Opening Of Β-lactam Ring Research Articles

Insights into the degradation mechanisms and pathways of cephalexin during homogeneous and heterogeneous photo-Fenton processes

The widespread occurrence of antibiotics in the environment poses a potential threat to human health. The photo-Fenton process has demonstrated better degradation performance compared with the conventional wastewater treatment processes. In this study, the degradation of cephalexin was evaluated comparatively by homogeneous (Fe2+/H2O2/UV) and heterogeneous (MoS2@Fe/H2O2/UV) photo-Fenton processes. Key influencing factors affecting photo-Fenton performance were assessed, confirming the optimum Fe2+ concentration at 0.2016 mg L−1 and H2O2/Fe2+ molar ratio at 6. Higher degradation efficiency (73.10%) and pseudo-first-order degradation rate constant (0.0078 min−1) were achieved with the assistance of MoS2@Fe as the heterogeneous catalyst. Completely different degradation products were identified in the homogeneous and heterogeneous photo-Fenton processes, with main degradation pathways proposed as β-lactam ring-opening, sulfoxide formation, demethylation, N-dealkylation, decarbonylation, hydroxylation and deamination in the Fe2+/H2O2/UV system and β-lactam ring-opening, hydroxylation, dehydration, amide hydrolysis, and demethylation and ring contraction in the MoS2@Fe/H2O2/UV system, respectively. The formation of newly identified products might root in the attack on cephalexin from active species (i.e., OH, h+, e−, O2−) photoinduced by the MoS2@Fe catalyst. Results also indicated the importance of understanding the underlying mechanisms and pathways to eliminate the antimicrobial activities of antibiotics in the future.

Antibiotic transformation in an anaerobic membrane bioreactor linked to membrane biofilm microbial activity

Although extensive research to date has focused on enhancing removal rates of antibiotics from municipal wastewaters, the transformation products formed by anaerobic treatment processes remain understudied. The present work aims to examine the possible roles that the different microbial communities of an anaerobic membrane bioreactor (AnMBR) play in the transformation of antibiotics during wastewater treatment. As part of this work, sulfamethoxazole, erythromycin, and ampicillin were added in separate stages to the influent of the AnMBR at incremental concentrations of 10, 50, and 250 μg/L each. Antibiotic-specific transformation products detected during each stage, as identified by high resolution LC-MS, are reported herein. Results suggest that both isoxazole (sulfamethoxazole) and β-lactam (ampicillin) ring opening could be facilitated by the AnMBR's bioprocess. Microbial community analysis results indicated that relative activity of the system's suspended biomass consistently shifted towards syntrophic groups throughout the duration of the experiment. Notable differences were also observed between the suspended biomass and the AnMBR's membrane biofilms. Membrane-attached biofilm communities showed high relative activities of several specific methanogenic (Methanothrix and Methanomethylovorans), syntrophic (Syntrophaceae), and sulfate-reducing (Desulfomonile) groups. Such groups have been previously identified as involved in the formation of the antibiotic degradation products observed in the effluent of the AnMBR. The activity of these communities within the biofilms likely confers certain advantages that aid in the biotransformation of the antibiotics tested.

Degradation of antibiotic Cephalosporin C in different water matrices by ionizing radiation: Degradation kinetics, pathways, and toxicity

Cephalosporin antibiotics are ubiquitous emerging pollutants in various aquatic environments due to their extensive production and application. Herein, the radiolytic degradation of antibiotic Cephalosporin C (CEP-C) in different water matrices was comprehensively investigated using gamma radiation at various experimental conditions. The results revealed that CEP-C oxidation obeyed pseudo first-order kinetics, and 100%, 94.9%, 67.0%, 44.6% and 34.5% removal of CEP-C with 10–200 mg/L was achieved at 0.4 kGy, respectively. The degradation was faster at higher absorbed dose and acidic conditions (pH = 3.5). The inorganic anions, including SO42−, NO3−, and HCO3−, had negative influence on the degradation of CEP-C, the corresponding rate constant decreased from 4.603 to 3.667, 1.677 and 2.509 kGy−1 respectively in the presence of SO42−, NO3−, and HCO3−. The analysis of intermediate products indicated that CEP-C was oxidized to generate about 10 intermediate products. Besides, it was inferred that the thioether sulfur oxidation, β-lactam ring opening, acetyl dissociation from dihydrothiazine ring and D-α-aminohexylamide group abscission were the major reaction mechanisms of CEP-C degradation by gamma radiation. Importantly, the antibacterial activity of CEP-C could be completely vanished by gamma radiation alone, while more toxic intermediate products might be formed. Addition of hydrogen peroxide and peroxymonosulfate could significantly improve the CEP-C degradation, and reduce the toxicity of intermediates of CEP-C degradation. Similar degradation behavior was observed in the groundwater and wastewater, implying that ionizing radiation can be used for degradation of Cephalosporin in water and wastewater.

