleophilic component D substrate (Kone.) Product Yield% tyrosine ethyl ester (0.05 M) tyrthyrOEt 9 10 phenylalanine ethyl ester (0.1 M) phepheOEt 10 tyrosine ethyl glycol ester (0.05 M) tyrthyrOEtOH 1 methionine methyl ester (0.1 M) methomethome 3 15 a) 15 m, 1 mM EDTA 20 25 30

Example 9 25

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Carboxypeptidase Y a) Catalyzed synthesis of LL dipeptides with side chain protected carboxy terminal amino acids with 5 L-TyrOEt (50 mM) as substrate component and side chain protected L amino acids and amides as nucleophiles

Nucleophil (Kone.) PH Medium Product Yield 10 Acetamidomethyl (1 M) 8.5 Water iyrCys (-SAcm) 0H 10 acetamidomethyl cysteine amide (0.4 M) 8.5 Water TyrCys (-SAcm) NH2 12

TyrCys (-SAcm) OH 1 O -benzyl-aspartic acid (0.1 M) b) 9.0 30% DMSO TyrAsp (OBzl) OH 13 ε Trifluoroacetyl-lysine (0.1 M) b) 8.5 30% DMSO TyrLys (Tfa) OH 12 r-tertbutylglutamic acid amide (0.1 M) b) 8.0 30% DMSO TyrGluiOtBu) 3 TyrGlu (0tBu) OH 12 r-methyl-glutamic acid (0.3 M) 8 , 5 Water TyrGlu (OMe) OH 20 r-Methylglutamic acid (0.3 M) 8.5 Water TyrGlu (0Et) OH 18 25_____

a) 10 am, 1 mM EDTA

b) 25 mM substrate, 20 µm 35

Example 10 26

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Carboxypeptidase Y a) Catalyzed synthesis of DL dipeptides with side chain protected carboxyterminal amino acids 5 with D-Tyr0Et '(50 mM) as substrate component and side chain protected L amino acids and amides as nucleophiles

Nucleophil (Con.) PH Medium Product Yield% 10 Acetamidomethyl cysteine (1 M) 8.5 Water tyrCys (-SAcm) CH 75

Acetamidomethyl cysteine amide (0.4 M) 8.5 Water tyrCys (-SAcm) NH2 71 tyrCys (-SAcm) OH 6 O -benzyl aspartic acid (0.1 Mb) 9.0 30% DMSO tyrAsp (OBzl) OH 54 Trifluoroacetyl-lysine (0.1 M) b 8.5 30% DMSO tyrLys (Tfa) OH 51 r-tertbutyl glutamic acid amide (0.1 M) b 8.0 30% DMSO tyrGlu (0tBu) NH2 42 tyrGlu (0tBu) 0H 10 20 ι-methyl glutamic acid (0.3 M) 8.5 Water tyrGlu (0Me) OH 75 T-ethyl glutamic acid (0.3 M) 8.5 Water tyrGlu (0Et) 0H 71 25 a) 10 µm, 1 mM EDTA ° 25 mM substrate, 20 m 35

Example 11 27

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Carboxypeptidase Y a) Catalyzed synthesis of LL dipeptides with side chain protected amino-terminal amino acid residues 5 from side chain protected substrate components (25 mM) and L-methionine (0.3) as nucleophilic component in 30% DMSO at pH

9.0

Substrate Product Yield 10% L-Aspartic Acid Dibenzyl Ester Asp (OBzl) MetOH 65 L-Glutamic Acid Dibenzyl Ester c) Glu (OBzl) MetOH 70 15 a) 20 am, 1 mM EDTA, reaction time> 20 hp) at 35% turnover c) At 60 % turnover 20 25 30 35

Example 12 28

DK 163365 B

Carboxypeptidase Y a) Catalyzed synthesis of dipeptide esters and amides containing ω-amino acids from L- and D-amino acid ester substrates at 50 mM in water with? -Alanine and? -Alaninamide as nucleophiles

Sub-Nucleophil (Cone.) PH Product Exchanged exchange 10% TyrOEt & Alanine methyl ester (0.2 M) 8.5 tyrBAlaOMe 51 tyrOEt / 5-Alanine methyl ester (0.5 M) 8.5 tyrBAlaOMe 72 pheOEt 5-Alanine methyl ester (0.5 M) 9.0 pheBalaOMe 83 15 PheOEt E-Alanine methyl (0.5 M) 9.0 PheBAlaOMe 15 pheOEt J5-Alanine amide (0.5 M) 9.0 pheBAlaNH2 70

