Shujiro Seo

Studies on fungal metabolites—XXXI: Anthraquinonoid colouring matters of Penicillium islandicum sopp and some other fungi (−)luteoskyrin, (−)rubroskyrin, (+)rugulosin and their related compounds

Abstract The structures of (+)rugulosin, (−)luteoskyrin and (−)rubroskyrin have been reexamined by NMR and new structures 18, 19 and 20 proposed respectively. Their absolute structures were established on the basis of the X-ray analysis of (+)dibromodehydrotetrahydrorugulosin (27). The minor analogous metabolites, (−)4a-oxyluteoskyrin (31) of P. islandicum and (+)4a-oxyrugulosin (32) of P. brunneum, have been formulated. On oxidation of (−)luteoskyrin and (+)rugulosin with pertrifluoroacetic acid (−)4a,4a′-dioxyluteoskyrin (33) and (+>4a,4a′-dioxyrugulosin (34) were formed, while with MnO2 (+)4a,4a′-dehydrorugulosin (35) was obtained. The structures of lumiluteoskyrin (37), and deoxylumiluteoskyrin (38), photooxidation products of luteoskyrin and deoxyluteoskyrin, respectively, have been elucidated.

Carbon-13 nmr spectra of 5β-steroidal sapogenins. Reassignment of the F-ring carbon signals of (25S)-spirostans

Abstract All 13 C NMR signals of 5β-steroidal sapogenins were assigned; in particular, the F-ring carbon signals were reassigned using deuteriated diotigenin ( 12 ) derivatives and the INEPT (insensitive nuclei enhanced by polarization transfer) spectra.

Studies on fungal metabolites—XXXII: A renewed investigation on (−) flavoskyrin and its analogues

Abstract 1-Oxo-1,2,3,4-tetrahydroanthraquinone ( 4a ), and its 8-hydroxy-( 4b ) and 8-hydroxy-6- methyl ( 4c ) derivatives were dimerized to the compounds formulated as ( 6a ), ( 6b ) and ( 6e ), respectively. The structure of 6a was confirmed by X-ray crystallographic analysis. By the analogy with these dimers and NMR spectral analysis, a revised structure ( 7 ) was proposed for (−) flavoskyrin, a yellow metabolite of Penicillium islandicum NRRL 1175. A biosynthetic scheme involving Diels-Alder type cyclo-addition (π4s + π2s) was proposed for (−) flavoskyrin.

Purification of squalene-2,3-epoxide cyclases from cell suspension cultures of Rabdosia japonica Hara

Microsomes prepared from cell suspension cultures of Rabdosia japonica Hara showed activities for cyclizing squalene‐2,3‐epoxide into cycloartenol, β‐amyrin and α‐amyrin in the presence of Triton X‐100. These activities were efficiently solubilized by treatment with Triton X‐100 and separated by chromatography on hydroxy apatite, DEAE‐cellulose, isoelectric focusing and gel filtration. The purified cycloartenol cyclase showed a single band on SDS‐polyacrylamide gel electrophoresis with Mr = 54000, while β‐amyrin cyclase gave a single band with Mr = 28000.

Effect on ergosterol biosynthesis of a fungicide, SSF-109, in Botrytis cinerea

Abstract Treatment of Botrytis cinerea with a novel fungicide SSF-109, (dl)-cis-1-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)cyclo-heptanol (0.45μgml−1), gave five 14α-methyl sterols, 24-methylene-24(25)-dihydrolanosterol, 24-methylene-24(25)-dihydrolanosten-3-one, obtusifoliol, obtusifolione, and 14α-methylfecosterol, together with ergosterol and ergosta-5,8,22-trien-3β-ol. SSF-109 was found to inhibit the biosynthesis of ergosterol at the 14α-demethylation step.

Biosynthesis of sitosterol, cycloartenol, and 24-methylenecycloartanol in tissue cultures of higher plants and of ergosterol in yeast from [1,2-13C2]- and [2-13C2H3]-acetate and [5-13C2H2]MVA

The [1,2]-methyl migrations postulated in the ‘biogenetic isoprene rule’ proposed by Ruzicka et al. have been verified by 13C n.m.r. spectroscopy in the biosynthesis of cycloartenol (10a), 24-methylenecycloartanol (11a), and sitosterol (12a) using cultured cells of higher plants,Rabdosia japonica and Physalis peruviana, and of ergosterol (14a) in yeast fed with [1,2-13C2] acetate. The [1,2]-hydride shifts from C-17 to C-20, and C-13 to C-17 have also been demonstrated in the biosynthesis of sitosterol (12b) in R. japonica and of ergosterol (14b) in yeast fed with [2-13C2H3]acetate. The [1,2]-hydride shift from C-9 to C-8 has also been verified in 24-methylenecycloartanol (11b) fed [2-13C2H3]acetate to tissue cultures of Trichosanthes kirilowii Maxim. var. japonica. In the side-chain formation of 24-methylenecycloartanol (11b) and ergosterol (14b), a [1,2]-hydride (deuteride) shift from C-24 to C-25 is observed. Conversely, no deuterium atom at C-24 or C-25 is observed in sitosterol (12b) formation. Both C-11 and C-12 of sitosterol (12c) labelled as 13C-2H2 and 13C-2H1H, biosynthesized from [5-13C2H2]MVA in R. japonica suggest that squalene is released from an enzyme and the following oxidation does not distinguish a terminal double bond of one farnesyl moiety from the other to form epoxysqualenes (8A) and (8B).

