ENZYMATIC PROCESS

Abstract: A process for the production of a dipeptide ester of formula: R1-R2- alpha -OR3, where R1 is an acidic amino acid, or an acidic amino acid in N-terminal blocked form, R2 is an amino acid residue and R3 is an alkyl group preferably a C1-4 alkyl group and most preferably a methyl, ethyl, or propyl group; a substituted alkyl group e.g. a trifluoromethyl group; an aryl group, e.g. a benzyl group; or a substituted aryl group, comprising the step of reacting together an ester of R1 and a compound of formula R2- alpha -OR3, where R2 and R3 are as defined above in the presence of a serine or cysteine endopeptidase at a pH at which aminolysis is effected.

Description: ENZYMATIC PROCESS Field of the Invention The present invention relates to a novel enzymatic process useful in the production of dipeptide sweeteners. In particular, it relates to an enzymatic step in a process for the production of L-Aspartic acid-oc- -phenylalanine-methyl ester and derivatives thereof. Background to the Invention The family of dipeptide sweeteners are currently used in many aspects of the food, brewing and pharmaceutical industries. The present invention provides a convenient, economically viable process for the production of dipeptides which may be operated on a large scale. The process of the invention is particularly useful for the production of L-Aspartic acid-«-L-phenylalanine methyl ester (L-Asρ-«-L-PheOMe, aspartame) and analogues thereof and substituted N-phenylcarbamoyl derivatives of aspartame-like compounds such as those described in European Patent Application No. EP 107597. Current manufacturing processes for aspartame involve enzymatic and/or organic chemistry techniques. The peptide chemistry approach commonly involves the coupling of N-blocked L-aspartic acid anhydride and L-phenylalanine methyl ester to generate two isomeric forms of aspartame, N-blocked L-Asρ-«-L-PheOMe and N-blocked L-Asp-β-L-PheOMe. The two isomers are formed because aspartic acid has, in addition to the <r-carboxyl group common to natural amino acids, a side chain β-carboxyl group. When the reaction is carried out under the most favourable conditions, the ratio of isomers is of the order of 80% <r to 20% β . The β-isomer has a bitter taste associated with it which therefore necessitates its removal from the mixture. It would be desirable to form only the «—isomer thereby avoiding the expense of this purification step. The conversion of the starting material to the desired product without the formation of the β-isomer would also result in a saving of raw material. This is especially important with respect to the phenylalanine methyl ester starting material which is an expensive raw material. Attempts have been made to overcome this problem by chemically blocking the β-carboxyl group and using a coupling compound to assist in peptide bond formation. This approach is however costly and has associated with it an undesirable reduction in yield at the side chain protection step. An alternative method has been developed by Toyo Soda Ltd. (Isowa et a^, Tetrahedron Letters C1979) 28.2611-2612 and US Patent No. 4436925) involving both enzymatic and organic chemistry steps. In this approach N-blocked-L-Aspartic acid and L-PheOMe are coupled by a theπαodynamic reversal of peptide bond hydrolysis which is catalysed by a metalloproteinase, thermolysin. There are, however, a number of associated disadvantages. Hydrolysis of the peptide bond is thermodynamically highly favoured and in order, therefore, to achieve good yields the reaction requires high concentrations of substrate and enzyme to drive it in the 'reverse' direction. The reaction times associated with this process are lengthy thereby adversely affecting productivity. In order for the process to work in high yield it may be necessary to remove the product, for example, by carrying out the reaction in the presence of an immiscible co-solvent in which the product is more soluble. In an attempt to make the process more economically viable other workers have immobilised the thermolysin on an insoluble matrix and packed this into a reaction column (Oyama and Kihara, Che tech Feb. (1984) 100-105; Oyama et al (1981) J. Org. Chem. 46. 