Technical field

The present invention relates to an improved immobilized Penicillin G acylase. Furthermore, the invention relates to the preparation of )3-lactam antibiotics by enzymatic aeylation of the parent amino ,9-lactam nucleus with the corresponding acylat¬ ing agent using said immobilized enzyme.

Background and field of the invention

Enzymatic production of semisynthetic 0-lactam antibiotics by aeylation of the parent amino β-lactam moiety with an acti- vated side chain acid derivative, such as an amide or an ester, is known from Dutch patent 158847, European patent applications 339751 and 473008, international patent applications WO 92/01061 and WO 93/12250, U.S. patent 3816253, and West German patent documents 2163792 and 2621618. The enzymes used in the art are in most cases penicillin acylases obtained from Escherichia coli and are immobilized on various types of water-insoluble materials. A drawback of the known enzymatic methods for the produc¬ tion of amoxycillin, ampicillin, cephadroxil, cephalexin, and cephradine is the high cost due to the selectivity of the immo- bilized enzyme. Said immobilized enzymes are capable of condens¬ ing activated side chain derivatives such as D(-)-phenylglycine amide (PGA), D(-)-phenylglycine methyl ester (PGM), D(-)-4- hydroxyphenylglycine amide (HPGA) , D(-)-4-hydroxyphenylglycine methyl ester (HPGM) , D(-)-2,5-dihydro-phenylglycine amide (DPGA), and D(-)-2,5-dihydrophenylglycine methyl ester (DPGM) with amino β-lactams such as 6-amino-penicillanic acid (6-APA) , 7-aminocephalosporanicacid (7-ACA) , 7-amino-3-chloro-3-cephem- 4-carboxylic acid (7-ACCA) , 7-aminodesacetoxycephalosporanic acid (7-ADCA) and 7-amino-3-[ (Z)-l-propenyl]-3-cephem-4-car- boxylic acid. On the other hand, said immobilized enzymes will also hydrolyse the activated side chain derivatives to worthless side chain acids. Also, the desired product hydrolyses to form

side chain acid and the parent amino ,9-lactam. A high ratio between synthesis and hydrolysis will lower the cost of acti¬ vated side chain derivative.

From international patent application WO 93/12250 it is known that the ratio synthesis/hydrolysis for cephadroxil and cephalexin synthesis by Escherichia coli penicillin G acylase immobi¬ lized on Eupergit PCA is strongly dependent on the reaction con¬ ditions such as pH, concentration of reactants and temperature. The influence of the nature of the carrier material on the ratio synthesis/hydrolysis however, has not been taught.

From European patent 222462 it is known that amino groups can be introduced onto the carrier material by adding amino- polymers such as alginate amine, chitosan pectin, or poly¬ ethylene imine to the base gelling constituent of the carrier. Surprisingly, it has been found that immobilization of Escherichia coli penicillin G acylase on a carrier consisting of a gelling agent and a polymer containing free amino groups gives an enzymatic catalyst with superior characteristics regarding the ratio synthesis/hydrolysis in the condensation reaction of activated side chain derivatives with amino 0-lactams as com¬ pared to penicillin G acylases immobilized on other carriers.

Summary of the invention

The present invention provides Penicillin G acylase immobi¬ lized on a carrier comprising a gelling agent and a polymer containing free amino groups. Preferably the polymer is selected from the group consisting of alginate amine, chitosan, pectin, or polyethylene imine, and more preferably, the gelling agent is gelatin. Furthermore, by applying such an immobilized enzyme, an improved process for the preparation of a _/ 9-lactam derivative by an enzymatic reaction of the parent amino _/ 5-lactam with the corresponding acylating agent has been provided for.

Specific embodiments

Examples of ,3-lactam derivatives that may be produced by the process of this invention are amoxycillin, ampicillin, cephaclor, cephadroxil, cephprozil, cephalexin, and cephradine.

The acylase activity is independent of the substituents at the 3-position of the cephem compounds, e.g. hydrogen, halogen, (lower) alkoxy, methyl or methyl substituted with, for instance, (lower) alkoxy, (lower) alkanoyloxy, halogen, S-R5 (where R5 is (lower) alkyl, (lower) alkanoyl or an optionally substituted heterocyclic ring) , N+-Rg (where Rg is (lower) alkyl or an optionally substituted heterocyclic ring) . By lower is meant 1-6 carbon atoms. A heterocyclic ring is defined as an unsatu¬ rated ring structure comprising at least one nitrogen, sulphur or oxygen atom.

