The transfer of hydrogen from C-24 to C-25 in ergosterol biosynthesis

1. A convenient synthesis of 3-hydroxytrisnorlanost-8-en-24-al and its conversion into [24-(3)H]lanosterol and [26,27-(14)C(2)]lanosterol is described. 2. A method for the efficient incorporation of lanosterol into ergosterol by the whole cells of Saccharomyces cerevisiae is also described. 3. It is shown that in the biosynthesis of ergosterol from doubly labelled lanosterol the C-24 hydrogen atom of lanosterol is retained in ergosterol. 4. On the basis of unambiguous degradations it is shown that the C-alkylation step in ergosterol biosynthesis is accompanied by the migration of a hydrogen atom from C-24 to C-25. 5. The mechanism for the biosynthesis of the ergosterol side chain is presented. 6. Mechanisms of other C-alkylation reactions are also discussed.     

Confirmation of the Relative Stereochemistry at C-23 and C-24 of Tautomycin

Degradation product including the moiety for C-19 through to C-25 of tautomycin were synthesized. This synthesis confirmed the relative stereochemistry at C-23 and C-24 of tautomycin.     

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.     

Nuclear magnetic resonance and infrared spectra of delta-24- and C-24 saturated steroids

The infrared (IR) and nuclear magnetic resonance (NMR) spectra of eight Delta(24)-steroids and nine C-24 saturated steroids were examined. NMR spectra allow unambiguous assignment of the biologically important Delta(24)-bond; introduction of a Delta(24)-bond causes the appearance of peaks at Delta 1.60 and 1.68 associated with the C-26, C-27 isopropylidene methyls, while C-24 saturated steroids of the cholestane series possess peaks at Delta 0.82 and 0.91 associated with the C-26, C-27 gem-dimethyls. IR spectra show a good correlation between the introduction of a Delta(24)-bond and a marked decrease in intensity of a band at 1365 cm(-1). NMR and IR spectra also allow an inference about the presence and location of nuclear double bonds in Ring B of cholesterol precursors.     

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.     

The detergent Solulan C-24 reveals properties of the olfactory adenylate cyclase system.

We have been developing the use of plasma-membrane-bound fluorescent probes to measure the pH values at the surfaces of living chondrocytes. For this purpose, three lipophilic pH indicators were made by covalently binding the xanthene dyes fluorescein, eosin or dichlorofluorescein to the amino group of phosphatidylethanolamine. The probes were incorporated into phospholipid vesicles and the effect of pH on the fluorescence was characterized. Fluorescence was measured at a single emission wavelength during excitation at two wavelengths, and the ratio of the intensities was calculated. The experimentally observed pKobs. values were determined by fitting the fluorescence ratios to the Henderson-Hasselbalch equation. All three probes acted as pH indicators, and the eosinyl-, dichlorofluoresceinyl- and fluoresceinylphosphatidylethanolamines had pKobs. values of 3.5, 6.3 and 7.5 respectively. At physiological salt concentrations, changes in the composition of the vesicle membrane had little effect on these values. We concluded that these probes were promising candidates for the measurement of pH values at cell surfaces.     

甾醇C-24甲基转移酶和甾醇C-8异构酶在酿酒酵母麦角甾醇生物合成中的调控作用

摘要：【目的】研究ERG6基因编码的甾醇C-24甲基转移酶和ERG2基因编码的甾醇C-8异构酶在酿酒酵母麦角甾醇生物合成代谢中的调控作用。【方法】通过PCR扩增克隆到酿酒酵母甾醇C-8异构酶的编码序列及其终止子序列,以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体,以磷酸甘油酸激酶基因PGK1启动子为上游调控元件构建了酵母菌表达质粒pPERG2;同时,在本实验室已构建的ERG6表达质粒pPERG6的基础上,构建了ERG2和ERG6共表达的重组质粒pPERG6-2。将表达质粒转化酿酒酵母单倍体菌株YS58,依据营养缺陷互补筛选到重组菌株YS58(pPERG2)和YS58(pPERG6-2)。通过紫外分光光度法和气相色谱法分析重组菌株甾醇组分和含量。【结果】在ERG6高表达的重组酵母菌中,甾醇中间体和终产物麦角甾醇的含量均比对照菌高;而在ERG2高表达的酵母菌株中,无论甾醇中间体,还是麦角甾醇的含量均明显降低。ERG6和ERG2共表达重组菌株YS58(pPERG6-2)的麦角甾醇含量是对照菌株YS58(YEp352)的1.41倍,是ERG2单独高表达菌株YS58(pPERG2)的1.92倍,是ERG6单独高表达菌株YS58(pPERG6)的1.12倍。【结论】本研究首次证明甾醇C-24甲基转移酶催化的反应是酿酒酵母麦角甾醇合成代谢途径中的一个重要的限速步骤,该酶活性提高不但补偿了ERG2高表达对甾醇合成的负效应,而且使麦角甾醇含量进一步提高,为构建麦角甾醇高产酵母工程菌株提供了实验依据。     

A facile synthesis of C-24 and C-25 oxysterols by in situ generated ethyl(trifluoromethyl)dioxirane

