Synthesis Of Β-lactam Antibiotics Research Articles

Characterization and engineering of cephalosporin C acylases to produce 7-Aminocephalosporanic acid

7-Aminocephalosporanic acid (7-ACA) is the core nuclei for the synthesis of β-lactam antibiotics. Cephalosporin C acylases (CCAs) are a class of enzymes that enable the hydrolysis of cephalosporin C (CPC) to 7-ACA. In this study, we identified, characterized and engineered new CCAs to prepare 7-ACA. The best mutant A14 (L159S/A419V) was obtained after several rounds of semi-rational enzyme design using the combinatorial active-site saturation testing (CAST) and iterative evolution. A 2.8-fold increase in specific enzymatic activity (6.1 U/mg) was obtained compared to the wild-type enzyme. The engineered variant also enabled the hydrolysis of a variety of β-lactam antibiotics including Penicillin G and phenylacetyl-7-aminodeacetoxycephalosporanic acid (G-7-ADCA). Overall, this study discovered new CCAs from distinct bacterial origins and enabled the synthesis of various core β-lactam antibiotics nuclei.

Enlarging the substrate binding pocket of penicillin G acylase from Achromobacter sp. for highly efficient biosynthesis of β-lactam antibiotics

Penicillin G acylase (PGA) is a key biocatalyst for the enzymatic production of β-lactam antibiotics, which can not only catalyze the synthesis of β-lactam antibiotics but also catalyze the hydrolysis of the products to prepare semi-synthetic antibiotic intermediates. However, the high hydrolysis and low synthesis activities of natural PGAs severely hinder their industrial application. In this study, a combinatorial directed evolution strategy was employed to obtain new PGAs with outstanding performances. The best mutant βF24G/βW154G was obtained from the PGA of Achromobacter sp., which exhibited approximately a 129.62-fold and a 52.55-fold increase in specific activity and synthesis/hydrolysis ratio, respectively, compared to the wild-type AsPGA. Thereafter, this mutant was used to synthesize amoxicillin, cefadroxil, and ampicillin; all conversions > 99% were accomplished in 90–135 min with almost no secondary hydrolysis byproducts produced in the reaction. Molecular dynamics simulation and substrate pocket calculation revealed that substitution of the smallest glycine residue at βF24 and βW154 expanded the binding pocket, thereby facilitating the entry and release of substrates and products. Therefore, this novel mutant is a promising catalyst for the large-scale production of β-lactam antibiotics.

De Novo Biosynthesis of D-p-Hydroxyphenylglycine by a Designed Cofactor Self-Sufficient Route and Co-culture Strategy.

d-p-Hydroxyphenylglycine (D-HPG) is an important intermediate for the synthesis of β-lactam antibiotics with an annual market demand of thousands of tons. Currently, the main production processes are via chemical approaches. Although enzymatic conversion has been investigated for D-HPG production, synthesis of the substrate DL-hydroxyphenylhydantoin is still chemically based, which suffers from high pollution and harsh reaction conditions. In this study, one cofactor self-sufficient route for D-HPG production from l-phenylalanine was newly designed and the artificial pathway was functionalized by selecting suitable enzymes and adjusting their expressions in strain Pseudomonas putida KT2440. Notably, a new R-mandelate dehydrogenase from Lactococcus lactis with relatively high activity under pH neutral conditions was successfully mined to demonstrate the biosynthetic pathway in vivo. The performance of the engineered P. putida strain was further increased by enhancing cellular NAD availability and blocking l-phenylalanine consumption. Coupled with the l-phenylalanine producer, Escherichia coli strain ATCC 31884, a stable and interactive co-culture process was also developed by engineering a "cross-link auxotrophic" system to produce D-HPG directly from glucose. Thus, this study is the first approach for the de novo biosynthesis of D-HPG by engineering a non-natural pathway and lays the foundation for further improving the efficiency of D-HPG production via a green and sustainable route.

Silver(I)-catalyzed hydroamination of (3S, 4R)-4-acetoxy-3-[(R)-1-tert-butyldimethylsiloxy)ethyl]azetidine-2-one derivatives for the synthesis of carbapenem skeleton

From (3S, 4R)-4-acetoxy-3-[(R)-1-tert-butyldimethylsiloxy)ethyl]azetidine-2-one (AOSA), a common precursor for the synthesis of β-lactam antibiotics, zinc-mediated Barbier-type reaction and subsequent silver(I)-catalyzed hydroamination were conducted to build the carbapenem skeleton bearing carboxylate ester at the C-3 position. Depending on catalysts used, a new type of rearrangement was observed. It was also affected by reaction time and the quantities of catalysts.

