Investigation of enzymes and solvents in the production process of 6-ammonium penicillanic acid (6-APA) in industry to reduce costs and improve production conditions.

In this study, the optimization of the amount of enzyme consumed in the enzymatic phase of substitution of butanol solvent instead of methanol in the powder washing phase after filtration was investigated. To perform this study, different amounts of the enzyme penicillin G amidase (PGA) were tested in reactions with the same conditions. The highest efficiency was observed in the reaction that the ratio of penicillin powder to the amount of enzyme was 2:1. In this reaction, for every 100 g of penicillin consumed, 50 g of the PGA was used. Replacement of butanol instead of methanol after filtration, the powder obtained from this step was washed with butanol instead of methanol and the powder obtained from this step was examined after drying. The resulting solvent powder was very small and the drying speed of the powder increased compared to the time of methanol usage. Optimizing the amount of enzyme in this process due to the high cost of the enzyme made this reaction more economically viable at the end of this study. In this study, for the first time, butanol was used as a suitable substitute for methanol and the ratio of enzyme use to penicillin powder was optimized. This research deals with the future perspective in the field of research in this regard.

Optimization of aqueous two-phase systems for the production of 6-aminopenicillanic acid in integrated microfluidic reactors-separators.

Aqueous two-phase systems (ATPSs) were screened for the production of 6-aminopenicillanic acid (6-APA) catalyzed by penicillin acylase, followed by the extractive separation of 6-APA from the reaction mixture. The key point of this study was to find an ATPS exhibiting a large difference in the partition coefficients of the biocatalyst and reaction products. Several ATPSs based on polyethylene glycol (PEG)/phosphate, PEG/citrate, and PEG/dextran were tested. We found that an ATPS consisting of 15 wt% of PEG 4000, 10 wt% of phosphates, 75 wt% of water (pH value 8.0 after dissolution) provided optimal separation of 6-APA from the enzyme. While the 6-APA was mainly found in the top PEG phase, the free enzyme favored the bottom salt-rich phase. This ATPS also fulfils other important requirements: (i) high buffering capacity, reducing an undesirable pH decrease due to the dissociation of phenylacetic acid (the side product of the reaction), (ii) a relatively low cost of the ATPS components, (iii) the possibility of electrophoretic transport of fine droplets as well as the reaction products for both the acceleration of phase separation and the enhancement of 6-APA concentration in the product stream. Extraction experiments in microcapillary and batch systems showed that the transport of 6-APA formed in the salt-rich phase to the corresponding PEG phase could occur within 30 s. The experimental results described form a base of knowledge for the development of continuously operating integrated microfluidic reactors-separators driven by an electric field for the efficient production of 6-APA.

99m Tc-tazobactam, a novel infection imaging agent: Radiosynthesis, quality control, biodistribution, and infection imaging studies.

The radiolabeled drug 99m Tc-tazobactam (99m Tc-TZB) was developed and assessed as an infection imaging agent in Pseudomonas aeruginosa and Salmonella enterica infection-induced animal models by comparing with inflammation induced animal models. Radiosynthesis of 99m Tc-TZB was assessed while changing ligand concentration, reducing agent concentration, pH, and reaction time while keeping radioactivity constant (~370 MBq). Percent labeling of the resulting complex was measured using paper chromatography and instant thin layer chromatography. The analysis of the 99m Tc-TZB complex indicated >95% labeling yield and electrophoresis revealed complex is neutral in nature. The biodistribution study also showed predominantly renal excretion; however liver, stomach, and intestine also showed slight tracer agent uptake. The agent significantly accumulated in Pseudomonas aeruginosa and Salmonella enterica infection induced tissues 3.58 ± 0.26% and 2.43 ± 0.42% respectively at 1 hour postinjection. The inflamed tissue failed to uptake noticeable activity at 1 hour time point. The scintigraphic study results were found in accordance with biodistribution pattern. On the basis of our preliminary results, the newly developed 99m Tc-TZB can be used to diagnose bacterial infection and to discriminate between infected and inflamed tissues.

