Steric effects at C-20 and C-24 on the metabolism of sterols by Tetrahymena pyriformis

Cultures of Tetrahymena pyriformis were incubated with various sterols and the extent of dehydrogenation at C-7 and C-22 was determined. The sterols incubated were desmosterol, 22-dehydrodesmosterol, 24-methyldesmosterol, 24 alpha-methylcholesterol (campesterol), 24-methylene-cholesterol, isohalosterol (26,27-bisnorcampesterol, also known as 24,24-dimethylchol-5-en-e beta-ol, a naturally occurring C26-sterol), and 20-isohalosterol. 20-Isohalosterol was not metabolized, while products with delta 7- and delta 22-bonds were formed from isohalosterol and all of the other sterols studied. This confirms an earlier conclusion, based on results with 20-isocholesterol and cholesterol, that inversion of the configuration from 20(R) to 20(S) completely prevents metabolism both in the nucleus and the side chain. On the other hand, changes in the electronics or stereochemistry at C-24 had a direct affect only on metabolism in the side chain. The presence of a methyl group at C-24 reduced the yield of metabolites with a delta 22-bond relative to those with a delta 7-bond producing an accumulation of 7-dehydro metabolite. A double bond at position-24 counteracted this steric effect, presumably by enhancing the rate of dehydrogenation, and a delta 24(28)-bond was more effect than was a delta 24(25)-bond.     

Assigning stereodiversity of the 27-Me group of furostane-type steroidal saponins via NMR chemical shifts

Applicability of (13)C and (1)H NMR chemical shifts for the assignment of the 25R/25S configuration of the 27-methyl group in the case of furostane-type steroidal saponins has been investigated. A comparative study of (13)C NMR data suggest that chemical shift values for C-20, C-21, C-22, C-23, C-24, C-25, C-26 and C-27 resonances were not much influenced by R/S configuration of the 27-Me group, thus reflecting limited application of (13)C NMR chemical shifts for such stereochemical determinations. In contrast, (1)H NMR chemical shifts (delta(a), delta(b)) for geminal protons of glycosyloxy methylene (H(2)-26) exhibit pronounced dependence and the difference (Delta(ab)=delta(a)-delta(b)) among their chemical shifts [Delta(ab)= or <0.48 for 25R; Delta(ab)= or >0.57 for 25S] seems to be of general applicability for ascertaining 25R/25S orientation of the 27-methyl group of furostane-type steroidal saponins.     

Sterols of some Clerodendrum species (Verbenaceae): occurrence of the 24 alpha- and 24 beta-epimers of 24-ethylsterols lacking a delta 25-bond

The configurations at C-24 of 24-alkylsterols of six samples of Clerodendrum species (Verbenaceae) - the aerial parts of C. fragrans, C. inerme, C. infortunatum, C. scandens, and C. siphonanthus, and the seeds of C. infortunatum - were examined by NMR. All samples contained 24 beta-ethylsterols possessing a delta 25-bond, clerosterol and 22-dehydroclerosterol, as the dominant sterol components. The other 24-ethylsterols lacking a delta 25-bond, 24-ethyl-22-dehydrocholestanol, 24-ethylcholesterol, and 24-ethyl-22-dehydrocholesterol, which were present as minor components, were shown to be mixtures of the 24 alpha- and 24 beta-epimers, with the 24 alpha-epimers predominating in all cases. Four minor 24-methyl-sterols, 24-methylcholestanol, 24-methylcholesterol, 24-methyl-22-dehydrocholesterol, and 24-methyllathosterol, were shown to be C-24 epimeric mixtures, whereas two others, 24-methyl-22,25-bisdehydrocholesterol and 24-methyl-22-dehydrolathosterol, were found to be present only as the 24 beta-epimers. This is the first report of the occurrence of 24 beta-ethyl-22-dehydrocholestanol in higher plants.     

Sterol methyltransferase2: purification, properties, and inhibition

Expression of the Arabidopsis sterol methyltransferase2 (SMT2) cDNA in Escherichia coli yields a native protein, when purified to homogeneity, has the predicted molecular mass ca. 40 kDa on SDS-PAGE and recognizes native sterols synthesized by Arabidopsis with a Delta(24(25))-bond (cycloartenol; K(m) 35 microM and k(cat) 0.001s(-1)) and Delta(24(28))-bond (24(28)-methylenelophenol; K(m) 28 microM and k(cat) 0.01 s(-1)). Cycloartenol was converted to a single olefinic product-24(28)-methylenecycloartanol whereas 24(28)-methylenelophenol was converted to a mixture of three stereochemically related products with the Delta(24(28))Z-ethylidene, Delta(24(28))E-ethylidene, and Delta(25(27))-24 beta-ethyl side chains. Structural determinants essential to activity were the nucleophilic features at C-3 and C-24. The double bond position in the sterol substrate influenced catalytic efficiency according to the order: side chain, Delta(24(24))相似文献   

