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Aims: Production of minor asukamycin congeners and its new derivatives by combination of targeted genetic manipulations with specific precursor feeding in the producer of asukamycin, Streptomyces nodosus ssp. asukaensis. Methods and Results: Structural variations of manumycins lie only in the diverse initiation of the ‘upper’ polyketide chain. Inactivation of the gene involved in the biosynthesis of cyclohexanecarboxylic acid (CHC) turned off the production of asukamycin in the mutant strain and allowed an increased production of other manumycins with the branched end of the upper chain. The ratio of produced metabolites was further affected by specific precursor feeding. Precursor-directed biosynthesis of a new asukamycin analogue (asukamycin I, 28%) with linear initiation of the upper chain was achieved by feeding norleucine to the mutant strain. Another asukamycin analogue with the unbranched upper chain (asukamycin H, 14%) was formed by the CHC-deficient strain expressing a heterologous gene putatively involved in the formation of the n-butyryl-CoA starter unit of manumycin A. Conclusions: Combination of the described techniques proved to be an efficient tool for the biosynthesis of minor or novel manumycins. Significance and Impact of the Study: Production of two novel asukamycin derivatives, asukamycins H and I, was achieved. Variations appeared in the upper polyketide chain, the major determinant of enzyme-inhibitory features of manumycins, affecting their cancerostatic or anti-inflammatory features.

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.

The lincomycin biosynthetic gene lmbX was deleted in Streptomyces lincolnensis ATCC 25466, and deletion of this gene led to abolition of lincomycin production. The results of complementation experiments proved the blockage in the biosynthesis of lincomycin precursor 4-propyl- l -proline. Feeding this mutant strain with precursor derivatives resulted in production of 4′-butyl-4′-depropyllincomycin and 4′-pentyl-4′-depropyllincomycin in high titers and without lincomycin contamination. Moreover, 4′-pentyl-4′-depropyllincomycin was found to be more active than lincomycin against clinical Staphylococcus isolates with genes determining low-level lincosamide resistance.

Robust and scalable chemoenzymatic procedure for obtaining gram amounts of pure silybin A and B within one week is described. It consists of the preparation of 23-O-β-pentaacetyl-galactopyranosides of silybin, their separation by silica gel chromatography, deprotection, and enzymatic sugar removal with β-galactosidase.

Two new carbasugars (9 and 10) were isolated from Streptomyces lincolnensis DSM 40355 along with streptol (valienol, 8), gabosine I (valienone, 14), and glucosylglycerate. The reported (1)H and (13)C assignments are based on 1D ((1)H, (13)C, 1D-TOCSY, homodecoupling) and 2D (gCOSY, J-resolved, TOCSY, ROESY, gHSQC, gHMBC) NMR techniques and electrospray ionization FT mass spectrometry (ESI FTMS).

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.

The structures of two new cyclosporins were elucidated by NMR and MS methods as cyclo[-MeBmt(1)-Abu(2)-Sar(3)-MeLeu(4)-Val(5)-MeLeu(6)-Ala(7)-d-Ala(8)-MeLeu(9)-MeNva(10)-MeVal(11)-] and cyclo[-MeBmt(1)-Abu(2)-Sar(3)-MeLeu(4)-Abu(5)-MeLeu(6)-Ala(7)-d-Ala(8)-MeLeu(9)-MeLeu(10)-MeVal(11)-].

Flavonolignan silybin ( 1 ) present in the seeds of the milk thistle ( Silybum marianum ) is widely used in human therapy of liver dysfunctions and as a hepatoprotectant thanks to its dual function: it acts as a highly effective radical scavenger (antilipoperoxidant) and also as an antioxidant. Molecular mechanisms of antiradical action of 1 and even functional groups responsible for this activity are not well known so far. Silybin forms during in vitro reaction with stable radicals (e.g., DPPH) or with enzyme laccase ( Trametes pubescens ) complex mixtures of oligomeric and polymeric products whose structural analysis is virtually impossible. Methylation of 7-OH in 1 yields under laccase-mediated oxidation C–O and C–C dimers in the ratio ca. 1:2.5. Using this approach, the hydroxyl groups responsible for antiradical activity of silybin (20-OH group) were determined and the molecular mechanism of the E-ring antiradical activity was explained.

