Steroid Compounds from Far Eastern Starfishes <Emphasis Type="Italic">Henricia aspera</Emphasis> and <Emphasis Type="Italic">H. tumida</Emphasis>

Six new natural compounds were isolated from two Far Eastern starfish species, Henricia aspera and H. tumida, collected in the Sea of Okhotsk. Two new glycosylated steroid polyols were obtained from H. aspera: asperoside A and asperoside B, which were shown to be (20R,24R, 25S)-3-O-(2,3-di-O-methyl-β -D-xylopyranosyl)-24-methyl-5α-cholest-4-ene-3β, 6β,8,15α,16β,26-hexaol and (20R, 24R,25S,22E)-3-O-(2,4-di-O-methyl-β-D-xylopyranosyl)-24-methyl-5α-cholest-22-ene-3β,4β,6β,8,15α,26-hexaol, respectively. Two other glycosylated polyols, tumidoside A, with the structure elucidated as (20R, 22E)-3-O-(2,4-di-O-methyl-β -D-xylopyranosyl)-26,27-dinor-24-methyl-5α-cholest-22-ene-3β,4β,6β,8,15α,25-hexaol, and tumidoside B, whose structure was elucidated as (20R,24S)-3-O-(2,3-di-O-methyl-β-D-xylopyranosyl)-5α-cholestan-3β,4β,6β,8,15α,24-hexaol, were isolated from the two starfish species. (20R, 24S)-5α-Cholestan-3β,6β,15α,24-tetraol and (20R, 24S)-5α-cholestan-3β,6β,8,15α,24-pentaol were identified only in H. tumida. The known monoglycosides henricioside H1 and laeviuscolosides H and G were also identified in both species.     

Trofosides A and B and other cytostatic steroid-derived compounds from the far east starfish <Emphasis Type="Italic">Trofodiscus über</Emphasis>

Three new polar steroids identified as trofoside A, 20R,24S)-24-O-(3-O-methyl-β-D-xylopyranosyl)-3β,6α,8,15β,24-pentahydroxy-5α-cholestane, its 22(23)-dehydro derivative (trofoside B), and 15-sulfooxy-(20R,24S)-5α-cholestane-3β,6β,8,15α,24-pentaol sodium salt, were isolated fromTrofodiscus über starfish extracts collected in the Sea of Ohotsk. Two known compounds, trofoside A aglycone, (20R,24S)-3β,6α,8,15β,24-pentahydroxy-5α-cholestane, and triseramide, (20R,24R,25S,22E)-24-methyl-3β6α,8,15β-tetrahydroxy-5α-cholest-22-en-27-oic acid (2-sulfoethyl)amide sodium salt, were also found. The structures of the isolated polyoxysteroids were established from their spectra. Minimal concentrations causing degradation of unfertilized egg-cells of the sea-urchin Strongylocentrotus intermedius(C _min) and terminating the cell division at the stage of the first division (C _min embr.), as well as the concentrations causing 50% immobilization of sperm cells (OC₅₀) and inhibiting their ability to fertilize egg-cells by 50% (IC₅₀) were determined for the isolated compounds. Of three compounds highly toxic in embryos and sea-urchin sperm cells, the polyol with a sulfo group in the steroid core was the most active; two glycosides with monosaccharide chains located at C3 and C24 atoms were less toxic. Note that all the compounds with the spermiotoxic activities differently affected the embryo development. The positions of monosaccharide residues in the core considerably influence the compound activity. For example, both mono-and double chained glycosides with the monosaccharide fragment at C3 and fragments at C3 and C4 atoms are active against sea-urchin sperm cells and embryos, whereas the C24 glycosylated trofoside A does not affect embryos and displays a poor spermiotoxicity.     

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.     