NMR spectra of C-24 isomeric sterols

Cholesterol and four pairs of C-24 isomeric sterols, campesterol-22,23-dihydrobrassicasterol, α-spinasterol-chondrillasterol, stigmasterol-poriferasterol, and sitosterol-22,23-dihydroporiferasterol were studied by NMR spectroscopy and their spectra are presented. The NMR spectra of three of the pairs of isomeric sterols recorded at 100 MHz could be differentiated from each other, although at 60 MHz only the spectra of campesterol (24α-methylcholesterol) and 22,23-dihydrobrassicasterol (24β-methylcholesterol) showed differences. Sitosterol and 22,23-dihydroporiferasterol, the pair of sterols that showed no differences in their NMR spectra are readily differentiated by the physical properties of their acetates. The practical application of NMR spectroscopy to several problems concerning the C-24 isomeric sterols is demonstrated.     

Absolute configuration at C-24 of 5 beta-ranol, a principal bile alcohol of the bullfrog

The stereochemistry of the hydroxyl group at C-24 in 5 beta-ranol (27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,26-pentol) a principal bile alcohol of the bullfrog which is structurally related to the major human urinary bile alcohol, 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol, is described. Two isomers (IIIa and IIIb) at C-24 of 27-nor-5 beta-cholest-25-ene-3 alpha,7 alpha,12 alpha, 24-tetrol were synthesized from cholic acid (I) by the conversion to 3 alpha, 7 alpha, 12 alpha-triacetoxy-5 beta-cholan-24-al (II) followed by a Grignard reaction with vinylmagnesium bromide. The absolute configurations at C-24 of the unsaturated tetrols (IIIa and IIIb) were elucidated as S and R, respectively, by means of the difference of the reactivity to Sharpless oxidation, a stereoselective epoxidation. Catalytic hydrogenation of each delta 25-tetrol (IIIa or IIIb) gave (24R)- or (24S)-27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha, 24-tetrol (IVa or IVb). The configurations at C-24 of two isomeric 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-27-nor-5 beta-cholestan-26-oic acids (Va and Vb) were determined as S and R, respectively, by means of their conversion into the saturated tetrols (IVa and IVb) of known absolute configurations by a Kolbe electrolytic coupling with acetic acid. The lithium aluminum hydride reduction product of the 24R-acid (Vb) was identical with the naturally occurring 5 beta-ranol, hence 5 beta-ranol has the 24R configuration.     

3,3]-Claisen rearrangements in 24alpha-methyl steroid synthesis. Application to campesterol, crinosterol, and Delta25-crinosterol side chain construction

This paper elaborates an improved synthesis of crinosterol and campesterol starting from stigmasterol. The proposed approach is based on Claisen rearrangement of Delta23-22-allylic alcohols with various configurations of the 22-hydroxy group and geometry of the Delta23-double bond. It allows complete use of the starting steroid for preparing 24alpha-methyl derivatives. It was possible to partially control the stereochemistry at C-25. Hydrogenation of the Delta22-double bond was shown to proceed with partial isomerization of the C-24 alkyl substituent. The Ireland ester enolate variant of the Claisen rearrangement was demonstrated to be useful for preparing 24alpha-methyl steroids containing the Delta22,25-system.     

Studies on the C-24 configurations of Δ7-sterols in theseeds of Cucurbita maxima

Uncertainties surrounding the structures of the Δ⁷-sterols in the seeds of Cucurbita maxima have been resolved. Seven components were found by TLC, GLC, HPLC, mass spectrometry and ¹H NMR. They were 24β-ethyl-5α-cholesta-7,22,25(27)-trien-3β-ol, 24β-ethyl-5α-cholesta-7,25(27)-dien-3gb-ol, avenasterol, spinasterol, 24-dihydrospinasterol, 24ζ-methyllathosterol and 25(27)-dehydrofungisterol. The ¹H NMR spectra indicated that the sterols with an ethyl substituent at C-24 occurred in the absence of their C-24 epimers. This seems to be the first instance of the detection of 25(27)-dehydrofungisterol in a higher plant.     

Efficient preparation of steroidal 5,7-dienes of high purity.

