Zur biogenese des aza-oxa-spiran-systems der steroidalkaloide vom spirosolan-typ in Solanaceen

5α,6-³H₂-Solacongestidine and 5α,6-³ H₂-(22S)-dihydrosolacongestidine administered to Solanum dulcamara as well as 16-³H₂-(22S: 25R)-22,26-epimino- cholest-5-en-3β-ol (25-isodihydroverazine) and 7α-³H-(22S: 25R)-22,26-epimino-cholest-5-en-3β,16β-diol administered to Solanum laciniatum were converted to coladulcidine and solasodine, respectively. These results are discussed in relation to spirosolane alkaloid biosynthesis.     

Polyhydroxylated steroids from an endophytic fungus, Chaetomium globosum ZY-22 isolated from Ginkgo biloba

A novel polyhydroxylated C₂₉-sterol, 25ξ-methyl-22-homo-5α-cholest-7,22-diene-3β,6β,9α-triol, designated globosterol (1), together with one known tetrahydroxylated ergosterol (22E, 24R)-ergosta-7,22-diene-3β,5α,6β,9α-tetraol (2) has been isolated from the cultures of an endophytic fungus, Chaetomium globosum ZY-22 originated from the plant Ginkgo biloba. The structures and relative configurations of 1 and 2 were established on the basis of extensive spectroscopic analyses including 1D and 2D NMR (¹H-¹H COSY, HSQC, HMBC, and NOESY) experiments and comparison with the literature. Globosterol (1) possesses an unprecedented 25-methyl Δ²²-C₁₀-side chain and Δ⁷-3β,6β,9α-hydroxy-steroid nucleus, which represents the first example for C₂₉-steroids of the group.     

Polyhydroxylated steroid compounds from the Far Eastern starfish <Emphasis Type="Italic">Distolasterias nipon</Emphasis>

Two new steroid glycosides: distolasteroside D₆, (24S)-24-O-(β-D-xylopyranosyl)-5α-cholestane-3β,6α,8,15β,16β,24-hexaol, and distolasteroside D₇, (22E,24R)-24-O-(β-D-xylopyranosyl)-5α-cholest-22-ene-3β,6α,8,15β,24-pentaol were isolated along with the previously known distolasterosides D₁, D₂, and D₃, echinasteroside C, and (25S)-5α-cholestane-3β4β,6α,7α,8,15α,16β,26-octaol from the Far Eastern starfish Distolasterias nipon. The structures of new compounds were elucidated by NMR spectroscopy and MALDI TOF mass spectrometry. Like neurotrophins, distolasterosides D₁, D₂, and D₃ were shown to induce neuroblast differentiation in a mouse neuroblastoma C1300 cell culture.     

Determination of estriol, estriol sulfate, progesterone and neutral steroid mono- and disulfates in umbilical cord blood plasma

Fractions of unconjugated steroids, and steroid mono- and disulfates were isolated from cord plasma, and the concentrations of estriol, estriol sulfate, progesterone, 13 neutral steroid monosulfates (MoS) and 10 neutral steroid disulfates (DiS) were determined by gas-liquid chromatography. The mean concentrations in 30 cord plasma samples at term after normal pregnancy and delivery were as follows (μg/100 ml of free steroid ±standard deviation): estriol 16±5; estriol monosulfate 135±43; progesterone 59±19; dehydroepiandrosterone MoS 76±23; 5-androstene-3_β,17_α-diol DiS 279±77; 5-androstene-3_β,17_β-diol DiS 211±109; 16_α-hydroxydehydroepiandrosterone MoS 305±97; 16_β-hydroxy-dehydroepiandrosterone DiS 8±25; 33,17_β-dihydroxy-5-androsten-16-one MoS 37±16, DiS 29±15 5-androstene-3_β,16_α,17_β-triol MoS 25±9; 5-androstene-3_β,16_β,17_α-triol DiS 31±14; pregnenolone MoS 4±33; 5-pregnene-3_β,20_α-diol MoS 41±14, DiS 68±43; 16_α-hydroxypregnenolone MoS 101±42; 17-hydroxypregnenolone MoS 56±30; 21-hydroxypregnenolone DiS 26±15; 5-pregnene-3_β,20_α,21-triol MoS 37±18; 5_α-pregnane-3_α,20_α-diol MoS 21±10, DiS 54±21; 5_α-pregnane-3_β,20_α-diol MoS 18±9, DiS 7±39; 5_β-pregnane-3_α,20_α-diol MoS 17±7; 5_α-pregnane-3_α,20_α,21-triol MoS 110±56, DiS 22±19.The total amount of steroid monosulfates in the cord plasma pool was 1 mg/100 ml and that of steroid disulfates 0.5 mg/100 ml. 3_β-Hydroxy-Δ⁵-steroids predominated. Considerable amounts of saturated c₂₁ steroids were also detected. No statistically significant differences were found in the concentrations of any of the steroids studied, when a group of male and female fetuses were compared.     

