WO1998056945A1

WO1998056945A1 - PROCESS FOR ENZYMATICALLY PREPARING A β-LACTAM ANTIBIOTIC AND THIS ANTIBIOTIC

Info

Publication number: WO1998056945A1
Application number: PCT/EP1998/003629
Authority: WO
Inventors: Thomas Van Der Does; Riesk Hendrik Kuipers; Adrianus Franciscus Michael Slobbe; Harold Monro Moody
Original assignee: Gist Brocades BV; DSM NV
Current assignee: Koninklijke DSM NV; DSM Delft BV
Priority date: 1997-06-10
Filing date: 1998-06-10
Publication date: 1998-12-17
Anticipated expiration: 1999-12-10
Also published as: AU8799498A

Abstract

The invention relates to a process for enzymatically preparing a β-lactam antibiotic from a β-lactam nucleus or a salt thereof and a precursor for a side chain, wherein a large amount of enzyme is used, which amount is at least twice the amount that is normally used.

Description

PROCESS FOR ENZYMATICALLY PREPARING A /?-LACTAM ANTIBIOTIC AND THIS ANTIBIOTIC

The invention relates to a process for enzymatically preparing a β-lactam antibiotic and to a β-lactam antibiotic obtainable thereby.

Nowadays, β-lactam antibiotics, such as penicillin and cephalosporin antibiotics, play an important role in medicine. This class of antibiotics comprises a great 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 group to the so-called side chain via a linear amide bond. The term β-lactam antibiotics as used herein includes fermentation products, such as penicillin G, penicillin V, cephalosporin C, isopenicillin N, intermediate products such as adipyl-6-aminopenicillanic acid (adipyl-6-APA), adipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA), adipyl-7-aminocephalosporanic acid (adipyl-7-ACA), adipyl-7-aminodesacetylcephalosporanic acid (adipyl-7-ADAC),

3-carboxyethylthiopropionyl-7-aminodesacetoxycephalosporanic acid,

2-carboxylethylthioacetyl-7-aminodesacetoxycephalosporanic acid and

3-carboxyethylthiopropionyl-7-aminodesacetoxycephalosporanic acid, and semi-synthetic products such as Ampicillin, Amoxycillin, Cephalexin, Cephacior and Cephadroxyl.

Conventionally, β-lactam antibiotics have been prepared in chemical procedures. However, such chemical methods have a number of grave disadvantages. They comprise many complex reactions, in which by-products which give rise to effluent and purification problems are formed. Also, because many steps have to be performed in order to obtain the final antibiotic product, the overall yield is quite low.

Recently, there has been a lot of interest for so called enzymatic semi-synthetic routes to β-lactam antibiotics. These routes involve enzymatically catalyzed processes and lack many of the disadvantages of the conventional synthetic methods for preparing β-lactam antibiotics. The enzymatic catalyzed reactions are highly selective, thus the production of many by-products, and the effluent and purification problems, which result therefrom, are avoided. Furthermore, enzymatic processes can be performed in aqueous environment.

The semi-synthetic routes mostly start from fermentation products such as isopenicillin N, penicillin G, penicillin V and cephalosporin C, which are enzymatically converted to a β-lactam nucleus, for instance in a manner as has been disclosed in K. Matsumoto, Bioprocess. Technol., J_6, ( 1 993), 67-88, J.G. Shewale & H. Sivaraman, Process Biochemistry, August 1 989, 1 46-1 54, T.A. Savidge, Biotechnology of Industrial Antibiotics (Ed. E.J. Vandamme) Marcel Dekker, New York, 1 984, or J.G. Shewale et al., Process Biochemistry International, June 1 990, 97-103. The obtained β-lactam nucleus is subsequently converted to the desired antibiotic by coupling to a suitable side chain, as has been described in inter alia EP-A-0 339 751 , JP-A-53 005 1 85 and CH-A-640 240. By making different combinations of side chains and β-lactam nuclei, a variety of penicillin and cephalosporin antibiotics may be obtained. For example, a D-(-)-phenylglycine side chain may be attached to a 6-aminopenicillanic acid (6-APA) nucleus, in a reaction with a suitable derivative of said D-(-)-phenylglycine acid, to yield Ampicillin, or to a 7-aminodesacetoxycephalosporanic acid (7-ADCA) nucleus to yield Cephalexin.

