Theoretical Calculations of β-Lactam Antibiotics. Part VI. AM1 calculations of alkaline hydrolysis of clavulanic acid

The gas-phase basic hydrolysis of clavulanic acid ( a ) was studied by using the AM1 semi-empirical method. The results obtained show that the hydroxyethylidene side chain at C(2) is pivotal to the stability of the different reaction products involved. The products with an open oxazolidine ring are more stable than those with a closed ring fused to the β-lactam ring. This behaviour differs from that of penicillins and cephalosporins where the most stable degradation products are those with an intact thiazolidine or dihydrothiazine ring, respectively, fused to the β-lactam ring. The different chemical reactivity of clavulanic acid relative to penicillins and cephalosporins could explain the disparate behaviour of the latter two types of compound towards β-lactamases. Once the acyl-enzyme intermediate of clavulanic acid has been formed, it can evolve with cleavage of the oxazolidine ring to form a difficult to deacylate compound.     

Study of the Alkaline Hydrolysis of the Azetidin-2-one Ring by ab initio Methods: Influence of the solvent

A comprehensive study of the alkaline hydrolysis of the β-lactam ring of azetidin-2-one was carried out using ab initio molecular-orbital calculations at the RHF/6-31 + G* level. The influence of the solvent on this reaction was investigated by using the reaction field method (SCRF); the solvent was found to suppress the interference of some gas-phase reactions and allow the presence of a transition state to be detected as the nucleophile approaches the β-lactam ring. The transition state corresponds to a structure where the OH^? group lies at a distance of 1.927 Å from the C?O group of the β-lactam ring and exhibits a potential barrier of 13.6 kcal/mol.     

Alkaline hydrolysis of cephaloridine: An 1H?NMR study. Temperature dependence of the rate constants

A kinetic study on the basic hydrolysis of cephaloridine at pD= 10.5 was carried out by using the ¹H? NMR technique. Epimerization at H₇, a nucleophilic attack of hydroxyl ion on the β-lactam carbonyl group followed by the release of the pyridine molecule, and isomerization of the double bond at position 3 in the dihydrothiazine ring were the major reactions observed. Based on the results obtained, it should be emphasized that the presence of a pyridine group at 3′ results in a slightly increased formation constant for the exo methylene compound relative to other cephalosporins with different substituents at that position. The activation energy for the epimerization constant and the cleavage of the β-lactam ring at pD 10.5 was 21.2 kcal/mol. © 1993 John Wiley & Sons, Inc.     

HPLC and 1H-NMR Studies of Alkaline Hydrolysis of Some 7-(Oxyiminoacyl)cephalosporins

Alkaline hydrolysis (pH 10.5) of the three 7-(oxyiminoacyl)cephalosporins 1a–c (cefuroxime, ceftazidime, and ceftriaxone) was studied at 37° using HPLC and ¹H-NMR techniques. The 7-epicephalosporin 2 , the 3-methylidene compound 3 , and the 6-epimer 4 of the 3-methylidene compound 3 were identified for each cephalosporin as the major degradation products under the conditions used; ceftazidime ( 1b ) yielded also the Δ²-isomer 5b (Scheme 1). A kinetic scheme was developed to account for the production of these compounds, and the different kinetic constants involved in the process were calculated. The experimental results show that the presence of a pyridinio group at position C–C(3) favours the appearance of the Δ²-isomer, which was detected mainly in cephalosporins bearing an ester function at C(4). The presence of an oxyimino group at C? CONH? C(7) facilitates epimerization at C(7) (→ 2 ), whereas that of an electron-withdrawing group at C? C(3) results in a increased formation constant for the 3-methylidene compound 3 . The 3-methylidene compounds 3a–c produced by the three cephalosporins on cleavage of the β-lactam ring all underwent epimerization at C(6) to yield the corresponding 6-epimer 4 .     

Theoretical calculations of β-lactam antibiotics. III. AM1, MNDO,and MINDO/3 calculations of hydrolysis of β-lactam compound (azetidin-2-one ring)

Semiempirical AM1, MINDO/3, and MNDO methods have been used in the study of the alkaline hydrolysis of β-lactam antibiotics through a base-catalyzed, acyl-cleavage, bimolecular mechanism. In this work, the hydroxyl ion has been chosen as nucleophilic agent and the azetidin-2-one ring like a model of β-lactam antibiotic. The MINDO/3 method does not predict correctly the energies of small rings. This, together with the fact that, like MNDO, it cannot detect the occurrence of hydrogen bonds, gives rise to uncertain estimates of energy barriers. The AM1 method can be considered the most suitable for studying the hydrolysis of β-lactam compounds.     