5241-5242; Nakaniεhi et al (1985) Biotechnology 3 ^ 459-464). This is, however, not believed to be an efficient process to operate on a large scale since the N-blocked aspartame precipitates into the pores of the matrix lowering the activity of the column. Additionally, the calcium essential for enzyme activity leaches out, thereby causing loss of activity of the thermolysin. The production of N-blocked dipeptide esters using serine and cysteine peptidases has been described (Autumn Conference Chem. Soc. Japan (1976) p482-486; US Patent No. 425836). The reactions involved were thermodynamic reversal procedures analogous to, and suffering from similar limitations and disadvantages as, the thermolysin catalysed reaction described above. Carboxypeptidases are exopeptidases which cleave the C-terminal bond of proteins and peptides. Serine and cysteine carboxypeptidases in addition to cleaving peptide bonds can also cleave C-terminal ester and amide bonds. Serine and cysteine proteases form a covalent substrate-enzyme intermediate which is subject to subsequent nucleophilic attack. Normally water attacks to give net hydrolysis but stronger nucleophiles can compete with this reaction resulting in formation of a new chemical bond. This type of reaction is known as aminolysis. European Patent Application No. EP 17485A discloses a process which includes production of N-blocked dipeptide esters by reacting an N-terminal blocked amino acid ester with a non N-blocked amino acid ester using a carboxypeptidase enzyme. The use of a broad specificity serine carboxypeptidase such as carboxypeptidase Y to effect an aminolysiε reaction where both the substrate and thenucleophile and hence the dipeptide product of the a inolysis reaction are esters has two major disadvantages for the formation of dipeptide ester sweeteners. Firstly, since both the product of the aminolyεis reaction and the nucleophile are substrates for the carboxypeptidase, polymerisation of the nucleophile onto the carboxyl end of the dipeptide product occurs resulting in the formation of tripeptides and larger polypeptides. Secondly, the C-terminal ester group which is an essential part of the dipeptide sweetener is hydrolysed by the enzyme thereby destroying the desired sweet taste. The production of dipeptide ester sweeteners using the above approach would not, therefore, be practical. Several publications disclose the use of proteases for peptide bond formation (for example see: Widmer et al in Peptides 1984 (1984) ed. U. Ragnarsson, 193-200; Schellenberger et al in Peptides 1984 (1984) ed. U. Ragnarsson, 201-204 and Jakubke et al (1985) Angew. Chem. Int. Ed. Engl. 24. (2) 85-93). In particular, in a study of the enzymatic synthesis of fragments of mouse epidermal growth factor, Widmer et al have used a number of enzymes including, CPDY, trypsin, chymotrypsin and V8 protease to catalyse peptide bond formation via an aminolysis reaction mechanism. The peptide bonds formed were however between peptide fragments and there is no teaching in this reference to make dipeptide ester sweeteners. Jakubke et al J. loc. cit. also discuss the use of proteolytic enzymes for the formation of peptide bonds. This reference does not, however, teach the use of endopeptidases to synthesise dipeptide ester sweeteners. Staphylococcal V8 protease has previously been used as a catalyst for the formation of a peptide bond between two polypeptides in a theπnodynamic reversal of hydrolysis (J. Cell. Biochem. Suppl. 