PheOEt O-Alanine amide (0.5 M) 9.0 PheBAlaNH2 3

20 a) 20 µm, 1 mM EDTA

25 30 35

Example 13 29

DK 163365 B

Carboxypeptidase Y a) Catalyzed synthesis of LL and DL dipeptides with hydroxyalkyl esters of L and D tyrosine and 5 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% 10___

TyrOEtOH 0 TyrMetOH 10

TyrOEtOH 40 TyrMetOH 8

TyrOEtOH 60) TyrMetOH 5 TyrOEtOH 0 TyrMetOH 56 15 PheOEtOH 0 PheMetOH 16

a) 5 nm, 1 mM EDTA

b) 10 μπι, 1 mM EDTA

20 25 30 35

Example 14 30

DK 163365 B

Synthesis of LL dipeptides catalyzed by barley and wheat carboxypeptidases at 50 mM starting L substrate ester concentration ve pH pH 8.0 in water and L amino acids as nucleophilic components

Enzyme Substrate Nucleophil (Wife) Product Yield% 10 _ CPD-MII3) PheOEt Methionine (0.4 M) PheMetOH 10 CPD-W * 3) PheOEt Arginine (0.8 M) PheArgOH 8

A) 20 tun, 1 mM EDTA, 2 M NaCl b) 10 um, 1 mM EDTA

Example 15 Synthesis of DL Dipeptides Catalyzed by Barley and Wheat Carboxypeptidases at 50 mM Starting Concentration of D-Phe Nylalanine Ethyl Ester at pH 8.0 in Water and Free L-Amino Acids as Nucleophil Components 25 Enzyme Substrate Nucleophil (Kone.) Product Yield te% CPD-MIIa) pheOEt Methionine (0.4 M) pheMetOH CPD-Wb) pheOEt Arginine (0.8 M) pheArgOH 13 30 CPD-Vjh) pheOEt Methionine (0.4 M) pheMetOH 40 a) 20 μΐη , 1 HIM EDTA, 2 M NaCl b) 10 µm, 1 mM EDTA, 2 M NaCl, Reaction time> 20 h, 35 turnover less than 50%

Example 16 31

DK 163365 B

Preparation of L, L-tyrosylcysteine, TyrCysOH Process L-tyrosine ethyl ester hydrochloride (1.5 g, 6 mmol) and L-cysteine (15.2 g, 125 mmol) are dissolved in 110 ml of 0.1 M KCl solution containing 1 mM EDTA. The pH is adjusted to 8.0 10 with triethylamine. The reaction is started by adding 7.5 ml of carboxypeptidase Y solution (16 mg / ml), and the pH is maintained at 8.0 throughout the course of the reaction by adding triethylamine with continuous stirring at room temperature. The remainder of the substrate (13.5 g, 54 mmol) is added 15 in 1.5 g portions over one hour. After 0.5 hours, the precipitation of tyrosine begins and after 3.5 hours the reaction is stopped by heating to 45 ° C for 20 minutes.

The tyrosine formed is filtered off and the filtrate is purified by R-preparative HPLC (Waters Prep. L C / system 500A) using two columns (5.7 x 30 cm) packed with 60 µm g particles and 50 mM acetic acid as eluent. The collected fractions containing pure product are evaporated in vacuo to dryness with the addition of absolute ethanol 25 several times. The residue is slurried in diethyl ether, after which 3.88 g of L, L-tyrosine cysteine (14 mmol, 22%) can be isolated by filtration and drying.

Identification 30

Chloride and acetate could not be detected. I.e. the product is available as zwitterion.

Amino acid analysis after acidic hydrolysis and derivation of Cys 35 with acrylonitrile gave:

Tyr (1.00)

Cys (1.08) 32

DK 163365 B

Purity TLC showed only a stain after derivation of the cysteine 5 side chain with acrylonitrile on silica gel 60-F using ninhydrin detection (Rf 0.73, eluent: ethyl acetate, butanol, acetic acid and water (1: 1: 1: 1 :)).