Kinetic Study on Cephamycin C Degradation

Cephamycin C (CepC) is a β-lactam antibiotic that belongs to the cephalosporin class of drugs. This compound stands out from other cephalosporins for its greater resistance to β-lactamases, which are enzymes produced by pathogenic microorganisms that present a major mechanism of bacterial resistance to β-lactam antibiotics. Cephamycin C is produced by the bacterium Streptomyces clavuligerus. Knowledge about the stability of the compound under different values of pH is important for the development of the process of production, extraction, and purification aimed at obtaining higher yields. Therefore, the stability of cephamycin C under different pH levels (2.2, 6.0, 7.0, 7.6, and 8.7) at 20 °C was evaluated in this study. Ultrafiltered broth from batch fermentations of S. clavuligerus was used in the trials. The results indicated that cephamycin C is a more stable compound than other β-lactam compounds such as penicillin and clavulanic acid. A higher degradation rate was observed at very acidic or basic pH levels, while this rate was lower at quasi-neutral pH levels. After 100 h of trial, the initial CepC showed 46 % degradation at pH 2.2, 71 % degradation at pH 8.7, and varied from 15 to 20 % at quasi-neutral pH levels.     

Metallo-β-lactamase-catalyzed hydrolysis of cephalosporins: some mechanistic insights into the effect of heterocyclic thiones on enzyme activity

The hydrolysis of β-lactam antibiotics using zinc-containing metallo-β-lactamases (mβl) is one of the major bacterial defense systems. These enzymes can catalyze the hydrolysis of a variety of antibiotics including the latest generation of cephalosporins, cephamycins, and imipenem. It is shown in this paper that the cephalosporins having heterocyclic -SR side chains are less prone to mβl-mediated hydrolysis than the antibiotics that do not have such side chains. This is partly due to the inhibition of enzyme activity by the thione moieties eliminated during hydrolysis. When the enzymatic hydrolysis of oxacillin was carried out in the presence of heterocyclic thiones such as MTT, MDT, DMETT, and MMA, the catalytic activity of the enzyme was inhibited significantly by these compounds. Although the heterocyclic -SR moieties eliminated from the β-lactams upon hydrolysis undergo a rapid tautomerism between thione and thiol forms, these compounds act as thiolate ligands toward zinc(II) ions. The structural characterization of two model tetranuclear zinc(II) thiolate complexes indicates that the -SR side chains eliminated from the antibiotics may interact with the zinc(II) metal center of mβl through their sulfur atoms.     

Evolution of class C β-lactamases: factors influencing their hydrolysis and recognition mechanisms

The most common bacterial resistance mechanism to β-lactam antibiotics is the production of β-lactamases. So far, β-lactamases have been classified into four different classes, three of them (A, C and D) have a serine in the active site as the nucleophilic group, which attacks to lactam antibiotic. Despite the large number of kinetic and theoretical studies and many native and complexed β-lactamases crystal structures, the mechanism by which they act is not well understood. The aim of this review is to show the different hypotheses which have been proposed to explain the hydrolysis mechanisms for class A and C lactamases and to cast light onto the interactions between the antibiotic and the Enterobacter cloacae P99 (a class C β-lactamase) in the Henry-Michaelis complex formed previous to the serine attack. Knowledge of these crucial points is essential for obtaining new β-lactam antibiotics not vulnerable to β-lactamases in order to minimize bacterial resistance.     

Rapid determination of cephalosporins with an immobilized enzyme reactor and sequential subtractive spectrophotometric detection in an automated flow-injection system

A flow-injection system is described for the rapid automated determination of different cephalosporins in aqueous solution. Measurement is based on a selective enzymatic cleavage of the cephalosporin-β-lactam ring in a small immobilized enzyme reactor, which contains highly purified cephalosporinase covalently bound to activated glass beads. Samples are injected into a phosphate buffer stream. The opening of the β-lactam ring is accompanied by a significant decrease in the ultraviolet (u.v.) absorbance. The difference between the absorbances of untreated and degraded cephalosporin is measured at 254 nm and is related linearly to cephalosporin concentration over the range 10–800 μg ml^?1. Several commercially available cephalosporins including some β-lactamase-resistant types were examined. The accuracy of the enzyme reactor/flow injection system was evaluated by comparison with h.p.l.c. results; the correlation was good. The relative standard deviation, evaluated from 15 consecutive injections of the same sample, was 0.7%; 2 μg ml^?1 cephalosporin C was the minimal detectable concentration. A single determination took about 2 min; sample throughput is 30 h^?1. Different β-lactamases were examined for enhancement of substrate selectivity.     