The known enzymatic methods for preparing β-lactam antibiotics all involve the preparation of a β-lactam intermediate and the subsequent coupling thereof to a suitable side chain, e.g. in a reaction with a suitable precursor for the desired side chain. References for enzymatic synthesis are: T.A. Savidge, Biotechnology of Industrial Antiobiotics (Ed. E.J. Vandamme) Marcel Dekker, New York 1 984, J.G. Shewale et al., Process Biochemistry International, June 1 990 97-1 03, E.J. Vandamme, Advances in Applied Microbiology, 2J_, (1 977), 89-1 23 and E.J. Vandamme, Enzyme Microb. Technol., 5_, ( 1 983), 403-41 6. In addition, new routes have been disclosed, which show the direct fermentative production of 7-ADCA, 7-ADAC and 7-ACA, in EP-A-0 540 21 0, WO-A-93/08287, WO-A-95/041 48 and WO- A-95/041 49.

A problem that is often encountered in the enzymatic conversion of β-lactam nuclei to β-lactam antibiotics is the degradation of one or more of the used reactants. Particularly the β-lactam nucleus starting material often decomposes under the reaction conditions usually applied for the conversion reaction. As a result of these undesired degradation reactions, up to 1 0% of the β-lactam antibiotic production may be lost. Thus, the efficiency and economic feasibility of the enzymatic preparation of β-lactam antibiotics is adversely affected by the degradation reactions.

It has now been found that the negative effect of the above mentioned degradation reactions can be significantly reduced by using a large amount of enzyme in the enzymatic reaction of a β-lactam nucleus and a precursor for a side chain. Accordingly, the invention provides a process for enzymatically preparing a β-lactam antibiotic from a β-lactam nucleus or a salt thereof and a precursor for a side chain, wherein a large amount of enzyme is used, which amount is at least twice the amount that is normally used.

Surprisingly, it has been found that the loss of β-lactam antibiotic production may be reduced to about 1 -2%. In addition, it has been found that the productivity of the conversion process, expressed as the production of β-lactam antibiotic per litre reaction mixture per hour, is greatly increased by applying the teaching of the present invention.

By a large amount of enzyme in accordance with the invention, an amount is intended which significantly exceeds the amounts which are generally applied for enzymatic conversions. More in detail, by a large amount of enzyme, an amount is intended which amount is at least twice the amount that is normally used. Due to the high activity and specificity of enzymes on the one hand and the cost of enzymes on the other hand, it is common practice to normally use enzymes in very small amounts. These amounts are often referred to as "catalytic amounts" and lie in the range of 0.1 to 5 mol%, based on one of the reactants in question. In accordance with the invention, it has been found that by using an amount of enzyme which significantly exceeds these usual catalytic amounts, viz. an amount which is at least twice the amount that is normally used, say about 1 0 mol%, the disadvantageous degradation of reactants can be greatly reduced. An "amount" of enzyme as used herein may be expressed in terms of mass or volume enzyme in relation to the amount of reaction mixture, or in terms of enzyme activity.

Preferably, the enzyme is present in an amount of at least about 60 ASU per litre reaction mixture. The unit ASU, or Amoxicillin Synthesis Unit, is defined herein as the activity required for formation of 1 g amoxicillin trihydrate per hour at 20 ° C and a pH of approximately 6.3, from 6-APA and D-4-hydroxyphenylgiycine methyl ester.

Thus, in accordance with this embodiment of the invention, the amount of enzyme to be used is defined in terms of activity. In a process for enzymatically preparing ampicillin, cefaclor, cephalexin, cephadroxil, cephadrine, epicillin, cefamandole, or in particular amoxicillin, it is further preferred that the enzyme is present in an amount of at least about 60 ASU, preferably at least about 1 00 ASU, more preferably at least about 1 20 ASU, per litre reaction mixture. In another preferred embodiment, the enzyme is present in an amount of at least about 200 grams wet nett weight of enzyme per litre reaction mixture, preferably about 250 grams wet nett weight of enzyme per litre reaction mixture, which usually corresponds to about 20 vol.%, more preferably about 25 vol.%, based on the reaction volume. The upper limit of the amount of enzyme to be used is determined by the amount which can physically be introduced in a reaction vessel wherein the process is to be performed and at which amount of enzyme there remains sufficient space for the other reactants present in a process according to the invention. In practice, this amount will be between about 330 and about 500 grams wet nett weight of enzyme per litre reaction mixture.

