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WO1998048037A1 - Procede de regulation de la solubilite d'une betalactamine - Google Patents

Procede de regulation de la solubilite d'une betalactamine Download PDF

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Publication number
WO1998048037A1
WO1998048037A1 PCT/EP1998/002457 EP9802457W WO9848037A1 WO 1998048037 A1 WO1998048037 A1 WO 1998048037A1 EP 9802457 W EP9802457 W EP 9802457W WO 9848037 A1 WO9848037 A1 WO 9848037A1
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Prior art keywords
acid
lactam
optionally substituted
solution
lactam nucleus
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PCT/EP1998/002457
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English (en)
Inventor
Erik De Vroom
Dirk Schipper
Jan Metske Van Der Laan
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Dsm N.V.
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Priority to AU76487/98A priority Critical patent/AU7648798A/en
Publication of WO1998048037A1 publication Critical patent/WO1998048037A1/fr

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    • 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
    • C12P35/04—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin by acylation of the substituent in the 7 position
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D499/00—Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
    • 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
    • C12P35/02—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin by desacylation of the substituent in the 7 position
    • 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
    • C12P37/00—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin
    • C12P37/04—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin by acylation of the substituent in the 6 position
    • 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
    • C12P37/00—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin
    • C12P37/06—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin by desacylation of the substituent in the 6 position

