7- ACA, from cephalosporin

APA from penicillin G, 7-ACA from cephalosporin C, 7-ADCA from desaacetoxy cephalosporin G Biotransformation in steroids, e.g. cortexolone to hydrocortisone and prednisolone Food additives Lactic Acid (now a bulk chemical for making polylactate). Citric acid, L-Glutamate, L-Lysine, etc. Vitamines C, B2, B12 Acarbose (antidiabetic drug)... 

Although cephalosporin C is divisable into a-aminoadipic acid, cysteine, and valine, the actual mechanism whereby Cephalosporium sp. incorporates the three amino acids into cephalosporin C has not been established, Arnstein and Morris isolated 8 (a-aminoadipyl) cysteinyl valine from mycelia of Penicillium chrysogenum and suggested that the tripeptide is a precursor in all penicillin biosynthesis.. This same tripeptide also appears to be found in the intracellular pool of Cephalosporium sp.- The final postulated step in the biosynthesis of penicillin is an acyl transfer reaction, or the production of 6-aminopeni-cillanic acid if precursor is not added. Cephalosporium sp. apparently do not produce sidechain amidases or acyl transferases, and no 7-ACA has been reported found in the fermentation. Thus, to obtain clinically useful antibiotics, chemical manipulation of cephalosporin C is necessary. Synthesis of many 7-acyl derivatives was possible once a practical cleavage reaction made available large amounts of 7-ACA from cephalosporin C. of these derivatives, sodium cephalothin was the first... 

Therefore, a way had to be found of obtaining 7-ACA from cephalosporin C by chemical hydrolysis. This is not an easy task. After all, a secondary amide has to be hydrolysed in the presence of a highly reactive (3-lactam ring. Normal hydrolytic procedures are not suitable and so a special method had to be worked out as shown in Fig. 10.44. 

Various methods are known to produce 7-ACA from cephalosporin C (Ceph C) by removing the a-aminoadipyl side-chain. They can be classified into three types, the chemical process, a two-step enzymatic process and an enzymatic process in which the side-chain is directly removed from Ceph C. Today two processes are running commercially on an industrial scale, the classical chemical process and the modern two-step biocatalytic process (Fig. 2). Until now the favorable direct process is less effective, because of low conversion. 

Figure 12.2-6. Cbemo-enzymatic production of 7-ACA from cephalosporin C.

Vicenzi and Hansen (16) have developed an economical process for the enzymatic oxidation of cephalosporin C to GL-T-ACA at a pilot plant scale utilizing nonviable whole cells of T. variabihs containing high levels of DAO. Prior to use, the whole cells were permeabilized with a 25% acetone-water solution and then incubated at pH 11, which served to selectively deactivate catalase. The treated cells were utilized within a cross-flow filter-re actor" that allowed easy and economical recycling of the cells for repeated use. The overall yield of GL-7-ACA from cephalosporin C was 90-95% (16),... 

To explore the potentialities of semisynthetic cephalosporins it became imperative to find a method for the production of 7-ACA from cephalosporin C in a yield much higher than that obtained by acid hydrolysis. We confidently expected that an enzyme would be found that would catalyze the removal of the 8-(D-a-aminoadipyl) side chain the same view was expressed by Dr. Karl Folkers and Dr. Denkewalter who visited us in Oxford in 1959. In any event, widespread searches for such... 

It was almost immediately recognised that the deacylated product, 7-aminocephalosporanic add (7-ACA, Figure 6.16), would be of similar importance as was 6-APA in the development of new penidllins. However, 7-ACA, the cephalosporin equivalent of 6-APA, could not be found in fermentations of Cephalosporin acremonium. In Figure 6.15 we have shown that penicillin acylase hydrolyses the acyl residue from natural cephalosporins. Up to a point this is true. These acylases will, however, only work with a limited range of acyl residues. It now seems that nature does not provide for acylases or transacylases that have the capacity to remove or change the D-a-aminoadipyl side chain of cephalosporin C efficiently in a single step. Widespread search for such an enzyme still remains unsuccessful. 

