Design of Ratio-Fluorescence Nanohybrid Based on Radix Hedysari Green-Synthesized CDs and GSH-AuNCs for Sensitive Detection of Cefodizime Sodium in Urine Sample.

A dual-emission ratio-fluorescent sensing nanohybrid based on Radix Hedysari green-synthesized carbon quantum dots (CDs) and glutathione-functionalized gold nanoclusters (GSH-AuNCs) had been developed for the determination of cefodizime sodium (CDZM). The designed fluorescence nanohybrid had two significant fluorescence emission peaks at 458 nm and 569 nm when excited at 360 nm, which was attributed to the CDs and GSH-AuNCs. With the addition of CDZM, the fluorescence at 458 nm was slightly weakened while the fluorescence at 569 nm was enhanced obviously. Based on the relationship between the I569/I458 fluorescence intensity ratio and the concentration of CDZM, the designed nanohybrid exhibited a good linearity range of 1.0-1000.0 µM and the limit of detection (LOD) was 0.19 µM. The method was finally applied in the detection of CDZM in urine, showing the potential applications in complicated biological samples.

Attaching Metal-Containing Moieties to ß-Lactam Antibiotics: The Case of Penicillin and Cephalosporin.

Procedures for the preparation of transition metal complexes having intact bicyclic cepham or penam systems as ligands have been developed. Starting from readily available 4-azido-2-azetidinones, a synthetic approach has been tuned using a copper-catalyzed azide-alkyne cycloaddition between 3-azido-2-azetinones and alkynes, followed by methylation and transmetalation to Au(I) and Ir(III) complexes from the mesoionic carbene Ag(I) complexes. This methodology was applied to 6-azido penam and 7-azido cepham derivatives to build 6-(1,2,3-triazolyl)penam and 7-(1,2,3-triazolyl)cepham proligands, which upon methylation and metalation with Au(I) and Ir(III) complexes yielded products derived from the coordination of the metal to the penam C6 and cepham C7 positions, preserving intact the bicyclic structure of the penicillin and cephalosporin scaffolds. The crystal structure of complex 28b, which has an Ir atom directly bonded to the intact penicillin bicycle, was determined by X-ray diffraction. This is the first structural report of a penicillin-transition-metal complex having the bicyclic system of these antibiotics intact. The selectivity of the coordination processes was interpreted using DFT calculations.

[Discovery of a New Siderophore Cephalosporin, Cefiderocol].

Cefiderocol is a novel siderophore-conjugated cephalosporin with a catechol residue acting as an iron chelator. Cefiderocol forms a chelating complex with ferric iron and is transported rapidly into bacterial cells through iron-uptake systems. As a result, cefiderocol shows good activity against Gram-negative bacteria, including carbapenem-resistant isolates that are causing significant global health issues. Cefiderocol has been approved for clinical use in the United States and Europe, where it is being used to treat infection caused by carbapenem-resistant Gram-negative pathogens.

Green synthesis of chitosan-encapsulated CuO nanocomposites for efficient degradation of cephalosporin antibiotics in contaminated water.

The synthesis and characterization of chitosan encapsulated copper oxide nanocomposites (CuNPs) using plant extracts for the photocatalytic degradation of second-generation antibiotics, cefixime and cefuroxime, were investigated. The study revealed that the presence of diverse chemical components in the plant extract significantly influenced the size of the CuNPs, with transmission electron microscopy (TEM) showing spherical shapes and sizes ranging from 11-35 nm. The encapsulation process was confirmed by an increase in size for certain samples, indicating successful encapsulation. X-ray photoelectron spectroscopy (XPS) analysis further elucidated the chemical makeup, confirming the valency state of Cu2+ and the presence of Cu-O bonding, with no contaminants detected. Photocatalytic activity assessments demonstrated that the copper oxide nanocomposites exhibited significant degradation capabilities against both antibiotics under UV light irradiation, with encapsulated nanocomposites (EnCu30) showing up to 96.18% degradation of cefuroxime within 60 min. The study highlighted the influence of chitosan encapsulation on enhancing photocatalytic performance, attributed to its high adsorption capability. Recycling studies confirmed the sustainability of the Cu nanocomposites, maintaining over 89% degradation rate after five consecutive cycles. This research underscores the potential of green-synthesized CuNPs as efficient, stable photocatalysts for the degradation of harmful antibiotics, contributing to environmental sustainability and public health protection.