Definitions

  • the invention relates to a method for controlling the solubility of a /3-lactam nucleus.
  • 3-lactam antibiotics such as penicillin and cephalosporin antibiotics comprises a great variety of compounds, all having their own activity profile.
  • 3-lactam antibiotics consist of a nucleus, the so-called ⁇ - lactam nucleus, which is linked through its primary amino group to the so-called side chain via a linear amide bond.
  • j ⁇ -Lactam nuclei are very important intermediates in the preparation of semi-synthetic penicillin and cephalosporin antibiotics.
  • the routes to prepare these semi-synthetic penicillins and cephalosporins mostly start from fermentation products such as penicillin G, penicillin V and Cephalosporin C, which are converted to the corresponding /3-lactam nuclei, for instance in a manner as is disclosed in K. Matsumoto, Bioprocess. Techn. , .16, (1993), 67-88, J.G. Shewale & H. Sivaraman, Process Biochemistry, August 1989, 146-154, T.A. Savidge, Biotechnology of Industrial Antibiotics (Ed. E.J. Vandamme) Marcel Dekker, New York, 1984, or J.G. Shewale et al . , Process Biochemistry International, June 1990, 97-103.
  • 3-lactam nuclei which are employed as precursor for several antibiotics are 6-aminopenicillanic acid (6-APA) , 7-aminocephalosporanic acid (7-ACA), 3-chloro-7- aminodesacetoxydesmethylcephalosporanic acid (7-ACCA) , 7-amino- desacetylcephalosporanic acid (7-7ADAC) , and 7-amino- desacetoxycephalosporanic acid (7-ADCA) .
  • the /3-lactam nuclei are converted to the desired antibiotic by coupling to a suitable side chain, as has been described in inter alia EP 0 339 751, JP-A-53005185 and CH-A-640 240.
  • a suitable side chain as has been described in inter alia EP 0 339 751, JP-A-53005185 and CH-A-640 240.
  • D- (-) -phenylglycine or a suitable derivative thereof, such as an amide or ester, may be attached to any of 7-ACA, 7-ACCA, 7-ADCA and 6-APA to produce Cephaloglycin, Cefaclor, Cephalexin or Ampicillin respectively.
  • Other examples of often employed side chains are D- (-) -4-hydroxyphenylglycine, 2-cyanoacetic acid and 2- (2-amino-4-thiazolyl) -2 - methoxyiminoacetic acid.
  • a fermentative process has been disclosed for the production of 7-7 ⁇ DCA and 7-ACA, involving the fermentative production of N-substituted /3-lactams, such as adipyl - 7 -ADCA or adipyl-7-ACA by a recombinant Penicillium chrysogenum strain capable of expressing a desacetoxycephalosporanic acid synthase (DAOCS) also known as "expandase” from a transgene (EP 0 532 341, EP 0 540 210, WO 93/08287, WO 95/04148, WO 95/04149) .
  • DOCS desacetoxycephalosporanic acid synthase
  • the expandase takes care of the expansion of the 5-membered ring of certain ⁇ -acylated penicillanic acids, thereby yielding the corresponding ⁇ -acylated desacetoxycephalosporanic acids.
  • the final step in the disclosed process comprises contacting the acyl-7-ADCA with a suitable acylase, whereby the acyl side chain is removed and the desired 7-ADCA or 7-ACA product is formed.
  • ADCA have to be isolated by crystallization before they may be converted to /3-lactam antibiotics.
  • a major drawback of crystallization of /3-lactam nuclei is loss of product in the mother liquor, which can amount to 10% of the theoretic yield, depending on the solubility of the product in question.
  • Simple recovery procedures such as extraction of the mother liquor with an organic solvent cannot be applied because of the amphoteric nature of the /3-lactam nucleus, which is an amino acid and thus has a tendency to readily dissolve in aqueous environment at any pH value .
  • the invention provides a method for controlling the solubility of a /3-lactam nucleus having the general formula (I)
  • R 0 is hydrogen or C x _ 3 alkoxy
  • Y is CH 2 , oxygen, sulfur, or an oxidized form of sulfur
  • R x is hydrogen, hydroxy, halogen, C ⁇ _ 3 alkoxy, optionally substituted, optionally containing one or more heteroatoms, saturated or unsaturated, branched or straight C ⁇ alkyl, preferably methyl, optionally substituted, optionally containing one or more heteroatoms, C 5 , 8 cycloalkyl, optionally substituted aryl or heteroaryl, or optionally substituted benzyl, or of a salt of said /3-lactam nucleus, wherein the amphoteric properties of said /3-lactam nucleus or the salt thereof are modified.
  • the present invention provides a method which may be applied in optimizing the yield of the intermediate /3-lactam nucleus, such as 6-APA, 7-ACA, 7-ACCA, 7-ADAC or 7-ADCA, in the industrial production of cephalosporin and penicillin antibiotics.
  • the invention has proven to be very useful in cases wherein the carboxylic acid function of a /3-lactam nucleus is protected by an ester group, such as a p-nitrobenzylester, a tertiary butylester, a p-methoxy benzylester, a benzhydrylester or an allylester.
  • an ester group such as a p-nitrobenzylester, a tertiary butylester, a p-methoxy benzylester, a benzhydrylester or an allylester.
  • a /3-lactam nucleus wherein the carboxylic acid function bears an ester group, such as a tertiary butyl group (t-butyl)