Biosynthesis of polar steroids from the Far Eastern starfish Patiria (=Asterina) pectinifera. Cholesterol and cholesterol sulfate are converted into polyhydroxylated sterols and monoglycoside asterosaponin P1 in feeding experiments

For the first time, it is experimentally established that the dietary cholesterol and cholesterol sulfate are biosynthetic precursors of polyhydroxysteroids and related low molecular weight glycosides in starfishes. These deuterium labeled precursors were converted into partly deuterated 5α-cholestane-3β,6α,7α,8,15α,16β,26-heptaol, 5α-cholestane-3β,4β,6α,7α,8,15β,16β,26-octaol, and steroid monoside asterosaponin P₁ in result of feeding experiments on the Far Eastern starfish Patiria (=Asterina) pectinifera. The incorporations of deuterium were established by MS and NMR spectroscopy. Scheme of the first stages of biosynthesis of polar steroids in these animals was suggested on the basis of inclusion of three from six deuterium atoms and determination of their positions in biosynthetic products, when [2,2,3,4,4,6-²H₆]cholesterol 3-sulfate was used as precursor. It was also shown that labeled cholesterol is transformed into Δ⁷-cholesterol (lathosterol) in digestive organs and gonads of the starfish.     

Side chain functionalization of cholesterol in the biosynthesis of solasodine in Solanum laciniatum

(25R)-26-Amino-cholesterol-[7α-³H], (25R)-26-amino-5-cholestene-3β,16β-diol-[7α-³H] and (25R)-26-acetylamino-5-cholestene-3β,16β-diol-[7α-³H] administered to Solanum laciniatum were converted into solasodine. The results indicate that in the biosynthesis of solasodine the introduction of nitrogen occurs immediately after the hydroxylation at C-26 and before a further oxidation of the side chain of cholesterol. The next step after the amination at C-26 is not hydroxylation at the 16β-position but probably the functionalization of C-22.     

Triterpenoid saponins from Aesculus sylvatica W. Bartram

16 triterpenoid saponins including two new compounds were isolated from the seeds of A esculus sylvatica W. Bartram. The two new saponins were assigned as 3-O-[β-D-glucopyranosyl-(1 → 2)]-α-L-arabinofuranosyl-(1 → 3)-β-D-glucuronopyranosyl-21,22-O-ditigloyl-3β,16α,21β,22α,24,28 hexahydroxyolean-12-ene (aesculioside S1, 1) and 3-O-[β-D-glucopyranosyl-(1 → 2)]-α-L-arabinofuranosyl-(1 → 3)-β-D-glucuronopyranosyl-21-O-tigloyl-22-O-angeloyl 3β,16α,21β,22α,24,28-hexahydroxyolean-12-ene (aesculioside S2, 2). Aesculioside S1 and S2 displayed moderate cytotoxicity against human non-small cell lung cancer cells (A549) and prostate cancer cells (PC3) (GI₅₀ ranged from 8.7 to 18.2 μM). The structural analysis of the saponins isolated from Aesculus supports the taxonomic placement of A. sylvatica under the section Pavia of Aesculus genus.     