Culture-free detection of ß-lactamase-Producing bacteria in urinary tract infections using a paper sensor.

Developing simple, inexpensive, fast, sensitive, and specific probes for antibiotic-resistant bacteria is crucial for the management of urinary tract infections (UTIs). We here propose a paper-based sensor for the rapid detection of ß-lactamase-producing bacteria in the urine samples of UTI patients. By conjugating a strongly electronegative group -N+(CH3)3 with the core structures of cephalosporin and carbapenem antibiotics, two visual probes were achieved to respectively target the extended-spectrum/AmpC ß-lactamases (ESBL/AmpC) and carbapenemase, the two most prevalent factors causing antibiotic resistance. By integrating these probes into a portable paper sensor, we confirmed 10 and 8 cases out of 30 clinical urine samples as ESBL/AmpC- and carbapenemase-positive, respectively, demonstrating 100% clinical sensitivity and specificity. This paper sensor can be easily conducted on-site, without resorting to bacterial culture, providing a solution to the challenge of rapid detection of ß-lactamase-producing bacteria, particularly in resource-limited settings.

Discovery of YFJ-36: Design, Synthesis, and Antibacterial Activities of Catechol-Conjugated ß-Lactams against Gram-Negative Bacteria.

Cefiderocol is the first approved catechol-conjugated cephalosporin against multidrug-resistant Gram-negative bacteria, while its application was limited by poor chemical stability associated with the pyrrolidinium linker, moderate potency against Klebsiella pneumoniae and Acinetobacter baumannii, intricate procedures for salt preparation, and potential hypersensitivity. To address these issues, a series of novel catechol-conjugated derivatives were designed, synthesized, and evaluated. Extensive structure-activity relationships and structure-metabolism relationships (SMR) were conducted, leading to the discovery of a promising compound 86b (Code no. YFJ-36) with a new thioether linker. 86b exhibited superior and broad-spectrum in vitro antibacterial activity, especially against A. baumannii and K. pneumoniae, compared with cefiderocol. Potent in vivo efficacy was observed in a murine systemic infection model. Furthermore, the physicochemical stability of 86b in fluid medium at pH 6-8 was enhanced. 86b also reduced potential the risk of allergy owing to the quaternary ammonium linker. The improved properties of 86b supported its further research and development.

Production of AmpC ß-Lactamase and Development of a Competitive Array for Discriminative Determination of Cephalosporins in Milk.

In this study, AmpC ß-lactamase of Escherichia coli was expressed, and its intermolecular interaction mechanisms with 15 cephalosporins (CPs) were studied by using a molecular docking technique. Results showed that this enzyme mainly interacted with the ß-lactam ring of these CPs, and the key contacting amino acids were Ser80 and Ser228. The AmpC ß-lactamase was combined with 5 horseradish peroxidase-labeled conjugates to develop a direct competitive array on a microplate for determination of 15 drugs in milk. Due to the use of principal component analysis method to analyze the data, this method could discriminate the 15 drugs at the concentration as low as 10 ng/mL. The detection results for the unknown milk samples were consistent with those obtained by the liquid chromatography-mass spectrometry method. As a general comparison, this method is better than the previous antibody-based and receptor-based detection methods for CPs. This is the first paper reporting a competitive array for discriminative determination of a class of small-molecule substances.

Preparation of pH-responsive block copolymers for separation of cephalosporin antibiotics by open-tubular capillary electrochromatography.

