6-aminopenicillanic Acid Research Articles

6-Aminopenicillanic acid revisited: A combined solid state NMR and in silico refinement

13C/15N (experimental and ab initio) solid-state NMR was used to achieve an affordable way to improve hydrogen refinement of 6-aminopenicillanic acid (6-APA) structure. The lattice effect on the isotropic chemical shifts was probed by using two different magnetic shielding calculations: isolated molecules and periodic crystal structure. The electron density difference maps of optimized and non-optimized structures were calculated in order to investigate the interactions inside the 6-APA unit cell. The 13C and 15N chemical shifts assignments were unambiguously stablished. In addition, some of the literature 13C resonances ambiguities could be properly solved.

Partial Least-Squares and Linear Support Vector Regression Chemometric Methods for Simultaneous Determination of Amoxicillin Trihydrate and Dicloxacillin Sodium in the Presence of Their Common Impurity.

Two multivariate chemometric models, namely, partial least-squares regression (PLSR) and linear support vector regression (SVR), are presented for the analysis of amoxicillin trihydrate and dicloxacillin sodium in the presence of their common impurity (6-aminopenicillanic acid) in raw materials and in pharmaceutical dosage form via handling UV spectral data and making a modest comparison between the two models, highlighting the advantages and limitations of each. For optimum analysis, a three-factor, four-level experimental design was established, resulting in a training set of 16 mixtures containing different ratios of interfering species. To validate the prediction ability of the suggested models, an independent test set consisting of eight mixtures was used. The presented results show the ability of the two proposed models to determine the two drugs simultaneously in the presence of small levels of the common impurity with high accuracy and selectivity. The analysis results of the dosage form were statistically compared to a reported HPLC method, with no significant difference regarding accuracy and precision, indicating the ability of the suggested multivariate calibration models to be reliable and suitable for routine analysis of the drug product. Compared to the PLSR model, the SVR model gives more accurate results with a lower prediction error, as well as high generalization ability; however, the PLSR model is easy to handle and fast to optimize.

A breakthrough in enzyme technology to fight penicillin resistance-industrial application of penicillin amidase.

Enzymatic penicillin hydrolysis by penicillin amidase (also penicillin acylase, PA) represents a Landmark: the first industrially and economically highly important process using an immobilized biocatalyst. Resistance of infective bacteria to antibiotics had become a major topic of research and industrial activities. Solutions to this problem, the antibiotics resistance of infective microorganisms, required the search for new antibiotics, but also the development of derivatives, notably penicillin derivatives, that overcame resistance. An obvious route was to hydrolyse penicillin to 6-aminopenicillanic acid (6-APA), as a first step, for the introduction via chemical synthesis of various different side chains. Hydrolysis via chemical reaction sequences was tedious requiring large amounts of toxic chemicals, and they were cost intensive. Enzymatic hydrolysis using penicillin amidase represented a much more elegant route. The basis for such a solution was the development of techniques for enzyme immobilization, a highly difficult task with respect to industrial application. Two pioneer groups started to develop solutions to this problem in the late 1960s and 1970s: that of Günter Schmidt-Kastner at Bayer AG (Germany) and that of Malcolm Lilly of Imperial College London. Here, one example of this development, that at Bayer, will be presented in more detail since it illustrates well the achievement of a solution to the problems of industrial application of enzymatic processes, notably development of an immobilization method for penicillin amidase suitable for scale up to application in industrial reactors under economic conditions. A range of bottlenecks and technical problems of large-scale application had to be overcome. Data giving an inside view of this pioneer achievement in the early phase of the new field of biocatalysis are presented. The development finally resulted in a highly innovative and commercially important enzymatic process to produce 6-APA that created a new antibiotics industry and that opened the way for the establishment of over 100 industrial processes with immobilized biocatalysts worldwide today.

