The class of P-lactam antibiotics, such as penicillin and cephalosporin antibiotics comprises a great variety of compounds, all having their own activity profile. In general, P-lactam antibiotics consist of a nucleus, the so-called p- lactam nucleus, which is linked through its primary amino group to the so-called side chain via a linear amide bond.

P-Lactam nuclei are very important intermediates in the preparation of semi-synthetic penicillin and cephalosporin antibiotics. The routes to prepare these semi-synthetic penicillins and cephalosporins mostly start from fermentation products such as penicillin G, penicillin V and Cephalosporin C, which are converted to the corresponding P-lactam nuclei, for instance in a manner as is disclosed in K. Matsumoto, Bioprocess. Techn., 16, (1993), 67-88, J.G. 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 -lactam nuclei which are employed as precursor for several antibiotics are 6-aminopenicillanic acid (6-APA), 7-aminocephalosporanic acid (7-ACA), 3-chloro-7- aminodesacetoxydesmethylcephalosporanic acid (7-ACCA), 7-amino- desacetylcephalosporanic acid (7-ADAC), and 7-amino- desacetoxycephalosporanic acid (7-ADCA).

The -lactam nuclei are converted to the desired antibiotic by coupling to a suitable side chain, as has been described in inter alia EP 0 339 751, JP-A-53005185 and CH-A-640 240. By making different combinations of side chains and -lactam

nuclei, a variety of penicillin and cephalosporin antibiotics may be obtained, all having their own activity profiles.

For example, D- (-)-phenylglycine, or a suitable derivative thereof, such as an amide or ester, may be attached to any of 7-ACA, 7-ACCA, 7-ADCA and 6-APA to produce Cephaloglycin, Cefaclor, Cephalexin or Ampicillin respectively. Other examples of often employed side chains are D- (-) -4-hydroxyphenylglycine, 2-cyanoacetic acid and 2-(2-amino-4-thiazolyl)-2- methoxyiminoacetic acid.

A fermentative process has been disclosed for the production of 7-ADCA and 7-ACA, involving the fermentative production of N-substituted -lactams, such as adipyl-7-ADCA or adipyl-7-ACA by a recombinant Penicillium chrysogenum strain capable of expressing a desacetoxycephalosporanic acid synthase (DAOCS) also known as "expandase" from a transgene (EP 0 532 341, EP 0 540 210, WO 93/08287, WO 95/04148, WO 95/04149). The expandase takes care of the expansion of the 5-membered ring of certain N-acylated penicillanic acids, thereby yielding the corresponding N-acylated desacetoxycephalosporanic acids. The final step in the disclosed process comprises contacting the acyl-7-ADCA with a suitable acylase, whereby the acyl side chain is removed and the desired 7-ADCA or 7-ACA product is formed.

It is commonly accepted that P-lactam nuclei, such as 7- ADCA, have to be isolated by crystallization before they may be converted to -lactam antibiotics. A major drawback of crystallization of -lactam nuclei is loss of product in the mother liquor, which can amount to 10% of the theoretic yield, depending on the solubility of the product in question. Simple recovery procedures such as extraction of the mother liquor with an organic solvent cannot be applied because of the amphoteric nature of the -lactam nucleus, which is an amino acid and thus has a tendency to readily dissolve in aqueous environment at any pH value.

In view of these problems, it is an object of the invention to provide a method for controlling the solubility of a -lactam nucleus.

Surprisingly, it has been found that the solubility of a P-lactam nucleus may very efficiently be controlled by modifying its amphoteric properties.

Description of the invention Accordingly, the invention provides a method for controlling the solubility of a -lactam nucleus having the general formula (I) wherein K0 is hydrogen or C13 alkoxy; Y is CH2, oxygen, sulfur, or an oxidized form of sulfur; and Z is wherein R, is hydrogen, hydroxy, halogen, C13 alkoxy, optionally substituted, optionally containing one or more heteroatoms, saturated or unsaturated, branched or straight C1s alkyl, preferably methyl, optionally substituted, optionally containing one or more heteroatoms, C5s cycloalkyl, optionally substituted aryl or heteroaryl, or optionally substituted benzyl, or of a salt of said -lactam nucleus, wherein the amphoteric properties of said -lactam nucleus or the salt thereof are modified.

By modifying the amphoteric properties of a P-lactam nucleus, present in the mother liquor of a crystallization process, a product is obtained, which may efficiently be recovered from a mother liquor, for instance in a process as described hereinabove (EP-A-0 532 341). Subsequently, the recovered product may be reconverted to a -lactam nucleus, after isolation from the mother liquor by extraction. Thus, the present invention provides a method which may be applied in

optimizing the yield of the intermediate p-lactam nucleus, such as 6-APA, 7-ACA, 7-ACCA, 7-ADAC or 7-ADCA, in the industrial production of cephalosporin and penicillin antibiotics.

In addition, the invention has proven to be very useful in cases wherein the carboxylic acid function of a -lactam nucleus is protected by an ester group, such as a p-nitrobenzylester, a tertiary butylester, a p-methoxy benzylester, a benzhydrylester or an allylester. Until now, a great disadvantage of handling an ester protected -lactam nucleus has been that it no longer dissolves in water because its amphoteric character has been eliminated. Consequently, it is difficult, if not impossible, to use enzymes for further conversions of said protected P-lactam nucleus, since enzymatic conversions usually have to be performed in aqueous systems. In accordance with the invention, it has proven to be possible to affect the solubility of a -lactam nucleus to an extent that a -lactam nucleus, wherein the carboxylic acid function bears an ester group, such as a tertiary butyl group (t-butyl), can be rendered soluble in water.

The starting material in the method according to the invention is a -lactam nucleus having the above general formula (I) or a salt thereof, wherein the symbols have the meanings as defined hereinabove. In the context of the invention, an oxidized form of sulfur is meant to include groups such as sulfoxide and sulfone. By optionally substituted alkyl, cycloalkyl, aryl, heteroaryl and benzyl, groups are intended, which have substituents such as alkyl groups of from 1 to 3 carbon atoms. Optionally substituted nitrogen includes primary, secondary and tertiary amine groups, which may be substituted with for instance alkyl groups of from 1 to 3 carbon atoms.

