Biosynthesis of cholest-5-ene-3beta, 24-diol (cerebrosterol) by bovine cerebral cortical microsomes

The presence of cholest-5-ene-3β, 24-diol (cerebrosterol) in samples of human, bovine, and rabbit brains has been established by isolation of the sterol therefrom and identification by comparison of physical properties. Cholest-5-ene-3β, 24-diol was present at the level of 66.5 sg/g of dried tissue in human brain, 42.9 μg/g in cattle brain, and 89.5 pg/g in rabbit brain. Cholest-5-ene-3β, 24-diol was the only readily detectable hydroxycholesterol derivative in these brain tissues and was concentrated in the 105,000 g pellet (microsomal fraction) of both human and bovine cerebral cortex, with no demonstrable amounts of the sterol present in nuclear or mitochondrial fractions. Incubation of [1,2-³H]- or of [4-¹⁴C]-cholesterol with the 105,000 g microsomal pellet from bovine cerebral cortical homogenates demonstrated 0.1-0.38 per cent conversions to radioactive cholest-5-ene-3β, 24-diol, isolated and purified as the 3β, 24-dibenzoate. The bioconversion required oxygen, and a stimulation of hydroxylation by added NADPH₂ was demonstrated. Our observations establish that a sterol 24-hydroxylase system is present in bovine cerebral cortex.     

Synthesis of polyhydroxysterols (IV): synthesis of 24-methylene-cholesta-3beta,5alpha,6beta,19-tetrol, a cytotoxic natural hydroxylated sterol

The cytotoxic, polyhydroxylated sterol 24-methylene-cholesta-3beta,5alpha,6beta,19-tetrol (1), previously isolated from the soft corals Nephthea albida and N. tiexieral verseveldt, was synthesized using stigmasterol as the starting material by 10 steps in 9% overall yield. The spectral data and physical constants of 1 were identical with those of the natural product. This is the first report of the synthesis of 1.     

Azasterol inhibitors in yeast. Inhibition of the 24-methylene sterol delta24(28)-reductase and delta24-sterol methyltransferase of Saccharomyces cerevisiae by 23-azacholesterol.

The effects of 23-azacholesterol on sterol biosynthesis and growth of Saccharomyces cervisiae were examined. In the presence of 0.2, 0.5, and 1 micron 23-azacholesterol, aerobically-growing yeast produced a nearly constant amount of ergosta-5,7,22,24(28)-tetraenol (approx. 36% of total sterol) and slowly accumulated zymosterol with a concommitant decline in ergosterol synthesis. Growth and total sterol content of yeast cultures treated with 0.2-1 micron 23-azacholesterol were similar to that of the control culture. Yeast cultures treated with 5 and 10 micron 23-azacholesterol produced mostly zymosterol (58-61% of total sterol), while ergosta-5,7,22,24(28)-tetraenol production declined to less than 10% of total sterol. The observed changes in the distribution of sterols in treated cultures are consistent with inhibition of 24-methylene sterol 24(28)-sterol reductase (total inhibition at 1 micron 23-azacholesterol) and of 24-sterol methyltransferase (71% inhibition at 10 micron 23-azacholesterol). Yeast cultures treated with 10 micron 23-azacholesterol were found to contain 4,4-dimethylcholesta-8,14,24-trienol and 4alpha-methylcholesta-8,14,24-trienol, which were isolated and characterized for the first time.     

Synthesis of polyhydroxysterols (I): synthesis of 24-methylenecholest-4-en-3beta,6beta-diol, a cytotoxic natural hydroxylated sterol

Starting from stigmasterol (2), 24-methylenecholest-4-en-3beta, 6beta-diol (1), a cytotoxic natural dihydroxylated sterol, was synthesized via 10 steps in 20% overall yield. The introduction of a side-chain of sterol was achieved by solid-liquid phase-transfer Wittig reaction using (3-methyl-2-oxo)butyltriphenylarsonium bromide (12) and K(2)CO(3). Construction of the steroidal nucleus was finished by the addition of 3beta-acetoxycholest-5,6-en-24-one (7) with NBA in dioxane under ambient temperature and by the elimination of 3beta, 6beta-diacetoxy-5a-bromocholestane-24-one (9). The spectral data of the synthetic product (1) are completely consistent with those of the natural compound (1).     

Synthesis of polyhydroxysterols (III): synthesis and structural elucidation of 24-methylenecholest-4-en-3beta,6 alpha-diol

Using stigmasterol as the starting material, 24-methylenecholest-4-en-3beta,6 alpha-diol (2) was synthesized in eight steps in 13% overall yield. The introduction of the sterol side-chain was carried out using (3-methyl-2-oxobutyl)-triphenylarsonium bromide (11) and K(2)CO(3) in a solid-liquid phase-transfer Wittig reaction. Construction of the steroidal nucleus was finished by oxidation of 24-methylenecholest-5-en-3beta-ol (9) with pyridinium chlorochromate (PCC) in dichloromethane at ambient temperature and by reduction of 24-methylenecholest-4-en-3,6-dione (10) with NaBH(4) in the presence of CeCl(3).7H(2)O.     