Diphenylmethyl deprotection in continuous flow and its application in synthesis of β-lactam antibiotics

Diphenylmethyl deprotection in continuous flow and its application in synthesis of β-lactam antibiotics

Kinetic model development for α-amino ester hydrolase (AEH)-catalyzed synthesis of β-lactam antibiotics

α-Amino ester hydrolases (AEH) are a class of enzymes highly specific for catalyzing the synthesis and hydrolysis of amides and esters of α-amino acids. They are particularly promising catalyst candidates as an alternative to penicillin G acylase (PGA) for synthesis of a variety of β-lactam antibiotics, including cephalexin, cefadroxil, amoxicillin, and ampicillin. In this study, X. campestris pv. campestris AEH and two previously established quadruple variants designed for enhanced stability (N186D/A275P/V622I/E143G termed QVG and N186D/A275P/V622I/E143H termed QVH) were characterized in terms of their kinetic properties. AEH appears to have highest substrate specificity for cephalosporins, particularly cephalexin. The optimum pH value for activity was found to be between pH 6.5 – 6.75 for synthesis of amoxicillin, cephalexin, and cefadroxil, while synthesis selectivity consistently increased with decreasing pH value. Evidence for substrate inhibition preventing the β-lactam nucleus from binding and forming the corresponding amide is discussed. Time-course kinetic data across a wide range of substrate concentrations was found to corroborate the presence of substrate inhibition, and five possible kinetic models are derived to explain AEH-catalyzed cephalexin synthesis kinetic profiles. A best fit model that incorporates both competitive substrate inhibition and partial substrate inhibition was selected based on the Akaike Information Criterion (AIC) and quality of fit at high substrate concentration. Finally, progress of the synthesis of beta-lactams in a single CSTR was simulated using the best fit kinetic model and a Pareto optimal front was constructed for fractional yield, productivity, and substrate conversion to evaluate potential reactor configurations.

Efficient synthesis of β-lactam antibiotics with in situ product removal by a newly isolated penicillin G acylase.

A penicillin G acylase (PGA) from Achromobacter xylosoxidans PX02 was newly isolated, and site-directed mutagenesis at three important positions αR141, αF142, βF24 was carried out for improving the enzymatic synthesis of β-lactam antibiotics. The efficient mutant βF24A was selected, and the (Ps/Ph)ini (ratio between the initial rate of synthesis and hydrolysis of the activated acyl donor) dramatically increased from 1.42-1.50 to 23.8-24.1 by means of the optimization of reaction conditions. Interestingly, the efficient enzymatic synthesis of ampicillin (99.1% conversion) and amoxicillin (98.7% conversion) from a high concentration (600mM) of substrate 6-APA in the low acyl donor/nucleus ratio (1.1:1) resulted in a large amount of products precipitation from aqueous reaction solution. Meanwhile, the by-product D-phenylglycine was hardly precipitated, and 93.5% yield of precipitated ampicillin (561mM) and 94.6% yield of precipitated amoxicillin (568mM) were achieved with high purity (99%), which significantly simplified the downstream purification. This was the first study to achieve efficient β-lactam antibiotics synthesis process with in situ product removal, with barely any by-product formation. The effect enzymatic synthesis of antibiotics in aqueous reaction solution with in situ product removal provides a promising model for the industrial semi-synthesis of β-lactam antibiotics.

Poly-lysine supported cross-linked enzyme aggregates of penicillin G acylase and its application in synthesis of β-lactam antibiotics

Penicillin G acylase (PGA) from Providencia rettgeri PX04 (PrPGA) was utilized to synthesize β-lactam antibiotics. Poly-lysine supported cross-linked enzyme aggregates (PL-CLEAs) were prepared using PGA. Addition of poly-lysine significantly increased retention of PGA activity in CLEAs, with a decrease in the synthesis/hydrolysis (S/H) ratio. PL-CLEAs with 0.56 mg/mL poly-lysine retained 83% of free PGA activity, and displayed a higher S/H ratio than that of the free enzyme. Both PL-CLEAs and CLEAs exhibited high pH and thermal stabilities. PL-CLEAs possessed the best stability profile, and the lowest α value [(kcat/Km)Ps/(kcat/Km)AD], and was most effective at amoxicillin synthesis. A >94% yield of amoxicillin was achieved using a D-HPGME/6-APA ratio of 1.2:1 (240 mM, 200 mM), with fed-batch addition of D-HPGME. PL-CLEAs displayed excellent operational stability during amoxicillin synthesis. Over 97% of initial conversion was retained after twenty rounds of catalysis. PL-CLEAs exhibited greater potency than CLEAs in practical catalysis, permitting a higher concentration of reactants.