A new method for the oxidation of ergoline alcohols to aldehydes was found (TFFA–DMSO, −78 °C, then DIPEA). Structural features of ergolines required for successful C7–C8 double bond introduction via Polonovski–Potier reaction of respective 6-N-oxides were defined and experimentally confirmed: (i) the presence of electron-withdrawing group at C-8; (ii) trans-diaxial orientation of N6–O and C7–H bonds (both requirements are fulfilled for dihydrolyserg-17-al and its 2,4-dinitrophenyl hydrazone prepared in this work).

Streptomyces caelestis DSM 40084 produces two osmolytes, viz. 2-O-(alpha-D-glucopyranosyl)-zeta-glyceric acid (GG) and trehalose. Both compounds were isolated and identified by nuclear magnetic resonance spectroscopy and mass spectrometry. A very sensitive regulation of the cell osmolytes was demonstrated in exponentially growing cultures. The intracellular levels of GG and trehalose increased 2x in response to a step change of medium osmolarity caused by 0.3% NaCl. 1H NMR analysis of the cell extracts did not confirm the presence of additional osmolytes. GG is a S. caelestis metabolite commonly released from the cells; its concentration reached 3 g/L during the cultivation in a yeast extract--(NH4)2SO4-glycerol medium. This is the first report on the occurrence of the ionic osmolyte GG in the genus Streptomyces and on its free excretion to the medium.

We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion-exchange and hydrophobic interaction chroma ...

The fungal enzyme pyranose dehydrogenase (PDH) (EC 1.1.99.29) purified to apparent homogeneity from culture media of Agaricus meleagris catalyzed the substrate-dependent C-1, C-2, C-3, C-1,3′ or C-2,3(′) (di)oxidation of a number of mono- and disaccharides with 1,4-benzoquinone as an electron acceptor. d -Ribose, d -allose, d -gulose and d -talose were oxidized to the corresponding aldonic acids. l -Arabinose was converted exclusively to 2-dehydro- l -arabinose ( l -erythro-pentos-2-ulose) whereas d -xylose underwent competing C-2 and C-3 oxidation followed by dioxidation to 2,3-didehydro- d -arabinose ( d -glycero-pentos-2,3-diulose). The major final oxidation products of maltose and cellobiose were the novel compounds 2,3′-didehydromaltose and 2,3′-didehydrocellobiose (α- and β- d -ribo-hexos-3-ulopyranosyl-(1 → 4)- d -arabino-hexos-2-ulose), formed via 2- and 3′-monooxidation intermediates. Minor concomitant (di)oxidation at C-1,(3′) to the corresponding bionic acids also took place. Maltotriose was preferentially oxidized at C-3″ of the terminal glucopyranosyl unit and at C-1 of the reducing moiety. The structures of these sugar oxidation products were established by in situ 1D and 2D NMR spectroscopy and ESI–MS. Based on HPLC analysis, conversions of (glycosyl)aldoses in non-buffered systems were nearly quantitative within 3–24 h, depending on the substrate. As the enzyme allows an easy access to highly reactive di- or tricarbonyl sugars, it might become a useful catalyst in carbohydrate chemistry.

Large series of O-alkyl derivatives (methyl and benzyl) of silybin and 2,3-dehydrosilybin was prepared. Selective alkylation of the silybin molecule was systematically investigated. For the first time we present here, for example, preparation of 19-nor-2,3-dehydrosilybin. All prepared silybin/2,3-dehydrosilybin derivatives were tested for cytotoxicity on a panel of drugs sensitive against multidrug resistant cell lines and the ability to inhibit P-glycoprotein mediated efflux activity. We have identified effective and relatively non-cytotoxic inhibitors of P-gp derived from 2,3-dehydrosilybin. Some of them were more effective inhibitors at concentrations lower than a standard P-gp efflux inhibitor cyclosporin A. Another group of 2,3-dehydrosilybin derivatives also had better inhibitory effects on P-gp efflux but a cytotoxicity comparable with that of parent 2,3-dehydrosilybin. Structural requirements for improving inhibitory activity and reducing toxicity of 2,3-dehydrosilybin were established. Effect of E-ring substitution as well as an influence of the substituent size at the C-7-OH position of A-ring on P-gp-inhibitory activity was evaluated for the first time in this study.