Optionally substituted methyl is meant to include a methyl group and various substituted methyl groups such as CHpDq, wherein D is a halogen and p and q are integers of which the sum equals 3.

Formula (I) is intended to encompass all P-lactam nuclei as disclosed in "Cephalosporins and Penicillins, Chemistry and Biology", Ed. E.H. Flynn, Academic Press, 1972, pages 151-166,

and "The Organic Chemistry of P-Lactams", Ed. G.I. Georg, VCH, 1992, pages 89-96, which are incorporated herein by reference.

Preferred are those starting materials wherein R1 represents a CH2-E or CH=CH-E group, wherein E is hydrogen, hydroxy, halogen, C13 alkoxy, optionally substituted, optionally containing one or more heteroatoms, saturated or unsaturated, branched or straight C1s alkyl, optionally substituted, optionally containing one or more heteroatoms C58 cycloalkyl, optionally substituted aryl or heteroaryl, or optionally substituted benzyl.

Suitable salts of the -lactam nuclei to be converted in a method according to the invention include any non-toxic salt, such as an alkali metal salt (e.g. lithium, potassium, sodium), an alkali earth metal salt (e.g. calcium, magnesium), an ammonium salt, or an organic base salt (e.g. trimethylamine, triethylamine, pyridine, picoline, dicyclohexylamine, N,N' dibenzyl diethylene diamine).

Generally, the P-lactam nucleus is a cephalosporanic acid, a penicillanic acid, or a salt thereof. Most preferred -lactam nuclei are 6-aminopenicillanic acid (6-APA), 7-amino- cephalosporanic acid (7-ACA), 3-chloro-7-aminodesacetoxy- desmethylcephalosporanic acid (7-ACCA), 7-aminodesacetylcephalo- sporanic acid (7-ADAC), or 7-aminodesacetoxycephalosporanic acid (7-ADCA) , as these are precursors for the penicillin and cephalosporin antibiotics which have the most advantageous activity profiles.

The -lactam nucleus starting material is preferably obtained from N-substituted p-lactam fermentation products such as penicillin G, penicillin V, cephalosporin C, adipyl-7-ADCA, <BR> <BR> <BR> 3-carboxyethylthiopropionyl-7-ADCA,2-carboxylethylthioacetyl -7- ADCA, 3-carboxyethylthiopropionyl-7-ADCA, adipyl-7-ACA, 3- <BR> <BR> <BR> carboxyethylthiopropionyl-7-ACA, 2 -carboxylethylthioacetyl-7-ACA or 3-carboxyethylthiopropionyl-7-ACA by enzymatic conversion by the action of a penicillin acylase. A suitable penicillin acylase is an enzyme that may be isolated from various naturally occurring micro-organisms, such as fungi and bacteria. Organisms that have been found to produce penicillin acylase are, for

example, Acetobacter, Aeromonas, Alcaligenes, Aphanocladium, Bacillus sp., Cephalosporium, Escherichia, Flavobacteriurn, Kluyvera, Mycoplana, Protaminobacter, Providentia, Pseudomonas or Xanthomonas species.

In accordance with the invention, by modifying the amphoteric properties of a compound is meant modifying the tendency of a compound to show both acidic and basic behaviour.

In other words, by modifying the amphoteric properties of a compound the charge distribution of the compound is affected.

It has now been found that the amphoteric properties of a -lactam nucleus or a salt thereof may very suitably be modified by subjecting it to the action of at least one dicarboxylate acylase. In this preferred embodiment, a method according to the invention is an enzymatic, selective and efficient process, in other words a method which does not result in effluent problems or involve expensive chemicals. Surprisingly, it has been found that a dicarboxylate acylase is capable of enzymatically modifying the amphoteric properties of a P-lactam nucleus at very low concentrations, such as normally occur in mother liqours of industrial crystallization processes.

A suitable dicarboxylate acylase to be used according to the invention is an enzyme that may be isolated from various naturally occurring micro-organisms, such as fungi and bacteria.

Such micro-organisms can be screened for enzymes with the desired dicarboxylic acid specificity by monitoring the hydrolysis of suitable substrates. Such suitable substrates may be e.g. chromophores such as succinyl-, glutaryl- or adipyl-p- nitroanilide. Also, the hydrolysis of the corresponding N- substituted p-lactams may be used for identifying the required enzymes.

Organisms that have been found to produce dicarboxylate acylase are Alcaligenes, Arthrobacter, Achromobacter, Aspergillus, Acinetobacter, Bacillus and Pseudomonas species.

More in particular, the following species produce highly suitable dicarboxylate acylases: Achromobacter xylosooxidans, Arthrobacter viscosis, Arthrobacter CA128, Bacillus CA78, Bacillus megaterium ATCC53667, Bacillus cereus, Bacillus laterosporus J1, 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 vesicularis B965, Pseudomonas syringae, Ps putida ATCC17390, Ps aeroginosa NCTC 10701, Proteus vulgaris ATCC9634, Ps fragi DSM3881, and B. subtilus IFO3025.

The dicarboxylate acylase may be obtained from the micro- organism by which it is produced in any suitable manner, for example as is described for the Pseudomonas sp SE83 strain in US 4,774,179. Also, the genes for e.g. SE83 or SY77 dicarboxylate acylases may be expressed in a different suitable host, such as E.coli as has been reported by Matsuda et al. in J. Bacteriology, 169, (1987), 5818-5820 for the SE83 strain, and in US 5,457,032 for the SY77 strain.

The enzymes isolated from the above sources are often referred to as glutaryl acylases. However, the side chain specificity of the enzymes is not limited to the glutaryl side chain, but comprises also smaller and larger dicarboxyl side chains. Some of the dicarboxylate acylases also express gamma- glutamyl transpeptidase activity and are therefore sometimes classified as gamma-glutamyl transpeptidases.

The dicarboxylate acylase may be used as the free enzyme, but also in any suitable immobilized form, for instance as has been described in EP 0 222 462. In addition, it is possible to use functional equivalents of the enzyme, wherein for instance properties of the enzyme, such as pH dependence, thermostability or specific activity may be affected by chemical modification or cross-linking, without significant consequences for the activity, in kind, not in amount, of the enzyme in a method according to the invention. Also, functional equivalents such as mutants or other derivatives, obtained by classical means or via recombinant DNA methodology, biologically active parts or hybrids of the enzymes may be used. In some cases, modification, chemical or otherwise, may be beneficial in a method according to the invention, as is part of the standard knowledge of the person skilled in the art.

