Process for preparing cephalosporanic acid derivatives using...

Details Process for preparing cephalosporanic acid derivatives using... Process for preparing cephalosporanic acid derivatives using...

2002-04-19

2004-05-04

Lilling, Herbert J. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound having a 1-thia-5-aza-bicyclo

C540S224000, C540S229000

Reexamination Certificate

active

06730497

ABSTRACT:

INTRODUCTION
The invention relates to a process for preparing 3-cephalosporin C derivatives which are used in the preparation of &bgr;-lactam antibiotics. In particular the invention relates to an enzymatic process for the preparation acid (3-thiolated-7-ACA) using &agr;-ketoacid intermediates. The &agr;-ketoacids or &agr;-oxoacids are important biopharmaceutical compounds.
Oxoacids of essential amino acids are gaining importance as nutraceuticals (Pszcola, D E,
Food Technol.
52, 30, 1998) as well as therapeutic agents for treating nitrogen accumulation disorders (Schaefer et al.,
Kidney Int. Suppl.
27, S136, 1989; Buto et al,
Biotechnol. Bioeng.
44, 1288, 1994). Another important application is the production of 7-amino cephalosporanic acid (Savidge, T A; In
Biotechnology of Industrial Antibiotics
, p 171, Marcel Dekker, New York, 1984) from cephalosporin C (3-acetoxymethyl-7&bgr;-(D-5-amino-5-carboxypentanamido) ceph-3-em-4-carboxylic acid). The transformation can be carried out by a D-amino acid transaminase from
Bacillus licheniformis
ATCC 9945, which converts cephalosporin C with &agr;-ketoacids into &agr;-ketoadipyl-7-ACA and the corresponding D-&agr;-amino acid, as described in DE 3447023 (Hoechst). This conversion is a transamination, the amino group of cephalosporin C being converted non-oxidatively into the keto group, without the release of hydrogen peroxide. However there is a low level of activity of this enzyme, as described in EP 0315786.
Chemical methods for the preparation of 3-thiolated-7-ACA cephalosporanic acid derivatives are known (U.S. Pat. No. 3,367,933; BE 718,824), however they have disadvantages such as low temperature reaction conditions, the use of costly and toxic solvents or reagents and chemical instability of intermediates which makes the processes difficult on an industrial scale.
To overcome the drawbacks of the chemical route to 7-ACA, alternative enzymatic cleavage of cephalosporin C has been described. Direct one-step removal of the lateral 7′-aminoadipic side-chain of cephalosporin C is possible by using specific cephalosporin acylases (FR 2,241,557; U.S. Pat. No. 4,774,179; EP 283,248; WO 9512680; WO 9616174). These processes, however, are often not reproducible and are characterised by low yields and lengthy reaction times as described in U.S. Pat. No. 5,296,358. No industrial application of this technology (single-step conversion of cephalosporin C to 7-ACA) has been reported at this time (Parmar et al, Crit. Rev. Biotechnol. 18, 1, 1998).
On the other hand, processes that transform the cephalosporin C into 7-ACA by means of two enzymatic steps are important from an industrial point of view. The first stage consists of using a D-amino acid oxidase (E.C. 1.4.3.3, hereinbelow indicated as DAAO) from different sources (
Trigonopsis variabilis
, GB 1,272,769;
Rhodotorula gracilis
, EP 0,517,200; or
Fusarium solari M-
0718, EP 0,364,275). DAAO oxidises the lateral D-5-amido-carboxypentanoyl chain of cephalosporin C in the presence of molecular oxygen, to produce 7&bgr;-(5-carboxy-5-oxopent-amido)-ceph-3-em-carboxylic acid (or &agr;-ketoadipyl-7-aminocephalosporanic acid, hereinbelow indicated as &agr;-ketoadipyl-7-ACA) and hydrogen peroxide, which chemically decarboxylate the &agr;-ketoadipyl-7-ACA to 7&bgr;-(4-carboxy butanamido)-ceph-3-em-4-carboxylic acid (or glutaryl-7-aminocephalo-sporanic acid, hereinbelow indicated as GL-7-ACA).
In a second stage, a specific acylase for GL-7-ACA, glutaryl-7-ACA acylase (E.C. 3.5.1.3), is used, for example that of a Pseudomonas type microorganism (U.S. Pat. No. 3,960,662, EP 0496993) over expressed in
E. coli
, which deacylates the GL-7-ACA into 7-amino-ceph-3-em4-carboxylic acid (or 7-amino cephalosporanic acid, hereinbelow indicated as 7-ACA).
This two-step enzymatic process for obtaining 7-ACA has been used on an industrial scale (Conlon et al. Biotechnol. Bioeng. 46, 510,1995).
