Bufadienolides from bulbs of Urginea lydenburgensis (Hyacinthaceae: Urgineoideae)

Bulbs of the ethnomedicinal hyacinthac Urginea lydenburgensis have yielded two bufadienolides, 16beta-acetoxy-3beta,14beta-dihydroxy-19-formyl-bufa-4,20,22-trienolide (scillicyanosidin) and 4beta,8beta,11alpha,14beta-tetrahydroxybufa-5,20,22-trienolide-12-one, 2alpha,3beta-O-4,6-dideoxy-L-glucose (lydenburgenin).     

1alpha,3beta,5beta-trihydroxy-24-methylenecholestan-6-one: a novel steroid from a soft coral Sinularia gibberosa

A novel steroid, 1alpha,3beta,5beta-trihydroxy-24-methylenecholestan-6-one (gibberoketosterol) (1), along with four known steroids, was isolated from the lipophilic extracts of a Taiwanese soft coral Sinularia gibberosa. The structure of the new metabolite was determined on the basis of extensive spectral analyses and chemical reaction. The relative stereochemistry of gibberoketosterol was established by the NOESY experiments and analysis of the pyridine-induced deshielding effect of the axial hydroxy groups. Gibberoketosterol is the first example of 1alpha,3beta,5beta-trihydroxy-6-oxosteroids isolated from natural sources and was found to exhibit a moderate cytotoxicity against the growth of Hepa59T/VGH cancer cells.     

Identification and characterization of key substructures involved in the early folding events of a (beta/alpha)8-barrel protein as studied by experimental and computational methods

A number of studies have examined the structural properties of late folding intermediates of (beta/alpha)8-barrel proteins involved in tryptophan biosynthesis, whereas there is little information available about the early folding events of these proteins. To identify the contiguous polypeptide segments important to the folding of the (beta/alpha)8-barrel protein Escherichia coli N-(5'-phosphoribosyl)anthranilate isomerase, we structurally characterized fragments and circularly permuted forms of the protein. We also simulated thermal unfolding of the protein using molecular dynamics. Our fragmentation experiments demonstrate that the isolated (beta/alpha)(1-4)beta5 fragment is almost as stable as the full-length protein. The far and near-UV CD spectra of this fragment are indicative of native-like secondary and tertiary structures. Structural analysis of the circularly permutated proteins shows that if the protein is cleaved within the two N-terminal betaalpha modules, the amount of secondary structure is unaffected, whereas, when cleaved within the central (beta/alpha)(3-4)beta5 segment, the protein simply cannot fold. An ensemble of the denatured structures produced by thermal unfolding simulations contains a persistent local structure comprised of beta3, beta4 and beta5. The presence of this three-stranded beta-barrel suggests that it may be an important early-stage folding intermediate. Interactions found in (beta/alpha)(3-4)beta5 may be essential for the early events of ePRAI folding if they provide a nucleation site that directs folding.     

Structure determinants and substrate recognition of serine carboxypeptidase-like acyltransferases from plant secondary metabolism

Structures of the serine carboxypeptidase-like enzymes 1-O-sinapoyl-beta-glucose:L-malate sinapoyltransferase (SMT) and 1-O-sinapoyl-beta-glucose:choline sinapoyltransferase (SCT) were modeled to gain insight into determinants of specificity and substrate recognition. The structures reveal the alpha/beta-hydrolase fold as scaffold for the catalytic triad Ser-His-Asp. The recombinant mutants of SMT Ser173Ala and His411Ala were inactive, whereas Asp358Ala displayed residual activity of 20%. 1-O-sinapoyl-beta-glucose recognition is mediated by a network of hydrogen bonds. The glucose moiety is recognized by a hydrogen bond network including Trp71, Asn73, Glu87 and Asp172. The conserved Asp172 at the sequence position preceding the catalytic serine meets sterical requirements for the glucose moiety. The mutant Asn73Ala with a residual activity of 13% underscores the importance of the intact hydrogen bond network. Arg322 is of key importance by hydrogen bonding of 1-O-sinapoyl-beta-glucose and L-malate. By conformational change, Arg322 transfers L-malate to a position favoring its activation by His411. Accordingly, the mutant Arg322Glu showed 1% residual activity. Glu215 and Arg219 establish hydrogen bonds with the sinapoyl moiety. The backbone amide hydrogens of Gly75 and Tyr174 were shown to form the oxyanion hole, stabilizing the transition state. SCT reveals also the catalytic triad and a hydrogen bond network for 1-O-sinapoyl-beta-glucose recognition, but Glu274, Glu447, Thr445 and Cys281 are crucial for positioning of choline.     

