A PROCESS FOR PRODUCING SULPHOXIDE ENRICHED IN THE (R) OR THE (S) ENANTIOMER

Abstract: A process for producing sulphoxide enriched in the (R) or the (S) enantiomer as required comprises contacting a racemic mixture of such sulphoxide with the enzyme DMSO reductase derived from a bacterium selected according to the enantiomer required from the class consisting of enteric bacteria and Rhodobacter.

Description: A process for Droduciπg sulphoxide enriched in the (R) or the (S. enantiomer. THIS INVENTION relates to sulphoxides and in particular to the production of enantiomerically enriched chiral sulphoxides. The article by Abo et al. (Biosci.Biotech.Biochem, 51(3), 596-597, 1994) describes the enantioselective reduction to sulphide of (s) -methyl phenyl sulphoxide by dimethyl sulphoxide reductase from Rhodobacter sphaeroides f.s. denitrificans. It has now been found that (1) this reduction can be conveniently effected using the enzyme from other Rhodobacter or from a non- photosynthetic bacterium: and (2) that the enantiospecificity of Rhodobacter is generally opposite to that of enteric bacteria. As a result, it is now practicable to effect enrichment in either the (R) or the (S) enantiomer, according to which is required e.g. for subsequent synthetic steps. Previously it was known that the enzyme from enteric bacteria would reduce dimethyl sulphoxide (DMSO) to dimethyl sulphide, but not that it was capable of enantiomeric selectivity. According to the invention in its first aspect a process for producing a sulphoxide enriched in the (R) or the (S) enantiomer as required is characterised by contacting a racemic mixture of such sulphoxide with the enzyme DMSO reductase derived from a bacterium selected from the class consisting of enteric bacteria and Rhodobacter. According to the invention in its second aspect a process for producing an enatiomerically enriched sulphoxide is characterised by contacting a corresponding racemic sulphoxide mixture with the enzyme DMSO reductase derived from a bacterium selected from the class consisting of enteric bacteria, Rhodobacter cansiiiat-iis and other varieties of Rhodobacter. the enzyme derived from said other varieties of Rhodobacter being provided by supplying whole cells. The sulphoxide suitably has the general formula: R 1 - SO - R 2 where R 1 and R 2 are carbon-linked organic groups sufficiently different from one another to give (R) or (S) chirality. R 1 and R 2 may be hydrocarbon groups or heterocyclic groups. Such hydrocarbon groups may be aliphatic, cycloaliphatic, aromatic or araliphatic (including heterocyclic-aliphatic) . Such heterocyclic groups may for example contain a 5-membered ring or 6- membered ring, said ring containing up to 3 or 4 hetero- atoms selected from nitrogen, oxygen and sulphur. Very suitably the linkage to sulphur in the starting sulphoxide is from a carbon atom adjacent to nitrogen in such a heterocyclic ring. The heterocyclic ring linked to sulphur in the sulphoxide may carry one or more fused hydrocarbon and/or heterocyclic rings. Typically R 1 is Cj - C 6 alkyl and R 2 is a phenyl or alkylphenyl; or R 1 is monocyclic and R 2 is bicyclic, especially when one or both of R 1 and R 2 are heterocyclic. Either or both of R 1 and R 2 can carry one or more substituent groups. The groups R 1 and R 2 and their substituents may be chosen to afford a level of biological, especially medicinal, activity enhanced by reason of enantiomeric enrichment. The process may use enzyme separated from the bacterium or may use whole cells. In a modification of the invention in its second aspect, whole cells of any Rhodobacter are used. Particular examples of enteric bacteria (Enterobacteriaceae) are Escherichia (such as E.coli) . Proteus (such as P.mirabilis and P.vulσaris1 and Salmonella; and also Schewanella (such as S.putrifaciens) . These bacteria and the enzyme derived therefrom have the special property