Steroid compounds from two pacific starfish of the genus <Emphasis Type="Italic">Evasterias</Emphasis>

Three new steroid glycosides (evasteriosides C, D, and E) along with six known compounds were isolated from two Pacific starfish of the genus Evasterias. Evasterioside C from E. retiferacollected from the Sea of Japan was identified as (20R, 22E)-3-O-(β-D-xylopyranosyl)-24-nor-5α-cholest-22-ene-3β,6β,15α,26-pentaol 26-sulfate sodium salt. The structures of evasteriosides D and E from E. echinosoma (collected from the Gulf of Shelichov, the Sea of Okhotsk) were established as (20R, 24S)-24-O-(β-D-glucopyranosyl)-5α-cholestane-3β,6α,8,15β,24-pentaol and (20R,24S)-3,24-di-O-(β-D-xylopyranosyl)-cholest-4-ene-3β,6β,8,15α,24-pentaol, respectively. In addition, the known compounds pycnopodiosides A and C, luridoside A, 5α-cholestane-3β,6α,8,15β,16β,26-hexaol. 5α-Cholestane-3β,6α,8,15β,24-pentaol 24-sulfate sodium saltand marthasterone sulfate sodium salt were identified in E. echinosoma. The structures of the isolated compounds were established on the basis of spectroscopic analyses, using 1D and 2D NMR techniques, mass spectrometry, and some chemical transformations.     

Organic Solvent-Tolerant Marine Microorganisms as Catalysts for Kinetic Resolution of Cyclic β-Hydroxy Ketones

Chiral cyclic β-hydroxy ketones represent key motifs in the production of natural products of biological interest. Although the molecules are structurally simple, they require cumbersome synthetic steps to get access to them and their synthesis remains a challenge in organic chemistry. In this report, we describe a straightforward approach to enantiomerically enriched (R)- and (S)-3-hydroxycyclopentanone 2a, (R)- and (S)-3-hydroxycyclohexanone 2b, and (R)- and (S)-3-hydroxycycloheptanone 2c involving a transesterification resolution of the racemates using whole cells of marine microorganisms as catalysts and vinyl acetate the acyl donor and solvent. Twenty-six strains from a wide collection of isolates from marine sediments were screened, and seven strains were found to markedly catalyze the resolution in an asymmetric fashion. Using the strain Serratia sp., (R)-2a was isolated in 27% yield with 92% ee and (S)-2a in 65% yield with 43% ee, corresponding to an E-value of 37; (R)-2b was isolated in 25% yield with 91% ee and (S)-2b in 67% yield with 39% ee, corresponding to an E-value of 40; and (R)-2c was isolated in 30% yield with 96% ee and (S)-2c in 63% yield with 63% ee, corresponding to an E-value of 75.     

One pot simultaneous preparation of both enantiomer of β-amino alcohol and vicinal diol via cascade biocatalysis

Objectives

To investigate the efficiency of a new cascade biocatalysis system for the conversion of R, S-β-amino alcohols to enantiopure vicinal diol and β-amino alcohol.

Results

An efficient cascade biocatalysis was achieved by combination of a transaminase, a carbonyl reductase and a cofactor regeneration system. An ee value of > 99% for 2-amino-2-phenylethanol and 1-phenyl-1, 2-ethanediol were simultaneously obtained with 50% conversion from R, S-2-amino-2-phenylethanol. The generality of the cascade biocatalysis was further demonstrated with the whole-cell approaches to convert 10–60 mM R, S-β-amino alcohol to (R)- and (S)-diol and (R)- and (S)-β-amino alcohol in 90–99% ee with 50–52% conversion. Preparative biotransformation was demonstrated at a 50 ml scale with mixed recombinant cells to give both (R)- and (S)-2-amino-2-phenylethanol and (R)- and (S)-1-phenyl-1, 2-ethanediol in > 99% ee and 40–42% isolated yield from racemic 2-amino-2-phenylethanol.

Conclusions

This cascade biocatalysis system provides a new practical method for the simultaneous synthesis of optically pure vicinal diol and an β-amino alcohol.

