Publications by authors named "Jan Sýkora"

85 Publications

Early Detection of Pancreatic Cancer in Type 2 Diabetes Mellitus Patients Based on H NMR Metabolomics.

J Proteome Res 2021 03 22;20(3):1744-1753. Epub 2021 Feb 22.

Department of Internal Medicine, 1st Faculty of Medicine of Charles University and Military University Hospital, Prague 6 16902, Czech Republic.

The association of pancreatic cancer with type 2 diabetes mellitus was investigated by H NMR metabolomic analysis of blood plasma. Concentration data of 58 metabolites enabled discrimination of pancreatic cancer (PC) patients from healthy controls (HC) and long-term type 2 diabetes mellitus (T2DM) patients. A panel of eight metabolites was proposed and successfully tested for group discrimination. Furthermore, a prediction model for the identification of at-risk individuals for future development of pancreatic cancer was built and tested on recent-onset diabetes mellitus (RODM) patients. Six of 59 RODM samples were assessed as PC with an accuracy of more than 80%. The health condition of these individuals was re-examined, and in four cases, a correlation to the prediction was found. The current health condition can be retrospectively attributed to misdiagnosed pancreatogenic diabetes or to early-stage pancreatic cancer.
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http://dx.doi.org/10.1021/acs.jproteome.0c00990DOI Listing
March 2021

Hemodialysis vascular access dysfunction and its endovascular treatment.

Vnitr Lek 2020 ;66(6):14-18

Hemodialysed patients with end stage renal disease are reliant to proper function of vascular access - mostly arterio-venous fistula (AVF). AVF patency is jeopardized by stenosis formation, which needs to be treated before thrombosis. Angioplasty is primarily indicated and prolongs vascular access patency. High pressure balloons and cutting balloons aid to high technical success rate. Angioplasty needs to be repeated in order to maintain long term patency. Drug-eluting balloons prolong long term patency. Stents and stentgrafts are seldom used in inoperable patients and in selected locations. Technical advances allowed endovascular treatment in AVF thrombosis followed by angioplasty of culprit lesion. Vascular access endovascular occlusion is alternative for surgery in inoperable patients. In patients with residual renal function or iodine allergy, intervention could be ultrasound guided or carbon dioxide could be used without the need of iodine contrast.
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January 2021

Nuclear Magnetic Resonance Aerosolomics: A Tool for Analysis of Polar Compounds in Atmospheric Aerosols.

ACS Omega 2020 Sep 3;5(36):22750-22758. Epub 2020 Sep 3.

Institute of Chemical Process Fundamentals, v.v.i., The Czech Academy of Sciences, Rozvojová 1/135, Prague 6 165 02, Czech Republic.

Nuclear magnetic resonance aerosolomics was proposed as a new approach to the analysis of the water-soluble organic compound fraction in aerosol particulate matter. The identification of individual compounds is based on a comparison of precise chemical shifts in the H NMR spectrum with the signals in the standards library. For this purpose, Chenomx metabolomics software and a comprehensive spectra library of 150 compounds known from chemistry of aerosols were used. This approach enabled the identification of 60 compounds in real aerosol samples collected at a suburban site in Prague. Using the metabolomic spectra library, three new compounds were identified in aerosols for the first time, and an association of four other compounds to the atmospheric particulate matter was confirmed. The obtained concentration profiles of all identified chemical individuals were subsequently subjected to advanced statistical analysis. NMR aerosolomics clearly differentiates between summer and winter aerosol samples via multivariate statistical analysis and revealed some interesting trends in composition, according to aerosol particle size. Furthermore, the univariate statistical analysis was applied to highlight compounds responsible for the group separation, and possible sources of these compounds were suggested.
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http://dx.doi.org/10.1021/acsomega.0c01634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7495480PMC
September 2020

Oxidative Photocyclization of Aromatic Schiff Bases in Synthesis of Phenanthridines and Other Aza-PAHs.

Int J Mol Sci 2020 Aug 15;21(16). Epub 2020 Aug 15.

Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, USA.

The oxidative photocyclization of aromatic Schiff bases was investigated as a potential method for synthesis of phenanthridine derivatives, biologically active compounds with medical applications. Although it is possible to prepare the desired phenanthridines using such an approach, the reaction has to be performed in the presence of acid and TEMPO to increase reaction rate and yield. The reaction kinetics was studied on a series of substituted imines covering the range from electron-withdrawing to electron-donating substituents. It was found that imines with electron-withdrawing substituents react one order of magnitude faster than imines bearing electron-donating groups. The H NMR monitoring of the reaction course showed that a significant part of the isomer in the reaction is transformed into isomer which is more prone to photocyclization. The portion of the isomer transformed showed a linear correlation to the Hammett substituent constants. The reaction scope was expanded towards synthesis of larger aromatic systems, namely to the synthesis of strained aromatic systems, e.g., helicenes. In this respect, it was found that the scope of oxidative photocyclization of aromatic imines is limited to the formation of no more than five -fused aromatic rings.
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http://dx.doi.org/10.3390/ijms21165868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461585PMC
August 2020

Sulphonamidic Groups as Electron-Withdrawing Units in Ureido-Based Anion Receptors: Enhanced Anion Complexation versus Deprotonation.