It is preferred that an enzyme is used, which has an activity of at least about 300, preferably more than about 400 ASU per kilogram nett wet weight. It was found that an enzyme having the above specified activity is particularly suitable for suppressing the degradation of any reactants in the preparation of a β-lactam antibiotic, when used in a large amount.

According to the invention, a β-lactam antibiotic is prepared starting from a β-lactam nucleus or a salt thereof. Although in principle any β-lactam nucleus is suitable, a β-lactam nucleus having the general formula

( i ) wherein

Ro is hydrogen or C₁.3 alkoxy; Y is CH₂, oxygen, sulphur, or an oxidized form of sulphur; and

Z is

wherein Ri is hydrogen, hydroxy, halogen, d-₃ alkoxy, optionally substituted, optionally containing one or more heteroatoms, saturated or unsaturated, branched or straight C1.5 alkyl, optionally substituted, optionally containing one or more heteroatoms, C₅.₈ cycloalkyi, optionally substituted aryl or heteroaryl, or optionally substituted benzyl, is preferably used as starting material.

In the context of the invention, an oxidized form of sulphur is meant to include groups such as sulfoxide and sulphone. By optionally substituted alkyl, cycloalkyi, aryl, heteroaryl and benzyl, groups are intended, which have substituents such as alkyl groups of from 1 to 3 carbon atoms, halogen or hydroxy.

Formula ( I ) is intended to encompass all β-lactam nuclei as disclosed in "Cephalosporins and Penicillins, Chemistry and Biology", Ed. E.H. Flynn, Academic Press, 1 972, pages 1 51 -1 66, and "The Organic Chemistry of β-Lactams", Ed. G.I. Georg, VCH, 1 992, pages 89-96, which are incorporated herein by reference. Preferred are those starting materials wherein R represents a CH₂-D or CH = CH-D group, wherein D is hydrogen, hydroxy, halogen, d.₃ alkoxy, optionally substituted, optionally containing one or more heteroatoms, saturated or unsaturated, branched or straight d-₅ alkyl, optionally substituted, optionally containing one or more heteroatoms C₅.₈ cycloalkyi, optionally substituted aryl or heteroaryl, or optionally substituted benzyl.

A suitable salt of a β-lactam nucleus to be converted in accordance with the invention is 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, Λ ,Λ '-dibenzyl diethyiene diamine).

Generally, the β-lactam nucleus is a cephalosporanic acid, a penicillanic acid, or a salt thereof. Most preferred β-lactam nuclei are 6-aminopenicillanic acid (6-APA), 7-aminocephalosporanic acid (7-ACA), 3-chloro-7-aminodesacetoxydesmethylcephalosporanic acid (7-ACCA), 7-aminodesacetylcephalosporanic acid (7-ADAC), 7-aminodesacetoxycephalo- sporanic acid (7-ADCA), or

7-amino-3-[[( 1 -methyl- 1 -H-tetrazol-5-yl)thio]methyl]-3-cephem-4-carboxylic acid (7-ATCA), as these are precursors for the penicillin and cephalosporin antibiotics which have the most advantageous activity profiles.

The β-lactam nucleus that is used to prepare a β-lactam antibiotic in a method according to the invention may be obtained by enzymatic hydrolysis of a fermentation product, such as penicillin V, penicillin G, isopenicillin N or cephalosporin C, or a ring enlarged analogue thereof, such as V-DCA or G-DCA, or a derivative thereof, as for instance has been described in EP-A-0 532 341 .

When a β-lactam nucleus is used in a method according to the invention, which has been obtained by enzymatic hydrolysis of a fermentation product as mentioned above, it is preferred that any by-products resulting therefrom, such as phenylacetic acid or phenoxyacetic acid, are removed to an extent, which is sufficient for the efficiency of the enzymatic preparation of a β-lactam antibiotic to be not unacceptably adversely affected by the presence of said by-products. The person skilled in the art will be able to determine to what extent said by-products need to be removed in order to obtain the most economically feasible process.