Preferably, the P-lactam nucleus or the salt thereof is subjected to the action of the dicarboxylate acylase in the presence of at least one suitable substrate for said dicarboxylate acylase. A suitable substrate, in this context, is intended to mean a substrate which is recognized by the enzyme.

Preferred substrates to be used are dicarboxylic acids or salts thereof. A dicarboxylic acid may be used in its acidic form or in the form of a salt, such as an ammonium, lithium, potassium or sodium salt. It is an additional advantage of the invention that no activation step of the dicarboxylic acid is necessary.

Dicarboxylic acids which have proven to be highly successful in this embodiment of the invention are those represented by the general formula (II) wherein 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, 4 or 5, and A is CH=CH, C-C, CHB, C=O, optionally substituted nitrogen, oxygen, sulfur or an optionally oxidized form of sulfur, and B is hydrogen, halogen, hydroxy, C13 alkoxy, or optionally substituted methyl.

Highly preferred dicarboxylic acids to be used in this embodiment of the invention are those having general formula (II) wherein the sum of m and n equals from 1 to 5. It was found, that these dicarboxylic acids are recognized by the dicarboxylate acylase to be used very efficiently.

The most preferred dicarboxylic acids are succinic acid, glutaric acid, adipic acid, 3- (carboxymethylthio)propionic acid, <BR> <BR> <BR> trans-p- hydromuconic acid, pimelic acid and 3,3' -thiodipropionic acid.

It was found, that in accordance with this preferred embodiment of the invention, a method is provided wherein a - lactam nucleus is converted into an N-substituted P-lactam. By

this conversion, the amphoteric properties of the -lactam nucleus are modified in that a carboxylic acid function is introduced and a primary amine function is converted to a secondary amine function.

With regard to the conversion of the -lactam nucleus to an N-substituted P-lactam, it is to be noted that known methods for this conversion have proven to be not suitable in industrial production processes of penicillin and cephalosporin antibiotics.

For example, synthetic procedures for converting P-lactam nuclei to N-substituted p-lactam nuclei, as has been reviewed in Recl. Trav. Chim. Pays-Bas 112, (1993), 66-81, involve the use of organic solvents such as dichloromethane, which causes effluent problems. Also, many complicated reactions, for instance for introducing and removing protective groups, have to be performed.

Although enzymatic processes have been reported wherein the use of organic solvents and protecting groups can be circumvented, the known enzymatic methods for conversion of p- lactam nuclei to N-substituted p-lactam nuclei are also not suitable in industrial production processes of penicillin and cephalosporin antibiotics. This is due to the fact that an additional chemical step has to be performed in order to obtain activated derivatives of the required N-substituent.

Enzymatic routes to N-substituted -lactam nuclei in aqueous systems by acylation of the parent P-lactam nucleus moiety with an activated N-substituent derivative, such as an amide or an ester, have been reported in inter alia Austrian patent 243 986, Dutch patent 158 847, European patent applications 0 339 751 and 0 473 008, international patent applications WO 92/01061 and WO 93/12250, U.S. patent 3,816,253 and German patents 21 63 792 and 26 21 618.

Furthermore, in contrast to the conventional chemical methods for converting P-lactam nuclei into N-substituted p- lactam nuclei, the method of this preferred embodiment of the invention does not lead to dimerization; in other words only one p-lactam nucleus molecule is attached to one dicarboxylic acid molecule.

Of course, the invention also encompasses an N-substituted P-lactam obtainable by any of the methods disclosed hereinabove.

The reaction conditions for the preparation of an N- substituted P-lactam according to the invention depend on various parameters, in particular the type of reagents, the concentration of reagents, reaction time, titrant, temperature, pH, enzyme concentration, and enzyme morphology. Given a specific N- substituted P-lactam that is to be prepared using a given dicarboxylate acylase, the person skilled in the art will be able to suitably choose the optimum reaction conditions.

It has, however, been found that the optimum reaction temperature in a method according to the invention lies between 0 and 800C, preferably between 10 and 500C. The optimum pH for preparing an N-substituted P-lactam according to the invention lies between 4.5 and 9.0, preferably 5.5 and 6.5. In this regard, it is to be noted that is highly preferable to perform a method according to the invention in aqueous environment, because thus the use of organic solvents, which would lead to effluent problems, is circumvented. Moreover, the dicarboxylate acylase enzyme has proven to catalyze the conversion reaction most efficiently in an aqueous environment.

Generally, the reagents will be present in amounts ranging between 0.01, preferably 0.5, and 3 mol per kilogram reaction mixture, preferably 2 mol per kilogram 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 millimole of substrate into product within one hour, about 500 to 3000 enzyme reaction units should be applied, wherein an enzyme reaction unit is defined as the amount of enzyme which converts one micromole of substrate into product in one minute under conditions which represent the actual process conditions.

In general, for the conversion of a certain amount of substrate in one hour, the enzyme dosage should be preferentially between 50 and 300 kUnits per mole. However, usually a larger excess of activity is dosed in order to compensate for any losses which may occur during the process.

Suitable titrants are inorganic acids, such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, or organic acids, such as formic acid, acetic acid, and so forth, or the dicarboxylate acid which is to be attached to the p-lactam nucleus in a method according to the invention. Titrant concentration may vary between 0.01 and 8 M, depending on the scale of the reaction and the solubility of the acid.

In a preferred embodiment, the invention provides a method for preparing a P-lactam nucleus from an N-substituted p-lactam obtained as disclosed hereinabove, wherein the N-substituted p- lactam is subjected to the action of a dicarboxylate acylase.

Surprisingly, the dicarboxylate acylase as defined hereinabove has been found to be capable of catalyzing both the conversion of a p-lactam nucleus into an N-substituted P-lactam and the opposite reaction of an N-substituted P-lactam to the corresponding P-lactamnucleus ef ficiently . It was found that the action of the dicarboxylate acylase may be influenced by choosing the appropriate reaction conditions, in particular the pH. While the conversion of a p-lactam nucleus to an N-substituted P-lactam is effected by a dicarboxylate acylase under acidic conditions, the reverse reaction transpires at higher pH values, preferably from 6.5 to 9, more preferably from 7 to 8.