     

Two new steroid glycosides from the Far East starfish <Emphasis Type="Italic">Hippasteria kurilensis</Emphasis>

Two new steroid glycosides were isolated from the Far East starfish Hippasteria kurilensis collected in the Sea of Okhotsk. They were characterized as (22E,24R)-3-O-(2-O-methyl-β-D-xylopyranosyl)-24-O-[2-O-methyl-β-D-xylopyranosyl-(1→5)-α-L-arabinofuranosyl]-5α-cholest-22-ene-3β,4β,6α,7α,8,15β,24-heptaol (kurilensoside I) and (24S)-3-O-(2-O-methyl-β-D-xylopyranosyl)-24-O-(α-L-arabinofuranosyl)-5α-cholestane-3β,4β,6β,15α,24-pentaol (kurilensoside J). In addition, the earlier known glycosides linkosides F and L1, leviusculoside G, forbeside L, desulfated echinasteroside, and granulatoside A were isolated and identified. The structures of the new compounds were established with the help of two-dimentional NMR spectroscopy and mass- spectrometry.     

Genotypical features in the expression of allozymes of β-specific hydrolase of carboxylic esters in wild-type <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The expression level of electrophoretically separated S- and F-allozymes of β-specific esterase (EC 3.1.1.2) in genotypes of wild-type Drosophila melanogaster (males and females) that are monozygous or heterozygous with respect to the locus β-Est is determined by means of computerized densitometry; α-naphthylacetate, β-naphthylacetate, and α-naphthylpropionate are used as the substrates. The intensity of the expression of the esterase is judged from the quantity of reaction product created as a result of simultaneous azo coupling between naphthol and diazonium in 4, 24, 44, and 64 min incubation times. Reliable differences in the expressions of the S- and F-allozymes as a function of the structure of the β-Est locus of genotypically distinct individuals are established. In all the variant experiments, a higher level of summary activity of the S- and F-allozymes of the β-esterase of the heterozygotes by comparison with the individual activity of the F-and S-allozymes of the corresponding homozygotes was demonstrated, independently of the sex of the Drosophila individual. A comparative estimate of the temporal dynamics of the expression of in vitro allozymes of the dominant homozygotes (β-Est _S /β-Est _S ), heterozygotes (β-Est _S /β-Est _F ), and recessive homozygotes (β-Est _F /β-Est _F ) is performed. Possible mechanisms for the occurrence of heterosis according to the character of expression of S- and F-allozymes of β-esterase on the basis of the theory of biochemical enrichment of heterozygote genotypes are considered.     

Pancreatic lipase inhibitory activity of monogalactosyldiacylglycerols isolated from the freshwater microalga <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis>

Chemical investigation of the freshwater microalga Chlorella sorokiniana led to the isolation of a monogalactosyldiacylglycerol (MGDG)-rich fraction possessing dose-dependent inhibitory activity against pancreatic lipase activity. The MGDG-rich fraction contains 12 MGDGs identified by LC/HRMS analysis. Among them, three MGDGs were new compounds, namely, (2S)-1-O-(7Z,10Z-hexadecadienoyl)-2-O-(7Z,10Z,13Z-hexadecatrienoyl)-3-O-β-D-galactopyranosylglycerol (1), (2S)-1-O-linoleoyl-2-O-(7Z,10Z-hexadecadienoyl)-3-O-β-D-galactopyranosylglycerol (6), and (2S)-1-O-oleoyl-2-O-(7Z,10Z-hexadecadienoyl)-3-O-β-D-galactopyranosylglycerol (8). The major galactolipids were isolated by semipreparative HPLC and tested for their effect toward pancreatic lipase inhibitory activity. All the tested MGDGs showed significant reduction of pancreatic lipase activity indicating possible beneficial use for management of lipase-related disorders such as obesity.     