  • the starting material in the method according to the invention is a /3-lactam nucleus having the above general formula (I) or a salt thereof, wherein the symbols have the meanings as defined hereinabove.
  • an oxidized form of sulfur is meant to include groups such as sulfoxide and sulfone.
  • alkyl, cycloalkyl, aryl, heteroaryl and benzyl, groups are intended, which have substituents such as alkyl groups of from 1 to 3 carbon atoms.
  • substituents such as alkyl groups of from 1 to 3 carbon atoms.
  • Optionally substituted nitrogen includes primary, secondary and tertiary amine groups, which may be substituted with for instance alkyl groups of from 1 to 3 carbon atoms .
  • Optionally substituted methyl is meant to include a methyl group and various substituted methyl groups such as -CH p D q , wherein D is a halogen and p and q are integers of which the sum equals 3.
  • Formula (I) is intended to encompass all /3-lactam nuclei as disclosed in "Cephalosporins and Penicillins, Chemistry and Biology", Ed. E.H. Flynn, Academic Press, 1972, pages 151-166, and "The Organic Chemistry of /3-Lactams", Ed. G.I. Georg, VCH, 1992, pages 89-96, which are incorporated herein by reference.
  • E is hydrogen, hydroxy, halogen, C._ 3 alkoxy, optionally substituted, optionally containing one or more heteroatoms, saturated or unsaturated, branched or straight C 1 _ 5 alkyl, optionally substituted, optionally containing one or more heteroatoms C 5 _ 8 cycloalkyl, optionally substituted aryl or heteroaryl, or optionally substituted benzyl .
  • Suitable salts of the /3-lactam nuclei to be converted in a method according to the invention include 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, N, N' - dibenzyl diethylene diamine) .
  • an alkali metal salt e.g. lithium, potassium, sodium
  • an alkali earth metal salt e.g. calcium, magnesium
  • an ammonium salt e.g. trimethylamine, triethylamine, pyridine, picoline, dicyclohexylamine, N, N' - dibenzyl diethylene diamine
  • the /3-lactam nucleus is a cephalosporanic acid, a penicillanic acid, or a salt thereof.
  • Most preferred /3-lactam nuclei are 6-aminopenicillanic acid (6-APA) , 7-amino- cephalosporanic acid (7-ACA) , 3-chloro-7-aminodesacetoxy- desmethylcephalosporanic acid (7-ACCA) , 7-aminodesacetylcephalo- sporanic acid (7-/ADAC) , or 7-aminodesacetoxycephalosporanic acid
  • the /3-lactam nucleus starting material is preferably obtained from N-substituted /3-lactam fermentation products such as penicillin G, penicillin V, cephalosporin C, adipyl-7-ADCA, 3 -carboxyethylthiopropionyl-7-ADCA, 2 -carboxylethylthioacetyl-7- ADCA, 3-carboxyethylthiopropionyl-7-ADCA, adipyl-7-ACA, 3- carboxyethylthiopropionyl-7-ACA, 2 -carboxylethylthioacetyl-7-ACA or 3-carboxyethylthiopropionyl-7-ACA by enzymatic conversion by the action of a penicillin acylase.
  • N-substituted /3-lactam fermentation products such as penicillin G, penicillin V, cephalosporin C, adipyl-7-ADCA, 3 -carboxyethy
  • a suitable penicillin acylase is an enzyme that may be isolated from various naturally occurring micro-organisms, such as fungi and bacteria. Organisms that have been found to produce penicillin acylase are, for example, Aceto acter, Aeromonas , Alcaligenes, Aphanocladium, Bacillus sp . , Cephalosporium, Escherichia , Flavobacterium, Kluyvera, Mycoplana, Protaminobacter, Providentia, Pseudomonas or Xanthomonas species.
  • by modifying the amphoteric properties of a compound is meant modifying the tendency of a compound to show both acidic and basic behaviour.
  • amphoteric properties of a compound the charge distribution of the compound is affected. It has now been found that the amphoteric properties of a /3-lactam nucleus or a salt thereof may very suitably be modified by subjecting it to the action of at least one dicarboxylate acylase.
  • a method according to the invention is an enzymatic, selective and efficient process, in other words a method which does not result in effluent problems or involve expensive chemicals.
  • a dicarboxylate acylase is capable of enzymatically modifying the amphoteric properties of a /3-lactam nucleus at very low concentrations, such as normally occur in mother liqours of industrial crystallization processes.
  • a suitable dicarboxylate acylase to be used according to the invention is an enzyme that may be isolated from various naturally occurring micro-organisms, such as fungi and bacteria. Such micro-organisms can be screened for enzymes with the desired dicarboxylic acid specificity by monitoring the hydrolysis of suitable substrates.
  • suitable substrates may be e.g. chromophores such as succinyl-, glutaryl- or adipyl-p- nitroanilide.
  • the hydrolysis of the corresponding N- substituted /3-lactams may be used for identifying the required enzymes.
  • Organisms that have been found to produce dicarboxylate acylase are Alcaligenes, Arthrobacter, Achromobacter, Aspergillus, Acinetobacter, Bacillus and Pseudomonas species. More in particular, the following species produce highly suitable dicarboxylate acylases : Ac ro-noJbacter xylosooxidans , Arthrobacter viscosis, Arthrobacter CA128, Bacillus CA78, Bacillus megaterium ATCC53667, Bacillus cereus , Bacillus la terosporus Jl, Paecilomyces C2106, Pseudomonas diminuta sp N176, Pseudomonas diminuta sp V22, Pseudomonas paucimobilis , Pseudomonas diminuta BL072 , Pseudomonas strain C427 , Pseudomonas sp SE83, Pse
