|
(11) | EP 0 000 932 B1 |
| (12) | EUROPEAN PATENT SPECIFICATION |
|
|
| (54) |
Production of N-acyl-thienamycins Herstellung von N-acyl-Thienamycinen Production de N-acyl-thiénamycines |
|
|
|||||||||||||||||||||||
| Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). |
DESCRIPTION
[0002] More particularly, the present invention provides a process for the preparation of N-acyl-thienamycins which process comprises bringing thienamycin and acyl compounds into contact with penicillin amidohydrolases (E.C. 3.5.1.11) capable of producing the N-acylated thienamycin.
[0003] By penicillin amidohydrolases capable of producing the N-acyl-thienamycins is meant the whole cells of the microorganism that produces an enzyme or mixture of enzyme extracted from these organisms, that causes the acyl compound to react with thienamycin to give the N-acylated thienamycins.
[0005] According to the present invention, N-acyl thienamycins having the following structure:
wherein R is an acyl group, are produced by reacting the compound thienamycin, having the following structure:
with an acyl compound in an aqueous medium in the presence of a penicillin amidohydrolase (E.C.3.5.1.11; E.C. = International Commission of Enzyme Nomenclature) capable of producing the N-acylated thienamycin.
[0006] Examples of acyl compounds are carboxylic acids, amides thereof, peptides thereof or lower (C1-e)alkyl esters thereof.
DETAILED DESCRIPTION OF THE INVENTION:
[0008] The process of preparing the N-acyl-thienamycins is by reacting the appropriate acyl compound, either as the carboxylic acid, an amide thereof, a peptide thereof or a lower alkyl ester thereof with thienamycin either in fermentation broth or an intermediate state of purity. Since the resulting derivatives can be more susceptible to isolation techniques employing organic solvents, they permit recovery of the thienamycin nucleus with higher efficiency from fermentation broths and concentrates. Once the derivatized thienamycin is recovered from the broth or solution, the acyl group can be removed in order to regenerate the thienamycin.
[0009] In the generic representation of the compounds of the present invention, (I above), the acyl radical R, can be derived from a saturated or unsaturated, substituted or unsubstituted aliphatic with greater than 5 carbon atoms, aromatic or arylaliphatic carboxylic acid or carbothioic acid. One group of acyl radicals can be represented by the general formula.
wherein X is 0 or S and R' represents a straight or branched chain alkyl group containing from 5-10 carbon atoms, aryl, aryloxy, typically 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 loweralkyl or aryl such as phenyl), alkyl or alkoxy groups having 1-6 carbon atoms, halo, such as CI, Br, F and I, cyano, carboxy, nitro, 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 1-6 carbon atoms, amidino, guanidino, N-substituted guanidino and guanidino lower alkyl. Representative examples of such acyl groups that might be mentioned are those wherein R' is benzyl, aminobenzyl, phenoxymethylene, p-hydroxybenzyl, n-amyl, n-heptyl, 3- or 4-nitrobenzyl, phenylethyl, β,β-diphenylethyl, methyldiphenylmethyl, triphenylmethyl, 2-methoxyphenyl, 2,6-dimethoxyphenyl, 2,4,6-trimethoxyphenyl, D - 4 - N - benzoylamino - 4 - carboxy - n - butyl, p - aminobenzyl, o-aminobenzyl, m-aminobenzyl, p-dimethylaminobenzyl, 2 - ethoxy - 1 - napthyl, 4-guanidinomethylphenyl, 4-guanidinomethylbenzyl, 4-guanidinobenzyl, 4-guanidinophenyl, o-sulfobenzyl, p-carboxymethylbenzyl, p-carbamoylmethylbenzyl, m-fluorobenzyl, m-bromobenzyl, p-chlorobenzyl, p-methoxybenzyl, 1-naphthylmethyl, 2-phenylvinyl, 2-phenylethynyl, phenyl, o-methoxyphenyl, o-chlorophenyl, o-phenylphenyl, or p-aminomethylbenzyl.