Synthesis of some heterofunctionalized penicillanic acid derivatives and investigation of their biological activities.

6-Substituted amino-penicillanic acid esters were synthesized starting with 6-apa. The compounds containing a 1,3-thiazole- or 1,3-thiazolidinone nucleus linked to the penicillanic acid skeleton via a hydrazino linkage were obtained from 6-apa. The treatment of carbonylamino and carbonothioylamino compounds with 4-chlorophenacyl bromide or ethyl bromoacetate gave 6-bis{4-[1,3-thiazol(idinone)amino]benzoyl}amino derivatives of 6-apa. Benzyl derivatives were synthesized in several steps, starting with 4-aminobenzoyl chloride. The treatment of 4-{[3-benzyl-4-oxo-1,3-thia(oxa)zolidin-2-ylidene]amino}benzoyl chlorides with 6-apa in ethanolic solution produced the 6-[bis(4-{[3-benzyl-4-oxo-1,3-thiazolidin-2-ylidene]amino}benzoyl)amino] derivative of penicillanic acid, while the reaction of the same intermediates in DMF gave the mono-substituted amino derivative of 6-apa. The synthesized compounds were screened for their biological activities, and some of them were found to possess good to moderate antimicrobial activity. Moreover, some of the compounds displayed antiurease, anti-ß-lactamase, and/or antilipase activities.

Recent advances in the synthesis of 1-monosubstituted 1,2,3-triazoles.

1-Monosubstituted 1,2,3-triazoles have widespread applications in diverse fields, and their preparation has attracted increasing attention. This review mainly covers the modern advances in the synthesis of these heterocycles based on different methods including the substitution of 1H-1,2,3-triazole and cycloaddition reactions involving moieties, such as acetylene, substituted acetylene,and vinyl compounds, among others.

A process to produce penicillin G acylase by surface-adhesion fermentation using Mucor griseocyanus to obtain 6-aminopenicillanic acid by penicillin G hydrolysis.

The production of extracellular and mycelia-associated penicillin G acylase (maPGA) with Mucor griseocyanus H/55.1.1 by surface-adhesion fermentation using Opuntia imbricata, a cactus, as a natural immobilization support was studied. Enzyme activity to form 6-aminopencillanic acid (6-APA) from penicillin G was assayed spectrophotometrically. The penicillin G hydrolysis to 6-APA was evaluated at six different times using PGA samples recovered from the skim milk medium at five different incubation times. Additionally, the effect of varying the penicillin G substrate concentration level on the PGA enzyme activity was also studied. The maximum reaction rate, V (max), and the Michaelis constant, K (M), were determined using the Michaelis-Menten model. The maximum levels for maPGA and extracellular activity were found to be 2,126.50 international unit per liter (IU/l; equal to 997.83 IU/g of support) at 48 h and 755.33 IU/l at 60 h, respectively. Kinetics of biomass production for total biomass showed a maximum growth at 60 h of 3.36 and 2.55 g/l (equal to 0.012 g of biomass per gram of support) for the immobilized M. griseocyanus biomass. The maPGA was employed for the hydrolysis of penicillin G to obtain 6-APA in a batch reactor. The highest quantity of 6-APA obtained was 226.16 mg/l after 40-min reaction. The effect of substrate concentration on maPGA activity was evaluated at different concentrations of penicillin G (0-10 mM). K(M) and V(max) were determined to be 3.0 x 10(-3) M and 4.4 x 10(-3) mM/min, respectively.

Synthesis and biological evaluation of penem inhibitors of bacterial signal peptidase.

We report the first synthesis of a 5S penem, known to bind bacterial type I signal peptidase, from the commercially available and inexpensive 6-aminopenicillanic acid. We report the first in vivo activity of the compound and use structure-activity relationship studies to begin to define the determinants of signal peptidase binding and also to begin to optimize the penem as an antibiotic.