Biotin-Labelled Clavulanic Acid to Identify Proteins Target for Haptenation in Serum: Implications in Allergy Studies.

Clavulanic acid (CLV) and amoxicillin, frequently administered in combination, can be independently involved in allergic reactions. Protein haptenation with β-lactams is considered necessary to activate the immune system. The aim of this study was to assess the suitability of biotinylated analogues of CLV as probes to study protein haptenation by this β-lactam. Two synthetic approaches afforded the labeling of CLV through esterification of its carboxylic group with a biotin moiety, via either direct binding (CLV-B) or tetraethylenglycol linker (CLV-TEG-B). The second analogue offered advantages as solubility in aqueous solution and potential lower steric hindrance for both intended interactions, with the protein and with avidin. NMR reactivity studies showed that both CLV and CLV-TEG-B reacts through β-lactam ring opening by aliphatic amino nitrogen, however with different stability of resulting conjugates. Unlike CLV conjugates, that promoted the decomposition of clavulanate fragment, the conjugates obtained with the CLV-TEG-B remained linked, as a whole structure including biotin, to nucleophile and showed a better stability. This was a desired key feature to allow CLV-TEG-B conjugated protein detection at great sensitivity. We have used biotin detection and mass spectrometry (MS) to detect the haptenation of human serum albumin (HSA) and human serum proteins. MS of conjugates showed that HSA could be modified by CLV-TEG-B. Remarkably, HSA preincubation with CLV excess only reduced moderately the incorporation of CLV-TEG-B, which could be attributed to different protein interferences. The CLV-TEG-B fragment with opened β-lactam was detected bound to the 404–430HSA peptide of the treated protein. Incubation of human serum with CLV-TEG-B resulted in the haptenation of several proteins that were identified by 2D-electrophoresis and peptide mass fingerprinting as HSA, haptoglobin, and heavy and light chains of immunoglobulins. Taken together, our results show that tagged-CLV keeps some of the CLV features. Moreover, although we observe a different behavior in the conjugate stability and in the site of protein modification, the similar reactivity indicates that it could constitute a valuable tool to identify protein targets for haptenation by CLV with high sensitivity to get insights into the activation of the immune system by CLV and mechanisms involved in β-lactams allergy.

C-N and C-O Bond Formation in Copper-Catalyzed/Mediated sp3 C-H Activation: Mechanistic Studies from Experimental and Computational Aspects.

Mechanistic studies on Cu-catalyzed/mediated sp3 C-H amidation and acetoxylation are investigated from experimental and computational aspects. The concerted metalation-deprotonation (CMD) mechanism rather than a radical-involved pathway is proved to occur in amidation and acetoxylation reactions, and this is the rare example of the CMD mechanism involved in the more challenging sp3 C-H activations. Theoretical calculations demonstrated that CMD is the rate-determining step either for methylic or benzylic positions in amidation and acetoxylation reactions, and intermolecular nucleophilic addition of acetate anions is more favorable than the ring opening of β-lactams and intramolecular acetoxylation. These mechanistic studies on the divergent and condition-dependent product formation are critical for developing Cu-promoted C-H functionalization via the CMD mechanism.

Fabrication of multi-walled carbon nanotubes and carbon black co-modified graphite felt cathode for amoxicillin removal by electrochemical advanced oxidation processes under mild pH condition.

