The biotransformation of methyl ent-15-oxokaur-16-en-19-oate by Rhizopus stolonifer and Mucor plumbeus

Incubation of methyl ent-15-oxokaur-16-en-19-oate with Rhizopus stolonifer and Mucor plumbeus gave methyl ent-7β,11-dihydroxy-15-oxokauran-19-oate and methyl ent-7β,16β-dihydroxy-15-oxokauran-19-oate, respectively.     

The biotransformation of a gibberellin 20→19-lactone by Gibberella fujikuroi

Incubation of ent-7,19-dihydroxygibberell- 16-en-20-oic acid 20→19-lactone with Gibberella fujikuroi led to hydroxylation at the 3- and 19-positions and to oxidation of the 7-alcohol.     

A simple and rapid method for the differentiation of C-13 manoyl oxide epimers in biologically important samples using GC-MS analysis supported with NMR spectroscopy and computational chemistry results

There is confusion in the literature related to the characterization of biologically important C13 manoyl oxide epimers by using their mass spectrometric data. Furthermore a method for calculating the epimeric purity has not been established. In this work mixtures of manoyl oxide C13 epimers and pure ent-13-epi-manoyl oxide have been isolated from plant extracts. The GC–MS analysis allows the characterization of each stereoisomer in a sample of natural origin: the ratio of intensities of peaks m/z 275 : 257 is lower in ent-13-epi-manoyl oxide than in manoyl oxide. NMR spectroscopy is used to give experimental evidence and simple calculations were performed to support the MS data. On the basis of these results the characterization of the two stereoisomers and the calculation of their ratio in plant extracts and essential oils can be done in a routine basis. The biological activity evaluation of mixtures with different epimeric composition of manoyl oxide showed that the ratio of the two epimers is important for their antibacterial activity. Ent-13-epi-manoyl oxide seems to be more active than its epimeric congener against Gram-positive bacteria.     

Structure elucidation of the core octasaccharide from Citrobacter PCM 1487 with the aid of 500-MHz, two-dimensional phase-sensitive correlated, relayed-coherence transfer, double-quantum, triple-quantum filtered, and N.O.E. H-n.m.r. spectra

The main features of the primary structure of the octasaccharide, - -Glcp-(1→2)-- -Glcp-(1→2)-[- -GalpNAc- (1→3)]-- -Galp-(1→3)-- -Glcp-(1→3)-[- -Hepp-(1→7)]-- -Hepp-(1→3)-- -Hep, have been determined in the ab initio manner by ¹H-n.m.r. spectroscopy without resorting to biochemical methods of analysis. Several nontypical interresidue n.O.e. values point to a preferred solution conformation of the molecule.     

Synthesis of the tetrasaccharide repeating-unit of the lipopolysaccharide isolated from pseudomonas maltophilia

The title tetrasacharide having the structure 3-O-Me-β- -Xylp-(1→4)-- -Rhap-(1→4)-- -Rhap-(1→2)- -Rhap was obtained by reaction of the -acetobromo derivative of 4-O-(3-O-methyl-β- -xylopyranosyl)- -rhamnopyranose and benzyl 3,4-di-O-benzyl-2-O-(2,3-O-isopropylidene-- -rhamnopyranosyl)-- -rhamnopyranoside, followed by removal of the protecting groups. The synthesised compounds were characterised on the basis of n.m.r. data.     

Feeding deterrent activity of enantiomeric isoalantones

The antifeeding activities on three storage pest insects of alantolactone, isoalantolactone and ent-isoalantolactone were measured and compared.     

Reactivity of {(Ph3P)Pt[μ-η-H---SiH(Ar)]}2 (Ar=2-isopropyl-6-methylphenyl) with phosphines. X-ray crystal structure of trans-{(dppe)Pt[μ-SiH(Ar)]}2

The dinuclear Pt---Si complex {(Ph₃P)Pt{μ-η²-H---SiH(IMP)]}₂ (trans-1a–cis-1b=3:1; IMP=2-isopropyl-6-methylphenyl) reacted with basic phosphines such as 1,2-bis(diphenylphosphino)ethane (dppe) and dimethylphenylphosphine (PMe₂Ph) to afford different dinuclear Pt---Si complexes with loss of H₂, {(P)₂Pt[μ-SiH(IMP)]}₂ [P=dppe, trans-2a (major), cis-2b (trace); PMe₂Ph, 3 (trans only)]. Complexes 2 and 3 were characterized by multinuclear NMR spectroscopy and X-ray crystallography (2a). In contrast, the reaction of 1a,b with the sterically demanding tricyclohexylphosphine (PCy₃) afforded {(Cy₃P)Pt{μ-η²-H---SiH(IMP)]}₂ (trans-4a–cis-4b 2:1) analogous to 1a,b where the central Pt₂Si₂(μ-H)₂ core remains intact but the PPh₃ ligands have been replaced by PCy₃. Complexes 4a and 4b was characterized by multinuclear NMR and IR spectroscopies.     