9B (1985) 127). There is no teaching in this reference to the use of Staphylococcal V8 protease to produce dipeptide esters or to exploit the aminolysis reaction mechanism. The present invention provides an improved process for the formation of dipeptide esters particularly dipeptide ester sweeteners. Summary of the Invention According to the invention we therefore provide a process for the production of a dipeptide ester of formula (I) R -R -«-OR_, (I) 1 2 3 where R is an acidic amino acid, or an acidic amino acid in ^,-terminal blocked form, R is an amino acid residue and R is an alkyl group preferably a C alkyl group and most preferably a methyl, ethyl, or propyl group; a substituted alkyl group e.g. a trifluoromethyl group; an aryl group, e.g. a benzyl group; or a substituted aryl group, comprising the step of reacting together an ester of R and a compound of formula (II) where R and R are as defined above in the presence of a 2 3 serine or cysteine endopeptidase at a pH at which aminolysis is effected. The process of the present invention exploits the fact that serine and cysteine proteases use similar reaction mechanisms which are fundamentally different to the mechanisms used by the metalloproteinases. A covalent substrate-enzyme intermediate is formed by the serine or cysteine protease and this is then normally attacked by water to give net hydrolysis but stronger nucleophiles such as, for example, the amino group of L-PheOMe can compete with this reaction resulting in aminolysis and formation of a new chemical bond. At alkaline pH serine and cysteine proteases will break ester bonds more efficiently than peptide bonds. This means that by employing a serine or cysteine protease with restricted specificity under appropriate conditions an N-blocked amino acid ester and a non N-blocked amino acid ester can be combined to form an N-blocked dipeptide ester that will accumulate without the need to be continually removed. The term amino acid as used herein denotes naturally occurring and non-naturally occurring amino acids. The amino acid residues R and R„ are linked by a peptide 1 2 bond between the = carboxylic acid group of R and the = amino group of R . 2 As used herein the symbol « denotes that the linkage is to the «= carbon of the amino acid residue. The group R may be any acidic amino acid or a salt thereof such as, for example, gluta ic acid or amino malonic acid and is preferably aspartic acid. The group R may be any amino acid residue or a salt thereof, such as, for example, methionine or tyrosine and is preferably phenylalanine. The R ester may be an alkyl ester preferably a C alkyl ester and most preferably a methyl, ethyl, or propyl ester; a substituted alkyl ester e.g. a trifluoromethyl ester; an aryl ester e.g. a benzyl ester; or a substituted aryl ester. The R ester may be an "--ester group or a mixture of «- and side chain esters. Since only the «-ester will participate in the reaction, unreacted side chain ester may easily be recovered, processed and recycled. R and R are preferably in L-isomeric form. In order to produce dipeptide ester sweeteners, the groups R and R should be chosen according to the teaching of the art, see for example, Fujino et al_ (1976) (Chem. Pharm. Bull. 24 2112-2117). The group R may be blocked at the N-terminus with any suitable blocking group which may if desired, be removed after dipeptide formation using physiochemical methods well known in the art (see for example, Peptide Synthesis eds. Bodanszky and Ondetti (1966) Interscience Publishers) .without affecting the rest of the molecule. Alternatively, the N-terminal blocking group may be amenable to removal by enzymatic means as described for example in UK Patent Application No. GB 2160870. Suitable N-terminal blocking groups include for example, aryl-lower alkyl groups, such as diphenylmethyl or triphenylmethyl groups which may be optionally substituted by halogen, nitro, lower alkyl or lower alkoxy groups; aroyl groups such as benzoyl groups; acyl groups, such as formyl, acetyl, acetoacetyl, tri luoroacetyl and benzenesulphenyl groups; groups derived from carbonic acid or thiocarbonic acid, which are optionally substituted in the aromatic radical by halogen atoms, nitro groups or lower alkyl, lower alkoxy or lower carboalkoxy groups, for example carbobenzoxy, p-methoxycarbobenzoxy and p-chlorocarbobenzoxy; benzyloxycarbonyl groups; aliphatic oxycarbonyl groups, such as alkoxycarbonyl groups for example t-butoxycarbonyl; aliphatic oxycarbonyl groups