The acylating agent may be a derivative of D(-)-phenyl¬ glycine, D(-)-4-hydroxyphenylglycine or D(-)-2,5-dihydro-phenyl- glycine such as a lower alkyl (methyl, ethyl, n-propyl or isopropyl) ester or an amide which is unsubstituted in the -CONH ₂ group.

The corresponding amino ,9-lactam contains the same β-lactam nucleus as the ,9-lactam derivative prepared.

Generally, the reaction temperature of the process of this invention may vary between 0°C and 35°C. The optimal temperature depends on the substrates as has been mentioned in European patent application 473008 and has not been optimized in the comparative examples given. The suitable pH value depends on the nature and concentration of the substrates and is typically in the range of 5 to 9. For convenient operation control of pH is used. Suitable reaction times are from several minutes to sev¬ eral hours, in particular from 30 minutes to three hours.

In commercial processes involving the use of a catalyst e.g. an enzyme, the price of the catalyst is often an important parameter in the overall economy of the process. In such cases it is an advantage if the catalyst can be reused without loss of catalytic activity. To this end, it is advantageous to have the enzyme in a reusable form, for example, in immobilized or entrapped form. The following immobilized Escherichia coli penicillin acylases were investigated: Type A: Escherichia coli penicillin acylase isolated as described in international patent application WO 92/12782. Immobil¬ ization was carried out as described in European patent application No. 222462.

Type B: Commercially available immobilized Escherichia coli penicil¬ lin G acylase from Recordati, Italy, as described in European patent application No. 473008. Type C: Commercially available immobilized Escherichia coli penicil- lin G acylase from Boehringer Mannheim GmbH, Germany, known as Enzygel ^® . Suitable enzyme concentrations may be from 0.1 U.ml ^-1 to 100 U.ml ^-1 (1 U = one unit of enzyme activity, see below) . Using the process according to this invention, extraordinary high synthesis/hydrolysis ratio's can be obtained.

Definitions and methods of analysis

Enzyme activity As definition of penicillin G acylase activity the follow¬ ing is used: one unit (U) corresponds to the amount of enzyme that hydrolyses per minute 1 μmole penicillin G under standard conditions (100 g.l" ¹ penicillin G potassium salt, 0.05 M potas¬ sium phosphate buffer, pH 8.0, 28°C).

HPLC analysis

Procedure A (amoxycillin)

Sample: 1:10 Dilution using 25% acetonitrile in 2 mM potassium phosphate buffer, pH 5 Column: Chromsphere C18, 5 μm (100 x 3.0 mm)

Solvent: 25% acetonitrile in 12 mM potassium phosphate buffer containing 0.2% sodium dodecyl sulphate, pH 2.6 Flow: 1 l.min" ¹ Detection: 214 nm

Retention: HPG (1.9 min); HPGA (3.1 min); 6-APA (3.4 min); amoxycillin (4.8 min); HPGM (7.3 min)

Procedure B (cephalexin) Sample: 1:10 Dilution using 25% acetonitrile in 2 mM potassium phosphate buffer, pH 5 Column: Chromsphere C18, 5 μm (100 x 3.0 mm)

Solvent: 29% acetonitrile in 5 mM potassium phosphate buffer containing 0.2% sodium dodecyl sulphate, pH 3.1

Flow: 1 ml.min ^-1

Detection: 214 nm

Retention: PG (0.8 min); 7-ADCA (1.3 min); PGA (3.7 min); cephalexin (6.2 min); PGM (7.8 min)

Procedure C (cephradine) Sample: 1:150 Dilution using 3% 1-propanol in 50 mM phosphoric acid buffer, pH 3.0 Column: Nucleosil 120 3 C18 (250 x 4.0 mm) Solvent: Eluent A: 50 mM phosphoric acid buffer, pH 3.0 Eluent B: 50% eluent A, 50% acetonitrile Gradient: 0-5 min: 100% A; 5-10 min: from 100%

A to 70% A; 10-18 min: 70% A; 18-18.1 min: from 70% A to 100% A. Flow: l ml.min ^"1 Detection: 220 nm Retention: 7-ADCA (5.3 min); DPG (6.0 min); DPGA

(9.1 min); DPGM (15.9 min); cephradine (18.5 min)

