6-aminopenicillanic acid , hydrolysis

Figure 6.14 Enzymatic side chain cleavage of penicillins. 6-Aminopenicillanic acid, a valuable intermediate for the production of various semi-synthetic penicillins, can be obtained through enzyme-mediated hydrolysis of the phenylacety group of penicillin G or the phenoxyacetyl group of penicillin V. The active site of the enzyme recognises the aromatic side chain and the amide linkage, rather than the penidllin nucleus. Chemical entitles other than penicillins are therefore often good substrates, as long as they contain the aromatic acetamide moiety.

Penicillin G acylase (PGA, EC 3.5.1.11, penicillin G amidase) catalyzes the hydrolysis of the phenylacetyl side chain of penicillin to give 6-aminopenicillanic acid. PGA accepts only phenylacetyl and structurally similar groups (phenoxyacetyl, 4-pyridylacetyl) in the acyl moiety of the substrates, whereas a wide range of structures are tolerated in the amine part [100]. A representative selection of amide substrates, which have been hydrolyzed in a highly selective fashion, is depicted in Figure 6.36. 

Fig. 13. Outlet concentration profiles from a batch chromatographic bioreactor for enzyme catalyzed hydrolysis. Both products, i. e., 6-aminopenicillanic acid (open triangle) and phenylacetic acid (open circle), are separated resulting in a very high conversion. The penicillin G profile is presented as a dashed line (Reprinted with permission from [183])...

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. 

Facilitated transport of penicilHn-G in a SLM system using tetrabutyl ammonium hydrogen sulfate and various amines as carriers and dichloromethane, butyl acetate, etc., as the solvents has been reported [57,58]. Tertiary and secondary amines were found to be more efficient carriers in view of their easy accessibility for back extraction, the extraction being faciUtated by co-transport of a proton. The effects of flow rates, carrier concentrations, initial penicilHn-G concentration, and pH of feed and stripping phases on transport rate of penicillin-G was investigated. Under optimized pH conditions, i. e., extraction at pH 6.0-6.5 and re-extraction at pH 7.0, no decomposition of peniciUin-G occurred. The same SLM system has been applied for selective separation of penicilHn-G from a mixture containing phenyl acetic acid with a maximum separation factor of 1.8 under a liquid membrane diffusion controlled mechanism [59]. Tsikas et al. [60] studied the combined extraction of peniciUin-G and enzymatic hydrolysis of 6-aminopenicillanic acid (6-APA) in a hollow fiber carrier (Amberlite LA-2) mediated SLM system. 

When H2O deacetylates the acyl-enzyme, phenylacetic acid is formed. When nucleophiles other than H2O deacylate the acyl-enzyme, a new condensation product, in this case phenylacetyl-O-R or phenylacetyl-NH-R is formed. By definition the hydrolysis of these condensation products can be catalyzed by the same enzyme that catalyzes their formation in equation 10.1. Thus, when the acyl-enzyme is formed from phenylacetyl-glycine or phenylacetyl-O-Me, this gives rise to an alternative process to produce Penicillin G, in addition to the thermodynamically controlled (= equilibrium controlled) condensation of phenylacetic acid and 6-aminopenicillanic acid (6-APA). This reaction that involves an activated side chain is a kinetically controlled (= rate controlled) process where the hydrolase acts as a transferase (Kasche, 1986 1989). 

The optimum yield of a condensation product is obtained at the pH where Ka has a maximum. For peptide synthesis with serine proteases this coincides with the pH where the enzyme kinetic properties have their maxima. For the synthesis of penicillins with penicillin amidase, or esters with serine proteases or esterases, the pH of maximum product yield is much lower than the pH optimum of the enzymes. For penicillin amidase the pH stability is also markedly reduced at pH 4-5. Thus, in these cases, thermodynamically controlled processes for the synthesis of the condensation products are not favorable. When these enzymes are used as catalysts in thermodynamically controlled hydrolysis reactions an increase in pH increases the product yield. Penicilhn hydrolysis is generally carried out at pH about 8.0, where the enzyme has its optimum. At this pH the equiUbrium yield of hydrolysis product is about 97%. It could be further increased by increasing the pH. Due to the limited stability of the enzyme and the product 6-aminopenicillanic acid at pH>8, a higher pH is not used in the biotechnological process. 

