Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/62—Carboxylic acid esters

Definitions

• the present invention relates to a process for the preparation of an intermediate which is useful in the preparation of compounds having antiviral activity.
• EP-A-141927 (Beecham Group p.I.e.) describes the compound, penciclovir and its use as an antiviral agent and EP-A-182024 (Beecham Group p.I.e.) describes its pro-drug, famciclovir.
• Penciclovir and famciclovir are of formulae (A) and (B) respectively:
• Q is a leaving group, such as hydroxy or halo, for example iodo or bromo.
• the present invention provides a process for the preparation of a compound of formula (II) as hereinbefore defined, which process comprises the treatment of a compound of formula (I) , as hereinbefore defined, with a microbial hydrolase.
• Suitable microbial hydrolases are to be found in microorganisms which include Penicillia and Bacteria, in particular Penicillium frequentans IMI 92265.
• the organisms are grown in a nutrient broth or other suitable medium at a temperature of 10-50°C, preferably around 20-40°C, according to the nature of the organism employed.
• the reactions can be carried out in this medium, or in an alternative medium or salt solution after separation and transfer of the cells.
• purified or partially purified enzymes can be used after cell disruption and fractionation of the cells.
• the reaction is carried out at a pH in the range 3-10, preferably 5.0-8.0, usually at a temperature of 10-40°C, or 20-40°C.
• the enzyme transformation may be improved in yield in the presence of ammonium sulphate, preferably 1 molar.
• enzyme in cell bound or cell-free form can be immobilised and re-used.
• the substrate concentration may be increased to 1% or greater w/v.
• a continuous production method can be envisaged, passing the substrate over the immobilised enzyme in a column, loop reactor or similar reactor.
• the product is purified by extraction into a suitable solvent following which it may optionally be chromatographed.
• the process has the advantage that minimal amounts of by-product of formula (III) are produced.
• Two loopsful of mycelium from a slope of Penicillium frequentans IMl 92265 were mixed in 4ml of 0.02% Tween 80 in water and 2ml of the suspension was used to inoculate 40ml of seed medium in a 250ml Erlenmeyer flask.
• the seed medium comprised (% w/v) cornsteep liquor (4%); treacle (2.6%); CaC0 3 (0.5%) and distillers' solubles (Scotafeed) (2%) in deionised water adjusted to pH 5.2. After shaking at 240rpm at 28 ⁇ C for 72h, 1.5ml of the seed broth was used to inoculate 40ml of production medium in 250ml Erlenmeyer flasks.
• This medium comprised (% w/v) glucose (3%), nutrient broth (0.8%); yeast extract (0.2%) and malt extract (0.3%) in deionised water adjusted to pH 5.6. These flasks were incubated, with shaking at 240rpm, at 28°C, for 72h.
• the triacetate of formula (I) was added to cultured shake flasks of Penicillium frequentans IMl 92265, produced as in example 1, to give a final substrate concentration of 4mg ml- .
• the flasks were then shaken at 240rpm and 28 ⁇ C and finally assayed for products after centrifugation and extraction of the supernatant with chloroform.
• the organic phase was assayed by hplc. After incubation of the substrate with the microorganism for 6hr, a 15% conversion of su,;strate to diacetate product (II) was indicated and the ratio of product (II) to its regioisomer (III) was 91:9. After 9h incubation a 6% conversion to product was indicated and the ratio of product (II) to its regioisomer (III) was 97:3.
• the biomass was resuspended to 650ml in 0.1M Tris buffer at pH 7.5 and the cells disrupted using a French press (1250psi) in 60ml batches of cell slurry.
• esterase preparation was obtained by the procedure described in example 3 with a protein concentration of 3.56mg ml . A 250 ⁇ l aliquot of this was added to each of two mixtures of 5mg of triacetate (I) in 125 ⁇ l of 4M ammonium sulphate solution in 12mM Tris buffer pH 7.5 and to the other reaction was added 125 ⁇ l 12mM Tris buffer pH 7.5.
• Gel immobilised enzyme was prepared as described in example 6 immobilising 5 mg of protein per ml of gel, and 144 ml of this was washed with an equal volume of O.IM Tris buffer, pH 7.5, containing IM ammonium sulphate. To the gel was added
• the reaction mixture was shaken at 14°C for 5h before centrifugation and removal of the supernatant.
• the immobilised enzyme was resuspended in 0.1M Tris buffer, pH 7.5, containing IM ammonium sulphate and stored at 4°C. The same batch was reused as described above on a further nine occasions giving similar results in each case.

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• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• Microbiology (AREA)
• Biotechnology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Enzymes And Modification Thereof (AREA)
• Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

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• 1990
  • 1990-03-01 GB GB909004647A patent/GB9004647D0/en active Pending
• 1991
  • 1991-02-21 CA CA002076628A patent/CA2076628A1/en not_active Abandoned
  • 1991-02-21 WO PCT/GB1991/000275 patent/WO1991013162A1/en not_active Ceased
  • 1991-02-21 JP JP3505507A patent/JPH05503428A/ja active Pending
  • 1991-02-21 AU AU73362/91A patent/AU645543B2/en not_active Ceased
  • 1991-02-21 EP EP91904921A patent/EP0518902A1/de not_active Withdrawn
  • 1991-02-27 ZA ZA911435A patent/ZA911435B/xx unknown
  • 1991-02-27 NZ NZ237234A patent/NZ237234A/en unknown
  • 1991-02-27 PT PT96900A patent/PT96900A/pt not_active Application Discontinuation
  • 1991-02-27 IE IE066791A patent/IE910667A1/en unknown

Non-Patent Citations (1)

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