Glycosyl azides are new efficient donors for glycosidases. Their high water solubility facilitates transglycosylations with comparable or better yields than common O-glycosides. The azido group totally changes the β-GalNAc-ase/β-GlcNAc-ase ratio in β-N-acetylhexosaminidases (from the usual 0.3–1.0 to

Mollugin was isolated from the extract of Rubia tinctorum roots. Lucidin was obtained by semisynthesis from xanthopurpurin. Mollugin crystallises in space group P-1 (No. 2), a = 8.5857(7), b = 11.473(4), c = 15.024(1) A, α = 77.64(2)∘, β = 89.36(1)∘, γ = 89.71(2)∘, and V = 1445.5(5) A3, lucidin crystallises in space group P21/c (No.14), a = 16.800(6), b = 9.637(2), c = 7.073(7) A, β = 98.01(5)∘, and V = 1134(1) A3.

Carboxylic acids derived from silybin (1) and 2,3-dehydrosilybin (2) with improved water solubility were prepared by selective oxidation of parent compounds and a new inexpensive method for preparation of 2,3-dehydrosilybin from silybin was developed and optimised. The antioxidative properties of the above-mentioned compounds and of side product 3a from oxidation of compound 1 were determined by cyclic voltammetry, free radical scavenging (DPPH, superoxide) assays, and by inhibition of in vitro generated liver microsomal lipid peroxidation. Dehydrogenation at C(2)–C(3) in flavonolignans (silybin vs 2,3-dehydrosilybin; silybinic acid vs 2,3-dehydrosilybinic acid) strongly improved antioxidative properties (analogously as in flavonoids taxifolin vs quercetin). Thus, in antioxidative properties, dehydrosilybin was superior to silybin by one order, but its water solubility is too low for application in aqueous milieu. On the other hand, 2,3-dehydrosilybinic acid is a fairly soluble derivative with antilipoperoxidation and antiradical activities better than that of silybin.

Quinone-dependent pyranose dehydrogenase presents a new tool for versatile conversions of numerous carbohydrates to their di- and tricarbonyl derivatives. This enzyme purified from the basidiomycete Agaricus meleagris catalysed dioxidation of several aromatic β- d -glucopyranosides and a β- d -xylopyranoside into the corresponding 3,4-didehydro-β- d -aldopyranosides (β- d -aldopyranosid-3,4-diuloses) in high yields, typically >80% for 4-nitrophenyl glycosides. These new compounds were doubly hydrated in aqueous solution. According to in situ NMR investigations, the reaction intermediates were the corresponding 3- and 4-dehydro compounds. The analogous anomeric α-glycosides underwent one-step oxidation only at C-3 to 3-dehydro-α- d -aldopyranosides (α- d -pyranosid-3-uloses).

The quinone-dependent pyranose dehydrogenase (PDH) purified from culture media of the basidiomycete fungus Agaricus xanthoderma catalyzed the simultaneous oxidation of lactose to β- d -galactopyranosyl-(1 → 4)- d- arabino -hexos-2-ulose (2-dehydrolactose) and lactobiono-1,5-lactone, with the latter spontaneously hydrolyzing to lactobionic acid. These products were identified by MS and in situ NMR spectroscopy. C-2 oxidation at the reducing moiety of lactose was confirmed by analysis of N,N -diphenylhydrazone derivatives of the reaction products. The proportions of C-1 to C-2 oxidation varied according to the source of PDH, ranging from almost exclusive formation of lactobionic acid to 2-dehydrolactose as a major product. The highest selectivity for C-2 was exhibited by PDH from A. xanthoderma and A. meleagris . Yields of 2-dehydrolactose in laboratory transformations monitored by HPLC approached 75%. This dicarbonyl derivative may serve as a key intermediate in a potential process for adding value to lactose by its (chemo)enzymatic isomerization to lactulose. A minor product in the PDH–lactose reaction mixtures formed on prolonged incubations with excess of oxidant was identified through its hydrazone derivative as β- d -galactopyranosyl-(1 → 4)- d- threo -hexos-2,3-diulose (2,3-didehydrolactose).

A careful choice of the reaction conditions (solvent, enzyme, acylating agent) allowed an efficient regioselective acylation of N-acetylhexosamines. 6-O-Acyl derivatives of 2-acetamido-2-deoxy- d -glucopyranose (GlcNAc), 2-acetamido-2-deoxy- d -galactopyranose (GalNAc) and 2-acetamido-2-deoxy- d -mannopyranose (ManNAc) have been isolated from regioselective esterifications catalysed by the protease subtilisin in CH3CN–DMSO 8:2 in good yields.

