Recent Publications, 2014-2021

Recent Original Papers, 2023-24

1. P. Jirasek, A. Jusku, J. Frankova, M. Urbankova, D. Diabelko, F. Ruzicka, B. Papouskova, K. Chytilova, J. Vrba, J. Havlasek, K. Langova, J. Storch, I. Voborna, V. Simanek, J. Vacek, Phytocannabinoids and gingival inflammation: Preclinical findings and a placebo-controlled double-blind randomized clinical trial with cannabidiol, J. Periodontol. Res. DOI: 10.1111/jre.13234 (in press)
2. F. Fadaei, M. Tortora, A. Gessini, C. Masciovecchio, J. Vigna, I. Mancini, A. Mele, J. Vacek, B. Minofar, B. Rossi, Local and cooperative structural transitions of double-stranded DNA in choline-based deep eutectic solvents, Int. J. Biol. Macromol., 256, 128443, 2024
3. J. Vacek, M. Zatloukalová, V. Dorčák, M. Cifra, Z. Futera, V. Ostatná, Electrochemistry in sensing of molecular interactions of proteins and their behavior in an electric field, Microchim. Acta, 190, 442, 2023
4. J. Havlasek, J. Vrba, M. Zatloukalova, B. Papouskova, M. Modriansky, J. Storch, J. Vacek, Hepatic biotransformation of non-psychotropic phytocannabinoids and activity screening on cytochromes P450 and UDP-glucuronosyltransferases, Toxicol. Appl. Pharmacol., 476, 116654, 2023
5. J. Vacek, M. Zatloukalova, E. Bartheldyova, D. Reha, B. Minofar, K. Bednarova, D. Renciuk, J. Coufal, M. Fojta, J. Zadny, A. Gessini, B. Rossi, J. Storch, M. Kabelac, Hexahelicene DNA-binding: Minor groove selectivity, semi-intercalation and chiral recognition, Int. J. Biol. Macromol., 250, 125905, 2023
6. D. Novak, M. Zatloukalova, L. Jedinak, B. Komarkova, M. Kabelac, J. Vacek, Preparation, stability, and molecular modeling of nitro-oleic acid/cyclodextrin complexes under aqueous conditions, J. Mol. Liq., 383, 122020, 2023
7. M. Zatloukalova, L. Poltorak, R. Bilewicz, J. Vacek, Lipid-based liquid crystalline materials in electrochemical sensing and nanocarrier technology, Microchim. Acta, 190, 187, 2023

J. Hrbac, P. Jakubec, V. Halouzka, P. Matejka, M. Pour, J. Kopecky, J. Vacek, The permselective layer prepared onto carbon and gold surfaces by electropolymerization of phenolic cyclopentenedione-nostotrebin 6, Electrochem. Commun. 38, 53–56, 2014 (PDF)

In this study, we report that electrooxidation of phenolic cyclopentenedione, 2,2′-bis[4,5-bis(4-hydroxybenzyl)-2-(4-hydroxyphenyl)- cyclopent-4-ene-1,3-dione] results in the formation of nonconducting films which make uniform, defect-free and stable layers with permselective properties on gold and carbon surfaces. The electropolymerization process proceeds in neutral aqueous medium at potentials higher than + 0.6 V (vs. Ag/AgCl, 3 M KCl). The polymer prepared was characterized by electrochemical methods, quartz crystal microbalance, Fourier transform Raman and infrared spectroscopies, and scanning electron microscopy. The polymeric film can be used for electrode coating and thus preparation of an anti-interference barrier, for example, in dopamine microelectrode sensor construction. The results presented here are the first evidence for the preparation of phenolic cyclopentenedione-based functional film.

M. Zatloukalova, J. Vacek, V. Simanek, Electrochemical Behaviour of Alkaloids: Detection and Interaction with DNA and Proteins, Heterocycles, 88, 879-897, 2014 (PDF)

Electroanalysis can provide valuable information on the molecular interactions, biological activity and pharmacological potential of alkaloids. This article summarizes current knowledge on the electrochemical properties of a broad spectrum of alkaloids using polarography and voltammetry. Alkaloid interactions with DNA and proteins using electrochemical techniques are also described. More than 100 references published in the last 70 years are reviewed.

