Antimicrobial macrozones interact with biological macromolecules via two-site binding mode of action: Fluorimetric, NMR and docking studies.

Macrozones are novel conjugates of azithromycin and thiosemicarbazones, which exhibit very good in vitro antibacterial activities against susceptible and some resistant bacterial strains thus showing a potential for further development. A combination of spectrometric (fluorimetry, STD and WaterLOGSY NMR) and molecular docking studies provided insights into atomic details of interactions between selected macrozones and biological receptors such as E. coli ribosome and bovine serum albumin. Fluorimetric measurements revealed binding constants in the micro-molar range while NMR experiments provided data on binding epitopes. It has been demonstrated that both STD and WaterLOGSY gave comparable and consistent results unveiling atoms in intimate contacts with biological receptors. Docking studies pointed towards main interactions between macrozones and E. coli ribosome which included specific π - π stacking and hydrogen bonding interactions with thiosemicarbazone part extending down the ribosome exit tunnel. The results of the docking experiments were in fine correlation with those obtained by NMR and fluorimetry. Our investigation pointed towards a two-site binding mechanism of interactions between macrozones and E. coli ribosome which is the most probable reason for their activity against azithromycin-resistant strains. Much better activity of macrozone-nickel coordinated compound against E. coli ribosome compared to other macrozones has been attributed to the higher polarity which enabled better bacterial membrane penetration and binding of the two thiosemicarbazone units thus additionally contributing to the overall binding energy. The knowledge gained in this study should play an important role in anti-infective macrolide design in the future.

Deep reinforcement learning classification of sparkling wines based on ICP-MS and DOSY NMR spectra.

An approach that combines NMR spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS) and advanced tensor decomposition algorithms with state-of-the-art deep learning procedures was applied for the classification of Croatian continental sparkling wines by their geographical origin. It has been demonstrated that complex high-dimensional NMR or ICP-MS data cannot be classified by higher-order tensor decomposition alone. Extension of the procedure by deep reinforcement learning resulted in an exquisite neural network predictive model for the classification of sparkling wines according to their geographical origin. A network trained on half of the sample set was able to classify even 94% of all samples. The model can particularly be useful in cases where the number of samples is limited and when simpler statistical methods fail to produce reliable data. The model can further be exploited for the identification and differentiation of sparkling wines including a high potential for authenticity or quality control.

Improvement of the Nutraceutical Profile of Brewer's Spent Grain after Treatment with Trametes versicolor.

Brewer's spent grain (BSG) is an important secondary raw material that provides a readily available natural source of nutraceuticals. It finds its largest application as animal feed and part of the human diet, while the future perspective predicts an application in the production of value-added products. In order to investigate a sustainable BSG treatment method, two BSG samples (BSG1 and BSG2) were evaluated as substrates for the production of hydrolytic (xylanase, ß-glucosidase and cellulase) and lignolytic enzymes (laccase, manganese peroxidase and lignin peroxidase) by solid-state fermentation (SSF) with Trametes versicolor while improving BSG nutritional value. The biological treatment was successful for the production of all hydrolytic enzymes and laccase and manganese peroxidase, while it was unsuccessful for the production of lignin peroxidase. Because the two BSGs were chemically different, the Trametes versicolor enzymes were synthesized at different fermentation times and had different activities. Consequently, the chemical composition of the two BSG samples at the end of fermentation was also different. The biological treatment had a positive effect on the increase in protein content, ash content, polyphenolic compounds, and sugars in BSG1. In BSG2, there was a decrease in the content of reducing sugars. Cellulose, hemicellulose, and lignin were degraded in BSG1, whereas only cellulose was degraded in BSG2, and the content of hemicellulose and lignin increased. The fat content decreased in both samples. The safety-related correctness analysis showed that the biologically treated sample did not contain any harmful components and was therefore safe for use in nutritionally enriched animal feed.

Interactions of Aminopropyl-Azithromycin Derivatives, Precursors in the Synthesis of Bioactive Macrozones, with E. coli Ribosome: NMR and Docking Studies.