Chempluschem 2020 07;85(7):1401-1411

Department of Analytical Chemistry, Institute of Chemical Process Fundamentals of CAS v.v.i, Rozvojová 135, Prague 6, 16502, Czech Republic.

A sulphonamidic moiety was utilized as an electron-withdrawing group for enhancement of anion complexation features of urea-based receptors. A series of receptors varying in acidity of sulphonamidic and urea NH groups was synthesized and thoroughly tested. The individual complexation properties reflect deprotonation/complexation equilibrium in a given molecule as a function of the substitution. The receptors containing electron-donating groups in conjugation to the sulphonamidic moiety showed higher association constants towards H PO and carboxylate anions, while those containing electron-withdrawing groups inclined to deprotonation of sulphonamidic NH. The deprotonation issue can be avoided by alkylation at the early step of receptor synthesis or it can be utilized for insertion of suitable groups that enable its anchoring on various substrates to form more elaborated receptor structures.
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http://dx.doi.org/10.1002/cplu.202000326DOI Listing
July 2020

Photochemical Oxidation Specific to Distorted Aromatic Amines Providing -Diketones.

Org Lett 2020 May 30;22(10):3905-3910. Epub 2020 Apr 30.

Department of Advanced Materials and Organic Synthesis, Institute of Chemical Process Fundamentals of the Czech Academy of Sciences, v. v. i. Rozvojová 135, 165 02 Prague 6, Czech Republic.

A straightforward visible-light-promoted oxidation of aminohelicenes providing helical -diketones is described. It is shown that the oxidation of amines proceeds via [2 + 2]-cycloaddition reaction with singlet oxygen as an oxidizer and the reaction is specific to distorted aromatic systems. The versatility of the prepared diketones and tetraketones was proven in several heterocycle-forming reactions. The observed adjustment of the physicochemical properties of original molecules is valuable for further development of functional molecules based on helicenes.
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http://dx.doi.org/10.1021/acs.orglett.0c01190DOI Listing
May 2020

Synthesis of a Helical Phosphine and a Catalytic Study of Its Palladium Complex.

ACS Omega 2020 Jan 2;5(1):882-892. Epub 2020 Jan 2.

Institute of Chemical Process Fundamentals, v.v.i., The Czech Academy of Sciences, Rozvojová 1/135, 165 02 Prague 6, Czech Republic.

In this study, 9-(diphenylphosphanyl)[7]helicene was prepared as a suitable ligand for the subsequent synthesis of palladium complexes. The corresponding PdLCl complex was then successfully obtained in both racemic and enantiopure forms. The PdLCl complex emerges exclusively in the arrangement showing dynamic interconversion between its homo- and heterochiral forms as evidenced by P NMR. The arrangement was ultimately confirmed by X-ray crystallography using single crystals of the homochiral complex. Additionally, the PdLCl complex was subjected to screening of its catalytic activity in a Suzuki-type reaction of aryl bromides with aryl boronic acids showing fair yields of the resulting biaryls. However, the final asymmetric reactions catalyzed by the optically pure PdLCl complex provided targeted binaphtyls only in negligible enantiomeric excess.
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http://dx.doi.org/10.1021/acsomega.9b03830DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6964522PMC
January 2020

Light-Induced Nanosecond Relaxation Dynamics of Rhenium-Labeled Azurins.

J Phys Chem B 2020 02 27;124(5):788-797. Epub 2020 Jan 27.

J. Heyrovský Institute of Physical Chemistry , Czech Academy of Sciences , Dolejškova 3 , CZ-182 23 Prague , Czech Republic.

Time-resolved phosphorescence spectra of Re(CO)(dmp) and Re(CO)(phen) chromophores (dmp = 4,7-dimethyl-1,10-phenanthroline, phen = 1,10-phenanthroline) bound to surface histidines (H83, H124, and H126) of azurin mutants exhibit dynamic band maxima shifts to lower wavenumbers following 3-exponential kinetics with 1-5 and 20-100 ns major phases and a 1.1-2.5 μs minor (5-16%) phase. Observation of slow relaxation components was made possible by using an organometallic Re chromophore as a probe whose long phosphorescence lifetime extends the observation window up to ∼3 μs. Integrated emission-band areas also decay with 2- or 3-exponential kinetics; the faster decay phase(s) is relaxation-related, whereas the slowest one [360-680 ns (dmp); 90-140 ns (phen)] arises mainly from population decay. As a result of shifting bands, the emission intensity decay kinetics depend on the detection wavelength. Detailed kinetics analyses and comparisons with band-shift dynamics are needed to disentangle relaxation and population decay kinetics if they occur on comparable timescales. The dynamic phosphorescence Stokes shift in Re-azurins is caused by relaxation motions of the solvent, the protein, and solvated amino acid side chains at the Re binding site in response to chromophore electronic excitation. Comparing relaxation and decay kinetics of and suggests that electron transfer (ET) and relaxation motions in the W122 mutant are coupled. It follows that nanosecond and faster photo-induced ET steps in azurins (and likely other redox proteins) occur from unrelaxed systems; importantly, these reactions can be driven (or hindered) by structural and solvational dynamics.
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http://dx.doi.org/10.1021/acs.jpcb.9b10802DOI Listing
February 2020

Concurrent Compression of Phospholipid Membranes by Calcium and Cholesterol.