of reducing (R)alkyl- aryl and alkyl-pyridyl sulphoxides preferentially. This specificity is believed to be not general to all sulphoxides represented by the general formula. For some combinations of R 1 and R 2 it is to be expected that the enteric enzyme will reduce the (S) sulphoxide preferentially. In this event the invention provides for the use of the Rhodobacter enzymes. Conversely; whereas the Rhodobacter enzyme reduces (S) alkyl-aryl and alkyl- pyridyl sulphoxides preferentially, recourse is to the enteric enzyme for RVR 2 combination when (R) reduction takes place. In the following Examples the microorganisms were: Escherichia coli - ATCC 33694 (The abbreviation ATCC herein refers to the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America) ; Proteus irabilis NCIMB 8268 and Proteus yulqarjs NCIMB 67 (NCIMB - National Collection of Industrial and Marine Bacteria, 23 St Machar Drive, Aberdeen AB2 1RY) ; Rhodobacter capsulatus DSM 938 - (DSM - Deutsche Sammlung von Mikroorganismen, Mascheroder Weg lb, D-38124, Braunschweig, Germany) . In each table of results "ee" represents enantiomeric excess, which is given by ee = (S)-(R) (R)-(S) x 100 or [when (R) exceeds (S)] x 100 (S)+(R) (S)+(R) where (S) and (R) are the measured concentrations of the respective enantiomer in mg/ml in the mixture at the end of the reaction. EXAMPLE 1 Asymmetric Reduction of Methyl-p-Tolyl Sulphoxide (MPTSO) by Enteric Bacteria The basal culture medium had the following composition (g/1): Na H 2 P0 4 , 1.56; K 2 HP0 4 , 1.9; (NH 4 ) 2 S0 4 , 1.8; MgS0 4 .7H 2 0, 0.2; Fe Cl 3 , 0.005; Na 2 Mo0 4 .2H 2 0, 0.001; casamino acids, 1.5. This was after autoclaving supplemented with the following components (g/1) : glycerol (for Escherichia coli) or glucose (for Proteus mirabilis) . 5; thiamine HCl, 0.03; nicotinic acid, 0.007. DMSO (70 mM) was added to the medium to serve as terminal electron acceptor for anaerobic respiration and as inducer for DMSO reductase. Trace elements solution (lml/1) was also added to the medium. The stock trace elements solution contained (g/1) : CuS0 4 .5H 2 0, 0.02; MnS0 4 .4H 2 0, 0.1; ZnS0 4 .7H 2 0, 0.1; CaCOj, 1.8. Microorganisms were grown under essentially anaerobic conditions in 1 litre screw capped flasks containing 800 ml of medium at 35°C for 48 h on a rotary shaker. Following growth, the organisms were recovered by centrifugation and then washed by resuspending in an equal volume of potassium phosphate buffer (100 mM, pH 7.6) and recentrifuging. Finally the organisms were resuspended in potassium phosphate buffer (100 mM pH 7.6) to give a dry cell weight concentration of 5g/l. Portions of the cell suspension (50 ml) were transferred to a temperature controlled reaction vessel maintained at 35°C and fitted to an autotitrator to maintain the pH at 7.6 by titration with sodium hydroxide (0.5M). The reaction mixture was stirred gently and the reaction started by the addition of racemic MPTSO (1.5g/l) and glucose (lOg/l) as energy source. Samples were taken (1ml) at intervals, cells removed and the supernatants analysed for extent of reduction and enantiomeric excess of residual MPTSO. For determination of the extent of reaction cell-free supernatants were diluted with HPLC eluent and analysed under the following conditions: Column Spherisorb S5 0DS2 (250 x 4.6 mm) Eluent acetonitrile (50%)+sodium phosphate buffer, pH 7.6, 25 mM (50%) Flowrate 0.8 ml/min Detection UV, 270 nm Retention Times MPTSO, 5 min methyl-p-tolyl sulphide, 18 min For determination of enantiomeric excess (Tables 1, 2) cell-free supernatants were extracted with an equal volume of dichloromethane; the solvent layer was recovered, evaporated to dryness and the residue redissolved in HPLC eluent (95% hexane, 5% ethanol) prior to analysis under the following conditions. Column Chiralcel OD (250 x 4.6 mm) Eluent Hexane (95%) ethanol (5%) Flowrate 0.8 ml/min Detection UV, 270 nm