Hydrogen peroxide (H2O2) electrogenerated via two-electron oxygen reduction reaction at cathode plays an important role in electrochemical advanced oxidation processes for organic pollutants removal from wastewater. Herein, multi-walled carbon nanotubes and carbon black co-modified graphite felt electrode (MWCNTs-CB/GF) was prepared as an efficient cathode for H2O2 electrogeneration and amoxicillin removal by anodic oxidation with hydrogen peroxide (AO-H2O2) and electro-Fenton (EF) under mild pH condition. Besides, the physicochemical and electrochemical properties of MWCNTs-CB/GF were characterized by scanning electron microscopy, N2 adsorption and desorption experiment, contact angle measurement, X-ray photoelectron spectroscopy, and linear sweep voltammetry. Compared with GF, MWCNTs-CB/GF showed a higher H2O2 generation of 309.0mgL-1 with a current efficiency of 60.9% (after 120min) and more effective amoxicillin removal efficiencies of 97.5% (after 120min) and 98.7% (after 30min) in AO-H2O2 and EF (with 0.5mM Fe2+) processes, under the condition of current density 12mAcm-2 and initial pH5.5. Meanwhile, the TOC removal efficiency was 45.2% during EF process after 120min. Anodic oxidation, H2O2 oxidation, and methanol capture indicated that ∙OH generated via electro-activation reaction at MWCNTs-CB/GF and Fenton reaction in solution played the dominant role in amoxicillin removal. Moreover, the TOC removal was associated with ∙OH generated during Fenton reaction in the solution. The major intermediates of AMX degradation by EF process were identified using LC-MS and the possible degradation pathways were proposed containing of β-lactam ring opening, hydroxylation reaction, decarboxylation reaction, methyl groups in the thiazolidine ring oxidation reaction, bond cleavage, and rearrangement processes. All of the above results proved that MWCNTs-CB/GF was an excellent cathode for AMX degradation under mild pH condition.

Self-assembled cyclodextrin-based nanoparticles for meropenem stabilization

Meropenem is a carbapenem antibiotic with ultrabroad spectrum of activity and active against bacterial strains resistant to other antibiotics. The drug is characterized by fast renal elimination and high instability in aqueous solution where it undergoes β-lactam ring opening with consequent loss of biological activity. The purpose of this study was to develop cyclodextrin (CD) based nanoparticles (NPs) for increased meropenem bioavailability and stability in aqueous solutions. Self-assembly nanoparticles are formed by carboxymethylcellulose (CMC) and quaternary amino βCD polymer (QAβCDp), NPs of CMC-QAβCDp with relatively narrow size distribution profile are spontaneously formed in aqueous solution. The NPs were characterized by UV-VIS spectroscopy, DLS size measurements, Zeta potential and NMR spectroscopy. The kinetic stability studies revealed that meropenem loaded NPs can slow down the β-lactam antibiotic hydrolysis at room temperature by nearly 30% while at 4 °C the hydrolysis is 63% slower. The NPs with mean hydrodynamic diameter of about 135 nm displayed size stability during three-month storage at 4 °C. Incorporation of meropenem into the NPs increased the drug permeation through semi-permeable membranes suggesting that the NPs could have positive effect on meropenem permeation through biological membranes.

Torquoselective Mechanochemical Activation of the Staudinger Reaction To Form β-Lactams

The Staudinger reaction yielding β-lactam rings via [2 + 2] cycloaddition is a torquoselective reaction where the stereochemistry of the product can be steered by suitable substituents. Although the mechanochemical ring-opening of β-lactams has been investigated recently, the force-assisted synthesis of this important functional four-ring motif remains unexplored. As it will be computationally demonstrated, mechanochemical activation greatly reduces the barrier of the rate-limiting ring-closure step while, at the same time, preserves its torquoselectivity. This finding strongly suggests that strained four-membered rings can be readily incorporated in chain molecules using sonication techniques that greatly enhance reactivity while conserving selectivity.

A New Pathway for Protein Haptenation by β-Lactams.

The covalent binding of β-lactams to proteins upon photochemical activation has been demonstrated by using an integrated approach that combines photochemical, proteomic and computational studies, selecting human serum albumin (HSA) as a target protein and ezetimibe (1) as a probe. The results have revealed a novel protein haptenation pathway for this family of drugs that is an alternative to the known nucleophilic ring opening of β-lactams by the free amino group of lysine residues. Thus, photochemical ring splitting of the β-lactam ring, following a formal retro-Staudinger reaction, gives a highly reactive ketene intermediate that is trapped by the neighbouring lysine residues, leading to an amide adduct. For the investigated 1/HSA system, covalent modification of residues Lys414 and Lys525, which are located in sub-domains IIIA and IIIB, respectively, occurs. The observed photobinding may constitute the key step in the sequence of events leading to photoallergy. Docking and molecular dynamics simulation studies provide an insight into the molecular basis of the selectivity of 1 for these HSA sub-domains and the covalent modification mechanism. Computational studies also reveal positive cooperative binding of sub-domain IIIB that explains the experimentally observed modification of Lys414, which is located in a barely accessible pocket (sub-domain IIIA).