[0010] The preferred compounds that can be utilized in this invention that fit the above acyl compound description are compounds of phenyl acetic acid, p-hydroxyphenyl acetic acid, p-aminophenyl acetic acid, 3-hexenoic acid and the N-glycyl and methyl esters of the above preferred carboxylic acids.
[0011] Some examples of these preferred compounds are methylphenyl acetate, methyl p-hydroxyphenyl acetate, methyl p-aminophenyl acetate, methyl 3-hexenoate, N-glycylphenyl acetate, N-glycyl p-hydroxyphenyl acetate, N-glycyl p-aminophenyl acetate and N-glycyl 3-hexenoate. Also included within this preferred group are the amide, N-glycyl or methyl ester of phenylglycine. Some examples of these preferred compounds are N-glycyl phenylglycinate and methyl phenylglycinate.
[0012] It is known in the art that penicillin amido-hydrolases used on an industrial scale to catalyze the hydrolytic removal of the side chain of penicillin to give the nucleus 6-aminopenicillanic acid (6-APA) can also be used to catalyze the reverse reaction. In the reverse (or synthetic) reaction, 6-APA plus acyl compound in the presence of penicillin amidohydrolase yield penicillin analogs. The synthetic reaction is generally promoted by the use of acyl compounds in the form of their lower alkyl (1-4 carbon atoms) esters present at high concentration and in excess of the 6-APA nucleus. That penicillin amidohydrolase is also capable of generating N-acylated thienamycins from thienamycin and appropriate acyl compounds is surprising. The process of this invention may be conducted by reacting the starting material of the general formula II along with the appropriate acyl compound as the carboylic acid, an amide thereof, a peptide thereof or a lower alkyl ester thereof, in the presence of the enzyme from an extract of a cultured broth, the filtrate or fermentation product of the Escherichia coli culture or a powder of the enzyme in an aqueous solution. Alternatively, the enzyme may be immobilized by adsorption or chemical reaction to an insoluble supporting structure such as glass, cellulose or agarose, and used to generate N-acylated thienamycins either by contacting it (in the presence of appropriate acyl compounds as the carboxylic acid, an amide thereof, a peptide thereof or a lower alkyl ester thereof) in suspension or by percolation through a bed of immobilized enzyme preparation.
[0013] The enzyme is capable of producing N-acyl thienamycins from thienamycin present or produced in fermentation broths as well as from isolated thienamycin.
[0014] More particularly, the acylation of thienamycin takes place in the presence of an enzyme of the microorganism of the genus Escherichia coli which is capable of producing the N-acylated thienamycins.
[0015] 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. For example, glucose, sucrose, glycerol, starch, and oils used for cultivation as a carbon source and peptone, buillion, corn steep liquor, yeast extract, meat extract, fish meal, defatted soybean, and wheat embryo as a nitrogen source may be employed. If required, other additives may be employed in combination with the above. It is an advantage but not a necessity to include phenylacetic acid or its salts or derivatives in fermentation media.
[0016] As a cultivation method, Escherichia coli is usually shaken or agitated under aeration. Cultivation temperature may range from 23-27°C. Cultivation period is usually 20-28 hours.
[0017] 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 precipitated with appropriate solvents, salted out or dialyzed or otherwise purified. It may be used free in solution or immobilized on an appropriate surface.
[0018] A method utilized in the present invention is that of utilizing the whole cell amidohydrolase preparation. By this method, after cultivation, the culture is centrifuged to obtain the whole cells for subsequent reaction.
[0019] The following are given for illustration purposes only and are not to be construed as limiting the scope of the present invention in any way.