  • the dicarboxylate acylase may be obtained from the microorganism by which it is produced in any suitable manner, for example as is described for the Pseudomonas sp SE83 strain in US 4,774,179. Also, the genes for e.g. SE83 or SY77 dicarboxylate acylases may be expressed in a different suitable host, such as E. coli as has been reported by Matsuda et al . in J . Bacteriology, 169, (1987) , 5818-5820 for the SE83 strain, and in US 5,457,032 for the SY77 strain.
  • the enzymes isolated from the above sources are often referred to as glutaryl acylases.
  • the side chain specificity of the enzymes is not limited to the glutaryl side chain, but comprises also smaller and larger dicarboxyl side chains.
  • Some of the dicarboxylate acylases also express gamma- glutamyl transpeptidase activity and are therefore sometimes classified as gamma-glutamyl transpeptidases .
  • the dicarboxylate acylase may be used as the free enzyme, but also in any suitable immobilized form, for instance as has been described in EP 0 222 462.
  • functional equivalents of the enzyme wherein for instance properties of the enzyme, such as pH dependence, thermostability or specific activity may be affected by chemical modification or cross-linking, without significant consequences for the activity, in kind, not in amount, of the enzyme in a method according to the invention.
  • functional equivalents such as mutants or other derivatives, obtained by classical means or via recombinant DNA methodology, biologically active parts or hybrids of the enzymes may be used.
  • the /3-lactam nucleus or the salt thereof is subjected to the action of the dicarboxylate acylase in the presence of at least one suitable substrate for said dicarboxylate acylase.
  • a suitable substrate in this context, is intended to mean a substrate which is recognized by the enzyme.
  • Preferred substrates to be used are dicarboxylic acids or salts thereof.
  • a dicarboxylic acid may be used in its acidic form or in the form of a salt, such as an ammonium, lithium, potassium or sodium salt. It is an additional advantage of the invention that no activation step of the dicarboxylic acid is necessary.
  • Dicarboxylic acids which have proven to be highly successful in this embodiment of the invention are those represented by the general formula (II)
  • X is (CH 2 ) m -A- (CH 2 ) n , wherein m and n are the same or different and are chosen from the group of integers 0, 1, 2, 3, 4 or 5, and
  • dicarboxylic acids to be used in this embodiment of the invention are those having general formula (II) wherein the sum of m and n equals from 1 to 5. It was found, that these dicarboxylic acids are recognized by the dicarboxylate acylase to be used very efficiently.
  • dicarboxylic acids are succinic acid, glutaric acid, adipic acid, 3- (carboxymethylthio) propionic acid, trans-/. -hydromuconic acid, pimelic acid and 3,3' -thiodipropionic acid.
  • a method is provided wherein a ⁇ - lactam nucleus is converted into an N-substituted /3-lactam.
  • the amphoteric properties of the /3-lactam nucleus are modified in that a carboxylic acid function is introduced and a primary amine function is converted to a secondary amine function.
  • known methods for this conversion have proven to be not suitable in industrial production processes of penicillin and cephalosporin antibiotics.
  • the method of this preferred embodiment of the invention does not lead to dimerization; in other words only one /3-lactam nucleus molecule is attached to one dicarboxylic acid molecule .
  • the invention also encompasses an N- substituted
  • reaction conditions for the preparation of an N- substituted /3-lactam 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 N- substituted /3-lactam that is to be prepared using a given dicarboxylate acylase, the person skilled in the art will be able to suitably choose the optimum reaction conditions.
  • the optimum reaction temperature in a method according to the invention lies between 0 and 80°C, preferably between 10 and 50°C.
  • the optimum pH for preparing an N- substituted /3-lactam according to the invention lies between 4.5 and 9.0, preferably 5.5 and 6.5.
  • the dicarboxylate acylase enzyme has proven to catalyze the conversion reaction most efficiently in an aqueous environment.
  • the reagents will be present in amounts ranging between 0.01, preferably 0.5, and 3 ol per kilogram reaction mixture, preferably 2 mol per kilogram reaction mixture, in both steps.
  • Suitable enzyme concentrations are chosen such that the total reaction time does not exceed 4 hours.
  • about 500 to 3000 enzyme reaction units should be applied, wherein an enzyme reaction unit is defined as the amount of enzyme which converts one micromole of substrate into product in one minute under conditions which represent the actual process conditions.
  • the enzyme dosage should be preferentially between 50 and 300 kUnits per mole. However, usually a larger excess of activity is dosed in order to compensate for any losses which may occur during the process .
