Hydrolysis of benzylpenicillin

H. Chaimovich, V. R. Correia, P. S. Araujo, M. V. Aleixo, I. M. Cuccovia, A Quantitative Analysis of the Effect of Hexadecyl-Trimethylammonium Bromide Micelles on the Rate of Alkaline Hydrolysis of Benzylpenicillin , J. Chem. Soc., Perkin Trans. 2 1985, 925-928. 

Extracts containing benzylpenicillin were prepared for analysis in buffer at pH 6.5 at 25°C, the rate constant for the hydrolysis of benzylpenicillin under these conditions is 1.7 x 10" s. What is the maximum length of time the solutions can be stored before analysis so that no more than 1 % decomposition occurs. 

Relative Rates of Hydrolysis of Various Substrates (Rate of Hydrolysis of Benzylpenicillin = 100)... 

A further example is seen in the rapid hydrolysis of benzylpenicillin (3.6) by cop-per(n) which is thought to proceed through an intermediate of type 3.7. Notice that in this case the activation of the carbonyl group to nucleophilic attack seems to be through... 

Figure 3-14. The copper(u)-directed hydrolysis of benzylpenicillin (3.6) showing the postulated intermediate, 3.7.

During the same period, Sheehan was working toward a total synthesis of penicillins. In 1958, he announced the synthesis of 6-amino-penicillanic acid (6-APA) and its utility for the preparation of new penicillins by acylation (67, 68). (Almost 10 years earlier, this substance had been postulated to be an intermediate in the biosynthesis of penicillins (69, 70). Prior Japanese literature also contained clear suggestions that it had been formed by enzymatic hydrolysis of benzylpenicillin (71) and in fermentations carried out in the absence of side chain precursors... 

Figure 77. pH-rate profile for hydrolysis of benzylpenicillin catalyzed by 3,6-bis(dimethylaminomethyl)catechol at 31.5°C. (Reproduced from Ref. 378 with permission.)... 

It is important to understand the mechanism of the cleavage of the (3-lactam ring catalyzed by 3-lactamase involving a zinc atom as an active site, because the zinc-containing enzyme is a factor in bacterial resistance to the 0-lactam antibiotics such as penicillin, cephalosporin, and so on. The first functional model for (3-lactamase was reported by Kimura and coworkers. " They demonstrated that the zinc complex involving 1,4,7,10-tetra-azacyclododecane ([12]aneN4) forms the [Zn—OH]" species under neutral conditions, and it accelerated the hydrolysis of benzylpenicillin in aqueous solution. In contrast, the native P-lactamase has two zinc atoms in the active site. From the three-dimensional structure infor-... 

We use the hydrolysis of A into P and Q as an illustration. Examples are the hydrolysis of benzylpenicillin (pen G) or the enantioselective hydrolysis of L-acetyl amino acids in a DL-mixture, which yields an enantiomerically pure L-amino acid as well as the unhydrolysed D-acetyl amino acid. In concentrated solutions these hydrolysis reactions are incomplete due to the reaction equilibrium. It is evident that for an accurate analysis of weak electrolyte systems, the association-dissociation reactions and the related phase behaviour of the reacting species must be accounted for precisely in the model [42,43]. We have simplified this example to neutral species A, P and Q. The distribution coefficients are Kq = 0.5 and Kp = K = 2. The equilibrium constant for the reaction K =XpXQ/Xj = 0.01, where X is a measure for concentration (mass or mole fractions) compatible with the partition coefficients. The mole fraction of A in the feed (z ) was 0.1, which corresponds to a very high aqueous feed concentration of approximately 5 M. We have simulated the hydrolysis conversion in the fractionating reactor with 50-100 equilibrium stages. A further increase in the number of stages did not improve the conversion or selectivity to a significant extent. Depending on the initial estimate, the calculation requires typically less than five iterations. 

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. 

The pH-rate profile for the hydrolysis of benzylpenicillin is shown in Fig. 9 (Gensmantel et al., 1978). There is no significant spontaneous hydrolysis but the P-lactam does undergo an acid catalysed degradation. Also shown in Fig. 9 is the pH-rate profile for the hydrolysis of cephaloridine. There are two immediate differences cephalosporins exhibit a spontaneous pH-independent hydrolysis and are less reactive towards acid than penicillins by a factor of about 10 (Proctor et al., 1982). 

