Process for the fermentative production of cephalosporin

Details Process for the fermentative production of cephalosporin Process for the fermentative production of cephalosporin

2000-11-16

2003-04-22

Berch, Mark L (Department: 1624)

Organic compounds -- part of the class 532-570 series

Organic compounds

Heterocyclic carbon compounds containing a hetero ring...

C540S301000, C540S205000

Reexamination Certificate

active

06552185

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a process for the preparation of cephalosporins and cephalosporin derivatives. More in particular, the present invention relates to the recovery of cephalosporins and derivatives thereof from complex mixtures of cephalosporins and other beta-lactam compounds. The invention is also concerned with recovery of deacylated cephalosporins from mixtures of beta-lactam compounds and side chains, such as hose obtainable by enzymatic side-chain removal.
BACKGROUND OF THE INVENTION
Semi-synthetic routes to prepare cephalosporins mostly start from fermentation products such as penicillin G, penicillin V and Cephalosporin C, which are converted to the corresponding &bgr;-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. The obtained &bgr;-lactam nuclei are subsequently converted to the desired antibiotic by coupling to a suitable side chain, as has been described in inter alia ED 0 339 751, JP-A-53005185 and CH-A-640 240. By making different combinations of side chains and &bgr;-lactam nuclei, a variety of penicillin and cephalosporin antibiotics may be obtained.
7-Amino desacetoxy cephalosporanic acid (7-ADCA) and 7-aminocephalosporonic acid (7-ACA) are known to be the most important intermediates for the production of antibiotics used in the pharmaceutical industry. 7-ADCA is for example obtained by chemical or enzymatic cleavage (deacylation) of phenylacetyldesacetoxy cephalosporanic acid yielding 7-amino desacetoxy cephalosporanic acid and phenyl acetic acid.
Phenylacetyldesacetoxy cephalosporanic acid is normally produced by chemical treatment of penicillin G sulfoxide, which is formed from penicillin G. In this production process a large amount of chemicals are required to ensure that the desired reaction take place. This is both expensive and places a heavy burden on waste management. Moreover, the total yield of the process, is not very high.
To overcome some of the drawbacks of the chemical process a fermentative process has been disclosed for the production of 7-ADCA, 7-amino desacetyl cephalosporanic acid (7-ADAC) and 7-ACA, involving fermentative production of N-substituted &bgr;-lactams, such as adipyl-7-ADCA, adipyl-7-ADAC or adipyl-7-ACA by a recombinant
Penicillium chzysogenum
strain capable of expressing a desacetoxycephalosporanic acid synthetase (DAOCS) also known as “expandase” from a transgene (EP 0 532 341, EP 0 540 210, WO 93/08287, WO 95/04148). The expandase takes care of the expansion of the 5-membered ring of certain N-acylated penicillanic acids, thereby yielding the corresponding N-acylated desacetoxycephalosporanic acids.
In order to yield the economically most important non-acylated cephalosporins, such as 7-ADCA, 7-ADAC and 7-ACA, the acyl groups are enzymatically removed with a suitable acylase.
Known processes for recovering chemically or enzymatically produced penicillanic and cephalosporanic acids are not effective for the recovery of the N-substituted &bgr;-lactam intermediates and deacylated amino-&bgr;-lactams. The main problem with the recovery of the fermentatively produced cephalosporin compounds mentioned above is the complexity of the broth, or culture filtrate. The broth usually comprises various penicillanic acids, such as alpha-aminoadipyl-6-penicillanic acid, alpha-hydroxyadipyl-6-penicillanic acid, 6-aminopenicillanic acid (6-APA), various cephalosporanic acids including alpha-aminoadipyl- and hydroxyadipyl-7-ADCA and a lot of proteinaceous material. Known recovery procedures do not give an acceptable quality of the cephalosporanic acid product in terms of purity. In deacylation this leads to problems in terms of reduced enzyme half-life, slower bioconversion rate and more expenses in the recovery after bioconversion and/or unacceptable contaminant levels. Moreover, after deacylation, such impurities prevent or at least hamper the recovery of the desired deacylated cephalosporin compound of the desired specifications.