The β-lactam nucleus starting material is in accordance with the invention reacted with a precursor for a side chain. This precursor for a side chain is chosen such that, starting from a certain β-lactam nucleus starting material, a β-lactam antibiotic having a desired activity profile is obtained.

Preferred precursors for a side chain in accordance with the invention are D-(-)-phenylglycine, D-(-)-4-hydroxyphenylglycine,

D-(-)-2,5-dihydrophenylglycine, 2-thienylacetic acid,

2-(2-amino-4-thiazolyl)-2-methoxyiminoacetic acid, α-(4-pyridylthio)acetic acid, 3-thiophenemalonic acid, 2-cyanoacetic acid, and D-mandelic acid. The precursor for a side chain is preferably an activated form, such as an ester or amide, of the side chain. Suitable esters in this respect are for instance lower alkyl esters, such as methyl, ethyl, n-propyl or isopropyl esters. Suitable amides are in this respect for instance amides which are unsubstituted or substituted at the -CONH₂ group. The precursor for a side chain may also be a salt, e.g. an HCI or an H₂S0₄ salt, of the side chain, or of an ester or amide thereof. It is possible to use the precursor in an activated form, but it is also possible that an activated form is formed in situ.

The enzyme which is used for enzymatically preparing a β-lactam antibiotic in accordance with the invention may be any enzyme catalyzing the reaction in question. Suitable enzymes are for instance those that have been referred to as penicillin amidase or penicillin acylase in the literature, and those that have been classified as E.C. 3.5.1 .1 1 . The enzyme may also be a so-called ampicillin hydrolase, acylase or amidase. In this connection, reference is made to Hakko to Koqyo 38 ( 1 980), 21 6 et seg., the contents of which are incorporated herein by reference.

Organisms that have been found to produce penicillin acylase are, for example, Acetobacter, Aeromonas, Alcaligenes, Aphanocladium, Bacillus sp., Cephalosporium, Escherichia, Flavobacterium, Kluyvera, Mycoplana, Protaminobacter, Pseudomonas or Xanthomonas species. Enzymes derived from Acetobacter pasteurianum, Alcaligenes faecalis, Bacillus megaterium, Escherichia coli, Kluyvera citrophila and Xanthomonas citrii have particularly proven to be successful in a method according to the invention.

The enzyme may be used as free enzyme, but it is preferred to use it in a suitable reusable form, such as an entrapped or immobilized form, for instance as has been described in WO-A-97/04086. 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 is affected by chemical modification or cross-linking, without significant consequences for the activity, in kind, not in amount, of the enzymes in a method according to the invention. Also, functional equivalents such as mutants or other derivatives, obtained by classic 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.

The reaction conditions applied in a method 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 β-lactam nucleus that is to be reacted with a given precursor for a side chain, using a given enzyme, to a given β-lactam antibiotic, 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 about -1 0 and about 50 ° C, preferably between about -5 and about 40 ° C. It is to be noted, that the higher the temperature chosen for the enzymatic preparation of a β-lactam antibiotic according to the invention, the higher the productivity of said process. However, it has been found that at lower temperatures, the degradation of any reactants is more efficiently suppressed. The skilled person will be able to choose the optimum temperature for a process in accordance with the invention, taking into account the effect on the productivity of the conversion reaction on the one hand, and the extent wherein any reactants are degraded on the other hand.

The optimum pH in the preparation of a β-lactam antibiotic according to the invention lies between about 4.5 and about 9.0, preferably between about 5.5 and about 8.5. In this regard, it is to be noted that is highly preferable to perform a method according to the invention in aqueous environment, because in that way the use of organic solvents, which would lead to effluent problems, is circumvented. Moreover, the enzyme has proven to catalyze the conversion reaction most efficiently in an aqueous environment. If desired, however, it is also possible to add an organic cosolvent to the reaction system.

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.