Electrochemical treatment of penicillin, cephalosporin, and fluoroquinolone antibiotics via active chlorine: evaluation of antimicrobial activity, toxicity, matrix, and their correlation with the degradation pathways.

Antibiotics are pharmaceuticals widely consumed and frequently detected in environmental water, where they can induce toxic effects and development of resistant bacteria. Their structural variety makes the problem of antibiotics in natural water more complex. In this work, six highly used antibiotics (at 40μmol L-1) belonging to three different classes (penicillins, cephalosporins, and fluoroquinolones) were treated using an electrochemical system with a Ti/IrO2 anode and a Zr cathode in the presence of NaCl (0.05μmol L-1). The attack of electrogenerated active chlorine was found to be the main degradation route. After only 20min of treatment, the process decreased more than 90% of the initial concentration of antibiotics, following the degradation order: fluoroquinolones>penicillins>cephalosporins. The primary interactions of the degrading agent with fluoroquinolones occurred at the cyclic amine (i.e., piperazyl ring) and the benzene ring. Meanwhile, the cephalosporins and penicillins were initially attacked on the β-lactam and sulfide groups. However, the tested penicillins presented an additional reaction on the central amide. In all cases, the transformations of antibiotics led to the antimicrobial activity decreasing. On the contrary, the toxicity level showed diverse results: increasing, decreasing, and no change, depending on the antibiotic type. In fact, due to the conservation of quinolone nucleus in the fluoroquinolone by-products, the toxicity of the treated solutions remained unchanged. With penicillins, the production of chloro-phenyl-isoxazole fragments increased the toxicity level of the resultant solution. However, the opening of β-lactam ring of cephalosporin antibiotics decreased the toxicity level of the treated solutions. Finally, the application of the treatment to synthetic hospital wastewater and seawater containing a representative antibiotic showed that the high amount of chloride ions in seawater accelerates the pollutant degradation. In contrast, the urea and ammonium presence in the hospital wastewater retarded the removal of this pharmaceutical.

Hybrid Potential Simulation of the Acylation of Enterococcus faecium l,d-Transpeptidase by Carbapenems.

The l,d-transpeptidases, Ldts, catalyze peptidoglycan cross-linking in β-lactam-resistant mutant strains of several bacteria, including Enterococcus faecium and Mycobacterium tuberculosis. Although unrelated to the essential d,d-transpeptidases, which are inactivated by the β-lactam antibiotics, they are nevertheless inhibited by the carbapenem antibiotics, making them potentially useful targets in the treatment of some important diseases. In this work, we have investigated the acylation mechanism of the Ldt from E. faecium by the carbapenem, ertapenem, using computational techniques. We have employed molecular dynamics simulations in conjunction with QC/MM hybrid potential calculations to map out possible reaction paths. We have focused on determining the following: (i) the protonation state of the nucleophilic cysteine of the enzyme when it attacks; (ii) whether nucleophilic attack and β-lactam ring-opening are concerted or stepwise, the latter occurring via an oxyanion intermediate; and (iii) the identities of the proton acceptors at the beginning and end of the reaction. Overall, we note that there is considerable plasticity in the mechanisms, owing to the significant flexibility of the enzyme, but find that the preferred pathways are ones in which nucleophilic attack of cysteine thiolate is concerted with β-lactam ring-opening.

Boron-doped diamond oxidation of amoxicillin pharmaceutical formulation: Statistical evaluation of operating parameters, reaction pathways and antibacterial activity

The electrochemical oxidation of a commercial amoxicillin formulation over a boron-doped diamond (BDD) anode was investigated. The effect of initial COD concentration (1–2 g/L), current density (30–50 mA/cm2), treatment time (15–90 min), initial pH (3–9) and electrolyte concentration (2–4 g/L NaCl) on COD removal was assessed through a factorial design methodology. For the range of conditions in question, the first three single effects, as well as the interaction between COD and time were the most important ones in terms of mass of COD removed.Liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) was employed to identify major transformation by-products (TBPs); thirteen compounds were detected as TBPs of AMX electrochemical degradation, while several others appear in the original formulation. AMX degradation occurs though the following pathways: (i) hydroxylation mainly in the benzoic ring, (ii) opening of β-lactam ring followed by decarboxylation, hydroxylation and re-arrangement, and (iii) bond cleavage between the carbons of amino and amide groups. Furthermore, the process is accompanied by the release of several ions, i.e. nitrate, sulfate and ammonium.The antibiotic activity of AMX up to 1000 mg/L was tested against Klebsiella pneumoniae and Enterococcus faecalis reference strains; both bacteria are completely inactivated at this concentration but the activity is reduced substantially at lower concentrations. Oxidized samples still exhibit some antibacterial activity (50–60%) which is due to TBPs and active chlorine species present in the liquid phase. The latter are generated from chloride ions and enhance considerably AMX degradation rates.