Synthese der “repeating unit” der O-spezifischen kette des lipopolysaccharides aus Aeromonas salmonicida

8-Methoxycarbonyloctyl 2-azido-4,6-O-benzylidene-2-deoxy-β-d-mannopyranoside reacted with 2,3,4-tri-O-acetyl-α-l-rhamnopyranosyl bromide to give a disaccharide from the which the glycosyl-acceptor 8-methoxycarbonyloctyl 2-azido-4,6-O-benzylidene-2-deoxy-3-O-(2,4,-di-O-acetyl-α-l-rhamnopyranosyl)-β-d-manno pyranoside (19) was obtained. This glycosyl-acceptor with 2,3,4,6-tetra-O-benzyl-α-d-glucopyranosyl chloride to give trisaccharide derivative 22 and with 2,3,6-tri-O-(α-²H₂)benzyl-4-O-(2,3,4,6-tetra-O-(α-²H₂)benzyl-α-d-glucopyranosyl)-α-d-glucopyranosyl chloride to give tetrasaccharide derivative 29. Deblocking of 22 yielded 8-methoxycarbonyloctyl O-(α-d-glucopyranosyl)-(1→3)-O-α-l-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-d-mannopyranoside and deblocking of 29 8-methoxycarbonyloctyle O-α-d-glucopyranosyl-(1→4)-O-α-d-glucopyranosyl-(1→3)-O-α-l-rhamnopyranosyl- (1→3)-2-acetamido-2-deoxy-β-d-mannopyranoside. Both oligosaccharides represent the “repeating unit” of the O-specific chain of the lipopolysaccharide from Aeromonas salmonicida.     

Method for the complete separation of radioactively labeled human γ-globin chains from pre-βA chains on CM-cellulose chromatography: Use of cold carrier globin chains

Carboxymethyl (CM)-cellulose chromatography of globins is the technique used most frequently in analysis of hemoglobin synthesis. However, if this method is to be reliable in cases where only small amounts of fetal hemoglobin (α₂γ₂) compared to adult hemoglobin (α₂β₂^A) are synthesized, it is important to obtain a clean separation of γ chains from pre-β^A chains. In the past, it has been found that small amounts of pre-β^A chains tend to elute with the γ chains. Radioactively labeled γ chains can be completely and reproducibly separated from small amounts of labeled pre-β^A chains by the addition of unlabeled β^J chains (Hb J Baltimore = β16 Gly → Asp) which elute at the same position as the pre β^A chains, thus increasing the quantity of this peak and allowing a clean separation from the γ chains.     

Alkaloids from Melodinus yunnanensis

Ten monoterpenoid indole alkaloids, namely meloyine, 19S-methoxytubotaiwine N₄-oxide, 16,19-epoxy-Δ¹⁴-vincanol, 14β-hydroxymeloyunine, meloyunine, Δ¹⁴-vincamenine N₄-oxide, 16β,21β-epoxy-vincadine, 14β,15β-20S-quebrachamine, 3-oxo-voaphylline, 2α,7α-dihydroxy-dihydrovoaphylline, and 32 known alkaloids were isolated from leaves and twigs of Melodinus yunnanensis. Their structures were elucidated based on 1- and 2-D NMR, FTIR, UV, and MS spectroscopic data. Meloyine I showed weak cytotoxic activity against four human cancer cell lines: MCF-7 breast cancer, SMMC-7721 hepatocellular carcinoma, HL-60 myeloid leukemia, and A-549 lung cancer.     

Pendulaosides A and B. Two acylated triterpenoid saponins from Harpullia pendula seed extract