Example 1
[0020] Fifty ml. 2.5% yeast extract containing 0.08% neutralized (with NaOH) phenylacetic acid in a 25- ml. Erlenmeyer flask is inoculated with tube of lyophilized culture of Escherichia coli N.C.I.B. 8743. This flask is shaken at 25°C at 240 rpm for 24 hours. A 35-ml. portion is centrifuged at 7500 rpm for 15 minutes. The supernatant is discarded and the pellet resuspended in 18 ml. distilled water. The solution is centrifuged at 7500 rpm for 15 minutes. The supernatant is discarded and the pellet take up in 1 ml. of 0.05M potassium phosphate buffer, pH 7.4 to yield a whole cell amidohydrolase preparation which is then stored at 0°C.
[0021] The following reaction mixtures are incubated 18 hours at 23°C.
(1) A 1 0-pl. portion of whole cell amido hydrolase preparation plus 30 fLl. of an approximately 1 mg./ml. solution of thienamycin plus 1 ,ul. neutralized 0.04M phenyl acetic acid.
(2) A 10-µl. portion of whole cell amidohydrolase plus 30 µl. 50mM potassium phosphate buffer, pH 7.4 plus 1 ,ul. neutralized 0.04M phenyl acetic acid.
(3) A 10-,ul. portion of 0.05M potassium phosphate buffer, pH 7.4 plus 30 µl. of an approximately 1 mg./ml. solution of thienamycin.
[0022] After the 18 hours of incubation, 5-µl. aliquots of the reaction mixtures are applied to a cellulose- coated TLC plate, which is developed in EtOH:H20. 70:30. After air drying, the TLC plate is placed on a Staphylococcus aureus ATCC 6538P assay plate for 5 minutes.
[0023] 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 plus 0.2% yeast extract 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./l. Difco yeast extract at 47°C. to 48°C., to make a composition containing 33.2 ml. or the suspension per liter of agar. Forty mi. 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 day maximum).
1. A process for the preparation of N-acyl thienamycins having the following structure:
wherein R is an acyl radical characterised by bringing the compound thienamycin having the following structure:
into contact with an acyl compound in an aqueous medium with a penicillin amidohydrolase (E.C. 3.5.1.11) capable of producing the N-acylated thienamycin.
wherein R is an acyl radical characterised by bringing the compound thienamycin having the following structure:
into contact with an acyl compound in an aqueous medium with a penicillin amidohydrolase (E.C. 3.5.1.11) capable of producing the N-acylated thienamycin.
2. A process according to Claim 1 wherein the penicillin amidohydrolase from Escherichia
coli, N.C.I.B. 8743 is used.
3. A process according to Claim 1 wherein the acyl radical is represented by the formula:
wherein X is 0 or S and R' represents a straight or branched chain alkyl group containing from 5-10 carbons, or an aryl, or aryloxy group containing 6-10 carbon atoms.
wherein X is 0 or S and R' represents a straight or branched chain alkyl group containing from 5-10 carbons, or an aryl, or aryloxy group containing 6-10 carbon atoms.
4. A process according to Claim 1 wherein the acyl radical is represented by the formula:
wherein X is 0 or S and R' represents a straight or branched chain alkyl group containing from 5-10 carbons, or an aryl, or aryloxy group containing 6-10 carbon atoms and wherein said groups are substituted with radicals selected from hydroxy, mercapto, alkyl, alkoxy, halo, amino, nitro, and carboxy.
wherein X is 0 or S and R' represents a straight or branched chain alkyl group containing from 5-10 carbons, or an aryl, or aryloxy group containing 6-10 carbon atoms and wherein said groups are substituted with radicals selected from hydroxy, mercapto, alkyl, alkoxy, halo, amino, nitro, and carboxy.
5. A process according to Claim 1 wherein the acyl compound is selected from saturated
or unsaturated aliphatic with greater than 5 carbons, aromatic or arylaliphatic carboxylic
acids or carbothioic acids, amides thereof, peptides thereof or lower alkyl esters
thereof.
6. A process according to Claim 1 wherein the acyl compound is selected from aryl
carboxylic acids and lower alkyl esters thereof.
