6- APA from penicillin

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)... 

Penicillin amidase is used industrially to produce 6-aminopenicillanic acid (6-APA) from penicillin G or V (see section 4.5). Acid is produced during the process and this will inactivate the enzyme. One way of overcoming this problem is by using a fixed bed reactor with immobilized enzyme. The substrate is pumped very rapidly... 

Enzymes are typically produced using fermentation. They cover a wide variety of applications (see Table 14.5) including their use in the production of antibiotics (as noted above with the use of penicillin acylases to produce 6-APA from penicillin). 

In Bgure 6.11 we indicated that penidllin acylases selectively hydrolysed the secondary amide link, releasing 6 aminopenidllanic add (6-APA). Although these enzymes could be used to produce 6-APA from penicillin G, initially, the vulnerability and hirfi costs of enzymatic deacylation were impcnlant reasons to search for alternative, diemical processes. 

Production of 6-amino penicillanic acid (6-APA) from penicillin G or V by the action of penicillin amidase from E. coli, Bacillus megaterium, or Bovista plumba (Toyo Jozo Inc., Asahi Chemical Industry Co., Ltd., Fujisawa Pharmaceutical Co., Gist-Brocades/DSM, Novo-Nordisk, Pfizer, and others). Annual world production of 6-APA 6000 tons, used for the manufacture of semisynthetic penicillins. 

Figure 11. Operational stability functions of E. coli cells immobilized in epoxy carrier for consecutive batch operation in the production of 6 APA from penicillin G. Top, experimental data bottom, model calculations for the superposition of 6 APA formation and conversion dependence catalyst deactivation reaction in the diffusion controlled regime.

Penicillin acylase Bacillus megaterium Formation of 6-APA from penicillin for... 

Penicillin is but one of a series of closely related compounds isolated from fermentation broths of Penicillium notatum. This compound, also known as penicillin G (1-1) or benzyl penicillin, is quite unstable and quickly eliminated from the body. Initial approaches to solving these problems, as noted above, consisted of preparing salts of the compound with amines that would form tight ion pairs that in effect provided a controlled release of the active dmg. Research on fermentation conditions aimed at optimizing fermentation yields succeeded to the point where penicillin G or penicillin V (26-1), in which the phenylacetyl group is replaced by phenoxyacetyl, is now considered a commodity chemical. Another result of this research was the identification of fermentation conditions that favored the formation of the deacylated primary amine, 6-aminopenicillanic acid (2-4) or 6-APA, a compound that provided the key to semisynthetic compounds with superior pharmaceutical properties than the natural material. An elegant procedure for the removal of the amide side chain proved competitive with 6-APA from fermentation. This method, which is equally applicable to penicillin V, starts by conversion of the acid to the corresponding silyl ester (2-1). Treatment of that compound with phosphoms pentachloride in the... 

The natural penicillins, primarily G and V, have a relatively narrow spectrum. They act mostly on gram-positive organisms. The fact that proper selection of precursors could lead to new variations in the penicillin side chain offered the first source of synthetic penicillins. Penicillin V, derived from a phenoxy-acetic acid precursor, attracted clinical use because of its greater acid tolerance, which made it more useful in oral administration. Also, the widespread use of penicillin eventually led to a clinical problem of penicillin-resistant staphylococci and streptococci. Resistance for the most part involved the penicillin-destroying enzyme, penicillinase, which attacked the beta-lactam structure of the 6-aminopenicillanic acid nucleus (6-APA). Semisynthetic penicillins such as ampicillin and carbenicillin have a broader spectrum. Some, such as methicillin, orafi-cillin, and oxacillin, are resistant to penicillinase. In 1984, Beecham introduced Augmentin, which was the first combination formulation of a penicillin (amoxicillin) and a penicillinase inhibitor (clavulanic acid). Worldwide production of semisynthetic penicillins is currently around 10,000 tons/year, the major producers are Smith Kline Beecham, DSM, Pfizer, and Toyo Jozo. 

In the classical chenaical process, this could be accomplished by a consecutive multistep procedure in a batch reactor, using 3 ton trimethylsilyl chloride, 8 ton N,N-dimethylaniline, 6 ton phosphorus pentachloride and 1.6 ton ammonia for the production of 5 ton 6-APA from 10 ton Penicillin G. In addition, 50 ton dichloromethane and 40 ton of butanol were used as solvents. Part of the reaction sequence was carried out at -40°C, leading to 30 MWh cooling energy costs. 

An example of a commercial process that uses a hydrolase in the hydrolytic mode is the production of the antibiotic intermediate 6-APA by Gist-brocades [13], discussed above in Section 7.2.1 (Fig. 7.1). The biocatalyst is a penicillin acylase, which generates 6-APA from the starting material penicillin G in one step at a moderate temperature in water. The atom utilization of the enzymic process is much higher than that of the corresponding chemical process [14]. 

For the penicillins a one-step biocatalytic route for the production of 6-aminopenicil-lin acid (6-APA) from the fermentation product penicillin (Pen G or Pen V) was well known. Beginning at the end of 1970s the chemical process (almost similar to the... 

Later, chemical methods and more efficient enzyme-mediated methods were devised that could selectively remove the side-chain of penicillin G to produce 6-APA. These two commodities, 6-APA and penicillin G, thus became the high tonnage products of the fermentation process. Modem fermenters now range in capacity from 100,000 to 200,000 L and can provide 40 g L l of penicillin G (compared with less than 20 mg L 1 in 1941). 

