Synthesis, characterization and SAR studies of bis(imino)pyridines as antioxidants, acetylcholinesterase inhibitors and antimicrobial agents.

In this study we synthesized a series of sixteen bis(imino)pyridines (BIPs) starting from 2,6-diaminopyridine and various aromatic aldehydes, and evaluated their antioxidant, antibacterial, antifungal and acetylcholinesterase (AChE) inhibitory activity. The chemical structures were elucidated by FTIR, elemental analysis, ESR and HRMS. 1H and 13C NMR spectra couldn't be acquired due to the formation of stable, carbon-centered radical cations in a solution, as confirmed by ESR spectroscopy and DFT calculations. The in vitro antioxidant potency was evaluated using four assays: free radical scavenging activity (DPPH and ABTS), reducing power and total antioxidant capacity assay. BIPs demonstrated excellent antioxidant properties, and two derivatives proved to be more potent than reference antioxidants (ascorbic acid and Trolox) in all assays. DFT calculations on ωB97XD/6-311++g(d,p) level of theory provided valuable insights into the radical scavenging mechanism of BIPs. For hydroxyl-substituted BIPs, hydrogen atom transfer (HAT) is a predominant mechanism, while the single electron transfer coupled with proton transfer (SET-PT) governs the antioxidant activity of other derivatives. Intramolecular hydrogen bonding (IHB) plays an important role in the mechanism of antioxidant activity as revealed by noncovalent interaction analysis and rotational barrier calculations. The spin density of radical cations is localized on carbon atoms of a pyridine ring, which corroborates with g-factors and multiplicity obtained from ESR analysis. The most potent BIP exhibited moderate inhibitory activity toward AChE (IC50 = 20 ± 4 µM), while molecular docking suggested binding at the peripheral anionic site of AChE with the MMFF94 binding enthalpy of -43.4 kcal/mol. Moderate in vitro antimicrobial activity of BIPs have been determined against several pathogenic bacterial strains: Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and clinical isolate of methicillin resistant S. aureus (MRSA). The antifungal activity of BIPs toward Candida albicans was also confirmed. The similarity ensemble approach combined with molecular docking suggested leucyl aminopeptidase as the probable antimicrobial target for the three most potent BIP derivatives.

Immobilization of horseradish peroxidase onto kaolin.

Kaolin showed as a very perspective carrier for the enzyme immobilization and it was used for the adsorption of horseradish peroxidase (HRP). The effects of the enzyme concentration and pH on the immobilization efficiency were studied in the reaction with pyrogallol and anthraquinone dye C.I. Acid Violet 109 (AV 109). In addition, Fourier transform infrared spectroscopy, scanning electron microscopy and analysis by Brunauer-Emmett-Teller were performed for kaolin, thermally activated kaolin and the immobilized enzyme. It has been shown that 0.1 IU of HRP-kaolin decolorized 87 % of dye solution, under the optimal conditions (pH 5.0, temperature 24 °C, dye concentration 40 mg/L and 0.2 mM of H2O2) within 40 min. The immobilized HRP decolorization follows the Ping Pong Bi-Bi mechanism with dead-end inhibition by the dye. The biocatalyst retained 35 ± 0.9 % of the initial activity after seven cycles of reuse in the decolorization reaction of AV 109 under optimal conditions in a batch reactor. The obtained kinetic parameters and reusability study confirmed improvement in performances of k-HRP compared to free, indicating that k-HRP has a great potential for environmental purposes.

Decolorization of anthraquinonic dyes from textile effluent using horseradish peroxidase: optimization and kinetic study.

Two anthraquinonic dyes, C.I. Acid Blue 225 and C.I. Acid Violet 109, were used as models to explore the feasibility of using the horseradish peroxidase enzyme (HRP) in the practical decolorization of anthraquinonic dyes in wastewater. The influence of process parameters such as enzyme concentration, hydrogen peroxide concentration, temperature, dye concentration, and pH was examined. The pH and temperature activity profiles were similar for decolorization of both dyes. Under the optimal conditions, 94.7% of C.I. Acid Violet 109 from aqueous solution was decolorized (treatment time 15 min, enzyme concentration 0.15 IU/mL, hydrogen peroxide concentration 0.4 mM, dye concentration 30 mg/L, pH 4, and temperature 24°C) and 89.36% of C.I. Acid Blue 225 (32 min, enzyme concentration 0.15 IU/mL, hydrogen peroxide concentration 0.04 mM, dye concentration 30 mg/L, pH 5, and temperature 24°C). The mechanism of both reactions has been proven to follow the two substrate ping-pong mechanism with substrate inhibition, revealing the formation of a nonproductive or dead-end complex between dye and HRP or between H2O2 and the oxidized form of the enzyme. Both chemical oxygen demand and total organic carbon values showed that there was a reduction in toxicity after the enzymatic treatment. This study verifies the viability of use of horseradish peroxidase for the wastewaters treatment of similar anthraquinonic dyes.

Immobilization of lipase from Candida rugosa on Sepabeads(®): the effect of lipase oxidation by periodates.

The objective of this paper was the investigation of a suitable Sepabeads(®) support and method for immobilization of lipase from Candida rugosa. Three different supports were used, two with amino groups, (Sepabeads(®) EC-EA and Sepabeads(®) EC-HA), differing in spacer length (two and six carbons, respectively) and one with epoxy group (Sepabeads(®) EC-EP). Lipase immobilization was carried out by two conventional methods (via epoxy groups and via glutaraldehyde), and with periodate method for modification of lipase. The results of activity assays showed that lipase retained 94.8% or 87.6% of activity after immobilization via epoxy groups or with periodate method, respectively, while glutaraldehyde method was inferior with only 12.7% of retention. The immobilization of lipase, previously modified by periodate oxidation, via amino groups has proven to be more efficient than direct immobilization of lipase via epoxy groups. In such a way immobilized enzyme exhibited higher activity at high reaction temperatures and higher thermal stability.

Detailed solvent, structural, quantum chemical study and antimicrobial activity of isatin Schiff base.

The ratios of E/Z isomers of sixteen synthesized 1,3-dihydro-3-(substituted phenylimino)-2H-indol-2-one were studied using experimental and theoretical methodology. Linear solvation energy relationships (LSER) rationalized solvent influence of the solvent-solute interactions on the UV-Vis absorption maxima shifts (νmax) of both geometrical isomers using the Kamlet-Taft equation. Linear free energy relationships (LFER) in the form of single substituent parameter equation (SSP) was used to analyze substituent effect on pKa, NMR chemical shifts and νmax values. Electron charge density was obtained by the use of Quantum Theory of Atoms in Molecules, i.e. Bader's analysis. The substituent and solvent effect on intramolecular charge transfer (ICT) were interpreted with the aid of time-dependent density functional (TD-DFT) method. Additionally, the results of TD-DFT calculations quantified the efficiency of ICT from the calculated charge-transfer distance (DCT) and amount of transferred charge (QCT). The antimicrobial activity was evaluated using broth microdilution method. 3D QSAR modeling was used to demonstrate the influence of substituents effect as well as molecule geometry on antimicrobial activity.