Langmuir 2019 09 23;35(35):11358-11368. Epub 2019 Aug 23.

J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences , 182 23 Prague 8 , Czech Republic.

Regulation of cell metabolism, membrane fusion, association of proteins with cellular membranes, and cellular signaling altogether would not be possible without Ca ions. The distribution of calcium within the cell is uneven with the negatively charged inner leaflet of the plasma membrane being one of the primary targets of its accumulation. Therefore, we decided to map the influence of Ca on the properties of lipid bilayers closely resembling natural lipid membranes. We combined fluorescence spectroscopy (analysis of time-resolved emission spectra of Laurdan probe and derived parameters: integrated relaxation time related to local lipid mobility, and total emission shift reflecting membrane polarity and hydration) with molecular dynamics simulations to determine the effect of the increasing CaCl concentration on model lipid membranes containing POPC, POPS, and cholesterol. On top of that, the impact of calcium on the plasma membranes isolated from HEK293 cells was investigated using the steady-state fluorescence of Laurdan. We found that calcium increases rigidity of all the model lipid membranes used, elevates their thickness, increases lipid packing and ordering, and impedes the local lipid mobility. All these effects were to a great extent similar to those elicited by cholesterol. However, the changes of the membrane properties induced by calcium and cholesterol seem largely independent from each other. At sufficiently high concentrations of calcium or cholesterol, the steric effects hindered a further alteration of membrane organization, i.e., the compressibility limit of membrane structures was reached. We found no indication for mutual interaction between Ca and cholesterol, nor competition of Ca ions and hydroxyl groups of cholesterol for binding to phospholipids. Fluorescence measurements indicated that Ca adsorption decreases mobility within the carbonyl region of model bilayers more efficiently than monovalent ions do (Ca ≫ Li > Na > K > Cs). The effects of calcium ions were to a great extent mitigated in the plasma membranes isolated from HEK293 cells when compared to the model lipid membranes. Noticeably, the plasma membranes showed remarkably higher resistance toward rigidification induced by calcium ions even when compared with the model membranes containing cholesterol.
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http://dx.doi.org/10.1021/acs.langmuir.9b00477DOI Listing
September 2019

Ascorbigen A-NMR identification.

Magn Reson Chem 2019 12 30;57(12):1084-1096. Epub 2019 Jun 30.

Institute of Chemical Process Fundamentals of the CAS, Prague 6, Czech Republic.

The connectivities of all atoms in ascorbigen A, an important metabolite, were determined unambiguously for the first time. The connectivity between carbon atoms was established by 2D INADEQUATE, and one-bond C- C coupling constants were determined for all pairs of directly connected carbon atoms except for two strongly coupled carbon pairs. The C- C coupling in one of the pairs was proved by a modification of standard INADEQUATE; however, the signals from the other pair were too weak to be observed. The connectivity within the two strongly coupled C-C pairs was confirmed by a combination of COSY and gHSQC; the latter experiment also identified all C-H bonds. The proton nuclear magnetic resonance ( H NMR) spectra in dry dimethyl sulfoxide allowed identification and assignment of the signals due to NH and OH protons. The derived structure, 3-((1H-indol-3-yl)methyl)-3,3a,6-trihydroxytetrahydrofuro[3,2-b]furan-2(5H)-one, agrees with the structure suggested for ascorbigen A in 1966. The density functional theory (DFT) calculations showed that among 16 possible stereoisomers, only two complied with the almost zero value of the measured J(H6-H6a). Of the two stereoisomers, 3S,3aS,6S,6aR and 3R,3aR,6R,6aS, the latter was excluded on synthetic grounds. The nuclear Overhauser effect measurements unveiled close proximity between H2' proton of the indole and the H6a proton of the tetrahydrofuro[3,2-b]furan part. Detailed structural interpretation of the measured NMR parameters by means of DFT NMR was hampered by rotational flexibility of the indole and tetrahydrofuro[3,2-b]furan parts and inadequacy of Polarizable Continuum Model (PCM) solvent model.
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http://dx.doi.org/10.1002/mrc.4890DOI Listing
December 2019

STRONG INADEQUATE, an experiment for detection of small J(C,C) couplings in symmetrical molecules.

Magn Reson Chem 2019 12 1;57(12):1107-1120. Epub 2019 Jul 1.

Department of Analytical Chemistry, Institute of Chemical Process Fundamentals of the CAS, v. v. i., Prague 6, Czech Republic.

A new version of the two-dimensional INADEQUATE experiment was designed for detection of small couplings between equivalent carbon atoms separated in the molecule by several bonds, where other techniques fail due to rich line splitting and mutual peak cancellation in many molecules. As the proposed method is suitable for detection of couplings in strongly coupled systems in general, we propose the name STRONG INADEQUATE in the paper. Similar to other methods for detection of couplings between equivalent carbons, the STRONG INADEQUATE experiment utilizes one-bond carbon-proton coupling for creation of the effective chemical shift differences. The STRONG INADEQUATE experiment works superbly for J , where n ≥ 3. Then the F1 pattern is reduced to a simple antiphase doublet with J separation, and this pattern is also preserved when a symmetrical HC···C'H' system is coupled to other protons. Even in the measurement of J couplings, the STRONG INADEQUATE experiment generates a much simpler pattern than the original pulse sequences for measurement of couplings between equivalent carbons.
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http://dx.doi.org/10.1002/mrc.4897DOI Listing
December 2019

Preparation and Physicochemical Properties of [6]Helicenes Fluorinated at Terminal Rings.

