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)
  • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
  • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
  • C12N9/84—Penicillin amidase (3.5.1.11)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/823—Acetobacter
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/83—Arthrobacter
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/832—Bacillus
  • Y10S435/839—Bacillus subtilis
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/848—Escherichia
  • Y10S435/849—Escherichia coli
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/873—Proteus
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/911—Microorganisms using fungi

Definitions

• R is selected from the group consisting of the acyl moieties of phenylacetic and phenoxyacetic acid;
• R and R' and R; are each selected from the group consisting of hydrogen, alkanoyloxymethyl having from three to eight carbon atoms, l-(alkanoyloxy)ethyl having from four to nine carbon atoms and phthalidyl;
• R is selected from the group consisting of R' trialkylsilyl having up to four carbon atoms in each alkyl group and a tetrazolylpenam nitrogen protecting group which is defined hereinafter;
• R is selected from the group consisting of R';, and trialkylsilyl having up to four carbon atoms in each alkyl group;
• R is selected from the group consisting of R hydrogen, and an amine protecting group which is defined hereinafter;
• the Prior Art Benzyland phenoxymethylpenicillin are produced in fermentation processes by a wide variety of organ- -isms. These are the most common of the naturallyoccurring penicillins.
• Semisynthetic penicillins differ from the naturally-occurring penicillins usually only in the nature of the N-acyl group. These are prepared by deacylating benzylpenicillin or phenoxymethylpenicillin to form 6-amino-2,2-dimethylpenicillanic acid and phenylacetic or phenoxyacetic acid respectively and then reacylating said penicillanic acid with the desired acyl moiety; both of these processes have been carried out by microbial methods. Many valuable semisynthetic penicillins have been produced in this manner.
• the present invention is directed to the enzymatic deacylation of penicillin tetrazoles. Since penicillin tetrazoles were unknown in the art prior to Ser. No. 407,097, there is obviously no prior art on the deacylation of penicillin tetrazoles. The enzymatic deacylation of benzyl and phenoxymethylpenicillin has been effected in the past. However, because enzymes are known to be highly specific, one skilled in the art would in no way expect that the same enzymes effective on these substrates would be effective with the corresponding tetrazoles. The deacylation of penicillins has been reviewed in Cephalosporins and Penicillins, ed. E. H. Flynn, Academic Press (New York, 1972).
• a process for the enzymatic deacylation of penicillin tetrazoles of the structure wherein R R and R are as previously defined.
• the process is also applicable to the salts of these compounds.
• the process comprises dispersing said penicillin tetrazoles in water at a concentration of at least 0.1% by weight, maintaining the pH of the reaction medium between about 5 and 9 and the temperature between 5 and 50C. while contacting said penicillin tetrazole with a deacylase selected as described below until the reaction to form the product with the general structural formula I or II wherein R, is hydrogen is substantially complete.
• R or R are subject to solvolysis in water or mild alkaline solutions'such as those wherein R or R is trialkylsilyl and certain of the tetrazolylpenam nitrogen protecting groups
• a product will be obtained wherein R or R is hydrogen.
• the penicillin tetrazole concentration is between about 0.1 and 20.5% by weight
• the temperature is maintained between 25 and 45C.
• the pH between 7.0 and 8.8.
• the deacylase is introduced into the reaction medium by a member of the group comprising bacteria, whole bacterial cells immobilized on a matrix, extracts isolated from said bacteria, fungi, whole fungi cells immobilized on a matrix, extracts isolated from said fungi, enzymes from said bacteria or fungi and said enzymes immobilized on a matrix.
• a member of the group comprising bacteria, whole bacterial cells immobilized on a matrix, extracts isolated from said bacteria, fungi, whole fungi cells immobilized on a matrix, extracts isolated from said fungi, enzymes from said bacteria or fungi and said enzymes immobilized on a matrix.
• Microorganisms of the genera Proteus Escherichia, kluyvera, Acetobacter, Aerobacter, Arthrobacter, Bacillus and Cryptococcus which successfully deacylate penicillin tetrazoles are disclosed.
• Said deacylases are also acylases for benzylpenicillin and the salts thereof when R, is the acyl moiety of phenylacetic acid and are acylases for phenoxymethylpenicillin and the salts thereof when R, is the acyl moiety of phenoxyacetic acid. Further, it has been surprisingly found that said deacylases are actually more active on the tetrazolylpenam substrate than they are on the corresponding penicillin substrate.