Two new acylated triterpenoid saponins named pendulaosides A and B as well as the known phenolic compounds methyl gallate, gallic acid, 1,2,3,6-tera-O-galloyl-β-d-glucose and 1,2,3,4,6-penta-O-galloyl-β-d-glucose, were isolated from the seeds of Harpullia pendula. The structures of pendulaosides A and B were determined using extensive 1D and 2D NMR analysis and mass spectrometry as well as acid hydrolysis, as 3-O-β-d-glucopyranosyl-(1→2)-[α-L-arabinofuranosyl-(1→3)]-β-d-glucuronopyranosyl-22-O-angeloyl-3β,16α,22α,24β,28-pentahydroxylolean-12-ene and 3-O-β-d-glucopyranosyl-(1→2)-[α-L-arabinofuranosyl-(1→3)]-β-d-glucuronopyranosyl-16-O-(2-methylbutyroyl)-3β,16α,22α,24β,28-pentahydroxylolean-12-ene, respectively. To the best of our knowledge the two triterpene parts 22-O-angeloyl-3β,16α,22α,24β,28-pentahydroxylolean-12-ene and16-O-(2-methylbutyroyl)-3β,16α,22α,24β,28-pentahydroxylolean-12-ene have never been characterized before. The two isolated saponins were assayed for their in-vitro cytotoxic activity against the three human tumor cell lines HepG2, MCF7 and PC3. The results showed that pendulaoside A exhibited moderate activity on PC3 cell line with IC50value equal to 13.0 μM and weak activity on HepG2 cell line with IC50 value equal to 41.0 μM. Pendulaoside B proved to be inactive against the three used cell lines.     

Noroleanane saponins from Celmisia petriei

Two biologically active noroleanane saponins from Celmisia petriei are identified as 3-O-(α-l-arabinopyranosyl (1 → 6)-β-d-glucopyranosyl (1 → 2)-α-l-arabinopyranosyl), 2β,17,23-trihydroxy-28-norolean-12-en-16-one and its 2″-O-acetyl derivative. ¹³C NMR and T₁ measurements allowed the determination of the sugar sequence and the majority of the linkage positions, but gave ambiguous results for the inner arabinose sugar. The structure of camellenodiol is revised to 3β,17-dihydroxy-28-norolean-12-en-16-one.     

The fatty acid and sterol composition of two marine dinoflagellates

The fatty acid, sterol and chlorophyll pigment compositions of the marine dinoflagellates Gymnodinium wilczeki and Prorocentrum cordatum are reported. The fatty acids of both algae show a typical dinoflagellate distribution pattern with a predominance of C₁₈, C₂₀ and C₂₂ unsaturated components. The acid 18:5ω3 is present at high concentration in these two dinoflagellates. G. wilczeki contains a high proportion (93.4%) of 4-methyl-5α-stanols including 4,23,24-trimethyl-5α-cholest-22E-en-3β-ol (dinosterol), dinostanol and 4,23,24-trimethyl-5α-cholest-7-en-3β-ol reported for the first time in dinoflagellates. The role of this sterol in the biosynthesis of 5α-stanols in dinoflagellates is discussed. P. cordatum contains high concentrations of a number of δ ²⁴⁽²⁸⁾-sterols with dinosterol, 24-methylcholesta-5,24(28)-dien-3β-ol, 23,24-dimethylcholesta-5,22E-dien-3β-ol, 4,24-dimethyl-5α-cholest-24(28)-en-3β-ol and a sterol identified as either 4,23,24-trimethyl- or 4-methyl-24-ethyl-5α-cholest-24(28)-en-3β-ol present as the five major components. The role of marine dinoflagellates in the input of both 4-methyl- and 4-desmethyl-5α-stanols to marine sediments is discussed.     

Polyoxygenated ent-kauranes and water-soluble conjugates in seed of Phaseolus coccineus

C-α and C-β, previously isolated from seed of Phaseolus coccineus, are shown respectively to be the bis-O-isopropylidene and the 16,17-mono-O-isopropylidene derivatives of ent-6α,7α,16β,17-tetrahydroxykauranoic acid. By GC-MS characterization of the products of acidic, basic and enzymatic hydrolysis, water soluble conjugates of the following compounds have been shown to occur in P. coccineus seed: GA₈, GA₁₇, GA₂₀, GA₂₈, ent-6α,7α,13-trihydroxykaurenoic acid, ent-6α,7α,17-trihydroxy-16β-kauranoic acid, ent-6α,7α,16β,17-tetrahydroxykauranoic acid, 7β,13-dihydroxykaurenolide and abscisic acid.     