J Org Chem 2019 02 7;84(4):1980-1993. Epub 2019 Feb 7.

Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry , Palacký University , Hněvotínská 3 , 775 15 Olomouc , Czech Republic.

The first racemization-stable helicene derivatives fluorinated at terminal rings, 1,2,3,4-tetrafluoro[6]helicene (6) and 1,2,3,4,13,14,15,16-octafluoro[6]helicene (15), were synthesized via the Wittig reaction followed by oxidative photocyclization in an overall yield of 41% of 6 and 76% of 15. The changed electronic structure in fluorinated helicenes was reflected in a slight shift of UV-vis absorption, fluorescence excitation, and emission spectra maxima when compared to unsubstituted [6]helicene. Cyclic voltammetry revealed a moderate decrease in the HOMO-LUMO gap with increasing fluorination. The specific rotation of tetrafluoro[6]helicene 6 enantiomers was found to be approximately 25% lower than that of unsubstituted [6]helicene. The theoretical study of the racemization barrier suggested a reasonable shift toward higher energy with increasing fluorination. The increasing fluorination also significantly affected the intermolecular interactions in the crystal lattice. The observed CH···F interactions led to the formation of 1D-molecular chains in the crystal structures of both fluorinated helicenes.
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http://dx.doi.org/10.1021/acs.joc.8b02870DOI Listing
February 2019

Proton detection of carbon-carbon couplings in symmetrical molecules: Analytical explanation, SYMONA pulse sequence.

J Magn Reson 2019 Jan 5;298:107-114. Epub 2018 Dec 5.

Institute of Chemical Process Fundamentals of the CAS, v. v. i., Rozvojova 135, 165 02 Prague 6, Czech Republic.

The approach to the measurement of one-bond indirect spin-spin coupling constants between equivalent nuclei was revisited. The analytical formulas for development of the density matrix of strongly coupled symmetrical HC-C'H' spin systems were derived and the optimal duration of polarization delay in the original 2QHMBC pulse sequence is discussed. Based on the analytical formulas a new version of a robust indirect detection experiment, called SYMONA (SYmmetrical MOlecules Natural Abundance double-quantum experiment), was proposed for carbon-carbon coupling constants detection in symmetrical molecules. Additionally, application of the SYMONA experiment to more complicated spin systems than isolated HC-C'H' is discussed.
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http://dx.doi.org/10.1016/j.jmr.2018.12.002DOI Listing
January 2019

Helicene-SPP-Based Chiral Plasmonic Hybrid Structure: Toward Direct Enantiomers SERS Discrimination.

ACS Appl Mater Interfaces 2019 Jan 20;11(1):1555-1562. Epub 2018 Dec 20.

Group of Advanced Materials and Organic Synthesis, Institute of Chemical Process Fundamentals , Czech Academy of Sciences , Rozvojová 135 , 165 02 Prague , Czech Republic.

Achieving chiral plasmon response based on the combination of achiral plasmonic nanostructures with highly chiral surrounding medium represents an attractive way for creation of hybrid optically active plasmonic materials. In this work, we present an attractive design and fabrication of chiral plasmon substrates based on a surface plasmon-polariton-supported structure coupled with extremely optically active helicene enantiomers. Such approach allows us to excite chiral plasmon waves and to design optically active surface-enhanced Raman spectroscopy substrates. Its further combination with standard Raman spectroscopy makes possible enantioselective detection/recognition of optical enantiomers with detection limits below those of standard spectral techniques. The chiral optical response of new plasmonic system was observed and controlled by the optical rotation of helicenes. Without necessity of previous chiral separation or implementation of sophisticated experimental equipment, we were able to estimate the concentration of enantiomers in their mixture by using left- or right-handed chiral plasmon substrates.
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http://dx.doi.org/10.1021/acsami.8b15520DOI Listing
January 2019

Molecular Gating of an Engineered Enzyme Captured in Real Time.

J Am Chem Soc 2018 12 14;140(51):17999-18008. Epub 2018 Dec 14.

J. Heyrovsky Institute of Physical Chemistry of the ASCR , v. v. i., Dolejskova 3 , 182 23 Prague 8 , Czech Republic.

Enzyme engineering tends to focus on the design of active sites for the chemical steps, while the physical steps of the catalytic cycle are often overlooked. Tight binding of a substrate in an active site is beneficial for the chemical steps, whereas good accessibility benefits substrate binding and product release. Many enzymes control the accessibility of their active sites by molecular gates. Here we analyzed the dynamics of a molecular gate artificially introduced into an access tunnel of the most efficient haloalkane dehalogenase using pre-steady-state kinetics, single-molecule fluorescence spectroscopy, and molecular dynamics. Photoinduced electron-transfer-fluorescence correlation spectroscopy (PET-FCS) has enabled real-time observation of molecular gating at the single-molecule level with rate constants ( k = 1822 s, k = 60 s) corresponding well with those from the pre-steady-state kinetics ( k = 1100 s, k = 20 s). The PET-FCS technique is used here to study the conformational dynamics in a soluble enzyme, thus demonstrating an additional application for this method. Engineering dynamical molecular gates represents a widely applicable strategy for designing efficient biocatalysts.
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http://dx.doi.org/10.1021/jacs.8b09848DOI Listing
December 2018

Roughness of Transmembrane Helices Reduces Lipid Membrane Dynamics.

iScience 2018 Dec 20;10:87-97. Epub 2018 Nov 20.