• the product of said enzymatic deacylation is a compound of the general structural formula III or IV wherein R is hydrogen. It should be noted that in those cases wherein R or R are subject to solvolysis in aqueuus solution, such as those wherein R or R are trialkylsilyl or certain tetrazolylpenam nitrogen protecting groups noted hereinafter, R or R may be displaced by hydrogen to yield a product wherein not only R, but also R or R are hydrogen.
• penicillin deacylases should exhibit either no activity or significantly diminished activity with penicillin tetrazoles rather than natural penicillins as a substrate. In fact, it has been surprisingly found not only that all the deacylases are active on a tetrazole substrate but also that activities equal to and, in some instances, threefold greater are observed with the tetrazole substrate.
• tetrazolylpenam nitrogen protecting group is intended to connote all groups known, or obvious, to one skilled in the art, which can be used (a) to permit the synthesis of the compounds of formula III, wherein R is an amino protecting group and R is the said tetrazolylpenam nitrogen protecting group, by the process starting with 6-(protected amino)penicillanic acid described hereinafter; and (b) can be removed from a compound of formula I, wherein R, is an acyl group and R is the said tetrazolylpenam nitrogen protecting group, or from a compound of formula III, wherein R is selected from the group consisting of hydrogen and an amino protecting group, and R is the said tetrazolylpenam nitrogen protecting group, using a method wherein the penam ring system remains substantially intact.
• the tetrazolylpenam nitrogen protecting group is required in order to protect the nitrogen atom which ultimately becomes N-l of the tetrazole ring in the said compounds of formulae 1 through VI, during the conversion of a 6-(protected amino)penicillanic acid into a compound of formula III. It is the ability of the tetrazolylpenam nitrogen protecting group to perform a specific function rather than its precise chemical structure, which is important; and the novelty of the antibacterial agents of the invention does not depend upon the structure of the protecting group. Selection and identification of appropriate protecting groups can be made readily and easily by one skilled in the art.
• An example of a typical tetrazolylpenam 'nitrogen protecting group is -CH2CH /Y wherein Y is an electron-withdrawing group, and Y is either hydrogen or a further electron-withdrawing group, which can be the same as or different from Y.
• the function of the electron-withdrawing group is to render a hydrogen atom, on the carbon atom to which Y and Y are attached, sufficiently acidic that the group is removable in a retrograde Michael reaction.
• Such a reaction is well-known in the art. For example consult House, Modern Synthetic Reactions, W. A. Benjamin, Inc. New York/Amsterdam, 1965, page 207.
• Typical electron-withdrawing groups are cyano, alkoxycarbonyl having from two to seven carbon atoms, phenoxycarbonyl, alkylsulfonyl having from one to six carbon atoms, phenylsulfonyl and SO -NR R wherein R and R are each selected from the group consisting of hydrogen, alkyl having from one to four carbon atoms, phenyl and benzyl.
• R and R are each selected from the group consisting of hydrogen, alkyl having from one to four carbon atoms, phenyl and benzyl.
• a particularly convenient configuration for this protecting group is that wherein Y is hydrogen; and preferred values for Y are alkoxycarbonyl having from two to seven carbon atoms and phenylsulfonyl.
• Such a grouping can be removed by mild hydrolysis, such as mild alkaline hydrolysis, or treatment with a nucleophile, such as an amine, or a thiol or thiolate anion.
• R particularly convenient values are alkyl having from one to six carbon atoms, benzyl, phenyl" and substituted phenyl, for example, phenyl substituted by up to two moieties each selected from the group consisting of nitro, fluoro, chloro, bromo, alkyl having from one to four carbon atoms and alkoxy having from one to four carbon atoms.
• a still further tetrazolylpenam nitrogen protecting group which can be used is a grouping of formula SO R,,.
• a yet further tetrazolylpenam nitrogen protecting group which can be used is Particularly convenient configurations for this protecting group are 11 and I R13 I wherein R and R are each selected from the group consisting of hydrogen, hydroxy, nitro, fluoro, chloro, bromo, iodo, alkyl having from one to six carbon atoms, alkoxy having from one tosix carbon atoms, al kanoyloxy having from two to seven carbon atoms, formyloxy, alkoxymethoxy having from two to seven carbon atoms, phenyl and benzyloxy;
• R is selected from the group consisting of hydrogen, alkyl having from one to four carbon atoms and phenyl;
• R and R are each selected from the group consisting of hydrogen and methyl
• X is selected from the group consisting of oxygen and sulfur.
