MXPA98010759A - A METHOD FOR PREPARING A&bgr;-LACTAM ANTIBIOTIC - Google Patents

Abstract

The invention relates to a method for preparing a'beta'-lactam antibiotic, wherein an N-substituted'beta'-lactam, having general formula (I), wherein R0 is hydrogen or C1-3 alkoxy;Y is CH2, oxygen, sulfur, or an oxidized form of sulfur;Z is (a), (b), (c) or (d), wherein R1 is hydrogen, hydroxy, halogen, C1-3 alkoxy, optionally substituted, optionally containing one or more heteroatoms, saturated or unsaturated, branched or straight C1-5 alkyl, preferably methyl, optionally substituted, optionally containing one or more heteroatoms C5-8 cycloalkyl, optionally substituted aryl or heteroaryl, or optionally substituted benzyl;and X is (CH2)m-A-(CH2)n, wherein m and n are the same or different and are chosen from the group of integers 0, 1, 2, 3 or 4, and A is CH=CH, CC, CHB, C=O, optionally substituted nitrogen, oxygen, sulfur or an optionally oxidized form of sulfur, and B is hydrogen, halogen, hydroxy, C1-3 alkoxy, or optionally substituted methyl, or a salt thereof, is contacted with at least one dicarboxylate acylase, or a functional equivalent thereof, and reacted with a precursor for a side chain of the'beta'-lactam antibiotic in the presence of at least one penicillin acylase, or a functional equivalent thereof.

Description

r METHOD FOR THE PRERJBPCIOTIF OF;. AN ANTIBIOLET ICO BETA U XPM Background of the Invention The invention relates to a method for the preparation of a β-lactam antibiotic. The class of β-lactam antibiotic, such as the antibiotics penicillin and cephalosporin, comprise a large variety of compounds, all having their own activity profile. In general, β-lactam antibiotics consist of a nucleus, the so-called β-lactam nucleus, which is linked through its primary amino groups to the so-called side chain by means of an ineal 1-amide bond. The ß-lactam nuclei are very important intermediary groups in the preparation of penicillin and cephalosporin semisynthetic antibiotics. The routes for preparing these penicillins and cephalosporins are isynthetic start mainly from the fermentation products such as penicillin G, penicillin V and Cephalosporin C, which are converted into the corresponding ß-lactam nuclei REF: 28992, for example in a manner as described in K. Matsumoto, Bioprocess, Techn., 16 (1993), 67-88, JG Shewale & H. Sivaraman, Process Biochemistry, August 1989, 146-154, T.A. Savidge, Biotechnology of Industrial Antibiotics (Ed. E.J. Vandamme) Marcel Dekker, New York, 1984, or J.G. Shewale et al., Process Biochemistry, International, June 1990, 97-103. Examples of ß-lactam nuclei that are "used as a precursor for the various antibiotics are 6-aminophenicillanic acid (6-APA), 7-aminocephalosporanic acid (7-ACA), 3-chloro-7-aminodeoacetoxydesmethyl-cephalosporanic acid ( 7-ACCA), 7-amino-desacetyl-cephalosporanic acid (7-ADAC), and 7-amino-deacetoxycephalosporanic acid (7-ADCA) The ß-lactam nuclei are converted to the desired antibiotic by coupling to a suitable side chain , as has been described, among others, in European Patent EP 0 339 751, Japanese Patent JP 53005185 and Swiss Patent CH 640 240. Through the realization of different combinations of side chains and ß-lactam nuclei, it is possible to obtain a variety of penicillin and cephalosporin antibiotics, having all their own activity profiles, for example, D- (-) -phenylglycine, or an appropriate derivative thereof, such as an amide or ester, can be coupled to any of 7-ACA, 7-ACCA, 7-ADCA and 6-APA to produce Cefalogl icina, Cefaclor, Cephalexin or Ampicillin, respectively. Other examples of the frequently used side chains are D - (-) - 4- "hydrophenylglycine, 2-cyanoacetic acid and 2- (2-amino-4-thiazolyl) -2-ethoxy-oxatoic acid. the preparation of the β-lactam antibiotics involves all the preparation of a β-lactam nucleus and the subsequent coupling thereof to an appropriate side chain The references for enzymatic synthesis are: TA Savidge, Biotechnology of Industrial Antibiotics (Ed. EJ Vandam e) Marcel Dekker, New York 1984, JG Shewale et al., Process Biochemistry International, June 1990 97-103, EJ Vandamme, Advances in Applied Microbiology, 21, (1997), 89-123 and EJ Vandamme, Enzyme Microb. Technology, .5, (1983), 403-416 In addition, new routes have been described, which show the direct fermentative production of 7-ADCA and 7-ACA, in EP 0 532 341, EP 0 540 210, WO 93/08287, WO 95/04148 and WO 95/04149. One of these methods is that the coupling reaction of the side chain starts from the ß-lactam nucleus, which has to be isolated before the coupling reaction. In the isolation of the β-lactam nucleus, which is usually carried out by crystallization, up to about 10% of the theoretical yield is lost. Due to the amphoteric nature of the β-lactam nucleus, it dissolves rapidly in an aqueous environment at any pH value, and a large part of the production of the β-lactam nucleus is lost in the mother liquor of crystallization. The present invention overcomes the above disadvantage by introducing the side chain in a reaction starting from a material other than a β-lactam nucleus.