HPLC purity: 99.5% (nucleosil 7 Cg, 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

Preparation of L, L-methionylmethionine, MetMetOH Process 20 L-methionine ethyl ester hydrochloride (24.6 g, 115 mmol) and L-methionine (20.6 g, 138 mmol) are dissolved in 190 ml of 0.1 M KCl solution containing 1 mM EDTA. The pH is adjusted to 9.0 with sodium hydroxide solution and the reaction is started by adding 14.2 ml of carboxypeptidase Y solution (20 mg / ml). The reaction is stirred overnight at room temperature and the pH is kept constant at 9.0 by the addition of sodium hydroxide solution with the aid of a PH state. The pH is adjusted to 3.0 with HCl solution at the end of the reaction.

30

Precipitated methionine is filtered off and the filtrate is purified by R-preparative HPLC as described in Example 16. Collected fractions containing pure product are concentrated by evaporation and finally lyophilized. There was thus obtained 10.6 g (37.8 mmol, 33%) of L-methionyl-L-methionine as a white amorphous powder.

33

DK 163365 B

Identification

Chloride could not be detected and acetate only in an amount of 1.9% (by weight). I.e. the product is predominantly in zwitterion form. Amino acid analysis after acid hydrolysis detected methionine but no free methionine in unhydrolyzed samples.

Purity 10 HPLC purity: 99.8 (nucleosil 7 Clg, 0.1 M ammonium phosphate, pH 3.0 / acetonitrile, 220 run).

Quantization by reaction with trinitrobenzenesulfonic acid (TNBS) and UV detection: 92.5%.

Water content according to Karl Fisher: 1.5%.

Example 18 20

Preparation of L, L-tyrosylvalinamide, TyrValN

Process 25 Dissolve L-tyrosine ethyl ester hydrochloride (16.0 g, 65 mmol) and L-valinamide hydrochloride (60 g, 390 mmol) in 1150 ml of water and 65 ml of 20 nM EDTA are added. The pH is adjusted to 9.0 with sodium hydroxide solution. The reaction is started by adding 12 ml of carboxypeptidase Y solution (20 30 mg / ml). The reaction is stirred at room temperature for four hours and the pH is kept constant at 9.0 by the addition of sodium hydroxide solution. After completion of the reaction, the reaction is stopped by raising the pH to 11.

The denatured enzyme is filtered off and the reaction mixture is diluted and purified by subsequent anion exchange on a Dowex A61x4 column and cation exchange on a Dowex 34

DK 163365 B

A650Wx4 column using sodium and ammonium acetate salt gradients, respectively, and finally desalted. Product fractions are combined and evaporated in vacuo to dryness with the addition of absolute ethanol several times, yielding 11.2 g of L, L-tyrosylvalinamide (40 mmol, 62%) as a white powder.

Identification 10 1.8% (by weight) of acetate, i.e. the product is predominantly in zwitterionic form.

Amino acid analysis after acid hydrolysis gave the following result: 15

Tyr (1.01)

Choice (0.99)

Purity 20 HPLC purity: 99.5 (Novapak C ^ g, 0.1 M ammonium phosphate containing alkyl sulfonate, pH 4.5 / acetonitrile, 220 nm).

UV quantization: 97.8% (293 nm, tyrosine absorbance in 0.1 M NaOH).

Example 19

Preparation of D, L-tyrosyl-arginine, D, L-tyrArgOH 30

Process 105 g of L-arginine (105 g, 603 mmol) are dissolved in 400 ml of water. The pH is adjusted to 9.0 with HCl solution. D-35 tyrosine ethyl ester hydrochloride (6.1 g, 25 mmol) and 5 ml of 0.1 M EDTA are added and the pH is adjusted to 9.0. The reaction is started by adding 7 ml of carboxypeptidase Y solution 35

DK 163365 B

(20 mg / ml) and the pH is kept constant at 9.0 by the addition of NaOH solution. The reaction is quenched after four hours by adjusting the pH to 3 with HCl solution.

The reaction mixture is diluted and purified by cation exchange on a Dowex A650Wx4 column using an ammonium acetate · salt gradient and finally salted. The collected product fractions are concentrated by evaporation and finally lyophilized to give 6.45 g of D, L-10 tyrosyl-arginine (19 mmol, 77%) as a white powder.

Identification

Acetate and chloride could not be detected. I.e. product 15 is present as zwitterion.

Amino acid analysis after acid hydrolysis gave the following results:

Arg (1.03) Tyr (0.97)

Purity HPLC purity: 99.5% (Novapak Clg, 0.1 M ammonium phosphate 25 with alkylsulfonic acid, pH 4.5 / acetonitrile, 220 nm).

