DESCRIPTION OF THE INVENTION:
[0001] This invention relates to a process for producing N-acyl-thienamycins.
[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 certain penicillin amidohydrolases wherein N-acyl- thienamycin derivatives
are produced.
[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 enzymes
extracted from these organisms, that causes the acyl compound to react with thienamycin
to give the N-acylated thienamycins.
[0004] Thienamycin is a new antibiotic (U.S. Patent No. 3,950,357). The teachings of U.S.
Patent No. 3,950,357 are incorporated herein by reference.
[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, either the carboxylic acid, an amide thereof, a peptide thereof
or a lower alkyl ester thereof, in the presence of penicillin amidohydrolases of the
category designated E.C. 3.5.1.11 by the International Commission of Enzyme Nomenclature.
DETAILED DESCRIPTION OF THE INVENTION:
[0006] The method of preparing the N-acyl-thienamycins is by reacting the appropriate acyl
compound, either as tne carboxylic acid, an amide therof, a peptide therof 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.
[0007] 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 O 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 Cl, 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, guanidino lower alkyl and the like. 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,
phenethyl, B,B-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-l-napthyl,
4-guanidinomethylphenyl, 4- guanidinomethylbenzyl, 4-guanidinobenzyl, 4- guanidinophenyl,
2,6-dimethoxy-4-guanidino, o-sulfobenzyl, p-carboxymethylbenzyl, p-carbamoyl- methylbenzyl,
m-fluorobenzyl, m-bromobenzyl, p-chlorobenzyl, p-methoxybenzyl, 1-naphthylmethyl,
2-phenylvinyl, 2-phenylethynyl, phenyl, o-methoxyphenyl, o-chlorophenyl, o-phenylphenyl,
or p-amino- methylbenzyl.
[0008] The preferred compounds that can be utilized in this invention that fit the above
acyl compound description are 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.
[0009] 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.
[0010] It is known in the art that penicillin amidohydrolases 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 carboxylic 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.
[0011] The enzyme is capable of producing N-acyl thienamycins from thienamycin present or
produced in fermentation broths as well as from isolated thienamycin.
[0012] 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.
[0013] 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. More specifically, glucose, sucrose, glycerol, starch, oils used
for cultivation and the like as a carbon source and peptone, buillion, corn steep
liquor, yeast extract, meat extract, fish meal, defatted soybean, wheat embryo and
the like as a nitrogen source may be employed. If required, other additives may be
employed in combination with the above. It is an advantage but no a necessity to include
phenylacetic acid or its salts or derivatives in fermentation media.
[0014] As a cultivation method, Escherichia coli is usually shaken or agitated under aeration.
Cultivation temperature may range from about 23-27°C. Cultivation period is usually
20-28 hours.
[0015] 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.
[0016] 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.
[0017] 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
[0018] Fifty ml. 2.5% yeast extract containing 0.08% neutralized (with NaOH) phenylacetic
acid in a 250-ml. Erlenmeyer flask is inoculated with a tube of lyophilized culture
of MB-2929 (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 taken 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.
[0019] The following reaction mixtures are incubated 18 hours at 23°C.
(1) A 10-µl. portion of whole cell amidohydrolase preparation plus 30 µl. of an approximately
1 mg./ml. solution of thienamycin plus 1 µl. 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 µl. neutralized 0.04M phenyl acetic acid.
(3) A 10-µl. portion of 0.05M potassium phosphate buffer, pH 7.4 plus 30 µl. of an
approximately 1 mg./ml. solution of thienamycin.
[0020] 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:H
20, 70:30. After air drying, the TLC plate is placed on a Staphylococcus aureus ATCC
6538P assay plate for 5 minutes.
[0021] 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 mi. 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 day maximum).
[0022] The TLC plate is removed and the assay plate incubated overnight at 37°C.
[0023] The following bioactive spots are observed:
(1) Rf 0.39-0.45 and 0.8;
(2) no bioactive spots;
(3) Rf 0.39-0.45.
[0024] The bioactive spots at R
f 0.39-0.45 are due to thienamycin. The bioactive spot at R
f 0.8 is due to N-phenylacetyl thienamycin.
1. A method for the preparation of N-acyl thienamycins having the following structure:

wherein R is an acyl radical is produced by bringing the compound thienamycin having
the following structure:

into contact with an acyl compound in an aqueous medium with a penicillin amidohydrolase
capable of producing the N-acylated thienamycin.
2. A process according to Claim 1 wherein the penicillin amidohydrolase is designated
3.5.1.11 from Escherichia coli, N.C.I.B. 8743.
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 groups containing
from 5-10 carbons, aryl, aryloxy groups 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 groups containing
from 5-10 carbons, aryl, aryloxy groups containing 6-10 carbon atoms and wherein said
groups are substituted with radicals selected from the group consisting of hydroxy,
mercapto, alkyl, alkoxy, halo, amino, nitro and carboxy.
5. A process according to Claim 1 wherein the acyl compound is selected from the group
consisting of carboxylic acids, an amide thereof, a peptide thereof or a lower alkyl
ester thereof.
6. A process according to Claim 1 wherein the acyl compound is selected from the group
consisting of aryl carboxylic acids and lower alkyl esters thereof.
7. A process according to Claim 5 wherein the acyl compound is selected from the group
consisting of 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.
8. A process according to Claim 7 wherein the acyl compound is selected from the group
consisting of phenyl acetic acid, p-hydroxyphenyl acetic acid and p-aminophenyl acetic
acid.
9. A process according to Claim 7 wherein the acyl compound is phenyl acetic acid.