New beta-lactam antibiotics. Cephem derivatives with electron withdrawing substituents at position 3 (author's transl)]

New β-lactam antibiotics. Cephem derivatives with electron withdrawing substituents at position 3 Oxidation of the 3-formyl-2-cephem compound 1 according to Corey [6] gave 2-cephem-3-carboxylic esters 4a, b, c (Scheme 1), which proved to be useful intermediates for the synthesis of cephalosporins bearing in position 3 a methoxycarbonyl group (10a, b, c, d / Scheme 2) or a carboxy group (20, 25, 30/Schemes 3, 4). The 3-formyl-3-cephem compounds 31a, b could be transformed into cyano- (33a) or methoxyiminomethyl- (36a, c, d) cephems (Scheme 5), which represent further examples of cephalosporins with electron withdrawing groups in position 3.     

Ab initio study of β-lactam antibiotics. II. Potential energy surface for the amidic CN bond breaking in the 3-cephem + OH− reaction and comparison with the β-lactam + OH− reaction

The potential energy surface for the β-lactam amidic CN bond breaking in the 3-cephem + OH^? reaction was investigated by using the ab initio Hartree—Fock method with a 9s6p/7s3p/3s basis set. The investigated reaction is a model of the reaction between an antibiotic cephalosporin and an enzymatic nucleophilic group, this last reaction being related to the mode of action of β-lactam antibiotics. The minimum-energy reaction path is characterized by a tetrahedral intermediate ≈ 116 kcal mol^?1 more stable than the reagents, by a barrier which corresponds to the partial breaking of the amidic bond and is ≈ 7 kcal mol^?1 above the intermediate and by a product ≈ 31 kcal mol^?1 more stable than the intermediate. The analysis of the wavefunction along the reaction path and the comparison with the β-lactam + OH^? reaction pointed out the role of electron-withdrawing groups on the height of the barrier and the role of intramolecular hydrogen bonds on the structure and energy of the product. The calculations suggest a model of the antibiotic activity of cephalosporins which is compared with previous qualitative pictures.     

Assessment of the absolute configuration of a series of (3R)-3-hydroxy-3-alkyl-β-lactams

The absolute stereochemistry of a series of (3R)-3-hydroxy-3-alkyl-β-lactams has been determined by circular dichroism and NMR spectroscopies. The sign of the circular dichroism band between 250 and 220 nm was related to the stereochemistry by applying the β-lactam sector rule. The NMR analysis unambiguously determines the relative configuration at C3 and C4 of the β-lactam ring. The reliability of the method has been proved by X-ray analysis of two of the examined compounds. The obtained results are in agreement with the mechanism proposed for the employed synthetic route.     

The efficiency of C-4 substituents in activating the beta-lactam scaffold towards serine proteases and hydroxide ion

The presence of a leaving group at C-4 of monobactams is usually considered to be a requirement for mechanism-based inhibition of human leukocyte elastase by these acylating agents. We report that second-order rate constants for the alkaline hydrolysis and elastase inactivation by N-carbamoyl monobactams are independent of the pKa of the leaving group at C-4. Indeed, the effect exerted by these substituents is purely inductive: electron-withdrawing substituents at C-4 of N-carbamoyl-3,3-diethylmonobactams increase the rate of alkaline hydrolysis and elastase inactivation, with Hammett pI values of 3.4 and 2.5, respectively, which indicate the development of a negative charge in the transition-states. The difference in magnitude between these pI values is consistent with an earlier transition-state for the enzymatic reaction when compared with that for the chemical process. These results suggest that the rate-limiting step in elastase inactivation is the formation of the tetrahedral intermediate, and that beta-lactam ring-opening is not concerted with the departure of a leaving group from C-4. Monobactam sulfones emerged as potent elastase inhibitors even when the ethyl groups at C-3, required for interaction with the primary recognition site, are absent. For one such compound, a 1 : 1 enzyme-inhibitor complex involving porcine pancreatic elastase has been examined by X-ray crystallography and shown to result from serine acylation and sulfinate departure from the beta-lactam C-4.     

Theoretical Study of the Alkaline Hydrolysis of Tricyclic Carbapenem

Alkaline hydrolysis is a good test to evaluate the properties of β‐lactam compounds and derivatives. In this work, a RHF/6‐31+G*//RHF/6‐31+G* theoretical study of the mechanism controlling the alkaline hydrolysis of sanfetrinem was conducted. The geometric properties of this compound are consistent with an intrinsic reactivity similar to that of other β‐lactams including penicillins and cephalosporins. Also, similarly to cephalosporins, the MeO group provides an alternative route for the hydrolysis mechanism.     

QM/MM studies of monozinc β-lactamase CphA suggest that the crystal structure of an enzyme-intermediate complex represents a minor pathway

QM/MM studies of the hydrolysis of a β-lactam antibiotic molecule (biapenem) catalyzed by a monozinc β-lactamase (CphA) have revealed the complete reaction mechanism and shown that an experimentally determined enzyme-intermediate complex is a stable intermediate or product in a minor pathway.     