Description of the invention An object of the invention is to provide a method for the preparation of a β-lactam antibiotic, wherein the side chain is introduced in a reaction starting from a material other than a β-lactam nucleus. A further objective of the invention is to provide a method for the preparation of a β-lactam antibiotic, which method can suitably be combined with the known enzymatic processes starting from the fermentation products such as penicillin G or Cephalosporin C. Another objective More of the present invention is to provide a method for preparing a β-lactam antibiotic, which method is a clean, efficient and economically feasible process, in other words whose method does not result in effluent problems or involve expensive chemicals. It has been found that the requirements of the above objectives can be met in a method for the preparation of a β-lactam antibiotic, wherein an N-substracted β-lactam has the general formula (I) (I) where R0 is hydrogen or alkoxy of 1 to 3 carbon atoms And it is CH2, oxygen, sulfur, or an oxidized form of sulfur; Z is where Ri is hydrogen, hydroxyl, halogen, alkoxy of 1 to 3 carbon atoms, alkyl of 1 to 5 carbon atoms, straight or branched, saturated or unsaturated, optionally substituted, or optionally containing one or more heteroatoms, preferably methyl, cycloalkyl from 5 to 8 carbon atoms, optionally substituted, optionally containing one or more optionally substituted heteroatoms, aryl or heteroaryl, or optionally substituted benzyl; and X is (CH2) m_A- (CH) 2) n, where m and n are the same or different and are chosen from the group of integers 0, 1, 2, 3 or 4, and A is CH = CH, C = C, CHB, C = 0, optionally substituted nitrogen, oxygen, sulfur or an optionally oxidized form of the sulfur, and B is hydrogen, halogen, hydroxyl, alkoxy of 1 to 3 carbon atoms, or optionally substituted methyl, or a salt of they are contacted with at least one dicarboxylate acylase, or a functional equivalent thereof, and are reacted with a precursor for a side chain of the β-lactam antibiotic in the presence of at least one penicillin acylase or a functional equivalent thereof. Surprisingly, it has been found that β-lactam antibiotics can be efficiently prepared by introducing the side chain of the β-lactam antibiotic in a reaction starting from an N-substimated β-lactam, and where Two enzymes that have different substrates are used. In the process of the invention, it is not necessary to recover the intermediates, for example, the product of the first enzymatic reaction, before the application of the second enzyme. Because the N-substigated β-lactams can also be prepared from the fermentation products, such as penicillin G, penicillin V, cephalosporin C, adiply-7-ADCA, 3-carboxyetho 1 -thopropionyl-7-ADCA , 2-carboxylethylthioacetyl-7-ADCA, 3-carboxyethylthiopropionyl-7-ADCA, adipyl-7-ACA, 3-carboxyethylthiopropionyl-7-ACA, 2-carboxylethylthioacetyl-7-ACA and 3-carboxyethylthiopropionyl-7-ACA, a A great advantage of the invention lies in the fact that it is now possible to enzymatically prepare the β-lactam antibiotics, starting from such fermentation products, without the isolation of a β-lactam core intermediate, whose isolation causes a significant loss of the product. A method according to the invention is a clean and highly specific process. This means that by-products are hardly generated or generated, which could cause effluent and / or purification problems. In addition, a method according to the invention does not require the use of complex and expensive reagents, which are difficult to use due to their sensitivity. Surprisingly, it has been found that no significant effect of enzymatic inhibition occurs in a method according to the invention. Hitherto it has been believed that transacylation using one or two enzymes in the preparation of the β-lactam antibiotics is not possible due to an enzymatic inhibition effect. It is expected that in the transacylation reaction phenylacetic acid or phenoxyacetic acid could be formed, whose acids act as inhibitors for certain enzymes, as reported by U. Schommer et al., Applied and