Regiospecific syntheses of 6alpha-(1R-Hydroxyoctyl)penicillanic acid and 6beta-(1R-hydroxyoctyl)penicillanic acid as mechanistic probes of class D beta-lactamases.

The unique hydrophobic surface patches in class D beta-lactamases presented an opportunity for designing two compounds, 6alpha-(1R-hydroxyoctyl)penicillanic acid and 6beta-(1R-hydroxyoctyl)penicillanic acid, as mechanistic probes of these enzymes. In a sequence of three synthetic steps from benzhydryl 6,6-dibromopenicillanate, the targeted compounds were prepared in a stereospecific manner.

Enzymatic hydrolysis of penicillin for 6-APA production in three-liquid-phase system.

A dodecane/thermosensitive polymer/water three-liquid-phase system was introduced for enzymatic hydrolysis of penicillin G (Pen G) for 6-aminopenicillanic acid (6-APA). The enzyme was covalently attached to the terminal of PEO-PPO-PEO polymer (L63), which would be transferred into a polymer coacervate phase at high temperature above its "cloud point". 6-APA was primarily resided in the aqueous phase due to its zwitterionic nature. More than 70% phenylacetic acid (PAA) was transferred into the organic phase using trioctylmethylammonium hydroxide and trihexyl-(tetradecyl)phosphonium bis 2,4,4-trimethylpentylphosphinate ionic liquids (Cyphos IL-104) mixture at pH 5.5, while most of Pen G resided in water. As a result, high operational pH was permitted in three-liquid-phase system, which leads to higher enzymatic activity (120 IU at 40 degrees C) and stability (enzymatic half-time up to 55 h at 60 degrees C) in comparison with the value in butyl acetate/water two-phase system. On the other hand, two products in three-liquid-phase system might be automatically separated from the enzyme sphere into different phases at the same time, which facilitated the reaction equilibrium towards the product's side with 6-APA productivity of 80% at 42 degrees C, pH 5.5.

Direct diazo-transfer reaction on beta-lactam: synthesis and preliminary biological activities of 6-triazolylpenicillanic acids.

In this study we report the first example of a direct diazo-transfer reaction on readily available 6-aminopenicillanates to give 6-azidopenicillanates in high yield. Subsequent Cu(I)-catalyzed Huisgen cycloaddition between these 6-azidopenicillanates and assorted terminal alkynes facilely furnished 6-triazolylpenicillanic acids. Preliminary biological screening indicates that these triazolylpenicillanic acids possess low to moderate antibacterial activities.

Improving the industrial production of 6-APA: enzymatic hydrolysis of penicillin G in the presence of organic solvents.

The hydrolysis of penicillin G in the presence of an organic solvent, used with the purpose of extracting it from the culture medium, may greatly simplify the industrial preparation of 6-APA. However, under these conditions, PGA immobilized onto Eupergit displays very low stability (half-life of 5 h in butanone-saturated water) and a significant degree of inhibition by the organic solvent (30%). The negative effect of the organic solvent strongly depended on the type of solvent utilized: water saturated with butanone (around 28% v/v) had a much more pronounced negative effect than that of methylisobutyl ketone (MIBK) (solubility in water was only 2%). These problems were sorted out by using a new penicillin G acylase derivative designed to work in the presence of organic solvents (with each enzyme molecule surrounded by an hydrophilic artificial environment) and a suitable organic solvent (MIBK). Using such solvent, this derivative kept its activity unaltered for 1 week at 32 degrees C. Moreover, the enzyme activity was hardly inhibited by the presence of the organic solvent. In this way, the new enzyme derivative thus prepared enables simplification of the industrial hydrolysis of penicillin G.

Continuous penicillin G hydrolysis in an electro-membrane reactor with immobilized penicillin G acylase.

Penicillin G (2%, w/v in phosphate buffer, pH 8) was hydrolysed in a flow-through, miniature electro-membrane reactor with the penicillin G acylase immobilized in 5% (w/v) polyacrylamide (diam. 10 mm, thickness 2.6 mm, enzyme activity 24 U ml(-1)). The conversion of penicillin G increased from 0.15 to almost 0.5 when the electric current applied to the reactor was changed from -600 to +600 A/m2 with a substrate residency of 1 h.