The calculated rate constant for benzylpenicillin at 30°C (Proctor et al., 1982) is 6.30 x 10 s which is about 6000 times greater than the spontaneous hydrolysis of benzylpenicillin with an ionised carboxylate at... 

Rate constants for the hydrolysis of benzylpenicillin catalysed by oxygen bases are given in Table 6. The corresponding Bronsted plot is shown in Fig. 10 its non-linearity is indicative of a change in mechanism. The Bronsted P-value for weak bases is 0.39 and probably represents general base catalysed hydrolysis. The reaction with alkoxide ions is nucleophilic. The... 

Fig. 10 Br0nsted plot of the oxygen-anion catalysed hydrolysis of benzylpenicillin at 30°C. The steep slope represents nucleophilic catalysis whilst the less steep one for weakly basic anions represents general base catalysis... 

Transition-metal ions cause an enormous increase in the rate of hydrolysis of penicillins and cephalosporins (Gensmantel et al., 1978, 1980 Cressman et al., 1969). For example, copper(ri) ions can enhance the rate of hydrolysis of benzylpenicillin 10 -fold, a change in the half-life from 11 weeks to 0.1 seconds at pH 7. In the presence of excess metal ions, the observed apparent first-order rate constants for the hydrolysis of the 3-lactam derivatives are first order in hydroxide ion but show a saturation phenomenon with respect to the concentration of metal ion which is indicative of the formation of an antibiotic/metal ion complex. A kinetic scheme is shown in (3), where M is... 

Summary of the rate and association constants for the metal-ion catalysed hydrolysis of benzylpenicillin and cephaloridine in water at 30°C (/ = 0.5 M) ... 

The micelle catalysed hydrolysis of penicillins in alkaline solution is unusual because it involves the reaction between two anions, the hydroxide ion and the negatively charged benzylpenicillin (Gensmantel and Page, 1982a). The rate of the hydroxide-ion catalysed hydrolysis of benzylpenicillin decreases approximately three-fold in micellar solutions of itself (Hong and Kosten-bauder, 1975). 

Fig. 11 Observed pseudo first-order rate constants for the hydrolysis of benzylpenicillin at the concentrations shown as a function of cetyltrimethylammonium bromide (CTAB) concentration at 30°C (Gensmantel and Page, 1982a)...

The acid catalysed degradation of penicillins is inhibited in cationic micelles of cetyltrimethylammonium bromide (Tsuji et al., 1982) and, as expected, neither anionic micelles of sodium dodecylsulphate nor polyoxyethylene lauryl ether promote the hydroxide-ion catalysed hydrolysis of benzylpenicillin (Gensmantel and Page, 1982a). In the presence of cetyltrimethylammonium bromide (CTAB) the pseudo first-order rate constants for the alkaline hydrolysis increase rapidly with surfactant concentration once... 

The kinetic evidence implies that there must be some binding between the benzylpenicillin anion and the micelles of CTAB and this has been shown spectroscopically (Chaimovich et ai, 1985). The maximum rate acceleration in the alkaline hydrolysis of benzylpenicillin by CTAB micelles is about 50. 

Added salts decrease the rate of the CTAB micelle catalysed alkaline hydrolysis of benzylpenicillin (Gensmantel and Page, 1982a). The salt effect can be considered to be due to competitive binding of the anions with the micelle. Increasing the unreactive anion concentration displaces hydroxide ion bound in the Stern layer leading to a reduction in the observed rate. 

It is interesting to note that the hydroxide ion catalysed hydrolysis of benzylpenicillin involves rate-limiting attack whereas alkoxide ions react with benzylpenicillin with rate-limiting breakdown of the tetrahedral intermediate. This could result from the intermediate formed by hydroxide ion attack either breaking down to reactants or products slower or faster, respectively, than [69]. 

Benzylpenicillin isocyanate has a /S-Iactam ring that could be hydrolyzed by the enzyme. When benzylpenicillin isocyanate binds to the catalytic site and hydrolysis and inactivation proceed simultaneously, these reactions should take place through the same reversible complex (E-BPI) as represented above. Although the rate was over 100 times less than that with benzylpenicillin, the hydrolysis of benzylpenicillin isocyanate did occur with concomitant inactivation of the enzyme. The apparent Michaelis constant, k-iks)/ki, was coincident with K (400 fJiM) obtained in the inactivation reaction described. Other penicillin isocyanates may be hydrolyzed in a similar manner by yS-lactamase. 

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