Suitable titrants are inorganic acids and bases, such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, ammonium hydroxide, and so forth, or organic acids, such as formic acid, acetic acid, succinic acid, adipic acid, glutaric acid and so forth. Titrant concentration may 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 obtainable by a method as disclosed hereinabove. Particularly preferred are β-lactam antibiotics, wherein the β-lactam nucleus starting material and the precursor for a side chain are chosen such that the β-lactam antibiotic is ampicillin, amoxicillin, cefaclor, cephalexin, cephadroxil, cephadrine, epicillin, or cefamandole.

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

EXAMPLES

Definitions and procedures

Assemblase™ is an immobilized Escherichia coli penicillin acylase from

E. coli ATCC 1 1 105 as described in patent application WO 97/04086, which is incorporated herein by reference. Immobilization was carried out as described in European patent application No. 0 222 462, which is also incorporated herein by reference, using gelatin and chitosan as gelling agents and glutaric dialdehyde as crosslinking agent. Final activity of the Escherichia coli penicillin acylase was determined by the amount of enzyme added to the activated beads.

The Amoxicillin Synthesis activity of pen acylases was determined as follows: 6-APA (purified as described in Example 6, 6.48 g, 30 mmoles), D-4-hydroxyphenylglycin methyl ester (6.26 g, 35 mmoles) and phenylacetic acid (0.65 mg) were suspended in water. Total volume was 1 00-x ml, wherein x is the amount of enzyme (in grams) to be tested. The mixture was thermostated to 20 ° C. The incubation was started by addition of x g enzyme. During incubation, samples were taken and the amount of amoxicillin formed was determined by HPLC analysis. Up until about 80 mM, formation of amoxicillin was linear with time. In this time interval, the amoxicillin formation rate can be expressed in g amoxicillin trihydrate per hour. Dividing this amoxicillin formation rate by x/1000 gives the specific activity of the enzyme in ASU per kg enzyme (ASU: Amoxicillin Synthesis Unit). 1 ASU is defined as the activity required for formation of 1 g amoxicillin trihydrate per hour under conditions mentioned in this example at a pH of approximately 6.3 without adding any acid or base.

EXAMPLE 1 Preparation of a solution of D-phenylglycineamide.¹/-; H₂SO₄

D-Phenylglycineamide (301 .6 g, 2.00 moles) was suspended in water

(650 g) at 5 ° C. The mixture was stirred and sulphuric acid (assay 96%,

1 02.1 g, 1 .00 mol) was added in the course of 1 hour. The mixture was cooled during addition of sulphuric acid, and the temperature was kept below

25 ° C.

EXAMPLE 2 Preparation of ampiciilin A cylindrical vessel (volume 1 .5 I, diameter 1 1 cm) equipped with a sieve bottom with 180 μm slots, thermometer and pH electrode was charged with Assemblase™ (300 g nett wet weight, specific activity 400 ASU per kg nett wet weight). A separate vessel was charged with 6-APA ( 1 31 .6 g, 0.600 moles), D-phenylglycineamide (30.2 g, 0.200 moles) and water (400 ml). The temperature of this mixture was adjusted to 1 0 ° C. The mixture was stirred during 1 5 minutes at 1 0 ° C, and subsequently transferred to the vessel with the sieve bottom. Remnants were rinsed to the vessel with the sieve bottom with water ( 100 ml). The contents of the vessel with the sieve bottom were stirred, and during 283 minutes 423.7 g of the solution prepared as described in Example 1 (containing 0.8 mol D-phenylglycineamide. ¹/ H₂SO₄) was added at constant rate. During addition of this solution the temperature was kept at 10 ° C, the pH was approximately 6.3. Stirring of the mixture was continued, and 328 minutes after addition of the reagents to the vessel with the sieve bottom, 3 M sulphuric acid was added at such a rate that the pH was maintained at pH = 6.3. 540 minutes after addition of the reagents to the vessel with the sieve bottom, the amount of ampicillin was at its maximum. 3 M Sulphuric acid was added until the pH was 5.6. At this point the reaction mixture contained: 575 mol ampicillin (96% yield based on 6-APA)

1 5 mmol 6-APA 50 mmol D-phenylglycineamide 365 mmol D-phenylglycine The volume was 1 400 ml. The productivity was 46 mmol ampicillin/l/h.