Retention Times Methyl-p-tolyl sulphide, 5.9 min (R) MPTSO, 15.5 min (S) MPTSO, 17.1 min TABLE 1 : ______ Time (R) - MPTSO (S) - MPTSO e.e (h) (mg/ml) Yield (mg/ml) Yield (%) (%) (%) 0 0.74 98.7 0.74 98.7 0 4 0.27 36.0 0.63 85.1 40 20 0 0 0.45 60.8 100 TABLE 2 : P. irabllle Time (R) - MPTSO (S) - MPTSO e.e (h) (mg/ml) Yield (mg/ml) Yield (%) (%) (%) 0 0.74 98.7 0.74 98.7 0 4 0.08 10.9 0.72 96.2 80 20 0 0 0.58 76.9 100 Both E.coli ATCC 33694 and P. mirabilis NCIMB 8268 were able enantioselectively to reduce MPTSO leaving optically pure (S) - MPTSO in high yield. In principle the sulphide product can be recovered, reoxidised and recycled through the process. EXAMPLE 2 Asymmetric Reduction of MPTSO by Whole Cells of Rh P flobttcttr c f tptul f ttug Rhodobacter capsulatus was grown under the same conditions as described in Example 1 except that the casa ino acids were replaced by yeast extract (2 g/1) and the carbon source was glucose (10 g/1) . Following growth, the cells were recovered by centrifugation, washed in potassium phosphate buffer (25 mM, pH 7.6) and finally resuspended in the same buffer to give a dry cell weight concentration of 4 g/1. Glucose (10 g/1) was added to the suspension. A portion of cell suspension (100 ml) was transferred to a 250 ml conical flask and 50 mg of racemic MPTSO added. The mixture was gently shaken at 35°C. After 15 min the cells were removed by centrifugation and the supernatant analysed as described in Example 1. Analysis indicated the presence of optically pure (R) MPTSO in 80% yield. EXAMPLE 3 Purification of DMSO Reductaae from Rhodobacter caosulatua and use in the Resolution of MPTSO The "RCV" medium used in this Example had the following composition (per litre of deionised water or as mM) : DL-malic acid 4 g (NH«) 2 S0 4 1 g K/P0 4 buffer 10 mM MgS0 4 . H 2 0 120 mg CaCl 2 .2H 2 0 75 mg Sodium EDTA 20 mg FeS0 4 . 7H 2 0 24 mg Thiamine hydrochloride 1 mg Trace element soln. 1 ml Trace element (per 250 ml) H 3 B0 3 0 . 7 g MnS0 4 . H 2 0 398 mg Na 2 Mo0 4 .2H 2 0 188 mg ZnS0 4 .7H 2 0 60 mg Cu (N0 3 ) 2 . 3H 2 0 10 mg Cells of R. capsulatus DSM 938 were grown phototrophically in 25 1 carboys containing RCV medium (minus malate) supplemented with 30 mM propionate and 50 mM DMSO between two banks of 100 W tungsten bulbs. Cells were harvested by cross-flow filtration followed by centrifugation at 10,000 rpm in a Beckman GSA rotor for 30 min. The pellets were washed once with 50 mM Tris/HCl pH 8.0 and resuspended to approximately 2g/ml in 50 mM Tris/HCl pH 8.0 + 0.5 M sucrose and 1.5 mM EDTA (STE buffer) . Lysozyme was added at 0.6 mg/ml and the suspension (approximately 11) was stirred at 30°C for 15 min. After centrifugation as described above the supernatant (periplasmic fraction) was decanted off and brought to 50% saturation with ammonium sulphate. Following centrifugation the supernatant was brought to 70% saturation and re-centrifuged. The 50-70% pellet was resuspended in a minimum volume of 50 mM Tris/HCl pH 8.0 and dialysed against 3 x 100 volumes of the same buffer before being concentrated to approximately 5 ml by ultra- filtration through an Amicon PM 10 membrane. The concentrated sample was brought to 150 mM NaCl, charged onto a Sephacryl S200 gel filtration column (column volume = 510 ml) and eluted with 700 ml of 50 mM Tris/HCl pH 8.0 + 150 mM NaCl. Peak enzyme activity eluted at approximately 320 ml (data not shown) and peak fractions were pooled and concentrated as above. The resolution reaction was carried out under the following conditions. A gas tight glass syringe was filled to 3 ml with degassed buffer (50 mM Tris/HCl pH 8.0 containing 10 mM ethylenediaminetetraacetic acid - EDTA) . To this was added flavin mononucleotide (FMN) and racemic MPTSO to give final concentrations of 250 μM and 3 g/1 respectively. The syringe was then illuminated with a 100 watt tungsten bulb to initiate the photo catalysed reduction