Environment Microbiology, (February 1984), 307-312 and by A.L. Margolin and collaborators in Biochim. Biophys. Acta, 616, (1980), 283-289. The starting material in a method according to the invention is a N-substissue β-lactam having the above-mentioned general formula (I), or a salt thereof. In the above definitions of the various symbols in formula (I), it is understood that an oxidized form of sulfur includes groups such as sulfoxide and sulfone. By optionally substituted alkyl groups is meant cycloalkyl, aryl, heteroaryl and benzyl, which have substituents such as alkyl groups of 1 to 3 carbon atoms. The optionally substituted nitrogen includes primary, secondary and tertiary amine groups, which may be substituted for example with alkyl groups of 1 to 3 carbon atoms. By "optionally substituted methyl" is meant a methyl group and various substituted methyl groups such as -CHpDq, wherein D is a halogen and p and p are integers of which the sum is equal to 3. The formula (I) is intended to encompass the ß -N-substudylactams, which are based on any ß-lactam nucleus described in "Cephalosporins and Penicillins, Chemistry and Biology", Ed. EH Flynn, Academic Press, 1972, pages 151-166 and "The Organic Chemistry of ß-lactams", Ed. G.I. Georg, VHC, 1992, pages 89-96, "which are incorporated by reference herein The preferred starting materials are those in which Ri represents a group CH2-E or CH = CH-E, wherein E is hydrogen, hydroxyl, halogen, alkoxy of 1 to 3 carbon atoms, alkyl of 1 to 5 carbon atoms, linear or branched, optionally substituted, optionally containing one or more heteroatoms, saturated or unsaturated, cycloalkyl of 5 to 8 carbon atoms optionally substituted , optionally containing one or more optionally substituted heteroatoms, aryl or heteroaryl, or optionally substituted benzyl Suitable salts of the initial N-substimated β-lactam material include any non-toxic salt, such as an alkali metal salt (eg example, sodium or potassium), an alkaline earth metal salt (for example calcium or magnesium), an ammonium salt, or an organic base salt (for example trimethylamine, triethylamine, pyridine, picoline, iclohexylamine, N, N'-dibenzyldiet and lendiamine). The initial N-substituted β-lactam material having the general formula (I) can be prepared enzymatically, for example in a method as described in EP 0 532 341, WO 95/04148 or WO 95/04149. Preferred starting materials are N-glutaryl-, N-succinyl-, N-adipyl-, N- 3- (carboxymethyl thio (propionyl-, N-trans-β-hydromuconyl-, N-pimenyl- or N- 3, 3 The iodipropioni 1-ß-lactam, or salts thereof The initial materials based on these dicarboxylic acids are efficiently converted by the enzymes used according to the invention The additionally preferred starting materials are 6-aminopenicillanic acid N- substituted (6-APA), 7-aminocephalosporanic acid N-substituted (7-ACA), N-substituted 3-chloro-7-aminodes acetoxidesmethocephalosporanic acid (7-ACCA), 7-aminodesacet-ilcephalosporanic acid N-substituted (7- ADAC), or the N-substituted 7-aminodesacetoxycephalosporanic acid (7-ADCA), since these N-substituted β-lactams result in β-lactam antibiotics having the most advantageous activity profiles: A dicarboxy lato-acylase suitable with which the N-substi tuted ß-lactam is contacted, in a method according to the invention, it is an enzyme that can be isolated from various microorganisms of natural origin, such as fungi and bacteria. Such microorganisms can be selected for enzymes with the desired specificity of the carboxylic acid, by periodically verifying the hydrolysis of suitable substrates. Such suitable substrates can be for example chromophores such as succinyl-, glutaryl- or adipyl-p-nitroanilide. Also, the hydrolysis of the corresponding N-substituted β-lactams can be used to identify the required enzymes. It has been found that the optimum pH range for these enzymes falls between about 6, preferably about 7, and about 9, preferably about 8. The organisms that have been found to produce the dicarboxylate acylase are Al cali genes, Arthroba ct er, Achromoba ct er, Aspergillus, Acinetobacte, Bacillus and Pseudomonas.