  • Suitable titrants are inorganic acids, such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, or organic acids, such as formic acid, acetic acid, and so forth, or the dicarboxylate acid which is to be attached to the /3-lactam nucleus in a method according to the invention.
  • Titrant concentration may vary between 0.01 and 8 M, depending on the scale of the reaction and the solubility of the acid.
  • the invention provides a method for preparing a /3-lactam nucleus from an N- substituted /3-lactam obtained as disclosed hereinabove, wherein the N-substituted ⁇ - lactam is subjected to the action of a dicarboxylate acylase.
  • the dicarboxylate acylase as defined hereinabove has been found to be capable of catalyzing both the conversion of a /3-lactam nucleus into an N- substituted /3-lactam and the opposite reaction of an N- substituted /3-lactam to the corresponding /3-lactam nucleus efficiently.
  • the action of the dicarboxylate acylase may be influenced by choosing the appropriate reaction conditions, in particular the pH. While the conversion of a /3-lactam nucleus to an N- substituted /3-lactam is effected by a dicarboxylate acylase under acidic conditions, the reverse reaction transpires at higher pH values, preferably from 6.5 to 9, more preferably from 7 to 8.
  • the preparation of an N- substituted /3-lactam may be performed in order to recover a ⁇ - lactam nucleus from a crystallization mother liquor in an industrial process for preparing a /3-lactam nucleus. Subsequently, the obtained N- substituted /3-lactam may be reconverted to a /3-lactam nucleus by subjecting it to the action of a dicarboxylate acylase as described hereinabove.
  • the invention enables the optimization of the yield of an expensive and important intermediate in commercial preparation processes for penicillin and cephalosporin antibiotics.
  • the mother liquor of a crystallization process is treated with a dicarboxylic acid in the presence of a dicarboxylate acylase as defined hereinabove.
  • the resulting N-dicarboxyl /3-lactam may be recovered by extraction with an organic solvent at a pH chosen between 0 and 3. Subsequently, the N-dicarboxyl /3-lactam containing organic phase is back-extracted in water at high concentrations at neutral to slightly basic pH values.
  • the resulting aqueous solution is again treated with dicarboxylate acylase at pH 7-9 in order to liberate the required /3-lactam nucleus.
  • Isolation is effected by crystallization at a pH value close to the pK a value of the /3-lactam nucleus.
  • this procedure may be preceded by extraction of the liberated dicarboxylic acid using an organic solvent.
  • the above recovery process can advantageously be combined with an existing production process based on fermentatively prepared N-dicarboxyl /3-lactam nuclei.
  • a dicarboxylic acid is used that is already present as a result of a preceding process step (e.g. enzymatic deacylation of an N-dicarboxyl /3-lactam) .
  • the resulting N-dicarboxyl /3-lactam may be recovered by extraction, essentially as described above, preferably using the same solvent as used in the extraction step of the fermentation process.
  • the N-dicarboxyl /3-lactam containing organic phase is combined with the organic phase of the fermentation process and further processed as described above to give the desired /3-lactam nucleus, which may be reacted with an appropriate side chain to yield a penicillin or cephalosporin antibiotic.
  • a method according to the invention is highly advantageous when performed with the purpose of recovering /3-lactam nucleus from a crystallization mother liquor in a preparation process for /3-lactam nuclei.
  • the invention also encompasses the use of a dicarboxylate acylase for recovering a /3-lactam nucleus from a mother liquor obtained after a reaction wherein an N-acylated /3-lactam is converted into a ⁇ - lactam nucleus and a dicarboxylic acid in a process for preparing a /3-lactam antibiotic.
  • one unit (U) corresponds to ' the amount of enzyme that hydrolyses per minute 1 micromole N- adipyl - 7 - aminodesacetoxycephalosporanic acid under standard conditions (100 mM N-adipyl - 7 -aminodesacetoxycephalosporanic acid, 100 mM Tris buffer, pH 8.0, 37°C) .
  • Condensation reactions are carried out in water at constant pH values. To this end a Mettler DL21 titration apparatus equipped with an automatic burette and Brother M1509 printing device is used. Proceeding of conversions is monitored by consumption of acid. When the equilibrium at a choosen pH-value is reached, the reaction is either terminated or continued at a lower pH-value .
  • 6-APA (1.05 g; 4.41 mmol) and glutaric acid (0.58 g; 4.41 mmol) were suspended in water (9 ml) and the pH was adjusted to
  • 6-APA (5.94 g; 25.0 mmol) and glutaric acid (3.30 g; 25.0 mmol) were suspended in water (40 ml) and the pH was adjusted to 6.8 with a solution of NaOH in water (8 M) . The volume was adjusted to 50.0 ml and immobilized dicarboxylate acylase