Yet another advance in enzymatic processes, is disclosed in EP 0846695, in which solid glutaryl-7-ACA is reacted with a heterocyclic group that contains at least a nitrogen with or without a sulphur or oxygen atom to produce a 3-modified glutaryl-7-ACA. These 3-derivatives are enzymatically transformed to their corresponding 3-heterocyclic thiomethyl-7-ACA derivatives.
This procedure can be defined as an enzymatic-chemical-enzymatic (ECE) process, since the isolated GL-7-ACA comes from a bioconversion of solubilised cephalosporin C, then GL-7-ACA is reacted with the heterocyclic thiols and finally the 3-heterocyclic thio-derivative is enzymated with GL-7-ACA acylase. The problem with this method is the need to isolate GL-7-ACA, which given its high water solubility, is technically difficult and expensive, as described in WO 9535020.
An additional problem is that the enzyme can only be reused a few times due to the “poisoning” of the biocatalyst by the residual heterocyclic thiols. This poisoning effect is well documented with one of the thiols used, 5-methyl-1,3,4-thiadiazole-2-thiol (MMTD) (Won et al, App. Biochem. Biotech. 69, 1, 1998).
The oxidative deamination of the D-adipamido side chain of cephalosporin C under aerobic conditions into &agr;-ketoadipyl-7-ACA has been described using D-amino acid oxidase (D-AAO) from cell-free extracts (GB 1,2 72,769, Glaxo) or in toluene-activated (permeabilised) cells (GB 1,385,685) of the yeast
Trigonopsis variabilis
or
Rhodotorula glutinis
(EP 0517200). In this reaction, molecular oxygen acts as the electron acceptor and is converted to hydrogen peroxide, which chemically reacts with the &agr;-ketoadipyl-7-ACA producing its decarboxylation into glutaryl-7-ACA. In the presence of large quantities of the catalase produced for the above yeasts, the hydrogen peroxide is cleaved to water and molecular oxygen, rendering a mixture of &agr;-ketoadipyl-7-ACA and glutaryl-7-ACA. The &agr;-ketoadipyl-7-ACA is quite unstable (GB 1,385,685) and rapidly decomposes to unknown products and hence reduces the yield of glutaryl-7-ACA from 90 to 95% to 60 to 70%, depending on the yeast and strain (Parmar et al,
Crit. Rev. Biotechnol.
18, 1, 1998; Rietharst, W. and Riechert, A,
Chimia
53, 600, 1999). As a result no industrial application has been described.
There is therefore a need for an efficient and improved process for the preparation of 3-thiolated-7-ACA cephalosporanic acid derivatives on an industrial scale. In addition the isolation of stable &agr;-ketoacid derivatives which are important biopharmaceutical compounds would be beneficial.
STATEMENTS OF INVENTION
According to the invention there is provided a process for preparing cephalosporanic acid derivatives comprising the steps of:
enzymatically converting a 3-thiolated cephalosporin C compound of formula III:
into a 3-thiolated-&agr;-ketoadipyl-7-aminocephalosporanic acid derivative of formula IV:
wherein R is a heterocyclic group comprising at least a nitrogen atom.
Preferably the compound of formula III is enzymatically converted into a compound of formula IV by an immobilised enzyme system. Most preferably the enzyme system comprises co-immobilised D-Amino acid oxidase and catalase.
Preferably the enzymatic conversion is carried out in the presence of molecular oxygen, at a pressure of 1 to 5 bar absolute, a pH of from 6.5 to 8.0 and at a temperature of from 15 to 30° C. for a period of from 30 mins to 180 mins.
Preferably the process comprises the step of separating the enzyme system from the reaction mixture, preferably by filtration.
In one embodiment of the invention the process includes the step of purifying the compound of formula IV.
Most preferably the compound is purified using an adsorption column. Preferably the enzymes are co-immobilised using a suitable cross-linker agent in a suitable solid support. The enzymes may be in the form of crystals of a size suitable for use as a biocatalyst.
Preferably the enzymatic processes are carried out while maintaining the enzyme in dispersion in an aqueous substrate solution. Preferably the or each enzymatic process is carried out in a column. Most preferably

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