Difference in cytotoxicity of (22R)-cholest-5-ene-3 beta,7 alpha,22-triol and (22R)-cholest-5-ene-3 beta,7 beta,22-triol is not explained by different patterns of metabolites

Ehrlich ascites tumor cells in suspension culture were incubated with the plant-derived sterol isomers (22R)-cholest-5-ene-3 beta,7 alpha,22-triol and (22R)-cholest-5-ene-3 beta,7 beta,22-triol. Both sterols were 7-dehydroxylated by the neoplastic cells, and the product was identified as (22R)-22-hydroxycholesta-4,6-dien-3-one. At sub-toxic sterol concentrations the conversion of the 7 alpha-hydroxy compound was about 5 times higher than that of the 7 beta-isomer. At higher sterol concentrations the 7 beta-hydroxy compound caused growth inhibition of the Ehrlich ascites cells, whereas the 7 alpha-hydroxylated sterol was ineffective. The rate of 7 alpha-dehydroxylation was, however, too low to be considered a likely pathway for detoxification. No other lipid-extractable products were detected, and no water-soluble products with influence on cell proliferation were present. Thus, the cytotoxicity is probably attributed to a property of the 7 beta-hydroxyl group of the (22R)-cholest-5-ene-3 beta,7 beta,22-triol.     

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.     

The crystal structure of 3 beta-hydroxy-4 alpha, 23R, 24R-trimethyl-5 alpha-cholestane (23R, 24R-5 alpha-dinostanol) from dinoflagellates

An x-ray crystal structure determination of a dinostanol from the dinoflagellate Protoceratium reticulatum and zooxanthellae from Orbulina universa was completed. The novel sterol was shown to be 3 beta-hydroxy-4 alpha, 23R,24R-trimethyl-5 alpha-cholestane and may be one of the molecular fossils found in sediment cores from the deepest Black Sea trench.     

15 beta-Methyl-5 alpha,14 beta-cholest-7-ene-3 beta,15 alpha-diol. Synthesis, structure, and inhibition of sterol synthesis in animal cells

Treatment of 3 beta-benzoyloxy-14 alpha,15 alpha-epoxy-5 alpha-cholest-7-ene with methyl magnesium iodide gave, as the major product, 15 beta-methyl-5 alpha,14 beta-cholest-7-ene-3 beta,15 alpha-diol. The product was characterized as the free sterol and in the form of its 3 beta-acetoxy and 3 beta-p-bromobenzoate derivatives. Unambiguous assignment of structure was based upon X-ray analysis of the latter derivative. 15 beta-Methyl-5 alpha,14 beta-cholest-7-ene-3 beta,15 alpha-diol was found to be a potent inhibitor of sterol synthesis in cultured mammalian cells. The 15 beta-methyl-3 beta,15 alpha-dihydroxysterol caused a 50% reduction of the level of HMG-CoA reductase activity and a 50% reduction in the incorporation of labeled acetate into digitonin-precipitable sterols in L cells at a concentration of 3.0 x 10(-6) M.     

Synthesis of the marine epoxy sterol 9 alpha,11 alpha-epoxy-5 alpha-cholest-7-ene-3 beta,5,6 beta-triol

The synthesis of 9 alpha,11 alpha-epoxy-5 alpha-cholest-7-ene-3 beta,5,6 beta-triol (1), a highly oxygenated marine sterol containing a 9,11-epoxide moiety in the nucleus, is described. Epoxy sterol 1 was synthesized from cholesta-5,7-dien-3 beta-ol. Oxidation of this sterol with m-chloroperbenzoic acid followed by hydrolysis and acetylation furnished 5 alpha-cholest-7-ene-3 beta,5,6 alpha-triol 3,6-diacetate (2). Mercuric acetate dehydrogenation of diacetate 2, followed by oxidation with manganese dioxide and epoxidation with m-chloroper-benzoic acid, afforded 9 alpha,11 alpha-epoxy-3 beta,5-dihydroxy-5 alpha-cholest-7-en-6-one (5). Reduction of 5 with lithium aluminum hydride gave the desired compound 1. The structures of all synthetic intermediates were confirmed by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. A reassignment of resonances for carbons 1, 8, and 15 in the 13C NMR spectrum of 1, based on 2D-NMR correlation spectroscopy, has been accomplished.     

Aerobic isolation of an ERG24 null mutant of Saccharomyces cerevisiae.

