- Role of zinc(II) in β-lactamase II: A model study with a zinc(II)-macrocyclic tetraamine (1,4,7,10-tetraazacyclododecane, cyclen) complex
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Cleavage of the β-lactam ring of benzylpenicillin (1) by a zinc(II) complex of the macrocyclic tetraamine 1,4,7,10-tetraazacyclododecane (cyclen) (8) has been studied in aqueous solution as a functional model of a zinccontaining hydrolytic enzyme, β-lactamase II. β-Lactam hydrolysis by 8 yielding (5R)-benzylpenicilloate (9) is a second-order reaction (the second-order rate constant k is (4.1 ± 0.1) X 10-2M-1 s-1 at 25 °C and I = 0.10 (NaNO3)), and a plot of the hydrolysis rate vs pH (6.6-9.6) gives a sigmoidal curve with an inflection point at pH 7.9, which is identical to the pKa value for the ZnII-bound water of ZnII-cyclen-OH2 (8a). Thus, ZnII-cyclen-OH- (8b) must play a crucial role in the hydrolysis of the β-lactam. The activation energy Ea for the hydrolysis by 8b was determined to be 49 kJ mol-1, lower than the value of 61 kJ mol-1 by aqueous OH-ion. The lower Ea value for 8b is due to the acidic nature of ZnII that stabilizes the anionic tetrahedral intermediate 15. The hydrolysis is subject to anion inhibition by deprotonated succinimide-, SCN-, CH3COO-, or Cl- in the same order as their binding affinity for 8a. The ZnII-cyclen 8 simultaneously catalyzes the isomerization of (5R)-benzylpenicilloate (9) to the 5S-epimer 10 at pH 6.5-9.5. The pH dependency of the catalytic activity discloses that the reactive species is 8a and the kinetically obtained pKa value of 8.0 is almost the same as that obtained thermodynamically. The second-order rate constant kep with 8a is 2.5 ± 0.1 M-1 s-1 at 25 °C and I = 0.10 (NaNO3). Presumably the reaction involves C-S bond rupture due to coordination of ZnII-cyclen, followed by recombination to form 10. While the reaction mechanism of β-lactamase II is still not known in detail, analogies may be drawn with the common role of the ZnII-OH- species in other zinc enzymes such as carbonic anhydrase.
- Koike, Tohru,Takamura, Masahiro,Kimura, Eiichi
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p. 8443 - 8449
(2007/10/02)
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- Thiazolidine Ring Opening in Penicillin Derivatives. Part 1. Imine Formation
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The rate of epimerisation of (3S,5R,6R)-benzylpenicilloic acid at C-5 shows three distinct dependencies upon pH in aqueous solution.Below pH 6 the rate shows a sigmoidal dependence upon pH, whereas it is pH-independent between pH 6 and 12, and above pH 12 the rate is hydroxide-ion dependent.These different regions of pH dependence are interpreted in terms of three mechanistic pathways all of which involve opening the thiazolidine ring by C-S bond fission and re-closure to generate the epimer.At low pH the reaction occurs by unimolecular ring opening of the S-conjugate acid which is kinetically equivalent to the N-conjugate acid of pKa 5.14.The pH-independent pathway involves formation of a zwitterion by unimolecular opening of the neutral thiazolidine.At high pH the unprotonated imine intermediate is formed by concerted hydroxide-ion-catalysed ring opening.The mono- and di-methyl esters of benzylpenicilloate also epimerise at C-5.At low pH the rates are similar for all three compounds but above pH 6 the mono- and di-esters are, respectively, 21 and 1700 times less reactive than the dianion of the diacid.
- Davis, Andrew M.,Jones, Mark,Page, Michael I.
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p. 1219 - 1223
(2007/10/02)
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- A liquid chromatographic study of stability of the minor determinants of penicillin allergy: A stable minor determinant mixture skin test preparation
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Various skin test reagents supplying minor determinants for detecting penicillin hypersensitivity have been examined by high-performance liquid chromatography (HPLC) for composition and stability. HPLC systems capable of separating and determining the four diastereoisomers of benzyl-D-penicilloic acid and the two benzyl-D-penilloic acids were developed for this purpose. The 'simple skin test reagent', consisting of an aged partial alkaline hydrolysate of penicillin, is possibly an adequate source of (5R,6R)-benzyl-D-penicilloate whereas the 'simple skin test reagent,' consisting of aged aqueous solution of penicillin, is a questionable source of this compound. A modified Levine, Voss, Redmond, and Zolov minor determinant mixture (MDM) reagent and the components (5R,6R)-benzyl-D-penicilloate and (5R)-benzyl-D-penilloate have been found to be highly labile in aqueous solution, giving rise to a mixture of diastereoisomers. The tendency to epimerize at C-5 was a prominent feature of (5R,6S)- and (5S,6R)- as well as (5R,6R)-benzyl-D-penicilloic acids. The MDM reagent has been prepared in single-dose ampules as a dried, lyophilized powder that can be stored without change and used as needed. Lyophilized MDM has served as a satisfactory substitute for freshly prepared MDM in several individuals with MDM-positive history and, in a recent clinical study, evaluating the question of penicillin skin test sensitization. This convenient, stable, single-dose form of the MDM reagent should facilitate skin testing for penicillin sensitivity.