Electron paramagnetic resonance saturation studies of P-700 reaction center chlorophyll in plant photosynthesis

Electron paramagnetic resonance (EPR) power saturation and saturation recovery methods have been used to determine the spin lattice, T₁, and spin-spin, T₂, relaxation times of P-700⁺ reaction-center chlorophyll in Photosystem I of plant chloroplasts for 10 K T 100 K. T₁ was 200 μs at 100 K and increased to 900 μs at 10 K. T₂ was 40 ns at 40 K and increased to 100 ns at 10 K. T₁ for 40 K T 100 K is inversely proportional to temperature, which is evidence of a direct-lattice relaxation process. At T = 20 K, T₁ deviates from the 1/T dependence, indicating a cross relaxation process with an unidentified paramagnetic species. The individual effects of ascorbate and ferricyanide on T₁ of P-700⁺ were examined: T₁ of P-700⁺ was not affected by adding 10 mM ascorbate to digitonin-treated chloroplast fragments (D144 fragments). The P-700⁺ relaxation time in broken chloroplasts treated with 10 mM ferricyanide was 4-times shorter than in the untreated control at 40 K. Ferricyanide appears to be relaxing the P-700⁺ indirectly to the lattice by a cross-relaxation process. The possibility of dipolar-spin broadening of P-700⁺ due to either the iron-sulfur center A or plastocyanin was examined by determining the spin-packet linewidth for P-700⁺ when center A and plastocyanin were in either the reduced or oxidized states. Neither reduced center A nor oxidized plastocyanin was capable of broadening the spin-packet linewidth of the P-700⁺ signal. The absence of diplolar broadening indicates that both center A and plastocyanin are located at a distance at least 3.0 nm from the P-700⁺ reaction center chlorophyll. This evidence supports previous hypotheses that the electron donor and acceptor to P-700 are situated on opposite sides of the chloroplast membrane. It is also shown that the ratio of photo-oxidized P-700 to photoreduced centers A and B at low temperature is 2 : 1 if P-700 is monitored at a nonsaturating microwave power.     

Synthesis of 2,3-O-(propane-1,2-diyl)- and 2,3-O-(3-hydroxypropane-1,2-diyl)-cyclomaltoheptaose

2¹,3¹-O-(Propane-1,2-diyl)cyclomaltoheptaose has been prepared from 2-O-allylcyclomaltoheptaose by mercuration in trifluoroacetic acid, followed by reduction with sodium borohydride. 2-O-(2,3-Epoxypropyl)cyclomaltoheptaose, prepared from 2-O-allylcyclomaltoheptaose by oxidation with dimethyldioxirane, was converted into 2¹,3¹-O-(3-hydroxypropane-1,2-diyl)cyclomaltoheptaose by treatment with trifluoroacetic acid. Both derivatives containing fused 1,4-dioxane rings are mixtures of stereoisomers, in which the methyl and hydroxymethyl group, respectively, that is linked to this ring, occupies an axial or an equatorial position.     

Comparison of cytochrome P-450 species which catalyze the hydroxylations of the aromatic ring of estradiol and estradiol 17-sulfate

For identification of microsomal cytochrome P-450 (P-450) enzymes which catalyze 2- or 4-hydroxylations of estrogens in the rat liver, estradiol (E₂) and estradiol 17-sulfate (E₂-17-S) were selected as the substrates and incubated with various kinds of purified P-450 enzymes: PB-1, PB-2, PB-4 and PB-5 obtained from phenobarbital-treated male rats (Sprague-Dawley); MC-1 and MC-5 from 3-methylcholanthrene-treated male rats; and UT-1, UT-2, UT-4 and UT-5 from untreated animals. The reactions were carried out under the P-450-reconstructed system, and the resulting products were determined by HPLC using electrochemical detection. All the enzymes tested were shown to have varying degrees of catalytic activities for 2-hydroxylation of the two substrates; UT-1 and UT-2 had the highest activity. Of the induced P-450 enzymes, PB-2 and MC-1 showed fairly high catalytic activity for 4-hydroxylation of E₂. The P-450 enzymes obtained from the untreated male rats, especially UT-4, showed the highest catalytic activity for 4-hydroxylation of the two substrates. From these results and also from kinetic experiments, the P-450 enzymes which catalyze 2- and 4-hydroxylations of estrogen were considered to be different species. A part of E₂ was converted to such metabolites as estrone and those having a hydroxyl group at positions 6β, 15 or 16, each production of which was estimated to be catalyzed by single or multiple P-450s.     