• Still another tetrazolylpenam nitrogen protecting group which can be used is phenacyl or substituted phenacyl. Such a group is removed by reaction with a nucleophilic reagent, such as thiophenoxide.
• Typical phenacyl groups which can be used are those of formula wherein R is selected from the group consisting of by drogen, nitro, fluoro, chloro, bromo and phenyl.
• Amine protecting groups referred to above are all those known, or obvious, in the art, particularly the art of peptide synthesis. In general, their function is to prevent unwanted substitution of the amino group and the opening of the B'lactam ring. It should be possible to remove them under rather mild conditions when the reaction: scheme is complete but they should be stable under all other conditions encountered throughout the reaction scheme. Typical examples are the 2,2,2- trihaloethoxy carbonyls and substituted and unsubstituted triphenylmethyl group.
• a characteristic feature of compounds of formulas I, through VI wherein the tetrazole ring is hydrogen substituted is their ability to form salts.
• the salts By virture of the acidic nature of a S-monosubstituted tetrazole, said compounds have the ability to form salts with basic agents, and the salts, referred to generically as tetrazolate salts, are to be considered within the scope of this invention.
• the salts can be prepared by standard techniques, such as contacting the acidic and basic components, usually in a 1:1 molar ratio, in an aqueous, non-aqueous or partially aqueous medium, as appropriate.
• Basic agents which are suitably employed in salt formation belong to both the organic and inorganic types, and they include ammonia, organic amines, alkali metal hydroxides, carbonates, bicarbonates, hydrides and alkoxides, as well as alkaline earth metal hydroxides, carbonates, hydrides and alkoxides.
• bases are primary amines, such as npropylamine, n-butylamine, aniline, cyclohexylamine, benzylamine, p-toluidine and octylamine; secondary amines, such as diethylamine, N-methylaniline, morpholine, pyrrolidine and piperidien; tertiary amines, such as triethylamine, N,N-dimethylaniline, N- ethylpiperidien, N-methylmorpholine and 1,5- diazabicyclo[4.3.0]non-5-ene; hydroxides, such as sodium hydroxide, potassium hydroxide, ammonium hy droxide and barium hydroxide; alkoxides, such as sodium methoxide; carbonates, such as potassium carbonate and sodium carbonate; and bicarbonates, such as sodium bicarbonate and potassium bicarbonate.
• primary amines such as npropylamine, n-but
• salts of said compounds When therapeutic use in mammals is being contemplated for a salt of said compounds, it is of course essential to use a pharmaceutically-acceptable salt.
• other salts are useful for a variety of other purpopses; such as, for example, isolating and purifying individual compounds, changing the solubility characteristics of an individual compound, and for interconverting pharmaceutically-acceptable salts with their nonsalt counterparts.
• the process of the present invention finds its utility in the preparation of substantially pure 6-amino-2,2- dimethyl-3(5-tetrazolyl)penam.
• Semi-synthetic penicillins are currently prepared by the chemical acylation of 6-amino2,2-dimethyl-3-(carboxylic acid)penam.
• the tetrazole analogues of said semi-synthetic penicillins may be prepared using the product of the process of the instant invention as the substrate.
• o-aminopenicillanic acid is first contacted with the chloride of one of the above-mentioned amine protecting group in a reaction-inert anhydrous solvent in the presence of a suitable base such as triethylamine at from about 0 to 20C. for at least about minutes.
• a suitable base such as triethylamine at from about 0 to 20C.
• Reaction-inert solvents are those which are without substantially adverse effect on reactants and products under the conditions employed.
• a preferred amine protecting group is triphenylmethyl.
• the protected aminopenicillanic acid is then activated, usually by conversion to a mixed anhydride, and contacted with an amine of the formula RQNHQ at about 0C.
• the crude amide is isolated by in vacuo evaporation.
• the crude amide is then dissolved in pyridine and cooled to about -5C.
• Thionyl chloride is added dropwise and the reaction mixture allowed to warm to room temperature. The mixture is allowed to stand until the reaction to form the imino chloride is substantially complete.
• the crude product is isolated by in vacuo evaporation, redissolved in a reaction-inert solvent and cooled to about 5.