Silylation-desilylation of propargyl amides: rapid synthesis of functionalised aldehydes and β-lactams

Propargyl functionalised β-silylalkenals were easily prepared starting from suitable propargyl compounds by a silylformylation process. In particular the use of propargyl tosyl amides allowed the synthesis of α,β-unsaturated aldehydes through a two-step sequence of silylformylation-desilylation reactions. TBAF was employed to induce the desilylation process that was performed under very mild experimental conditions and occurred along with an elimination step of the tosylamido moiety affording 2-methylaryl-2-alkenals with good yields and stereoselectivity. When the tosyl amides were reacted with a hydrosilane in the presence of catalytic amounts of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) α-silylmethylene-β-lactams were synthesised through a silylcarbocyclisation process. A high chemoselectivity towards the β-lactam was observed when dialkyl propargyl amides were employed. The obtained β-lactams were easily transformed into the corresponding methylaryl-β-lactams by fluoride induced aryl migration-desilylation with total retention of configuration of the migrating group and complete stereoselectivity towards the more stable β-lactam (E)-isomer.     

Modeling β-lactam interactions in aqueous solution through combined quantum mechanics–molecular mechanics methods

In this article, we have carried out a series of theoretical computations intended to analyze the interactions of β-lactam compounds in aqueous solution. The final aim is to rationalize the influence of the medium on β-lactam antibiotics reactivity. In particular, the hydrolysis reaction has been studied because of the considerable interest due to its relationship with resistance mechanisms developed by bacteria. The study is extended to the simplest β-lactam molecule, propiolactam or 2-azetidinone, and to the corresponding hydroxylated complex (resulting from the addition of a hydroxyl anion to the carbonyl group) that plays a crucial role in hydrolysis processes. Molecular Dynamics simulations have been carried out using a hybrid quantum mechanics–molecular mechanics potential: the solute is described using the density functional theory, whereas water solvent molecules are treated classically. This represents a sophisticated computational level which, compared to usual force-field simulations, has the advantage of allowing a detailed analysis of solute's electronic properties. The discussion of results is focused on the role played by solute–solvent hydrogen bonds and solvent fluctuations on solute's structure. ©1999 John Wiley & Sons, Inc. J Comput Chem 20: 1401–1411, 1999     

A Selective Colorimetric Method for the Determination of Penicillins and Cephalosporins with α-Aminoacyl Functions

ABSTRACT

A simple, sensitive and selective colorimetric method is described for the assay of ampicillin, amoxicillin, cephalexin, cefadroxil and cefaclor in their pharmaceutical preparations. The method is based on measuring the color obtained when the alkaline degradation products of these agents are allowed to react with ascorbic acid. The factors affecting the color generation and determination were studied and optimized. The reaction is selective to β-lactam antibiotics having amino acid side-chains with free amino functions and thus allow interference-free quantitation of some preparations containing these agents in combination with other β-lactam agents. The procedure is also successfully adopted as stability-indicating method for cephalosporins. A tentative mechanism of the color reaction is proposed.     

Activating water: important effects of non-leaving groups on the hydrolysis of phosphate triesters

The high rate of spontaneous hydrolysis of tris-2-pyridyl phosphate (TPP) is explained by the activating effects of the non-leaving ("spectator") groups on P-OAr cleavage, and not by intramolecular catalysis. Previous work on phosphate-transfer reactions has concentrated on the contributions to reactivity of the nucleophile and the leaving group, but our results make clear that the effects of the non-leaving groups on phosphorus can be equally significant. Rate measurements for three series of phosphate triesters showed that sensitivities to the non-leaving groups are substantial for spontaneous hydrolysis reactions, although significantly smaller for reactions with good nucleophiles. There are clear differences between triaryl and dialkyl aryl triesters in sensitivities to leaving and non-leaving groups with the more reactive triaryl systems showing lower values for both β(LG) and β(NLG). Intramolecular catalysis of the hydrolysis of TPP by the neighbouring pyridine nitrogens is insignificant, primarily because of their low basicity.     

Kinetic and theoretical studies on the mechanism of alkaline hydrolysis of DNA

The reaction mechanism of alkaline hydrolysis of DNA has been investigated by kinetic analysis and density-functional-theory calculation. The rates of hydrolysis of thymidine 3'-monophosphate esters (including thymidylyl(3'-5')thymidine (Tp-OT)) monotonically decrease as the leaving groups get poorer. According to the theoretical calculation in which the solvent effects are incorporated, no intermediate is formed in the course of the reaction. In the alkaline hydrolysis of the activated Tp-OT analogues having good leaving groups, the 3',5'-cyclic monophosphate of thymidine is concurrently formed through the intramolecular attack by the 5'-alkoxide ion. In the hydrolysis of the native dinucleotide, however, this side reaction does not occur, since the transition state leading to the departure of its poor leaving group cannot be formed due to conformational restraint. These arguments are supported by the theoretical analysis on the hydrolysis of both dimethyl phosphate and its O(bridging)-->S substituted analogue.