M. Spulak, J. Pourova, M. Voprsalova, J. Mikusek, J. Kunes, J. Vacek, M. Ghavre, N. Gathergood, M. Pour, Novel bronchodilatory quinazolines and quinoxalines: Synthesis and biological evaluation, Eur. J. Med. Chem. 74, 65-72, 2014 (PDF)

A series of heterocyclic derivatives analogous to (−)vasicinone, in which the vasicinone C-ring was replaced with alkyl chain terminated by tertiary amine was prepared. N3, C4–O, C4–S or C4–N were used as the sites of attachment. The 4-[3-(1-piperidyl)propylsulfanyl]derivatives displayed bronchodilatory effect at low micromolar concentrations on isolated rat trachea, and low toxicity both on Balb/c 3T3 mouse fibroblast cells and in mice.

J. Vacek, J. Vrba, M. Zatloukalova, M. Kubala, Electrochemical oxidation of proteins using ionic liquids as solubilizers, adsorption solvents and electrolytes, Electrochim. Acta 126, 31–36, 2014 (PDF)

This study focuses on application of room temperature ionic liquids (RTILs) as solubilizers, adsorption solvents and supporting electrolytes for electrochemical analysis of human and bovine serum albumins, HSA and BSA. The proteins were analyzed by ex situ, adsorptive transfer, square-wave voltammetry (SWV) at a basal-plane pyrolytic graphite electrode after solubilization using imidazolium- and ammonium-based RTILs. The application of RTILs enabled SWV scan from 0 to +1.5 V (vs. Ag/AgCl/3 M KCl) without interference with the anodic response of the proteins. Concretely, Tyr (Y) and Trp (W) oxidation currents of HSA and BSA, peak Y&W around +0.85 V, were observed and characterized under different RTILs and RTIL/water conditions. The electrochemical data were supported by electrophoresis under denaturing and native conditions. These provided evidence for the structural changes and stability of the studied proteins in the presence of RTILs. The data acquired using BSA and HSA model proteins, could be used in further applications of RTILs in protein electrochemistry and for developing new protein sensing strategies.

E. Vavrikova, J. Vacek, K. Valentova, P Marhol, J. Ulrichova, M. Kuzma, V. Kren, Chemo-Enzymatic Synthesis of Silybin and 2,3-Dehydrosilybin Dimers, Molecules 19, 4115-4134, 2014 (PDF)

Divalent or multivalent molecules often show enhanced biological activity relative to the simple monomeric units. Here we present enzymatically and chemically prepared dimers of the flavonolignans silybin and 2,3-dehydrosilybin. Their electrochemical behavior was studied by in situ and ex situ square wave voltammetry. The oxidation of monomers and dimers was similar, but adsorption onto the electrode and cell surfaces was different. A 1,1-diphenyl-2-picrylhydrazyl (DPPH) and an inhibition of microsomal lipoperoxidation assay were performed with same trend of results for silybin and 2,3-dehydrosilybin dimers. Silybin dimer showed better activity than the monomer, while on the contrary 2,3-dehydrosilybin dimer presented weaker antioxidant/antilipoperoxidant activity than its monomer. Cytotoxicity was evaluated on human umbilical vein endothelial cells, normal human adult keratinocytes, mouse fibroblasts (BALB/c 3T3) and human liver hepatocellular carcinoma cell line (HepG2). Silybin dimer was more cytotoxic than the parent compound and in the case of 2,3-dehydrosilybin its dimer showed weaker cytotoxicity than the monomer.