An intensive bromination of 2,6-diaryl-4,4-diphenyl-4H-thiopyrans can lead through 3,5-dibromo derivatives to unexpected 2,8-diaryl-3-bromo- and 2,8-diaryl-3,5-dibromo-3a-phenyl-3aH-benzo[3,4]cyclopenta[1,2-b]thiophene as demonstrated on examples where the respective aryl groups are phenyl or 4-fluorophenyl. On the other hand, analogous spiro- [fluorene-9,4'-thiopyran]s do not exhibit the rearrangement evidently due to a rigid conformation of the fluorene moiety. The reaction mechanism for the rearrangement is proposed.

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 8α-(N3-histidyl)-FAD.

Pyranose oxidase (pyranose:O2 2‐oxidoreductase, EC 1.1.3.10) purified from mycelia of the basidiomycete fungi Trametes versicolor and Oudemansiella mucida catalyzed oxidation of d‐galactose successively at C‐2 and C‐3 to d‐threo‐hexos‐2,3‐diulose (2,3‐dehydro‐d‐galactose, 2,3‐diketo‐d‐galactose) in the yields up to 80%. The sites of oxidation were deduced from structures of the N,N‐diphenylhydrazone derivatives of the reaction products. Under the reaction conditions used, the diulose was susceptible to non‐enzymatic oxidative decarboxylation to d‐threo‐pentos‐2‐ulose (2‐dehydro‐d‐xylose, 2‐keto‐d‐xylose) in yields of 5–10%.

The quinone-dependent pyranose dehydrogenase (PDH) purified from mycelial extracts of the basidiomycete fungus Agaricus meleagris catalyzed oxidation of the non-reducing oligosaccharides sucrose, melezitose and erlose at C-3 of their terminal glucopyranosyl moiety while α,α-trehalose was double oxidized at both C-3 and C-3′. Analogously, using MS and in situ NMR spectroscopy, formation of C-3 carbonyl derivatives was also demonstrated with methyl-α- d -Glcp, methyl-β- d -Glcp and methyl-α- d -Galp. Yields of conversions performed at room temperature with 10 mM sugars and 1,4-benzoquinone as an electron acceptor in non-buffered systems were ≥90% within 3–20 h, depending on substrate.

The product ion mass spectra (collisionally induced dissociation mass spectra) of 12 different cyclosporins modified at every N-methylated amino acid residue with respect to cyclosporin A were compared and the effect of N-demethylation on ring opening mechanisms was evaluated. The four preferential protonation sites were identified in [MeBmt(1)]-cyclosporins. Three sites represented the N-methylated nitrogens of Sar(3), MeLeu(6) and MeLeu(9), while the remaining one represented the lactone group formed by the intramolecular N,O-acyl shift. Selective N-demethylation resulted either in the deletion of the entire fragment ion series or its substantial attenuation. The structures of three new natural cyclosporins in the study were supported by NMR data.

Mycelial cultures of 76 strains of lignocellulose-degrading basidiomycete fungi were screened for the activity of pyranose dehydrogenase, a novel sugar oxidoreductase recently detected in Agaricus bisporus. Of these fungi, 37 strains belonging to seven phylogenetically related genera of mostly litter-decomposing Agaricales were positive for the dehydrogenase, based on activity assays towards D-glucose with 1,4-benzoquinone or ferricenium ion as electron acceptors, and on TLC/HPLC analyses of the reaction products. Lack of activity with O2 as the oxidant, specificity for C-3 of D-glucose, and active extracellular secretion of the enzyme were used as criteria to differentiate pyranose dehydrogenase from pyranose 2-oxidase (EC 1.1.3.10), known to be produced by numerous wood-rotting fungi. Extracellular pyranose dehydrogenase from Macrolepiota rhacodes was heavily glycosylated. The enzyme was characterized as a 78-kDa flavoprotein under denaturing conditions and a 76-kDa native protein using gel filtration. This enzyme had a maximum extracellular activity of 4.1 U ml–1 in 39-day liquid cultures. It exhibited broad selectivity for sugar substrates and oxidized D-glucose (K m=1.82) exclusively at C-3 to 3-dehydro-D-glucose (D-ribo-hexos-3-ulose), in contrast to pyranose dehydrogenases from Agaricus species, which acted at both C-3 and C-2 of D-glucose. The N-terminal sequence, AVVYRHPDEL, showed significant similarity with that reported for A. bisporus.