In accordance with this embodiment, the preparation of an N-substituted p-lactam may be performed in order to recover a - lactam nucleus from a crystallization mother liquor in an industrial process for preparing a P-lactam nucleus.

Subsequently, the obtained N-substituted P-lactam may be reconverted to a P-lactam nucleus by subjecting it to the action of a dicarboxylate acylase as described hereinabove. Thus, the invention enables the optimization of the yield of an expensive and important intermediate in commercial preparation processes for penicillin and cephalosporin antibiotics.

In order to recover a P-lactam nucleus from a crystal- lization mother liquor by converting it to an N-substituted p- lactam in accordance with the invention, the mother liquor of a crystallization process is treated with a dicarboxylic acid in the presence of a dicarboxylate acylase as defined hereinabove.

The resulting N-dicarboxyl P-lactam may be recovered by

extraction with an organic solvent at a pH chosen between 0 and 3. Subsequently, the N-dicarboxyl P-lactam containing organic phase is back-extracted in water at high concentrations at neutral to slightly basic pH values. The resulting aqueous solution is again treated with dicarboxylate acylase at pH 7-9 in order to liberate the required P-lactam nucleus. Isolation is effected by crystallization at a pH value close to the PKa value of the P-lactam nucleus. Optionally, this procedure may be preceded by extraction of the liberated dicarboxylic acid using an organic solvent.

Of course, the above procedure may be repeated with the mother liquor obtained after the crystallization of the P-lactam nucleus.

The above recovery process can advantageously be combined with an existing production process based on fermentatively prepared N-dicarboxyl P-lactam nuclei. For reasons that will be evident to the skilled person, a dicarboxylic acid is used that is already present as a result of a preceding process step (e.g.

enzymatic deacylation of an N-dicarboxyl P-lactam). The resulting N-dicarboxyl P-lactam may be recovered by extraction, essentially as described above, preferably using the same solvent as used in the extraction step of the fermentation process. Subsequently, the N-dicarboxyl P-lactam containing organic phase is combined with the organic phase of the fermentation process and further processed as described above to give the desired P-lactam nucleus, which may be reacted with an appropriate side chain to yield a penicillin or cephalosporin antibiotic.

As will be clear from the above, a method according to the invention is highly advantageous when performed with the purpose of recovering P-lactam nucleus from a crystallization mother liquor in a preparation process for p-lactam nuclei. Hence, the invention also encompasses the use of a dicarboxylate acylase for recovering a P-lactam nucleus from a mother liquor obtained after a reaction wherein an N-acylated P-lactam is converted into a - lactam nucleus and a dicarboxylic acid in a process for preparing a P-lactam antibiotic.

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

EXAMPLES DEFINITIONS AND PROCEDURES Enzyme activity As definition of dicarboxylate acylase activity the following is used: one unit (U) corresponds to the amount of enzyme that hydrolyses per minute 1 micromole N-adipyl-7- aminodesacetoxycephalosporanic acid under standard conditions (100 mM N-adipyl-7-aminodesacetoxycephalosporanic acid, 100 mM Tris buffer, pH 8.0, 370C).

pH-stat procedure Condensation reactions are carried out in water at constant pH values. To this end a Mettler DL21 titration apparatus equipped with an automatic burette and Brother M1509 printing device is used. Proceeding of conversions is monitored by consumption of acid. When the equilibrium at a choosen pH-value is reached, the reaction is either terminated or continued at a lower pH-value.

NMR analysis Samples are quantitatively analyzed using 1H NMR spectroscopy with either a Bruker AMX 360 or a Bruker AMX 600 instrument. To an accurately weighed sample was added a known quantity of internal standard (i.e. maleic acid, in phoshate buffer pH 6.4).

The mixture was lyophilized and dissolved in D2O. Spectra were measured using 90° pulses; the FID was multiplied with an exponential function causing line-broadening of 0.2 Hz. The integral of the H5 or H6 proton and the internal standard were measured and the purity was calculated. The accuracy of this absolute method is considered to be better than 0.5% (from validation studies on similar compounds).

EXAMPLE 1 DIPOTASSIUM N-SUCCINYL-(6R,7R)-3-ACETOXYMETHYL-7-AMINO- CEPH-3-EM-4-CARBOXYLATE 7-ACA (2.72 g; 10.0 mmol) and succinic acid (1.18 g; 10.0 mmol) were suspended in water (30 ml) and the pH was adjusted to 7.2 at 370C with a solution of KOH in water (2 M). The pH was adjusted to 6.6 by the addition of succinic acid (0.10 g; 0.85 mmol). Immobilized dicarboxylate acylase (Pseudomonas SE-83, 10.0 g; 110 units.g-1) was added and the mixture was stirred at 370C for 1 h while maintaining the pH of the solution at 6.6 with a solution of HCl in water (1 M). The pH was lowered to 5.9 using a succinic acid (0.10 g; 0.85 mmol). After 15 h, the immobilized enzyme was removed by filtration, and part of the filtrate (68% by weight) was lyophilized to give 4.14 g of dipotassium N- <BR> <BR> <BR> <BR> succinyl- (6R, 7R) -3-acetoxymethyl-7-aminoceph-3-em-4-carboxylate as a light-yellow solid (purity 27% as determined by 360 MHz 1H NMR in D2O). Yield 36.4%.

EXAMPLE 2 DISODIUM N-GLUTARYL- 6B-AMINOPENICILLANATE 6-APA (1.05 g; 4.41 mmol) and glutaric acid (0.58 g; 4.41 mmol) were suspended in water (9 ml) and the pH was adjusted to 7.0 with a solution of NaOH in water (8 M). Dicarboxylate acylase (Pseudomonas SE-83, 2.0 ml; 444 units.ml-1) was added and the mixture was stirred at 200C for 3 h while maintaining the pH of the solution at 7.0 with a solution of HCl in water (1 M). The pH was lowered to 6.1 using a solution of HCl in water (6 M) and stirring was continued at this pH-value for 2.5 h. Again, the pH was lowered to 5.5 and stirring was continued at this pH-value for 2 h. The reaction mixture was filtered over a G3 glass sintered funnel and ultrafiltrated at 2000 rpm using an Amicon Centriprep-30 unit with a membrane cutoff of MW 30,000. The permeate was lyophilized to give 2.57 g of disodium N-glutaryl- 6 -aminopenicillanate as an off-white solid (purity 60% as determined by 600 MHz 1H NMR in D2O). Yield 93.4%.

