The thiolysis mechanism of benzylpenicillin has been determined by 1H-NMR and HPLC. The degradation of benzylpenicillin was accelerated when the thiol used presented a b-group capable of acting as a general acid catalyst, such as a-monothioglycerol, 2-mercaptoethanol and mercaptoethylamine. With these thiols, after the formation of the thioester, an intramolecular acyl transfer reaction occurs at a pH far below the pKa of the group acting as a general acid catalyst, which shows that the proton transfer has already occurred, probably in a concerted mechanism with the nucleophilic attack. Rate constants were calculated. The system can be taken as a simple model of the general acid catalyst in serine and cysteine proteases.

Thiolysis of benzylpenicillin has been investigated by HPLC and 1H-NMR techniques. Thiols catalyse the hydrolysis of benzylpenicillin through the formation of a thioester intermediate. The catalytically reactive form of the thiol has been demonstrated to be the thiolate anion. Variation of reactivity with changing basicity of the thiolate anion generates a Bronsted value of 0.96, indicating that the breakdown of the tetrahedral intermediate is the rate-limiting step, as occurs in aminolysis and alcoholysis. Solvent kinetic isotope effects of 2.2-2.4 indicate that the solvent, water, probably acts as a general acid catalyst in the breakdown of the tetrahedral intermediate. PM3 theoretical calculations support the proposal that breakdown of the tetrahedral intermediate ir rate-limiting. The experimental activation energies for the thiolysis of benzylpenicillin vary from 6.9 to 10.4 kcal/mol.

The alkaline hydrolysis of a thio-b-lactam in the gas phase was examined in the light of RHF and DFT ab initio calculations. The solvent effect was considered via IPCM computations. The tetrahedral intermediate for the thio-b-lactam studied is unstable, so the compound evolves directly to the corresponding thio-azethidin-2-one open-ring with cleavage of the C-S bond. The end-products obtained bear a carbamate group, which suggests that the thio-b-lactam might be an effective inhibitor for b-lactamases.

Various mechanisms for the alkaline hydrolysis of an aza-b-lactam in the gas phase were studied by ab initio calculations at the RHF/6-31+G^*//RHF/6-31+G^*, MP2/6-31+G^*//MP2/6-31+G^* and B3LYP/6-31+G^*//B3LYP/6-31+G^* levels. Solvent effects were considered via IPCM (isodensity polarizable continuum model) calculations at the IPCM/6-31+G^*//RHF/6-31+G^* level.<The alkaline hydrolysis of b-lactams begins with a nucleophilic attack of the hydroxyl ion on the carbonyl of the b-lactam ring. The tetrahedral intermediate thus formed undergoes cleavage of the C₇-N₄ bond to give the reaction end products. In addition to the typical cleavage reaction, the b-lactam studied can undergo opening at the C₇-N₆ bond (Scheme 1). Both processes have a similar activation energy that varies slightly depending on the particular computation method used. The most stable end products are those formed via the typical mechanism. In any case, both mechanisms yield products possessing a carbamate group, which suggests that the starting aza-b-lactam might be an effective inhibitor for b-lactamases.

Various mechanisms for the alkaline hydrolysis of oxo-b-lactam compound were analyzed on the basis of RHF/6-31+G*//RHF/6-31+G* calculations in order to identify potential differences with classical b-lactam antibiotics. Changes in the tetrahedral intermediate were studied via the cleavage not only of the bond between atoms at 7 and 4 as in classical b-lactam antibiotics but also of that between those at 7 and 6, which was previously put forward as a plausible pathway for the hydrolysis of these compounds. Cleavage of the 7–6 bond was found to be the energetically more favorable pathway in this compound. Opening of the b-lactam ring at the 7–6 bond yields especially stable carbamates, which suggests a potential inhibitory action in serine-b-lactamases. Based on the computations, those hydrolysis products where the five-membered ring is opened by cleavage of the C₅–S₁ bond are highly stable.