Stimuli-responsive block copolymers have exhibited their feasibility for drug delivery and analysis of biomolecules. However, study of the electrophoretic behavior of antibiotics by open tubular capillary electrochromatography (OT-CEC) based on smart block copolymers coatings is still a substantial challenge. Herein, we reported an OT-CEC protocol for analysis of cephalosporin antibiotics with pH-responsive block copolymers as coatings. By using the reversible addition-fragmentation chain-transfers radical polymerisation technique, the smart poly(styrene-maleic anhydride-acrylic acid) (P(St-MAn-AA)) was synthesized and subsequently chemical bonded onto the inner walls of amino-grafted capillaries. The pH induced changes in the stretch/curl states of P(St-MAn-AA) chains were used to generate an adjustable hydrophobic/hydrophilic interaction and hydrogen bonds between the polymer coatings and the analytes. The OT-CEC performance was evaluated by varying the monomer ratios, polymer coating amounts and layers, buffer concentrations and pH values. Baseline separation of the three-test antibiotics was achieved at pH 8.0. The proposed OT-CEC technique was further applied to the determination of rat serum antibiotics in the metabolic processes. The present work demonstrates an enhancement in antibiotics separation efficiency, and shows a great potential for the preparation of stimuli-responsive block copolymers coatings and in OT-CEC analysis of real samples in living bio-systems.

Electrochemical fingerprinting of cephalosporin antibiotics and its applications for investigations of hydrolysis behavior.

Cephalosporin, as one of the most widely used antibiotics, study of its hydrolysis process is important for predicting their environmental persistence. Two critical factors are considered has the first priority, which are hydrolysis rate constant (kh) and half-life (t1/2). To date, many efforts have been made by using various analytical techniques to obtain the data for calculating kh and t1/2. However, the typical techniques such as UV/vis spectrophotometry and liquid chromatography are of significant challenges like low accuracy and timely operations. Herein, we explored an electrochemical method by identifying the characteristic peaks with the same parent nuclear structure through square wave voltammetry (SWV). This proposed electrochemical fingerprinting was able to track the hydrolysis of intact cephalosporin molecules, ß-lactam ring, and transformation product. The kh and t1/2 of cefadroxil (CDX) under pH = 7 and 25 °C by electrochemical (0.0640 d-1 and 11.0 d) were consistent with those of high-performance liquid chromatography-UV/vis (HPLC-UV/vis) (0.0660 d-1 and 10.7 d). The t1/2 ranged from 3.40 to 36.2 d, 7.33 d-43.7 d and 9.63 d-45.3 d for base-catalyzed, neutral pH and acid-catalyzed hydrolysis hydrolyzed, respectively, indicating that base-catalyzed hydrolysis rates were the greatest under alkaline conditions. Meanwhile, hydrolysis rates increased 2.50-3.60-fold for every 10 °C raise in temperature. Besides, the electrochemical fingerprinting could realize cephalosporin and ß-lactam ring hydrolysis rates close to 100% in-situ hydrolysis process monitoring. This present work provides a powerful technology for understanding the environmental fate and predicting the environmental behavior of antibiotics with fast, high accuracy, specific recognition, and in situ monitoring.

Stereochemically altered cephalosporins as potent inhibitors of New Delhi metallo-ß-lactamases.

Antibiotic resistance caused by ß-lactamases, particularly metallo-ß-lactamases, has been a major threat to public health globally. New Delhi metallo-ß-lactamase-1 (NDM-1) represents one of the most important metallo-ß-lactamases; the production of NDM-1 in bacterial pathogen significantly reduces the efficacy of ß-lactam antibiotics, including life-saving carbapenems. Herein, we have demonstrated stereochemically altered cephalosporins as potent inhibitors against NDM-1, as well as mutants of NDM. The structure and activity relationship (SAR) study on over twenty cephalosporin analogues discloses the stereochemistry and the substituents on 7-position and 3'-position of cephalosporin are critical to suppress the activity of NDM-1 and the optimal compound 1u exhibited an IC50 of 0.13 µM. Furthermore, a crystal complex of NDM-1 and 1u has been obtained, suggesting this cephalosporin derivative inhibits enzyme activity by the formation of a relatively stable hydrolytic product-NDM-1 intermediate. The discovery in this study may pave the way to turn cephalosporin, a natural substrate of ß-lactamase, into an effective NDM-1 inhibitor to combat antibiotic resistance.

Specific detection of IMP-1 ß-lactamase activity using a trans cephalosporin-based fluorogenic probe.