EXAMPLE 3 DISODIUM N-GLUTARYL-6 -AMINOPENICILLANATE 6-APA (5.94 g; 25.0 mmol) and glutaric acid (3.30 g; 25.0 mmol) were suspended in water (40 ml) and the pH was adjusted to 6.8 with a solution of NaOH in water (8 M). The volume was adjusted to 50.0 ml and immobilized dicarboxylate acylase (Pseudomonas SE-83, 15.0 g; 96 units.gl) was added and the mixture was stirred at 200C for 6 h while maintaining the pH of the solution at 6.8 with a solution of HCl in water (1 M). The pH was lowered to 5.8 using a solution of HC1 in water (6 M) and stirring was continued at this pH-value for 2 h. The immobilized enzyme was removed by filtration, washed with water (50 ml) and the filtrate was lyophilized to give 10.78 g of disodium N- glutaryl-6P-aminopenicillanate as a white solid (purity 60% as determined by 600 MHz 1H NMR in D2O). Yield 69.2%.

EXAMPLE 4 DIAMMONIUM N-GLUTARYL- (6R, 7R) -3 -ACETOXYMETHYL-7 -AMINO- CEPH-3-EM-4-CARBOXYLATE 7-ACA (5.44 g; 20.0 mmol) and glutaric acid (2.64 g; 20.0 mmol) were suspended in water (40 ml) and the pH was adjusted to 7.0 at OOC with a 25% solution of ammonia in water. The temperature was adjusted to 300C and immobilized dicarboxylate acylase (Pseudomonas SE-83, 10.0 g; 96 units.g-') was added and the mixture was stirred at 300C for 3 h while maintaining the pH of the solution at 6.6 with a solution of HCl in water (1 M) . The pH was lowered to 5.9 using a solution of HCl in water (6 M).

After 4.5 h, the immobilized enzyme was removed by filtration, and part of the filtrate (69% by weight) was lyophilized to give 6.79 g of diammonium N-glutaryl-(6R,7R)-3-acetoxymethyl-7- aminoceph-3-em-4-carboxylate as a light-yellow solid (purity 40% as determined by 600 MHz 1H NMR in D2O). Yield 46.8%.

EXAMPLE 5 DIAMMONIUM N-GLUTARYL- (6R, 7R) -3 -ACETOXYMETHYL-7 -AMINO- CEPH-3-EM-4-CARBOXYLATE 7-ACA (1.36 g; 5.00 mmol) and glutaric acid (0.66 g; 5.00 mmol) were suspended in water (10 ml) and the pH was adjusted to

7.0 at OOC with a 25% solution of ammonia in water. The temperature was adjusted to 150C and immobilized dicarboxylate acylase (Acinetobacter sp., Boehringer Mannheim, 10.0 g; 12 units.gl) was added and the mixture was stirred at 150C for 16 h while maintaining the pH of the solution at 6.7 with a solution of HCl in water (1 M). The immobilized enzyme was removed by filtration, and part of the filtrate (39% by weight) was lyophilized to give 2.16 g of diammonium N-glutaryl-(6R,7R)-3- acetoxymethyl-7-aminoceph-3-em-4-carboxylate as a light-yellow solid (purity 26% as determined by 600 MHz 1H NMR in D2O) . Yield 68.5%.

EXAMPLE 6 DIPOTASSIUM N-ADIPYL-6 -AMINOPENICILLANATE 6-APA (10.99 g; 50.8 mmol) and adipic acid (7.31 g; 50.0 mmol) were suspended in water (80 ml). The pH was adjusted to 6.9 with a solution of NaOH in water (8 M). The volume was adjusted to 100.0 ml with water and immobilized dicarboxylate acylase (Pseudomonas SE-83, 33.3 g; 96 units.g') was added and the mixture was stirred at 200C for 3 h while maintaining the pH of the solution at 6.7 with a solution of HC1 in water (1 M). The pH was lowered to 6.0 using a solution of HCl in water (6 M) and the mixture was stirred for 2 h while maintaining the pH at 6.0.

Again, the pH was lowered to 5.5 and stirring was continued at this pH-value for 2 h. Immobilized dicarboxylate acylase was removed by filtration. A small portion of the filtrate (5 ml) was lyophilized and analyzed using 360 MHz 1H NMR in D2O which indicated that 75% of the starting 6P-aminopenicillanic acid was converted into N-adipyl-6P-aminopenicillanic acid. The pH of the remaining bulk of the filtrate was adjusted to 4.0 using a solution of HCl in water (6 M) and the filtrate was extracted with diethyl ether (3 x 150 ml). The pH was adjusted to 3.0 and the aqueous phase was extracted with 1-butanol (3 x 200 ml) . The combined organic phases were evaporated under reduced pressure to a volume of 200 ml and a solution of potassium 2- ethylhexanoate in 1-butanol (1.1 M; 105 ml) was added. The white precipitate was stirred for 30 min at OOC and collected by filtration, washed with 1-butanol (1 cake volume) and acetone (2

cake volumes). The product was dried under vacuum to give 12.34 g white product. Purity 65%, yield 37.5%.

EXAMPLE 7 DIPOTASSIUM N-ADIPYL-6 -AMINOPENICILLANATE, OBTAINED FROM INDUSTRIAL 6-APA MOTHER LIQUOR A sample from an industrially obtained 6-APA motherliquor (1.0 1 containing 2.7 g 6-APA) was evaporated at 370C under reduced pressure (32 mm Hg) to a volume of 810 ml. To 50 ml of this solution (containing approx. 0.8 mmol 6-APA) was added adipic acid (438 mg; 3.00 mmol). At 100C, the pH was adjusted to 5.8 with a solution of KOH in water (2 M) . Immobilized dicarboxylate acylase (Pseudomonas SE-83, 3.0 g; 110 units.g') was added and the mixture was stirred at 100C for 15 h while maintaining the pH of the solution at 5.8 with a solution of HCl in water (1 M) . Immobilized dicarboxylate acylase was removed by filtration. Part of the filtrate (89% of the reactor volume by weight) was lyophilized to give 2.01 g of white product. The product contains 8% dipotassium N-adipyl-6P-aminopenicillanate as determined by 360 MHz 1H NMR in D2O. The overall yield based upon 6-APA present in the original mother liquor is therefor 53.7%.