 

A comprehensive study of the elimination reaction of the pyrazolidinone ring in gas phase was carried out from ab initio calculations performed using a 6-31+G* basis set. All the structures studied were also optimized by using Moller-Plesset's perturbation theory. In the gas phase, the attack of a hydroxyl group on a carbonyl compound may involve an interaction between the hydroxyl ion and a proton, followed by elimination of the proton and formation of a water molecule. This reaction may interfere with the nucleophilic attack to the carbonyl group, which is related to the mechanism of enzymatic acylation in lactams. The elimination reaction on the pyrazolidinone ring begins with the withdrawal of the hydroxyl group from the initial product of the nucleophilic attack (a tetrahedral intermediate), which is followed by the abstraction of a proton from the pyrazolidinone ring to produce a water molecule. We identified three possible conformations of the tetrahedral intermediate of the nucleophilic attack on the pyrazolidinone ring and studied the elimination reaction for each structure.

Various potential mechanisms for the alkaline hydrolysis of an oxo-b-lactam in the gas phase (Scheme 1) were examined in the light of ab initio data obtained at the RHF/6-31+G^*//RHF/6-31+G^* and MP2/6-31+G^*//MP2/6-31+G^* levels. The influence of the solvent was also examined from IPCM (isodensity polarizable continuum model) computations at the RHF/6-31+G^* level. In penicillins and cephalosporins, alkaline hydrolysis begins with a nucleophilic attack on the carbonyl group of the b-lactam ring, which is followed by cleavage of the C₇–N₄ bond. In the oxo-b-lactam studied, the process additionally involves cleavage of the C₇–O₆ bond in the ring. In fact, this cleavage is subject to a very small activation energy: as little as 0.21 kcal/mol versus the 14.15 kcal/mol for the typical cleavage energy (based on MP2/6-31+G^*//MP2/6-31+G^* calculations) for C₇–N₄ bond. In addition, the hydrolysis end products are more stable than those resulting from the typical cleavage. Consequently, the alkaline hydrolysis involving cleavage of the C₇–O₆ bonds is kinetically and thermodynamically more favorable than the classical hydrolysis mechanism for peni­cillins and cephalosporins. This suggests that oxo-b-lactams might act as b-lactamase inhibitors.

Various mechanisms for the alkaline hydrolysis of aza-b-lactam, oxo-b-lactam and thio-b-lactam compounds were analysed on the basis of PM3 calculations in order to identify potential differences with classical b-lactam antibiotics. Changes in the tetrahedral intermediate were studied via the cleavage not only of the bond between atoms at 7 and 4 as in classical b-lactam antibiotics but also of that between those at 7 and 6, which was previously put forward as a plausible pathway for the hydrolysis of these compounds. Cleavage of the 7–6 bond was found to be the energetically more favourable pathway in the three compounds studied, both in the gas phase and in solution. Opening of the b-lactam ring at the 7–6 bond yields especially stable carbamates, which suggests a potential inhibitory action in serine-b-lactamases. Based on the computations, those hydrolysis products where the five-membered ring is opened by cleavage of the C₅–S₁ bond are highly stable.

Semiempirical calculations (PM3) have been used to investigate the reaction mechanism (BAC2) of the alkaline hydrolysis of N-methylazetidin-2-one. This mechanism involves the nucleophilic attack of a hydroxyl ion on the carbonyl carbon to give a tetrahedral complex followed by cleavage of the C-N bond and proton transfer to form the final product. The influence of the solvent in this process has been analyzed using the supenmolecular approach with a water solvation sphere of 20 molecules around the solute. The results obtained have been compared with those based on a continuum treatment of the solvent with semiempirical and ab initio methodology. The potential barrier of 17.5 kcal/mol due to the attack of the nucleophile is very close to the experimental value (16.1 kcal/mol) and the final product is about 52 and 27 kcal/mol more stable than the reactives and the tetrahedral intermediate, respectively