Protected forms of dehydroepiandrosterone, delta 5 cholenic acid, (25R)-26-hydroxycholesterol and diosgenin were converted to the corresponding delta 5,7 dienes by successive treatment with 1,3-dibromo-5,5-dimethylhydantoin (dibromantin), tetrabutylammonium bromide and tetrabutylammonium fluoride. The crude products, which contained the delta 5,7 species contaminated by minor amounts of the delta 5 and delta 4,6 steroids, were purified by silica gel-AgNO3 chromatography to give the following steroids in approximately 99% purity and at least 50% yield: 3 beta-acetoxyandrosta-5,7-dien-17-one, methyl 3 beta-acetoxychola-5,7-dien-24-oate, (25R)-3 beta,26-diacetoxycholesta-5,7-diene and (25R)-3 beta-acetoxyspirosta-5,7-diene. Analogous treatment of acetate derivatives of pregnenolone and stigmasterol gave 3 beta-acetoxypregna-5,7-dien-20-one and 3 beta-acetoxystigmasta-5,7,22-triene in approximately 50% yield but of lower purity. Full 1H and 13C NMR assignments are given for seven delta 5,7 steroid acetates and the corresponding delta 5 starting materials. Coupling constants for rings A, B and C of delta 5,7 steroids are presented and stereochemical assignments have been made for the following 1H NMR signals: the C-11 protons of delta 5,7 steroids, the C-16 protons of sterols and bile acids, the C-22 and C-23 protons of bile acid esters and the C-28 protons of stigmasterol derivatives.     

Determination of the anomeric specificity of the Escherichia coli CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase by 13C NMR spectroscopy

[99%, 1-13C]- and [90%, 2-13C]3-deoxy-D-manno-octulosonic acid (KDO) were prepared enzymatically and used to determine the anomeric specificity of the CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyl transferase (CMP-KDO synthetase) by 13C NMR spectroscopy. Addition of CMP-KDO synthetase to reaction mixtures containing either 1-13C- or 2-13C-labeled KDO resulted in rapid CMP-KDO formation which was accompanied by a substantial decrease in the 13C-enriched resonances of the beta-pyranose form of KDO relative to the resonances of other KDO species in solution, demonstrating that the beta-pyranose is the preferred substrate. Concomitant with the production of CMP-KDO was the appearance of peaks at 174.3 and 101.4 ppm when [1-13C]- and [2-13C]KDO, respectively, were used as substrates. The correspondence of these resonances to the enriched carbons in CMP-KDO was confirmed by the expected 3-bond (3JP,C-1 = 6.9 Hz) and 2-bond coupling (2JP,C-2 = 8.3 Hz) between the labeled carbons and the ketosidically linked phosphoryl group. A large coupling (3J = 5.7 Hz) was observed in proton-coupled spectra of CMP-[1-13C]KDO between carbon 1 and the axial proton at carbon 3 of KDO. The magnitude of this coupling constant supports a diaxial relationship between these two groups and, along with chemical shift data, indicates that KDO retains the beta-configuration when linked in CMP-KDO.     

Characterization of the structure of a 4-methyl-delta 8,24-cholestadien-3 beta-ol isolated from rat skin

A new sterol has been isolated from the skin of rats treated with triparanol. Its chromatographic behavior on silicic acid-Celite columns and in gas-liquid chromatographic systems indicated it to be a 4-methyl-Delta(8,24)-cholestadien-3beta-ol. The specific rotation, the delayed color reaction with Liebermann-Burchard reagent, and the nuclear magnetic resonance (NMR) data support the Delta(8(9))-unsaturation. Previous workers have shown that triparanol treatment results in an accumulation of Delta(24)-unsaturated sterols in animal tissues. Consonant with this observation, the infrared, NMR, and mass spectrometric data confirm the presence of a C-24(25) unsaturated side chain in this sterol.     

The 220 MHz NMR spectra of phytosterols

The 220MHz NMR spectra of forty two steroids are reported. Eight pairs of C-24 epimers (24α- and 24β) and two pairs of double bond isomers (cis and trans) can be distinguished by this technique. The influence of substituents, solvents and stereochemistry on methyl group chemical shifts is discussed.     