EXAMPLE 8 DIPOTASSIUM N-ADIPYL-(6R,7R)-7-AMINO-3-METHYLCEPH-3- EM-4-CARBOXYLATE, OBTAINED FROM SYNTHETIC 7-ADCA MOTHER LIQUOR (5 G/L) To a sample from a synthetically prepared 7-ADCA motherliquor (1.0 1 containing 5.0 g 7-ADCA) was added adipic acid (20.0 g; 137 mmol). To 50 ml of this solution was added immobilized dicarboxylate acylase (Pseudomonas SE-83, 5.0 g; 110 units.g-') . The mixture was stirred at 400C for 3 h while maintaining the pH of the solution at 6.1 with a warm (400C) solution of adipic acid in water (0.3 M). Immobilized dicarboxylate acylase was removed by filtration. Part of the filtrate (87% of the reactor volume by weight) was lyophilized to give 1.65 g of white product. The product contains 21% dipotassium N-adipyl- (6R,7R) -7-amino-3-methylceph-3-em-4-

carboxylate as determined by 360 M11z 1H NMR in D2O. The overall yield based upon 7-ADCA present in the original liquid is therefor 82.9%.

EXAMPLE 9 DIPOTASSIUM N-ADIPYL- (6R,7R) -7-AMINO-3-METHYLCEPH-3- EM-4-CARBOXYLATE, OBTAINED FROM SYNTHETIC 7-ADCA MOTHER LIQUOR (5 G/L, IN THE PRESENCE OF 1-BUTANOL) To a sample from a synthetically prepared 7-ADCA motherliquor (1.0 1 containing 5.0 g 7-ADCA and 16.2 g of 1- butanol) was added adipic acid (20.0 g; 137 mmol). To 50 ml of this solution was added immobilized dicarboxylate acylase (Pseudomonas SE-83, 5.0 g; 110 units.g-'). The mixture was stirred at 400C for 3 h while maintaining the pH of the solution at 6.1 with a warm (400C) solution of adipic acid in water (0.3 M).

Immobilized dicarboxylate acylase was removed by filtration. Part of the filtrate (80% of the reactor volume by weight) was lyophilized to give 1.65 g of white product. The product contains 21% dipotassium N-adipyl- (6R,7R) -7-amino-3-methylceph-3-em-4- carboxylate as determined by 360 MHz 1H NMR in D2O. The overall yield based upon 7-ADCA present in the original liquid is therefore 84.9%.

EXAMPLE 10 DIPOTASSIUM N-ADIPYL- (6R,7R) -7-AMINO-3-METHYLCEPH-3- EM-4-CARBOXYLATE, OBTAINED FROM SYNTHETIC 7-ADCA MOTHER LIQUOR (2.5 G/L) To a sample from a synthetically prepared 7-ADCA motherliquor (1.0 1 containing 2.5 g 7-ADCA) was added adipic acid (10.0 g; 69 mmol). To 50 ml of this solution was added immobilized dicarboxylate acylase (Pseudomonas SE-83, 5.0 g; 110 units.g') . The mixture was stirred at 100C for 15 h while maintaining the pH of the solution at 6.1 with a warm (400C) solution of adipic acid in water (0.3 M). Immobilized dicarboxylate acylase was removed by filtration. Part of the filtrate (85% of the reactor volume by weight) was lyophilized to give 0.85 g of white product. The product contains 17% dipotassium N-adipyl-(6R,7R)-7-amino-3-methylceph-3-em-4-

carboxylate as determined by 360 MHz 1H NMR in D2O. The overall yield based upon 7-ADCA present in the original liquid is therefor 70.6%.

EXAMPLE 11 DISODIUM N-ADIPYL-( 4R, 6R, 7R ) -7-AMINO-3-METHYLENECEPHAM-4- CARBOXYLATE (4R, 6R, 7R) -7-Amino-3-methylenecepham-4-carboxylic acid (1.12 g; 5.0 mmol) was suspended in water (9 ml) and the pH was adjusted to 6.3 with a solution of NaOH in water (8 M). Adipic acid (0.73 g; 5.0 mmol) was added and the pH was adjusted to 6.0 and immobilized dicarboxylate acylase (Pseudomonas SE-83, 4.4 g; 96 units.gl) was added and the mixture was stirred at 200C for 3 h while maintaining the pH of the solution at 6.0 with a solution of HC1 in water (1 M). The pH was lowered to 5.5 using a solution of HC1 in water (6 M) and stirring was continued at this pH-value for 2 h. Immobilized dicarboxylate acylase was removed by filtration. The filtrate was lyophilized to give 2.32 <BR> <BR> <BR> g o f disodiumN-adipyl- (4R,61?,7R) -7P-amino-3-methylenecepham-4- carboxylate (purity 60% as determined by 360 MMz 1H NMR in D2O).

Y-ield 72.1%.

EXAMPLE 12 DILITHIUM N-ADIPYL- (6R,7R) -3-ACETOXYMETHYL-7-AMINO- CEPH- 3 -EM-4 -CARBOXYLATE 7-ACA (4.09 g; 15.0 mmol) and adipic acid (2.20 g; 15.0 mmol) were suspended in water (70 ml) and the pH was adjusted to 7.5 at 0°C with a solution of LiOH in water (4 M). The temperature was adjusted to 100C, the pH was adjusted to 6.5, and immobilized dicarboxylate acylase (Pseudomonas SE-83, 20.0 g; 96 units.g-1) was added and the mixture was stirred at 100C for 16 h while maintaining the pK of the solution at 6.5 with a solution of HCl in water (1 M). The immobilized enzyme was removed by filtration, and part of the filtrate (58% by weight) was lyophilized to give 4.35 g of dilithium N-adipyl-(6R,7R)-3- acetoxymethyl-7-aminoceph-3-em-4-carboxylate as a light-yellow solid (purity 37% as determined by 600 MMz 1H NMR in D2O) . Yield 44.8%.