A complete study on the acidic hydrolysis of the azetidin-2-one ring, an essential component of ß-lactam compounds, has been carried out by means of ab initio calculations by using a 6-31G** type basis set. The former reaction has been studied by means of an A-l type unimolecular mechanism, characterized by an N-protonation followed by an opening of the ring and further addition of water to the carbonyl group. The system involving the azetidin-2-one ring, the H30+ ion and a water molecule has been considered, where three transition states have been identified, being the barriers corresponding to the addition of H+ to the nitrogen and the addition of water to the carbonyl group being practicalty negligible (first and third reaction steps, respectively). The energy barrier for the opening of the ring (second reaction step) was 14.23 kcal/mol

A complete study of basic hydrolysis of the pyrazolidinone ring by ab initio calculations at RHF/6-31+G*//RHF/6-31+G* and MP2/6-31+G*//MP2/6-31+G* has been carried out.

The alkaline hydrolysis has been studied through a B_AC2 mechanism, characterized by a nucleophilic attack of the hydroxyl group on the carbonyl of the g-lactam ring, formation of the tetrahedral intermediate and cleavage of the C2-N3 bond to yield the final reaction product. In the gas phase, the interaction of OH- with the carbonyl carbon to form a tetrahedral intermediate takes place without any barrier height.

Two possible mechanisms have been considered for the transfer of the hydroxyl hydrogen to the nitrogen of the g-lactam. A stepwise mechanism involving the cleavage of the C2-N3 bond and subsequent transfer of the hydrogen to the g-lactam nitrogen, and a concerted mechanism. MP2/6-31+G*//MP2/6-31+G* barrier height are 32.72 and 25.64 kcal/mol respectively.

The elimination reaction, which in the gas phase may interfere with the nucleophilic attack, has also been studied.

A study of the alkaline hydrolysis of a g-lactam compound (pyrazolidinone ring) by ab-initio calculations (RHF/6-31+G*//RHF/6-31+G* and MP2/6-31+G*//MP2/6-31+G*) in gas phase has been carried out. The alkaline hydrolysis has been studied through a B_AC2 mechanism, with an initial attack of a hydroxyl group at the carbonyl of the pyrazolidinone, formation of the tetrahedral intermediate and cleavage of the C₂-N₃ bond. Two possible mechanisms have been considered for the transfer of the hydroxyl hydrogen to the nitrogen of the pyrazolidinone, the first one concerted and a second stepwise mechanism. The value of the activation energy for the C-N bond cleavage happens to be greater in the stepwise process than in the concerted mechanism (34.79 kcal/mol and 27.58 kcal/mol by MP2/6-31+G*//MP2/6-31+G* calculations respectively). The water-assisted hydrolysis of the pyrazolidinone ring has been also studied, being observed a slightly decrease of the potential barrier in the opening of the tetrahedral intermediate. The activation energy of this process is of 21.62 kcal/mol (MP2/6-31+G*//MP2/6-31+G*).

The rate of degradation of 6-epi-ampicillin in acidic, neutral, and alkaline aqueous solutions was followed at 35ºC and an ionic strength of 0.5 mol dm^-3 (KCl) by high-performance liquid chromatography (HPLC) and spectrophotometric assays. Pseudo-first-order rate constants were determined in a variety of buffer solutions, and the overall pH-rate profile was obtained by extrapolation to zero buffer concentration. The hydrolysis of 6-epi-ampicillin is subject to acid and hydroxide-ion catalysis and, for a penicillin, an unusual pH-independent reaction. Intramolecular general base-catalyzed hydrolysis by the side chain amido group is proposed to explain the enhanced rate of neutral hydrolysis of 6-epi-ampicillin and cephalosporins. The b-lactam of 6-epi-ampicillin also undergoes intramolecular aminolysis by nucleophilic attack of the 6-a side chain amino group to give a stable piperazine-2,5-dione derivative. The low effective molarity for intramolecular aminolysis of only 40 M is partly attributed to the unfavorable trans to cis isomerization about the 6-amide side chain required for ring closure. Theoretical calculations show that the intramolecular aminolysis of 6-epi-ampicillin nucleophilic attack occurs from the a-face of the b-lactam ring with an activationenergy of 14.4 kcal/mol.