Procedure D (cephaclor) Sample: 1:150 Dilution using 3% 1-propanol in 50 mM phosphoric acid buffer, pH 3.0 Column: Nucleosil 120 3 C18 (250 x 4.0 mm) Solvent: Eluent A: 50 mM phosphoric acid buffer, pH 3.0 Eluent B: 50% eluent A, 50% acetonitrile Gradient: 0-5 min: 100% A; 5-10 min: from 100%

A to 70% A; 10-18 min: 70% A; 18-18.1 min: from 70% A to 100% A. Flow: 1 ml. in ^"1 Detection: 220 nm Retention: 7-ACCA (3.2 min); PG (3.8 min); PGA (5.6 min); cephaclor (14.9 min)

Procedure E (ampicillin)

Sample: 1:200 Dilution using 33% acetonitrile in 3.4 mM potassium phosphate buffer, pH 6.9

Column: Chromsphere C18, 5 μm (100 x 3.0 mm) Solvent 30% Acetonitrile in 5 mM potassium phosphate buffer containing 0.1% sodium dodecyl sulphate, pH 3.0

Flow: 1 ml.min ^'1

Detection: 214 nm

Retention: PG (1.0 min); 6-APA (1.3 min); PGA (2.6 min); ampicillin (4.5 min); PGM (5.8 min)

Example 1

Synthesis of amoxycillin from 6 -APA and HPGA using immobilized Escherichia coli penicillin G acylase

To an aqueous solution (50 ml) containing 10 mM HPGA and 30 mM 6-APA is added 50 U of immobilized Escherichia coli penicillin G acylase at 21°C. The pH is adjusted to 6.0 and the reaction is allowed to proceed under a nitrogen atmosphere with pH con¬ trol using a 0.05 M solution of H ₂ SO ₄ in water. At different time intervals (see tables below) samples are analyzed according to procedure A as described above. The molar ratio synthesis/ hydrolysis (S/H) is calculated from the results thus obtained.

Time (min) 5 10 15 20 25 30 60 90 120

S/H-ratio 1.1 1.3 1.3 1.4 1.2 1.2 1.2 1.1 1.1

Table 1.1 Synthesis of amoxycillin using type A enzyme

Time (min) 18 60 90 110 150 180

S/H-ratio 0.6 0.7 0.7 0.7 0.6 0.5

Table 1.2 Synthesis of amoxycillin using type B enzyme

Time (min) 18 30 60 90 120

S/H-ratio 0.7 0.7 0.6 0.6 0.5

Table 1.3 Synthesis of amoxycillin using type C enzyme

Example 2 synthesis of amoxycillin from 6-APA and HPMG using immobilized

Escherichia coli penicillin G acylase

To an aqueous solution (50 ml) containing 10 mM HPGM and 30 mM 6-APA is added 50 U of immobilized Escherichia coli penicillin G acylase at 21°C. The pH is adjusted to 6.0 and the reaction is allowed to proceed under a nitrogen atmosphere with pH con¬ trol using a 0.05 M solution of H ₂ SO ₄ in water. At different time intervals (see tables below) samples are analyzed according to procedure A as described above. The molar ratio synthesis/ hydrolysis (S/H) is calculated from the results thus obtained.

Time (min) 10 20 40 60

S/H-ratio 1.6 1.4 1.3 1.2

Table 2.1 Synthesis of amoxycillin using type A enzyme

Example 3 synthesis of cephalexin from 7-ADCA and PGA using immobilized Escherichia coli penicillin G acylase

To an aqueous solution (50 ml) containing 10 mM PGA and 30 mM 7-ADCA is added 50 U of immobilized Escherichia colipenicillin G acylase at 21°C. The pH is adjusted to 7.0 and the reaction is allowed to proceed under a nitrogen atmosphere with pH control using a 0.05 M solution of H ₂ SO ₄ in water. At different time intervals (see tables below) samples are analyzed according to procedure B as described above. The molar ratio synthesis/ hydrolysis (S/H) is calculated from the results thus obtained.

Time (min) 5 10 20 30

S/H-ratio 6.5 4.2 3.4 2.4

Table 3.1 Synthesis of cephalexin using type A enzyme

Time (min) 5 10 20 30

S/H-ratio 1.0 0.9 0.8 0.7

Table 3.2 Synthesis of cephalexin using type B enzyme

Example 4

Synthesis of cephradine from 7-ADCA and DPGM.HCl using immobi¬ lized Escherichia coli penicillin G acylase

To an aqueous solution (120 ml) containing 300 mM DPGM.HCl and 300 mM 7-ADCA is added immobilized Escherichia coli penicillin G acylase (units as given in tables) . The pH is adjusted to the value given in the tables below and the reaction is allowed to proceed under a nitrogen atmosphere. At different time intervals samples are analyzed according to procedure C as described above. The molar ratio synthesis/hydrolysis (S/H) is calculated from the results thus obtained.