Quantum Chemical Modeling of Molecule 6-Aminopenicillanic Acid (6-Apa), of Model of 6-Apa-Phosgene and 6-Apa-Acetyl Chloride

For the first time a quantum-chemical method PM3 (Parametric Method 3) in semi empirical approach appreciated geometric and electronic parameters of the new acylated form the molecules 6-APA (6-aminopenicillanic acid), identified patterns of change according to the nature of the acylating agent. In general, the process of acylation of the molecule 6-APA by acylating agents (phosgene and acetyl chloride) leads to much larger changes in new systems of electronic structure than in its spatial structure. In this electronic structure analysis indicates that the acylation of 6-APA of the molecule both acylating agents leads to formation of more stable in the plan hydrolytic cleavage of form of this acid. This fact is a prediction of the characters and is positive for future synthesis of new antibiotics on the basis of 6-APA. Significant absolute value and the sign of the negative enthalpy of formation calculated by PM3, for both systems 6-APA-phosgene 6-APA-acetyl chloride indicate their high thermodynamic stability. The most stable in terms of the thermodynamic system is the 6-APA-acetyl chloride.

Degradation Kinetics and Mechanism of a β-Lactam Antibiotic Intermediate, 6-Aminopenicillanic Acid, in a New Integrated Production Process

In an effort to promote sustainability and to reduce manufacturing costs, the traditional production process for 6-aminopenicillanic acid (6-APA) has been modified to include less processing units. The objectives of this study are to investigate the degradation kinetics of 6-APA, to propose a reasonable degradation mechanism, and to optimize the manufacturing conditions within this new process. A series of degradation kinetic studies were conducted in the presence of impurities, as well as at various chemical and physical conditions. The concentrations of 6-APA were determined by high-performance liquid chromatography. An Arrhenius-type kinetic model was established to give a more accurate prediction on the degradation rates of 6-APA. A hydrolysis degradation mechanism is shown to be the major pathway for 6-APA. The degradation mechanisms and the kinetic models for 6-APA in the new system enable the design of a good manufacturing process with optimized parameters.

Efficient cascade synthesis of ampicillin from penicillin G potassium salt using wild and mutant penicillin G acylase from Alcaligenes faecalis

To avoid isolation and purification of the intermediate 6-aminopenicillanic acid (6-APA), a two-enzyme two-step cascade synthesis of ampicillin from penicillin G was established. In purely aqueous medium, penicillin G hydrolysis and ampicillin synthesis were catalyzed by immobilized wild-type and mutagenized penicillin G acylases from Alcaligenes faecalis (Af PGA), respectively (Fig. 1). The βF24 G mutant Af PGA (the 24th Phenylalanine of the β-subunit was replaced by Glycine) was employed for its superior performance in enzymatic synthesis of ampicillin. By optimizing the reaction conditions, including enzyme loading, temperature, initial pH and d-PGME/6-APA ratio, the conversion of the second step of ampicillin synthesis reached approximately 90% in 240min and less than 1.7mole d-PGME were required to produce 1mole ampicillin. Overall, in a 285min continuous two-step procedure, an ampicillin yield of 87% was achieved, demonstrating the possibility of improving the cascade synthesis of ampicillin by mutagenized PGA, providing an economically efficient and environmentally benign procedure for semi-synthetic penicillins antibiotics synthesis.

In situ one-pot preparation of superparamagnetic hydrophilic porous microspheres for covalently immobilizing penicillin G acylase to synthesize amoxicillin