The structure and interactions of several aminopropyl-azithromycin derivatives (1a-c) have been studied by using NMR spectroscopy and docking calculations. Compounds 1a-c are precursors in the synthesis of macrozones, novel bioactive azithromycin-thiosemicarbazone conjugates active against some resistant bacterial strains. Today, bacterial resistance is considered as one of the major threats to human health. Knowledge on drug binding mode and conformations is one of the key factors in the process of designing molecules to fight resistance. In solution state, compounds 1a and 1c exist in the 3-endo-folded-out conformation, while 1b adopts a classical folded-out conformation. 13C and 15N CPMAS NMR spectra pointed towards similar structures in the solid state. The transferred NOESY NMR spectra confirmed binding to the E. coli ribosome and suggest that dominant conformations in the bound state resemble those in the free one. STD experiments identified reactive groups of 1a-c in close contact with the ribosome resembling binding epitopes observed for the related 15-membered macrolides. Docking studies revealed that the studied compounds bind to the same ribosome binding pocket similarly to erythromycin in the crystal state, and that the binding is achieved through H-bonds and van der Waals interactions. The bound conformation is the same as determined by NMR. STD enhancements observed for methylene protons in the aminopropyl side chain indicate additional interactions which contribute to the overall binding energy.

Advanced Methods for Studying Structure and Interactions of Macrolide Antibiotics.

Macrolide antibiotics are macrocyclic compounds that are clinically used and prescribed for the treatment of upper and lower respiratory tract infections. They inhibit the synthesis of bacterial proteins by reversible binding to the 23S rRNA at or near the peptidyl transferase center. However, their excellent antibacterial profile was largely compromised by the emergence of bacterial resistance. Today, fighting resistance to antibiotics is one of the greatest challenges in medicinal chemistry. Considering various physicochemical properties of macrolides, understanding their structure and interactions with macromolecular targets is crucial for the design of new antibiotics efficient against resistant pathogens. The solid-state structures of some macrolide-ribosome complexes have recently been solved, throwing new light on the macrolide binding mechanisms. On the other hand, a combination of NMR spectroscopy and molecular modeling calculations can be applied to study free and bound conformations in solution. In this article, a description of advanced physicochemical methods for elucidating the structure and interactions of macrolide antibiotics in solid state and solution will be provided, and their principal advantages and drawbacks will be discussed.

Condensation reaction between carbohydrazide and salicylaldehyde: in-line vibrational spectroscopy monitoring and characterization of the reaction products in solution and solid state.

The condensation reaction between carbohydrazide and salicylaldehyde was monitored in-line by using vibrational NIR and Raman spectroscopies and statistical methods. Prior to in-line data analysis the reaction products were fully characterized in solution and solid state in order to check the potential of the in-line approach as a tool for in-process Schiff bases reaction control. It was demonstrated that a combination of vibrational spectroscopy and principal component analysis made it possible to detect and identify the reaction products, e.g. mono(salicylidene)carbohydrazide (1) and bis(salicylidene)carbohydrazide (2) in different solvents, and to determine the reaction end points in real time. Owing to complexity of the reaction mixtures and band overlapping, it was not possible to determine the relative ratio of the reaction products in-line. The off-line analysis showed that 1 was predominant in methanol while the highest portion of 2 was obtained in ethanol.

Fast real-time monitoring of entacapone crystallization and characterization of polymorphs via Raman spectroscopy, statistics and SWAXS.

Crystallization of the drug entacapone from binary solvent mixtures was monitored in situ using a Raman optical probe. The recorded Raman spectra and statistical analysis, which included the principal components method and indirect hard modeling made it possible to estimate the starting point of crystallization, to assess crystallization temperatures and to provide information on the polymorphic content of the mixture. It was established that crystallization temperatures were proportional to the volume content of the solvent in mixtures. The samples were also evaluated off-line via Raman spectroscopy and SWAXS. The collected data showed the presence of forms b and g in all solvent mixtures. In a toluene/methanol 30:70 mixture, in addition to forms b and g, at least one of the forms A, D or a was also indicated by SWAXS. The results have shown that the presence of a particular polymorph is strongly dependent on the nature and portion of the solvent in the binary solvent mixture.

In-line reaction monitoring of entacapone synthesis by Raman spectroscopy and multivariate analysis.

In-line Raman spectroscopy and multivariate analysis were used to monitor Knoevenagel condensation reaction, the final step in preparation of drug entacapone. By applying a fiber optical Raman probe immersed into a reaction vessel Raman spectra of the reaction mixture were recorded in situ during the entacapone synthesis in toluene, heptane and isobutyl acetate. Due to the complexity of the measured spectra, the obtained data were analyzed and interpreted by means of principal component analysis. It has been shown that progress of this reaction can be monitored in real-time and reaction end points can be determined in different solvents. The reaction was found to be the fastest in heptane due to the lower loss of the catalyst. For a comparison the reaction was independently monitored by off-line Raman spectroscopy and liquid chromatography which confirmed the results obtained in-line. The results presented here have shown that this in-line approach can be used as a fast, non destructive and reliable method to monitor the Knoevenagel reaction in real time. The knowledge gained in this study can further be exploited for the industrial process control.