Pyranose dehydrogenase purified to homogeneity from the mycelia of the basidiomycete fungus Agaricus bisporus catalyzed the oxidation of d -xylose at C-2 to d - threo -pentos-2-ulose (2-keto- d -xylose) and successively at C-3 to d - glycero -pentos-2,3-diulose (2,3-diketo- d -xylose) using 1,4-benzoquinone as an electron acceptor. The sites of oxidation were deduced from the spectroscopic analysis (MS, NMR) of the N , N -diphenylhydrazone derivatives of the reaction products.

The titanocene–bis(trimethylsilyl)acetylene (BTMSA) complexes Cp′2Ti[η2-C2(SiMe3)2] (Cp′C5H5−nMen; n=0–5) react with acetone azine Me2CNNCMe2 (AA) in two different ways depending on the number of Me substituents at the Cp′ ligands (n). For n=0–2, BTMSA is replaced by AA, which then undergoes an oxidative addition accompanied by a proton transfer, affording titana-2-isopropyl-4-methyl-2,3-diazacyclopent-3-ene complexes A0–A2. For n=3–5, replacement of the acetylene is followed by the splitting of AA to give either (C5Me5)2Ti(III)(NCMe2) (C5) or the Ti(IV) (Cp′A)2Ti complexes B3 and B4 for n=3 and 4, respectively. The intramolecularly bridging Cp′A ligand 1-(η5-2,3,5-trimethylcyclopentadienide)-2-(η1-amide)-2,2′-dimethylethane arises from a formal insertion of the CN bond of isopropylidene amide into a CH bond of one methyl group of the cyclopentadienyl ligands before or after splitting of AA. Crystal structures of C5 and (η5:η1-C5HMe3CH2CMe2NH)2Ti(IV) (B4) were determined.

Five 6'-deoxoergopeptines were prepared in 51-68% yield by selective reduction of parent alkaloids with lithium aluminium hydride in tetrahydrofuran at low temperature. New compounds were characterized by mass spectrometry and NMR spectroscopy. The conformation of the peptide part in starting compounds and reduced derivatives is discussed on the basis of crystal structure determination of 6'-deoxo-9,10-dihydroergotamine dihydrate butan-2-one solvate as a representative member of the series.

Enzymatic synthesis of lysergol β-D-glucuronide (2) using ovine microsomal uridine 5'-diphosphoglucuronyl transferase and uridine 5'-diphosphoglucuronic acid (Na3-salt) is demonstrated.

The pure diastereomers of 4-fluoroglutamine and 4-fluoroisoglutamine were prepared from the corresponding 4-fluoroglutamic acids. Glutamic decarboxylase treatment of the acids led to chiral 2-fluoroGABA.

Acid-catalyzed degradation of cyclosporin A was studied in various solvents and products of reaction were monitored by HPLC. Identification of amino acids and their chirality were determined after hydrolysis and derivatization by GC-MS. Cyclosporin H was isolated as the principal product and its structure was determined by X-ray diffraction: Cyclosporin H- diethyl ether-water (1 : 0.5 : 1) crystallizes in the monoclinic space groupI2witha= 12.338(2) Å,b= 18.963(2) Å,c= 34.074(3) Å, β = 96.47(2)°,Z= 4, andV= 7 921.4(17) Å3.

p-Nitrophenyl β-chitobioside (2) was prepared from p-nitrophenyl 2-acetamido-2--deoxy-β-D-glucopyranoside (1) using β-N-acetylhexosaminidase from Aspergillus oryzae. As minor by-product a (1→6)-linked regioisomer (3a) was formed. Addition of (NH4)2SO4 and organic co-solvents (acetonitrile, dioxane) substantially increased the yield.

Pyranose 2-oxidase, a homotetrameric FAD-flavoprotein from the fungus Trametes multicolor, catalyzes the oxidation of 1→6 disaccharides gentiobiose (β-D-Glcp-(1→6)-D-Glc), melibiose (α-D-Galp-(1→6)-D-Glc), and isomaltose (α-D-Glcp-(1→6)-D-Glc) at C-2 of their reducing moiety. The products were identified as their hydrazone derivatives.