substituted by for example a halogen atom e.g. a 1,1,1 trichloroethyloxycarbonyl group or a fluorenyl methoxy carbonyl group; carbamoyl, phenylthiocarbamoyl or phenylcarbamoyl groups which may be substituted by for example an acyl group, a thioacyl group or a cyano group. Particularly preferred blocking groups are carbobenzoxy, phenylthiocarbamoyl and phenylcarbamoyl groups which may if desired be substituted by, for example, a halogen atom e.g. a fluorine atom; an alkyl carbonyl group, e.g. a methyl carbonyl group; an alkoxycarbonyl group, e.g. a methoxy or ethoxycarbonyl group; a cyano group; a nitro group; or a sulphonyloxy group. Particularly preferred serine or cysteine endopeptidases are Staphylococcal V8 protease (EC.3. .21.19) , members of the subtilisin (EC.3.4.21.14) family, cucumisin (EC.3. .21.25) and ^a i (EC.3. .22.2) and those serine or cysteine endopeptidases with similar substrate specificities to Staphylococcal V8 protease (EC.3. .21.19). The cysteine or serine endopeptidases may be modified, for example, genetically or chemically, but must still have similar substrate specificity to Staphylococcal V8 protease (EC.3. .21.19) . The process of the invention is carried out at a pH at which the serine or cysteine endopeptidase effects an aminolysis reaction. The pH is generally alkaline, such as, for example, pH 7 to 12 and is preferably in the range 8 to 10. The serine or cysteine endopeptidase may be employed in a soluble form. Alternatively, it may be employed in an insoluble or in an immobilised form thereby increasing enzyme stability and facilitating operation of the process. The concentration of enzyme used in the process may be in the range molar to nanomolar and is preferably at nano olar to micromolar concentration. The concentration of the esterified R amino acid is preferably in the range micromolar to millimolar and the concentration of compounds of formula (II) is preferably in the range molar to millimolar. The yield of the process is generally proportional to the concentration of compounds of formula (II) and it may therefore be advantageous to use such compounds at as high a concentration as is practicable. The serine or cysteine endopeptidase used in the process of the invention may conveniently be produced by a culture of a strain of microorganism capable of producing the desired enzyme either naturally or after genetic manipulation using techniques well known in the art. The cysteine or serine endopeptidase may be either partially or completely purified from the cells or culture medium using methods well known in the art. The process of the reaction is carried out at a temperature at which the cysteine or serine endopeptidase shows maximal activity o o and stability such as, for example, 30 C - 40 C. The reaction is preferably carried out in an aqueous environment, but may, if desired, be performed in the presence of a water miscible solvent or in an aqueous water miscible/water immiscible solvent mixture or in an aqueous environment in the presence of an immiscible solvent. The solvent may have the effect of increasing the reaction yield. The term solvent includes also supercritical fluids. The process of the invention results in the formation of only the desired =—isorαer of the dipeptide thereby avoiding the uneconomic production and removal of the unwanted side chain ester. The process of the invention makes use of an enzymatic reaction which is thermodynamically favoured. This results in a fast reaction time and the requirement for relatively low concentrations of enzyme. This may have a beneficial effect on the cost involved in the production of dipeptide ester sweeteners using the process of the invention. Additionally the lack of the requirement for an immiscible co-solvent, the stability of the enzyme used in the process of the reaction and the absence of enzyme cofactor requirements make the process of the invention convenient to operate on a large scale in a batch or continuous mode. In addition, the process of the invention couples L-isomers with high specificity and it may