Kinetic and Structural Characterization of the Interaction of 6-Methylidene Penem 2 with the β-Lactamase from Mycobacterium tuberculosis.

Mycobacterium tuberculosis is intrinsically resistant to most β-lactam antibiotics because of the constitutive expression of the blaC-encoded β-lactamase. This enzyme has extremely high activity against penicillins and cephalosporins, but weaker activity against carbapenems. The enzyme can be inhibited by clavulanate, avibactam, and boronic acids. In this study, we investigated the ability of 6-methylidene β-lactams to inhibit BlaC. One such compound, penem 2, inhibited BlaC more than 70 times more efficiently than clavulanate. The compound forms a covalent complex with BlaC as shown by mass spectrometry. Crystallization of the complex revealed that the bound inhibitor was covalently attached via the Ser70 active site residue and that the covalently, acylated form of the inhibitor had undergone additional chemistry yielding a 4,7-thiazepine ring in place of the β-lactam and a thiazapyroline ring generated as a result of β-lactam ring opening. The stereochemistry of the product of the 7-endo-trig cyclization was the opposite of that observed previously for class A and D β-lactamases. Addition of penem 2 greatly synergized the antibacterial properties of both ampicillin and meropenem against a growing culture of M. tuberculosis. Strikingly, penem 2 alone showed significant growth inhibition, suggesting that in addition to its capability of efficiently inhibiting BlaC, it also inhibited the peptidoglycan cross-linking transpeptidases.

Synthesis of new penicillin derivatives as drug-like molecules for biological screening

Abstract Chemical modification of penicillin β-lactam ring was made. Six thiazolidine amides were produced through N4-C7 β-lactam ring opening of penicillin V methyl ester with various aliphatic, aromatic, and heterocyclic primary amines. Five 8-hydroxypenillic acid derivatives with side chains of methyl, propyl, benzyl, and diethylaminoethyl groups were yielded via β-lactam ring rearrangement from 6-aminopenicillanic acid (6-APA). Parallel synthetic methods were used for the alkylation of 8-hydroxypenillic acid and β-lactam ring opening of penicillin V methyl ester. The biological activities of the compounds were evaluated.

Studies on the formation and forming mechanism of the related substance E in potassium clavulanate production by HPLC-MS/MS

The objective of this study was to investigate the formation and forming mechanism of the related substance E in potassium clavulanate production. The impurity with retention time of 11.1 min in potassium clavulanate final product was confirmed as the related substance E by high performance liquid chromatography with tandem mass spectrometric detection (HPLC-MS/MS).The related substance E analysis during the production of clavulanic acid showed that this impurity could be formed during both the fermentation and purification processes, especially in the later fermentation stage, filtration concentration and back-extraction procedure. Clavulanic acid was the precursor of the related substance E. Studies on its forming mechanism showed that the related substance E was formed by the combination of the imino group of one molecule of clavulanic acid with the carboxyl group of another molecule of clavulanic acid with the opening of β-lactam ring. Results of a multi-factor orthogonal test confirmed that the concentration of clavulanic acid was the dominant factor to accelerate the reaction, while the temperature was another contributing factor. The pH 5.0-6.5 had little impact on the generation of the related substance E.

Structure-selective hot-spot Raman enhancement for direct identification and detection of trace penicilloic acid allergen in penicillin

Trace penicilloic acid allergen frequently leads to various fatal immune responses to many patients, but it is still a challenge to directly discriminate and detect its residue in penicillin by a chemosensing way. Here, we report that silver-coated gold nanoparticles (Au@Ag NPs) exhibit a structure-selective hot-spot Raman enhancement capability for direct identification and detection of trace penicilloic acid in penicillin. It has been demonstrated that penicilloic acid can very easily link Au@Ag NPs together by its two carboxyl groups, locating itself spontaneously at the interparticle of Au@Ag NPs to form strong Raman hot-spot. At the critical concentration inducing the nanoparticle aggregation, Raman-enhanced effect of penicilloic acid is ~60,000 folds higher than that of penicillin. In particular, the selective Raman enhancement to the two carboxyl groups makes the peak of carboxyl group at C6 of penicilloic acid appear as a new Raman signal due to the opening of β-lactam ring of penicillin. The surface-enhanced Raman scattering (SERS) nanoparticle sensor reaches a sensitive limit lower than the prescribed 1.0‰ penicilloic acid residue in penicillin. The novel strategy to examine allergen is more rapid, convenient and inexpensive than the conventional separation-based assay methods.