The reactions of glycosidases with pyridoxine were used for testing their ability to make new glycosidic bonds. Of 35 glycosidases examined, some exhibited regiospecificity towards one primary alcoholic group; glycosylation of phenolic hydroxyl group was not observed. A series of new glycosides of pyridoxine, 2-acetamido-2-deoxy-β-D-glucopyranosides, α-D-manno- pyranosides, and one α-D-galactopyranoside were prepared and completely characterized by MS and NMR.

The structures of O-acetyl-(4R)-4-(E-2-butyl)-4,N-dimethyl-L-threonyl-cyclosporin A (1) and O-acetyl-(4R)-4-[E-2-(4-bromobutyl)]-4,N-dimethyl-L-threonyl-cyclosporin A (2) were determined by X-ray diffraction methods and compared with the structure of related cyclosporins. In contrast to expectation, neither the acetylation nor the subsequent bromination of 1 affects the conformation and packing of cyclosporins in the solid state. Both compounds are isomorphous and crystallize in the orthorhombic space group P212121 with a = 12.936(2) Å, b = 15.590(2) Å, c = 36.280(3) Å, and a = 12.916(3) Å, b = 15.675(4) Å, c = 36.715(7) Å, for 1 and 2, respectively.

(Z)-6-(4-Hydroxy-3-methylbut-2-en-1-ylamino)purine, (Z)-zeatin, and the isomeric (Z)-isozeatin, both free from the E isomers, were prepared using the cycloaddition of the in situ generated tert-butyl nitrosoformate on isoprene. A mixture of tert-butyl 5(and 4)-methyl-3,6-dihydro-2H-1,2-oxazine-2-carboxylates thus formed was deprotected and reductively cleaved to (Z)-4-amino-2(and 3)-methylbut-2-en-1-ols, which were chromatographically separated and finally alkylated with 6-chloropurine to give the title compounds.

4-O-(2-Acetamido-2-deoxy-β-D-glucopyranosyl)-2-acetamido-2-deoxy-D-mannopyranose (2) was prepared from diacetylchitobiose (1) by Lobry de Bruyn-Alberda van Ekenstein rearrangement under catalysis of Ca(OH)2. The disaccharide 2 shows about ten times higher affinity than 1 towards NKR-P1 protein acting as a crucial activating receptor of rat natural killer cells (leukocytes). Chitotriose (3) can be epimerized analogously to give GlcNAcβ1-4GlcNAcβ1-4ManNAc (4) and its binding properties to NKR-P1 are also about ten times higher than those of 3.

The quinone-dependent sugar oxidoreductase, pyranose dehydrogenase purified from the basidiomycete Agaricus bisporus catalyzed C-2 and C-3 oxidation of d -Glc to d - arabino -hexos-2-ulose (2-keto- d -Glc) and, preferentially, d - ribo -hexos-3-ulose (3-keto- d -Glc). The two aldoketoses accumulated only transiently in the reaction mixture being continually converted to the same end-product, d - erythro -hexos-2,3-diulose (2,3-diketo- d -Glc). d -Gal was oxidized by pyranose dehydrogenase and 1,4-benzoquinone exclusively at C-2 to produce d - lyxo -hexos-2-ulose (2-keto- d -Gal). Structures of the enzyme reaction products were deduced from their N , N -diphenylhydrazone derivatives.

A new natural cyclosporin, [Ala2, Val11]cyclosporin, has been isolated from the submerged culture of Mycelium sterilae MS 2929. Its structure was deduced from the NMR and mass spectral data.

[ 3 H]-Cyclosporin A labeled at the first amino acid ([ω- 3 H-MeBmt 1 ]CS) has been prepared by tritium gas hydrogenolysis of [O-acetyl-ω-bromo-MeBmt 1 ]CS followed by deprotection with methanolic sodium methoxide. The requisite bromo intermediate was prepared by bromination of [O-acetyl-MeBmt 1 ]CS with N-bromosuccinimide. After synthesis and purification, 2.45 mg of [ω 3 H-MeBmt 1 ]CS with a total activity of 327 MBq were produced with a radiochemical purity >97 %, and specific activity of 160 GBq/mmol (4.3 Ci/mmol).