Phytosterol biosynthesis pathway in Mortierella alpina

The Zygomycetes fungus Mortierella alpina was cultured to growth arrest to assess the phytosterol biosynthesis pathway in a less-advanced fungus. The mycelium was found to produce 13 sterols, but no ergosterol. The sterol fractions were purified to homogeneity by HPLC and their identifies determined by a combination of GC-MS and 1H NMR spectroscopy. The principal sterol of the mycelium was cholesta-5, 24-dienol (desmosterol) (83%), with lesser amounts of 24beta-methyl-cholesta-5,25(27)-dienol (codisterol) (2%), 24-methyldesmosterol (6%), 24(28)-methylene cholesterol (3%) and lanosterol (3%) and several other minor compounds (3%). The total sterol accounted for approximately 0.07% of the mycelial dry wt. Mycelium fed methionine-methyl-2H3 for 6 days, generated 3 2H-24-methyl(ene) sterols, [C28-2H2]24(28)-methylenecholesterol, [C28-2H3]24-methylcholesta-5,24-dienol and [C28-2H3]24beta-methyl-cholesta-5,25(27)-dienol. The formation of the 24-methyl sterols seems to be catalyzed by the direct methylation of a common Delta24-acceptor sterol thereby bypassing the intermediacy of an isomerization step for rearrangement of the Delta24(28)-bond to Delta25(25)-position as operates in Ascomycetes fungi and all plants.     

Highly active analogs of 1alpha,25-dihydroxyvitamin D(3) that resist metabolism through C-24 oxidation and C-3 epimerization pathways

The secosteroid hormone 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is metabolized in its target tissues through modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the main side chain modification pathway is initiated by hydroxylation at C-24 of the side chain and leads to the formation of the end product, calcitroic acid. The C-23 and C-26 oxidation pathways, the minor side chain modification pathways are initiated by hydroxylations at C-23 and C-26 of the side chain and lead to the formation of the end product, calcitriol lactone. The C-3 epimerization pathway, the newly discovered A-ring modification pathway is initiated by epimerization of the hydroxyl group at C-3 of the A-ring to form 1alpha,25(OH)(2)-3-epi-D(3). A rational design for the synthesis of potent analogs of 1alpha,25(OH)(2)D(3) is developed based on the knowledge of the various metabolic pathways of 1alpha,25(OH)(2)D(3). Structural modifications around the C-20 position, such as C-20 epimerization or introduction of the 16-double bond affect the configuration of the side chain. This results in the arrest of the C-24 hydroxylation initiated cascade of side chain modifications at the C-24 oxo stage, thus producing the stable C-24 oxo metabolites which are as active as their parent analogs. To prevent C-23 and C-24 hydroxylations, cis or trans double bonds, or a triple bond are incorporated in between C-23 and C-24. To prevent C-26 hydroxylation, the hydrogens on these carbons are replaced with fluorines. Furthermore, testing the metabolic fate of the various analogs with modifications of the A-ring, it was found that the rate of C-3 epimerization of 5,6-trans or 19-nor analogs is decreased to a significant extent. Assembly of all these protective structural modifications in single molecules has then produced the most active vitamin D(3) analogs 1alpha,25(OH)(2)-16,23-E-diene-26,27-hexafluoro-19-nor-D(3) (Ro 25-9022), 1alpha,25(OH)(2)-16,23-Z-diene-26,27-hexafluoro-19-nor-D(3) (Ro 26-2198), and 1alpha,25(OH)(2)-16-ene-23-yne-26,27-hexafluoro-19-nor-D(3) (Ro 25-6760), as indicated by their antiproliferative activities.     

Use of S-Mosher acid as a chiral solvating agent for enantiomeric analysis of some trans-4-(4-fluorophenyl)-3-substituted-1-methylpiperidines by means of NMR spectroscopy.

S-Mosher acid 1 induced chemical-shift differences (Delta delta) in NMR spectra of chiral trans-4-fluorophenyl-3-substituted-1-methylpiperidines. The magnitude of Delta delta in (1)H and (19)F NMR spectra varied with the substituent at position 3 of the piperidine ring. The magnitudes of Delta delta observed for certain protons and for the fluorine in the 4-fluorophenyl group were sufficiently large to allow determination of enantiomeric composition.     

Identification of 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 xi, 25 xi,26-hexol and partial characterization of the bile alcohol profile in urine

The nature of the bile alcohols present in urine of an infant with neonatal cholestasis has been investigated. Urine was extracted with Sep-Pak C18 cartridges and a glucuronide fraction was isolated by ion exchange chromatography on Lipidex-DEAP. Following enzymatic hydrolysis and purification on Lipidex-DEAP, the bile alcohols were isolated by high performance liquid chromatography. Fourteen compounds were studied by a combination of microchemical reactions and capillary column gas-liquid chromatography-mass spectrometry. Both C26 and C27 bile alcohols were present. Among the former, three additional isomers of the previously identified 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 xi,25 xi-pentol were detected. A new C26 bile alcohol, 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 xi,25 xi,26 -hexol, was identified, and a 27-norcholestane-pentolone with hydroxyl groups at C-24 and C-25 and a keto group in the ring system was partially characterized. The C27 bile alcohols consisted of cholestanepentols, -tetrolones, and -pentolones. 5 beta-Cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol (5 beta-bufol), one of its isomers and an isomer of cholestane-3,7,12,24,26-pentol were present. Two cholestanetetrolones and two cholestanepentolones having the keto group in the ring system were partially characterized. The hydroxyl groups in the side chain of the tetrolones were at C-24,26 and C-25,26, respectively, whereas the pentolones had hydroxyl groups at C-24,25 and C-25,26, respectively. The excretion of glucuronidated bile alcohols in urine is suggested to reflect an alternative metabolism of intermediates in the normal biosynthesis of bile acids.     