Age-related changes of bile acid metabolism in rats

Cholesterol and bile acid leves were examined in young (8 weeks), middle-aged (12 months) and old (24 months) germ-free male rats, and young (8 weeks) and middle-aged (12 months) conventional male rats. The plasma cholesterol levels were higher in the aged rats, being more marked in the conventional rats. The liver cholesterol levels also increased with age and the increases were almost identical for both groups. No age-related changes were found in the biliary bile acid secretion, the pool size and distribution of bile acids in the bile, small intestine and large intestine, nor in the turnover frequency of bile acids, but the pool size in the young and middle-aged germ-free rats was much larger than that in the conventional rats. The turnover frequency was less in the germ-free rats. The bile acid synthesis presumed from the fecal bile acid excretion decreased in the aged germ-free rats but not in the conventional rats. A most remarkable age-related change was found in the bile acid composition; cholic acid increased and beta-muricholic acid derived from chenodeoxycholic acid in the rat decreased by aging, resulting in an increase of the CA/CDCA ratio (bile acids belonging to the cholic acid group/bile acids to the chenodeoxycholic acid group) in the bile, feces and pool. These results suggest that cholic acid synthesis increases while chenodeoxycholic acid synthesis is impaired by aging in rats.

Stereoselective synthesis of the C-24 and C-25 stereoisomeric pairs of 24-ethyl-26-hydroxy- and 24-ethyl-[26-2H]sterols and their Δ22-derivatives: reassignment of 13C n.m.r. signals of the pro-R and the pro-S methyl groups at C-25 of 24-ethylsterols

Two C-24 and C-25 epimeric pairs of 24-ethyl-26-acid sterol derivatives (22)–(25) were stereoselectively synthesized via an ester-enolate Claisen rearrangement of the (22R)-(20) and (21) and (22S)-23E-ene derivatives (18) and (19). The absolute configuration at C-24 and C-25 of methyl (22E,24S,25S)-6β-methoxy-3α,5-cyclo-5α-stigmast-22-en-26-oate (22a) and (22E,24R,25R)-6β-methoxy-3α,5-cyclo-5α-stigmast-22-en-26-oic acid (24) were confirmed by X-ray crystallography. Four C-24 and C-25 epimeric pairs of 24-ethyl-[26-2H]-(34)–(37) and 24-ethyl-26-hydroxy-sterols (42)–(45) and their Δ22-derivatives (38)–(41) and (46)–(49) have been synthesized in order to study the stereochemistry in the biosynthesis of (24R)- and (24S)-24-ethylsterol side-chains. The data showed that C-26 and C-27 arise biosynthetically from C-6 and C-2 of MVA, respectively.

Biosynthesis of campesterol and dihydrobrassicasterol in cultured cells of Amsonia elliptica

Abstract Reversed phase HPLC analysis of sterols of cultured cells of Amsonia elliptica showed that campesterol is the major sterol. Campesterol and its 24-epimer, dihydrobrassicasterol, were isolated from suspension cultures of A. elliptica incubated with l -[ Me - 2 H 3 ]methionine. Mass spectral analysis showed that both 24-epimeric 24-methylcholesterols retained two deuterium atoms. This result indicates that campesterol and dihydrobrassicasterol are biosynthesized via an intermediate with the 24-methylene side-chain.

Synthesis of (25R)-[26-2H1]cholesterol and 1H n.m.r. and h.p.l.c. resolution of (25R)- and (25S)-26-hydroxycholesterol

Yamogenin acetate (1) was isolated from crude diosgenin acetate and converted into (25S)-26-hydroxycholesterol (6a). The absolute configuration at C-25 of (6a) was determined by X-ray crystallography. (25R)-[26-2H1] Cholesterol (10) was prepared by reduction of the 26-tosyloxy group by LiAl2H4. Reverse-phase h.p.l.c. resolution without derivatiration was developed for the diastereoisomers, (25R)- and (25S)-26-hydroxycholesterol. The (+)- or (–)-MTPA esters of these diastereoisomers showed distinctive 1H n.m.r. signals for 26-H.