Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

• the present invention relates to a novel enzymatic process useful in the production of dipeptide sweeteners.
• it relates to an enzymatic step in a process for the production of L-Aspartic acid- ⁇ -L-phenylalanine-methyl ester and derivatives thereof.
• 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-As ⁇ - ⁇ -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.
• thermolysin 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.
• other workers have immobilised the thermolysin on an insoluble matrix and packed this into a reaction column (Oyama and Kihara, Chemtech Feb. (1984) 100-105; Oyama et al (1981) J. Org. Chem. 46 5241-5242; Nakanishi 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.
• 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.
• 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.
• a broad specificity serine carboxypeptidase such as carboxypeptidase Y to effect an aminolysi ⁇ reaction where both the substrate and thenucleophile and hence the dipeptide product of the aminolysis reaction are esters has two major disadvantages for the formation of dipeptide ester sweeteners.
• 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.
• the present invention provides an improved process for the formation of dipeptide esters particularly dipeptide ester sweeteners.
• R 1 is an acidic amino acid, or an acidic amino acid in N-terminal blocked form
• R 2 is an amino acid residue
• R 3 is an alkyl group preferably a C 1-4 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 R 1 and a compound of formula (II)
• R 2 and R 3 are as defined above in the presence of a 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.
• nucleophiles such as, for example, the amino group of L-PheOMe
• 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.
• amino acid as used herein denotes naturally occurring and non-naturally occurring amino acids.
• the amino acid residues R 1 and R 2 are linked by a peptide bond between the ⁇ carboxylic acid group of R 1 and the ⁇ amino group of R 2 .
• ⁇ denotes that the linkage is to the ⁇ carbon of the amino acid residue.
• the group R 1 may be any acidic amino acid or a salt thereof such as, for example, glutamic acid or amino malonic acid and is preferably aspartic acid.
• the group R 2 may be any amino acid residue or a salt thereof, such as, for example, methionine or tyrosine and is preferably phenylalanine.
• the R 1 ester may be an alkyl ester preferably a C 1-4 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 1 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 1 and R 2 are preferably in L-isomeric form.
• the groups R 1 and R 2 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 1 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.
• 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, trifluoroacetyl 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
• 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.
• a halogen atom e.g
• Particularly preferred serine or cysteine endopeptidases are Staphylococcal V8 protease (EC.3.4.21.19), members of the subtilisin (EC.3.4.21.14) family, cucumisin (EC.3.4.21.25) and papain (EC.3.4.22.2) and those serine or cysteine endopeptidases with similar substrate specificities to Staphylococcal V8 protease (EC.3.4.21.19).
• cysteine or serine endopeptidases may be modified, for example, genetically or chemically, but must still have similar substrate specificity to Staphylococcal V8 protease (EC.3.4.21.19).
• 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 nanomolar to micromolar concentration.
• the concentration of the esterified R 1 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 and stability such as, for example, 30 oC- 40 oC.
• 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.
• solvent includes also supercritical fluids.
• the process of the invention results in the formation of only the desired ⁇ - isomer 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.
• 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.
• R 1 and R 2 may be converted to the same or different physiologically acceptable salt forms using methods well known in the art. If R 1 and R 2 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 1 are known in the art and may conveniently be produced from the corresponding anhydride by reaction with a primary alcohol of formula (III)
• R 4 may be, for example an alkyl group, e.g. a methyl or 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 both the ⁇ - and side chain ester forms. These need not be separated since only the ⁇ -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 and treated, e.g. with a high pH solution, to remove the ester group thereby regenerating the R 1 starting material.
• Compounds of formula (II) may be prepared using methods well known in the art or may be commercially available.
• 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.
• CBz N-carbobenzoxy
• 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.
• 50mM ⁇ -N-benzoyl-L-aspartic acid- ⁇ -methyl ester (in 50% v/v 1,4-dioxane) was diluted 1 in 10 in 100mM sodium borate buffer ⁇ H9.0,or 100mM 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 warming the solution to 37 oC, and by the addition of
• Staphylococcal V8 protease (EC.3.4.21.19) to give a final enzyme concentration of 10 ⁇ M. 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 h.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.
• 10mMM ⁇ -N-carbobenzoxy-L-aspartic acid- ⁇ methyl ester and 350 mM L-phenylalanine-methyl ester were incubated with 10 ⁇ M 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.
• concentrations of ⁇ -N-carbobenzoxy-L-aspartic acid and ⁇ -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 1mM ⁇ -N-carbobenzoxy-L-aspartic acid standard (i.e. assuming that the extinction coefficients for ⁇ -N-carbobenzoxy-L-aspartic acid and phenylalanine methyl ester are approximately equal).
• 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- ⁇ -methyl ester and the enzyme concentration was reduced to 1 ⁇ M.
• 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.
• 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 ⁇ -N-carbobenzoxy-L-aspartic acid- ⁇ -benzyl ester as the substrate than for the thermolysin process described in example 7 of US patent 4436925.
• 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 ⁇ -N-carbobenzoxy-L-aspartic acid ⁇ - L-phenylalanine methyl ester is 428; thus these results confirm that the novel peak does correspond to this compound.

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• 1986
  • 1986-09-04 GB GB868621377A patent/GB8621377D0/en active Pending
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