• At least an equivalent amount of a suitable azide as trimethylsilyl azide is then added with stirring and the reaction-mixture allowed to warm to room temperature. Stirring is continued until the reaction to form the protected tetrazole is substantially complete. Excess azide is then destroyed by the addition of a suitable aqueous base. The waterimmiscible layer containing the product is then separated, extracted with brine, treated with a desiccant and concentrated to dryness to afford the crude product. The product may be purified by liquid chromatography. The product may be converted to one of Formula III wherein R is hydrogen by treating it with p-toluenesulfonic acid in a reaction-inert solvent such as acetone. A product of pharmaceutical grade purity may be prepared by the acylation-deacylation cycle described below.
• Compounds of Formula III wherein R is hydrogen may be prepared from those of Formula III wherein R is hydrogen and R is not, by treating them with trifluoroacetic acid at about 40C. for at least 30 minutes. This reaction yields 6-amino-2,2-dimethyl-3-(5- tetrazolyl)penam.
• the contaminants are probably products of the polymerization of the removed tetrazole nitrogen protecting group.
• purification required a series of extractions followed by lyophilization and another series of extractions. Finally, the product must be recrystallized. It has now been found that a pharmaceutical grade product may be prepared by the acyIation-deacylation cycle described below.
• Compounds of Formula IV wherein R is other than hydrogen may be prepared by contacting a 6-protected amino-2,2-dimethyl-3-(S-tetrazolyl)penam with a compound of the formula R Cl in a reaction-inert solvent usually in the presence of a base at about room temperature until the reaction is substantially complete.
• N,N-dimethylformamide is suitable as a solvent and triethylamine as a base. This reaction affords a mixture of isomers of Formulas III and IV which may be separated by thin layer chromatography.
• crude 6-amino-2,2- dimethyl-3-(5-tetra2olyl)penams with or without substituents on the tetrazole ring are first acylated with phenylacetyl chloride or phenoxyacetyl chloride.
• the crude material is first suspended in water and brought into solution by the slow addition of aqueous base.
• the pH is then lowered to about 7 with a mineral acid and the suspension clarified by suction filtration.
• Approximately a 10 percent molar excess of phenylacetyl or phenoxyacetyl chloride is then added with stirring while maintaining the pH between about 6 and 7 with dilute aqueous base. The mixture is stirred for about four hours at room temperature.
• the mixture is then cooled to about 10C., its pH adjusted to about 2 with hydrochloric acid and it is then extracted several times with chloroform.
• the combined organic extracts are then poured into about a 6:1. hexane/ether mixture.
• the white precipitate which forms is then filtered, washed with hexane and dried to afford substantially pure benzylor phenoxymethylpenicillin tetrazole.
• the benzylor phenoxymethylpenicillin tetrazole is dispersed in water at concentration of at least about 0.1% and less than about 20% by weight.
• the pH is then adjusted with acid or base so that it is in the range of about 5 to 9 and preferably in the range of about 7.0 to 8.8 by the addition of a suitable acid or base such as hydrochloric acid or sodium hydroxide.
• the deacylase activity is introduced as whole cells, immobilized whole cells, cellular extracts, enzyme concentrates, substantially pure enzymes or immobilized enzymes. These modes of introduction along with others are'well known to those skilled in the art or zymurgy.
• the weight ratio of cells to said tetrazoles should be in the range of about 0.2 to 10.
• the amount introduced should exhibit about 10 to 100 units of activity against benzylpenicillin for each gram of said tetrazole' present in the reaction mixture.
• Said mixture is then incubated aerobically at a temperature of about to 50C. and preferably between about 25 and 45C. while maintaining the pH in the range of about to 9 and preferably about 7.0 to 8.8 by the addition of a suitable acid or base until the reaction to form the 6-amino-2,2-dimethyl-3-( 5- tetrazolyl)penam is substantially complete.
• the progress of the reaction may be monitored by employing one of the thin layer chromatography systems listed below along with appropriate blanks. Semiquantitative estimates of the yield of 6-aminopenicillin tetrazole can also be made from these chromatograms.
• the product is isolated by first acidifying the reaction mixture to a pH of about 2 with a suitable acid such as hydrochloric acid and extracting the acidified solution several times with a suitable water-immiscible organic solvent such as ethyl acetate.