Z. Sevcikova, M. Pour, D. Novak, J. Ulrichova, J. Vacek, Chemical Properties and Biological Activities of Cyclopentenediones: A Review, Mini-Rev. Med. Chem. 14, 322-331, 2014 (PDF)

Cyclopentenediones (CPDs) are secondary metabolites of higher plants, fungi, algae, cyanobacteria and bacteria. A common denominator of CPDs is the cyclopent-4-ene-1,3-dione skeleton (1), which is modified by several functional groups. The heterogeneity of these substitutions is reflected in around one hundred CPDs reported to date. Most of the derivatives were isolated primarily from plant sources. Synthetic analogues were then prepared with new biological activities and more interesting pharmacological potential. Antifungal substances called coruscanones (2, 3) are the most studied of the CPDs. Other intensely investigated CPDs include lucidone (4), linderone (5), asterredione (6), involutone (7), nostotrebin 6 (8), TX-1123 (9), G2201-C (10), madindolines (11, 12) and many others. In addition to antibacterial and antifungal effects, a broad spectrum of biological activities for CPDs has been reported in the past two decades, especially anti-inflammatory, cytostatic and specific enzyme inhibitory activities. The CPD skeleton has been identified in a number of substances isolated from the plant kingdom; hence, CPDs can be referred to as a new group of natural bioactive substances. The main goal of this review is to define CPDs with respect to basic chemistry, isolation, synthetic approaches and description of their biological effects. Special attention is given to a detailed view into biological activities of CPDs in vitro and their phamacological potential.

R. Vecerkova, L. Hernychova, P. Dobes, J. Vrba, B. Josypcuk, M. Bartosik, J. Vacek, Investigation of protein FTT1103 electroactivity using carbon and mercury electrodes. Surface-inhibition approach for disulfide oxidoreductases using silver amalgam powder, Anal. Chim. Acta 830, 23–31, 2014 (PDF)

Recently, it was shown that electrochemical methods can be used for analysis of poorly water-soluble proteins and for study of their structural changes and intermolecular (protein–ligand) interactions. In this study, we focused on complex electrochemical investigation of recombinant protein FTT1103, a disulfide oxidoreductase with structural similarity to well described DsbA proteins. This thioredoxin-like periplasmic lipoprotein plays an important role in virulence of bacteria Francisella tularensis. For electrochemical analyses, adsorptive transfer (ex situ) square-wave voltammetry with pyrolytic graphite electrode, and alternating-current voltammetry and constant-current chronopotentiometric stripping analysis with mercury electrodes, including silver solid amalgam electrode (AgSAE) were used. AgSAE was used in poorly water-soluble protein analysis for the first time. In addition to basic redox, electrocatalytic and adsorption/desorption characterization of FTT1103, electrochemical methods were also used for sensitive determination of the protein at nanomolar level and study of its interaction with surface of AgSA microparticles. Proposed electrochemical protocol and AgSA surface-inhibition approach presented here could be used in future for biochemical studies focused on proteins associated with membranes as well as on those with disulfide oxidoreductase activity.

E. Stejskalova, P. Horakova, J. Vacek, R. P. Bowater, M. Fojta, Enzyme-linked electrochemical DNA ligation assay using magnetic beads, Anal. Bioanal. Chem. 406, 4129–4136, 2014 (PDF)

DNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for applications in biomedical research. Here we present an enzyme-linked electrochemical assay based on two terminally tagged probes forming a nicked junction upon hybridization with a template DNA. Nicked DNA bearing a 5' biotin tag is immobilized on the surface of streptavidin-coated magnetic beads, and ligated product is detected via a 3' digoxigenin tag recognized by monoclonal antibody-alkaline phosphatase conjugate. Enzymatic conversion of napht-1-yl phosphate to napht-1-ol enables sensitive detection of the voltammetric signal on a pyrolytic graphite electrode. The technique was tested under optimal conditions and various situations limiting or precluding the ligation reaction (such as DNA substrates lacking 5′-phosphate or containing a base mismatch at the nick junction, or application of incompatible cofactor), and utilized for the analysis of the nick-joining activity of a range of recombinant Escherichia coli DNA ligase constructs. The novel technique provides a fast, versatile, specific, and sensitive electrochemical assay of DNA ligase activity.