Malonylated flavonol glycosides from the petals of Clitoria ternatea

Three flavonol glycosides, kaempferol 3-O-(2"-O-alpha-rhamnosyl-6"-O-malonyl)-beta-glucoside, quercetin 3-O-(2"-O-alpha-rhamnosyl-6"-O-malonyl)-beta-glucoside, and myricetin 3-O-(2",6"-di-O-alpha-rhamnosyl)-beta-glucoside were isolated from the petals of Clitoria ternatea cv. Double Blue, together with eleven known flavonol glycosides. Their structures were identified using UV, MS, and NMR spectroscopy. They were characterized as kaempferol and quercetin 3-(2(G)- rhamnosylrutinoside)s, kaempferol, quercetin, and myricetin 3-neohesperidosides, 3-rutinosides, and 3-glucosides in the same tissue. In addition, the presence of myricetin 3-O-(2"-O-alpha-rhamnosyl-6"-O-malonyl)-beta-glucoside was inferred from LC/MS/MS data for crude petal extracts. The flavonol compounds identified in the petals of C. ternatea differed from those reported in previous studies.     

The in vivo metabolism of 7 beta, 17-dimethyltestosterone-6,7-3H.

7 beta, 17-Dimethyltestosterone (17 beta-hydroxy-7 beta, 17-dimethyl-4-androsten-3-one) (I) was given to three subjects in oral doses of 400 mg per day for ten days. The initial dose contained the steroid tritiated in the 6 and 7 positions. Plasma levels and urinary excretion patterns were followed in all three subjects. Isolations were done on the urine, plasma, and stools of one patient. From the urine 7 beta, 17-dimethyl- 5 alpha-androstane-3 beta,17 beta-diol (VI) was isolated from the nonhydrolyzed fractions. Unchanged (I), 7 beta,17-dimethyl-5 beta-androstane-3 alpha,17 beta-diol (III) and 7 beta, 17-dimethyl-5 beta-androstane-3 beta,17 beta-diol (IV) were isolated from the nonhydrolyzed and enzyme-hydrolyzed fractions. 7 beta,17-dimethyl-5 alpha-androstane-3 alpha,17 beta-diol (V) was isolated from the enzymatic fractions. From the stools were isolated unchanged (I), (III), (IV), (V), and (VI). Unchanged (I) and its 5 alpha-dihydro derivative (17 beta-hydroxy-7 beta,17-dimethyl-5 alpha-androstan-3-one) (II) were identified in the plasma. The total recovery of radioactivity in the one patient on whom the isolations were done was 57%; 40% from the urine and 17% from the stools.     

Sesquiterpenoids of Torilis japonica fruit

From the methanolic extract of Torilis japonica D. C. fruit (Umbelliferae), two eudesmane-type sesquiterpenoids were isolated together with five previously described sesquiterpenoids. From the results of spectral analyses, they were characterized as 4(15)-eudesmene-1beta,5alpha-diol and 4alpha,15-epoxyeudesmane-1beta,6alpha-diol, respectively. The absolute stereostructures of these sesquiterpenoids were elucidated by the modified Mosher's method.     

Ericifolin: an eugenol 5-O-galloylglucoside and other phenolics from Melaleuca ericifolia

Ericifolin, an eugenol 5-O-beta-(6'-O-galloylglucopyranoside) possessing the naturally unknown phenolic moiety, 5-hydroxyeugenol, together with the two new phenolics, 2-O-p-hydroxybenzoyl-6-O-galloyl-(alpha/beta)-4C1-glucopyranose and 3-methoxyellagic acid 4-O-rhamnopyranoside have been isolated from the antibacterial leaves extract of Melaleuca ericifolia. In addition, 19 known phenolics were also separated and characterized. All structures were elucidated on the basis of analysis of 1H, 13C NMR, HMQC, HMBC and FTMS spectral data.     