Biotechnological applications of penicillin acylases: state-of-the-art.

This review describes the most recent developments in the biotechnological applications of penicillin acylases. This group of enzymes is involved mainly in the industrial production of 6-aminopenicillanic acid and the synthesis of semisynthetic beta-lactam antibiotics. In addition, penicillin acylases can also be employed in other useful biotransformations, such as peptide synthesis and the resolution of racemic mixtures of chiral compounds. Particular emphasis is placed on advances in detection of new enzyme specificities towards other natural penicillins, enzyme immobilization, and optimization of enzyme-catalyzed hydrolysis and synthesis in the presence of organic solvents.

Synthesis and biological evaluation of 6-bromo-6-substituted penicillanic acid derivatives as beta-lactamase inhibitors.

The synthesis of a selected set of 6-bromopenicillanic acid derivatives with an additional C6 substituent is reported. All these substances were tested as inhibitors of class A and C beta-lactamase enzymes derived from Escherichia coli (TEM-1) and E. cloacae (P99). As 6-(1-hydroxyethyl) derivatives 4c and 6c were found to be weak beta-lactamase inhibitors, they were further investigated in combination with amoxicillin against a series of beta-lactamase-producing bacterial strains. Some structure-activity relationships are discussed.

Synthesis of new C-6 alkyliden penicillin derivatives as beta-lactamase inhibitors.

New penicillin, penicillin sulfone and sulfoxide derivatives bearing a C-6-alkyliden substituent were prepared. Their chemical synthesis, in vitro antibacterial activity and inhibition properties against two selected enzymes representing Class A and C beta-lactamases are reported. Compounds 3a-c, 7a-c were able to inhibit either TEM-1 (a Class A enzyme, from Escherichia coli) or P-99 (a Class C enzyme, from E. cloacae), or both enzymes, when tested in competition experiments using nitrocefin as the reporter substrate. However, when tested in combination with amoxicillin, the same compounds did not show synergistic effects against E. coli and E. cloacae strains producing TEM-1 and P99 enzymes, respectively. This finding is most likely related to poor penetration through the bacterial cell wall, as shown by using a more permeable isogenic E. coli strain. Interestingly, a synergistic effect against a strain of S. aureus which produces PC1-enzyme (a Class A beta-lactamase) was observed for compound 3a when used in combination with amoxicillin.

Inhibition of beta-lactamases by 6,6-bis(hydroxylmethyl)penicillanate.

beta-Lactamases of classes A and C are the two most prevalent resistant determinants to beta-lactam antibiotics among bacterial pathogens. Both these enzymes pursue different mechanisms for their catalytic processes, highlighted by the fact that the hydrolytic water molecule in each approaches the ester of the intermediary acyl-enzyme species from the opposite ends. 6,6-Bis(hydroxylmethyl)penicillanate was designed as an inhibitor that would impair the approach of the hydrolytic water molecule in either of these enzymes upon formation of the acyl-enzyme species. The design, synthesis, and kinetic evaluation of this inhibitor are disclosed herein.

Synthesis and antibacterial activity of 5-nitrofuryl and 3-methoxy-2-nitrophenyl derivatives of 6 beta-aminopenicillanic, 7 beta-aminocephalosporanic and 7 beta-aminodesacetoxy-cephalosporanic acids.