More particularly, the following species produce highly suitable dicarboxylate acylases: Achromobacter xylosooxidans, Arthrobacter viscosis, Arthrobacter CA128, Bacillus CA78, Bacillus megaterium ATCC53667, Bacillus cereus, Bacillus laterosporus Jl, Paecilomyces C2106, Pseudomonas diminuta sp N176- Pseudomonas diminuta sp V22, Pseudomonas paucimobilis, Pseudomonas diminuta BL072, Pseudomonas strain C427, Pseudomonas sp SE83, Pseudomonas sp SE495, Pseudomonas ovalis ATCC950, Comamonas sp SY77, Pseudomonas GK 16, Pseudomonas SY-77-1, Pseudomonas sp A14, Pseudomonas vesicular B965, Pseudomonas syringae, Ps putida ATCC17390, Ps aeroginosa NCTC 10701, Proteus vulgaris ATCC9634, Ps fragi DSM3881, and B. subtilus IFO3025. The dicarboxylase acylase can be obtained from the microorganism by which it is produced in any suitable manner, for example as described for strain SE83 of Pseudomonas sp strain SE83 in US Pat. No. 4,774,179. Also, genes for example for the dicarboxylate acylases SE83 or SY77 can be expressed in a different suitable host, such as E. coli, as reported by Matsuda et al. In J. Bacteriology 169, (1987), 5818-5820 for the strain SE83, and in the US Patent US 5,457,032 for the strain SY77. Enzymes isolated from the above sources are frequently referred to as glutaryl acylases. However, the side chain specificity of the enzymes is not limited to the glutaryl side chain, but also comprises larger and smaller dicarboxylic side chains. Some of the dicarboxylate acylases also express gamma-glutamyl-t ranspeptidase activity and are therefore sometimes referred to as gamma-glutamyl transpeptidases. A suitable penicillin acylase, with which the ß-lactam N-subs tute is brought into contact in a method according to the invention, is an enzyme that can be isolated from various microorganisms of natural origin, such as fungi and bacteria . Such microorganisms can be selected for enzymes with the desired specificity in a periodic monitoring or verification test analogous to one described for dicarboxylate acylase. Of these enzymes it was found that the optimum pH falls between about 4, preferably about 5, and about 7, preferably about 6. Organisms that have been found to produce penicillin acylase are, for example, the species of Acetobacter, Aeromonas, Alcaligenes, Aphanocladium, Bacillus sp, Cephalosporium, Escherichia, Fia vobacterium, Kluyvera,? Fycoplana, Protaminobacter, Providentia, Pseudomonas or Xantiiomonas. The enzymes derived from Acetoacet pasteurioanuiíi, Alcaligenes faecalis, Bacillus megaterium, Escherichia coli, Providentia rettgeri and Xanthomonas citrii have proved to be particularly successful in a method according to the invention. In the literature, penicillin acylases have also been referred to as penicillin amidases. The dicarboxylate acylase and the penicillin acylase or penicillin acylase can be used as free enzymes, but also in any immobilized, suitable form, for example as described in European Patent EP 0 222 462 and in the WO international document. 97/04086. It is possible to carry out a method according to the invention, wherein both enzymes are immobilized on a carrier, and where the enzymes are immobilized on different carriers. Furthermore, it is possible to use functional equivalents of one or both of the enzymes, where, for example, the properties of the enzymes, such as pH dependence, thermostability or specific activity can be affected by chemical modification or by the crosslinking, without significant consequence for the activity, in the type, not in the quantity, of the enzymes in a method according to the invention. Also, functional equivalents such as mutants or other derivatives, obtained by classical means or by means of recombinant DNA methodology, biologically active or hybrid parts of the enzymes, can also be used. In some cases, the modification, chemical or otherwise, may be beneficial in a method according to the invention, as it is part of the standard knowledge of the person skilled in the art. The precursor for a side chain of the β-lactam antibiotic to be prepared in a method according to the invention can be any compound that is recognized by the penicillin acylases defined above, and which leads to a product of the kind of the ß-lactam antibiotics. Preferably, the substrate is chosen from the group of D- (-) phenylglycine, D - (-) - 4-hydroxyphenylglycine, D- (-) -2,5-dihydrophenylglycine, 2-t-eneyl-acetic acid, 2- ( 2-amino-4-thiazolyl) -2-methoxyiminoacetic acid, α- (4-pyridyl-1-thio) -acetic acid, 3-t-isophenamonic acid or 2-thienyl-acetic acid, and derivatives thereof, since these substrates lead to β-lactam antibiotics that have the most advantageous activity profile. Suitable derivatives of these substrates are esters and amides, wherein the side chain molecule is connected to an alkyl group of 1 to 3 carbon atoms through an ester or amide linkage. In a method according to the invention, the dicarboxy lato-acylase, the precursor for the β-lactam side chain and penicillin acylase can be added to the initial N-substituted β-lactam material, either jointly or separately. Preferably, the enzymes are added together to the N-substituted β-lactam and to the precursor for the side chain. In a preferred embodiment of the invention, a process is carried out without the isolation and / or purification of any intermediates which may at one time or another be present in the reaction mixture. In this way, no product is lost in a process of isolation or purification. In a highly preferred embodiment of the invention, a process is carried out as a process in a single container. By "single-vessel process" is meant any process in which the entire process is carried out in a reactor vessel. In other words, major reaction components are not essentially removed from the reactor vessel at any time during the time in which a method according to the invention is carried out. The advantages of this modality will be evident for the expert person. The conditions applied in a method according to the invention depend on various parameters, in particular the type of the reagents, the concentration of the reactants, the reaction time, the titrant, the temperature, the pH, the enzymatic concentration , and of the enzymatic morphology. Given a specific N-substituted β-lactam to be converted to a given β-lactam antibiotic, using a given dicarboxylate-acylase and a given penicillin-acylase, the person skilled in the art will be able to properly choose the conditions of optimal reaction.