Structural analysis of a novel N-carbamoyl-d-amino acid amidohydrolase from a Brazilian Bradyrhizobium japonicum strain: In silico insights by molecular modelling, docking and molecular dynamics

In this work we performed several in silico analyses to describe the relevant structural aspects of an enzyme N-Carbamoyl-d-amino acid amidohydrolase (d-NCAase) encoded on the genome of the Brazilian strain CPAC 15 (=SEMIA 5079) of Bradyrhizobium japonicum, a nonpathogenic species belonging to the order Rhizobiales. d-NCAase has wide applications particularly in the pharmaceutical industry, since it catalyzes the production of d-amino acids such as D-p-hydroxyphenylglycine (D-HPG), an intermediate in the synthesis of β-lactam antibiotics. We applied a homology modelling approach and 50 ns of molecular dynamics simulations to predict the structure and the intersubunit interactions of this novel d-NCAase. Also, in order to evaluate the substrate binding site, the model was subjected to 50 ns of molecular dynamics simulations in the presence of N-Carbamoyl-d-p-hydroxyphenylglycine (Cp-HPG) (a d-NCAase canonical substrate) and water-protein/water-substrate interactions analyses were performed. Overall, the structural analysis and the molecular dynamics simulations suggest that d-NCAase of B. japonicum CPAC-15 has a homodimeric structure in solution. Here, we also examined the substrate specificity of the catalytic site of our model and the interactions with water molecules into the active binding site were comprehensively discussed. Also, these simulations showed that the amino acids Lys123, His125, Pro127, Cys172, Asp174 and Arg176 are responsible for recognition of ligand in the active binding site through several chemical associations, such as hydrogen bonds and hydrophobic interactions. Our results show a favourable environment for a reaction of hydrolysis that transforms N-Carbamoyl-d-p-hydroxyphenylglycine (Cp-HPG) into the active compound D-p-hydroxyphenylglycine (D-HPG). This work envisage the use of d-NCAase from the Brazilian Bradyrhizobium japonicum strain CPAC-15 (=SEMIA 5079) for the industrial production of D-HPG, an important intermediate for semi-synthesis of β-lactam antibiotics such as penicillins, cephalosporins and amoxicillin.

Johnson Matthey to offer Gusev Ester Hydrogenation Catalysts via an exclusive global licensing agreement with GreenCentre Canada

From (3S, 4R)-4-acetoxy-3-[(R)-1-tert-butyldimethylsiloxy)ethyl]azetidine-2-one (AOSA), a common precursor for the synthesis of β-lactam antibiotics, zinc-mediated Barbier-type reaction and subsequent silver(I)-catalyzed hydroamination were conducted to build the carbapenem skeleton bearing carboxylate ester at the C-3 position. Depending on catalysts used, a new type of rearrangement was observed. It was also affected by reaction time and the quantities of catalysts.

Efficient synthesis of β-lactam antibiotics with very low product hydrolysis by a mutant Providencia rettgeri penicillin G acylase.

Penicillin G acylase (PGA) was isolated from Providencia rettgeri PX04 (PrPGApx04) and utilized for the kinetically controlled synthesis of β-lactam antibiotics. Site-directed mutagenesis was performed to increase the process efficiency. Molecular docking was carried out to speculate the key mutant positions corresponding with synthetic activity, which resulted in the achievement of an efficient mutant, βF24G. It yielded higher conversions than the wild-type enzyme in the synthesis of amoxicillin (95 versus 17.2%) and cefadroxil (95.4 versus 43.2%). The reaction time for achieving the maximum conversion decreased from 14 to 16h to 2-2.5h. Furthermore, the secondary hydrolysis of produced antibiotics was hardly observed. Kinetic analysis showed that the (kcat/Km)AD value for the activated acyl donor D-hydroxyphenylglycine methyl ester (D-HPGME) increased up to 41 times. In contrast, the (kcat/Km)Ps values for the products amoxicillin and cefadroxil decreased 6.5 and 21 times, respectively. Consequently, the α value (kcat/Km)Ps/(kcat/Km)AD, which reflected the relative hydrolytic specificity of PGA for produced antibiotics with respect to the activated acyl donor, were only 0.028 and 0.043, respectively. The extremely low hydrolytic activity for the products of the βF24G mutant enabled greater product accumulation to occur during synthesis, which made it a promising enzyme for industrial applications.

