Valerije Vrček

Thermal and enzymatic recovering of proteins from untanned leather waste.

The laboratory trials of a process to treat untanned leather waste to isolate valuable protein products are presented. In this comparative study, both thermal and enzymatic treatments of leather waste were performed. The enzymatic method utilizes commercially available alkaline protease at moderate temperatures and for short periods of time. The concentration of the enzyme was 500 units per gram of leather waste which makes the method cost-effective. Amino acid composition in the hydrolysate obtained by the enzyme hydrolysis of untanned leather waste is determined. Chemical and physical properties of protein powder products from untanned leather waste were evaluated by spectrophotometric and chromatographic methods and by use of electron microscope. The results of microbiological assays confirm that these products agree to food safety standards. This relatively simple treatment of untanned leather waste may provide a practical and economical solution to the disposal of potentially dangerous waste.

Antibacterial Properties of Metallocenyl-7-ADCA Derivatives and Structure in Complex with CTX-M β-Lactamase

A series of six novel metallocenyl-7-ADCA (metallocenyl = ferrocenyl or ruthenocenyl; 7-ADCA = 7-aminodesacetoxycephalosporanic acid) conjugates were synthesized and their antibacterial properties evaluated by biochemical and microbiological assays. The ruthenocene derivatives showed a higher level of inhibition of DD-carboxypeptidase 64-575, a Penicillin Binding Protein (PBP), than the ferrocene derivatives and the reference compound penicillin G. Protein X-ray crystallographic analysis revealed a covalent acyl-enzyme complex of a ruthenocenyl compound with CTX-M β-lactamase E166A mutant, corresponding to a similar complex with PBPs responsible for the bactericidal activities of these compounds. Most interestingly, an intact compound was captured at the crystal-packing interface, elucidating for the first time the structure of a metallocenyl β-lactam compound that previously eluded small molecule crystallography. We propose that protein crystals, even from biologically unrelated molecules, can be utilized to determine structures of small molecules.

Prereactive complexes in chlorination of benzene, triazine, and tetrazine: a quantum chemical study.

In order to perform a complete search for prereactive complexes between arenes and chlorine, the stochastic search method was employed. Stationary points are optimized at B3LYP, M05-2X, and MP2 levels, while improved energetics are calculated using the B2PLYP-D method, which includes corrections important for accurate description of dispersion forces. New intermediates were located and their mechanistic relevance has been discussed. It has been suggested that, at least in the gas-phase, the T-shaped complex precedes the formation of classical benzene/chlorine π-complex. No σ-complex is found on the energy surface, unless explicit counterions are included in calculations. Neither π- nor σ-complexes were located on the reactant side of chlorination of triazine, but only linear and T-shaped complexes were identified as stable minima. These structures represent important prereactive complexes for chlorination of triazine. In the case of tetrazine, which is unlikely to undergo direct chlorination, only two complexes (resting and T-shaped) were located.

Rearrangemements in piperidine-derived nitrogen-centered radicals. A quantum-chemical study.

Hydrogen migration reactions in piperidine radicals and their protonated counterparts were studied by quantum chemical calculations. G3B3 and G3(MP2)-RAD levels of theory were used as reference procedures in order to evaluate the efficiency of other computational models. In the gas phase, the 1,4-[N<-->C]-H and 1,4-[C<-->C]-H shifts are the most feasible rearrangements in the piperidine radical cation and neutral piperidine radical, respectively. However, if one explicit water molecule is well placed to facilitate hydrogen migrations, the 1,2-[N<-->C]-H shift becomes the most favorable process in both cases. Three different water-catalyzed [N<-->C]-H shift mechanisms were considered for piperidine radical cation, and only one is found to be operative in the case of neutral piperidine radical. We found that explicit solvation and protonation of piperidine-derived radicals strongly influence the overall mechanism of hydrogen migration reactions.

Chlorination of N-methylacetamide and amide-containing pharmaceuticals. Quantum-chemical study of the reaction mechanism.

Chlorination of amides is of utmost importance in biochemistry and environmental chemistry. Despite the huge body of data, the mechanism of reaction between amides and hypochlorous acid in aqueous environment remains unclear. In this work, the three different reaction pathways for chlorination of N-methylacetamide by HOCl have been considered: the one-step N-chlorination of the amide, the chlorination via O-chlorinated intermediate, and the N-chlorination of the iminol intermediate. The high-level quantum chemical G3B3 composite procedure, double-hybrid B2-PLYPD, B2K-PLYP methods, and global hybrid M06-2X and BMK methods have been employed. The calculated energy barriers have been compared to the experimental value of ΔG(#)298 ≈ 87 kJ/mol, which corresponds to reaction rate constant k(r) ≈ 0.0036 M(-1) s(-1). Only the mechanism in which the iminol form of N-methylacetamide reacts with HOCl is consistent (ΔG(#)298 = 87.3 kJ/mol at G3B3 level) with experimental results. The analogous reaction mechanism has been calculated as the most favorable pathway in the chlorination of small-sized amides and amide-containing pharmaceuticals: carbamazepine, acetaminophen, and phenytoin. We conclude that the formation of the iminol intermediate followed by its reaction with HOCl is the general mechanism of N-chlorination for a vast array of amides.