M. Kubala, J. Geleticova, M. Huliciak, M. Zatloukalova, J. Vacek, M. Sebela, Na+/K+-ATPase inhibition by cisplatin and consequences for cisplatin nephrotoxicity, Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech Repub. 158, 194-200, 2014 (PDF)

Aims: Cisplatin is a widely used chemotherapeutic. However, it is associated with numerous adverse effects. The aim of our study was examination of cisplatin interaction with Na⁺/K⁺-ATPase (NKA, the sodium pump). This enzyme is of crucial importance for all animal cells and particularly for the kidney, which is frequently damaged during chemotherapy. Methods: The entire NKA was isolated from porcine kidney. Its large cytoplasmic segment connecting transmembrane helices 4 and 5 (C45), was heterologously expressed in E.coli (wild-type or C367S mutant). The ATPase activity was evaluated according to the inorganic phosphate production and the interaction of isolated C45 with cisplatin was studied using chronopotentiometry and mass spectrometry. Results: Our experiments revealed that cisplatin can inhibit NKA. The finding that other platinum-based drugs with a low nephrotoxicity, carboplatin and oxaliplatin, did not inhibit NKA, suggested that NKA/cisplatin interaction is an important factor in cisplatin adverse effects. The inhibitory effect of cisplatin could be prevented by preincubation of the enzyme with reduced glutathione or DTT. Using chronopotentiometry and mass spectrometry, we found that cisplatin is bound to C45. However, our mutagenesis experiment did not confirm that the suggested Cys367 could be the binding site for cisplatin. Conclusion: Unintended interactions of drugs present serious limitations to treatment success. Although a large number of membrane pumps have been identified as potential targets of cisplatin, vis-a-vis nephrotoxicity, NKA inhibition seems to be of crucial importance. Experiments with isolated large cytoplasmic segment C45 revealed that it is the main target of cisplatin on NKA and that the reaction with cysteine residues plays an important role in cisplatin/NKA interactions. However, further experiments must be performed to identify the interacting amino acid residues more precisely.

J. Hrbac, V. Halouzka, L. Trnkova, J. Vacek, eL-Chem Viewer: A Freeware Package for the Analysis of Electroanalytical Data and Their Post-Acquisition Processing, Sensors 14, 13943-13954, 2014 (PDF)

In electrochemical sensing, a number ofvoltammetric or amperometric curves are obtained which are subsequently processed, typically by evaluating peak currents and peak potentials or wave heights and half-wave potentials, frequently after background correction. Transformations of voltammetric data can help to extract specific information, e.g., the number of transferred electrons, and can reveal aspects of the studied electrochemical system, e.g., the contribution of adsorption phenomena. In this communication, we introduce a LabView-based softwarepackage, 'eL-Chem Viewer',which is for the analysis of voltammetric and amperometric data, and enables their post-acquisition processing using semiderivative, semiintegral, derivative,integral and elimination procedures. The software supports the single-click transfer of peak/wave current and potential data to spreadsheet software, a feature that greatly improves productivity when constructing calibration curves, trumpet plots and performing similar tasks. eL-Chem Viewer is freeware and can be downloaded from www.lchem.cz/elchemviewer.htm

J. Hrbac, J. Storch, V. Halouzka, V. Cirkva, P. Matejka, J. Vacek, Immobilization of helicene onto carbon substrates through electropolymerization of [7]helicenylthiophene, RSC Adv. 4, 46102–46105, 2014 (PDF)

In this work,monomeric 3-([7]helicen-9-yl)thiophene was synthesized and used for the preparation of a polymeric film by electrosynthesis. The polymer prepared was characterized using Fe(CN)6 3
/4
and Ru(NH3)6 3+/2+ redox probes, scanning electron microscopy and vibrational spectroscopy. The electropolymerization approach presented here provides a new option for the immobilization of helicenes onto solid supports.