Synthesis of an isomeric mixture (24RS,25RS) of sodium scymnol sulfate

This is the first reported multistep synthesis of the shark bile sterol sodium scymnol sulfate epimeric at the C-24 hydroxyl and C-27 sulfate positions. The starting cholic acid was protected as the tetrahydropyran ether (THP) derivative, reduced to the C-24 alcohol and oxidized to the protected aldehyde. This aldehyde was then coupled with methyl 3-hydroxypropionate using 2equiv. of lithium diethylamide at -65 degrees C to produce methyl (24RS,25RS)-24,27-dihydroxy-3alpha,7alpha,12alpha,tris[(tetrahydropyran-2-yl)oxy]-5beta-cholestan-26-oate. After protecting the 24 and 27 hydroxyls as the THP derivatives, this fully protected ester was then reduced to the monoalcohol. The monoalcohol was sulfated using the sulfur trioxide-triethylamine complex in dimethylformamide. The protective THP groups were removed with methanolic HCl and the sulfate was converted to the sodium salt with sodium ethoxide in methanol. This general synthetic scheme has application to produce a range of monosulfated sterols.     

Codisterol and other Δ5-sterols in the seeds of Cucurbita maxima

A substantial amount (ca 18%) of the sterol found in the seeds of Cucurbita maxima had a Δ-bond and consisted of seven components. They were identified as 25(27)-dehydroporiferasterol, clerosterol, isofucosterol, stigmasterol, sitosterol, campesterol and codisterol. The C-24 configuration of each of the sterols was unequivocally established by a ¹H NMR spectral comparison with authentic standards. This is the first time codisterol has been found in a higher plant and also the first time the structures and configurations of the Δ⁵-sterols from a Cucurbitaceae species have been clearly characterized.     

Proton magnetic resonance assay of total and taurine-conjugated bile acids in bile.

Biliary bile acids, coexisting with phospholipid and cholesterol, are partly conjugated with taurine. In the present report we show that total and taurine-conjugated bile acids in bile can be simultaneously and quantitatively measured by high-resolution (1)H-nuclear magnetic resonance ((1)H-NMR) spectroscopy. We used a 7.05-Tesla NMR spectrometer to obtain the (1)H-NMR spectra of model and biological biles. Only addition of trimethylsilyl-3-propionic acid sodium salt-D(4) (TSP) to each sample as an internal standard was required in preparation for (1)H-NMR measurement. In (1)H-NMR spectra of rat bile, peaks of C-18 methyl protons of bile acids and of C-25 methylene protons on the taurine moiety of taurine-conjugated bile acids were detected at 0.7 ppm and 3.1 ppm, respectively. Peak areas, of C-18 and C-25 peaks, increased in proportion to the concentrations of bile acids or taurine-conjugated bile acids, even in the presence of phospholipid and cholesterol. The accuracy of NMR measurement of total and taurine-conjugated bile acids was confirmed by comparing the results of NMR with those of enzyme-fluorimetry.The results clearly demonstrate that (1)H-NMR spectroscopy can be applied to the quantitative determination of total and taurine-conjugated bile acids in bile without troublesome preparative steps.     

A new approach to the side chain formation of 24-alkyl-22-hydroxy steroids: application to the preparation of early brassinolide biosynthetic precursors

A new synthetic route to 22S-hydroxy-24R-methyl steroids has been developed and applied for the preparation of cathasterone, (22S)-hydroxycampesterol, and 6-deoxocathasterone, which are precursors in the early stages of the biosynthesis of brassinolide. The construction of the steroid side chain with the correct stereochemistry at C-24 is based on the use of Claisen rearrangement. The introduction of the 22-hydroxyl group has been achieved by epoxidation of the Δ(22)-double bond, nucleophilic opening of the intermediate mesyl epoxide with sodium sulfide, and desulfurization of the formed tetrahydrothiophenes with Raney nickel.