of flavin mono nucleotide FMN. The reaction was then started by the addition of DMSO reductase (3.6 μg of protein). Samples (1 ml) were analysed as described in Example 1. Results are shown in Table 3. TABLE 3 Time (R) - MPTSO (S) - MPTSO ee (%) (min) (g/1) (g/i) 3 1.49 1.46 1.0 15 1.30 1.00 13.0 30 1.29 0.65 32.9 50 1.27 0.31 60.8 70 1.31 ND 100 ND - Not Detected Optically pure (R) -MPTSO was produced in 87% yield. EXAMPLE 4 Asymmetric Reduction of Ethyl( -pyridyl) sulphoxide (EPSO) by Enteric Bacteria The culture medium had the same composition as in Example 1 except that yeast extract (2 g/1) replaced casamino acids and glucose (10 g/1) was provided as carbon and energy source for both E.coli and P.vulσaris and glycerol (10 g/1) for a second E.coli culture. Microorganisms were grown under essentially anaerobic conditions in 500 ml screw capped flasks containing 400 ml of medium at 35°C for 48 h. The optical density of the culture at the end of growth was: P.vulσaris 0.96; E.coli (glucose) 1.05; E.coli (glycerol) 0.54, as measured at 600 um using a 1 cm light path. Following growth, the organisms were recovered by centrifugation, washed by resuspending in 200 ml of anaerobic potassium phosphate buffer (100 mM, pH 7.6), recentrifuged and resuspended in 70 ml of the same buffer supplemented with glucose (10 g/1) . Portions of the cell suspensions (20 ml) were transferred to 50 ml serum bottles containing EPSO (52 mg) sealed with rubber stoppers the headspace flushed with nitrogen then incubated at 35°C without agitation but with gentle shaking twice a day. Samples (2 ml) were taken at intervals, cells removed and the supernatants analysed for extent of reduction and enantiomeric excess. For determination of the extent of reaction cell-free supernatants were diluted with HPLC eluent and analysed under the conditions described in Example 1 except that the eluent flow rate was 1.0 ml/min. Retention times EPSO: 2.4 min ethyl (2-pyridyl) sulphide : 6.5 min For determination of enantiomeric excess (Table 4) cell-free supernatants (1 ml) were extracted with dichloromethane (5 ml) and the recovered organic layer was dried with anhydrous sodium sulphate. The dry organic layer was evaporated under reduced pressure and the residue was redissolved in HPLC eluent (1 ml) for analysis under the following conditions. Column Chiralcel OB (250 x 4.6 mm) eluent hexane (85%), 2-propanol (15%) flow rate 0.8 ml/min detection UV, 270 nm retention times ethyl (2-pyridyl) sulphide 9.5 min EPSO 16.5 min 29.5 min TABLE 4 E.coli Time p.vulqaris Glucose Grown Glycerol Grown (h) Sulphoxide ee* Sulphoxide ee* Sulphoxide ee* remaining (%) remaining (%) remaining (%) (mg/ml) (mg/ml) (mg/ml) 1 1.9 32 nt 10 2.2 16 3.5 1.3 100 1.6 70 1.7 48 6 nt nt 1.3 100 1.5 68 23 1.3 100 1.0 100 1.1 100 10 nt - not tested * of peak eluting at 16.5 min Note: The starting EPSO was obtained by reacting ethanethiol with 2-chloro-pyridine and oxidising the resulting thio-ester with sodium meta periodate. EXAMPLE 5 Asymmetric Reduction of Ethyl (2-pyridyl) Sulphoxide (EPSO) by Rhodobacter caosulatus The culture medium had the same composition as in Example 4 with glucose (10 g/1) as carbon and energy source. The microorganism was grown as described in Example 4 except that the culture was illuminated by tungsten bulbs (3 x 60 watt) placed 40 cm from the culture flask. The optical density at the end of 48 h growth was 1.4, measured as in Example 4. Following growth the cells were recovered by centrifugation, washed by resuspending in anaerobic potassium phosphate buffer (100 mM, pH 7.6 : 200 ml for the cells from 1200 ml of culture) , and resuspended in 110 ml of the same buffer supplemented with glucose (10 g/1). A portion of the cell suspension (20 ml) was transferred to a 50 ml serum bottle containing EPSO (64 mg) , sealed with a rubber stopper, the headspace flushed with nitrogen and incubated at 35°C. Samples (2 ml) were taken at intervals, cells removed by centrifugation and the supernatants analysed for extent of sulphoxide reduction and sulphoxide enantiomeric excess (Table 5) as described in Example 4. TABLE 5 Time Sulphoxide ee* (h) remaining (%) (mg/ml) 1 1.82 74 3 1.74 84 5 1.69 90 21 1.36 100 * of peak eluting at 29.5 min. EXAMPLE 6 Asymmetric Reduction of Methoxymethylphenylsulphoxide (MMPSO) by Enteric Bacteria The culture medium used had the same composition at that in Example 4, with glucose (10 g/1) as carbon and energy source for both E.coli and P.vulσaris. Microorganisms were grown as described in Example 4. Optical densities at 48 h growth were: P.vulσaris 0.46; E.coli 0.6. Cells from 400 ml of culture were recovered by centrifugation, washed in 200 ml of anaerobic potassium phosphate buffer (100 mM, pH 7.6) and resuspended in 80 ml of the same buffer supplemented with glucose (10 g/1) . Cell suspensions (20 ml) were dispensed into 50 ml serum bottles containing MMPSO (50 mg) , sealed with rubber stoppers, the headspace flushed with nitrogen, and incubated at 35°C. Samples (2 ml) were taken at intervals, cells removed by centrifugation and the supernatants analysed for extent of sulphoxide reduction and enantiomeric excess of residual sulphoxide. For determination of the extent of sulphoxide reduction cell- free supernatants were diluted with HPL-C eluent and analysed under the conditions described in Example 1 except that the eluent flow rate was 1.0 ml/min. Retention times MMPSO : 2.6 min Methoxymethylphenylsulphide : 8.3 min For determination of enantiomeric excess (Table 6) cell-free supernatants (1 ml) were prepared as described in Example 4 for analysis under the conditions described in Example 1. Retention times: methoxymethylphenylsulphide : 6.1 min MMPSO 24.3 min 28.7 min P.vulσaris E.COli Time Sulphoxide ee* Sulphoxide ee* (h) remaining (%) remaining (%) (mg/ml) (mg/ml) 1 2.5 5 2.4 3 3 2.4 14 2.3 12 5 2.2 25 1.9 32 21 1.9 49 1.6 52 * of peak eluting at 28.7 min Note: the starting sulphoxide was obtained by oxidising commercially available methoxymethylphenylsulphide with m- chloroperbenzoic acid. EXAMPLE 7 Asymmetric Reduction of Methoxymethylphenylsulphoxide (MMPSO) by Rhodobacter caosulatus The culture was grown and prepared for the biotransformation as described in Example 5. A portion of cell suspension (20 ml) was added to a 50 ml serum bottle containing MMPSO (46 mg) and the reaction and analysis performed as described in Example 6. Results are shown in Table 7. TABLE 7 Time Sulphoxide ee* (h) remaining (%) (mg/ml) 1 2.0 28 3 1.9 32 5 1.8 36 21 1,5 53 of peak eluting at 24 . 3 min . EXAMPLE 8 Asymmetric Reduction of Methylthiomethyl Methylsuphoxide (MTMSO) by Enteric Bacteria The cultures were grown and prepared for the biotransformation as described in Example 6. A portion of cell suspension (20 ml) was added to a 50 ml serum bottle containing MMSMS (48 mg) . The serum bottle was sealed with a rubber stopper, the headspace flushed with nitrogen and incubation carried out at 35°C without agitation but with gentle shaking prior to sampling. Samples (2 ml) were taken at intervals, cells removed by centrifugation and supernatants analysed for extent of reduction and enantiomeric excess. For determination of extent of reduction cell-free supernatants were diluted with an equal volume of aqueous 2,3-butanediol (2 g/1) and analysed by gas chromatography: 2 ,3-butanediol was used as internal standard. Gas chromatography was performed under the following conditions: column DB5 (J&W Scientific) 28m x 0.328 mm film 1.0 micron carrier gas helium gas pressure 8.0 psi chromatograph Perkin Elmer 8500 detector flame ionisation temperature program 150°C for 3 min then rising at 20°C/min to 200°C then held at 200°C for 2 min retention times butane-2,3-diol, 2.16 min bis(methylthio)methane, 2.68 min MTMSO, 4.9 min Samples were prepared and analysed for