However, it has now been found that the optimum reaction temperature in a method according to the invention falls between 0 and 80 ° C, preferably between 10 and 50 ° C. The optimum pH in the preparation of a β-lactam antibiotic according to the invention falls between 4.5 and 9.0. In this regard, it should be noted that it is highly preferable to perform a method according to the invention in an aqueous environment, because in this way the use of organic solvents is avoided, which could lead to effluent problems. In addition, dicarboxylate acylase and penicillin acylase have proven to be enzymes that catalyze the conversion reaction more efficiently in an aqueous environment. In general, the reagents will be present in amounts in the range of between 0.01, preferably 0.5, and 3 mol per kilogram of reaction mixture, preferably 2 mol per kilogram of reaction mixture, in both steps. Suitable enzyme concentrations are chosen such that the total reaction time does not exceed 4 hours. For the conversion of 10 mmol of substrate into product within one hour, approximately 500 to 3000 units of enzymatic reaction should be applied, where an enzyme reaction unit is defined as the amount of enzyme which converts a micromole of substrate to product in one minute, under conditions which represent the effective conditions of the process. In general, for the conversion of a certain amount of substrate in one hour, the enzyme dose should preferably be between 50 and 300 kUnits per mole. However, a large excess of activity is usually dosed in order to compensate for any losses that may occur during the process. Suitable titrants are acids and inorganic bases, such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, ammonium hydroxide, and so on, or organic acids such as formic acid, acetic acid, succinic acid, adipic acid, glutaric acid and so on. The concentration of the titrant can vary between 0.01 and 8 M, depending on the scale of the reaction and the solubility of the titrant. Of course, the invention also encompasses a β-lactam antibiotic which can be obtained by the methods described hereinabove.