Department of Biophysical Chemistry, J. Heyrovsky Institute of Physical Chemistry of the Czech Academy of Sciences, Dolejškova 3, 182 23 Prague, Czech Republic. Electronic address:

The dynamics of cellular membranes is primarily determined by lipid species forming a bilayer. Proteins are considered mainly as effector molecules of diverse cellular processes. In addition to large assemblies of proteins, which were found to influence properties of fluid membranes, biological membranes are densely populated by small, highly mobile proteins. However, little is known about the effect of such proteins on the dynamics of membranes. Using synthetic peptides, we demonstrate that transmembrane helices interfere with the mobility of membrane components by trapping lipid acyl chains on their rough surfaces. The effect is more pronounced in the presence of cholesterol, which segregates from the rough surface of helical peptides. This may contribute to the formation or stabilization of membrane heterogeneities. Since roughness is a general property of helical transmembrane segments, our results suggest that, independent of their size or cytoskeleton linkage, integral membrane proteins affect local membrane dynamics and organization.
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http://dx.doi.org/10.1016/j.isci.2018.11.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277224PMC
December 2018

The use of styrene-maleic acid copolymer (SMA) for studies on T cell membrane rafts.

Biochim Biophys Acta Biomembr 2019 01 14;1861(1):130-141. Epub 2018 Aug 14.

Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Praha 4, Czech Republic. Electronic address:

An emerging alternative to the use of detergents in biochemical studies on membrane proteins is apparently the use styrene-maleic acid (SMA) amphipathic copolymers. These cut the membrane into nanodiscs (SMA-lipid particles, SMALPs), which contain membrane proteins possibly surrounded by their native lipid environment. We examined this approach for studies on several types of T cell membrane proteins, previously defined as raft or non-raft associated, to see whether the properties of the raft derived SMALPs differ from non-raft SMALPs. Our results indicate that two types of raft proteins, GPI-anchored proteins and two Src family kinases, are markedly present in membrane fragments much larger (>250 nm) than those containing non-raft proteins (<20 nm). Lipid probes sensitive to membrane fluidity (membrane order) indicate that the lipid environment in the large SMALPs is less fluid (more ordered) than in the small ones which may indicate the presence of a more ordered lipid L phase which is characteristic of membrane rafts. Also the lipid composition of the small vs. large SMALPs is markedly different - the large ones are enriched in cholesterol and lipids containing saturated fatty acids. In addition, we confirm that T cell membrane proteins present in SMALPs can be readily immunoisolated. Our results support the use of SMA as a potentially better (less artifact prone) alternative to detergents for studies on membrane proteins and their complexes, including membrane rafts.
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http://dx.doi.org/10.1016/j.bbamem.2018.08.006DOI Listing
January 2019

Diagnosis of pancreatic cancer viaH NMR metabolomics of human plasma.

Analyst 2018 Dec;143(24):5974-5978

Department of Analytical Chemistry, University of Chemistry and Technology Prague, Prague 6, Czech Republic.

Metabolic changes induced by pancreatic cancer were investigated by 1H NMR spectroscopy of plasma samples of patients and healthy controls. The acquired data were submitted to multivariate statistical analysis providing clear discrimination between both groups. The most significant differences were found in levels of 3-hydroxybutyrate and lactate. The obtained results (100% sensitivity, 90% specificity) clearly show the potential of 1H NMR spectroscopy in pancreatic cancer diagnosis. Therefore, the NMR-based metabolomics may contribute to the early diagnosis, prevention and/or therapy of diseases in the future. On the other hand, the number of samples in the presented pilot study is limited and has to be significantly increased in the future in order to obtain solid statistical models and to confirm the current findings.
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http://dx.doi.org/10.1039/c8an01310aDOI Listing
December 2018

Monitoring of nucleophosmin oligomerization in live cells.

Methods Appl Fluoresc 2018 Jun 29;6(3):035016. Epub 2018 Jun 29.

Department of Proteomics, Institute of Hematology and Blood Transfusion, U Nemocnice 2094/1, 128 20 Praha 2, Czechia.