Biosynthesis of gallotannins. Enzymatic conversion of 1,6-digalloylglucose to 1,2,6-trigalloylglucose

Cell-free extracts from Rhus typhina L. (staghorn sumach) leaves were found to catalyze the transfer of the galloyl moiety of -glucogallin (1-O-galloyl--D-glucose) to 1,6-di-O-galloyl--D-glucose, resulting in the specific formation of 1,2,6-tri-O-galloyl--D-glucose, an intermediate of gallotannin biosynthesis. The reaction product was unequivocally identified by co-chromatography with authentic references using reversed-phase high-performance liquid chromatography and by ¹H-nuclear-magnetic-resonance spectroscopy.Abbreviations HPLC high-performance liquid chromatography - NMR nuclear magnetic resonance     

Triterpenoidal glycosides from Justicia betonica

dFrom the aerial portion of Justicia betonica L., four triterpenoidal glycosides (justiciosides A-D) were isolated. Their structures were established through chemical and NMR spectroscopic analyses as olean-12-ene-1beta,3beta,11alpha,28-tetraol 28-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranoside, olean-12-ene-1beta,3beta,11alpha,28-tetraol 28-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranoside, 11alpha-methoxy-olean-12-ene-1beta,3beta,28-triol 28-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranoside, 11alpha-methoxy-olean-12-ene-1beta,3beta,28-triol 28-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranoside, respectively.     

Unique phenolic carboxylic acids from Sanguisorba minor

The unique phenolic carboxylic acids, 4,8-dimethoxy-7-hydroxy-2-oxo-2H-1-benzopyran-5,6-dicarboxylic acid and 2-(4-carboxy-3-methoxystyryl)-2-methoxysuccinic acid were isolated and identified from the whole Sanguisorba minor plant. The known phenolics, gallic acid; ellagic acid; quercetin-3-O-(6"-galloylglucose); beta-glucogallin; 2,3-hexahydroxydiphenoyl-(alpha/beta)-glucose; 1-galloyl-2,3-hexahydroxydiphenoyl-alpha-glucose together with its beta-isomer were also characterized. Structures were established by conventional methods of analysis and confirmed by NMR and ESI-MS spectral analysis.     

Biotransformations of 20-hydroxyecdysone and analogues by Curvularia lunata NRRL 2178

20-Hydroxyecdysone, the arthropod moulting hormone, was biotransformed by the fungus Curvularia lunata NRRL 2178 to the rare ecdysteroid, 2-dehydro-3-epi-20-hydroxyecdysone, and the novel 3alpha,9alpha-cyclo ecdysteroid analogue, (20R,22R)-3beta,14alpha,20,22,25-pentahydroxy-3alpha,9alpha-cyclo-5beta-cholest-7-en-2,6-dione in 14 and 44% yields, respectively. Ponasterone A and pterosterone were similarly biotransformed to the corresponding 2-dehydro-3-epi- and 3alpha,9alpha-cyclo-analogues.     

Semisynthetic lysogalactolipids of plant origin

Starting from the natural mono- and digalactosyl diglycerides, 1′-O-acyl-3′-O-β-d-galactopyranosyl-sn-glycerol and 1′-O-acyl-3′-O-(6-O-α-d-galactopyranosyl-β-d-galactopyranosyl)-sn-glycerol were synthesized. In an attempt to prepare the 2′-O-acyl-isomer, only a mixture of the 1′-and 2′-O-acyl-isomers was obtained.     

New cyclomaltoheptaose (beta-cyclodextrin) derivative 2-O-(2-hydroxybutyl)cyclomaltoheptaose: preparation and its application for the separation of enantiomers of drugs by capillary electrophoresis

A new soluble cyclomaltoheptaose (cyclodextrin) derivative, 2-O-(2-hydroxybutyl)cyclomaltoheptaose [2-O-(2-hydroxybutyl)-beta-cyclodextrin, 2-HB-beta-CD], was prepared and studied as an efficient chiral selector in the separation of racemic mixtures of drugs by capillary electrophoresis (CE). Results showed that 2-HB-beta-CD could provide higher separating capability than that of beta-CD and the similarly substituted 2-HP-beta-CD.     