The invention will now be elucidated by the following non-limiting examples.

EXAMPLES DEFINITIONS AND PROCEDURES Enzymatic activity As a definition of the acylase activity of penicillin G, the following were used: one unit (U) corresponds to the amount of enzyme that hydrolyzes 1 micromol of penicillin G per minute under standard conditions (100 grams of potassium salt of penicillin G) 1-1, 0.05 M potassium phosphate buffer, pH 8.0, 28 ° C). As a definition of the dicarboxylate acylase activity, the following were used: one unit (U) corresponds to the amount of enzyme that hydrolyzes 1 mmol of N-adipyl-7-ADCA per minute under standard conditions (N-adipil-7-ADCA 100 mM, 100 mM Tris buffer, pH 8.0, 37 ° C).

PH measurement A Mettler DL21 titration apparatus equipped with an automatic burette and a Brother M1509 printing device was used.

Analysis of High Resolution Liquid Chromatography (HPLC) For Amoxicillin: Column: Chromsphere C18, 5 mM (100 x 3.0 nM) Solvent: 25% acetonitrile in 12 mM potassium phosphate buffer containing 0.2% sodium dodecyl sulfate. Flow: 1 ml / min Detection: 214 nm For Cephalexin: Column: Chromsphere C18, 3 mM (100 x 4.6 mM) Solvent: 29% Acetonitrile in 14 mM potassium diacid phosphate buffer pH 3.0 with phosphoric acid Flow: 1 ml / min Detection: 254 nm EXAMPLE 1 AMOXICILLINE FROM N-ADIPIL-6β-AMINOPENICILANIC ACID AND METHYL ESTER OF D - (-) - 4-HYDROXYPHENYLGLYCINE To one of the N-adipyl-6β-aminopenicilinate "dipotassium (0.71 grams, purity 59%, 1.0 mmol) and methyl ester of D- (-) -4-hydroxyphenylglycine (0.45 grams, purity greater than 97%, 2.4 mmol) in water (10 ml) was added dicarboxylate acylase obtained from Ps and udomona s SE83 (1044 grams, 96 U.g "1) and penicillin acylase obtained from Es ch eri chi a col i (0.80 grams, 125 GB-1). The mixture was stirred at room temperature and the pH was maintained at 6.9 using a 1M solution of sodium hydroxide in water. The product formation was checked periodically using HPLC analysis. The results are shown in Table 1.