A fluorogenic probe for the specific detection of IMP-1 ß-lactamase activity has been developed. This imaging reagent features a unique trans-acetylamino cephalosporin as an enzymatic recognition moiety, exhibiting excellent selectivity to IMP-1 ß-lactamase over other ß-lactamases, including serine- and metallo-ß-lactamases. The selective activation of the probe by IMP-1 ß-lactamase leads to over 30-fold enhancement in the fluorescence intensity, which allows enzyme activity to be reported with high sensitivity.

Detection of an enzyme isomechanism by means of the kinetics of covalent inhibition.

Turnover of substrates by many enzymes involves free enzyme forms that differ from the stable form of the enzyme in the absence of substrate. These enzyme species, known as isoforms, have, in general, different physical and chemical properties than the native enzymes. They usually occur only in small concentrations under steady state turnover conditions and thus are difficult to detect. We show in this paper that in one particular case of an enzyme (a class C ß-lactamase) with specific substrates (cephalosporins) the presence of an enzyme isoform (E') can be detected by means of its different reactivity than the native enzyme (E) with a class of covalent inhibitors (phosphonate monoesters). Generation of E' from E arises either directly from substrate turnover or by way of a branched path from an acyl-enzyme intermediate. The relatively slow spontaneous restoration of E from E' is accelerated by certain small molecules in solution, for example cyclic amines such as imidazole and salts such as sodium chloride. Solvent deuterium kinetic isotope effects and the effect of methanol on cephalosporin turnover showed that for both E and E', kcat is limited by deacylation of an acyl-enzyme intermediate rather than by enzyme isomerization.

Novel Cephalosporin Conjugates Display Potent and Selective Inhibition of Imipenemase-Type Metallo-ß-Lactamases.

In an attempt to exploit the hydrolytic mechanism by which ß-lactamases degrade cephalosporins, we designed and synthesized a series of novel cephalosporin prodrugs aimed at delivering thiol-based inhibitors of metallo-ß-lactamases (MBLs) in a spatiotemporally controlled fashion. While enzymatic hydrolysis of the ß-lactam ring was observed, it was not accompanied by inhibitor release. Nonetheless, the cephalosporin prodrugs, especially thiomandelic acid conjugate (8), demonstrated potent inhibition of IMP-type MBLs. In addition, conjugate 8 was also found to greatly reduce the minimum inhibitory concentration of meropenem against IMP-producing bacteria. The results of kinetic experiments indicate that these prodrugs inhibit IMP-type MBLs by acting as slowly turned-over substrates. Structure-activity relationship studies revealed that both phenyl and carboxyl moieties of 8 are crucial for its potency. Furthermore, modeling studies indicate that productive interactions of the thiomandelic acid moiety of 8 with Trp28 within the IMP active site may contribute to its potency and selectivity.

Electrochemistry of Intact Versus Degraded Cephalosporin Antibiotics Facilitated by LC-MS Analysis.

The electrochemical detection of cephalosporins is a promising approach for the monitoring of cephalosporin levels in process waters. However, this class of antibiotics, like penicillins, is composed of chemically active molecules and susceptible to hydrolysis and aminolysis of the four membered ß-lactam ring present. In order to develop a smart monitoring strategy for cephalosporins, the influence of degradation (hydrolysis and aminolysis) on the electrochemical fingerprint has to be taken into account. Therefore, an investigation was carried out to understand the changes of the voltammetric fingerprints upon acidic and alkaline degradation. Changes in fingerprints were correlated to the degradation pathways through the combination of square wave voltammetry and liquid chromatography quadrupole time-of-flight analysis. The characteristic electrochemical signals of the ß-lactam ring disappeared upon hydrolysis. Additional oxidation signals that appeared after degradation were elucidated and linked to different degradation products, and therefore, enrich the voltammetric fingerprints with information of the state of the cephalosporins. The applicability of the electrochemical monitoring system was explored by the analysis of the intact and degraded industrial process waters containing the key intermediate 7-aminodeacetoxycephalosporanic acid (7-ADCA). Clearly, the intact process samples exhibited the expected core signals of 7-ADCA and could be quantified, while the degraded samples only showed the newly formed degradation products.