Phenolic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) at concentration 0.55 mM and 2.25 mM, respectively, added to cultures of Streptomyces cinnamonensis C-100-5 caused up to 380% higher production of polyether antibiotic monensin. S. cinnamonensis exhibits high tolerance to BHT, but it is more sensitive towards BHA. 5.5 mM BHA practically stopped growth, while 45 mM BHT still stimulated antibiotic production. Another antioxidants probucol and tocoferol acetate did not exert such positive effects. BHA was predominantly metabolised to 5-hydroxy-BHA. When BHA and BHT were applied simultaneously, the main transformation metabolite was 3,3′,5,5′-tetra-tert-butyl-stilbene-4,4′-quinone accompanied by 1,2-bis(3,5-di-tert-butyl-4-hydroxyphenyl)ethane.

Cell cultures of Papaver somniferum var. setigerum were shown to carry out the biotransformation of silybin into silybin -7-O-β- d -glucopyranoside .

Pergolide sulfoxide (1), a metabolite of pergolide (2), has been prepared by oxidation of 2 with hydrogen peroxide. Its structure was determined by X-ray diffraction and compared with the structures of related ergot alkaloids. Two molecules of pergolide (S)- and (R)-sulfoxide crystallize with one molecule of water in the orthorhombic space group P212121 with a = 12.216(3) Å, b = 13.733(2) Å, c = 22.186(6) Å, Z = 4, V = 3 722(2) Å3.

The complexes [(η5-C5Me4Ph)TiCl(μ-Cl)]2 (1) and [(η5-C5Bn5)TiCl(μ-Cl)]2 (2) (Bn = benzyl) were obtained by the reaction of TiCl3 and the corresponding alkali metal cyclopentadienides in a THF-toluene mixture. The X-ray diffraction single-crystal analysis revealed that both compounds form centrosymmetric dimers bridged via a pair of chloride ligands. Compound 1 crystallizes in the monoclinic space group P21/n (No. 14; a = 11.387(3) Å, b = 16.496(2) Å, c = 8.383(2) Å, β = 110.786(15)°, V = 1 472.2(6) Å3, Z = 2), and compound 2 in the monoclinic space group P21/c (No. 14; a = 18.967(5) Å, b = 10.7190(10) Å, c = 17.996(5) Å, β = 116.747(15)°, V = 3 267.2(13) Å3, Z = 2). Common features of the structures differ only negligibly. The Ti-Ti distance (in 1 3.3345(11) Å, in 2 3.374(2) Å) is considerably shorter than in paramagnetic dimeric titanocene monochlorides (3.91-3.95 Å). The compounds are diamagnetic, probably due to spin-paired d1 electrons.

Galactosyltransferase from bovine milk was found to be able to utilise UDP-Glc to transfer Glc onto GlcNAc and chitooligomers[-beta-GlcNAc-(1-->4)-]n, n = 2-4. beta-Glucosylated products were used in binding studies with NKR-P1A protein cloned from rat natural killer cells.

Agroclavine (1) was oxidized, by haloperoxidase from Streptomyces aureofaciens, to 2,3-dihydro-6,8-dimethyl-3β-acetoxy-8-ergolen-2-one (2) in the presence of sodium acetate and bromine ions. Acetate was incorporated into the above product during oxidation. In a propionate buffer, incorporation of propionate into the product was observed, yielding 2,3-dihydro-6,8-dimethyl-3β-propionoxy-8-ergolen-2-one (4). The biotransformation proceeded faster in the propionate buffer, affording also the higher oxidation product (4aS),(10bS)-7-amino-3,4,4a,5,6,10b-hexahydro-2,4-dimethyl-6-oxobenzo[f]quinoline (3).

The crystal structures of two ergot alkaloids ergocristam (I) and ergocristine acetone solvate (II), have been determined by X-ray diffraction and compared with their conformation in solution obtained from NMR data. The presence of D-proline in I essentially influences the conformation of the alkaloid tripeptide moiety which seems likely to explain why I does not undergo further enzymatical hydroxylation and is found as a terminal step of ergot alkaloid biosynthesis.

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.

N-Deoxyribosides of agroclavine (1), lysergol (2), and 9,10-dihydrolysergol (3) were prepared by SnCl4 -catalyzed N-glycosylation of their TMS derivatives with 1-chloro-3,5-di-O-toluoyl-2-deoxy-D-ribofuranose. None of the new compounds exhibited activity against HIV.