Water content according to Karl Fisher: 3.4% 30 35 36

DK 163365 B

Example 20

Preparation of D, L-phenylalanylmethionine, D, L-pheMetOH using immobilized carboxypeptidase Y 5

Immobiliseringsprocedure

Carboxypeptidase Y was immobilized on Eupergit C following the manufacturer's recommended procedure. Immobilization 10 was carried out in a phosphate buffer at pH of 7.5 and residual active gel groups were blocked with ethanolamine at pH 8.0 and subsequent washing.

93% of the enzyme was bound to the gel containing 2.5 mg of protein / ml. The Eupergit C-linked enzyme was stored in 10 mM PIPES, 1 mM EDTA, 0.05% hydroxybenzoic acid ethyl ester, pH 7.0 at 4 ° C.

Preparation Procedure 20 D-phenylalanine ethyl ester hydrochloride (5.7 g, 25 mmol) and L-methionine (29.8, 200 mmol) were dissolved in 400 mL of 0.

5 ml of 0.1 N EDTA was added and the pH was adjusted to 9.0 with sodium hydroxide solution to a reaction volume of 500 ml. The reaction mixture was stirred continuously and the pH was kept constant at 9.0 with sodium hydroxide solution while passing the solution over a column of immobilized CPD-Y at a flow rate of 3 ml / min. The column contained immobilized CPD-Y on Eupergit 30C, 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 CPD-Y. The circulation was continued for 10 hours. 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 lyophilized, 37

DK 163365 B

to give 3.5 g (12 mmol, 48%) of D, L-phenylalanylmethionine as a white amorphous powder.

Enzyme Preparation Stability 5

The experiment could be repeated several times with the same enzyme-gel preparation without remarkable decrease in turnover rate and comparable results. Thus, the enzyme is quite stable under the reaction conditions, which further improves the process economy.

product Identity

The product contained no chloride but 7.0% (by weight) of acetate and was thus partially in acetate form.

Amino acid analysis detected the absence of free amino acids and after acidic hydrolysis:

With (0.98) 20 Phe (1.03)

The specific optical rotation using sodium D line at 25 ° C and c = 0.25 in water was -128.9 ° 25.

HPLC: 99.6% (nucleosil 7 Cg, 0.1 M ammonium phosphate, pH

3 / acetonitrile, 220 nm).

30 Water content according to Karl Fisher: 2.8% 35

Example 21 38

DK 163365 B

Preparation of D, D-Phenylalanylphenylalanine ethyl ester hydrochloride, D, D-phepheOEt.HC1 5

Process D-Phenylalanine ethyl ester hydrochloride (2.5 g, 11 mmol) was dissolved in 45 ml of water and then 0.5 ml of 0.1 N EDTA was added. The pH was adjusted to 9.0 with sodium hydroxide solution, with the substrate initially included as a partially oily suspension. The reaction was started by adding 3.4 ml of carboxypeptidase Y solution (20 mg / ml) and stirred for 2.5 hours at room temperature.

The pH was maintained at 9.0 with sodium hydroxide solution. The reaction was stopped by adjusting the pH to 3 with HCl solution.

The reaction mixture was filtered and purified by R-preparative HPLC as described in Example 16.

The collected product fractions were concentrated by evaporation in vacuo and lyophilized with added HCl solution to give 0.49 g (1.3 mmol, 12%) as a white amorphous powder.

Identification

The product contained 9.2% (by weight) of chloride and no acetate. I.e. the product is present as hydrochloride.

Amino acid analysis after acid hydrolysis detected phenylalanine and no phenylalanine before acid hydrolysis.

The product could be further hydrolyzed with a base to give a product which was chromatographically different from D, L-PhePheOH and the product itself eluted 39

DK 163365 B

with an L, L-PhePheOEt standard in the HPLC system used.

Finally, the specific optical rotation using sodium D line at 25 ° C and c = 0.5 in acetic acid was determined to be -42.7 °. This compares with a pure L, L-PhePheOEt standard which gave + 52.1 ° under the same conditions. The difference here is believed to be due to the lower purity of the produced peptide.

Purity HPLC purity: 82.3% (nucleosil 7 Clg, 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 according to Karl Fisher: 7.5%.