 J. Vrba, B. Papouskova, M. Pyszkova, M. Zatloukalova, K. Lemr, J. Ulrichova, J. Vacek, Metabolism of palmatine by human hepatocytes and recombinant cytochromes P450, J. Pharm. Biomed. Anal. 102, 193 –198, 2015 (PDF)

In this study, we developed a new liquid chromatography–mass spectrometry (LC–MS) method foranalysis of the protoberberine alkaloid palmatine and its metabolites with separation performed on acyanopropyl-modified stationary phase. Palmatine (10 µM) was metabolized using suspensions of humanhepatocytes and human recombinant cytochrome P450 (CYP) enzymes. Our analyses using electrosprayionization-quadrupole time-of-flight mass spectrometry revealed that palmatine was relatively resis-tant to the metabolic activity of human hepatocytes and recombinant CYP enzymes. However, we foundthat the biotransformation of palmatine in human hepatocytes included O-demethylation or hydroxyla-tion, and that the product of palmatine demethylation was conjugated by glucuronidation or sulfation.Moreover, we found that human recombinant CYP2D6 and, to a lesser extent, CYP1A2 can mediate O-demethylation of palmatine. These results provide fundamental insights into the biotransformation ofpalmatine in human in vitro models and, together with the LC–MS method, can be applied for furtherstudies on the biotransformation of palmatine and other protoberberine alkaloids.

J. Storch, J. Zadny, T. Strasak, M. Kubala, J. Sykora, M. Dusek, V. Cirkva, P. Matejka, M. Krbal, J. Vacek, Synthesis and Characterization of a helicene-based imidazolium salt and its application in organic molecular electronics, Chem. Eur. J. 21, 2343-2347, 2015 (PDF)

Herein we demonstrate the synthesis of a helicene-based imidazolium salt. The salt was prepared by starting from racemic 2-methyl[6]helicene, which undergoes radical bromination to yield 2-(bromomethyl)[6]helicene. Subsequent treatment with 1-butylimidazole leads to the corresponding salt 1-butyl-3-(2-methyl[6]helicenyl)-imidazolium bromide. The prepared salt was subsequently characterized by using NMR spectroscopy and X-ray analysis, various optical spectrometric techniques, and computational chemistry tools. Finally, the imidazolium salt was immobilized onto a SiO₂ substrate as a crystalline or amorphous deposit. The deposited layers were used for the development of organic molecular semiconductor devices and the construction of a fully reversible humidity sensor.

M. Havlikova, M. Zatloukalova, J. Ulrichova, P. Dobes, J. Vacek, Electrocatalytic assay for monitoring methylglyoxal-mediated protein glycation, Anal. Chem. 87, 1757-1763, 2015 (PDF)

Protein glycation is a complex process that plays an important role in diabetes mellitus, aging, and the regulation of protein function in general. As a result, current methodological research on proteins is focused on the development of novel approaches for investigating glycation and the possibility of monitoring its modulation and selective inhibition. In this paper, a first sensing strategy for protein glycation is proposed, based on protein electroactivity measurement. Concretely, the label-free method proposed is based on the application of a constant-current chronopotentiometric stripping (CPS) analysis at Hg-containing electrodes. The glycation process was monitored as the decrease in the electrocatalytic protein signal, peak H, observed at highly negative potentials at around -1.8 V (vs Ag/AgCl_{3 M KCl}), which was previously ascribed to a catalytic hydrogen evolution reaction (CHER). Using this method, a model protein bovine serum albumin was investigated over 3 days of incubation with the glycation agent methylglyoxal in the absence or presence of the glycation inhibitor aminoguanidine (pimagedine). The electrochemical methodology presented here could open up new possibilities in research on protein glycation and oxidative modification. The methodology developed also provides a new option for the analysis of protein intermolecular interactions using electrochemical sensors, which was demonstrated by the application of a silver solid amalgam electrode (AgSAE) for monitoring the glycation process in samples of bovine serum albumin, human serum albumin, and lysozyme.