7. A process according to Claim 5 wherein the acyl compound is selected from phenyl
acetic acid, p-hydroxyphenyl acetic acid, p-aminophenyl acetic acid, 3-hexenoic acid,
methylphenyl acetate, methyl p-hydroxyphenyl acetate, methyl p-aminophenyl acetate,
methyl 3-hexenoate, N-glycylphenyl acetate, N-glycyl p-hydroxyphenyl acetate, N-glycyl
p-aminophenyl acetate, N-glycyl 3-hexenoate, N-glycyl phenylglycinate and methyl phenylglycinate.
1. Verfahren zur Herstellung von N-Acylthienamycinen der Strukturformel
in der R einen Acylrest bedeutet, dadurch gekennzeichnet, dass man Thienamycin der Strukturformel
in Kontakt mit einer Acylverbindung in einem wässrigen mit einer Penicillin-amidohydrolase (E.C. 3.5.1.11), die zur Bildung von N-acyliertem Thienamycin in der Lage ist, bringt.
in der R einen Acylrest bedeutet, dadurch gekennzeichnet, dass man Thienamycin der Strukturformel
in Kontakt mit einer Acylverbindung in einem wässrigen mit einer Penicillin-amidohydrolase (E.C. 3.5.1.11), die zur Bildung von N-acyliertem Thienamycin in der Lage ist, bringt.
2. Verfahren nach Anspruch 1, wobei Penicillin-amidohydrolase aus Escherichia coli,
N.C.I.B. 8743 verwendet wird.
3. Verfahren nach Anspruch 1, wobei der Acylrest die Formel aufweist
in der X 0 oder S bedeutet und R' eine geradkettige oder verzweigte Alkylgruppe mit 5 bis 10 Kohlenstoffatomen oder eine Aryl- oder Aryloxygruppe mit 6 bis 10 Kohlenstoffatomen bedeutet.
in der X 0 oder S bedeutet und R' eine geradkettige oder verzweigte Alkylgruppe mit 5 bis 10 Kohlenstoffatomen oder eine Aryl- oder Aryloxygruppe mit 6 bis 10 Kohlenstoffatomen bedeutet.
4. Verfahren nach Anspruch 1, wobei der Acylrest die Formel aufweist
in der X O oder S bedeutet und R' eine geradkettige oder verzweigte Alkylgruppe mit 5 bis 10 Kohlenstoffatomen oder eine Aryl- oder Aryloxygruppe mit 6 bis 10 Kohlenstoffatomen bedeutet und wobei diese Gruppen durch Hydroxy-, Mercapto-, Alkyl-, Alkoxy-, Halogen-, Amino-, Nitro- und/oder Carboxyreste substituiert sind.
in der X O oder S bedeutet und R' eine geradkettige oder verzweigte Alkylgruppe mit 5 bis 10 Kohlenstoffatomen oder eine Aryl- oder Aryloxygruppe mit 6 bis 10 Kohlenstoffatomen bedeutet und wobei diese Gruppen durch Hydroxy-, Mercapto-, Alkyl-, Alkoxy-, Halogen-, Amino-, Nitro- und/oder Carboxyreste substituiert sind.
5. Verfahren nach Anspruch 1, wobei es sich bei der Acylverbindung um gesättigte oder
ungesättigte aliphatische (mit mehr als 5 Kohlenstoffatomen), aromatische oder arylaliphatische
Carbonsäuren oder Thiocarbonsäuren oder um deren Amide, Peptide oder niedere Alkylester
handelt.
6. Verfahren nach Anspruch 1, wobei es sich bei der Acylverbindung um Arylcarbonsäuren
oder niedere Alkylester davon handelt.