A theoretical study of the gas-phase alkaline hydrolysis of penicillin G on the assumption of a BAC2 mechanism is reported. Various semiempirical methods were used to determine the influence of different parameterizations on the process. Among the most salient results obtained, the standard AM1 method predicted opening of the thiazolidine ring to yield the corresponding imine and enamine structures.

A complete study of the alkaline and acidic hydrolysis of the ß-lactam ring of azetidin-2-one was carried out using ab initio molecular orbital calculations at the RHF/6-3l+G* and RHF/6-31G** levels, respectively. Alkaline hydrolysis has been studied through a BAC2 mechanism characterized by a nucleophilic attack on the ß-lactam carbonyl group, formation of the tetrahedral intermediate and cleavage of the C-N bond until the formation of the final product of the reaction, this being the limiting step of the reaction. On the other hand, the acidic hydrolysis has been studied by means of a A- 1 type unimolecular mechanism, characterized by a nitrogen-protonation followed by an opening of the ring and further addition of water to the carbonyl group. The system involving the azetidin-2-one ring, the H30+ ion and a water molecule has been considered. Three transition states have been identified; the barriers corresponding to the addition of H+ to the nitrogen and the addition of water to the carbonyl group are practically negligible (first and third reaction steps, respectively).

A comprehensive study of the alkaline hydrolysis of the ß-lactam ring of azetidin-2-one was carried out using ob 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 sorne gas-phase reactions and allow the presence of a transition state to be detected as the nucleophile approaches the ß-lactam ring. The transition state corresponds to a structure where the OH- group lies at a distance of 1.927 A from the C=O group of the ß-lactam ring and exhibits a potential barrier of 13.6 kcal/mol.

We used semiempirical and ab initio calculations to investigate the nucleophilic attack of the hydroxyl ion on the ß-lactam carbonyl group. Both allowed us to detect reaction intermediates pertaining to proton-transfer reactions. We also used ab initio calculations and the PM3 semiempirical rnethod to investigate the influence of the solvent on the process. The AMSOL method predicts the occurrence of a potential energy barrier of 20.7 kcal mol-1 due to the desolvation of the hydroxyl ion in approaching the ß-lactam carbonyl group. Using the supermolecular approach and a water solvation sphere of 20 molecules around the solute, the potential energy barrier is lowered to 17.5 kcal/mol. Ab initio calculations using the SCRF method predict a potential energy barrier of 13.6 kcal/mol. These three values, especially the last two, are very close to the experimental value of 16.7 kcal/mol.

 

We used semi-empirical and ab initio calculations to investigate the nucleophilic attack of the OH ion on the ß-lactam carbonyl group. Both allowed us to detect reaction intermediates pertaining to proton-transfer reactions rather than the studied reaction. We also used the PM3 semi-empirical method to investigate the influence of the solvent on the process. The AMSOL method predicts the occurrence of a potential barrier of 20.7 kcal/mol due to the desolvation of the hydroxyl ion in approaching the ß-lactam carbonyl group. Using the supermolecular approach and a H20 solvation sphere of 20 molecules around the solute, the potential barrier is lowered to 17.5 kcal/mol, which is very close to the experimental value (16.7 kcal/mol).

 

The degradation of cefaclor (1), an oral cephalosporin antibiotic, was studied at 37ºC in a neutral aqueous medium by HPLC and 1H-NMR. Under these conditions, 1 underwent intramolecular aminolysis by the 7-side-chain NH2 group on the ß-lactam moiety to give a piperazine-2,5-dione. The most prominent peak in the HPLC profile of a degradation solution from 1 was isolated by prep. HPLC. Mechanistically, the formation of this degradation product cis-11 from 1 involves the contraction from a six-membered cephem ring to a five-membered ring, which presumably takes place via a common episulfonium ion intermediate 9 (see Scheme). Loss of the Cl-atom from 3-chloro-3-cephem is a general reaction subsequent to ¬ß-lactam ring opening.

