EP0000931B1 - Enzymatic cleavage of n-acyl-thienamycins - Google Patents

Enzymatic cleavage of n-acyl-thienamycins Download PDF

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Publication number
EP0000931B1
EP0000931B1 EP78100699A EP78100699A EP0000931B1 EP 0000931 B1 EP0000931 B1 EP 0000931B1 EP 78100699 A EP78100699 A EP 78100699A EP 78100699 A EP78100699 A EP 78100699A EP 0000931 B1 EP0000931 B1 EP 0000931B1
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EP
European Patent Office
Prior art keywords
thienamycin
acyl
amidohydrolase
carbon atoms
enzyme
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
Application number
EP78100699A
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German (de)
English (en)
French (fr)
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EP0000931A1 (en
Inventor
Jean Sawyer Kahan
Frederick Marvin Kahan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck and Co Inc
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Merck and Co Inc
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Publication date
Application filed by Merck and Co Inc filed Critical Merck and Co Inc
Publication of EP0000931A1 publication Critical patent/EP0000931A1/en
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D477/00—Heterocyclic compounds containing 1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. carbapenicillins, thienamycins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulphur-containing hetero ring
    • C07D477/10—Heterocyclic compounds containing 1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. carbapenicillins, thienamycins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulphur-containing hetero ring with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 4, and with a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2
    • C07D477/12—Heterocyclic compounds containing 1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. carbapenicillins, thienamycins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulphur-containing hetero ring with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 4, and with a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2 with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, attached in position 6
    • C07D477/16—Heterocyclic compounds containing 1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. carbapenicillins, thienamycins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulphur-containing hetero ring with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 4, and with a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2 with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, attached in position 6 with hetero atoms or carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 3
    • C07D477/20—Sulfur atoms