D. Riman, Z. Bartosova, V. Halouzka, J. Vacek, D. Jirovsky, J. Hrbac, Facile preparation of nanostructured coppercoated carbon microelectrodes for amperometric sensing of carbohydrates, RSC Adv. 5, 31245–31249, 2015 (PDF)

We report a novel method for fabricating nanostructured copper coated carbon cylindrical fiber microelectrodes and show the high efficiency of these electrodes in carbohydrate non-enzymatic and label-free amperometric sensing in both batch and flow-detection arrangements.

P. Hrouzek, A. Kapuscik, J. Vacek, K. Voracova, J. Paichlova, P. Kosina, L. Voloshko, S. Ventura, J. Kopecky, Cytotoxicity evaluation of large cyanobacterial strain set using selected human and murine in vitro cell models, Ecotox. Environ. Safety, 124, 177-185, 2016 (PDF)

The production of cytotoxic molecules interfering with mammalian cells is extensively reported in cyanobacteria. These compounds may have a use in pharmacological applications; however, their potential toxicity needs to be considered. We performed cytotoxicity tests of crude cyanobacterial extracts in six cell models in order to address the frequency of cyanobacterial cytotoxicity to human cells and the level of specificity to a particular cell line. A set of more than 100 cyanobacterial crude extracts isolated from soil habitats (mainly genera Nostoc and Tolypothrix) was tested by MTT test for in vitro toxicity on the hepatic and non-hepatic human cell lines HepG2 and HeLa, and three cell systems of rodent origin: Yac-1, Sp-2 and Balb/c 3T3 fibroblasts. Furthermore, a subset of the extracts was assessed for cytotoxicity against primary cultures of human hepatocytes as a model for evaluating potential hepatotoxicity. Roughly one third of cyanobacterial extracts caused cytotoxic effects (i.e. viability<75%) on human cell lines. Despite the sensitivity differences, high correlation coefficients among the inhibition values were obtained for particular cell systems. This suggests a prevailing general cytotoxic effect of extracts and their constituents. The non-transformed immortalized fibroblasts (Balb/c 3T3) and hepatic cancer line HepG2 exhibited good correlations with primary cultures of human hepatocytes. The presence of cytotoxic fractions in strongly cytotoxic extracts was confirmed by an activity-guided HPLC fractionation, and it was demonstrated that cyanobacterial cytotoxicity is caused by a mixture of components with similar hydrophobic/hydrophilic properties. The data presented here could be used in further research into in vitro testing based on human models for the toxicological monitoring of complex cyanobacterial samples.

The protective constituents of silymarin, an extract from Silybum marianum fruits, have been extensively studied in terms of their antioxidant and hepatoprotective activities. Here, we explore the electron-donor properties of the major silymarin flavonolignans. Silybin (SB), silychristin (SCH), silydianin (SD) and their respective 2,3-dehydroderivatives (DHSB, DHSCH and DHSD) were oxidized electrochemically and their antiradical/antioxidant properties were investigated. Namely, Folin-Ciocalteau reduction, DPPH and ABTS(+) radical scavenging, inhibition of microsomal lipid peroxidation and cytoprotective effects against tert-butyl hydroperoxide-induced damage to a human hepatocellular carcinoma HepG2 cell line were evaluated. Due to the presence of the highly reactive C3-OH group and the C-2,3 double bond (ring C) allowing electron delocalization across the whole structure in the 2,3-dehydroderivatives, these compounds are much more easily oxidized than the corresponding flavonolignans SB, SCH and SD. This finding was unequivocally confirmed not only by experimental approaches, but also by density functional theory (DFT) calculations. The hierarchy in terms of ability to undergo electrochemical oxidation (DHSCH~DHSD>DHSB>>SCH/SD>SB) was consistent with their antiradical activities, mainly DPPH scavenging, as well as in vitro cytoprotection of HepG2 cells. The results are discussed in the context of the antioxidant vs. prooxidant activities of flavonolignans and molecular interactions in complex biological systems.

