Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D501/00—Heterocyclic compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
• • 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
  • C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin

Definitions

• the invention relates to a process for the preparation of a ⁇ -lactam antibiotic by enzymatic acylation of a ⁇ -lactam core by means of an acylation agent.
• the enzyme to be applied should be suitable to be re-used many times (for instance more than 50 times, in particular more than 100 times) without much loss of activity.
• the object of the invention is to obtain a process for the enzymatic acylation of a ⁇ -lactam core whereby said drawback is eliminated.
• the enzyme activity in successive cycles depends on the concentration of the inorganic salts in the reaction mixture during the enzymatic acylation reaction, as well as on the sum of the concentrations of the ⁇ -lactam antibiotic and the ⁇ -lactam core.
• ⁇ -lactam cores that can be used in the process according to the invention are penicillic acid derivatives, for instance 6- aminopenicillic acid (6-APA), and cephalosporanic acid derivatives, for instance 7-aminocephalosporanic acid (7- ACA), 7-aminodesacetoxy-cephalosporanic acid (7-ADCA) and 7-amino-3-chlorocephalosporanic (7-ACCA) .
• penicillic acid derivatives for instance 6- aminopenicillic acid (6-APA)
• cephalosporanic acid derivatives for instance 7-aminocephalosporanic acid (7- ACA), 7-aminodesacetoxy-cephalosporanic acid (7-ADCA) and 7-amino-3-chlorocephalosporanic (7-ACCA) .
• Suitable acylation agents that can be used in the process according to the invention are for instance ⁇ - amino acid derivatives, in particular amides and esters of phenyl glycine, p-hydroxyphenyl glycine and dihydrophenyl glycine.
• any enzyme can be used that is suitable as catalyst in the coupling reaction.
• Such enzymes are for instance the enzymes that are known under the general designations 'penicillin amidase' and 'penicillin acylase'.
• suitable enzymes are enzymes derived from Acetobacter, Aeromonas, Alcali ⁇ enes, Aphanocladium, Bacillus SD. , Cephalosporium. Escherichia. Flavobacterium, Kluvvera, Mvcoplana, Protaminobacter, Pseudomonas and Xanthomonas, in particular Acetobacter pasteurianum, Alcali ⁇ enes faecalis, Bacillus me ⁇ aterium.
• Escherichia coli and Xanthomonas citrii Preferably an immobilized enzyme is used, since the enzyme can be easily re-used then. Immobilized enzymes are known as such and are commercially available. Highly suitable enzymes have appeared to be the Escherichia coli enzyme from Boehringer Mannheim GmbH, which is commercially available under the name 'Enzygel*', the immobilized Penicillin-G acylase from Recordati, the immobilized Penicilline-G acylase from Pharma Biotechnology Hannover, and an Escheria coli penicilline acylase isolated as described in WO-A-92/12782 and immobilised as described in EP-A-222462.
• the enzymatic acylation reaction is mostly carried out at a temperature between -5 and 35°C, in particular between 5 and 35°C preferably between 0 and 28°C, most preferably between 15 and 28°C.
• the pH at which the acylation reaction is carried out is mostly between 6 and 8.5.
• the optimum pH depends on, among other things, the antibiotic, since the stability and the solubility of the ⁇ -lactam antibiotic as well as the ⁇ -lactam core depend on the pH.
• the pH is preferably between 6.2 and 8.5, most preferably between 7 and 8; if a p-hydroxyphenyl glycine derivative is used as acylation agent the pH is preferably between 6 and 7.5, most preferably between 6 and 7.
• the enzyme activity is also pH-related.
• the acylation reaction is mostly carried out in water.
• the reaction mixture may also contain an organic solvent or a mixture of organic solvents, preferably less than 30 vol.%.
• organic solvents that can be used are alcohols with 1-7 carbon atoms, for instance a monoalcohol, in particular methanol or ethanol; a diol, in particular ethylene glycol or a triol, in particular glycerol.
• concentration of the ⁇ -lactam antibiotic and the concentration of the ⁇ -lactam core are chosen such that the sum of the two concentrations is between 200 and 800 mM.
• concentrations are chosen such that the sum of the two concentrations is between 300 and 700, most preferably between 300 and 600 mM.
• the molar ratio between the acylating agent and the ⁇ -lactam core is between 0.5:1 and 2:1, preferably between 0.7:1 and 1.3:1, most preferably between 0.8:1 and 1.2:1.
• the molar ratio between the acylating agent and the ⁇ -lactam core is preferably chosen such that the pH of the reaction mixture throughout the process does not exceed the above-mentioned limits, without titration with acid.
• inorganic salts will accumulate in the reaction mixture in the presence of anions, for instance Cl", S0 4 2 ", P0 4 3 ", N0 3 ". It has been found that if the concentration of inorganic salts becomes too high, for instance higher than 1000:n mM, where n is the valency of the anion, the enzyme activity declines strongly after several cycles.
• concentration of inorganic salts in the process according to the invention is therefore kept below 1000:n mM, preferably below 700:n mM, with n as defined above, which implies that for chlorides and nitrates n is 1 and for sulphates it is 2. In order to keep the concentration of inorganic salts low, it is preferred to ensure that the amount of inorganic salts formed during the enzymatic acylation reaction is restricted to a minimum.
• acylation agents are phenyl glycine and p-hydroxyphenyl glycine derivatives, for instance their amides or esters.
• these are often used in the form of the salt of a strong acid, for instance phenyl glycine amide.HC1, phenyl glycine amide.*$H 2 S0 4 , phenyl glycine methyl ester.HC1 or dihydrophenyl glycine methyl ester.HC1.
• the free amide or the free ester is preferably used in order to prevent the formation of additional inorganic salts as a result of neutralization of the salts of the acylation agents.
• Free amides in solid form are particularly suitable for this purpose.
• D-PGA D-phenyl glycine
• Enzyme recycling experiment with 600 mM of D-PGA and 400 mM of 7-ADCA, pH 7.5 - ⁇ 7.8, and titration with 12N H 2 S0 4 .
• Carried out analogously to example I with 20 g of wet enzyme and a feed consisting of 45.0 g of free D- PGA, 43.7 g of 7-ADCA, 1.8 g of D-PG, 413 ml of water and 5 ml of 25 mass% aqueous NH 4 0H.
• Initial pH - 7.6, final pH 7.8, kept constant by means of titration with 12N H 2 S0 4 . Reaction time: 2 hours.
• Enzyme recycling experiment with 600 mM of D-PGA.> $ H 2 S0 4 and 400 mM of 7-ADCA, pH 7.2 - ⁇ 7.5, in the presence of 100 mM of (NH 4 ) 2 S0 4 .
• Enzyme recycling experiment with 500 mM of D-PGA and 500 mM of 7-ADCA, pH 7.3 ⁇ 7.4, in the presence of 1000 mM of NH 4 C1.
• Example IX Enzyme recycling experiment with 400 mM of D-PGA and 400 mM Of 7-ADCA, pH 7.3 ⁇ * 7.8.

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• 1995
  • 1995-02-02 BE BE9500081A patent/BE1009071A3/nl not_active IP Right Cessation
• 1996
  • 1996-02-01 AU AU48462/96A patent/AU4846296A/en not_active Abandoned
  • 1996-02-01 WO PCT/NL1996/000052 patent/WO1996023897A1/en not_active Application Discontinuation
  • 1996-02-01 IN IN162MA1996 patent/IN181940B/en unknown
  • 1996-02-01 IN IN161MA1996 patent/IN182469B/en unknown
  • 1996-02-01 EP EP96904337A patent/EP0815256A1/de not_active Ceased

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