A computational drug repurposing approach in identifying the cephalosporin antibiotic and anti-hepatitis C drug derivatives for COVID-19 treatment.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused over 1.4 million deaths worldwide. Repurposing existing drugs offers the fastest opportunity to identify new indications for existing drugs as a stable solution against coronavirus disease 2019 (COVID-19). The SARS-CoV-2 main protease (Mpro) is a critical target for designing potent antiviral agents against COVID-19. In this study, we identify potential inhibitors against COVID-19, using an amalgam of virtual screening, molecular dynamics (MD) simulations, and binding-free energy approaches from the Korea Chemical Bank drug repurposing (KCB-DR) database. The database screening of KCB-DR resulted in 149 binders. The dynamics of protein-drug complex formation for the seven top scoring drugs were investigated through MD simulations. Six drugs showed stable binding with active site of SARS-CoV-2 Mpro indicated by steady RMSD of protein backbone atoms and potential energy profiles. Furthermore, binding free energy calculations suggested the community-acquired bacterial pneumonia drug ceftaroline fosamil and the hepatitis C virus (HCV) protease inhibitor telaprevir are potent inhibitors against Mpro. Molecular dynamics and interaction analysis revealed that ceftaroline fosamil and telaprevir form hydrogen bonds with important active site residues such as Thr24, Thr25, His41, Thr45, Gly143, Ser144, Cys145, and Glu166 that is supported by crystallographic information of known inhibitors. Telaprevir has potential side effects, but its derivatives have good pharmacokinetic properties and are suggested to bind Mpro. We suggest the telaprevir derivatives and ceftaroline fosamil bind tightly with SARS-CoV-2 Mpro and should be validated through preclinical testing.

Efficient enzymatic synthesis of cephalexin in suspension aqueous solution system.

An efficient method for the enzymatic synthesis of cephalexin (CEX) from 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and d-phenylglycine methyl ester (PGME) using immobilized penicillin G acylase (IPGA) as catalyst in a suspension aqueous solution system was developed, where the reactant 7-ADCA and product CEX are mainly present as solid particles. The effects of key factors on the enzymatic synthesis were investigated. Results showed that continuous feeding of PGME was more efficient for the synthesis of CEX than the batch mode. Under the optimized conditions, the maximum 7-ADCA conversion ratio of 99.3% and productivity of 200 mmol/L/H were achieved, both of which are much superior to the homogeneous aqueous solution system. Besides, IPGA still retained 95.4% of its initial activity after 10 cycles of enzymatic synthesis, indicating the excellent stability of this approach. The developed approach shows great potential for the industrial production of CEX via an enzyme-based route.

Separation and characterization of impurities and isomers in cefpirome sulfate by liquid chromatography/tandem mass spectrometry and a summary of the fragmentation pathways of oxime-type cephalosporins.

RATIONALE: Although the identification of degradation products of cefpirome sulfate has been reported, there has been no report concerning the impurities in bulk samples of this compound. To meet the requirements of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use, the structures of impurities whose content are over 0.1% need to be confirmed. Thus, characterization of the impurities in cefpirome sulfate bulk samples is critical for controlling the production of this drug. METHODS: The structures of cefpirome sulfate impurities were investigated using two-dimensional liquid chromatography (LC) coupled to electrospray ionization tandem mass spectrometry. In the first LC dimension, a Kromasil 100-5C18 column (4.6 mm × 250 mm, 5 µm) was used, and the mobile phases were 0.03 M ammonium dihydrogen phosphate solution and acetonitrile. In the second dimension, the column was a Shimadzu Shim-pack GISS C18 column (50 mm × 2.1 mm, 1.9 µm), and the mobile phases were 10 mM ammonium formate solution and methanol. An ion trap time-of-flight mass spectrometer operated in both positive and negative ion mode was employed in this study. RESULTS: Nine impurities and isomers in cefpirome sulfate, eight of which were previously unknown, were separated and characterized. Structures were proposed for the eight unknown compounds based on the MSn fragmentation data. The degradation behavior of cefpirome sulfate was also studied. CONCLUSIONS: Based on the characterization of impurities and isomers, this study could be used to improve the quality control of the cefpirome sulfate drug recommended in pharmacopoeias. The degradation behavior of cefpirome sulfate provides a basis for the selection of storage conditions.