EXAMPLE 13 DIPOTASSIUM N-ADIPYL- (6R17R) -7-AMINO-3-CHLOROCEPH-3-EM- 4-CARBOXYLATE 7-ACCA (352 mg; 1.50 mmol) and adipic acid (220 mg; 1.50 mmol) were suspended in water (15 ml) and the pH was adjusted to 7.5 at 100C with a solution of KOH in water (2 M). To the clear yellow solution immobilized dicarboxylate acylase (Pseudomonas SE-83, 3.0 g; 96 units.g') was added and the mixture was stirred at 100C for 20 h while maintaining the pH of the solution at 6.2 with a solution of HCl in water (1 M) . The immobilized enzyme was removed by filtration, and part of the filtrate (75% by weight) was lyophilized to give 910 mg of dipotassium N-adipyl- (6R, 7R) -7- amino-3-chloroceph-3-em-4-carboxylate as a light-yellow solid (purity 18% as determined by 360 MMz 1H NMR in D2O). Yield 33.8%.

EXAMPLE 14 DIPOTASSIUM N-ADIPYL- (6R17R) -7-AMINO-3-CHLOROCEPH-3-EM- 4-CARBOXYLATE 7-ACCA (352 mg; 1.50 mmol) and adipic acid (220 mg; 1.50 mmol) were suspended in water (15 ml) and the pH was adjusted to 7.5 at 100C with a solution of KOH in water (2 M). To the clear yellow solution immobilized dicarboxylate acylase (Acinetobacter sp., 24.0 g; 12 units.g-') was added and the mixture was stirred at 100C for 20 h while maintaining the pH of the solution at 6.2 with a solution of HCl in water (1 M). The immobilized enzyme was removed by filtration, and part of the filtrate (36% by weight) was lyophilized to give 2.14 g of dipotassiumN-adipyl- (6R, 7R) -7 - amino-3-chloroceph-3-em-4-carboxylate as a white solid (purity 6% as determined by 360 MMz 1H NMR in D2O). Yield 55.7%.

EXAMPLE 15 DIPOTASSIUM N-ADIPYL- (6R,7R) -7-AMINO-3-HYDROXYMETHYLCEPH-3- EM-4-CARBOXYLATE 7-ADAC (359 mg; 1.50 mmol) and adipic acid (250 mg; 1.71 mmol) were suspended in water (15 ml) and the pH was adjusted to 7.5 at 400C with a solution of KOH in water (2 M). The pH was adjusted to 5.8, and immobilized dicarboxylate acylase (Pseudomonas SE-83, 10.0 g; 96 units.g') was added and the

mixture was stirred at 400C for 4 h while maintaining the pH of the solution at 5.8 with a solution of HCl in water (1 M). The immobilized enzyme was removed by filtration, and part of the filtrate (53% by weight) was lyophilized to give 530 mg of <BR> <BR> <BR> dipotassiumN-adipyl- (61?, 71?) -7-amino-3-hydroxymethylceph3em4 carboxylate as a light-yellow solid (purity 28% as determined by 360 MMz 1H NMR in D2O) . Yield 42.6%.

EXAMPLE 16 DILITHIUM N-(3-METHYLADIPYL)- N- (3-METHYLADIPYL)-6 -AMINOPENICILLANATE AND DILITHIUM N-(4-METHYLADIPYL)- N- (4-METHYLADIPYL)-6 -AMINOPENICILLANATE (MIXTURE OF ISOMERS) 6-APA (2.20 g; 10.0 mmol) and 3-methyladipic acid (1.60 g; 10.0 mmol) were suspended in water (30 ml) and the pH was adjusted to 6.4 with a solution of LiOH in water (4 M). To the clear solution immobilized dicarboxylate acylase (Pseudomonas SE- 83, 10.0 g; 96 units.g1) was added and the mixture was stirred at 10°C for 16 h while maintaining the pH of the solution at 6.4 with a solution of HCl in water (1 M). The pH was lowered to 5.7 and stirring was continued at this pH-value for 3 h. The immobilized enzyme was removed by filtration and part of the filtrate (68% by weight) was lyophilized to give 3.23 g of white product. Analysis by 360 MMz 1H NMR in D2O indicated the presence of 10% dilithium N-(3-methyladipyl) -6P-aminopenicillanate and 38% N-(4-methyladipyl)-6 -aminopenicillanate. Combined yield is 61.6%.

EXAMPLE 17 DISODIUM N- (trans--HYDROMUCONYL) - 6 P - AMINOPENI C I LLANATE 6-APA (5.94 g; 25.0 mmol) and trans-P-hydromuconic acid (3.60 g; 25.0 mmol) were suspended in water (40 ml) and the pH was adjusted to 7.0 with a solution of NaOH in water (8 M). The volume was adjusted to 50.0 ml and immobilized dicarboxylate acylase (Pseudomonas SE-83, 15.0 g; 96 units.g-') was added and the mixture was stirred at 200C for 2 h while maintaining the pH of the solution at 7.0 with a solution of HCl in water (1 M) . The pH was lowered to 5.5 using a solution of HCl in water (6 M) and stirring was continued at this pH-value for 4 h. Again, the pH

was lowered to 5.3 and stirring was continued for 1 hour. The immobilized enzyme was removed by filtration, washed with water (50 ml) and the filtrate was lyophilized to give 12.0 g of disodium N- (trans-p-hydromuconyl) -6P-aminopenicillanate as a white solid (purity 38% as determined by 600 MMz 1H NMR in D2O).

Yield 47.2%.

EXAMPLE 18 DIPOTASSIUM N- (trans --HYDROMUCONYL) - 6-AMINOPENICILLANATE 6-APA (1.19 g; 5.00 mmol) and trans-P-hydromuconic acid (0.72 g; 5.00 mmol) were suspended in water (10 ml) and the pH was adjusted to 6.4 with a solution of KOH in water (2 M) . To the clear solution immobilized dicarboxylate acylase (Acinetobacter sp., 10.0 g; 12 units.g-') was added and the mixture was stirred at 100C for 28 h while maintaining the pH of the solution at 6.4 with a solution of HCl in water (1 M) . The immobilized enzyme was removed by filtration and part of the filtrate (53% by weight) was lyophilized to give 1.06 g of dipotassium N-(trans-P- hydromuconyl) -6P-aminopenicillanate as a white solid (purity 15% as determined by 360 MMz 1H NMR in D2O) . Yield 14.0%.