Oligomerization plays a crucial role in the function of nucleophosmin (NPM), an abundant nucleolar phosphoprotein. Two dual-color methods based on modern fluorescence confocal microscopy are applied for tracking NPM aggregates in live cells: cross-correlation Number and Brightness analysis (ccN&B) combined with pulsed interleaved excitation (PIE) and fluorescence-lifetime imaging microscopy (FLIM) utilizing resonance energy transfer (FRET). HEK-293T cells were transfected with mixture of plasmids designed for tagging with fluorescent proteins so that the cells express mixed population of NPM labeled either with eGFP or mRFP1. We observe joint oligomers formed from the fluorescently labeled NPM. Having validated the in vivo methods, we study an effect of substitutions in cysteine 21 (Cys21) of the NPM N-terminus on the oligomerization to demonstrate applicability of the methods. Inhibitory effect of mutations of the Cys21 to nonpolar Ala or to aromatic Phe on the oligomerization was reported in literature using in vitro semi-native electrophoresis. However, we do not detect any break-up of the joint NPM oligomers due to the Cys21 mutations in live cells. In vivo microscopy observations are supported by an in vitro method, the GFP-Trap immunoprecipitation assay. Our results therefore show importance of utilizing several methods for detection of biologically relevant protein aggregates. In vivo monitoring of the NPM oligomerization, a potential cancer therapy target, by the presented methods offers a new way to monitor effects of drugs that are tested as NPM oligomerization inhibitors directly in live cells.
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http://dx.doi.org/10.1088/2050-6120/aaccb9DOI Listing
June 2018

Xanthones Content in D. Don from Nepal.

Molecules 2018 05 3;23(5). Epub 2018 May 3.

Institute of Chemical Process Fundamentals, Czech Academy of Sciences, Rozvojová 135, 165 02 Prague, Czech Republic.

The medicinal plant D. Don was collected in Rasuwa District (Nepal) and the xanthone content of its ethyl acetate extracts was studied. The total amount of xanthones in determined by HPLC reaches almost 13 g of xanthones per 1 kg of dry matter. The identification of xanthones in was achieved by a combination of HPLC, LC⁻MS and LC⁻NMR. The final assignment of the individual chemical structures was provided by NMR, supported by preparative HPLC. In eight chromatographic peaks, four major xanthones were identified—1,3-dihydroxy-5,8-dimethoxyxanthone, 1-hydroxy-3,5,8-trimethoxyxanthone, bellidifolin (1,5,8-tri-hydroxy-3-methoxyxanthone), and decussatin (1-hydroxy-3,7,8-trimethoxyxanthone).
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http://dx.doi.org/10.3390/molecules23051067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102544PMC
May 2018

2-Bromo[6]helicene as a Key Intermediate for [6]Helicene Functionalization.

J Org Chem 2018 04 19;83(7):3607-3616. Epub 2018 Mar 19.

Institute of Chemical Process Fundamentals of the CAS, v. v. i. , Rozvojová 2/135 , Prague 6 , 165 02 , Czech Republic.

The synthesis of 2-bromo[6]helicene was revised and improved up to 51% yield. Its reactivity was thoroughly investigated, and a library of 17 different carbon, boron, nitrogen, phosphorus, oxygen and sulfur substituted derivatives was prepared. The racemization barrier for 2-bromo[6]helicene was determined, and the usage of enantiomers in the synthesis of optically pure helicenes was rationalized. The three most energy-demanding reactions using enantiomerically pure 2-bromo[6]helicene were tested in order to confirm the predicted enantiomeric excess.
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http://dx.doi.org/10.1021/acs.joc.7b03234DOI Listing
April 2018

6,7-dimethoxy-coumarin as a probe of hydration dynamics in biologically relevant systems.

Methods Appl Fluoresc 2018 Feb 12;6(2):025005. Epub 2018 Feb 12.

Department of Biophysical Chemistry, J. Heyrovský Institute of Physical Chemistry of the CAS, v. v. i., Dolejškova 2155/3, 182 23 Prague 8, Czechia.

Coumarin derivatives are well known fluorescence reporters for investigating biological systems due to their strong micro-environment sensitivity. Despite having wide range of environment sensitive fluorescence probes, the potential of 6,7-dimethoxy-coumarin has not been studied extensively so far. With a perspective of its use in protein studies, namely using the unnatural amino acid technology or as a substrate for hydrolase enzymes, we study acetyloxymethyl-6,7-dimethoxycoumarin (Ac-DMC). We investigate the photophysics and hydration dynamics of this dye in aerosol-OT (AOT) reverse micelles at various water contents using the time dependent fluorescence shift (TDFS) method. The TDFS response in AOT reverse micelles from water/surfactant ratio of 0 to 20 confirms its sensitivity towards the hydration and mobility of its microenvironment. Moreover, we show that the fluorophore can be efficiently quenched by halide ions. Hence, we conclude that the 6,7-dimethoxy-methylcoumarin fluorophore is useful for studying hydration parameters in biologically relevant systems.
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http://dx.doi.org/10.1088/2050-6120/aaaa05DOI Listing
February 2018

Internal dynamics in helical molecules studied by X-ray diffraction, NMR spectroscopy and DFT calculations.

Phys Chem Chem Phys 2017 Jan;19(4):2900-2907

Institute of Chemical Process Fundamentals, Czech Academy of Sciences, Rozvojová 135, 16502 Prague, Czech Republic.