Computational design of new enzymes for hydrolysis and synthesis of third-generation cephalosporin antibiotics.

Engineering active sites in inert scaffolds to catalyze chemical transformations with unnatural substrates is still a great challenge for enzyme catalysis. In this research, a p-nitrobenzyl esterase from Bacillus subtilis was identified from the structural database, and a double mutant E115A/E188A was designed to afford catalytic activities toward the hydrolysis of ceftizoxime. A quadruple mutant E115A/E188A/L362S/I270A with enhanced catalytic efficiency was created to catalyze the condensation reaction of ethyl-2-methoxy-amino-2-(2-aminothiazole-4-yl) acetate with 7-amino-3-nor-cephalosporanic acid to produce ceftizoxime in a fully aqueous medium. The catalytic efficiencies of the computationally designed mutants E115A/E188A/L362S/I270A and E115A/Y118 K/E188 V/I270A/L362S can be taken as starting points to further improve their properties towards the practical application in designing more ecology-friendly production of third-generation cephalosporins.

Mechanistic Insights into the Oxidative Ring Expansion from Penicillin N to Deacetoxycephalosporin C Catalyzed by a Nonheme Iron(II) and α-KG-Dependent Oxygenase.

Deacetoxycephalosporin C synthase (DAOCS) is a nonheme iron(II) and 2-oxoglutarate (α-KG)-dependent oxygenase that catalyzes the oxidative ring expansion of penicillin N (penN) to deacetoxycephalosporin C (DAOC). Earlier reported crystal structures of DAOCS indicated that the substrate penicillin binds at the same site of succinate, leading to the proposal of the unusual "ping-pong" mechanism. However, more recent data provided evidence of the formation of ternary DAOCS·α-KG·penN complex, and thus DAOCS should follow the usual consensus mechanism of α-KG-dependent nonheme iron(II) oxygenases. Nevertheless, how DAOCS catalyzes the ring expansion is unknown. In this paper, on the basis of the crystal structure, we constructed two reactant models and performed a series of combined quantum mechanics/molecular mechanics (QM/MM) calculations to illuminate the catalysis of DAOCS. The binding mode of substrate was found to be crucial in determining which hydrogen atom in two methyl groups is first abstracted and whether the second H-abstraction to be abstracted in the final desaturation step locates in a suitable orientation. The highly reactive FeIV-oxo species prefers to abstract a hydrogen atom from one of two methyl groups in penN to trigger the ring arrangement. After the H-abstraction, the generated methylene radical intermediate can easily initiate the ring arrangement. First, the C-S bond cleaves to generate a thiyl radical, which is in concert with the formation of the terminal C═C double bond; the newly generated thiyl radical then rapidly shifts to the more stable tertiary C atom to complete ring expansion. In the final step, the FeIII-OH species abstracts the second hydrogen to give the desaturated DAOC product. During the catalysis, no active site residue is directly involved in the chemistry, which implies that the other pocket residues except the coordinate ones with iron play a role only in anchoring the substrate.

ß-Lactamase triggered visual detection of bacteria using cephalosporin functionalized biomaterials.

We report the conjugation of a chromogenic cephalosporin ß-lactamase (ßL) substrate to polymers and integration into biomaterials for facile, visual ßL detection. Identification of these bacterial enzymes, which are a leading cause of antibiotic resistance, is critical in the treatment of infectious diseases. The ßL substrate polymer conjugate undergoes a clear to deep yellow color change upon incubation with common pathogenic Gram-positive and Gram-negative bacteria species. We have demonstrated the feasibility of formulating hydrogels with the ßL substrate covalently tethered to a poly(ethylene glycol) (PEG) polymer matrix, exhibiting a visible color change in the presence of ßLs. This approach has the potential to be used in diagnostic biomaterials for point-of-care detection of ßL-producing bacteria, helping combat the spread of drug resistant microbes.