• a suitable acid such as hydrochloric acid
• the pH of the aqueous layer is then raised about 4.5 with a suitable base such as sodium hydroxide and concentrated in vacuo to yield said 6-aminopenicillin tetrazole product.
• phenylacetic or phenoxyacetic acid may be isolated from the organic layer by evaporation in vacuo after drying over a suitable desiccant.
• the microorganisms of the instant invention are kept on agar slants.
• a water extract from the slant is first incubated in an inoculum medium for about 24 hours and a portion of the inoculum is then transferred to a fermentation medium which is then aerobically incubated for approximately 24 hours.
• Cells may be harvested as needed by centrifugation.
• the deacylase activity of these organisms may be increased by methods well-known to those skilled in the art of zymurgy such as culturing at about 25C. rather than 37C. and adding phenylacetic or phenoxyacetic acid to the culture medium.
• Said activity may be extracted from cells with solutions such as 0.2 M sodium chloride or sodium citrate.
• the enzyme may then be precipitated by a variety of reagents such as ammonium sulfate, calcium nitrate together with a quaternary ammonium salt or acetone. Said enzyme may then be purified by dialysis followed by lyophilization or solidliquid chromatography.
• reagents such as ammonium sulfate, calcium nitrate together with a quaternary ammonium salt or acetone.
• Said enzyme may then be purified by dialysis followed by lyophilization or solidliquid chromatography.
• the preparation of immobilized enzymes has been fully discussed by O. Zaborsky in Immobilized Enzymes, CRC Press (Cleveland, 1 973 Species of the following genera were found to exhibit deacylase activity for benzyland phenoxymethylpenicillin tetrazoles and their carbamoyl analogues: Proteus, Escherichia, Kluyvera, Acetobacter, Aerobacter, Arthrobacter, Bacillus and
• the organisms listed below were found to contain said deacylase: Proteus rettgeri, ATCC 9918; Proteus rettgeri, ATCC 31052; Escherichia coli, ATCC 9637; Escherichia coli, ATCC 31030; Kluyvera citrophilia, ATCC 21 285 Acetobacter cerinus, [F0 3268; Aerobacter Aerogenes, ATCC 31027; Arthrobacler tumescensyATCC 6947; Bacillus subtilis, ATCC 31028; Bacillus species, ATCC 31029; Cryptococcus albidus, ATCC 10666.
• ATCC 31028 was originally identified and registered as B. globigii. Later biochemical tests indicate that it is actually B. subtilis. There is reason to believe that this culture may be a subtransfer of ATCC 9372 but it has not been possible to prove or disprove this.
• ATCC 31029 was originally identified and registered as B. mesentericus. Tests later indicated that it actually is an unidentifiable species or mixture of species of the genus Bacillus. All of these organisms, except Cryptacoccus albz'dus which is a yeast or fungus, are bacteria.
• 278 ml. of triethylamine To a stirred slurry of2 l 6 g. of 6-aminopenicillanic acid in 1,500 ml. of anhydrous chloroform is added 278 ml. of triethylamine, and the mixture is then stirred at ambient temperature until a clear solution is obtained. This requires about 15 minutes.
• the solution is cooled to about 0C., and then 306 g. of triphenylmethyl chloride is added.
• the stirring is continued at about 0C. for 30 minutes, and then at ambient temperature for a further 24 hours.
• the mixture is cooled to about 0C.
• the total volume of 4-methoxybenzylamine added is 131 ml.
• the cooling bath is then removed, and the reaction is stirred for a further 1 hour.
• the chloroform solution is washed successively with five 2,000-ml. portions of water and one 2,000-ml. portion of saturated brine.
• the chloroform is finally dried using anhydrous sodium sulfate.
• the NMR spectrum (in CHCI of this product shows absorption bands at 4.70 ppm (singlet, C-3 hydrogen), 4.65 ppm (singlet, benzyl hydrogens), 4.30-4.60 ppm (multiplet, C-5 and C-6 hydrogens), 3.75 ppm (singlet, methoxy hydrogens), 1.57 ppm (singlet, C-2 methyl hydrogens) and 1.38 ppm (singlet, C-2 methyl hydrogens).