J. Vacek, M. Zatloukalova, J. Geleticova, M. Kubala, M. Modriansky, L. Fekete, J. Masek, F. Hubatka, J. Turanek, Electrochemical Platform for the Detection of Transmembrane
Proteins Reconstituted into Liposomes, Anal. Chem. 88, 4548−4556, 2016 (PDF)

 The development of new methods and strategies for the investigation of membrane proteins is limited by poor solubility of these proteins in an aqueous environment and hindered by a number of other problems linked to the instability of the proteins outside lipid bilayers. Therefore, current research focuses on an analysis of membrane proteins incorporated into model lipid membrane, most frequently liposomes. In this work, we introduce a new electrochemical methodology for the analysis of transmembrane proteins reconstituted into a liposomal system. The proposed analytical approach is based on proteoliposomal sample adsorption on the surface of working electrodes followed by analysis of the anodic and cathodic signals of the reconstituted proteins. It works based on the fact that proteins are electroactive species, in contrast to the lipid components of the membranes under the given experimental conditions. Electroanalytical experiments were performed with two transmembrane proteins; the Na⁺/K⁺ATPase that contains transmembrane as well as large extramembraneous segments and the mitochondrial uncoupling protein 1, which is a transmembrane protein essentially lacking extramembraneous segments. Electrochemical analyses of proteoliposomes were compared with analyses of both proteins solubilized with detergents (C₁₂E₈ and octyl-PoE) and supported by the following complementary methods: microscopy techniques, protein activity testing, molecular model visualizations, and immunochemical identification of both proteins. The label-free electrochemical platform presented here enables studies of reconstituted transmembrane proteins at the nanomolar level. Our results may contribute to the development of new electrochemical sensors and microarray systems applicable within the field of poorly water-soluble proteins.

 R. Sokolová, J. Tarábek, B. Papoušková, J. Kocábová, J. Fiedler, J. Vacek, P. Marhol, E. Vavříková, V. Křen, Oxidation of the Flavonolignan Silybin. In situ EPR Evidence of the Spin-Trapped Silybin Radical, Electrochim. Acta 205, 118-123, 2016 (PDF)

Oxidation of natural flavonolignan silybin in an aprotic environment was studied by electrochemical methods, electron paramagnetic resonance (EPR), UV–vis and IR in situ spectroelectrochemistry and chromatographic techniques. The presence of electrochemically generated silybin radical was proven using in situ EPR spectroelectrochemistry by spin trapping method with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO) as a spin trap reagent. The adduct of the silybin radical with BMPO was additionally unambiguously identified by UPLC-ESI-MS/MS. For the first time, the flavonolignan radical was detected upon electrochemical oxidation under such conditions. The one-electron oxidation of silybin leads to a hydroxylated silybin derivative. Its specific redox behaviour can reflect its bioefficiency.

B. Papouskova, M. Bernard, J. Ottenschlager, J. Karban, P. Velisek, J. Hrbac, J. Sykora, J. Storch, J. Vacek, A comprehensive LC/MS analysis of novel cyclopentenedione library, J. Pharm. Biomed. Anal, 128, 342-351, 2016 (PDF)

Cyclopentenediones (CPDs) are compounds with a variety of applications ranging from the preparation of functional polymers to the development of antimicrobial agents, suggesting the potential use of CPDs as novel bioactive compounds or drugs. For this reason, a detailed characterization of CPDs and the development of robust analytical methods for their trace analysis are being sought. Here we focused on the design and synthesis of a library of novelized benzylidene CPD derivatives that were consequently characterized by ultra-high performance liquid chromatography (UHPLC) on-line connected with tandem mass spectrometry (MS/MS). The library design was based on a 2-benzylidene-4-cyclopentene-1,3-dione skeleton substituted with a variety of hydroxy, methoxy, halogen, linear aliphatic, heterocyclic and saccharide moieties, primarily modulating the skeleton's hydrophobicity. The prepared CPDs were effectively ionized by positive/negative atmospheric pressure photoionization (APPI) and atmospheric pressure chemical ionization (APCI). After careful optimization of the dopant composition and flow rate, positive-mode APPI proved to be more sensitive than APCI. In negative mode, both ionization techniques gave similar results. Further, a detailed MS fragmentation study was performed, confirming the structure of the compounds and enabling positional isomers of CPDs to be differentiated on the basis of their collision spectra analysis. Finally, an optimization of the composition of the mobile phase and reversed-phased separation mode were done, followed by a selection of the most suitable UHPLC stationary phases, i.e. C18, C8 and phenyl. The applicability of the method was evaluated by the inclusion of the other two substances in the study, i.e. monomeric and dimeric bioactive CPDs, compound TX-1123 and nostotrebin 6 with cytostatic and antimicrobial activities, respectively. The results presented here could be used in further investigations of the chromatographic retention and MS behavior of CPDs, which could be utilized for their isolation, detailed characterization and analysis in biological systems.