Time (min) 26 62 75 106 120

Conversion (%) 40 63 63 58 54

S/H-ratio 12 4.0 2.9 2.0 1.9

Table 4.1 Synthesis of Cephradine atpH 7.5 using type A enzyme (12 U.ml ^'1 )

Time (min) 45 110 170 255

Conversion (%) 33 49 51 68

S/H-ratio 2.4 1.7 1.4 1.4

Table 4.2 Synthesis of Cephradine atpH 7.0 using type B enzyme (33 U.ml ^'1 )

Example 5

Synthesis of cephaclor from 7-ACCA and PGA using immobilized

Escherichia coli penicillin G acylase

To an aqueous solution (120 ml) containing PGA and 7-ACCA (concentrations and enzyme units as given in tables below) is added immobilized Escherichia coli penicillin G acylase. The pH is adjusted to 7.7 and the reaction proceeds with pH control using a 2.0 M solution of H ₂ S0 ₄ in water. At different time intervals (see tables below) samples are analyzed according to procedure D as described above. The molar ratio synthesis/hydrolysis (S/H) is calculated from the results thus obtained.

Time (min) 2 62 90

Conversion (%) 3 58 66

S/H-ratio 2.0 6.2 4.0

Table 5.1 Synthesis of cephaclor from PGA (0.5 M) and 7 -ACC A (0.6M) using type A enzyme (9 U.mt ¹ )

Time (min) 26 62 124 161 266

Conversion (%) 25 40 50 55 58

S/H-ratio 5.3 4.4 3.4 3.2 2.6

Table 5.2 Synthesis of cephaclor from PGA (0.6 M) and 7-A CCA (0.6 M) using type B enzyme (47 U.mt ¹ )

Example 6

Synthesis of ampicillin from 6-APA and PGA using immobilized Escherichia coli penicillin G acylase

To an aqueous solution (100 ml) containing 500 mM PGA and 300 mM 6-APA is added 100 U of immobilized Escherichia coli penicil¬ lin G acylase. The pH is adjusted to 7.5 and the reaction is allowed to proceed with pH control using a 6.0 M solution of HCl in water. At different time intervals samples are analyzed according to procedure E as described above. The conversion and

the molar ratio synthesis/hydrolysis (S/H) are calculated from the results thus obtained and given in the tables below.

Alginate amine (%) 0 1.0 2.0 3.0

Conversion (%) 5 5 10 5 5 10

Time (min) 115 54 116 151 68 135

S/H-ratio 2.4 4.6 3.5 3.9 3.9 2.9

Table 6.1.1 Synthesis of Ampicillin using type A enzyme (as polymer alginate amine has been used)

Chitosan (%) 0 1.0 1.5 2.0 2.5 3.0

Conversion (%) 5 5 10 5 10 5 10 5 10 5 10

Time (min) 115 34 73 22 51 26 62 30 57 26 52

S/H-ratio 2.4 2.5 2.6 2.4 2.4 2.1 2.1 2.5 2.0 3.4 3.4

Table 6.1.2 Synthesis of Ampicillin using type A enzyme (as polymer chitosan has been used)

Pectin (%) 0 2.0 3.0

Conversion (%) 5 5 10 5 10

Time (min) 115 65 133 45 94

S/H-ratio 2.4 2.4 1.9 3.5 2.7

Table 6.1.3 Synthesis of Ampicillin using type A enzyme (as polymer pectin has been used)

Polyethylene imine (%) 0 1.0 2.0 3.0

Conversion (%) 5 5 10 5 10 5 10

Time (min) 115 64 132 49 100 43 93

S/H-ratio 2.4 2.5 2.4 2.4 2.8 2.7 2.5

Table 6.1.4 Synthesis of Ampicillin using type A enzyme (as polymer polyethylene imine has been used)

Conversion (%) 5 10

Time (min) 43 92

S/H-ratio 2.3 2.4

Table 6.2 Synthesis of Ampicillin using type B enzyme

Conversion (%) 5 10

Time (min) 33 69

S/H-ratio 3.3 2.8

Table 6.3 Synthesis of Ampicillin using type C enzyme