Electron paramagnetic resonance study of intersubunit interactions in nitrosyl-deoxy asymmetrical hybrid haemoglobin

The asymmetrical nitrosyl-deoxy hybrid haemoglobin, (α^NOβ^NO), (α^deoxyβ^deoxy), was prepared by removing oxygen with sodium dithionite from a mixture of oxyhaemoglobin and nitrosylhaemoglobin (Cassoly, 1978). This asymmetrical hybrid exhibited a distinctive triplet hyperfine structure in the electron paramagnetic resonance spectrum. This triplet has been shown to arise predominantly from the nitrosyl haem of an α subunit which has a deoxy-like structure (Nagai et al., 1978). By removing one or two carboxyl-terminal residues by carboxypeptidase digestion before mixing, one can obtain asymmetrical nitrosyl-deoxy hybrid haemoglobins in which only one of the four subunits is specifically modified. Eight such modified derivatives were examined by e.p.r.². They were (desArgα^NOβ^NO) (α^deoxyβ^deoxy), (desArg-Tyrα^NOβ^NO) (α^deoxyβ^deoxy), (α^NOdesHisβ^NO) (α^deoxyβ^deoxy), (α^NOdesHis-Tyr β^NO) (α^deoxyβ^deoxy), (α^NOβ^NO) (desArgα^deoxyβ^deoxy), (α^NOβ^NO) (desArg-Tyrα^deoxyβ^deoxy), (α^NOβ^NO) (α^deoxydesHisβ^deoxy) and (α^NOβ^NO) (α^deoxydesHis-Tyrβ^deoxy), where desArg, desArg-Tyr, desHis and desHis-Tyr indicate that the amino acids were removed from the carboxyl terminus of the subunit.The e.p.r. spectra for these eight derivatives have a more or less reduced relative intensity of the triplet, indicating that the non-covalent bonds involving carboxyl-terminal residues which stabilize the structure of deoxyhaemoglobin (Perutz, 1970) must all be intact in the unmodified asymmetrical nitrosyl-deoxy hybrid haemoglobin, (α^NOβ^NO) (α^deoxyβ^deoxy). By comparing the relative intensity of the triplet we were able to examine the effect of modification of one specific carboxyl terminus on the nitrosyl haem in the α₁ subunit. The effect was not symmetric, but increased in the order α₁ < β₂ < β₁ < α₁ (suffices 1 and 2 as defined by Perutz (1965)). We attribute this order to the non-equivalence of intersubunit interactions.     

Steroidal saponins from the leaves of Cordyline fruticosa (L.) A. Chev. and their cytotoxic and antimicrobial activity

Three new steroidal saponins, spirosta-5,25(27)-diene-1β,3β-diol-1-O-α-l-rhamnopyranosyl-(1→2)-β-d-fucopyranoside (fruticoside H) 1, 5α-spirost-25(27)-ene-1β,3β-diol-1-O-α-l-rhamnopyranosyl-(1→2)-(4-O-sulfo)-β-d-fucopyranoside (fruticoside I) 2, and (22S)-cholest-5-ene-1β,3β,16β,22-tetrol 1-O-β-galactopyranosyl-16-O-α-l-rhamnopyranoside (fruticoside J) 3, together with the known quercetin 3-O-β-d-glucopyranoside, quercetin 3-O-[6-trans-p-coumaroyl]-β-d-glucopyranoside, quercetin 3-rutinoside, apigenin 8-C-β-d-glucopyranoside and farrerol, were isolated from the leaves of Cordyline fruticosa. Their structures were elucidated by spectroscopic techniques (¹H NMR, ¹³C NMR, HSQC, ¹H–¹H COSY, HMBC, TOCSY, NOESY), mass spectrometry (HRESIMS, Tandem MS–MS), chemical methods and by comparison with published data. Compounds 1 and 2 showed moderate cytotoxic activity against MDA-MB 231 human breast adenocarcinoma cell line, HCT 116 human colon carcinoma cell line, and A375 human malignant melanoma cell line, while compound 3 was not active. Compound 2 also showed a moderate antibacterial activity against the Gram-positive Enterococcus faecalis.     