Stereoselective and improved syntheses and anticancer testing of 3'-O-silatranylthymidines

A stereoselective synthesis of 3′-O-((R,R,R)-trimethylsilatranyl)thymidine (R,R,R-1) and synthesis of 3′-O-silatranylthymidine (5) via an improved silatranylation procedure using tetrakis(dimethylamino)silane are reported. Diastereomeric mixture 1 showed more activity than R,R,R-1 or 5 in a primary anticancer screen against breast, CNS, and lung cell lines; demonstrating the import of the configuration and presence, respectively, of the silatrane methyl groups for growth inhibition.     

Slippage of η-allenyl/propargyl to an η structure and tautomerization of η-propargyl to η-allenyl in ligand addition and substitution reactions of platinum(II) complexes

Reactions of [(PPh₃)₂Pt(η³-CH₂CCPh)]OTf with each of PMe₃, CO and Br⁻ result in the addition of these species to the metal and a change in hapticity of the η³-CH₂CCPh to η¹-CH₂CCPh or η¹-C(Ph)=C=CH₂. Thus, PMe₃ affords [(PMe₃)₃Pt(η¹-C(Ph)=C=CH₂)]⁺, CO gives both [trans-(PPh₃)₂Pt(CO)(η¹-CH₂CCPh)]⁺ and [trans-(PPh₃)₂Pt(CO)(η¹-C(Ph)=C=CH₂)]⁺, and LiBr yields cis-(PPh₃)₂PtBr(η¹-CH₂CCPh), which undergoes isomerization to trans-(PPh₃)₂PtBr(η¹-CH₂CCPh). Substitution reactions of cis- and trans-(PPh₃)₂PtBr(η¹-CH₂CCPh) each lead to tautomerization of η¹-CH₂CCPh to η¹-C(Ph)=C=CH₂, with trans-(PPh₃)₂PtBr(η¹-CH₂CCPh) affording [(PMe₃)₃Pt(η¹-C(Ph)=C=CH₂)]⁺ at ambient temperature and the slower reacting cis isomer giving [trans-(PPh₃)(PMe₃)₂Pt(η¹-C(Ph)=C=CH₂)]⁺ at 54 °C . All new complexes were characterized by a combination of elemental analysis, FAB mas spectrometry and IR and NMR (¹H, ¹³C{¹H} and ³¹P{¹H}) spectroscopy. The structure of [(PMe₃)₃Pt(η¹-C(Ph)=C=CH₂)]BPh₄·0.5MeOH was determined by single-crystal X-ray diffraction analysis.     

Enzymatic deacylation of l,2-diacyl-sn-glycero-3-phosphocholines to sn-glycerol-3-phosphocholine

This research was undertaken to study the enzymatic deacylation of l,2-diacyl-sn-glycero-3-phosphocholines (sn-1,2-PC) to sn-glycerol-3-phosphocholine (GPC); this compound could be an useful intermediate in the synthesis of “structured” sn-1,2-PC, after re-acylations of the two sn-positions of the glycerol backbone. The enzymatic reactions represent a valid alternative to the chemical deacylation that can be simply obtained in alkaline conditions. High conversion were achieved using a lipase selective for the sn-1-position of sn-1,2-PC (Lipozyme IM, from Mucor miehei) together with a Phospholipase A₂ from hog pancreas, enzyme selective for the sn-2-position; the best results were obtained carrying out the enzymatic reaction in a microemulsion system.     

Photooxidation of cytochrome b-559 in leaves and chloroplasts at room temperature

1. Light-induced absorbance changes of cytochrome b-559 and cytochrome f in the -band region were examined in leaves and in isolated chloroplasts.

2. Absorbance changes of cytochrome b-559 were not detected in untreated leaves or in most preparations of isolated chloroplasts. After treatment of leaves or chloroplasts with carbonyl cyanide m-chlorophenylhydrazone, high rates of photooxidation of cytochrome b-559 were obtained, both in far-red (>700 nm) and red actinic light. Cytochrome f was photooxidized in far-red light, but in red light it remained mainly in the reduced state. The initial rates of photooxidation of cytochrome b-559 in leaves or chloroplasts treated with carbonyl cyanide m-chlorophenylhydrazone were considerably decreased by 3-(3′,4′-dichlorophenyl)-1,1-dimethyl urea.

3. A slow photoreduction of cytochrome b-559 was observed in aged mutant pea chloroplasts in red light.

4. The results do not support the view that cytochrome b-559 is a component of the electron transport chain between the light reactions. It is suggested that cytochrome b-559 is located on a side path from Photosystem II, but with a possible additional link to Photosystem I.     