J. Cheel, K. Bogdanová, S. Ignatova, I.c. Garrard, P.C. Hewitson, M. Kolář, J. Kopecký, P. Hrouzek, J. Vacek, Dimeric cyanobacterial cyclopent-4-ene-1,3-dione as selective inhibitor of Gram-positive bacteria growth: Bio-production approach and preparative isolation by HPCCC, Algal Res. 18, 244–249, 2016 (PDF)

The need for new antimicrobial agents is greater than ever because of the emergence of multidrug resistance in common pathogens and incidence of new infections. Cyclopent-4-ene-1,3-diones (CPDs) have been reported as a new class of compounds with promising antimicrobial and antifungal properties. Herein we report the selective antibiotic properties of nostotrebin 6, a phenolic CPD produced biotechnologically by the culture of cyanobacterium Nostoc sp. str. Lukešová 27/97. High performance countercurrent chromatography (HPCCC) combined with gel permeation chromatography (GPC) was used for the isolation of nostotrebin 6 with a relatively high 0.53 ± 0.1% yield (calculated from dried biomass) and final purity higher than 96%. Nostotrebin 6 was tested for its antimicrobial and antifungal activities by using standard micro-dilution method, and the results were expressed as minimal inhibitory concentrations (MICs). Nostotrebin 6 unequivocally inhibited the growth of Gram-positive reference (Enterococcus faecalis CCM 4224, Staphylococcus aureus CCM 4223 and Staphylococcus aureus CCM 3953) and multidrug-resistant (Staphylococcus haemolyticus A/16568, Staphylococcus aureus MRSA 4591 and Enterococcus faecium VanA 419/ana) strains. Its strongest effect was exerted against the Gram-positive bacteria with MICs ranging between 6.25 and 15.6 μg/mL. There was no effect on Gram-negative strains tested and yeasts. Our results suggest that nostotrebin 6 could serve as basic nucleus for further design of novel antibiotic agents and demonstrate that the bio-production approach based on HPCCC/GPC isolation endpoint is an efficient methodology for obtaining nostotrebin 6 in multi-gram scale. Furthermore, the presented isolation method can be easily up-scaled to process kilograms of the cyanobacterial biomass.

V. Dorčák, M. Kabeláč, O. Kroutil, K. Bednářová, J. Vacek, Electrocatalytic monitoring of peptidic proton-wires, Analyst 141, 4554–4557, 2016 (PDF)

The transfer of protons or proton donor/acceptor abilities is an important phenomenon in many biomolecular systems. One example is the recently proposed peptidic proton-wires (H-wires), but the ability of these His-containing peptides to transfer protons has only been studied at the theoretical level so far. Here, for the first time the proton transfer ability of peptidic H-wires is examined experimentally in an adsorbed state using an approach based on a label-free electrocatalytic reaction. The experimental findings are complemented by theoretical calculations at the ab initio level in a vacuum and in an implicit solvent. Experimental and theoretical results indicated Ala₃(His–Ala₂)₆ to be a high proton-affinity peptidic H-wire model. The methodology presented here could be used for the further investigation of the proton-exchange chemistry of other biologically or technologically important macromolecules.