therefore be possible to use D/L mixtures of starting materials. Where it is desired to produce compounds of formula I in salt form, this may be achieved using methods well known in the art. If desired, when R has an acidic side chain both or either of R, and R may be converted to the same or different 1 2 physiologically acceptable salt forms using methods well known in the art. If R and R contain a free amino group then this may be converted to the same or different physiologically acceptable acid addition salt using methods well known in the art. See for example US Patents 4029701, 3714139, 4411925, 4031258 and 4029701 and European Patent Application No. EP 95772. Esters of R are known in the art and may conveniently be produced from the corresponding anhydride by reaction with a primary alcohol of formula (III) R, - OH (III) 4 c where R may be, for example an alkyl group, e.g. a methyl or => 4 ethyl group; a substituted alkyl group; an aryl group, e.g. benzyl; or a substituted aryl group. The reaction may be catalysed by the presence of a weak acid having a pKa value of around 4 such as, for example, acetic acid. The reaction forms 10 both the <*- and side chain ester forms. These need not be separated since only the < χ -isomer will undergo enzyme-catalysed aminolysis according to the process of the invention. The unreacted side chain ester can easily be separated from the dipeptide ester product produced by the process of the invention 5 and treated, e.g. with a high pH solution, to remove the ester group thereby regenerating the starting material. Compounds of formula (II) may be prepared using methods well known in the art or may be commercially available. The invention is further illustrated in the following non-limiting examples and with reference to the following Tables details of which are given below. Table 1: shows percentage yields of the enzyme-dependant hydrolysis and aminolysis products using N-benzoyl (Bz) L-Aspartic-acid- «-OMe as substrate i.e. N-Bz-L-Asp-OH and N-Bz-Aspartame. Table 2: shows percentage yields of the enzyme-dependant hydrolysis and aminolysis products using N-carbobenzoxy (CBz) blocked L-Aspartic acid esters as substrates i.e. N-CBz-L-Asp-OH and N-CBz-Aspartame. Table 3: shows comparison of reaction parameters of V8 protease catalysed reaction using CBz-Asp-OMe and CBz-Asp-OBz as substrates with those of the thermolysin catalysed reaction. Table 4: shows the conditions for reverse phase hplc analyses and retention times of substrates and products. Detailed Description of the Embodiments Example 1 g -N-BENZOYL-L-ASPARTIC ACID-°cMETHYL ESTER AS SUBSTRATE 50mM β -N-benzoyl-L-aspartic acid-«-methyl ester (in 50% v/v 1,4-dioxane) was diluted 1 in 10 in lOOmM sodium borate buffer ρH9.0,or lOOmM sodium carbonate/sodium hydrogen carbonate buffer pH9.0. Solid L-phenylalanine methyl ester was added to give a concentration of 250mM, and the pH was adjusted back to 9.0 with concentrated sodium hydroxide. The reaction was initiated by o warming the solution to 37 C, and by the addition of Staphylococcal V8 protease (EC.3. .21.19) to give a final enzyme concentration of 10μM. After various incubation times, aliquots were removed and further reaction was stopped by addition of one-fifth volume of 1% v/v trifluoroacetic acid/acetonitrile. These aliquots were analysed by reverse phase t p.l.c. using a C18 column (Rainin Microsorb, 15cm x 4.6mm id.) and the eluate was monitored for absorbance at 260nm. Table 4 shows the analysis conditions and gives the retention times for the various substrates and products. A novel peak was observed that was more hydrophobic than the substrate and its hydrolysis product. The percentage yields of this peak and those of substrate hydrolysis product peaks, expressed as areas of the peaks, are shown in Table 1. The identity of this peak was analysed as follows. The eluate containing this peak was collected, dried in vacuo. and then o hydrolysed in 6M hydrochloric acid for 16 hours at 105 C. The hydrolysate was dried iii vacuo and assayed by amino acid analysis on