The conformational behaviour of a prototype helical molecule, [6]helicene, and its derivatives was studied in solution and the solid state. Available crystal structures of [6]helicene revealed surprisingly large flexibility of this molecule and variable-temperature NMR experiments provided unusual temperature dependence of chemical shifts of hydrogen, carbon and fluorine atoms in the peripheral aromatic rings of [6]helicene and tetrafluoro[6]helicene. These chemical shift changes were interpreted as a consequence of the helicene 'pitch' opening with elevated temperature, and the experimental data were corroborated by DFT calculations of the chemical shift dependence on the helicene conformation and by variable-temperature DFT molecular dynamics simulations.
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http://dx.doi.org/10.1039/c6cp07552eDOI Listing
January 2017

Synthesis and in vitro cytotoxicity of acetylated 3-fluoro, 4-fluoro and 3,4-difluoro analogs of D-glucosamine and D-galactosamine.

Beilstein J Org Chem 2016 20;12:750-9. Epub 2016 Apr 20.

Institute of Chemical Process Fundamentals of the CAS, Rozvojová 135, 165 02 Praha, Czech Republic.

Background: Derivatives of D-glucosamine and D-galactosamine represent an important family of the cell surface glycan components and their fluorinated analogs found use as metabolic inhibitors of complex glycan biosynthesis, or as probes for the study of protein-carbohydrate interactions. This work is focused on the synthesis of acetylated 3-deoxy-3-fluoro, 4-deoxy-4-fluoro and 3,4-dideoxy-3,4-difluoro analogs of D-glucosamine and D-galactosamine via 1,6-anhydrohexopyranose chemistry. Moreover, the cytotoxicity of the target compounds towards selected cancer cells is determined.

Results: Introduction of fluorine at C-3 was achieved by the reaction of 1,6-anhydro-2-azido-2-deoxy-4-O-benzyl-β-D-glucopyranose or its 4-fluoro analog with DAST. The retention of configuration in this reaction is discussed. Fluorine at C-4 was installed by the reaction of 1,6:2,3-dianhydro-β-D-talopyranose with DAST, or by fluoridolysis of 1,6:3,4-dianhydro-2-azido-β-D-galactopyranose with KHF2. The amino group was introduced and masked as an azide in the synthesis. The 1-O-deacetylated 3-fluoro and 4-fluoro analogs of acetylated D-galactosamine inhibited proliferation of the human prostate cancer cell line PC-3 more than cisplatin and 5-fluorouracil (IC50 28 ± 3 μM and 54 ± 5 μM, respectively).

Conclusion: A complete series of acetylated 3-fluoro, 4-fluoro and 3,4-difluoro analogs of D-glucosamine and D-galactosamine is now accessible by 1,6-anhydrohexopyranose chemistry. Intermediate fluorinated 1,6-anhydro-2-azido-hexopyranoses have potential as synthons in oligosaccharide assembly.
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http://dx.doi.org/10.3762/bjoc.12.75DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4901990PMC
June 2016

A comprehensive LC/MS analysis of novel cyclopentenedione library.

J Pharm Biomed Anal 2016 Sep 2;128:342-351. Epub 2016 Jun 2.

Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, Olomouc 775 15, Czech Republic. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jpba.2016.05.048DOI Listing
September 2016

Probing the Ca(2+)-assisted π-π interaction during Ca(2+)-dependent protein folding.

Soft Matter 2016 Jan 22;12(2):531-41. Epub 2015 Oct 22.

Laboratory of Molecular Biology of Bacterial Pathogens, Institute of Microbiology of the ASCR, v.v.i., Videnska 1083, 14200 Prague, Czech Republic.

Protein folding is governed by a balance of non-covalent interactions, of which cation-π and π-π play important roles. Theoretical calculations revealed a strong cooperativity between cation-π involving alkali and alkaline earth metal ions and π-π interactions, but however, no experimental evidence was provided in this regard. Here, we characterized a Ca(2+)-binding self-processing module (SPM), which mediates a highly-specific Ca(2+)-dependent autocatalytic processing of iron-regulated protein FrpC secreted by the pathogenic Gram-negative bacterium Neisseria meningitidis. The SPM undergoes a Ca(2+)-induced transition from an intrinsically unstructured conformation to the compact protein fold that is ultimately stabilized by the π-π interaction between two unique tryptophan residues arranged in the T-shaped orientation. Moreover, the pair of tryptophans is located in a close vicinity of a calcium-binding site, suggesting the involvement of a Ca(2+)-assisted π-π interaction in the stabilization of the tertiary structure of the SPM. This makes the SPM an excellent model for the investigation of the Ca(2+)-assisted π-π interaction during Ca(2+)-induced protein folding.
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http://dx.doi.org/10.1039/c5sm01796cDOI Listing
January 2016

High Efficacy but Low Potency of δ-Opioid Receptor-G Protein Coupling in Brij-58-Treated, Low-Density Plasma Membrane Fragments.

PLoS One 2015 18;10(8):e0135664. Epub 2015 Aug 18.

Department of Biomathematics, Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic.