7. Verfahren nach Anspruch 5, wobei es sich bei der Acylverbindung um Phenylessigsäure,
p-Hvdroxvphenylessigsäure, p-Aminophenylessigsäure, 3-Hexensäure, Methylphenylacetat, Methyl-p-hydroxyphenylacetat,
Methyl-p-aminophenylacetat, Methyl - 3 - hexenoat, N-Glycylphenylacetat, N - Glycyl
- p - hydroxyphenylacetat, N - Glycyl - p - aminophenylacetat, N - Glycyl - 3 - hexenoat,
N - Glycyl - phenylglycinat oder Methyl-phenylglycinat handelt.
1. Un procédé pour la préparation de N-acylthiénamycines ayant la structure suivante:
dans laquelle R est un radical acyle, caractérisé en ce qu'on met en contact la thiénamycine ayant la structure suivante:
avec un composé acylé dans un milieu aqueux en présence d'une pénicilline amidohydrolase (E.C.3.5.1.11) capable de produire la thiénamycine N-acylée.
dans laquelle R est un radical acyle, caractérisé en ce qu'on met en contact la thiénamycine ayant la structure suivante:
avec un composé acylé dans un milieu aqueux en présence d'une pénicilline amidohydrolase (E.C.3.5.1.11) capable de produire la thiénamycine N-acylée.
2. Un procédé selon la revendication 1, dans lequel on utilise la pénicilline amidohydrolase
dérivée d'Escherichia coli N.C.I.B. 8743.
3. Un procédé selon la revendication 1, dans lequel le radical acyle est représenté
par la formule:
dans laquelle X est O ou S et R' représente un groupe alcoyle à chaîne droite ou ramifiée contenant de 5 à 10 atomes de carbone, un groupe aryle ou un groupe aryloxy contenant de 6 à 10 atomes de carbone.
dans laquelle X est O ou S et R' représente un groupe alcoyle à chaîne droite ou ramifiée contenant de 5 à 10 atomes de carbone, un groupe aryle ou un groupe aryloxy contenant de 6 à 10 atomes de carbone.
4. Un procédé selon la revendication 1, dans lequel le radical acyle est représenté
par la formule:
dans laquelle X est O ou S et R' représente un groupe alcoyle à chaîne droite ou ramifiée contenant de 5 à 10 atomes de carbone, un groupe aryle ou un groupe aryloxy contenant de 6 à 10 atomes de carbone et ces groupes sont substitués par des radicaux choisis parmi les radicaux hydroxy, mercapto, alcoyle, alcoxy, halogéno, amino, nitro et carboxy.
dans laquelle X est O ou S et R' représente un groupe alcoyle à chaîne droite ou ramifiée contenant de 5 à 10 atomes de carbone, un groupe aryle ou un groupe aryloxy contenant de 6 à 10 atomes de carbone et ces groupes sont substitués par des radicaux choisis parmi les radicaux hydroxy, mercapto, alcoyle, alcoxy, halogéno, amino, nitro et carboxy.
5. Un procédé selon la revendication 1, dans lequel le composé acyle est choisi parmi
les acides carboxyliques aliphatiques saturés ou non ayant plus de 5 atomes de carbone,
aromatiques ou aryl- aliphatiques ou les acides carbothioîques, leurs amides, leurs
peptides ou leurs esters d'alcoyle inférieur.
6. Un procédé selon la revendication 1, dans lequel le composé acylé est choisi parmi
les acides arylcarboxyliques et leurs esters d'alcoyle inférieur.
7. Un procédé selon la revendication 1, caractérisé en ce que le composé acylé est
choisi parmi l'acide phénylacétique, l'acide p-hydroxyphénylacétique, l'acide p-aminophénylacétique,
l'acide 3- hexénoïque, le phénylacétate de méthyle, le p-hydroxyphénylacétate de méthyl,
le p-aminophénylacétate de méthyle, le 3-hexénoate de méthyle, le phénylacétate de
N-glycyle, le p-hydroxyphénylacétate de N-glycyle, le p-aminophénylacétate de N-glycyle,
le 3-hexénoate de N-glycyle, le phénylglycinate de N-glycyle et le phénylglycinate
de méthyle.