Definitions

  • the enzyme, penicillin amidohydrolase of bacterial or fungal origin is used on an industrial scale to catalyze the hydrolytic removal of the side chain of penicillin to give the nucleus 6-amino penicillanic acid (6-APA) (see US-A-3,260,653).
  • 6-APA 6-amino penicillanic acid
  • This nucleus is the starting material for the synthesis of broad spectrum penicillins (semi-synthetic) which are prepared by acylation of the amino group.
  • a new antibiotic is thienamycin, (U.S. Patent No. 3,950,357).
  • a process for the preparation of thienamycin is known from DE-A-2652678 wherein one brings into intimate contact acetylamidothienamycin and an amidohydrolase, that is capable of hydrolysing the N-acetyl group.
  • N-acyl derivatives of thienamycin are unexpectedly cleaved by penicillin amidohydrolase.
  • the present invention provides a new process for conversion of N-acyl-thienamycins to thienamycin.
  • N-acyl-thienamycin having the following structure: wherein R' and R 2 are independently selected from the group consisting of hydrogen (R' and R 2 are not both hydrogen) and an acyl group is contacted by in an aqueous medium with penicillin amidohydrolases which are capable of removing the N-acyl moiety in order to form thienamycin.
  • Thienamycin has the structure: Thienamycin is a valuable antibiotic which is active against both gram-positive and gram-negative bacteria.
  • the preferred compounds of this invention are those wherein R' is hydrogen and R 2 is acyl.
  • the N-acyl derivatives may be prepared by any of the techniques well known in the art if the thienamycin has been isolated from the fermentation broth or solution. If the thienamycin has not been isolated from the fermentation broth, then the method of preparing the N-acyl-thienamycin is by reacting the appropriate acyl compound while the thienamycin is still in the broth or solution. Alternatively, the acyl group can be incorporated biosynthetically following supplementation of the fermentation broth with the parent acid or a derivative thereof. These derivatives, by virtue of their increased solubility in organic solvents and additional physical properties, provide alternative and more efficient routes for recovery of the thienamycin nucleus. Once the derivatized thienamycin is recovered from the broth or solution, it can be treated by the methods described in this invention in order to regenerate the thienamycin.
  • acyl radical is represented by the general formula: wherein X is O or S and R" represents a straight or branched chain alkyl or alkoxymethylene group containing from 5-10 carbon atoms; alkyl thio, aryl thio from 6-10 carbon atoms; aryloxymethylene, containing 6-10 carbon atoms.
  • Such above-listed groups can be unsubstituted or can be substituted by radicals such as OH, SH, SR (R is alkyl having 1 to 6 carbon atoms or aryl such as phenyl), alkyl or alkoxy groups having 1 to 6 carbon atoms, halo, such as Cl, Br, F and I, cyano, carboxy, sulfamino, carbamoyl, sulfonyl, azido, amino, substituted amino such as alkylamino including quaternary ammonium wherein the alkyl group comprises 1-6 carbon atoms, haloalkyl such as trifluoromethyl, carboxyalkyl, carbamoylalkyl, N-substituted carbamoylalkyl, wherein the alkyl moiety of the foregoing four radicals comprises 1 to 6 carbon atoms, amidino, guanidino, N-substituted guanidino, or guanidino lower
  • the preferred compounds that can be utilized in this invention that fit the above acyl radical description are phenylacetyl, p-hydroxyphenylacetyl, phenylglycyl, 2-thienylacetyl, phenoxyacetyl, n-propoxyacetyl and iso-butoxyacetyl.
  • penicillin amidohydrolases to convert penicillins into 6-aminopenicillanic acid (6-APA) is known in the art.
  • penicillin amidohydrolases to remove the N-acyl side chains of N-acylated theinamycin is surprising.
  • the process of this invention may be conducted by reacting the starting material of the general formula I with the enzyme extract from a cultured broth, the filtrate or fermentation product of the Escherichia coli culture or a powder of the enzyme in an aqueous solution.
  • the enzyme may be immobilized by adsorptiuon or chemical reaction to an insoluble supporting structure such as glass, cellulose or agarose, and used to hydrolyse the N-acyiated thienamycin either by contacting it in suspension, or by the percolation of the acylated material through a bed of the immobilized enyzme preparation.
  • an insoluble supporting structure such as glass, cellulose or agarose
  • the enzyme is capable of removing N-acyl moieties which were present or produced in fermentation broths as well as those'N-acyl groups introduced during isolation of the antibiotic or made by chemical synthesis techniques.
  • N-acylated thienamycin takes place in the presence of an enzyme of the microorganism of the genus Escherichia coli which is able to remove the acyl moiety to provide the antibiotic thienamycin.
  • the amidohydrolase enzyme for the production of the amidohydrolase enzyme by cultivation of the above-mentioned microorganism, there may be used various culture media commonly employed for the cultivation of a microorganism.
  • various culture media commonly employed for the cultivation of a microorganism.
  • glucose, sucrose, glycerol, starch, oils used for cultivation as a carbon source and peptone, buillion, corn steep liquor, yeast extract, meat extract, fish meal, defatted soybean, wheat embryo as a nitrogen source may be employed.
  • other additives may be employed in combination with the above. It is an advantage but not a necessity to include phenylaceteic acid or its salts or derivatives in fermentation media.
  • Escherichia coli is usually shaken or agitated under aeration.
  • Cultivation temperature may range from 23-27°C.
  • Cultivation period is usually 20-28 hours.
  • the amidohydrolase contained in the cultured broth or its extract may be utilized in the present process without any further purification.
  • the amidohydrolase enzyme may be precipiated with appropriate solvents, salted out or dialyzed or otherwise purified. It may be used free in solution or immobilized on an appropriate surface.
  • a method utilized in the present invention is that of utlizing the whole cell amidohydrolase preparation.
  • the culture is centrifuged to obtain the whole cells for subsequent reaction with the derivatized theinamycin.
  • reaction mixtures are incubated 18 hours at 23°C.
  • the assay plates are prepared as follows: An overnight growth of the assay organism, Staphylococcus aureus ATCC 6538P, in nutrient broth plus 0.2% yeast extract is diluted with nutrient broth to a suspension having 60% transmittance at a wavelength of 660 nm. This suspension is added to Difco nutrient agar supplemented with 2.0 g./I. Difco yeast extract at 47°C. to 48°C., to make a composition containing 33.2 ml. of the suspension per liter of agar. Forty ml. of this suspension is poured into 22.5 cm. x 22.5 cm. petri plates, and these plates are chilled and held at 4°C. until used (5 days maximum).
  • the TLC plate is removed and the assay plate incubated overnight at 37°C.
  • the additional bioactive spots present at R f 0.39 and R f 0.45 in the enzyme-treated reaction mixtures containing phenylacetyl thienamycin and N-(O-formyl)-1-mandeloyl thienamycin are due to thienamycin produced by amidohydrolase enzyme reaction.
  • a control containing buffer and enzyme alone produces no bioactive spots.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
EP78100699A 1977-08-19 1978-08-18 Enzymatic cleavage of n-acyl-thienamycins Expired EP0000931B1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US825883 1977-08-19
US05/825,883 US4162193A (en) 1977-08-19 1977-08-19 Enzymatic cleavage of N-acyl-thienamycins