- Ressler,Neag,Mendelson
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p. 448 - 454
(2007/10/02)
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- Epimerization of benzylpenicilloic acid in alkaline media
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5R,6R-Benzylpenicilloic acid was found to epimerize slowly in alkaline media to 5S,6R-benzylpenicilloic acid until equilibrium was established. Epimerization proceeded via the imine tautomer of penamaldic acid rather than the enamine form and was found to favor the 5S,6R-epimer at equilibrium. The conversion process was monitored using both reverse-phase high-performance liquid chromatography and NMR spectroscopy.
- Ghebre Sellassie,Hem,Knevel
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p. 125 - 128
(2007/10/02)
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- The Micelle-catalysed Hydrolysis of Benzylpenicillin
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Micelles of cetyltrimethylammonium bromide catalyse the alkaline hydrolysis of benzylpenicillin with a rate enhancement of ca. 50-fold.Howewer, the rate of reaction is inhibited by increasing concentrations of hydroxide ion and penicillin anion.A saturation phenomenon is observed with increased concentration of surfactant.Attempts are made to determine the binding- and rate-constants using existing kinetic models.These are not completely satisfactory and a model is proposed which assumes that both hydroxide ion and penicillin have to be bound to the micelle for reaction to occur.Bromide, chloride, acetate, fluoride, and benzylpenicilloate ions all inhibit the micellar catalysis.
- Gensmantel, Nigel P,Page, Michael I.
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p. 147 - 154
(2007/10/02)
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- The Chemical Reactivity of Penicillins and Other β-Lactam Antibiotics
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The rates of the acid catalysed hydrolysis of penicillins and cephalosporins are linear in Ho and, unlike other amides, show no rate maximum with increasing acidity.Electron-withdrawing substituents at C-6 in penicillins decrease the rate of hydrolysis with a ρI of ca. 4 and they decrease the rate when attached to the amine leaving group.The acylamido-group at C-6 in penicillins, but not at C-7 in cephalosporins, exhibits neighbouring group participation with a rate enhancement of ca. 103.The absence of penicillenic acid formation from benzylpenicillin in acidic solution is not due to the ionisation of the carboxy-group.These observations are rationalised by a scheme involving N-protonation and formation of an acylium ion intermediate.The alkaline hydrolysis of penicillins proceeds 102 faster than a β-lactam after correction for substituent effects.There is no evidence for substantial inhibition of amide resonance in the bicyclic β-lactam antibiotics, little evidence to indicate extra strain in these systems and no evidence that expulsion of the leaving group at C-3 in cephalosporins occurs in the transition state.
- Proctor, Philip,Gensmantel, Nigel P.,Page, Michael I.
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p. 1185 - 1192
(2007/10/02)
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- Intra- and Inter-molecular Catalysis in the Aminolysis of Benzylpenicillin
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The rate law for the aminolysis of benzylpenicillin in water is reported.The Bronsted β-values for the uncatalysed, the amine-catalysed, and hydroxide-ion catalysed reactions are 1.0, 1.09, and 0.96 respectively.This indicates that in the transition states for all three pathways the amine nucleophile contains a unit positive charge which is consistent with the formation of a tetrahedral intermediate.Intramolecular general base catalysis occurs with the reaction of ethylenediamine and, despite the importance of general base catalysis in the aminolysis reaction, the effective concentration of the catalysing base is only ca. 1M, which is attributed to the 'loose' transition state involved in intermolecular catalysis.Intramolecular general acid catalysis occurs with the reaction of ethylenediamine monocation.This suggests that nucleophilic attack takes place from the least hindered α-side.
- Morris, Jeffrey J.,Page, Michael I.
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p. 212 - 219
(2007/10/02)
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