Table 1 Time (hour) Adipyl-6-APA (mM) 6-APA (mM) Amoxicillin (mM) 0 121 0 0 0.5 81 13 4 1.0 80 12 7 EXAMPLE 2 CEFALEXIN FROM N-ADIPIL-7-AMINO-3-ME TILCEF-3-EM- -CARBOXYLATE AND FROM AMIDA FROM D - (-) - PHENYLGLYCINE To a solution of N-adipyl-7-amino-3-methyl-3-em-4-carboxylate (0.68 grams, purity of 97. 1%, 2.0 mmol) and the amide of D- (-) - phenylglycine (0.75 grams, purity 96%, 4.8 mmol) in water (20 ml), the dicarboxylate acylase obtained from Ps e udomon as SE83 (4.00 grams, 369 Ug-1) and penicillin acylase obtained from Es ch eri chi a col i (1.6 grams, 250 Ug "1) .The initial pH of the reaction mixture was 6.3, reaction mixture was stirred to 33 ° C, and after 30 minutes the HPLC analysis (pH = 6.8) showed the formation of Cephalexin. For HPLC analysis, 0.5 ml of the reaction mixture was taken from the reaction vessel, centrifuged from the filtrate, and a volume of 0.2 ml was made up to 50 ml with buffer solution pH 7. The results are shown in Table 2 Table 2 Time (hour) Adipyl-7-ADCA (mM) 7-ADCA (mM) Cephalexin (mM) 0 95 0 0 0.5 34 35 9.9 EXAMPLE 3 CEFALEXIN FROM N-ADIPIL-7-AMINO-3-METILCEF-3-EM-4 -CARBOXYLATE AND OF THE AMIDA OF D- (-) -FENILGLYCIN To a solution of N-adipyl-7-amino-3-methylcephef-3-e-carboxylate (0.68 grams, purity 97.1%, 2.0 mmol) in water (20 ml), the obtained dicarboxylate acylase was added to from Ps e udomon as SE83 (4.00 grams, 369 Ug "1) .The reaction mixture was stirred at 35 ° C and the pH was maintained at 8.0 by the use of a 2M solution of potassium hydroxide in water. about 1 hour, the content of the reaction was filtered and added to the filtrate combined amide of D- (-) - phenylglycine (0.75 grams, purity 96%, 4.8 mmol) and the acylase of penicillin obtained from Esterichia coli (1.6 grams, 250 Ug "x). The content of the reaction was maintained at 13 ° C and at the pH of 7.5 by the use of a 1M solution of hydrochloride in water. HPLC analysis showed the formation of cephalexin. For the HPLC analysis, 0.5 ml of the reaction mixture was taken from the reaction vessel, centrifuged and from the filtrate, a volume of 0.2 ml was constituted up to 25 ml with PH 7 buffer solution. The results are shown in Table 3 Table 3 Time (hour) Adipil-7-ADCA (mM) 7-ADCA (mM) Cef alexin (mM) 0 95 0 0 1.0 2.2 32 0 2.0 3.7 14 37 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (11)

1. A method for the preparation of a β-lactam antibiotic, characterized in that an ß-iactam N-subtitle, which has the general formula (I) wherein RT is hydrogen or alkoxy of 1 to 3 carbon atoms; And it is CHr, oxygen, sulfur, or an oxidized form of sulfur; Z is where R; is hydrogen, hydroxyl, halogen, alkoxy 1 to 3 carbon atoms, linear or branched, saturated or unsaturated alkyl of 1 to 5 carbon atoms, optionally substituted, or optionally containing one or more heteroatoms, preferably methyl, optionally substituted cycloalkyl of 5 to 8 carbon atoms, optionally containing one or more optionally substituted heteroatoms, aryl or heteroaryl, or optionally substituted benzyl; and X is (CH2) mA- (CH) 2) n, where m and n are the same or different and are chosen from the group of integers 0, 1, 2, 3 or 4, and A is CH = CH, C = C, CHB, C = 0, optionally substituted nitrogen, oxygen, sulfur or an optionally oxidized form of the sulfur, and B is hydrogen, halogen, hydroxyl, alkoxy of 1 to 3 carbon atoms, or optionally substituted methyl, or a salt of they are contacted with at least one dicarboxylate acylase, or a functional equivalent thereof, and are reacted with a precursor for a side chain of the β-lactam antibiotic in the presence of at least one penicillin acylase or a functional equivalent thereof. •

2. A method according to claim 1, characterized in that intermediary products are not isolated and / or purified.

3. A method according to claim 2, characterized in that it is carried out as a process in a single container.

4. A method according to any of the preceding claims, characterized in that the N-substituted β-lactam is an N-glutaryl-, N-succinyl-, N-adipyl-, N-3- (carboxymethylthio) propionyl-, N-trans -β-hydromuconyl-, -N-pimelyl- or N- 3, 3'-t iodipropioni 1-β-lactam, or a salt thereof.