A kinetic study on the alkaline hydrolysis of cefotaxime at pH 10.5 and 37 ºC has been carried out by using HPLC and 1H NMR. The main resulting degradation products have been isolated and identified. These include, apart from the well-known deacetylcefotaxime, the exocyclic methylene derivative, the 7-epimer of cefotaxime and the 7-epimer of deacetylcefotaxime. The kinetic constants involved in the process have been determined and according to the experimental results the attack of the hydroxyl group on the ester function bonded to the 3'-carbon is the fastest step in the proposed kinetic scheme. It should be emphasized that the base-catalyzed epimerization of the hydrogen at the 7 position clearly depends on the presence of a good electron-withdrawing group at C(3'). On the other hand, no hydrolysis of the amide at position 7 was detected.

The gas-phase basic hydrolysis of clavulanic acid (a) was studied by using the AM1 semi-ernpirical rnethod. 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 ß-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 ß-lactam ring. The different chemical reacivity of clavulanic acid relative to penicillins and cephalosporins could explain the disparate behaviour of the latter two types of compound towards ß-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.

We carried out a comprehensive theoretical study on the alkaline hydrolysis of the bicyclic system of penicillins (a four-member ring fused to a thiazolidine ring) on the basis of a BAC2 mechanism. We assayed the MINDO/3, MNDO, and AM1 semi-empirical calculation methods in order to determine their suitability for studying ß-lactam rings. Both the geometric and the energetic results obtained for the different intermediate states were compared with literature values - chiefly those determined by ab initio methods - with which they proved to be very consistent. The conformation of the carboxyl group at position 3 was found to be rather significant to the determination of the energy of the different reaction maxima and minima.

We studied the absorption spectrum of 7-aminocephalosporanic acid between 312 and 238 nm (32,000-42,000 cm-1) at different pH values. The spectrum was deconvoluted into log-normal curves that showed three different transitions; the first two bands are due to p- p transitions between the HOMO and LUMO, and from the HOMO to the LUMO+1, respectively. We also recorded the spectrum of the acid in dioxane/water mixtures and found band I to shift to shorter wavelengths with an increase in the medium polarity. Theoretical calculations allowed us to ascribe this behavior to the fact that the O4actam nitrogen atom possesses a high electron density in the ground state. As the medium polarity increases, so does the stability of this state, which results in the band being shifted to higher energy values. The theoretical calculations (MNDO) performed yielded 36.8 and 39.8 kK (1 kK = 10³ cm^-1) for the first two electron transitions, i.e., very close to the experimental values (~36 and 39 kK).

Semiempirical AM1, MINDO/3, and MNDO methods have been used in the study of the alkaline hydrolysis of b-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 ß-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 ß-lactam compounds.

The geometries of several cephalosporins were determined by the MINDO/3, MNDO and AM1 semiempirical calculation methods. The geometric parameters of the bicyclic system thus obtained (bond distances and angles, and dihedral angles) were compared with crystallographic and literature data. Of the three methods used, MINDO/3 provided the best estimations of the geometric values, while MNDO reproduced the pyramidal character of the ß-lactam nitrogen with the greatest accuracy and AM1 yielded an intermediate solution. All three methods were used to carry out a comprehensive conformational analysis of the bicyclic system. In this respect, MINDO/3 only detected one minimum, while both MNDO and AM1 yielded two minima, corresponding to two extreme situations (S1-up and C2-up).

MINDO/3, MNDO and AM1 calculations have been carried out in order to calculate the geometry of some penicillins. Theoretical bond lengths, bond angles and dihedral angles have been checked with crystallographic data. Results show that MINDO/3 is the best method for predicting the geometry of the bicyclic system. However, it is not very good in predicting the conformations and pyramidality of the ß-lactam nitrogen. In spite of its poor accuracy in predicting the geometry of the antibiotic, the MNDO method reproduces very well the pyrarnidality of the ß-lactam nitrogen. The AM1 method predicts the geometry of the system quite well, except for the S-C and C7-N4 bond lengths which are underestimated and overestimated respectively. In addition, AM1 is very good at treating conformations of the thiazolidine ring.