Figure 14 Repetitive batch hydrolysis of penicillin-G to 6-aminopenicillanic acid. Plot shows batch number versus time required for complete hydrolysis of starting material.

The penicillins, from the fungus Penicillium chryso-genum, are the oldest and most widely used antibiotics. They are formed through stepwise build-up from a tripeptide (ACV) derived from a-Amino adipic acid, cysteine, and valine. Successive oxidation steps form the p-lactam and close the thiazolidine ring to form isopenicillin N. Action of an acyltransferase then yields penicillin G (Fig. 47). Alternatively, hydrolysis of isopenicillin N (or penicillin G) yields 6-aminopenicillanic acid, a key precursor for the wide range of semisynthetic pencillins used therapeutically. 

Aminopenicillanic acid by hydrolysis of penicillin Bacterial/Fungal acylase... 

Amoxicillin (7) is a semisynthetic penicillin antibiotic. The penicillin portion is derived from fermentation of either penicillin-V or -G, and then the side chain is removed chemically to afford 6-aminopenicillanic acid (6-APA) [3,16], The D- 7-hydroxyphenylglycine is then attached as the new side chain—chemical and enzymatic methods are available to achieve this [17-21]. This amino acid is obtained by a classical resolution or by enzymatic hydrolysis of a hydantoin (Chapter 8) [22-26],... 

Here as with the tetracyclines, the solution of difficult chemical problems was a prerequisite to successful new drug discovery. At first the counterpart of 6-aminopenicillanic acid could be made only in very low yields by chemical hydrolysis. A practical enzymatic hydrolysis of cephalosporin C to 7-aminocephalosporanic acid (7-ACA) was not found. R. B. Morin and co-workers provided the elegant solution (Figure 21) (101), which made the preparation of 7-ACA and semisynthetic cephalosporins possible on a practical scale. The impetus to persevere in this... 

A. Classification All penicillins are derivatives of 6-aminopenicillanic acid and contain a beta-lactam ring structure that is essential for antibacterial activity. PeniciUin subclasses have additional chemical substituents that confer differences in antimicrobial activity, susceptibility to acid and enzymic hydrolysis, and biodisposition. 

Penicillin acylase is an extremely important enzyme for the industrial production of 6-aminopenicillanic acid and 7-amino 3-desacetoxicephalosporanic, as key intermediates of semi-synthetic (3-lactam antibiotics (Parmar et al. 2000). These precursors are now industrially produced mainly by hydrolysis of penicillin G and cephalosporin G with immobilized penicillin acylase, which have replaced the former cumbersome chemical processes almost completely (Bruggink 2001 Kallenberg et al. 2005), representing one of the most successful cases of industrial application of hydrolytic enzymes in bioprocesses. 

In the case of penicillin acylase, that catalyzes the hydrolysis of penicillin G into 6-aminopenicillanic acid and phenylacetic acid, which is inhibited by 6-aminopenicillanic acid non-competitively and by phenylacetic acid competitively, from Table 3.2 only K, Ki, K2, K2, and kcat have finite values. Then, from Eq. 3.41 ... 

Roche D, Prasad K, Repic O (1999) Enantioselective acylation of 3-aminoesters using penicillin G acylase in organic solvents. Tetrahed Lett 40 3665-3668 Rolinson GN, Batchelor ER, Butterworth D et al. (1960) Formation of 6-aminopenicillanic acid from penicillin by enzymatic hydrolysis. Nat Lond 187 236-237 Rolinson GN, Geddes AM (2007) The 50th anniversary of the discovery of 6-aminopenicillanic acid (6-APA). Internatl J Antimicrob Agents 29 3-8 Resell CM, Ferndndez-Lafuente R, Guisdn JM (1993) Resolution of racemic mixtures by synthesis reactions catalyzed by immobilized derivatives of the enzyme peniciUin G acylase. J Mol Catal 84 365-371... 