5. A method according to any of the preceding claims, characterized in that the β-lactam N-susbtituide is an N-substituted 6-aminopenicillanic acid (6-APA), 7-aminocephalosporanic acid (7-ACA), 3-chloro- 7-aminodesacetoxydesmethylcephalosporanic acid (7-ACCA), 7-aminodesacet-ilcephalosporanic acid (7-ADAC), or 7-aminodesacetoxycephalosporanic acid (7-ADCA), or a salt thereof.

6. A method according to any of the preceding claims, characterized in that the precursor for a side chain of the β-lactam antibiotic is D- (-) -phenylglycine, D - (-) - 4-hydroxyphenylglycine, D- (-) - 2,5-dihydrophenylglycine, 2-thienoacetic acid, 2- (2-amino-4-thiazolyl) -2-methoxy iminoacetic acid, α- (4-pyridylthio) acetic acid, 3-t-isophenmalonic acid, or 2-cyanoacic acid, or an amide or ester of the same.

7. A method according to any of the foregoing indications, characterized in that the dicarboxylate acylase is obtained from a species of Alcaligenes, Arthrobacter, Achromobacter, Aspergillus, Acinetobacter, Bacillus or Pseudomonas.

8. A method according to any of the preceding claims, characterized in that the penicillin acylase is obtained from a species Acetobacter, Aeromonas, Alcaligenes, Aphanocladium, Bacillus sp. , Cephalosporium, Escherichia, Flavobacterium, Kluyvera, Mycoplana, Protaminobacter, Providentia, Pseudomonas or Xanthomonas.

9. A method according to any of the preceding claims, characterized in that the N-substituted β-lactam is obtained by an enzymatic process starting from a fermentation product.

10. A method according to claim 9, characterized in that the fermentation product is penicillin G, penicillin V, Cephalosporin C, adipyl-7-ADCA, 3-carboxyethylthiopropionyl-7-ADCA, 2-carboxyethylthioacetyl-7-ADCA, and 3-carboxyethyl-thiopropionyl-7-ADCA, adipyl-7-ACA, 3-carboxyethylthiopropionyl-7-ACA, 2-carboxyethylthioacetyl-7-ACA and 3-carboxyethylthiopropionyl-7-ACA.

11. The use of a dicarboxylate acylase and a penicillin acylase to convert an N-substituted β-lactam to a β-lactam antibiotic. SUMMARY OF THE INVENTION The invention relates to a method for the preparation of a β-lactam antibiotic, wherein an N-substituted β-lactam has the general formula (I), wherein R 0 is hydrogen or alkoxy of 1 to 3 carbon atoms; And it is CH2, oxygen, sulfur or an oxidized form of sulfur; Z is (a), (b), (c) or d, wherein Rx is hydrogen, hydroxyl, halogen, alkoxy of 1 to 3 carbon atoms, alkyl of 1 to 5 carbon atoms, linear or branched, optionally substituted , optionally containing one or more heteroatoms, saturated or unsaturated, preferably methyl, optionally substituted C 5 -C 8 cycloalkyl optionally containing one or more optionally substituted heteroatoms, aryl or heteroaryl, or optionally substituted benzyl; and X is (CH2) mA- (CH2) n, where m and n are the same or different and are chosen from the group of integers 0, 1, 2, 3 or 4, and A is CH = CH, C = C, CHB, C = 0, optionally substituted nitrogen, oxygen, sulfur or an optionally oxidized form of the sulfur, and B is hydrogen, halogen, hydroxyl, alkoxy of 1 to 3 carbon atoms, or optionally substituted methyl, or a salt thereof , is contacted with at least one carboxylate acylase or a functional equivalent thereof, and is reacted with a precursor for a side chain of the β-lactam antibiotic in the presence of at least one penicillin acylase, or equivalent functional of it.