The chemical fate of paroxetine metabolites. Dehydration of radicals derived from 4-(4-fluorophenyl)-3-(hydroxymethyl)piperidine

Quantum chemical calculations have been used to model reactions which are important for understanding the chemical fate of paroxetine-derived radicals in the environment. In order to explain the experimental observation that the loss of water occurs along the (photo)degradation pathway, four different mechanisms of radical-induced dehydrations have been considered. The elimination of water from the N-centered radical cation, which results in the formation of an imine intermediate, has been calculated as the most feasible process. The predicted energy barrier (ΔG = 98.5 kJ mol(-1)) is within the barrier limits set by experimental measurements. All reaction intermediates and transition state structures have been calculated using the G3(MP2)-RAD composite procedure, and solvent effects have been determined using a mixed (cluster/continuum) solvation model. Several new products, which comply with the available experimental data, have been proposed. These structures could be relevant for the chemical fate of antidepressant paroxetine, but also for biologically and environmentally related substrates.

Reactions of the carbonyl group with nitroso compounds. The cases of pyruvic acid and acetaldehyde

Pyruvic acid and acetaldehyde react with substituted nitrosobenzenes to give the corresponding N-phenylacetohydroxamic acids. A mechanism for these reactions involving three sequential steps is proposed. The first step is the nucleophilic attack of the nitroso group on the carbonyl group, which leads to the formation of an unstable dipolar intermediate. This step is followed by proton transfer to the dipolar intermediate to form a more stable cationic intermediate, which, in the subsequent step, undergoes decarboxylation (in the case of pyruvic acid) or elimination of a proton, from the carbon of the nitrosocarbinolic group (in the case of acetaldehyde), leading to the final product, hydroxamic acid.The reaction of pyruvic acid includes an intramolecular reaction pathway, along with an acid-catalysed one. The experimental evidence obtained in support of such a description includes: (a) the order of reactivity of substituted nitrosobenzenes as demonstrated by the plot of log kobsvs. Hammett parameters with slope –1.97 in the case of pyruvic acid and –0.93 in the case of acetaicohyde; (b) the observation of acid-catalysed and ‘uncatalysed’ pathways in the reaction of pyruvic acid; (c)the observation of general acid catalysis in these reactions; (d) the observation of an inverse solvent deuterium isotope effect of 0.41 in the case of acetaldehyde; (e)the observation of a solvent deuterium isotope effect of ca. 1.0 in the acid-catalysed reaction, and solvent isotope effect of ca. 1.2 in the ‘uncatalysed’ reaction of pyruvic acid with nitrosobenzene.

Structural and electronic determinants of flavonoid binding to human serum albumin: An extensive ligand-based study

Flavonoids are ubiquitous plant metabolites that interfere with different biological processes in the human organism. After absorption they bind to human serum albumin (HSA), the most abundant carrier protein in the blood which also binds various hormones and drugs. Binding of flavonoids to HSA may impact their distribution, influencing the active concentration in the blood. To determine the most prominent features responsible for binding of 20 different flavonoid aglycones to the IIA region of HSA, in vitro fluorescence measurements and density functional theory (DFT) calculations were conducted. These results were then integrated to elucidate structure–affinity relationships. The presented results reveal that flavones and flavonoles bind most strongly to the IIA region of HSA. There are several electronic and structural determinants associated with flavonoid binding to this HSA region: high C3 nucleophilicity and partial charge of O4, high HOMO and LUMO energies, and coplanarity of AC and B rings. Both steric and electronic characteristics of flavonoids have a great impact on their binding to HSA, with hydrogen donor and acceptor properties and coplanarity being the most prominent.

Formation of hydroxamic acids promoted by metal ions. interaction of aldehyde carbonyl group with C-nitroso group in the presence of ferric ions

Abstract Formation of N-phenyl substituted hydroxamic acids in the reaction of formaldehyde with substituted mtrosobenzene is strongly catalysed by Fe3+ ions, which stabilize the transition state for the rate-controlling proton transfer from the carbon of nitrosocarbinolic cation intermediate leading to the product, hydroxamic acid

Reaction of pyruvic acid with nitrosobenzenes

Pyruvic acid reacts with nitrosobenzes in both acid-catalysed and uncatalysed reactions giving N-phenylacetohydroxamic acids.