Specificity of oxidation of linoleic acid homologs by plant lipoxygenases

The lipoxygenase-catalyzed oxidation of linoleic acid homologs was studied. While the linoleic acid oxidation by maize 9-lipoxygenase (9-LO) specifically produced (9S)-hydroperoxide, the dioxygenation of (11Z,14Z)-eicosadienoic (20:2) and (13Z,16Z)-docosadienoic (22:2) acids by the same enzyme lacked regio- and stereospecificity. The oxidation of 20:2 and 22:2 by 9-LO afforded low yields of racemic 11-, 12-, 14-, and 15-hydroperoxides or 13- and 17-hydroperoxides, respectively. Soybean 13-lipoxygenase-1 (13-LO) specifically oxidized 20:2, 22:2, and linoleate into (ω6S)-hydroperoxides. Dioxygenation of (9Z,12Z)-hexadecadienoic acid (16:2) by both 9-LO and 13-LO occurred specifically, affording (9S)- and (13S)-hydroperoxides, respectively. The data are consistent with the “pocket theory of lipoxygenase catalysis” (i.e. with the penetration of a substrate into the active center with the methyl end first). Our findings also demonstrate that the distance between carboxyl group and double bonds substantially determines the positioning of substrates within the active site.     

Highly enantioselective production of a chiral intermediate of sitagliptin by a novel isolate of <Emphasis Type="Italic">Pseudomonas pseudoalcaligenes</Emphasis>

Objective

To produce (S)-3-hydroxy-1-(3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-4-(2,4,5-trifluorophenyl)butan-1-one (S)-1 from 4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro [1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-1-(2,4,5-trifluorophenyl)butan-2-one (2) by microbial bioreduction.

Results

A new isolate of Pseudomonas pseudoalcaligenes reduced enantioselectively prochiral ketone 2 to chiral alcohol (S)-1. Whole cells of the bacterium were tolerant towards 20 % (v/v) DMSO and 10 g 2/l. Under the optimal conditions, the preparative-scale bioreduction yielded (S)-1 at 90 % yield and >99 % ee. Cells could be re-used with the yield and ee of product being 45 % and >99 %, respectively, after five cycles.

Conclusion

Bioreduction using whole cells of P. pseudoalcaligenes is an attractive approach to produce (S)-1, as a chiral intermediate of the anti-diabetic drug, sitagliptin.

     

Identification of 21 novel <Emphasis Type="Italic">S-RNase</Emphasis> alleles and determination of <Emphasis Type="Italic">S</Emphasis>-genotypes in 66 loquat (<Emphasis Type="Italic">Eriobotrya</Emphasis>) accessions

As observed in other self-incompatible species in the Pyrinae subtribe, loquat (Eriobotrya japonica) demonstrates gametophytic self-incompatibility that is controlled by the S-locus, which encodes a polymorphic stylar ribonuclease (S-RNase). This allows the female reproductive organ (style) to recognize and reject the pollen from individuals with the same S-alleles, but allows the pollen from individuals with different S-alleles to effect fertilization. The S-genotype is therefore an important consideration in breeding strategies and orchard management. In an attempt to optimize the selection of parental lines in loquat production, the S-RNase alleles of 35 loquat cultivars and their 26 progeny, as well as five wild loquat species, were identified and characterized in this study. The best pollinizer cultivar combinations were also explored. A total of 28 S-alleles were detected, 21 of which constituted novel S-RNase alleles. The S-haplotypes S₂ and S₆ were the most frequent, followed by S ₂₉ , S ₃₁ , S ₅ , S ₂₄ , S ₂₈ , S ₃₃ , S ₃₄ , S ₃₂ , and S ₁₅ , while the rare alleles S ₁ , S ₉ , S ₁₄ , S ₁₆ , S ₁₇ , S ₁₈ , S ₁₉ , S ₂₀ , S ₂₁ , S ₂₂ , S ₂₃ , S ₂₇ , and S ₃₅ were only observed in one of the accessions tested. Moreover, the S-genotypes of five wild loquat species (E. prinoides, E. bengalensis, E. prinoides var. dadunensis, E. deflexa, and E. japonica) are reported here for the first time. The results will not only facilitate the selection of suitable pollinators for optimal orchard management, but could also encourage the crossbreeding of wild loquat species to enhance the genetic diversity of loquat cultivars.     