  • CEPH-3-EM-4-CARBOXYLATE 7 -ACA (5.44 g; 20.0 mmol) and glutaric acid (2.64 g; 20.0 mmol) were suspended in water (40 ml) and the pH was adjusted to 7.0 at 0°C with a 25% solution of ammonia in water. The temperature was adjusted to 30°C and immobilized dicarboxylate acylase ( Pseudomonas SE-83, 10.0 g; 96 units. g "1 ) was added and the mixture was stirred at 30°C for 3 h while maintaining the pH of the solution at 6.6 with a solution of HCl in water (1 M) .
  • immobilized dicarboxylate acylase Pseudomonas SE-83, 10.0 g; 96 units. g "1
  • the immobilized enzyme was removed by filtration, and part of the filtrate (39% by weight) was lyophilized to give 2.16 g of diammonium N-glutaryl - ( 6R, 7R) -3- acetoxymethyl-7-aminoceph-3-em-4-carboxylate as a light-yellow solid (purity 26% as determined by 600 MHz X H ⁇ MR in D 2 0) . Yield 68.5%.
  • adipic acid (20.0 g; 137 mmol) .
  • immobilized dicarboxylate acylase Pseudomonas SE- 83 , 5.0 g; 110 units. g "1 ) .
  • the mixture was stirred at 40°C for 3 h while maintaining the pH of the solution at 6.1 with a warm (40°C) solution of adipic acid in water (0.3 M) .
  • Immobilized dicarboxylate acylase was removed by filtration.
  • Immobilized dicarboxylate acylase was removed by filtration. Part of the filtrate (80% of the reactor volume by weight) was lyophilized to give 1.65 g of white product.
  • the product contains
  • adipic acid (10.0 g; 69 mmol) .
  • immobilized dicarboxylate acylase Pseudomonas SE- 83 , 5.0 g; 110 units. g "1 ).
  • the mixture was stirred at 10°C for 15 h while maintaining the pH of the solution at 6.1 with a warm (40°C) solution of adipic acid in water (0.3 M) .
  • Immobilized dicarboxylate acylase was removed by filtration.
  • the immobilized enzyme was removed by filtration, and part of the filtrate (36% by weight) was lyophilized to give 2.14 g of dipotassium N-adipyl - (61?, 71?) - 7 - amino-3-chloroceph-3-em-4-carboxylate as a white solid (purity 6% as determined by 360 MHz X H ⁇ MR in D 2 0) . Yield 55.7%.
  • 6-APA (2.20 g; 10.0 mmol) and 3-methyladipic acid (1.60 g; 10.0 mmol) were suspended in water (30 ml) and the pH was adjusted to 6.4 with a solution of LiOH in water (4 M) .
  • immobilized dicarboxylate acylase Pseudomonas SE- 83, 10.0 g; 96 units. g "1 ) was added and the mixture was stirred at 10 °C for 16 h while maintaining the pH of the solution at 6.4 with a solution of HCl in water (1 M) .
  • the pH was lowered to 5.7 and stirring was continued at this pH-value for 3 h.
  • EXAMPLE 18 DIPOTASSIUM N- ( rans-/S-HYDROMUCO ⁇ YL) -6S-AMI ⁇ OPE ⁇ ICILLA ⁇ ATE 6-APA (1.19 g; 5.00 mmol) and trans-/3-hydromuconic acid (0.72 g; 5.00 mmol) were suspended in water (10 ml) and the pH was adjusted to 6.4 with a solution of KOH in water (2 M) . To the clear solution immobilized dicarboxylate acylase (Acinetobacter sp., 10.0 g; 12 units.
  • Acinetobacter sp. 10.0 g; 12 units.
  • METHYLCEPH-3 -EM-4-CARBOXYLATE 7-ADCA (3.21 g; 15.0 mmol) and trans-/3-hydromuconic acid (2.16 g; 15.0 mmol) were dissolved in water (40 ml) at pH 8.1 using a solution of NaOH in water (6 M) .
  • the volume was adjusted to 50.0 ml, the pH was brought to 6.5 and immobilized dicarboxylate acylase ( Pseudomonas SE-83, 25.0 g; 96 units. g ""1 ) was added and the mixture was stirred at 20°C for 3 h while maintaining the pH of the solution at 6.5 with a solution of HCl in water (1 M) .
  • 6-APA (1.19 g; 5.00 mmol) and trans, trans-rauconic acid (0.73 g; 5.00 mmol) were suspended in water (20 ml) and the pH was adjusted to 6.3 with a solution of LiOH in water (4 M) .
  • immobilized dicarboxylate acylase (Pseudomonas SE- 83, 20.0 g; 96 units. g "1 ) was added and the mixture was stirred at 30°C for 14 h while maintaining the pH of the solution at 6.3 with a solution of HCl in water (1 M) .
  • the immobilized enzyme was removed by filtration and part of the filtrate (16% by weight) was lyophilized to give 340 mg of dilithium N- ( trans, trans - muconyl) -6/3-aminopenicillanate as a light-yellow solid (purity 15% as determined by 360 MHz X H ⁇ MR in D 2 0) . Yield 18.3%.
  • 6-APA (2.19 g; 10.0 mmol) and pimelic acid (1.63 g; 10.0 mmol) were suspended in water (30 ml) and the pH was adjusted to 6.4 with a solution of KOH in water (2 M) .
  • immobilized dicarboxylate acylase Pseudomonas SE-83, 10.0 g; 110 units. gA was added and the mixture was stirred at 14 °C for 1.5 h while maintaining the pH of the solution at 6.4 with a solution of HCl in water (1 M) .
  • the pH was lowered to 6.2 and stirring was continued for 18 h. Again, the pH was lowered to 5.6 by the addition of pimelic acid (0.25 g; 1.5 mmol) .
  • METHYLCEPH-3 -EM-4-CARBOXYLATE 7-ADCA (3.21 g; 15.0 mmol) and 3 , 3 ' -thiodipropionic acid (97%, 2.76 g; 15.0 mmol) were dissolved in water (40 ml) at pH 8.1 using a solution of NaOH in water (6 M) .
  • the volume was adjusted to 50.0 ml, the pH was brought to 6.5 and immobilized dicarboxylate acylase ( Pseudomonas SE-83, 25.0 g; 96 units.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
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  • Microbiology (AREA)
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  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Cephalosporin Compounds (AREA)