Kinetic Characterization of Hydrolysis of Nitrocefin, Cefoxitin, and Meropenem by β-Lactamase fromMycobacterium tuberculosis

The constitutively expressed, chromosomally encoded β-lactamase (BlaC) is the enzyme responsible for the intrinsic resistance to β-lactam antibiotics in Mycobacterium tuberculosis. Previous studies from this laboratory have shown that the enzyme exhibits an extended-spectrum phenotype, with very high levels of penicillinase and cephalosporinase activity, as well as weak carbapenemase activity [Tremblay, L. W., et al. (2008) Biochemistry 47, 5312-5316]. In this report, we have determined the pH dependence of the kinetic parameters, revealing that the maximal velocity depends on the ionization state of two groups: a general base exhibiting a pK value of 4.5 and a general acid exhibiting a pK value of 7.8. Having defined a region where the kinetic parameters are pH-independent (pH 6.5), we determined solvent kinetic isotope effects (SKIEs) for three substrates whose kcat values differ by 5.5 orders of magnitude. Nitrocefin is a highly activated, chromogenic cephalosporin derivative that exhibits steady-state solvent kinetic isotope effects of 1.4 on both V and V/K. Cefoxitin is a slower cephalosporin derivative that exhibits a large SKIE on V of 3.9 but a small SKIE of 1.8 on V/K in steady-state experiments. Pre-steady-state, stopped-flow experiments with cefoxitin revealed a burst of β-lactam ring opening with associated SKIE values of 1.6 on the acylation step and 3.4 on the deacylation step. Meropenem is an extremely slow substrate for BlaC and exhibits burst kinetics in the steady-state experiments. SKIE determinations with meropenem revealed large SKIEs on both the acylation and deacylation steps of 3.8 and 4.0, respectively. Proton inventories in all cases were linear, indicating the participation of a single solvent-derived proton in the chemical step responsible for the SKIE. The rate-limiting steps for β-lactam hydrolysis of these substrates are analyzed, and the chemical steps responsible for the observed SKIE are discussed.

Diastereoselective synthesis of trans-trifluoromethyl-β-lactams and α-alkyl-β-trifluoromethyl-β-amino esters

The diastereoselective synthesis of β-lactams was examined from N-tosyl-1-chloro-2,2,2-trifluoroethylamine 3 and various nonactivated aliphatic acid chlorides in the presence of a Brønsted base. The mild reaction conditions allowed to get trifluoromethyl-β-lactams in good yields with high trans-diastereo selectivity. In addition, we also demonstrated that ring-opening of β-lactams easily provided α-alkyl-β-trifluoromethyl-β-amino esters.

ChemInform Abstract: β‐Lactam Ring Opening: A Useful Entry to Amino Acids and Relevant Nitrogen‐Containing Compounds

AbstractReview: 173 refs.

Prediction of bond length variation in classical β-lactam structures and enzymatic ring instability

Comparison of intrinsic, solvational, and enzymatic rates of β-lactam ring opening allows rate enhancement due to specific enzyme interactions with given β-lactam moieties to be identified and modeled. Enzymatic rates of β-lactam ring opening may be derived from potency data and are briefly reviewed. For widespread comparison with intrinsic instabilities, convenient criteria for accurate estimation of the latter are required. The conditions under which differences in intrinsic instabilities of β-lactams correlate closely with their ground-state properties have been defined. Theoretical prediction of substituent effects in β-lactam structures requires geometry optimization if accuracy with the biological instability data over even moderate ranges of activity is to be maintained. The β-lactam amidic C-N bond length is a powerful indicator of intrinsic ring instability. The contributions of the ring nuclei to enzymatic stability in classical β-lactam structures correlate with the predicted C-N bond lengths for both STO-3G and 4-31G bases to ˜0.4 kcal. This accuracy corresponds to a bond length sensitivity of ± 0.004 A. The limits of convergence of the theoretical estimates are of this order. The relative theoretical amidic C-N bond lengths offer a more precise scale for ranking intrinsic β-lactam instabilities than one employing experimental X-ray quantities, with their associated perturbations from crystal forces. A specific interaction of 6/7 acylamino substituents in penicillins and cephalosporins with pbp I enzymes is identified.