EXAMPLE 3 Preparation of ampicillin

A cylindrical vessel (volume 1 .5 I, diameter 1 1 cm) equipped with a sieve bottom with 1 80 μm slots, thermometer and pH electrode was charged with Assemblase™ (300 g nett wet weight, specific activity 400 ASU per kg nett wet weight). A separate vessel was charged with water (600 ml, T = 10 ° C), D-phenylglycineamide ( 143.3 g, 0.950 moles) and sulphuric acid (assay 96%, 22.6 g 0.221 moles). The mixture was stirred during 1 0 minutes at 10 ° C. The pH was now 7.2. Subsequently, 6-APA (1 31 .6 g, 0.600 moles) was added. The 6-APA/D-phenylglycineamide mixture was stirred during 1 5 minutes at 1 0 ° C, and subsequently the mixture was transferred to the vessel with the sieve bottom. Remnants were rinsed to the vessel with the sieve bottom with water ( 1 00 ml). The contents of the vessel with the sieve bottom were stirred at 10 ° C and the pH was maintained at pH = 6.3 with the aid of 3 M sulphuric acid. 300 minutes after addition of the reagents to the vessel with the sieve bottom the amount of ampicillin was at its maximum. 3 M Sulphuric acid was added until the pH was 5.6. At this point the reaction mixture contained:

575 mmol ampicillin (96% yield based on 6-APA) 1 5 mmol 6-APA

50 mmol D-phenylglycineamide

365 mmol D-phenylglycine The volume was 1400 ml. The productivity was 82 mmol ampicillin/l/h.

EXAMPLE 4

Preparation of ampicillin

A cylindrical vessel (volume 1 .5 I, diameter 1 1 cm) equipped with a sieve bottom with 1 80 μm slots, thermometer and pH electrode was charged with Assemblase™ (300 g nett wet weight, specific activity 400 ASU per kg nett wet weight). A separate vessel was charged with water (500 ml) and D-phenylglycineamide (143.2 g, 0.950 moles). The mixture was stirred and kept at 1 0 ° C, while 6-APA ( 1 31 .6 g, 0.600 moles) was added in portions in the course of 1 5 minutes. The pH was kept at pH = 7.0 by titration with 3 M sulphuric acid during addition of 6-APA. 54.5 ml 3 M sulphuric acid was required. The 6-APA/D-phenylglycineamide mixture was stirred during 1 5 minutes at 1 0 ° C and subsequently the mixture was transferred to the vessel with the sieve bottom. Remnants were rinsed to the vessel with the sieve bottom with water (100 ml). The contents of the vessel with the sieve bottom were stirred at 10 ° C and the pH was maintained at pH = 7.0 with the aid of 3 M sulphuric acid. 1 60 minutes after addition of the reagents to the vessel with the sieve bottom the amount of ampicillin was at its maximum. 3 M Sulphuric acid was added until the pH was 5.6 (147.6 ml 3 M sulphuric acid was added to the vessel with the sieve bottom). At this point the reaction mixture contained: 551 mmol ampicillin (92% yield based on 6-APA)

24 mmol 6-APA

50 mmol D-phenylglycineamide

330 mmol D-phenylglycine The volume was 1 400 ml. The productivity was 1 48 mmol ampiciliin/l/h.

EXAMPLE 5 Purification of 6-APA

Industrially produced 6-APA may contain phenylacetic acid. Phenylacetic acid retards the enzymatic conversion. Therefore, phenylacetic acid was removed by extraction.

A solution of 6-APA in dilute sulphuric acid (concentration 40 g 6-APA/l, pH = 1 .5) was extracted three times with n-butanol at 3 ° C. The solution was crystallized by aid of 25% 25% ammonia at pH =4.0 and 10 ° C. 6-APA was isolated by filtration, washed with water and dried. Analysis by HPLC and by GLC revealed that the phenylacetic acid content in 6-APA was < 2 ppm.

EXAMPLE 6 Synthesis of amoxicillin 6-APA (purified as described in example 5, 1 2.97 g, 60.0 mmoles) and D-4-hydroxyphenylglycine methyl ester (14.78 g, 81 .6 mmoles) and enzyme were suspended in water. A solution of phenylacetic acid in water (10 ml of a 0.01 3% by weight solution, 0.01 mmoles phenylacetic acid) was added in order to simulate industrial conditions where 6-APA often contains a certain amount of phenylacetic acid. By first purifying 6-APA and subsequently adding a known amount of phenylacetic acid, a 6-APA stock is obtained which may be used for experiments of which the results are to be compared, without a possible impact of differences in phenylacetic acid concentrations on those results.