20

Example 22

Preparation of D-tyrosyl-β-alanine, D-Tyr-β-Ala-OH via the ester D-tyrosyl-β-alanine methyl ester by enzymatic unblocking

Process 30 D-tyrosine ethyl ester (1.07 g, 5.1 mmol) and ε-alanine methyl ester (4.14 g, 40.1 mmol) are dissolved in 57 mL of 60 containing 60 mmol (22.3 mg) of disodium EDTA. and 3 mmol (363.3 mg) of TRIS. The reaction is started by adding 3.40 ml of a solution of carboxypeptidase-Y (353 µM) and maintained at 35 pH 8.3 throughout the course of the reaction.

After 2 hours, the pH is adjusted to 3.0 by the addition of 1 N

40

DK 163365 B

hydrochloric acid. The reaction mixture is diluted to 100 ml and purified by RP preparative HPLC (Waters Prep LC / System 500Ά) using a column (5.7 x 30 cm) packed with 60 µm C-18 particles and 50 mM acetic acid / ethanol-5 mixtures.

Collected fractions containing pure product were concentrated under reduced pressure and finally lyophilized.

In the process 0.78 g of D-tyrosyl-α-alanine methyl ester acetate (2.7 mmol, 58%) was obtained.

Purity HPLC purity: 98.0% (Nucleosic 7 C-18, 0.1 M ammonium phosphate, pH 3.0 / acetonitrile, 220 nm).

20 Identification

Animoic acid analysis showed the absence of free amino acids but after acidic hydrolysis the following result was obtained: 25 E-Ala (1.04) D-Tyr (0.96)

Process 30 The dipeptide methyl ester (0.65 g, 2.3 mmol) was dissolved in 40 ml H 2 O containing 4 ml 0.5 M phosphate buffer (pH 7.0). The reaction was started by adding 2.8 ml of a pig liver esterase solution (PLE, 357 µM) and maintained at pH 7.0 throughout the course of the reaction.

After 4 hours, the pH was adjusted to 3.0 by the addition of 10 N hydrochloric acid. The reaction mixture was diluted to 35

DK 163365B

41 70 ml and purified by RP Preparative HPLC (Waters Prep LC / System 500A) using a column (5.7 x 30 cm) packed with 60 »m C-18 particles and 50 mM acetic acid / ethanol mixtures .

5

Collected fractions containing pure product were concentrated under reduced pressure and finally lyophilized.

By this process 0.50 g of D-tyrosyl-10? -Alanine acetate (1.5 mmol, 65%) was obtained.

Identification

Amino acid analysis showed the absence of free amino acids, but after acidic hydrolysis the following result was obtained: i-Ala (1.04) D-Tyr (0.96) Acetate content by HPLC: 1.2% (w / w) . Chloride was not detected.

Specific optical rotation: -34.9 ° (C = 0.01 in 0.1 N HCL using the 20 ° C sodium D line).

25

Purity HPLC purity: 98.7% (Nucleosic 7 C-18, 0.1 M ammonium phosphate, pH 3.0 / acetonitrile, 220 nm).

30 35

Example 23 42

DK 163365 B

Preparation of b-alanyl-L-methionine catalyzed with g Λ carboxypeptidase-Y using b-alanine benzyl ester as a substrate component and free methionic acid as a nucleophilic component in water at 2 different pH values

Wife. of 10 Substrate Nucleophil pH Yield (%) 25 mM 0.4 M 8.0 5b) 25 mM 0.4 8.5 8 15 a) 50 nM CPD-Y, 2 mM Na2-EDTA.

b) Determined at less than 50% turnover.

20 25 30 35 43

DK 163365 B

Example 24

Preparation of dipeptides catalyzed with carboxy-α peptidases using D-amino acid ethyl esters 5 as substrate components and aminomethyl fos for acid (AMP) and 2-aminoethylsulfonic acid (2-AES) as nucleophilic components in water at various pH .

Enzyme Substrate Nucleophilic pH Yield (%) 10-CPD-Y D-tyr-OEt (50 mM) AMP (0.2 M) 9.3 2 CPD-W D-tyr-OEt (50 mM) AES (0.5 M) 8.5 4b) 15 a) 20 hM CPD-W, 2 mM Na 2 EDTA and 0.1 M NH 4 Cl (pH adjusted) b) Determined relative to hydrolysis via a standard at 20 less than 100% turnover.

25 30 35

Example 25 44

DK 163365 B

Preparation of L, L-TyrValNH2 catalyzed with carboxy peptidase Y in high organic solvent concentrations Using L-Tyrosine ethyl ester as substrate component and L-Valinamide as nucleophilic component at pH 8.5.