Wang T, Zhu H, Ma X et al. (2006) Structure-based stabihzation of an enzyme the case of penicillin acylase from Mcaligenes faecalis. Prot Pept Lett 13 177-183 Wang Z, Wang L, Xu JH et al. (2007a) Enzymatic hydrolysis of peniciUin G to 6-aminopenicillanic acid in cloud point system with discrete countercurrent experiment. Enzyme Microb Technol... 

Fig. 15.20. (top curve). 6-Aminopenicillanic acid before hydrolysis. (Holt and Stewart, 1965.)... 

Den Hollander et al. [ 14,16] investigated the enzymatic hydrolysis of penicillin G to phenylacetic acid and 6-aminopenicillanic acid in biphasic aqueous-organic systems without pH-control. In a preliminary study, the two phases were counter-currently contacted in a discrete manner, so that equilibriiun was reached in each stage. Sets of three and five shake flasks served to mimic equilibrium stages in the counter-current set-up. It was shown, that counter-current contact leads to significant improvement of the equilibrium conversion relative to the batch or co-current situation. When penicillin G was fed in an intermediate stage, either exit contained mainly one of the two products. This simplifies product recovery. 

Inorganic membranes, usually appUed when high temperatures or chemically active mixtures are involved, are made of ceramics [171,172], zirconia-coated graphite [173],silica-zirconia [174],zeolites [168], or porous glass [175] among others [176]. Ceramic membranes are steam sterilizable and offer a higher mechanical stability [134], thus they may be preferably used in aseptic fermentations, since some hollow fibers are only chemically sterilizable and not very suitable for reuse. Composite materials, in which glass fiber filters are used as support for the polymerization of acrylamide monomers, were developed for the hydrolysis of penicillin G in an electrically immobilized enzyme reactor. By careful adjustment of the isoelectric point of amphoteric membranes, the product of interest (6-aminopenicillanic acid) was retained in an adequate chamber, adjacent to the reaction chamber, while the main contaminant (phenyl acetic acid), was collected in a third chamber [120]. 

The jS-lactam nucleus is very unstable below pH 2 and above pH 9, which precludes a simple acid- or base-catalysed hydrolysis to liberate the ) -lactam nucleus 6-APA (6-aminopenicillanic acid) (34). Although this compound was known to be a minor component of the commercial PenkilUum acremonium fermentation for the natural penicillins themselves, this also was not a suitable method for its production. A major breakthrough came around 1960 when several groups noticed that various micro-organisms were able to hydrolyse the exo-cyclic amide bond specifically, without also destroying the amide in the -lactam ring (Scheme 6.12). The amidohy-... 

Aminopenicillanic acid (6 APS) is an important precursor for the organic synthesis of new P. The compound itself has no antibiotic activity it is isolated as a fermentation product from cultures of Pen-cillium chrysogenum, or prepared by the enzymatic hydrolysis of benzylpenicillin. Thousands of new P. have been prepared by the acylation of 6 APS, but only a few of these are therapeutically useful, e.g Penicillin V is relatively stable to acid and is not hydrolysed in the stomach, so that it may be administered in tablet form Ampicillin (the aminophenyl-acetyl derivative of 6 APS), has a wider spectrum of activity than most other R, including activity against various Gram-negative bacteria (Typhus, E. coli, etc.). 

The observed pseudo first-order rate coefficient for the hydrolysis of benzylpenicillin is first order in hydroxide ion up to 2 M sodium hydroxide. Above this concentration it begins to level off (Minhas and Page 1982). This is probably attributable to ionisation of the benzylamido side chain. Presumably hydroxide ion attack on the penicillin with a 6-amido anion side chain is retarded. In support of this, the observed rate constant for the hydrolysis of phenoxymethylpenicillin shows a non-linear dependence upon hydroxide ion above 0.1 M sodium hydroxide (Minhas and Page, 1982 Pratt et al., 1983). The more electron-withdrawing phenoxymethyl group decreases the pA j-value of the amide side chain to 13.3. The observed first-order rate eonstants for the hydrolysis of 6-aminopenicillanic acid are, as expected, linear in hydroxide ion concentration. 

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