Magnetic hydrophilic porous microspheres were successfully one-pot synthesized for the first time via in situ inverse suspension polymerization of glycidyl methacrylate, N,N′-methylene bisacrylamide and 2-hydroxyethyl methacrylate in the presence of Fe3+ and Fe2+ dispersed in formamide, which were denoted as magnetic Fe3O4-GMH microspheres. The morphology and properties of magnetic Fe3O4-GMH microspheres were characterized by SEM, VSM, XRD, FTIR, and so on. The formamide content had an important influence on the morphology of Fe3O4-GMH, and nearly perfectly spherical Fe3O4-GMH particles were formed when the amount of formamide was 15ml. The diameters of the microspheres were in the range of 100–200μm and Fe3O4-GMH exhibited superparamagnetic behavior with the saturation magnetization of 5.44emu/g. The specific surface area of microspheres was 138.7m2/g, the average pore diameter and pore volume were 15.1nm and 0.60cm3/g, respectively. The content of oxirane groups on Fe3O4-GMH was 0.40mmol/g. After penicillin G acylase (PGA) was covalently immobilized on Fe3O4-GMH microspheres, the catalytic performance for amoxicillin synthesis by 6-aminopenicillanic acid and d-hydroxyphenylglycine methyl ester was largely improved. As a result, 90.1% amoxicillin yield and 1.18 of the synthesis/hydrolysis (S/H) ratio were achieved on PGA/Fe3O4-GMH with ethylene glycol as solvent, but only 62.6% amoxicillin yield and 0.37 of the S/H ratio were obtained on free PGA under the same reaction conditions. Furthermore, the amoxicillin yield and S/H ratio were still kept at 88.2% and 1.06, respectively after the immobilized PGA was magnetically separated and recycled for 10 times, indicating that PGA/Fe3O4-GMH had a very good reusability.

Synthesis and antimicrobial activities of new higher amino acid Schiff base derivatives of 6-aminopenicillanic acid and 7-aminocephalosporanic acid

Novel β-lactam derivatives (1c-3c) (1d-3d) were produced by using 6-aminopenicillanic acid (6-APA), 7-aminocephalosporanic acid (7-ACA) and the higher amino acid Schiff bases. The synthesized compounds were characterized by elemental analysis, IR, 1H/13C NMR and UV–vis spectra. Antibacterial activities of all the higher amino acid Schiff bases (1a-3a) (1b-3b) and β-lactam derivatives were screened against three gram negative bacteria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii RSKK 02026), three gram positive bacteria (Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 07005, Bacillus subtilis ATCC 6633) and their drug-resistant isolates by using broth microdilution method. Two fungi (Candida albicans and Candida krusei) were used for antifungal activity.

In vivo kinetic analysis of the penicillin biosynthesis pathway using PAA stimulus response experiments

In this study we combined experimentation with mathematical modeling to unravel the in vivo kinetic properties of the enzymes and transporters of the penicillin biosynthesis pathway in a high yielding Penicillium chrysogenum strain. The experiment consisted of a step response experiment with the side chain precursor phenyl acetic acid (PAA) in a glucose-limited chemostat. The metabolite data showed that in the absence of PAA all penicillin pathway enzymes were expressed, leading to the production of a significant amount of 6-aminopenicillanic acid (6APA) as end product. After the stepwise perturbation with PAA, the pathway produced PenG within seconds. From the extra- and intracellular metabolite measurements, hypotheses for the secretion mechanisms of penicillin pathway metabolites were derived. A dynamic model of the penicillin biosynthesis pathway was then constructed that included the formation and transport over the cytoplasmic membrane of pathway intermediates, PAA and the product penicillin-G (PenG). The model parameters and changes in the enzyme levels of the penicillin biosynthesis pathway under in vivo conditions were simultaneously estimated using experimental data obtained at three different timescales (seconds, minutes, hours). The model was applied to determine changes in the penicillin pathway enzymes in time, calculate fluxes and analyze the flux control of the pathway. This led to a reassessment of the in vivo behavior of the pathway enzymes and in particular Acyl-CoA:Isopenicillin N Acyltransferase (AT).

Comparison of binding behavior for molecularly imprinted polymers prepared by hierarchical imprinting or Pickering emulsion polymerization.