reverse-phase h.p.l.c. using precolumn derivatisation with o-ρthalaldehyde as described by D.C. Turnell and J.D.H. Cooper (Clin. Chem. (1982) 28, 527-531). The analysis showed approximately equimolar amounts of aspartic acid and phenylalanine. This is consistent with the peak being < χ -N-benzoyl-L-aspartic acid-=-L-phenylalanine methyl ester. Table 1 le IncubaLion time 15 ins 30 mins 60 mins 240 mins « -. N-Bz- N-Bz- N-Bz N-Bz- N-Bz- N-Bz- N-Bz- N-Bz- hi L-Asp- Oil aspartame -A.sp-011 aspartame L-Asp- H aspartame L-Asp-OH aspartame (ft Borate pllQ.O 12 10 12 14 14 14 20 16 Carbonate pH9.0 16 12 12 10 16 14 24 20 π I Example 2 g -N-CARBOBENZOXY-L-ASPARTIC ACID- <=-METHYL ESTER AS SUBSTRATE lOrπM ec-N-carbobenzoxy-L-aspartic acid-α methyl ester and 350 M L-phenylalanine-methyl ester were incubated with 10 μM Staphylococcal V8 protease (EC. 3.4.21.19) at 30C in buffer or water at pH8.5 or 9.0 and the presence of 5% v/v 1,4 dioxane. The buffer was either bistris propane or bicine at 50mM. Aliquots were removed at intervals and further reaction was stopped by the addition of one-fifth volume of 1% v/v trifluoroacetic acid/acetonitrile. These aliquots were then assayed by reverse-phase h.p.l.c on a C18 column and the eluate was monitored for absorbance at 260mm. The analysis conditions and retention times of the peaks are given in Table 4. A novel product peak was observed with a retention time similar to that predicted for α-N-carbobenzoxy-L-aspartic acid-«-L-phenylalanine-methyl ester. The results, expressed as percentage yields of «-N-carbobenzoxy-L-aspartic acid-«-L-phenylalanine-methyl ester and the substrate hydrolysis product, «-N-carbobenzoxy-L-aspartic acid, are presented in Table 2. The concentrations of or-N-carbobenzoxy-L-aspartic acid and α-N-carbobenzoxy-L-aspartic acid «-methyl ester are calculated from the areas of the peaks by comparison to standards of known concentration. This could not be done for «-N-carbobenzoxy-L-aspartic acid ■"•"-phenylalanine methyl ester since standards were not available. Thus, in order to convert peak areas for this compound to approximate concentrations the area was divided by twice the area obtained for a lmM e c-N-carbobenzoxy-L-aspartic acid standard (i.e. assuming that the extinction coefficients for ec-N-carbobenzoxy-L-aspartic acid and phenylalanine methyl ester are approximately equal). Example 3 g -N-CARBOBENZOXY-L-ASPARTIC ACID cc-BENZYL ESTER The experimental conditions are identical to those described in Example 2, except that <∑-N-carbobenzoxy-L-aspartic acid-«- benzyl ester was substituted for «-N-carbobenzoxy-L-aspartic acid-o.-methyl ester and the enzyme concentration was reduced to IμM. The novel product peak observed by the reverse-phase hplc analysis in the presence of the protease and L-phenylalanine-methyl ester had a retention time identical to that of the novel product peak observed with *-N-carbobenzoxy-L~aspartic acid-«-methyl ester as substrate. (See Table 4). The results are presented in Table 2, Areas of the various compounds were converted to concentrations as described in Example 2. Table 3 shows the reaction parameters obtained under the conditions described in this example and the previous example, and compares them with those calculated for the thermolysin process from data given in US patent 4436925. With °--N-carbobenzoxy-L-aspartic acid- β --benzyl ester as the substrate the specific productivity is at least 50 fold greater than that obtained for the thermolysin process. The fractional conversion and volumetric production rate are both greater for the V8 protease catalyzed reaction with ot-N-carbobenzoxy-L-aspartic acid-«-benzyl ester as the substrate than for the thermolysin process described in example 7 of US patent 4436925. All four reaction parameters could be expected to improve for the V8 protease catalyzed reaction if a higher concentration of phenylalanine methyl ester was used, or the water activity was decreased by the presence of an organic solvent. In addition if a higher concentration of <*-N-carbobenzoxy-L-aspartic acid—x-benzyl