enantiomeric excess as described in Example 1 except that supernatants (1 ml) were extracted with 5 ml of dichloromethane and MTMSO was detected by UV absorption at 225 nm. Retention times - MTMSO, 31 min 32.5 min Results are shown in Table 8. TABLE 8 E.coli P.vulσaris Time Sulphoxide ee* Sulphoxide ee* (h) remaining (%) remaining (%) (mg/ml) (mg/ml) 1 2.23 10 1.61 15 3 1.53 18 1.17 30 5 1.16 40 0.81 64 21 0.28 100 0.64 100 * of peak eluting at 32.5 min EXAMPLE 9 Asymmetric Reduction of Methylthiomethyl methylsulphoxide (MTMSO) by Rhodobacter caosulatus The culture was grown and prepared for biotransformation as described in Example 5. A portion of cell suspension (20 ml) was transferred to a 50 ml serum bottle containing MMSMS (48 mg) and the bottle sealed with a rubber stopper and the headspace flushed with nitrogen. The mixture was incubated at 35°C. Analyses were performed as described in Example 7. Time Sulphoxide remaining ee* (h) (mg/ml) (%) 1 0.54 70 * of peak eluting at 31 min 94KHC05S/MS - 16 November 1995

Claims: CLAIMS 1 A process for producing sulphoxide enriched in the (R) or the (S) enantiomer as required characterised by contacting a racemic mixture of such sulphoxide with the enzyme DMSO reductase derived from a bacterium selected according to the enantiomer required from the class consisting of enteric bacteria and Rhodobacter. 2 A process for producing an enantiomerically enriched sulphoxide characterised by contacting a corresponding racemic sulphoxide mixture with the enzyme DMSO reductase derived from a bacterium selected from the class consisting of enteric bacteria Rhodobacter capsulatus and other varieties of Rhodobacter. the enzyme derived from said other varieties of Rhodobacter being provided by supplying whole cells. A process according to Claim 2 in which the bacterium is Rhodobacter capsulatus and the enzyme is separated before use. A process according to any of Claims 1 to 3 in which the sulphoxide has the formula: R 1 - SO - R 2 where R 1 and R 2 are carbon-linked hydrocarbon groups and/or heterocyclic groups sufficiently different from one another to give chirality. A process according to Claim 4 in which R 1 is C, - C 6 alkyl and R 2 is a phenyl or alkylphenyl; or R 1 is monocyclic and R 2 is bicyclic, especially when one or both of R 1 and R 2 are heterocyclic. A process according to any one of the preceding claims in which at least one of R 1 and R 2 is heterocyclic and the linkage to sulphur in the starting sulphoxide is from a carbon atom adjacent to a nitrogen atom. 7 A process according to any one of the preceding claims in which the enteric bacteria are selected from Escherichia (such as E.coli) . Proteus (such as P,mirabilis and P-vulgaris) and Salmonella: and Sς ewanellfl (such as S.putrifaei ns.. 8 A process according to Claim 6 in which the bacteria are selected from: Escherichia coli - ATCC 33694 (The abbreviation ATCC herein refers to the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America); Proteus mirabilis - NCIMB 8268 (NCIMB - National Collection of Industrial & Marine Bacteria, 23 St Machar Drive, Aberdeen AB2 1RY) ; and Proteus vulσaris - NCIMB 67. 9 A process according to any one of Claims 1 to 6 in which the bacterium is Rhodobacter capsulatus DSM 938 - (DSM - Deutsche Sammlung von Mikroorganismen, Mascheroder Weg lb, D-38124, Braunschweig, Germany) . 10 A process according to any one of the preceding claims including the further steps of: (a) recovering the wanted sulphoxide from the reaction product; (b) recovering the sulphide by-product from the reaction mixtures; (c) oxidising the sulphide to racemic sulphoxide; and (d) recycling the racemic sulphoxide to the enantiospecific reduction.

Inventors: HOLT ROBERT ANTONY, HOLT, ROBERT, ANTONY
Applicants: HOLT ROBERT ANTONY, ZENECA LTD, HOLT, ROBERT, ANTONY, ZENECA LIMITED
Application year: 1995
Main classification: C12P11
IPC codes: C12P11, C12P41