Synthesis of an Oxacepham derivative from 1,2-O-Cyclohexylidene-α-DXylofuranose

Carbohydrates have gained much attention as cheap chiral starting materials in stereocontrolled target oriented synthesis. β-lactam antibiotics still remain the main tool against bacterial infections but the search for new active compounds has not been the aim of reported investigations. Synthesis of β-lactam antibiotics from carbohydrate precursors played a special role owing to the importance of these classes of compounds in modern chemotherapy. In this report, we carried out this subject to synthesize β-lactam derivative (oxacepham) via [2 + 2] cycloaddition of chlorosulfonyl isocyanate (CSI) to vinyl ether derived from carbohydrate (α-D-glucose).

Cephalosporin-acid synthetase of Escherichia coli strain VKPM B-10182: Genomic context, gene identification, producer strain production

An enzyme of cephalosporin-acid synthetase produced by the E. coli strain VKPM B-10182 has specificity for the synthesis of β-lactam antibiotics of the cephalosporin acids class (cefazolin, cefalotin, cefezole etc.). A comparison of the previously determined genomic sequence of E. coli VKPM B-10182 with a genome of the parent E. coli strain ATCC 9637 was performed. Multiple mutations indicating the long selection history of the strain were detected, including mutations in the genes of RNase and β-lactamases that could enhance the level of enzyme synthesis and reduce the degree of degradation of the synthesized cephalosporin acids. The CASA gene--a direct homolog of the penicillin G-acylase gene--was identified by bioinformatics methods. The homology of the gene was confirmed by gene cloning and the expression and determination of its enzymatic activity in the reaction of cefazolin synthesis. The CASA gene was isolated and cloned into the original expression vector, resulting in an effective E. coli BL2l(DE3) pMD0107 strain producing CASA.

Draft Genome Sequence of Escherichia coli Strain VKPM B-10182, Producing the Enzyme for Synthesis of Cephalosporin Acids.

Escherichia coli strain VKPM B-10182, obtained by chemical mutagenesis from E. coli strain ATCC 9637, produces cephalosporin acid synthetase employed in the synthesis of β-lactam antibiotics, such as cefazolin. The draft genome sequence of strain VKPM B-10182 revealed 32 indels and 1,780 point mutations that might account for the improvement in antibiotic synthesis that we observed.

Modulation of the Microenvironment Surrounding the Active Site of Penicillin G Acylase Immobilized on Acrylic Carriers Improves the Enzymatic Synthesis of Cephalosporins

The catalytic properties of penicillin G acylase (PGA) from Escherichia coli in kinetically controlled synthesis of β-lactam antibiotics are negatively affected upon immobilization on hydrophobic acrylic carriers. Two strategies have been here pursued to improve the synthetic performance of PGA immobilized on epoxy-activated acrylic carriers. First, an aldehyde-based spacer was inserted on the carrier surface by glutaraldehyde activation (immobilization yield = 50%). The resulting 3-fold higher synthesis/hydrolysis ratio (vs/vh1 = 9.7 ± 0.7 and 10.9 ± 0.7 for Eupergit® C and Sepabeads® EC-EP, respectively) with respect to the unmodified support (vs/vh1 = 3.3 ± 0.4) was ascribed to a facilitated diffusion of substrates and products as a result of the increased distance between the enzyme and the carrier surface. A second series of catalysts was prepared by direct immobilization of PGA on epoxy-activated acrylic carriers (Eupergit® C), followed by quenching of oxiranes not involved in the binding with the protein with different nucleophiles (amino acids, amines, amino alcohols, thiols and amino thiols). In most cases, this derivatization increased the synthesis/hydrolysis ratio with respect to the non derivatized carrier. Particularly, post-immobilization treatment with cysteine resulted in about 2.5-fold higher vs/vh1 compared to the untreated biocatalyst, although the immobilization yield decreased from 70% (untreated Eupergit® C) to 20%. Glutaraldehyde- and cysteine-treated Eupergit® C catalyzed the synthesis of cefazolin in 88% (±0.9) and 87% (±1.6) conversion, respectively, whereas untreated Eupergit® C afforded this antibiotic in 79% (±1.2) conversion.

