Penicillin derivatives hydrolysis

Note that penicillins and structurally related antibiotics are frequently deactivated by the action of bacterial -lactamase enzymes. These enzymes also contain a serine residue in the active site, and this is the nucleophile that attacks and cleaves the P-lactam ring (see Box 7.20). The P-lactam (amide) linkage is hydrolysed, and then the inactivated penicillin derivative is released from the enzyme by further hydrolysis of the ester linkage, restoring the functional enzyme. The mode of action of these enzymes thus closely resembles that of the serine proteases there is further discussion in Box 7.20. 

Figure 3 The different fates of three acyl enzymes the -o-Ala-o-Ala derived PBP o-Ala acyl-enzyme (transpeptidation) the penicillin-derived /3-lactamase acyl-enzyme (hydrolysis) the /3-lactamase inactivator-derived /3-lactamase acyl-enzyme (hydrolysis and fragmentation to stable acyl-enzymes).

A survey of crystalline hydrates of /3-lactams was recently reported in order to probe the significance of crystallinity on the stability of reactive APIs [54], Compounds which contain the /3-lactam core, including penicillin derivatives, carbapenems and cephalosporins, are known to undergo hydrolysis both in solution and in the solid state [55, 56], Solid-state stability has been shown to be dependent on storage conditions and degree of crystallinity [57-59]. It has also been shown that even hydrates of /3-lactams, where the water is regarded as a potential reactant, can be stable in the solid state for years. 

The CTAB catalysed hydrolysis of penicillin derivatives appears to exhibit some degree of specificity. Increasing the hydrophobicity of the 6-P-side chain increases micellar catalysis. The association of the penicillin substrate with the micelle is presumably the result of interactions similar to those that give micelles stability relative to their monomeric form in aqueous solution hence the not unexpected increase in substrate binding with increased lipophilicity of the molecule. It appears that once the 6-p-side has been... 

Summary of the data for the hydroxide-ion catalysed hydrolysis of penicillin derivatives in the presence and absence of micelles of cetyltrirnethylammonium bromide at 30°C ... 

Second-order rate constant for hydrolysis in the absence of micelles, ionic strength I = 0.05 M Apparent sccond-ordcr rate constant for hydrolysis in the presence of CTAB micelles, 0.05M-NaOH and 2 x 10 M penicillin Apparent binding constant of penicillin derivative to micelle... 

The second example, which is also in daily use, is the specific hydrolysis of natural penicillin G by penicillin amidase in preparative scale, again through a chromatographic process (129). It produces a clean hydrolytic product (6-aminopenicillanic acid) free of possible contaminant and it can then be used to prepare chemically all kinds of semisynthetic penicillin derivatives. Unlike the rather unstable soluble enzyme, the insolubilized preparation shows no loss activity after up to 11 weeks of continuous operation at 37°C. Furthermore, the procedure is free from potentially allergy-inducing contaminants. 

Various chemical species influence the rates of hydrolysis of penicillins, e.g. metal ions (Cu >Zn >Ni Co ) (80JCS(P2)1725), carbohydrates (78MI51101), certain amine-containing catechol derivatives (69JPS1102) and /3-cyclodextrin (71JA767). Some of these even show some of the characteristics of enzyme-catalyzed hydrolyses. 

Stereoselective hydrolysis of racemic l-(//-phenylacetylamino) alkanephos-phonic acids performed in the presence of penicillin acylase under the kinetic resolution conditions gave both the unreacted substrates and the products - the corresponding 1-aminophosphonic acids in high yields and with full enantioselec-tivity. The unreacted A -acyl derivatives were hydrolysed chemically and in this way each enantiomer of the free acid was obtained (Scheme 5). ... 

In some cases enzymes can increase the rate of reaction by up to lO times. Carnell and Roberts (1997) have briefly discussed the scope of biotransformations that are used to make pharmaceuticals like penicillins, cephalosporines, erythromycin, lovastatin, cyclosporin, etc., and for food additives like citric acid, L-glutamate, and L-lysine. A very successful transformation by Zeneca has been that of benzene reduction, with Pseudomonase Putida, to dihydrocatechol and catechol the dihydro derivative is used to produce (+/-) pinitol. Fluorobenzene has been converted to fluorodihydrocatechol, an intermediate for pharmaceuticals. The highly stereo selective Bayer-Villeger reaction has been carried out with genetically engineered S-cerevisvae. Hydrolases have allowed enantioselective, and in some cases regioselective, hydrolysis of racemic esters. 