ester was used this would also be expected to improve all four reaction parameters (the concentration used in these examples is lOmM compared to 1.25M and 0.29M used in examples 7 and 19 of US patent 4436925). Table 2 Incubation Lime 15 mins 60 mius 240 mins CBz-L-Asp-OH CBz-Asp flame CBz-L-Asp-OH CBz-Aspartame CBz-L-Asp-OH CBz-Aspαrtame CBz-L-Asp-«- OMo 10»M enzyme pll8.5 26 9 61 39 58 42 pUO.O nd nd 34 37 50 47 pH8.5 28 15 62 38 58 42 ρllθ.0 16 I'I 46 44 51 49 pll8.5 14 13 48 48 48 52 pll9.0 11 13 44 44 49 51 C I Bis Li * is p opane ρllβ.5 69 31 66 34 nd nd Bistris propane ρll9.0 41 3 49 51 nd nd Bicine pllB.5 56 49 51 nd nd Bicine ρllQ.0 51 48 66 34 nd nd Water ρll8.5 56 4 52 48 nd nd Water pllQ.O 52 7 51 49 nd nd Example 4 Identification of novel peak as «-N-carbobenzoxy-L-asρartic acid-L-phenylalanine methyl ester by Fast atom bombardment mass spectrometry The experimental conditions were as described in Example 3. Table 4 shows the analysis conditions of the hplc analysis and the retention times for the peaks. Aliquots of the eluate from the hplc column corresponding to the novel peak were collected from several hplc runs. The pooled aliquots were dried in vacuo and redissolved in 5% v/v acetic acid. An aliquot of this was applied to the target of a VG Analytical ZAB high field mass spectrometer. A fast atom bombardment was carried out in the positive ion mode and an (M + H) molecular ion of m/z 429 was observed. The predicted molecular weight of acid "-"-L-phenylalanine methyl ester is 428; thus these results confirm that the novel peak does correspond to this compound. Example 5 c-N-carbobenzoxy-L-κlutamic acid g -benzyl ester as substrate lOmM <=-N-carbobenzoxy-L-glutamic acid <*-benzyl ester and 400mM phenylalanine methyl ester were incubated in the absence or presence of IμM Staphylococcal V8 protease (EC.3. .21.19) at o 30 C in 40mM Bis Tπs propane buffer pH 9.0, 10% v/v 1,4 dioxane. Aliquots were removed at 15 and 60 minutes and further reaction stopped by addition of acetonitrile and trifluoracetic acid to give 20% and 0.2% respectively. These aliquots were assayed by reverse-phase hplc. The conditions used for the analysis are given in Table 4 together with the retention times of the peaks. A novel peak was observed in the presence of enzyme which did not correspond to "--N-carbTsbenzoxy-L-glutamic acid. The retention time of this peak differed from that of <=-N-carbobenzoxy -L-aspartic acid-L-phenylalanine methyl ester by a similar amount to the difference between the retention times of «"-N-carbobenzoxy-L-asρartic acid =-benzyl ester and «-N- carbobenzoxy-L-glutamic acid β -benzyl ester. Thus, this peak is believed to correspond to «-I.-carbobenzoxy-L-glutamic acid- ^-phenylalanine methyl ester. Some of the hydrolysis product, <=-N-carbobenzoxy-L-glutamic acid was also observed. Example 6 L-Phenylalanine ethyl ester as the nucleophile lOniM cr_N-carbobenzoxy-L-aspartic acid α-benzyl ester and 400mM L-phenylalanine ethyl ester or L-phenylalanine methyl ester o were incubated with lμM Staphylococcal V8 protease at 30 C in 40mM Bis Tris propane HC1 buffer pH 9.0, 10% v/v, 1,4-dioxane. After 30 minutes the reaction mixtures were analysed by reverse-phase hplc after addition of acetonitrile and phenylmethyl sulphonyl fluoride to 20% and 5mM respectively. The analysis conditions and retention times of the peaks are given in Table 4. A novel peak was observed in the presence of enzyme and L-phenylalanine-ethyl ester which had a larger retention time than that of the peak observed with L-phrnylalanine-methyl ester. This novel peak is believed to correspond to °=-N-carbobenzoxy-L-aspartic acid-°--L- phenylalanine ethyl ester. Example 7 D-phenylalanine methyl ester as the nucleophile lOmM =-N-carbobenzoxy-L-aspartic acid ec-benzyl ester was incubated with either 400mM L-phenylalanine methyl ester or 400mM D-phenylalanine methyl ester in the presence of luM Staphylococcal V8 protease in 40mM Bis Tris propane buffer pH9, 10% v/v 1,4-dioxane. Aliquots were removed at 15, 60 and 240 minutes o after incubation at 30 C