Cytochrome P-450 distribution in rat liver and the effect of sodium phenobarbitone administration

A microspectrophotometric method for assaying cytochrome P-450 in fresh 24 μm unfixed cryostat sections of rat liver has been developed. When used to assay this cytochrome in sections of microsomal preparations it has yielded results equivalent to those obtained by the conventional spectrophotometric assay of the same preparations. Random measurements made throughout sections of liver have given mean values for cytochrome P-450 concentrations which are twice those measured in microsomes prepared from the livers of the same animals (not corrected for the yield in the homogenate).

Measurements of the cytochrome P-450 content of liver cells by the microspectrophotometric method show that in liver from male Wistar rats, cells nearer to the central veins contain up to twice as much cytochrome P-450 as those nearer to the portal tract (mean cell concentrations of 26.4 (±4.4) μmol/l and 17.5 (±3.0) μmol/l respectively). In the livers from similar rats, killed at the same time, but which had received 1 mg/ml sodium phenobarbitone in their drinking water for one week, the cells near the central vein contained up to five times as much cytochrome P-450 as those near the portal tract (mean cell concentrations of 77.3 (±25.0) μmol/l and 28.3 (±9.6) μmol/l respectively).

The results show a selective increase in cytochrome P-450 content by the cells in the centrilobular region after treatment with sodium phenobarbitone and a smaller increase by some of the cells in the periportal region.     

Separative preparation of the four stereoisomers of β-methylphenylalanine with N-carbamoyl amino acid amidohydrolases

The selective preparation of the four stereoisomers of β-methylphenylalanine (Mphe) from mixtures of the four stereoisomers of N-carbamoyl-β-methylphenylalanine (NCMphe) with N-carbamoyl amino acid amidohydrolases (carbamoylases) was developed. -Carbamoylase specifically hydrolyzed threo- -NCMphe with a little side activity toward erythro- -NCMphe, thus threo- -Mphe was produced with high optical purity from a mixture of the four stereoisomers of NCMphe. -Carbamoylase specifically produced threo- -Mphe from a mixture of the four stereoisomers of NCMphe. The erythro- -Mphe was obtained from erythro- -NCMphe which was prepared through diastereomer resolution by separative crystallization of benzoyl Mphe with a little side activity of -carbamoylase toward erythro- -NCMphe and the remaining erythro- -NCMphe was chemically hydrolyzed to erythro- -Mphe.     

Saponins from Albizzia lucida.

Three main saponins were isolated from the seeds of Albizzia lucida. Their structures were established by spectral analyses and chemical and enzymatic transformations as 3-O-[β- -xylopyranosyl(1→2)-- -arabinopyranosyl (1→6)] [β- -glucopyranosyl (1→2)] β- -glucopyranosyl echinocystic acid; 3-O-[- -arabinopyranosyl (1→6)][β- -glucopyranosyl (1→2)]-β- -glucopyranosyl echinocystic acid and 3-O-[β- -xylopyranosyl (1→2)-β- -fucopyranosyl (1→6)-2-acetamido-2-deoxy-β- -glucopyranosyl echinocystic acid, characterized as its methyl ester.     

Reducibility of cytochromes b in aerobic beef-heart mitochondria treated with antimycin

Under anaerobic conditions cytochrome b in beef-heart mitochondria is partially reduced in the presence of NADH, whereas other cytochromes are completely reduced. Addition of antimycin together with oxygen under these conditions causes an immediate reduction of cytochromes b-558, b-562 and b-566 and oxidation of cytochrome c. During the subsequent transient aerobic steady state cytochromes b-558 and b-566 are rapidly re-oxidized without changes in redox state of cytochrome c, but cytochrome b-562 remains reduced. When oxygen is consumed by the leak through or around the antimycin-inhibition site, cytochrome b-562 becomes oxidized with concomitant reduction of cytochrome c.

The cytochromes b in lyophilized beef-heart mitochondria are more readily accessible to electrons from NADH, and in the presence of antimycin and NADH a complete and stable reduction is obtained under both aerobic and anaerobic conditions. Gradual addition of rotenone under these conditions causes re-oxidation of cytochromes b in which oxidation of cytochromes b-558 and b-566 precedes that of cytochrome b-562.

It is concluded that (1) the effect of antimycin in the presence of oxygen involves all three cytochromes b, (2) the reducibility of the cytochromes b in the aerobic steady state of antimycin-treated mitochondria is dependent upon the potential of the substrate redox couple registered on the cytochromes, and (3) the midpoint potential of cytochrome b-562 in the presence of antimycin is higher than that of cytochrome b-558 or b-566.