A number of 5-nitrofuryl and 3-methoxy-2-nitrophenyl derivatives of 6 beta-aminopenicillanic (6 beta-APA), 7 beta-aminocephalosporanic (7 beta-ACA) and 7 beta-aminodesacetoxycephalosporanic (7 beta-ADCA) acids were synthesized by the method of mixed anhydrides or via Schiff bases. The chemical structures of the new compounds were confirmed by IR-, 1H-NMR and mass spectral data, obtained by negative ion electrospray ionization. The in vitro testing results indicated that all penicillins and cephalosporins prepared exhibited antibacterial activity equal to or in many cases considerably higher than those of ampicillin (CAS 69-53-4) and cephalexin (CAS 23325-78-2) against the Gram-positive microorganisms, excluding B. subtilis L2, B. subtilis HB2 and S. aureus 1/45 "Oxford". Their activity towards the two strains of Proteus mirabilis was also good being greater than that of cephalexin contrary to the demonstrated lower activity towards all strains of E. coli tested. The most active compounds which simultaneously possessed the broadest spectrum of antibacterial activity proved to be compounds 1 and 8 both bearing as a substituent a 5-nitrofuran group.

Synthesis and antibacterial activity of new arylamido derivatives of 6 beta-aminopenicillanic, 7 beta-aminocephalosporanic and 7 beta-aminodesacetoxycephalosporanic acids.

New semisynthetic penicillins and cephalosporins have been synthesized by acylation of 6 beta-aminopenicillanic, 7 beta-aminocephalosporanic and 7 beta-aminodesacetoxycephalosporanic acids with ortho-substituted aromatic acids, using the method of mixed anhydrides. The chemical structures of the compounds obtained were confirmed by elemental analysis and by IR- and 1H-NMR spectra. Antibacterial activities of the compounds were determined by the macrodilution susceptibility test in brain-heart infusion broth. Test organisms producing beta-lactamases: Bacillus subtilis L2, Bacillus subtilis HB2, Bacillus cereus 30, Bacillus subtilis 6633 ATCC, Bacillus mycoides 924; Staphylococcus aureus 1/45 "Oxford" as Gram-positive bacteria, and Escherichia coli 111, Escherichia coli K12/F-¿lac-/, Escherichia coli K12/F-¿lambda-/, Escherichia coli K12/F-¿lambda-¿lac+/ as Gram-negative bacteria. In general, the derivatives of 6 beta-aminopenicillanic acid were more active than 7 beta-aminocephalosporanic and 7 beta-aminodesacetoxycephalosporanic acid derivatives. Among all the compounds synthesized 6 beta-[4'-(dimethylamino)-azobenzene-2-amido]penicillanic acid and 6 beta-(N-phenylanthranilamido)penicillanic acid showed the best activity and were with the broadest spectrum of action.

Synthesis and evaluation of 2 beta-oxyimino and alkenylpenicillanic acid sulfone derivatives as beta-lactamase inhibitors.

The synthesis and in vitro synergies of 2 beta-alkenyl and oxyiminopenam sulfone derivatives are described. Most of the compounds synthesized exhibited good inhibitory activities and synergistic antibacterial activities with piperacillin and ceftriaxone, respectively, against several beta-lactamase producing strains. Particularly the 2 beta-alkenylpenam sulfone derivatives. 1e and 1g, showed good synergistic activity with ceftriaxone against Citrobacter freundi NIH 10018-68 and Proteus vulgaris 20. Also the compounds 2a, 2c, and 2f, 2 beta-oxyiminopenam sulfone derivatives, exhibited improved synergistic activity with piperacillin against Citrobacter freundi NIH 10018-68.

Synthesis and beta-lactamase inhibitory activity of new 6beta-cysteinesulfonamidopenicillanic acids.

New 6beta-cysteinesulfonamidopenicillanic acids and their sulfoxides were synthesized by sulfonylation of 6beta-aminopenicillanic acid or its (S)-sulfoxide with (R)-N-benzyloxycarbonylcysteinesulfonyl chloride ethyl ester (2a, 1b) and (R)-N-benzyloxycarbonylcysteinesulfonyl chloride benzyl ester (2a, 2b). The corresponding 6beta-cysteinesulfonamidopenicillanic acids sulfones 1c and 2c were prepared by oxidation of the sulfoxides 1b and 2b with potassium permanganate in aqueous medium. When combined with ampicillin some of the compounds reduced the minimal inhibitory concentrations of ampicillin against beta-lactamase producing strains.