SUMMARY OF THE INVENTION
The invention provides for a method for the recovery of a cephalosporanic acid compound of the general formula (I):
wherein
R
0
is hydrogen or C
1-3
alkoxy;
Y is CH
2
, oxygen, sulphur, or an oxidised form of sulphur;
R
1
is any of the groups selected from the group consisting of
hydrogen,
hydroxy,
halogen,
saturated or unsaturated, straight or branched alkyl (1-5 carbon atoms; optionally replaced by one or more heteroatoms), optionally substituted with hydroxy, halogen, aryl, alkoxy (1-3 carbon atoms), or acyl;
alkoxy (1-3 carbon atoms; optionally replaced by one or more heteroatoms), optionally substituted with hydroxy or halogen; or
cycloalkyl (3-8 carbon atoms) optionally substituted with hydroxy, halogen, amino;
aryl;
heteroaryl; and
R
2
is selected from the group consisting of adipyl (1,4-dicarboxybutane), succinyl, glutaryl, adipyl, pimelyl, surberyl, 2-(carboxyethylthio)acetyl, 3-(carboxy-ethylthio)propionyl, higher alkyl saturated and higher alkyl unsaturated dicarboxylic acids,
from a complex mixture comprising in addition to the compound of the general formula 6-aminopenicillanic acid (6-APA) and optionally one or more N-substituted &bgr;-lactam compounds,
comprising the steps of:
(a) acidifying the complex mixture to a pH below 6.5 and maintaining the mixture below said pH at a temperature of between 10° C. and 150° C.; and/or
(b) contacting the complex mixture with a carbon dioxide source; and
(c) recovering the cephalosporanic acid compound of the formula (I) from the mixture obtained after steps (a) and/or (b).
Preferably in step (a) the temperature is kept between about 50° C. and about 130° C., preferably between 70 and 120° C., for between 10 seconds and about 1 week and the pH is kept at or below pH 4.5. According to a preferred method the compound of formula (I) has been produced by fermentation of a micro-organism capable thereof, the complex mixture being a broth, a culture filtrate or any culture liquid derivable from the broth after fermentation.
Preferred compounds of the general formula (I) are selected from the group consisting of adipyl-7-ADCA, adipyl-7-ADAC and adipyl-7-ACA.
According to another aspect of the invention step (c) is performed by subjecting the mixture obtained after steps (a) and/or (b) to chromatography, preferably adsorption chromatography, more preferably Hydrophobic Interaction Chromatography.
According to another aspect of the invention the use of chromatography in a process or recovering a cephalosporin compound according to formula (I) is provided, preferably by adsorption chromatography, more preferably Hydrophobic Interaction Chromatography, still more preferably using Simulated Moving Bed technology.
According to yet another aspect of the invention a method is provided for making a compound of formula (II):
wherein
R
0
is hydrogen or C
1-3
alkoxy;
Y is CH
2
, oxygen, sulphur, or an oxidised form of sulphur;
R
1
is any of the groups selected from the group consisting of
hydrogen,
hydroxy,
halogen,
saturated or unsaturated, straight or branched alkyl (1-5 carbon atoms; optionally replaced by one or more heteroatoms), optionally substituted with hydroxy, halogen, aryl, alkoxy (1-3 carbon atoms), or acyl;
alkoxy (1-3 carbon atoms; optionally replaced by one or more heteroatoms), optionally substituted with hydroxy or halogen; or
cycloalkyl (3-8 carbon atoms) optionally substituted with hydroxy, halogen, amino; aryl;
heteroaryl;
comprising the steps of making a compound according to formula (I) wherein R
0
, Y and R
1
are as above and R
2
is selected from the group consisting of adipyl (1,4-dicarboxybutane), succinyl, glutaryl, adipyl, pimelyl, surberyl, 2-(carboxyethylthio)acetyl, 3-(carboxyethylthio)-propionyl, higher alkyl saturated and higher alkyl unsaturated dicar

Affiliated with

Also associated with

Rating

Process for the fermentative production of cephalosporin does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Process for the fermentative production of cephalosporin, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Process for the fermentative production of cephalosporin will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3096627