Abstract

L'invention concerne un procédé de régulation de la solubilité d'une bêtalactamine correspondant à la formule générale (I), ou d'un sel de celle-ci. Dans cette formule R0 représente hydrogène ou alcoxy C1-3, Y représente CH2, oxygène, soufre ou une forme oxydée de soufre, et Z représente (a), (b), (c) ou (d) où R1 représente hydrogène, hydroxy, halogène, alcoxy C1-3, alkyle C1-5 ramifié ou droit, éventuellement substitué et contenant, éventuellement un ou plusieurs hétéroatomes saturés ou insaturés, de préférence méthyle, cycloalkyle C5-8 éventuellement substitué et contenant le cas échéant un ou plusieurs hétéroatomes, aryle ou hétéroaryle éventuellement substitué, ou benzyle éventuellement substitué. L'invention est caractérisée en ce que les propriétés amphotères de cette bêtalactamine ou du sel de celle-ci sont modifiées.
PCT/EP1998/002457 1997-04-22 1998-04-22 Procede de regulation de la solubilite d'une betalactamine WO1998048037A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU76487/98A AU7648798A (en) 1997-04-22 1998-04-22 A method for controlling the solubility of a beta-lactam nucleus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP97201199 1997-04-22
EP97201199.3 1997-04-22