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. 

Kinetic studies of the unnatural 6-a -epimer of ampicillin, fi-ept-ampicillin (154), have revealed an intramolecular process not undergone by ampicillin (or other natural /3-substituted penicillins) At pH 6-9, intramolecular attack of the jS-lactam carbonyl group by the side-chain amino group of (154) yields a stable piperazine-2,5-dione derivative (155). Theoretical calculations show that the intramolecular aminolysis of 6-epi-ampicillin nucleophilic attack occurs from the a-face of the -lactam ring with an activation energy of 14.4kcalmor In other respects, the hydrolysis of the b-a-epimer is unexceptional. 

Chemical or enzymatic hydrolysis of this compound allows to obtain large quantities of 7-aminocephalosporanic acid. A number of semisynthetic beta-lactam cephalosporin antibiotics were created by acylating the amino group of the last with various acid derivatives (analogous to the semisynthetic penicillin series) and currently there are about 25,000 of them, of which about 100 are used in medicine. Unlike penicillins, semisynthetic cephalosporins are synthesized not only by expanding the spectrum of various acids by which 7-aminocephalosporanic acid is acylated, but also by internal modifications of aminocephalosporanic nucleus (Rj and Rj). 

Another interesting example of resolution through formation of diastereo-mers is the isolation of four stereoisomers of 3-amino-2-methyl-3-trifluoro-methyl butanoic acid [55]. In this process, the chemical-enzymatic method by the combination of chemical and enzymatic reaction is a very convenient. At first, -phenylacetyl derivatives 61a and 61b were prepared in excellent isolated yields via the Schotten-Baumann procedure. After these materials were hydrolysed with penicillin acylase (EC 3.5.1.11) from Escherichia coli until attainment of 50% conversion, enzymatically unconverted -phenylacetyl derivatives 62 a and 62 b (organic layer) and amino acids 63 b and 63 d (aqueous layer) were separated. Acidic hydrolysis of unconverted materials produced other stereoisomers 63 a and 63 c in high optical pure form. 

The term / -lactamase denotes an enzyme which catalyzes the hydrolysis of the amide bond in the /3-lactam ring of 6-amino-penicillanic acid (6-APA) or 7-amino-cephalosporanic acid (7-ACA) and of their A-acyl derivatives (2). Such derivatives are commonly referred to as penicillins [Fig. 1 (I) and cephalosporins Fig. 1 (III), (V), and (VII)], respectively. There is no evidence that any bond other than the amide bond in the intact nucleus of penicillin or cephalosporin is broken by the... 

A number of interesting studies of lactam hydrolysis have been published. The metal(II)-catalyzed hydrolysis of some penicillin and cephalosporin derivatives displays saturation kinetics.410"412 A 1 1 complex is formed between the metal ion and penicillin which undergoes base hydrolysis up to 10s times faster than the free ligand. The catalytic activity follows the order Cu,>ZnII>NiII = Co11. Coordination of penicillin to copper(II) is believed to occur via the /3-lactam nitrogen and the carboxylate group (121)4l0-4n but other sites have been proposed.413... 

Amoxicillin (21) is a semi-synthetic penicillin antibiotic. The penicillin portion is derived from fermentation of either penicillin-V or penicillin-G, and then the sidechain is removed to afford 6-APA. This transformation can be done chemically.69 207 The alternative, which is growing in importance, is to perform an enzymatic cleavage under mild conditions.208 The D-p-hydroxyphenylglycine is then attached as the new sidechain chemical and enzymatic methods are available to achieve this (Scheme 31.16).209 215 The phenylglycine amino acid is obtained by a resolution (Chapters 2, 7, and 25) or by enzymatic hydrolysis of a hydantoin (Chapter 2, 6, and 19).216-220... 

The different anhydride and thioanhydride derivatives may be regarded merely as intermediates useful for obtaining amides and esters. The penicillin anhydrides were synthesized long ago [105,106]. Some cephalosporin anhydrides too were described recently [106]. Cephalothin anhydride (72a) was reported to be effective against S. aureus Smith in a dose of 0.24 figlml. Assuming quantitative hydrolysis, the antimicrobial... 

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