Cephalosporins are a class of antibiotic produced via the intermediate 7-aminocephalos-poranic acid (7-ACA), or 7-aminodesacetoxycephalosporanic acid (7-ADCA). Directed evolution has been used to improve the activity of cephalosporin acylases to produce these intermediates from adipyl-7-ACA or cephalosporin C [68]. Using site-directed saturation mutagenesis and a selection system whereby the E. coli host is dependent on leucine liberated from derivatives of the cephalosporin side-chains, a mutant was found that increased the catalytic efficiency toward adipyl-7-ADCA by 36-fold. 

Production of 7-aminocephalosporanic acid (7-ACA) (and of 7-aminodesacetoxy cepha-losporanic acid (7-ADCA)) from cephalosporins by the action of cephalosporin C amidase from Pseudomonas sp. (Toyo Jozo Inc., Asahi Chemical Industry Co., Ltd., and others). Annual world production of 7-ACA 1000 tons and 7-ADCA 500 tons, used for the manufacture of semisynthetic cephalosporins. 

The reaction scheme for enzymatic synthesis of 7-ACA from c halosportn C is shown in Figure 1. The pathway involves the oxidative deamination of cephalosporin C into 7 P (5cephalosporanic acid (keto-AD-7-ACA) by DAO. A portion of the product further reacts nonenzymatically with hydrogen peroxide, which is formed as a by-product in the above reaction, to give GL-7-ACA. GL-7-ACA is hydrolyzed by GL-7-ACA acylase to 7-ACA. C7 side chains of cephalosporin C, keto-AD-7-ACA, and GL-7-ACA ate hydrolyzed by GC acylase to 7-ACA. However, the CC acylase activity for cephalo xirin C is low compared with that for GL-7-ACA. 

Figuie I The reaction scheme for enzymatic synthesis of 7-ACA from cef talosporm C. The sub-stiate specificities of cephalosporin acylases are described in the text. 

These results indicate that the biosynthetic pathways of 7-ACA and 7-ADACA from cephalosporin C and DAC via keto-AD-7-ACA and keto-AD-7-ADACA, and D-H AD-7-ACA and d-H-AD-7-ADACA, respectively, may exist in Hml78. A large amount of 7-ACA and 7-ADACA may be synthesized from cephalosporin C and DAC via those products in A. chrvsogenum Hml78. 

The role of p-lactam acylases in the manufacturing of semisynthetic cephalosporins and penicillins. In the left pathway, the production of 6-amino penicillanic acid (6-APA) from the fermentation product penicillin G is shown. In the right pathway, the production of 7-aminocephalosporanic acid (7-ACA) from the fermentation product cephalosporin-C is depicted... 

Figure 6.16 Production of 7-aminodeacetyicephalosporanic acid from 7-ACA using an immobilised acetyl esterase. Following enzymatic removal of the acetyl group from 7-ACA, a 3-hydroxymethyl cephalosporin is obtained that can serve as intermediate in the production of cefuroxime.

Cephalosporin C, obtained during the purification of cephalosporin N this is a true cephalosporin, and from it has been obtained 7-aminocephalosporanic acid (7-ACA Fig. 5.4), the starting point for new cephalosporins. 

Semi-synthetic penicillins are accessed from 6-aminopenicillanic acid, (6-APA), derived from fermented penicillin G. Starting materials for semi-synthetic cephalosporins are either 7-aminodesacetoxycephalosporanic acid (7-ADCA), which is also derived from penicillin G or 7-aminocephalosporanic acid (7-ACA), derived from fermented cephalosporin C (Scheme 1.10). These three key building blocks are produced in thousands of tonnes annually worldwide. The relatively labile nature of these molecules has encouraged the development of mild biocatalytic methods for selective hydrolysis and attachment of side chains. 