Microbial conversion of pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione acetates by Nocardioides simplex VKM Ac-2033D

The conversion of pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione 21-acetate (I) and 17,21-diacetate (VI) by Nocardioides simplex VKM Ac-2033D was studied. The major metabolites formed from I were identified as pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II) and pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione (IV). Pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione (III) and pregna-1,4,9(11)-triene-17alpha,20beta,21-triol-3-one (V) were formed in minorities. Biotransformation products formed from VI were pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17,21-diacetate (VII), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione (IV), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17-acetate (VIII), pregna-1,4,9(11)-triene-17alpha,20beta,21-triol-3-one (V). The conversion pathways were proposed including 1(2)-dehydrogenation, deacetylation, 20beta-reduction and non-enzymatic migration of acyl group from position 17 to 21. The conditions providing predominant accumulation of pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II) from I and pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17-acetate (VIII) from VI in a short-term biotransformation were determined.     

A new ecdysteroid with unique 9beta-OH and four other ecdysteroids from Silene italica ssp. nemoralis

A new natural ecdysteroid, 9beta,20-dihydroxyecdysone (1) and four related compounds 5alpha-20-hydroxyecdysone (2), 5alpha-2-deoxy-integristerone A (3), integristerone A (4) and 22-deoxy-integristerone A (5) were isolated from the herb of Silene italica ssp. nemoralis. Compound 1 is the C-9 epimer of the known 9alpha,20-dihydroxyecdysone (6) and represents a peculiar steroid skeleton. The structures of the compounds were elucidated by 1D and 2D NMR, IR and MS spectroscopy.     

Taxoid metabolism: Taxoid 14beta-hydroxylase is a cytochrome P450-dependent monooxygenase

The production of the anticancer drug Taxol in Taxus (yew) cell cultures is often accompanied by the formation of side-route polyoxygenated taxoid metabolites bearing a 14beta-hydroxyl group. The recent acquisition of several new semisynthetic taxoid intermediates enabled the screening of a family of Taxus cytochrome P450 cDNA clones for the 14beta-hydroxylase and additional taxoid oxygenases. The candidate cytochrome P450 clones were functionally expressed in yeast and tested by in vivo feeding of radiolabeled 5alpha-acetoxy-10beta-hydroxy taxadiene and 5alpha,13alpha-dihydroxy taxadiene. One clone efficiently and specifically transformed the 5alpha-acetoxy-10beta-ol, but not the 5alpha,13alpha-diol, to a more polar product with the chromatographic properties of a taxoid triol monoacetate, and the identity of this product was confirmed by spectroscopic means as 5alpha-acetoxy-10beta,14beta-dihydroxy taxadiene. Microsome preparation from the transformed yeast allowed characterization of this new hydroxylase, which was shown to resemble other cytochrome P450 taxoid hydroxylases with pH optimum at 7.5 and a K(m) value for the taxoid substrate of about 50 microM. Because Taxol is unsubstituted at C14, the 14beta-hydroxylase cannot reside on the pathway to the target drug but rather appears to be responsible for diversion of the pathway to 14-hydroxy taxoids that are prominent metabolites of Taxus cell cultures. Manipulation of this hydroxylase gene could permit redirection of the pathway to increase flux toward Taxol and could allow the preparation of 13alpha,14beta-hydroxy taxoids as new therapeutic agents.     

ent-Kauranoid derivatives from Sideritis moorei

Seven new ent-kauranoid derivatives ent-7alpha,18-dihydroxykaur-16-en-3-one, ent-18-acetoxy-3beta,7alpha-dihydroxykaur-15-en-17-al, ent-3beta-acetoxy-7alpha,18-dihydroxykaur-15-en-17-al, ent-18-acetoxy-3beta,7alpha,17-trihydroxykaur-15-ene, ent-3beta-acetoxy-7alpha,17,18-trihydroxykaur-15-ene, ent-18-acetoxy-3beta,7alpha,17-trihydroxy-15beta,16beta-epoxykaurane and ent-3beta-acetoxy-7alpha,17,18-trihydroxy-15beta,16beta-epoxykaurane have been isolated from Sideritis moorei. The structures of these compounds have been established by spectroscopic means and chemical correlations.