Publications (1)

Publication Number Publication Date
WO1998048037A1 true WO1998048037A1 (fr) 1998-10-29

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WO (1) WO1998048037A1 (fr)
ZA (1) ZA983396B (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007073947A2 (fr) 2005-12-28 2007-07-05 Dsm Ip Assets B.V. Acylases mutantes

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0532341A1 (fr) * 1991-09-11 1993-03-17 Gist-Brocades B.V. Nouveau procédé biologique de préparation de 7-AADC
WO1993008287A1 (fr) * 1991-10-15 1993-04-29 Merck & Co., Inc. Nouveaux procedes biologiques de preparation de 7-aca et de 7-adac
WO1995004148A1 (fr) * 1993-07-30 1995-02-09 Gist-Brocades B.V. Procede de production efficace de 7-adca par l'intermediaire du 2-(carboxyethylthio)acetyl-7-adca et du 3-(carboxymethylthio)propionyl-7-adca
WO1997035029A1 (fr) * 1996-03-15 1997-09-25 Antibioticos, S.A. Nouveau procede d'obtention de l'acide 6-amino-penicillanique (6-apa)
WO1997034902A1 (fr) * 1996-03-15 1997-09-25 Antibioticos, S.A. Procede de purification de 7-substitue-amino-desacetoxy-cephalosporines par l'emploi de membranes de filtration
WO1998002551A2 (fr) * 1996-07-16 1998-01-22 Gist-Brocades B.V. Procede ameliore de production de cephalosporines d'adipoyle

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0532341A1 (fr) * 1991-09-11 1993-03-17 Gist-Brocades B.V. Nouveau procédé biologique de préparation de 7-AADC
WO1993005158A1 (fr) * 1991-09-11 1993-03-18 Merck & Co., Inc. Nouveau procede biochimique pour la preparation de 7-adca
WO1993008287A1 (fr) * 1991-10-15 1993-04-29 Merck & Co., Inc. Nouveaux procedes biologiques de preparation de 7-aca et de 7-adac
WO1995004148A1 (fr) * 1993-07-30 1995-02-09 Gist-Brocades B.V. Procede de production efficace de 7-adca par l'intermediaire du 2-(carboxyethylthio)acetyl-7-adca et du 3-(carboxymethylthio)propionyl-7-adca
WO1997035029A1 (fr) * 1996-03-15 1997-09-25 Antibioticos, S.A. Nouveau procede d'obtention de l'acide 6-amino-penicillanique (6-apa)
WO1997034902A1 (fr) * 1996-03-15 1997-09-25 Antibioticos, S.A. Procede de purification de 7-substitue-amino-desacetoxy-cephalosporines par l'emploi de membranes de filtration
WO1998002551A2 (fr) * 1996-07-16 1998-01-22 Gist-Brocades B.V. Procede ameliore de production de cephalosporines d'adipoyle

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AGRIC. BIOL. CHEM., vol. 45, no. 10, 1981, pages 2231 - 2236 *
CHEMICAL ABSTRACTS, vol. 96, no. 1, 4 January 1982, Columbus, Ohio, US; abstract no. 2721, ICHIKAWA S. ET AL.: "7-Aminocephalosporanic acid production" XP002074817 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007073947A2 (fr) 2005-12-28 2007-07-05 Dsm Ip Assets B.V. Acylases mutantes
EP2851423A1 (fr) 2005-12-28 2015-03-25 DSM Sinochem Pharmaceuticals Netherlands B.V. Acylases de bêta-lactame de type II mutées

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ZA983396B (en) 1998-10-27
AU7648798A (en) 1998-11-13

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