• the NMR spectrum shows absorption bands at 7.25 ppm (multiplet, aromatic hydrogens), 5.50 ppm (broad singlet, benzyl hydrogens), 5.05 ppm (singlet, C-3 hydrogen), 4.40 ppm (broad singlet, C-5 and C-6 hydrogens), 3.80 ppm (singlet, methoxy hydrogens), 1.45 ppm (singlet, C-2 methyl hydrogens) and 0.70 ppm (singlet, C-2 methyl hydrogens).
• the NMR spectrum shows absorption bands at 7.20 ppm multiplet, aromatic hydrogens), 5.80 ppm (multiplet, benzyl hydrogens, C-5 hydrogen and C-3 hydrogens), 5.20 ppm (doublet, C-6 hydrogen), 3.75 ppm (singlet, methoxy hydrogens, 2.35 ppm (singlet, sulfonate methyl hydrogens), 1.70 ppm (singlet, C-2 methyl hydrogens) and 0.85 ppm (singlet, C 2 methyl hydrogens).
• the amine protecting group may be removed from the compounds of Example 1.
• the suspension and solvent is cooled to about 0C., and to it is then added, portionwise, 80 ml. of 2N sodium hydroxide, giving two clear phases.
• the pH of the aqueous phase at this point is about 2.7.
• the layers are separated, and the ether phase is discarded.
• the pH of the aqueous phase is raised to 4.1 with 2N sodium hydroxide.
• This aqueous phase is then washed with 100 ml. of ether and filtered. It is combined with the corresponding aqueous phases from four other identical experiments, and the total aqueous solu tion is lyophilized to give crude 6 -amino-2,2dimethyl- 3-( 5-tetrazolyl)penam.
• This crude product is slurried in a small amount of water and filtered off. lt is then resuspended in water and brought into solution by raising the pH to 7.4 by the addition of sodium hydroxide solution. The clear solution is extracted with ether and the extracts are discarded. The pH of the aqueous phase is adjusted to 4.1 using dilute hydrochloric acid, and the product which precipitates is filtered off. The infrared spectrum of the product shows an absorption at 1795 cm.
• the title compound may be prepared from the following compounds:
• benzylpenicillin tetrazole washed with hexane and dried over P to yield benzylpenicillin tetrazole.
• the benzylpenicillin tetrazole is used in the following example to provide a source of pure 6aminopenicillin tetrazole.
• any of the substituted tetrazole Th f ll i Organisms were f d to tetrazoles may be Similarly acylated except that in hibit deacylase activity with both benzyland phenoxthose Cases where the substituent is subject to hydroly- O ymethylpenicmin tetrazole.
• a product with an unsubstituted tetrazole ring will I p rettgeri ATCC 9918 be obtamed' P. rettgeri ATCC 31052
• EXAMPLE V E. coli ATCC 9637 E. coli ATCC 31030 Screening Microorganisms for Penicillin Deacylase K.
• the inoculum medium is prepared by separately auto- Y o veloped with ninhydrin or starch-iodine sprays or a claving solutions A and B at 126 C. for minutes and combination thereof. mixing them.
• BYF 300 (crude yeast extract) 0.55 g benzylpenicillin 0.67
• the broth was then incubated on a rotary shaker at dud 28C. After 24 hours, the inoculum medium (10 ml.) Compound was added to the fermentation broth (2 l.) contained in acid 019 -aminopenicillin tetrazole 0.32 a 4 l. fermenter.
• the fermentation broth is prepared by benzylpenicillin 0.71 mixing the components below and autoclaving for 60 benzylpenicmi" pheiioxymethylpenicillin 0.68
• the pH of the suspension was raised from 3.4 to 8.0 with IN
• the contents of the fermenter were then incubated 9 hydroxlde' A immPblhzed rettgen penio cillm deacylase (30 g, 200 units/g against benzylpeniin a water bath at 28 C., aerated at a rate of one volume I o 7 lume er minute and Stirred at 1750 RPM th cillin) was added and the mixture maintained at 37 perhvo p and a pH of 8.0 by the periodic addition of 1N sodium a t ree bladed impeller.
• Cells are generally harvested hydroxide The consumption of Sodium hydroxide by Cenmfugatlon after 24 hours stopped after 2 hours and the mixture was filtered.
• the Activity Screening immobilized enzyme cake was washed with water.
• the filtrate and wash were combined and acidified to a pH Intact, whole cells harvested as described above were contacted with a 0.25% by weight solution of benzylpenicillin tetrazole in water.
• the cell concentration ranged from about 1 to about 50 g/l.