EXAMPLE 19 DISODIUM N- (trans- -HYDROMUCONYL)-(6R,7R)-7-AMINO-3- METHYLCEPH-3-EM-4-CARBOXYLATE 7-ADCA (3.21 g; 15.0 mmol) and trans-P-hydromuconic acid (2.16 g; 15.0 mmol) were dissolved in water (40 ml) at pH 8.1 using a solution of NaOH in water (6 M). The volume was adjusted to 50.0 ml, the pH was brought to 6.5 and immobilized dicarboxylate acylase (Pseudomonas SE-83, 25.0 g; 96 units.g') was added and the mixture was stirred at 200C for 3 h while maintaining the pH of the solution at 6.5 with a solution of HCl in water (1 M) . The pH was lowered to 5.8 using a solution of HC1 in water (6 M) and stirring was continued at this pH-value for 3.5 h. The immobilized enzyme was removed by filtration and the filtrate (76% of the total reaction by weigth) was lyophilized to give 3.74 g of disodium N-( trans-P-hydromuconyl )-(6R, 7R)-7- amino-3-methylceph-3-em-4-carboxylate as a white solid (purity 19% as determined by 600 MMz lH NMR in D2O) . Yield 16.2%.

EXAMPLE 20 DILITHIUM N- (trans, trans-MUCONYL) 6 -AMINOPENICILLANATE 6-APA (1.19 g; 5.00 mmol) and trans,trans-muconic acid (0.73 g; 5.00 mmol) were suspended in water (20 ml) and the pH was adjusted to 6.3 with a solution of LiOH in water (4 M). To the clear solution immobilized dicarboxylate acylase (Pseudomonas SE- 83, 20.0 g; 96 units.g-') was added and the mixture was stirred at 300C for 14 h while maintaining the pH of the solution at 6.3 with a solution of HCl in water (1 M). The immobilized enzyme was removed by filtration and part of the filtrate (16% by weight) was lyophilized to give 340 mg of dilithium N-(trans,trans- muconyl)-6P-aminopenicillanate as a light-yellow solid (purity 15% as determined by 360 MMz 1H NMR in D2O) . Yield 18.3%.

EXAMPLE 21 DIPOTASSIUM N-PIMELYL- 6 -AMINOPENICILLANATE 6-APA (2.19 g; 10.0 mmol) and pimelic acid (1.63 g; 10.0 mmol) were suspended in water (30 ml) and the pH was adjusted to 6.4 with a solution of KOH in water (2 M). To the clear solution immobilized dicarboxylate acylase (Pseudomonas SE-83, 10.0 g; 110 units.g~l) was added and the mixture was stirred at 140C for 1.5 h while maintaining the pH of the solution at 6.4 with a solution of HCl in water (1 M). The pH was lowered to 6.2 and stirring was continued for 18 h. Again, the pH was lowered to 5.6 by the addition of pimelic acid (0.25 g; 1.5 mmol). After 3 h, the immobilized enzyme was removed by filtration and part of the filtrate (62% by weight) was lyophilized to give 3.52 g of dipotassium N-pimelyl-6P- aminopenicillanate as a white solid (purity 59% as determined by 360 MMz 1H NMR in D2O). Yield 77.6%.

EXAMPLE 22 <BR> <BR> <BR> <BR> DISODIUM N- (3,3'-THIODIPROPIONYL) - (6R,7R) -7-AMINO-3- <BR> <BR> <BR> <BR> <BR> <BR> METHYLCEPH-3 -EM-4 -CARBOXYLATE 7-ADCA (3.21 g; 15.0 mmol) and 3,3'-thiodipropionic acid (97%, 2.76 g; 15.0 mmol) were dissolved in water (40 ml) at pH 8.1 using a solution of NaOH in water (6 M). The volume was adjusted to 50.0 ml, the pH was brought to 6.5 and immobilized

dicarboxylate acylase (Pseudomonas SE-83, 25.0 g; 96 units.g-') was added and the mixture was stirred at 400C for 1 h while maintaining the pH of the solution at 6.5 with a solution of HC1 in water (1 M). The temperature was raised to 500C and stirring was continued for another 2.5 h. The pH was lowered to 6.2 using a solution of HC1 in water (6 M) and stirring was continued at this pH-value for 4 h. Finally, the reaction was allowed to proceed at pH 5.9 for 1 h and the immobilized enzyme was removed by filtration and the filtrate (72% of the total reaction by weigth) was lyophilized to give 5.14 g of disodium N-(3,3'- thiodipropionyl)-(6R,7R)-7-amino-3- methylceph-3-em-4-carboxylate as a yellow solid (purity 11% as determined by 360 MMz 1H NMR in D2O). Yield 12.8%.

EXAMPLE 23 <BR> <BR> <BR> <BR> DIPOTASSIUM N-SUBERYL- (4R, 6R,7R) - 7 - AMINO-3-METHYLENECEPHAM- 4 - CARBOXYLATE (4R,6R, 7R)-7-Amino-3-methylenecepham-4-carboxylic acid (1.12 g; 5.0 mmol) and suberic acid (1.12 g; 6.3 mmol) were suspended in water (20 ml) and the pH was adjusted to 6.2 with a solution of KOH in water (2 M). To the clear solution immobilized dicarboxylate acylase (Pseudomonas SE-83, 10.0 g; 110 units.g-') was added and the mixture was stirred at 100C for 63 h while maintaining the pH of the solution at 6.4 with a solution of HCl in water (1 M). The pH was lowered to 5.7 by adding suberic acid (0.25 g; 1.4 mmol) and stirring was continued for 1.5 h. The temperature was raised to 400C and stirring was continued for 2 h after which the immobilized enzyme was removed by filtration and part of the filtrate (60% by weight) was lyophilized to give 1.91 g of potassium N- suberyl-(4R,6R,7R)-7-amino-3-methylenecepham-4-carboxylate as a white solid (purity 5% as determined by 360 MMz 1H NMR in D2O) . Yield 7.1%.