Sterols with unusual nuclear unsaturation from three cultured marine dinoflagellates

Several new 4α-methyl sterols with unusual unsaturation in the Δ⁸⁽¹⁴⁾-or Δ¹⁴-positions, 4α,24S-dimethyl-5α-cholest-8 (14)-en-3β-ol, 4α-methyl-24ξ-ethyl-5α-cholest-8(14)-en-3β-ol, 4α-methyl-24(Z)-ethylidene-5α-cholest-8(14)- en-3β-ol, 4α,23 (or 22),24ξ-trimethyl-5α-cholesta-8(14),22-dien-3β-ol, 4α,24S(or 23ξ)-dimethyl-5α-cholest-14-en-3β-ol and 14-dehydrodinosterol, have been isolated from extracts of the cultured marine dinoflagellates Amphidinium carterae, A. corpulentum and Glenodinium sp. 4α-Methyl-24ξ-ethyl-5α-cholestan-3β-ol was isolated from the steryl ester fraction of Glenodinium sp. The structures of these new sterols are based upon extensive 360 MHz ¹H NMR and MS analyses.     

A simple method for preparation of valency hybrid hemoglobins, (α3+β2+)2 and (α2+β3+)2

The improved methods for the preparation of valency hybrid hemoglobins, (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ were presented. The (α³⁺β²⁺)₂ valency hybrid was separated from the solutions of partially reduced methemoglobin with ascorbic acid, by using CM 32 column chromatography. The (α²⁺β³⁺)₂ valency hybrid was also isolated from hemoglobin solutions, which were partially oxidized with ferricyanide, by chromatography on CM 32 column. These valency hybrid hemoglobins were found to be single on isoelectric focusing electrophoresis. Present procedures are very simple and are suitable for the bulk preparation of (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ valency hybrids.     

The synthesis of cholestane and furostan saponin analogues and the determination of sapogenin's absolute configuration at C-22

A facile and efficient way for the synthesis of cholestane and furostan saponin analogues was established and adopted for the first time. Following this strategy, starting from diosgenin, three novel cholestane saponin analogues: (22S,25R)-3β,22,26-trihydroxy-cholest-5-ene-16-one 22-O-[O-α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranoside] 11, (25R)-3β,16β,26-trihydroxy-cholest-5-ene-22-one 16-O-[O-α-l-rhamnopyranosyl-(1 → 2)-α-d-glucopyranoside] 14 and (25R)-3β,16β,26-trihydroxy-cholest-5-ene-22-one 16-O-[O-α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranoside] 17, three novel furostan saponin analogues: (22S,25R)-furost-5-ene-3β,22,26-triol 22-O-(α-d-glucopyranoside) 23, (22R,25R)-furost-5-ene-3β,22,26-triol 22-O-(α-d-glucopyranoside) 24 and (22S,25R)-furost-5-ene-3β,22,26-triol 22-O-[O-α-l-rhamnopyranosyl-(1 → 2)-α-d-glucopyranoside] 26, were synthesized ultimately. The structures of all the synthesized analogues were confirmed by spectroscopic methods. The S-chirality at C-22 of cholestane was confirmed by Mosher's method. The absolute configuration at C-22 of furostan saponin analogues was distinguished by conformational analysis combined with the NMR spectroscopy. The cytotoxicities of the synthetic analogues toward four types of tumor cells were shown also.     

The conversion of 5α-lanost-24-ene-3β,9α-diol and parkeol into poriferasterol by the alga Ochromon as malhamensis

The 4,4-dimethylsterols 4α-lanost-24-ene-3β,9α-diol-[2-³H₂] and parkeol-[2-³H₂] were synthesized from lanosterol and subsequently incubated with cultures of Ochromonas malhamensis. 5α-Lanost-24-ene-3β,9α-diol was converted into poriferasterol with three times the efficiency of parkeol. Clionasterol was also found to be labelled from both parkeol and 5α-lanost-24-ene-3β,9α-diol. No significant incorporation of radioactivity into sterols was obtained after feeding 5α-lanost-24-ene-3β,9α-diol to higher plants, the chlorophyte alga Trebouxia, yeast or a cell free homogenate of rat liver.