Principal Findings: HEK293 cells stably expressing PTX-insensitive δ-opioid receptor-Gi1α (C351I) fusion protein were homogenized, treated with low concentrations of non-ionic detergent Brij-58 at 0°C and fractionated by flotation in sucrose density gradient. In optimum range of detergent concentrations (0.025-0.05% w/v), Brij-58-treated, low-density membranes exhibited 2-3-fold higher efficacy of DADLE-stimulated, high-affinity [32P]GTPase and [35S]GTPγS binding than membranes of the same density prepared in the absence of detergent. The potency of agonist DADLE response was significantly decreased. At high detergent concentrations (>0.1%), the functional coupling between δ-opioid receptors and G proteins was completely diminished. The same detergent effects were measured in plasma membranes isolated from PTX-treated cells. Therefore, the effect of Brij-58 on δ-opioid receptor-G protein coupling was not restricted to the covalently bound Gi1α within δ-opioid receptor-Gi1α fusion protein, but it was also valid for PTX-sensitive G proteins of Gi/Go family endogenously expressed in HEK293 cells. Characterization of the direct effect of Brij-58 on the hydrophobic interior of isolated plasma membranes by steady-state anisotropy of diphenylhexatriene (DPH) fluorescence indicated a marked increase of membrane fluidity. The time-resolved analysis of decay of DPH fluorescence by the "wobble in cone" model of DPH motion in the membrane indicated that the exposure to the increasing concentrations of Brij-58 led to a decreased order and higher motional freedom of the dye.

Summary: Limited perturbation of plasma membrane integrity by low concentrations of non-ionic detergent Brij-58 results in alteration of δ-OR-G protein coupling. Maximum G protein-response to agonist stimulation (efficacy) is increased; affinity of response (potency) is decreased. The total degradation plasma membrane structure at high detergent concentrations results in diminution of functional coupling between δ-opioid receptors and G proteins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0135664PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4540457PMC
May 2016

Various extraction methods for obtaining stilbenes from grape cane of Vitis vinifera L.

Molecules 2015 Apr 8;20(4):6093-112. Epub 2015 Apr 8.

Institute of Chemical Process Fundamentals, Academy of Sciences of the Czech Republic, v.v.i., Rozvojová 2/135, 16502 Prague 6, Czech Republic.

Grape cane, leaves and grape marc are waste products from viticulture, which can be used to obtain secondary stilbene derivatives with high antioxidant value. The presented work compares several extraction methods: maceration at laboratory temperature, extraction at elevated temperature, fluidized-bed extraction, Soxhlet extraction, microwave-assisted extraction, and accelerated solvent extraction. To obtain trans-resveratrol, trans-ε-viniferin and r2-viniferin from grape cane of the V. vinifera variety Cabernet Moravia, various conditions were studied: different solvents, using powdered versus cut cane material, different extraction times, and one-step or multiple extractions. The largest concentrations found were 6030 ± 680 µg/g dry weight (d.w.) for trans-resveratrol, 2260 ± 90 µg/g d.w. for trans-ε-viniferin, and 510 ± 40 µg/g d.w. for r2-viniferin. The highest amounts of stilbenes (8500 ± 1100 µg/g d.w.) were obtained using accelerated solvent extraction in methanol.
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http://dx.doi.org/10.3390/molecules20046093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6272250PMC
April 2015

Site-specific analysis of protein hydration based on unnatural amino acid fluorescence.

J Am Chem Soc 2015 Apr 9;137(15):4988-92. Epub 2015 Apr 9.

‡Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic.

Hydration of proteins profoundly affects their functions. We describe a simple and general method for site-specific analysis of protein hydration based on the in vivo incorporation of fluorescent unnatural amino acids and their analysis by steady-state fluorescence spectroscopy. Using this method, we investigate the hydration of functionally important regions of dehalogenases. The experimental results are compared to findings from molecular dynamics simulations.
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http://dx.doi.org/10.1021/jacs.5b01681DOI Listing
April 2015

Cytotoxic Lipopeptide Muscotoxin A, Isolated from Soil Cyanobacterium Desmonostoc muscorum, Permeabilizes Phospholipid Membranes by Reducing Their Fluidity.

Chem Res Toxicol 2015 Feb;28(2):216-24

There is mounting evidence that cyanobacterial lipopeptides can kill mammalian cells, presenting a hazard to human health. Unfortunately, their mechanism of toxicity is poorly understood. We have isolated new cyclic undecalipopeptides muscotoxin A and B containing unique lipophilicresidue 3-amino-2,5-dihydroxydecanoic acid (5-OH Ahdoa). Muscotoxin B was not used for biological studies due to its poor yield. Muscotoxin A was cytotoxic to YAC-1, Sp/2, and HeLa cancer cell lines (LC(50) ranged from 9.9 to 13.2 μM after 24 h of exposure), causing membrane damage and influx of calcium ions. Subsequently, we studied this lytic mechanism using synthetic liposomes with encapsulated fluorescent probes. Muscotoxin A permeabilized liposomes composed exclusively of phospholipids, demonstrating that no proteins or carbohydrates present in biomembranes are essential for its activity. Paradoxically, the permeabilization activity of muscotoxin A was mediated by a significant reduction in membrane surface fluidity (stiffening), the opposite of that caused by synthetic detergents and cytolytic lipopeptide puwainaphycin F. At 25 °C, muscotoxin A disrupted liposomes with and without cholesterol/sphingomyelin; however, at 37 °C, it was selective against liposomes with cholesterol/sphingomyelin. It appears that both membrane fluidity and organization can affect the lytic activity of muscotoxin A. Our findings strengthen the evidence that cyanobacterial lipopeptides specifically disrupt mammalian cell membranes and bring new insights into the mechanism of this effect.
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http://dx.doi.org/10.1021/tx500382bDOI Listing
February 2015