Petr Sedmera

Mechanism of the antioxidant action of silybin and 2,3-dehydrosilybin flavonolignans: a joint experimental and theoretical study.

Flavonolignans from silymarin, the standardized plant extract obtained from thistle, exhibit various antioxidant activities, which correlate with the other biological and therapeutic properties of that extract. To highlight the mode of action of flavonolignans as free radical scavengers and antioxidants, 10 flavonolignans, selectively methylated at different positions, were tested in vitro for their capacity to scavenge radicals (DPPH and superoxide) and to inhibit the lipid peroxidation induced on microsome membranes. The results are rationalized on the basis of (i) the oxidation potentials experimentally obtained by cyclic voltammetry and (ii) the theoretical redox properties obtained by quantum-chemical calculations (using a polarizable continuum model (PCM)-density functional theory (DFT) approach) of the ionization potentials and the O-H bond dissociation enthalpies (BDEs) of each OH group of the 10 compounds. We clearly establish the importance of the 3-OH and 20-OH groups as H donors, in the presence of the 2,3 double bond and the catechol moiety in the E-ring, respectively. For silybin derivatives (i.e., in the absence of the 2,3 double bond), secondary mechanisms (i.e., electron transfer (ET) mechanism and adduct formation with radicals) could become more important (or predominant) as the active sites for H atom transfer (HAT) mechanism are much less effective (high BDEs).

Molecular mechanisms of silybin and 2,3-dehydrosilybin antiradical activity—role of individual hydroxyl groups

The flavonolignans silybin (1) and 2,3-dehydrosilybin (2) are important natural compounds with multiple biological activities operating at various cell levels. Many of these effects are connected with their radical-scavenging activities. The molecular mechanisms of the antioxidant activity of these compounds and even the functional groups responsible for this activity are not yet well known. Their mechanism can be inferred from the structures of the dimeric products obtained from radical-mediated reactions of selectively methylated derivatives of 1 and 2. The radical oxidation of 1 methylated at 7-OH and 2 methylated at both 3-OH and 7-OH yields C-C and C-O dimers that enable the molecular mechanism of their E-ring interaction with radicals to be elucidated and shows the importance of the 20-OH group in this respect. The pivotal role of the 3-OH group in the radical-scavenging activity of 2 was confirmed through the formation of another type of dimer from its selectively methylated derivative.

Identification of the covalent flavin adenine dinucleotide-binding region in pyranose 2-oxidase from Trametes multicolor.

We present the first report on characterization of the covalent flavinylation site in flavoprotein pyranose 2-oxidase. Pyranose 2-oxidase from the basidiomycete fungus Trametes multicolor, catalyzing C-2/C-3 oxidation of several monosaccharides, shows typical absorption maxima of flavoproteins at 456, 345, and 275 nm. No release of flavin was observed after protein denaturation, indicating covalent attachment of the cofactor. The flavopeptide fragment resulting from tryptic/chymotryptic digestion of the purified enzyme was isolated by anion-exchange and reversed-phase high-performance liquid chromatography. The flavin type, attachment site, and mode of its linkage were determined by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy of the intact flavopeptide, without its prior enzymatic degradation to the central aminoacyl moiety. Mass spectrometry identified the attached flavin as flavin adenine dinucleotide (FAD). Post-source decay analysis revealed that the flavin is covalently bound to histidine residue in the peptide STHW, consistent with the results of N-terminal amino acid sequencing by Edman degradation. The type of the aminoacyl flavin covalent link was determined by NMR spectroscopy, resulting in the structure 8alpha-(N(3)-histidyl)-FAD.

Titanocene-bis(trimethylsilyl)acetylene complexes: effects of methyl substituents at the cyclopentadienyl ligands on the structure of thermolytic products

Abstract The (C5H5−nMenTi[η5-C2(SiMe3)2] (n = 0–5) (1A–1F) complexes were prepared by the reduction of corresponding titanocene dichlorides with magnesium in THF in the presence of bis(trimethylsily)acetylene (BTMSA). All of them were characterized by spectroscopic methods and (C5HMe2Ti[η5-C2(SiMe3)2] (1E) by the X-ray crystal analysis. The complexes decompose at temperatures in the range 100–200°C. Those with n = 0–3 yield (μ-η5 : η5-fulvalene)(di-μ-hydrido)bis (η5-cyclopentadienyltitanium) (2A) and its methylated analogues (2B–2D) whereas BTMSA is released. The crystal structure of 2D showed that the hexamethylfulvalene ligand contains non-methylated carbon atoms in inner alternate positions. Complex 1E afforded a mixture of products. Among them only volatile isomers (η3:η4-1,2-dimethyl-4,5-dimethylcyclopenteny)(η5-tetramethylcyclopentadienyl)titanium (2Ea) and (η3:η4-1,3-dimethyl-4, 5-dimethylenecyclopentenyl)(η5-tetramethylcyclopentadienyl)titanium (2Eb) have been so far isolated as minor products. The C5Me5 comples 1F yields quantitatively (η3:η4-1,2,3-trimethyl-4,5-dimethylenecyclopentyl)(pentamethylcyclopentadienyl)titanium (2F) and BTMSA is hydrogenated to a mixture of cis- and trans-bis(trimethylsilyl)ethene.

Antioxidant and antiviral activities of silybin fatty acid conjugates

Two selective acylation methods for silybin esterification with long-chain fatty acids were developed, yielding a series of silybin 7-O- and 23-O-acyl-derivatives of varying acyl chain lengths. These compounds were tested for their antioxidant (inhibition of lipid peroxidation and DPPH-scavenging) and anti-influenza virus activities. The acyl chain length is an important prerequisite for both biological activities, as they improved with increasing length of the acyl moiety.