Publications (2)

Publication Number Publication Date
EP0000931A1 EP0000931A1 (en) 1979-03-07
EP0000931B1 true EP0000931B1 (en) 1982-04-14

Family

ID=25245138

Family Applications (1)

Application Number Title Priority Date Filing Date
EP78100699A Expired EP0000931B1 (en) 1977-08-19 1978-08-18 Enzymatic cleavage of n-acyl-thienamycins

Country Status (9)

Country Link
US (1) US4162193A (enrdf_load_stackoverflow)
EP (1) EP0000931B1 (enrdf_load_stackoverflow)
JP (1) JPS5446890A (enrdf_load_stackoverflow)
DE (1) DE2861737D1 (enrdf_load_stackoverflow)
DK (1) DK365478A (enrdf_load_stackoverflow)
ES (1) ES472701A1 (enrdf_load_stackoverflow)
IE (1) IE47315B1 (enrdf_load_stackoverflow)
IT (1) IT1106626B (enrdf_load_stackoverflow)
PT (1) PT68428A (enrdf_load_stackoverflow)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02135843U (enrdf_load_stackoverflow) * 1988-11-08 1990-11-13

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE1111778B (de) * 1959-04-18 1961-07-27 Bayer Ag Verfahren zur Herstellung von 6-Aminopenicillansaeure
GB939708A (en) 1961-06-01 1963-10-16 Beecham Res Lab Production of ª -hydroxybenzylpenicillin
CH475284A (de) * 1963-11-19 1969-07-15 Ciba Geigy Verfahren zur Herstellung von Desacetyl-7-amino-cephalosporansäure
JPS5417030B2 (enrdf_load_stackoverflow) * 1972-12-06 1979-06-27
GB1474519A (enrdf_load_stackoverflow) * 1973-05-14 1977-05-25
JPS5713280B2 (enrdf_load_stackoverflow) * 1974-03-11 1982-03-16
US3950357A (en) * 1974-11-25 1976-04-13 Merck & Co., Inc. Antibiotics
DK497476A (da) * 1975-11-24 1977-05-25 Merck & Co Inc Fremgangsmade til fremstilling af et antibiotisk stof
NL7800958A (nl) * 1977-02-11 1978-08-15 Merck & Co Inc Antibioticum desacetyl-dihydro 890 a9, werkwijze ter bereiding daarvan en farmaceutisch preparaat dat dit antibioticum bevat.

Also Published As

Publication number Publication date
IT1106626B (it) 1985-11-11
DK365478A (da) 1979-02-20
DE2861737D1 (en) 1982-05-27
EP0000931A1 (en) 1979-03-07
ES472701A1 (es) 1979-02-16
JPS5446890A (en) 1979-04-13
US4162193A (en) 1979-07-24
IE47315B1 (en) 1984-02-22
IT7850747A0 (it) 1978-08-16
PT68428A (en) 1978-09-01
IE781674L (en) 1979-02-19
JPS6210639B2 (enrdf_load_stackoverflow) 1987-03-07

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