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... 

Many semi-synthetic penicillins are made from 6-aminopenicillanic acid (6-APA, R = NH2>. 

APA may be either obtained directly from special Penicillium strains or by hydrolysis of penicillin Q with the aid of amidase enzymes. A major problem in the synthesis of different amides from 6-APA is the acid- and base-sensitivity of its -lactam ring which is usually very unstable outside of the pH range from 3 to 6. One synthesis of ampidllin applies the condensation of 6-APA with a mixed anhydride of N-protected phenylglydne. Catalytic hydrogenation removes the N-protecting group. Yields are low (2 30%) (without scheme). 

The only penicillins used in their natural form are benzylpenicillin (penicillin G) and phenoxymethylpenicillin (penicillin V). The remainder of penicillins in clinical use are derived from 6-APA and most penicillins having useful biological properties have resulted from acylation of 6-APA using standard procedures. 

Chemical Modification. Chemical modification of most positions in the penicillin nucleus have been carried out and these are summarized in Table 4. Apart from acylation of 6-APA, few of these modifications have proven profitable in terms of improving the biological properties of the derived penicillins. However, one of the modifications that has led to beneficial properties is substitution at the 6a-position. 

The acylation of 6-APA (Scheme 59) has been a very versatile way in which to generate new penicillin derivatives which differ from fermentation-produced penicillins in the 6-side chain. As will be discussed in Section 5.11.5.1, this approach has led to significant improvements in the therapeutic properties of penicillins, and, in fact, of the penicillins in medical use today, only benzylpenicillin and phenoxymethylpenicillin are produced directly by fermentation. 

The nature of the penicillin derivatives accessible by this "feeding" route was severely limited by the fact that the acylat-ing enzyme of the Penicillium molds would accept only those carboxylic acids which bore at least some resemblance to its natural substrates. A breakthrough in this field was achieved by the finding that rigid exclusion of all possible side-chain substrate from the culture medium afforded 6-APA as the main fermentation... 

Different strains of micro-organisms are responsible for the production of either penicillins or cephalosporins. In penicillin-producing strains, an acyltransferase enzyme system is present which can remove the side chain from isopenirillin N to give 6-aminopenicillanic acid (6-APA), and which can subsequently acylate 6-APA to generate various penicillins, the most important ones being penicillin G and V(see section 6.3, Table 6.2). 

Figure 6.15 Production of semi-synthetic penicillins (left) and cephalosporins (right), from enzymatically obtained intermediates 6-APA and 7-ADCA respectively.

In this process, penicillin G is first hydrolysed to 6-APA with the acylase derived from Kluyvera citwphila at a slightly alkaline pH (pH 75). Subsequently the 6-APA is incubated with an acylase derived from Pseudomonas mdanogenum and with DL-phenylglydne methyl ester at pH 55. This produces ampiciilin in reasonable yields only because of the specificity of the P. melanogenum enzyme. This enzyme does not react with penicillin G nor phenylacetic acid. 

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. 

Rasor and Tischer (1998) have brought out the advantages of enzyme immobilization. Examples of penicillin-G to 6-APA, hydrolysis of cephalospwrin C into 7-ACA, hydrolysis of isosorbide diacetate and hydrolysis of 5-(4-hydroxy phenyl) hydantom are cited. De Vroom (1998) has reported covalent attachment of penicillin acylase (EC 3.51.11) from E.Coli in a gelatine-based carrier to give a water insoluble catalyst assemblase which can be recycled many times, and is suitable for the production of semi-synthetic antibiotics in an aqueous environment. The enzyme can be applied both in a hydrolytic fashion and a synthetic fashion. 6-APA was produced from penicillin-G similarly, 7-ADCA was produced from desa acetoxycephalosporin G, a ring expansion product of penicillin G. 

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. 

There has also been extensive activity towards the replacement of the entire chemical route to 7-ADCA (Scheme 1.14) with a biocatalytic one. This is somewhat more complex than the above example, as the penicillin fermentation product requires ring expansion as well as side-chain hydrolysis in order to arrive at the desired nucleus. The penicillin nucleus can be converted to the cephalosporin nucleus using expandase enzymes, a process that occurs naturally during the biosynthesis of cephalosporin C by Acremonium chryso-genum and cephamycin C by Streptomyces clavuligems from isopenicUhn N (6-APA containing a 6-L-a-aminoadipoyl side chain). ... 

These defects have spurred attempts to prepare analogs. The techniques used have been (1) natural fermentation (in which the penicillin-producing fungus is allowed to grow on a variety of complex natural nutrients from which it selects acids for incorporation into the side chain), (2) biosynthetic production (in which the fermentation medium is deliberately supplemented with unnatural precursors from which the fungus selects components for the synthesis of "unnatural" penicillins), (3) semisynthetic production (in which 6-aminopenicillanic acid (2) is obtained by a process involving fermentation, and suitably activated acids are subsequently reacted chemically with 6-APA to form penicillins with new side chains) and (4) total synthesis (potentially the most powerful method for making deep-seated structural modifications but which is at present unable to compete economically with the other methods). 

An extremely important progress in the development of penicillins took place in 1959, when the penicillin nucleus, 6-aminopenicillanic acid (6-APA), was removed from the side chain and isolated from a culture of Penicillium chrysogenum. 

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