[6+2]Cycloadditions catalyzed by titanium complexes

Abstract The Ziegler catalyst TiCl4-Et2AlCl and the arenetitanium(II) complex (η6-C6H6)Ti(II)(AlCl4)2 induce [6 + 2]cycloaddition reactions of cycloheptatriene with dienes and acetylenes. Addition to 1,3-butadiene affords 7 - endo - vinyl - bicyclo[4.2.1]nona - 2,4 - diene (main product) and bicyclo[4.4.1]- undeca - 2,4,8 - triene, a product of [6+4]cycloaddition. Isoprene reacts similarly, yielding mainly 7- endo - isopropenyl - bicyclo[4.2.1]nona - 2,4 - diene. 2,3 - Dimethyl - 1,3 - butadiene gives 8,9dimethylbicyclo [4.4.1]undeca - 2,4,8 - triene, a product of [6 + 4]cycloaddition, while [6 + 2]cross-adducts are minor products. The reaction of cycloheptatriene with norbornadiene gives mainly hexacyclo[6.5.1.02,7.03,12.6,10.09,13]tetradec - 4 - ene via [6+2]cycloaddition followed by intramolecular Diels-Alder reaction. As a by-product, pentacyclo[7.5.0.02,7.03,5.048]tetradeca - 10,12 - diene is formed by a [2+2+2]mechanism. Addition of cycloheptatriene to diphenylacetylene and bis - (tri- methylsilyl)acetylene furnishes sustituted bicyclo[4.2.1]nona - 2,4,7 - trienes. Alkenes, E,E-2,4 - hexadiene and 1,3 - cyclooctadiene are unreactive. The [6+2]cycloaddition is made possible by coordination of cycloheptatriene to titanium, which changes the symmetry of the frontier orbitals in the triene. The reactivity of the trienophile is also enhanced by coordination to the catalyst.

Properties of pyranose dehydrogenase purified from the litter‐degrading fungus Agaricus xanthoderma

We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion‐exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose–methanol–choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi‐covalent flavinylation in PDH, 9‐S‐cysteinyl, 8‐α‐N3‐histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are d‐glucose, d‐galactose, l‐arabinose, and d‐xylose. As shown by in situ NMR analysis, d‐glucose and d‐galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino‐bis‐(3‐ethylbenzthiazolin‐6‐sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (kcat/Km). The enzyme may play a role in lignocellulose degradation.

Pyranose oxidase and pyranosone dehydratase: enzymes responsible for conversion of d-glucose to cortalcerone by the basidiomycete Phanerochaete chrysosporium

Pyranose oxidase (glucose 2-oxidase) and pyranosone dehydratase were purified 27.6- and 43.9-fold respectively from mycelial extracts of the fungus Phanerochaete chrysosporium using hydrophobic interaction, anion exchange and gel filtration chromatography. The enzymes appeared substantially homogeneous on SDS-PAGE and were comprised of identical subuntis with apparent Mr values of 69 000 and 99 000 for pyranose oxidase and pyranosone dehydratase, respectively. The apparent Mrs of the native enzymes, based on equilibrium ultracentrifugation, were 308 000 and 221 000. In coupled reactions, the enzymes catalyzed conversion of d-glucose via d-glucosone (d-arabino-2-hexosulose) to the antibiotic β-pyrone, cortalcerone. The latter compound was isolated as a diphenylhydrazone derivative and spectroscopically identified.

SYNTHESIS, CRYSTAL STRUCTURES AND SOME PROPERTIES OF DIMETHYLSILYLENE-BRIDGED ANSA-PERMETHYLTITANOCENE TI(IV), (III) AND (II) COMPLEXES

Dimethylsilylene-bridged complexes Me2Si(C5Me4)2TiCl (2), Me2Si(C5Me4)2Ti[η2-C2(SiMe3)2] (3) and Me2Si(C5H4)2Ti[η2-C2(SiMe3)2] (4) have been prepared by the general methods which are known for obtaining of analogous non-bridged titanocene complexes. X-ray crystal structures of Me2Si(C5Me4)2TiCl2 (1), 2, and 3 reveal that the dihedral angle between the least-squares planes of cyclopentadienyl rings increases in the order 2 < 3 < 1. Comparison with the structures of analogous (C5HMe4)2Ti and (C5Me5)2Ti compounds shows that the value of increases in the series (C5Me5)2Ti < (C5HMe4)2Ti < Me2Si(C5Me4)2Ti, e.g. in the bis(trimethylsilyl)acetylene complexes from 41.1° for (C5Me5)2Ti[η2-C2(SiMe3)2] (9) to 50.0° for (C5HMe4)2Ti[η2-C2(SiMe3)2] (8) and to 53.5° for 3. Compounds 3, 8 and 9 induce the head-to-tail dimerization of 1-hexyne with the selectivity of 72%, 21% and ca. 100% respectively. The discrepancy between the selectivities and the values of for 3 and 8 is accounted for by a larger flexibility of the titanocene skeleton in 8, affording a larger space for a non-specific coordination of 1-hexyne. The effects of the μ-Me2Si group in 2 and 3 on some of their properties are compared with the effects of Me and H substituents in the non-ansa compounds with controversial results. For instance, the affinity of 2 to 2-methyltetrahydrofuran approaches that of (C5H2Me3)2TiCl whereas the shift of the v(C≡C) vibration in 3 indicates a stronger metal-acetylene bond than in 9.

Cyclosporins from Tolypocladium terricola

New natural cyclosporins were isolated from the mycelium of surface cultivated fungus Tolypocladium terricola. The chemical structures of [Leu4] CS and [MeLeu1] CS = cyclosporin-J, were deduced from the NMR and mass spectral data. Biological activity of new cyclosporins is reported based on the proliferative mitogen stimulation test.