Comparing the methods for quantifying the culm surface area of bamboo, <Emphasis Type="Italic">Phyllostachys pubescens</Emphasis>

We compared several methods for quantifying the culm surface area (S) of one of the most common bamboos in Japan, Phyllostachys pubescens Mazel ex Houz. Nine sample culms of P. pubescens were felled, and the true S was determined by the fine resolution analysis of the culm form (S _FRA). The S was then calculated independently with the sectional measurement method from the successive diameters measured at equal intervals of one-twentieth (S ₂₀), one-tenth (S ₁₀), one-fifth (S ₅) and one-half (S ₂) of the total culm length. The S was also quantified geometrically from the total culm length and the diameter at breast height or at base by assuming that the culm form could be approximated by a cone (S _DBH and S _DAB). The S _FRA was compared with each of the computed S values. For S ₁₀, S ₅ and S ₂, both the mean relative bias (%BIAS) and relative root mean square error (%RMSE) decreased with an increase in the number of measured diameters. The %BIAS and %RMSE of the S ₁₀ were, respectively, comparable and smaller compared to those of S ₂₀. The bias of the S _DBH and S _DAB suggested that the cone assumption of the culm form was violated. In conclusion, we recommend that the S should be quantified from the successive diameters measured at equal intervals of one-tenth of the total culm length using the sectional measurement method.     

Interaction of gibberellic acid and 24-epibrassinolide in the regulation of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> seedling scotomorphogenesis

The effects of GA₃, 24-epibrassinolide (EBL), and their combination on morphogenesis of Arabidopsis thaliana (L.) Heynh seven-day-old seedlings were studied. Four plant lines were analyzed: wild type Ler and ga4-1 mutant, belonging to the Landsberg erecta ecotype and wild type Col and det2 mutant, both of the Columbia ecotype. In ga4-1 and det2, GA_4/1-and brassinosteroid-deficient mutants, the highest hypocotyl growth response to the lack of hormones was noted. The cotyledon shape and size were dependent on EBL, and the root length was both GA₃-and EBL-regulated, indicating organ specificities in the responses to these hormones. Simultaneous treatment of dark-grown plants with GA₃ and EBL exerted an additive stimulatory effect on the root growth of det2, reduced the inhibitory effect of EBL on hypocotyl elongation of ga4-1, and enhanced the effect of EBL on hypocotyl and cotyledon elongation of det2.     

<Superscript>18</Superscript>F-labeled tyrosine derivatives: Synthesis and experimental studies on accumulation in tumors and abscesses

Tyrosine derivatives labeled with a short-lived fluorine-18 isotope (T _1/2 110 min), namely 2-[¹⁸F]fluoro-L-tyrosine (FTYR) and O-(2′-[¹⁸F]fluoroethyl)-L-tyrosine (FET), promising radiopharmaceuticals (RPs) for positron emission tomography (PET), were obtained by asymmetric syntheses. Accumulation of FTYR and FET in the rat tumor “Glioma 35 rats tumor” and in abscesses induced in Wistar rats muscles was studied and compared with that of a well-known glycolysis radiotracer 2-[¹⁸F]fluoro-2-deoxy-D-glucose (FDG). It was shown that the relative accumulation indices of amino acid RPs were considerably lower than those of FDG. At the same time, tumor/muscle ratios were high enough (2.9 for FET and 3.9 for FTYR 120 min after injection) for reliable tumor visualization. The data obtained indicated a possibility in principle to use FTYR and FET for differentiated PET diagnostics of brain tumors and inflammation lesions. Of the tyrosine derivatives studied, FET seems to be the most promising agent due to a simple and easily automated method of preparation based on direct nucleophilic substitution of the leaving tosyloxy group of an enantiomerically pure Ni-(S)-BPS-(S)-Tyr(CH₂CH₂OTs) precursor by an activated [¹⁸F]fluoride.     