The aim of this study was the evaluation of the binding performances and selectivity of molecularly imprinted beads prepared toward several penicillins (i) by hierarchical bulk polymerization in the pores of template-grafted silica microbeads (hMIPs) and (ii) by Pickering emulsion polymerization in the presence of template-decorated silica nanobeads (pMIPs). 6-Aminopenicillanic acid was chosen as the common fragmental mimic template. Both approaches produced micron-sized polymeric beads with good recognition properties toward the target ligands whereas the selectivity pattern appeared quite different. The polymer prepared by the Pickering emulsion approach showed binding properties similar to imprinted beads prepared by hierarchical approach. Equilibrium binding constants changed their values from 0.1-0.2 × 10(6) (hMIPs) to 0.2-0.6 × 10(6) M(-1) (pMIPs), while the binding site densities changed from 3.7-4.8 (hMIPs) to 0.3-0.55 μmol/g (pMIPs). Compared to the hierarchical polymerization, Pickering emulsion polymerization represents a more practical approach when a template mimic needs to be used.

Beta-Lactamase Repressor BlaI Modulates Staphylococcus aureus Cathelicidin Antimicrobial Peptide Resistance and Virulence.

BlaI is a repressor of BlaZ, the beta-lactamase responsible for penicillin resistance in Staphylococcus aureus. Through screening a transposon library in S. aureus Newman for susceptibility to cathelicidin antimicrobial peptide, we discovered BlaI as a novel cathelicidin resistance factor. Additionally, through integrational mutagenesis in S. aureus Newman and MRSA Sanger 252 strains, we confirmed the role of BlaI in resistance to human and murine cathelidicin and showed that it contributes to virulence in human whole blood and murine infection models. We further demonstrated that BlaI could be a target for innate immune-based antimicrobial therapies; by removing BlaI through subinhibitory concentrations of 6-aminopenicillanic acid, we were able to sensitize S. aureus to LL-37 killing.

Efficient enzymatic synthesis of ampicillin by mutant Alcaligenes faecalis penicillin G acylase

Semi-synthetic β-lactam antibiotics (SSBAs) are one of the most important antibiotic families in the world market. Their enzymatic synthesis can be catalyzed by penicillin G acylases (PGAs). In this study, to improve enzymatic synthesis of ampicillin, site-saturating mutagenesis was performed on three conserved amino acid residues: βF24, αR146, and αF147 of thermo-stable penicillin G acylase from Alcaligenes faecalis (Af PGA). Four mutants βF24G, βF24A, βF24S, and βF24P were recovered by screening the mutant bank. Kinetic analysis of them showed up to 800-fold increased kcat/Km value for activated acyl donor D-phenylglycine methyl ester (D-PGME). When βF24G was used for ampicillin synthesis under kinetic control at industrially relevant conditions, 95% of nucleophile 6-aminopenicillanic acid (6-APA) was converted to ampicillin in aqueous medium at room temperature while 12% process time is needed to reach maximum product accumulation at 25% enzyme concentration compared with the wild-type Af PGA. Consequently, process productivity of enzymatic synthesis of ampicillin catalyzed by Af PGA was improved by more than 130 times, which indicated an enzyme viable for efficient SSBAs synthesis.

Screening reactivity of molecules 6-aminopenicillanic acid and its acylated forms according to the calculation results molecular modeling

Screening reactivity of molecules 6-aminopenicillanic acid and its acylated forms according to the calculation results molecular modeling

Fluorescent supramolecular tweezers for selective recognition of cephradine

Novel supramolecular molecular tweezers based on a biphenyl bis-triazole hexahydroquinoline system are designed and synthesized as a highly sensitive and selective fluorescent probe for recognizing and detecting cephradine in the presence of cefotaxime, amoxicillin, penicillin, 6-aminopenicillanic acid, diclofenac, pefloxacin, ceftriaxone, cefaclor and paracetamol at a pH of 7.7. The detection limit is calculated to be 1μM with a regression coefficient of 0.99. The binding ratio of the tweezers and cephradine is determined to be 1:1 according to ESI-MS analysis, which is further supported by the Job plot. The structure of the supramolecular molecular tweezers and its precursors are characterized by IR, NMR, mass spectroscopy and XRD.