• the pH was adjusted to and maintained at 8.0 with 1N sodium of 2.0 with 5N hydrochloric acid.
• the acidified solution was washed three times with ethyl acetate (150 ml each).
• phenylacetic acid (1.09 g, yield) mp 7173C.
• the nuclear magnetic resonance and infrared spectrum were identical to those of an authentic sample of phenylacetic acid.
• the aqueous base was adjusted to a pH of 4.5 with N sodium hydroxide and concentrated in vacuo at a temperature below 40C. to a final volume of 50 ml at which point a white solid precipitated.
• the mixture was cooled in an ice bath to 5C. and filtered.
• the solids were washed with ice water and acetone, and air dried to afford the title compound (1.54 g, 64% yield).
• the nuclear magnetic resonance and infrared spectra were identical to an authentic sample.
• immobilized enzymes isolated from the other microorganisms of interest in the instant invention, are used to deacylate benzyland phenoxymethylpenicillin tetrazoles.
• benzylpenicillin tetrazole (3.33 g, 9.29 m. moles) was contacted with a wet, immobilized enzyme isolated from P. rettgeri ATCC 31052. After 2 hours, the mixture was filtered and the title compound recovered (1.63 g, 73% yield). Spectrophotometric data were identical to those of an authentic sample.
• the mixture was centrifuged and the supemate adjusted to a pH of 2.0 with hydrochloric acid and extracted with ethyl acetate.
• the resulting aqueous solution was adjusted to a pH of 4.5 with sodium hydroxide and concentrated in vacuo to precipitate the title compound which was filtered and air dried to yield the title compound 1.74 g, 60% yield).
• the nuclear magnetic resonance spectrum was identical with that of an authentic sample of 6-aminopenicillin tetrazole.
• the mixture is then centrifuged and the supemate adjusted to a pH of 2.0 with hydrochloric acid and extracted with ethyl acetate.
• the resulting aqueous solution is adjusted to pH 4.5 with sodium hydroxide and concentrated in vacuo to precipitate the title compound which is filtered and dried in a vacuum.
• reaction mixture is extracted with three successive ml. portions of chloroform.
• the combined chloroform extracts are dried over anhydrous sulfated and evaporated in vacuo to give a yellow solid.
• This yellow solid is recrystallized two times from chloroform/ethyl acetate solution to give white, crystalline 6-amino-2,2-dimethyl-3-( 1-[4- methoxybenzyl1tetrazol-5 -yl)penam.
• R is the acyl moiety of phenylacetic or phenoxyacetic acid
• R is selected from the group consisting of hydrogen, trialkylsilyl having from one to four carbon atoms in each of said alkyl groups, alkanoyloxymethyl having from three to eight carbon atoms, 1- (alkanoyloxy )ethyl having from four to nine carbon atoms, phthalidyl and a tetrazolylpenam nitrogen protecting group;
• R is selected from the group consisting of hydrogen, trialkylsilyl having from one to four carbon atoms in each of the said alkyl groups, alkanoyloxymethyl having from three to eight carbon atoms, l-(alkanoyloxy)ethyl having from four to nine carbon atoms and phthalidyl;
• said process comprising dispersing said penicillin tetrazole in water at a concentration of at least about 0.1% by weight, adjusting the pH of resulting aqueous dispersion to a value between about 5 and 9, contacting the penicillin tetrazole with a deacylase, and maintaining the pH of the solution between about 5 and 9 and the temperature between about 5 and 50C. until the reaction is substantially complete, said deacylase being a deacylase for benzylpenicillin when R is the acyl moiety of phenylacetic acid and said deacylase being a deacylase for phenoxymethylpenicillin when R is the acyl moiety of phenoxyacetic acid.
• said diacylase is introduced into the reaction medium be a member of the group consisting of bacteria, whole bacterial cells immobilized on a matrix, extracts isolated from said bacteria, fungi, whole fungi cells immobilized on a matrix, extracts isolated from said fungi, and enzymes from said bacteria or fungi and said enzymes immobilized on a matrix.

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• 1974
  • 1974-12-04 US US529480A patent/US3905868A/en not_active Expired - Lifetime
• 1975
  • 1975-06-17 IE IE1356/75A patent/IE41561B1/en unknown
  • 1975-07-28 NL NL7508961A patent/NL7508961A/xx not_active Application Discontinuation
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