Water was added to a total of 1 20 ml. The mixture was stirred at 20 ° C, and the pH was monitored. At first the pH was 6.3. After some time, the pH increased to pH = 6.8, and subsequently the pH decreased to pH = 6.2. At this point (total reaction time 1 58 minutes) water was added to a total weight of 238 g. Immediately a weighed sample was taken. The sample was diluted in buffer, and analyzed by HPLC. The results obtained using different conditions are shown in Table I.

Table

" CLEC: The Cross-Linked Enzyme Crystal from penicillin acylase, obtained from Altus Biologies Inc., Cambridge, USA, Product Number: CEC-1 -G-S. Description ChiroClec-EC, lot nr. EC96-004.

Claims

1 . A process for enzymatically preparing a β-lactam antibiotic from a β-lactam nucleus or a salt thereof and a precursor for a side chain, wherein a large amount of enzyme is used, which amount is at least twice the amount that is normally used.

2. A process according to claim 1 , wherein the enzyme is present in an amount of at least about 60 ASU per litre reaction mixture, preferably at least about 1 00 ASU per litre reaction mixture.

3. A process according to claim 1 , wherein the enzyme is present in an amount of at least about 200 grams wet nett weight of enzyme per litre reaction mixture, preferably about 250 grams wet nett weight of enzyme per litre reaction mixture.

4. A process according to claim 1 -3, wherein the enzyme has an activity of at least about 300, preferably more than about 400 ASU per kilogram nett wet weight.

5. A process according to any of the preceding claims, wherein the β-lactam nucleus is represented by the general formula

( i ) wherein

Ro is hydrogen or d-₃ alkoxy;

Y is CH₂, oxygen, sulphur, or an oxidized form of sulphur; and

Z is

wherein Ri is hydrogen, hydroxy, halogen, d-3 alkoxy, optionally substituted, optionally containing one or more heteroatoms, saturated or unsaturated, branched or straight d_₅ alkyl, optionally substituted, optionally containing one or more heteroatoms, C₅.₈ cycloalkyi, optionally substituted aryl or heteroaryl, or optionally substituted benzyl.

6. A process according to claim 5, wherein the β-lactam nucleus is chosen from the group of 6-aminopenicillanic acid (6-APA), 7-aminocephaiosporanic acid (7-ACA), 3-chloro-7-aminodesacetoxydesmethyl- cephalosporanic acid (7-ACCA), 7-aminodesacetylcephalosporanic acid (7-ADAC), 7-aminodesacetoxycephalosporanic acid (7-ADCA), and 7-amino-3-[[(1 -methyl- 1 -H-tetrazol-5-yl)thio]methyl]-3-cephem-4-carboxylic acid (7-ATCA).

7. A process according to any of the preceding claims, wherein the precursor for a side chain is chosen from the group of D-(-)-phenylglycine, D-(-)-4-hydroxyphenylglycine, D-(-)-2,5-dihydrophenylglycine, 2-thienylacetic acid, 2-(2-amino-4-thiazolyl)-2-methoxyiminoacetic acid, α-(4-pyridyl- thio)acetic acid, 3-thiophenemalonic acid, 2-cyanoacetic acid, and D-mandelic acid, or an amide or ester thereof.

8. A process according to any of the preceding claims, wherein the enzyme is a penicillin acylase or a functional equivalent thereof.

9. A process according to claim 8, wherein the penicillin acylase or functional equivalent thereof is obtained from Acetobacter pasteurianum, Alcaligenes faecalis, Bacillus megaterium, Escherichia coli, Kluyvera citrophilia and Xanthomonas citrii.

1 0. A β-lactam antibiotic obtainable by any of the preceding claims.

1 1 . A β-lactam antibiotic according to claim 10, wherein the β-lactam antibiotic is ampicillin, amoxicillin, cefaclor, cephalexin, cephadroxil, cephadrine, epicillin, or cefamandole.