Effect of bed configuration of immobilized biocatalysts on penicillin G hydrolysis efficiency

The external and internal mass transfer of Penicillin G in the process of its enzymatic hydrolysis to 6-Aminopenicillanic acid under competitive and non-competitive inhibitions have been comparatively analyzed for a bioreactor with mobile bed vs. a stationary basket bioreactor, both with Penicillin amidase immobilized in Eupergit C. The Penicillin G mass transfer and hydrolysis enzymatic rates have been analyzed by means of the ratios’ values between the oxygen mass transfer coefficients, effectiveness factors, external mass flows and Penicillin G concentrations at the biocatalyst particle surface for the considered bioreactors. The results indicated that the bioreactor with mobile bed is more efficient especially for biocatalyst particles with diameter under 1.5 mm. For larger particles the performances of the two bioreactors become similar. Moreover, taking into consideration the external mass flow of Penicillin G and the number of enzymatic hydrolysis cycles, the basket bioreactor is recommended. The mathematical equations proposed are in good concordance with the experimental results, the average deviations varying from ±4.11% for the bioreactor with mobile bed of immobilized Penicillin amidase to ±5.03% for the basket bioreactor.

Hydrophilic porous magnetic poly(GMA-MBAA-NVP) composite microspheres containing oxirane groups: An efficient carrier for immobilizing penicillin G acylase

Abstract Magnetic hydrophilic polymeric microspheres containing oxirane groups were prepared by inverse suspension polymerization of glycidyl methacrylate (GMA), N, N′-methylene bisacrylamide (MBAA) and N-vinyl pyrrolidone (NVP) in the existence of formamide, which were denoted as magnetic poly(GMA-MBAA-NVP) microspheres. The magnetic poly(GMA-MBAA-NVP) microspheres were characterized by scanning electron microscopy (SEM), FT-IR spectroscopy, X-ray diffraction (XRD), vibrating sample magnetometer (VSM) and so on. The results showed that poly(GMA-MBAA-NVP) microspheres possessed well spherical shape, narrow size distribution, abundant porous structure, reactive oxirane groups and superparamagnetic properties. Formamide used in the present work served as a modifier, a dispersant and a porogen to form final porous polymer microspheres. The penicillin G acylase (PGA) was covalently immobilized onto the magnetic microspheres through the reaction between the amino groups of enzyme and the oxirane groups on the microspheres for producing 6-aminopenicillanic acid (6-APA). The effects of GMA/NVP ratio and crosslink density on the activity of immobilized PGA were investigated. The highest apparent activity, enzyme loading and coupling yield of immobilized PGA were 821 IU/g, 65.3 mg/g and 42.3% respectively when the mass ratio of GMA/NVP was 1:1 and crosslink density was 60%. Compared with the free PGA, immobilized PGA showed a wider range of pH value and reaction temperature. The relative activity and reaction rate of immobilized PGA remained almost constant after 20 recycles. The magnetic poly(GMA-MBAA-NVP) microspheres would be very promising carriers for immobilizing enzymes in industrial application.

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.

Catalytic Cycle of Penicillin Acylase from Escherichia coli: QM/MM Modeling of Chemical Transformations in the Enzyme Active Site upon Penicillin G Hydrolysis