Experiments were performed to compare the regioselective hydroxylation of the isopropyl C-H bond at C-25 in 5α-cholestan-3β-yl acetate by in situ generated dimethyldioxirane, methyl(trifluoromethyl)dioxirane, hexafluoro(dimethyl)dioxirane or ethyl(trifluoromethyl)dioxirane (ETDO). The dioxiranes were generated from the corresponding ketones and potassium peroxymonosulfate in aq. NaHCO₃, pH 7.5-8.0. Of the four dioxiranes examined, partially fluorinated, sterically bulky ETDO displayed the highest reactivity and regioselectivity. Using in situ generated ETDO, a facile, synthesis was developed for two naturally occurring oxysterols, i.e., 25-hydroxycholesterol, as well as its 3-sulfate (overall yield of the sulfate, 24%) and 24-oxocholesterol (16%), starting from cholesterol.     

The mechanism of alkylation at C-24 during clionasterol biosynthesis in Monodus subterraneus

Clionasterol isolated from Monodus subterraneus grown in the presence of methionine-[methyl-²H₃] contained four ²H atoms showing the participation of a 24-ethylidene sterol intermediate in its biosynthesis. Clionasterol isolated from M. subterraneus grown in the presence of mevalonic acid-[2-¹⁴C,(4R)-4-³H₁ had a ¹⁴C:₃H atomic ratio of 5:3 indicating that the 24-ethylidene sterol intermediate is reduced directly to clionasterol and not isomerized to a Δ²⁴-sterol which is then reduced.     

On the side-chain conformation of N-acetylneuraminic acid and its epimers at C-7, C-8, and C-7,8

The side-chain conformation of N-acetylneuraminic acid and analogs has been studied by n.m.r. spectroscopy. The results of the 1H-, 13C-n.m.r.-, and 1H-nuclear-Overhauser-enhancement measurements were used to distinguish between different local-minima conformations suggested by hard-sphere calculations. Attempts were made to correlate the major conformation determined for each compound with the behavior towards activation with N-acetylneuraminic acid-CMP-synthetase.     

Configuration at C-24 of sterols from the liverwort Palavicinnia lyellii

The 4-desmethylsterol fraction of the liverwort Palavicinnia lyellii is composed of 36% 24β-methylcholest-5-en-3β-ol (dihydrobrassicasterol), 16% 24α-methylcholest-5-en-3β-ol (campesterol), 33% 24α-ethylcholest-5-en-3β-ol (sitosterol) and 15% 24ξ-ethylcholesta-5,22-dien-3β-ol.     

The protein-synthesizing apparatus of a BHK-21 (C-13) cell culture and of its large-cell clone C-9

Cell line BHK-21 (C-13) and its large cell clone C-9 differ in morphology, karyotype and cultural properties. Clone C-9 is polyploid. It has been shown that C-9 clone cells display 2.5-3-fold excess in the nucleolus organizer region (NOR) in chromosomes, and 2-3 times higher intensity of protein synthesis and of ribosomal material content compared to the original line. Data of sedimentation analysis and of protein synthesis activity of the total ribosomal material in the cell-free translational system from rabbit reticulocytes allow to conclude that the quantity and size of polyribosomes in C-9 cells are higher than in cells of the original line. Apparently, the quantity of NOR-chromosomes reflect the activity of protein translation system of investigated cells.     

Control of fungal sterol C-24 transalkylation: importance to developmental regulation

When cultures of Gibberella fujikuroi are incubated with 24-epiiminolanosterol the introduction of a methyl group into sterol side chains at C-24 is blocked inducing a mycelial accumulation of lanosterol and 24-desalkylsterols, i.e., having the cholestane side chain. The altered sterol composition lead to aberrant mycelial membranes resulting in growth inhibition. A compensatory physiological response to the ensuing hyphal death was induction of asexual sporulation. The results are interpreted to imply that regulation of sterol C-24 transalkylation may be a mechanism to mediate life cycle events of fungi.     

甾醇C-24甲基转移酶基因在酿酒酵母中的内源表达及工程菌麦角甾醇的合成

通过高保真PCR克隆到含酿酒酵母甾醇C-24甲基转移酶基因编码序列及终止子序列的DNA片段ERG6, 以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体, 磷酸甘油酸激酶基因PGK1启动子为上游调控元件构建了酵母菌表达质粒pPERG6。通过同源重组, 以铜离子螯合蛋白基因CUP1替换染色体上ERG6基因内部序列获得ERG6破坏菌株YS58-erg6, 其中麦角甾醇的合成被阻断, 同时细胞的生长也受到明显抑制。表达质粒pPERG6转化破坏菌株YS58-erg6后, 不但使细胞恢复了合成麦角甾醇的能力, 细胞生物量也得到明显提高, 这说明表达质粒上的ERG6基因得到了功能性的表达。分别用载体质粒YEp352和表达质粒pPERG6转化酿酒酵母单倍体菌株YS58, 获得对照菌株YS58(YEp352)和重组菌株YS58(pPERG6)。重组菌株YS58(pPERG6) 生物量和麦角甾醇含量分别是对照菌YS58(YEp352)的1.23和1.32倍。可见甾醇C-24甲基转移酶基因的高表达可以增强酵母细胞麦角甾醇的合成能力。