Pistil-function breakdown in a new <Emphasis Type="Italic">S</Emphasis>-allele of European pear, <Emphasis Type="Italic">S</Emphasis><Subscript><Emphasis Type="Italic">21</Emphasis></Subscript>°, confers self-compatibility

European pear exhibits RNase-based gametophytic self-incompatibility controlled by the polymorphic S-locus. S-allele diversity of cultivars has been extensively investigated; however, no mutant alleles conferring self-compatibility have been reported. In this study, two European pear cultivars, ‘Abugo’ and ‘Ceremeño’, were classified as self-compatible after fruit/seed setting and pollen tube growth examination. S-genotyping through S-PCR and sequencing identified a new S-RNase allele in the two cultivars, with identical deduced amino acid sequence as S ₂₁ , but differing at the nucleotide level. Test-pollinations and analysis of descendants suggested that the new allele is a self-compatible pistil-mutated variant of S ₂₁ , so it was named S ₂₁ °. S-genotypes assigned to ‘Abugo’ and ‘Ceremeño’ were S ₁₀ S ₂₁ ° and S ₂₁ °S ₂₅ respectively, of which S ₂₅ is a new functional S-allele of European pear. Reciprocal crosses between cultivars bearing S ₂₁ and S ₂₁ ° indicated that both alleles exhibit the same pollen function; however, cultivars bearing S ₂₁ ° had impaired pistil-S function as they failed to reject either S ₂₁ or S ₂₁ ° pollen. RT-PCR analysis showed absence of S ₂₁ °-RNase gene expression in styles of ‘Abugo’ and ‘Ceremeño’, suggesting a possible origin for S ₂₁ ° pistil dysfunction. Two polymorphisms found within the S-RNase genomic region (a retrotransposon insertion within the intron of S ₂₁ ° and indels at the 3′UTR) might explain the different pattern of expression between S ₂₁ and S ₂₁ °. Evaluation of cultivars with unknown S-genotype identified another cultivar ‘Azucar Verde’ bearing S ₂₁ °, and pollen tube growth examination confirmed self-compatibility for this cultivar as well. This is the first report of a mutated S-allele conferring self-compatibility in European pear.     

The <Emphasis Type="Italic">Adh</Emphasis> locus and adaptation of <Emphasis Type="Italic">cn</Emphasis> and <Emphasis Type="Italic">vg</Emphasis> mutants in experimental populations of <Emphasis Type="Italic">Drosophila melanogaster MEIG</Emphasis>

A complex study on the adaptation of cn and vn mutants and the allozymes of alcoholdehydrogenase (ADH) was carried out in initially pure lines, and their panmixia populations during exchange of the mutant genotype with that of wild-type flies (C-S) and D) through saturating crossings. The relative adaptation of the genotypes was estimated by their effect on reproductive efficiency in the experimentally obtained population. Fecundity, lifespan, and the resistance of the studied genotypes to hyperthermia were investigated individually. It was shown that the high level of adaptation of the cn mutants and the low level of adaptation of the vg mutants was correlated with the presence of different ADH allozymes. In the studied population, the F-allozyme of ADH accompanied the vg mutation, while the S-allozyme of the enzyme was detected in cn mutants. Saturating crossings of C-S(Adh ^S)×vg(Adh ^F) and D(Adh ^F) × cn(Adh ^S), along with the parallel determination of the allele composition of the Adh locus, demonstrated that the complete substitution of the F-allozyme of ADH in the vg mutants by the S-allozyme in D flies, as well as the substitution of the S-allozyme of ADH in the cn mutants by the F-allozyme in D flies was realized only after the 15th–20th backcrosses. These results favor the coadaptation of cn and vg marker genes with alleles of the Adh locus and indicate the important role of the latter in the adaptation of genotypes. In the studied population, selection acted primarily against the vg mutants, which were inferior to the cn mutants, and heterozygote genotypes in indices of the main adaptation components.