Penicillin G acylase from Achromobacter sp. CCM 4824

Penicillin G acylase from Achromobacter sp. (NPGA) was studied in the enzymatic synthesis of β-lactam antibiotics by kinetically controlled N-acylation. When compared with penicillin acylase of Escherichia coli (PGA), the NPGA was significantly more efficient at syntheses of ampicillin and amoxicillin (higher S/H ratio and product accumulation) in the whole range of substrate concentrations. The degree of conversion of 6-aminopenicillanic acid to amoxicillin and ampicillin (160mM 6-APA, 350mM acyl donor methylester[Symbol: see text]HCl, pH6.3, 25°C, reaction time of 200min) with immobilized NPGA equaled 96.9% and 91.1%, respectively. The enzyme was highly thermostable with maximum activity at 60°C (pH8.0) and 65°C (pH6.0). Activity half-life at 60°C (pH8.0) and at 60°C (pH6.0) was 24min and 6.9h, respectively. Immobilized NPGA exhibited long operational stability with half-life of about 2,000 cycles for synthesis of amoxicillin at conversion conditions used in large-scale processes (230mM 6-APA, 340mM D-4-hydroxyphenylglycine methylester[Symbol: see text]HCl, 27.5°C, pH6.25). We discuss our results with literature data available for related penicillin acylases in terms of their industrial potential.

Changing the specificity of α-amino acid ester hydrolase toward para-hydroxyl cephalosporins synthesis by site-directed saturation mutagenesis

α-Amino acid ester hydrolases (AEHs) catalyze the synthesis of β-lactam antibiotics containing an α-amino group with decreased activity toward antibiotics with a p-hydroxyl group. The AEH gene from Xanthomonas rubrillineans was cloned and expressed in Escherichia coli. Based on the crystal structure of the AEH and cefprozil complex, 13 residues not directly involved in substrate recognition were mutated individually. The resulting ~1,300 mutants were screened for activity using cefprozil as a model product based on spectrophotometric assay in a 96-well format. Mutants with improved cefprozil synthetic activity revealed the particular importance of positions 87, 131 and 175 for specificity. The mutant V131S with the highest initial rates of synthesis toward three p-hydroxyl cephalosporins showed 23 %, 17 % and 64 % increase in maximum product accumulation of cefadroxil, cefprozil and cefatrizine, respectively.

Enzymatic synthesis of amoxicillin by penicillin G acylase in the presence of ionic liquids

A major obstacle for the industrial implementation of the enzymatic synthesis of β-lactam antibiotics is the limited yield of the product, due to undesirable hydrolytic reactions. This drawback can be partially avoided by reducing the water activity in the medium. Ionic liquids (ILs) have emerged as an alternative to conventional organic media due to their high thermal and chemical stability, negligible vapor pressure, non-flammability, and easy recycling. In this context, this paper assesses the catalytic activity of penicillin G acylase (E.C.3.5.1.11) in the synthesis of amoxicillin using different ILs, all based on the 1-butyl-3-methylimidazolium cation. An increase of 400% in selectivity (synthesis/hydrolysis, S/H ratio) was observed for the reactions carried out with BMI·PF6 as a cosolvent at 75% (vIL/vwater) when compared to the totally aqueous medium (using phosphate buffer). This figure reached 350% for BMI·NTf2, while for BMI·BF4 there was only a slight increase in selectivity. The highest conversion of the β-lactam nucleus (6-APA) was achieved using BMI·NTf2 as a cosolvent at 71% (v/v), more than 36% above the one in water. No deactivation of the enzyme after the reactions was observed in any of the ILs, and the physical integrity of the biocatalyst particles was maintained.

Optimization in the immobilization of penicillin G acylase by entrapment in xerogel particles with magnetic properties

Biocatalysis presents a sound alternative to chemical synthesis in the field of drug production, given the highly selective nature of biological catalysts. Penicillin G Acylase (PGA) from E. coli is currently used to hydrolyze penicillin G (PG) and catalyzes the synthesis of β-lactam antibiotics. In this work, particular emphasis is given to recent developments in penicillin G acylase immobilization, by entrapment simultaneously with nano-magnetic particles in a silica matrix. The sol–gel biocatalytic particles were prepared either by a conventional method (crushed powder) or by a more recent approach, based in an emulsion system using 150 mM AOT/isooctane, which allowed for the formation of spherical micro- and nanobeads. The effects on PGA activity of different sol–gel precursors, additives, enzyme concentration, aging, drying conditions and mechanical stability were evaluated. After these optimization studies, a mechanically stable carrier based on porous xerogels silica matrixes, starting from tetramethoxysilane (TMOS) with 65–67% PGA activity yield in these carriers allowed an immobilization yield of 74 mg protein gdry sol–gel−1 and 930 Ugdry sol–gel−1 for specific activity were obtained.

ChemInform Abstract: Exocyclic Bromination of N-Substituted β- and γ-Lactams.

Abstract Regioselective halogenation of the N-susbtituted lactams (3a)-(3d) affords the corresponding exocyclic bromides (4a)-(4d). The procedure provides a novel alternative route to N-(α-haloalkyl)-substituted lactams, which are of particular interest in the synthesis of β-lactam antibiotics.