Wife. of u \ «\ 10 substrate Nucleophil Solution m. 'Yield (%)' 25 mM 0.5 M 92% Glycerol 53 15 a) Determined at 80% reaction, 100 µM CPD-Y, 2 mM Na2-EDTA.

b) The solvent concentration is calculated as the ratio of water to organic solvent, excl. solids in solution.

25 30 35

Claims (13)

45 DK 163365 B Patent claims; A process for the preparation of dipeptide derivatives of the general formula HABY wherein A means an optionally side chain protected L or D-α amino acid residue or ω amino acid residue, and B means an optionally side chain protected L or Da amino carboxylic acid residue or o-amino acid residue. amino acid residues which may be the same or different from A, an L or D aminophosphonic acid residue or an L or D amino sulfonic acid residue and Y is OH or a C-terminal protecting group or acid addition salts and hydrates thereof, characterized by reacting a substrate component which is an amino acid derivative of formula R2 HA-OR1 or HAN \ R3 wherein A is as defined above, R1 is hydrogen, alkyl, aryl or aralkyl optionally substituted with hydroxy, nitro or halogen or an α-des-amino fragment of a and R 2 and R 3 are the same or different and each means hydrogen, alkyl, aryl or aralkyl optionally substituted with hydroxy, nitro or halogen, 30 a having a nucleophilic component which when A = B can be formed in situ and selected of (a) amino acids of formula OD 46 DK 163365 B HB-OH where B is as defined above 5 (b) amino acid amides of formula R2 / HBN \, R 10 where B is as defined above, R2 has the above and R3 has the meaning given above for R3, except that when R2 is hydrogen, then R3 may also mean hydroxy or amino (c) amino acid esters of formula HB-OR4 wherein B is as defined above and R4 is alkyl, aryl or aralkyl and (d) straight or branched chain the aminophosphonic acids or aminosulfonic acids of formula 25 NH and / or __ an enzyme-binding promoting salt and / or a complex-binding agent, maintaining during the reaction a substantially constant pH between 5 and 10.5, and if desired, a side chain protecting group present is deprotected. or a C-terminal protecting group and / or if desired transfers the obtained dipeptide derivative into an "acid addition salt or hydrate thereof. Process according to claim 1, characterized in that the carboxypeptidase used is yeast carboxypeptidase Y. 10 Process according to claim 2, characterized in that the carboxypeptidase used is purified by affinity chromatography over an affinity resin consisting of a polymeric resin backbone having a number of benzyl-succinyl groups attached. Method according to claim 1, characterized in that the carboxypeptidase used is carboxypeptidase α from wheat penicillocarboxypeptidase S-1 or S-2 from 20 Penicillium janthinellum, a carboxypeptidase from Aspergillus saitoi or Aspergillus oryzae; pineapple) powder. Process according to claims 1-4, characterized in that the carboxypeptidase used is chemically modified or is a biosynthetic mutant of a natural form. Method according to any one of the preceding claims, characterized in that the carboxypeptidase used is immobilized. 35 Process according to any one of the preceding claims, characterized in that an aqueous is used. B reaction solution or dispersion containing from 0 to 50% of an organic solvent. Process according to claim 7, characterized in that the organic solvent used is selected from alkanols, dimethyl sulfoxide, dimethylformamide, dimethoxyethane, ethylene glycol and ethyl acetate. Process according to any one of the preceding claims, characterized in that a D- or L-amino acid ester selected from benzyl esters or straight or branched C 1 -C 6 alkyl esters optionally substituted with hydroxy is used as the substrate component. , nitro or halogen. 15 Process according to any of the preceding claims, characterized in that as an nucleophilic component an amino acid or amino acid amide of formula 20 HB -OH or HB -NHR3 Μ f wherein R8 is hydrogen or C1-C4 alkyl is used, and B is an L-amino acid residue. 25 Process according to claim 1, characterized in that an ester of the formula is used as a nucleophile component Wherein H is an amino carboxylic acid residue and R 4 is C 1 -C 6 alkyl. Process according to any one of the preceding claims, characterized in that a C-terminal protecting group is present in the dipeptide formed and the group 49 DK 163365 B is enzymatically cleaved, preferably by the same carboxypeptidase enzyme used in the preceding reaction. . Process according to any one of the preceding claims, characterized in that one or more side chain protecting groups are present in the dipeptide formed and that the group or groups are enzymatically cleaved, preferably by an esterase, lipase or proteolytic enzyme. 15 20 25 30 35