Penicillin acylase from Escherichia coli is a unique enzyme that belongs to the recently discovered superfamily of N-terminal nucleophile hydrolases. It catalyzes selective hydrolysis of the side chain amide bond of penicillins and cephalosporins while leaving the labile amide bond in the β-lactam ring intact. Despite wide applications of penicillin acylase in the industry of β-lactam antibiotics and production of chiral amino compounds, its catalytic mechanism at atomic resolution has not yet been characterized. The complete cycle of chemical transformations of the most specific substrate of the enzyme, penicillin G, leading to formation of 6-aminopenicillanic and phenylacetic acids was modeled following quantum mechanics–molecular mechanics (QM/MM) calculations of the minimum energy reaction profile. The active site residues and the substrate were included in the QM part, and the rest of the system was treated applying molecular mechanics and classical force field parameters. The 3D structures in the enzyme active site corresponding to the noncovalent enzyme–substrate complex, the covalent acylenzyme intermediate, the noncovalent enzyme–product complex, the tetrahedral intermediates, and the respective transition states have been identified. QM/MM studies have shown that the α-amino group of the N-terminal catalytic βSer1 plays a key role in the catalytic machinery and directly assists its hydroxyl group in a proton relay at major stages of penicillin acylase catalytic mechanism, formation and hydrolysis of the covalent acylenzyme intermediate, which are characterized by close energy barriers. The βSer1 residue together with the oxyanion hole residues βAla69 and βAsn241 as well as βArg263 and βGln23 constitute a buried active site interaction network responsible for stabilization of tetrahedral intermediates, transition states, orientation of substrate and catalytic residues. βArg263 and βGln23 maintain the integrity of the catalytic machinery: βArg263 participates in orientation of the substrate as well as the α-amino group of βSer1 and coordinates the oxyanion hole residue βAsn241 across the whole catalytic cycle, whereas the backbone of βGln23 is responsible for orientation of both the βSer1 and the substrate. These results deliver insight into the earlier unknown ability of N-terminal amino acid to activate its own nucleophilic group directly as well as into organization of the stabilizing interaction network in penicillin acylase’s active site and will be used to design more effective enzyme variants for synthesis of new penicillins and cephalosporins.

ENGINEERING ASPECTS OF PENICILLIN G TRANSFER AND CONVERSION TO 6-AMINOPENICILLANIC ACID IN A BIOREACTOR WITH A MOBILE BED OF IMMOBILIZED PENICILLIN AMIDASE

This article presents studies on the external and internal mass transfers of penicillin G for 6-aminopenicillanic acid enzymatic production using a bioreactor with a stirred bed of immobilized penicillin amidase. By means of the substrate mass balance for a single particle of biocatalyst and considering the kinetic model adapted for competitive and noncompetitive inhibitions, specific mathematical models were developed for describing the profiles of penicillin G concentration in the outer and inner regions of biocatalyst and for estimating its mass flows in the liquid boundary layer surrounding the particle and inside the particle. The values of the mass flows are significantly influenced by the internal diffusion velocity and rate of the enzymatic conversion of substrate. These cumulated influences led to the appearance of an enzymatic inactive region near the particle center, its magnitude varying from 0 to 9.2% of the overall volume of particles.

Enzymatic hydrolysis of penicillin and in situ product separation in thermally induced reversible phase-separation of ionic liquids/water mixture

Enzymatic hydrolysis of penicillin G to produce 6-aminopenicillanic acid, key intermediate for the production of semisynthetic β-lactam antibiotics, is one of the most relevant example of industrial implementation of biocatalysts. The hydrolysis reaction is traditionally carried out in aqueous buffer at pH 7.5–8. However, the aqueous rout exhibits several drawbacks in enzyme stability and product recovery. In this study, several ionic liquids (ILs) have been used as media for enzymatic hydrolysis of penicillin G. The results indicated that hydrophobic ILs/water two-phase system were good media for the reaction. In addition, a novel aqueous two-phase system based on the lower critical solution temperature type phase changes of amino acid based ILs/water mixture was developed for in situ penicillin G hydrolysis and product separation. For instance, hydrolysis yield of 87.13% was obtained in system containing 30wt% [TBP][Tf-ILe] with pH control (pH 7.6). Since the phase-separation of this medium system can be reversible switched from single to two phases by slightly changing the solution temperature, enzymatic hydrolytic reaction and product recovery were more efficient than those of aqueous system. In addition, the ILs could be reused for at least 5 cycles without significant loss in hydrolysis efficiency.