Penicillin acylases or amidohydrolases, which cleave the amide side chain of penicillin G, have been known for almost 50 years. " As one of the first enzymes to be developed for use at scale in the pharmaceutical industry, penicillin G acylase (PGA) has often been used as a model system for academic studies from molecular biology to biochemical engineering. Despite extensive screening, however, for decades there was no equivalent enzyme to generate 7-ACA by cleaving the polar D-a-aminoadipoyl side chain from cephalosporin C. 

Lactams, i.e., penicillins and cephalosporins, represent the most important class of antibiotics. Penicillins consist of a common core, 6-aminopenicillanic acid (6-APA) and different side chains. Penicillin G (pen G), with a phenyl acetate side chain, and penicillin V (pen V), with a phenoxyacetate side chain, are fermented from the fungus Penicillium chrysogenum all the others are produced from 6-APA, which nowadays is produced mostly from pen G via penicillin G amidase (pen G amidase, pen G acylase, PGA, E.C. 3.5.1.11 Figure 7.33). Cephalosporins feature 7-aminocephalosporanic acid (7-ACA) or its deacetyl-form, 7-aminodesacetyl-cephalosporanic add (7-ADCA) as their common core cephalosporin C (Ceph C) is obtained through fermentation, and all the others are derived from 7-A(D)CA. 

Adding up the times of all steps, an industrial scale production takes roughly 3 weeks, of which 2 weeks are devoted to the fermentation and about 1 week is required for the downstream processing. Derivatization at positions 3 and 7 to yield an API is not included. Starting from 7-ACA, these processes may take 1 day each for the derivatization plus the time for purification, crystallization, and drying. The resulting bulk active cephalosporin can then be sterilized and formulated for marketing. 

A second purification strategy involves the substitution of the amine moiety on the a-aminoadipyl side chain at C-7. Two substituted derivatives, iV-2,4-dichlorobenzoyl CPC and tetrabromocarboxybenzoyl CPC, can be crystallized from the acidic aqueous solution. Alternatively, salts can be formed between the, /V-substituted derivatives and an organic base, such as dicyclohexylamine or dimethylbenzylamine, resulting in cephalosporin salts that are solvent extractable. Bristol-Myers Squibb uses a solvent-extractable process resulting in the isochlorobutylformate (ICBF) ester of CPC, termed cephalosporin D. Several extraction steps are usually necessary to achieve the desired final purity. iV-Substituted CPC salts containing small amounts of contaminants can be effectively converted to 7-ACA. 

About one-third of the commercial cephalosporins are derived from 7-ADCA. Due to the lower cost of penicillin, 7-ADCA is usually produced from penicillin G by ring expansion of a penicillin sulfoxide ester to yield a cephalosporin ester. The removal of the ester group is followed by cleavage of the phenylacetyl side chain to give 7-ADCA. Two-thirds of the commercial cephalosporins are derived from 7-ACA, that is produced from CPC by either chemical or enzymatic deacylation. 

Aminocephalosporanic acid (15, Scheme 9) is an important intermediate in the production of many semisynthetic cephalosporin antibiotics (66, 67). However, direct deacylation of cephalosporin C (13) to 15 by cephalosporin C acy-lase is unfavorable, so an enzymatic process is used involving D-amino acid oxidase (DAAO) oxidation of 13 to A-glutaryl-7-aminocephalosporanic acid (14, GL-7-ACA) followed by deacylation to 15 and glutaric acid, catalyzed by GL-7-ACA acylase from Pseudomonas sp. 130 (Scheme 9) (68, 69). GL-7-ACA acylase underwent pseudo first-order time-dependent inactivation by 7 3-bromoacetyl aminocephalos-poranic acid (16) (70). Dialysis did not regenerate enzyme activity, indicating irreversible inhibition. The rate of inactivation was lowered by the presence of either glutaric acid or 15,... 

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