and further reaction was stopped by addition of acetonitrile and trifluoroacetic acid to 20% and 0.2% respectively. These aliquots were analysed by reverse-phase hplc using the conditions described in Table 4. The retention times of the substrate and product peaks are also given in Table . In the presence of either the D- or the L- .somer a novel peak with retention time of 22.8 minutes was observed, corresponding to «-N-carbobenzoxy-aspartic acid-°--phenylalanine methyl ester. After 15 minutes incubation in the presence of either isoraer all the β -ϊl-carbobenzoxy-aspartic acid =-benzyl ester had been used. The amount of ^-N-carbobenzoxy-aspartic acid ■ ^-phenylalanine methyl ester formed with the D-isomer was 15% of that formed with the L-isomer. Thus, D-phenylalanine methyl ester will participate as the nucleophile in the aminolysis reaction, but at slower rate than does L-phenylalanine methyl ester. Table 3 Comparison of reaction parameters of V8 protease catalyzed reaction usinR CBz-Asp-OMe and CBz-Asp-OBz as substrates with those of the thermolysin catalyzed reaction CBz-Asp-OMe CBz-Asp-OBz Thermolysin CBz-Λsp CBz-Asp-PheOMe CBz-Asp CBz-Asp-PheOMe CBz-Asp-PheOMe d e Fractional Conversion (mole/mole) 0.50 0.47 0.51 0.49 0.12 0.61 V*' NJ Volumetric Titre (g/li) 1.3 2.0 1.3 2.2 119 144 ω "" Volumetric production m rate (at 15 mins)(g/li/hr) 0.9 1.6 4.4 7.4 2.5 36 in Specific productivity (g/li/nmole enzyme) 1.0 0.7 6.5 10.9 0.2 0.03 a = 1.0 ιιM V8 protease c = parameters calculated from examples 7(d) and 19(e) in b = 1 μM V8 protease US Patent No. 4436925 Table 4 Conditions for Reverse-Phase HPLC analyses and retention times of substrates and products Example Hplc Column Buffer Substrate Retn Nucleophile Retn Product Retn Hydrolysis Retn Number System System Time Time Time Product Time Gilson Hainin a Bonzoyl- 13.5 PheOMe 12.5 Benzoyl- 22.3 Benzoyl-Asp 9.1 microsorb 5u Asp- OMe Asp-PheOMe <0. 25cm) ϊ Gilson Kainin b CBz-Asp-OMe 16.3 PheOMe 11.5 CBz-Asp- 19.3 CBz-Asp 14.0 microsorb 5u PheOMe (0.4x25cm) • I Gilson Rainin b CBz-Asp-OBz 20.4 PheOMe 11.5 CBz-Asp- 19.3 CBz-Asp 14.0 microsorb 5u PheOMe l (0.4x25cm) Waters Super Pac 5u b CBz-Asp-OBz 23,7 PheOMe 15.6 CBz-Asp- 22.8 CBz-Asp 17.7 5 (0,4x ) PheOMe Waters Super Pac 5u b CBz-Glu-OBz 23,8 PheOMe 15.6 CBz-Glu- 22.8 CBZ-Glu 17.9 (0.4x ) PheOMe Waters Novapak 4u c CBz-Asp-OBz 19.7 PheOEt 13.5 CBz-Asp- 19.9 CBz-Asp 13.7 (0.4x15cm) PheOEt CBz-Asp-OBz 19.7 PheOMe 11.9 CBz-Asp- 19.0 CBz-Asp 13.6 PheOMe Waters Super Pac 5u b CBz-Asp-OBz 23.5 D-PheOMe 15.2 CBz-Asp-D- 22.8 CBz-Asp 17.7 (0.4x ) PheOMe CUz-Asp-OBz 23.5 L-PheOMe 15.4 CBz-Asp-L- 22.8 CBz-Asp 17.7 PheOMe a, 0.1% TI'A=A; 0.1% Tl-A/acetonitrile-^B b. 0,1% TI'A=Λ; 0,1% T.'A/acetonitrileaB c. 0,1% TFA, 02% pentane sulphonic acid=A 12%B 0 - 5 min 0%B 0 - 2 min acetonitrile=B 12-50%B 5 - ?2 min 0-80%B 2 - 25 min 0%B 0- 2 min 50-1.00%B 22 - 24 min 0-80%B 2-25 min

Claims: CLAIMS 1. A process for the production of a dipeptide ester of formula (I) R l " R 2 " α " ° R 3 (I) where is an acidic amino acid or an acidic amino acid in N-terminal blocked form, R is an amino acid residue and R is an alkyl group, a substituted alkyl group; an aryl group or a substituted aryl group, comprising the step of reacting together an ester of R_ and a compound of formula (II) R 2 - α - O (II) where R_, and R„ are as defined above in the presence of a 2 3 serine or cysteine endopeptidase at a pH at which aminolysis is e fected. 2. A process according to Claim 1 wherein the serine or cysteine endopeptidase is staphylococcal V8 protease. 3. A process according to Claim 1 wherein the pH is in the range 7 to 12. 4. A process according to Claim 1 wherein R is aspartic acid, R is phenylalanine and R is methyl. 5. A process according to Claim 1 wherein R and R are in L-isomeric form. 6. A process according to Claim 1 wherein the reaction is carried out at a temperature of 30 C to 40 C.

Inventors: BENNETT ALAN DAVID, BENNETT, ALAN, DAVID
Applicants: CELLTECH LTD, CELLTECH LIMITED
Application year: 1987
Main classification: C12P21
IPC codes: C12P21