The ERG24 gene, encoding the C-14 sterol reductase, has been reported to be essential to the aerobic growth of Saccharomyces cerevisiae. We report here, however, that strains with null mutations in the ERG24 gene can grow on defined synthetic media in aerobic conditions. These sterol mutants produce ignosterol (ergosta-8,14-dienol) as the principal sterol, with no traces of ergosterol. In addition, we mapped the ERG24 gene to chromosome XIV between the MET2 and SEC2 genes. Our results indicate that ignosterol can be a suitable sterol for aerobic growth of S. cerevisiae on synthetic media and that inactivation of ERG24 is only conditionally lethal.     

Microbial degradation of the phytosterol side-chain to 24-oxo products

A mutant of the potent sterol degrader Mycobacteriun fortuitum (ATCC6842) has been isolated which is defective in its ability to degrade both the steroid nucleus and sterol side-chains that are branched at the 24-position. Bioconversions of phytosterol mixtures by this mutant resulted in the accumulation of the novel 24-oxo intermediates 9-hydroxy-27-nor-4-cholestene-3,24-dione (II) and 9-hydroxy-26,27-dinor-4-cholestene-3,24-dione (III). Under the same conditions, cholesterol is degraded mainly to 9-hydroxy-4-androstene-3,17-dione (I) by this organism.     

Sterols of the unicellular algae Nematochrysopsis roscoffensis and Chrysotila lamellosa: isolation of (24E)-24-n-propylidenecholesterol and 24-n-propylcholesterol

Two rare C₃₀-sterols, (24E)-24-n-propylidenecholest-5-en-3β-ol and 24-n-propylcholest-5-en-3β-ol, and (24S)-24-ethylcholesta-5,22-dien- 3β-ol (stigmasterol) are the major sterols of Nematochrysopsis roscoffensis, a Chrysophyte of the Sarcinochrysidales order. This unique sterol composition is different from the sterol contents of other Chrysophytes and justifies the peculiar position of the Sarcinochrysidales, which are by some characteristics morphologically and biologically related to the Phaeophyceae. The presence of (24S)-24-methylcholesta-5,22-dien-3β-ol (24-epibrassicasterol) as a major sterol in Chrysotila lamellosa is in accordance with the few previous results obtained from other Prymnesiophyceae, although the presence of the other major sterol, (24R)-24-ethylcholesta-5,22-dien-3β-ol (poriferasterol) has never been reported in these algae.     

24-Methylene-25-methylcholesterol,a sterol from the seeds of Brassica juncea

A new sterol isolated from the seeds of Brassica juncea has been shown to be 24-methylene-25-methylcholesterol.     

Chemical studies of marine inverterbrates. XXIX 4 alpha-Methyl-3 beta 8-beta-dihydroxy-5 alpha-ergost-24(28)-en-23-one, a novel polyoxygenated sterol from the soft coral Litophyton viridis. (Coelenterata, Octocorallia, Alcyonacea).

A novel sterol, 4α-methyl-3β,8β-dihydroxy-5α-ergost-24(28)-en-23-one (I), has been isolated from the soft coral Litophyton viridis. Its structure and relative configuration has been established by X-Ray diffraction analysis.     

24-hydroxycholesterol levels in human brain

24-Hydroxycholesterol (cerebrosterol, cholest-5-ene-3β;, 24ξ-diol) occurs at low levels in human (Di Frisco , De Ruggieri and Ercoli , 1953; Ercoli and De Ruggieri , 1953a, 19536; Schubert , Rose and Burger , 1961; Van Lier and Smith , 1969 , 1970), equine (Ercoli , Di Frisco and De Ruggieri , 1953a; Ercoli and De Ruggieri , 1953a, 1953b; Fieser , Huang and Bhattacharyya , 1957) and bovine (Richter and Dannenberg , 1969) brain tissue. Only one of two possible C-24 epimeric alcohols appears to occur in human brain (Van Lier and Smith , 1970) and the sterol may be regarded as a true endogenous trace-level sterol and not as an artifact of autoxidation derived during isolation and analysis. As a phase of our continuing interests in the presence of trace-level polar sterols in human tissues (Van Lier and Smith , 1967 , 1969, 1970 , 1971a; Smith and Van Lier , 1970), we sought to measure levels of 24-hydroxycholesterol in different parts of human brain by gas chromatographic means. The present report deals with our measurements of 24-hydroxycholesterol in human cortex, subcortical white matter, midbrain, pons, and cerebellum.     

24-Methyl-23-dehydrocholesterol: a new sterol intermediate in C-24 demethylation from the nematodes Panagrellus redivivus and Caenorhabditis elegans?

Panagrellus redivivus produced 24-methyl-23-dehydrocholesterol as 4.0% of the 4-desmethylsterols when propagated in a medium containing campesterol as the dietary sterol. The re-examination of previous data revealed that Caenorhabditis elegans produced 1.8% 24-methyl-23-dehydrocholesterol when propagated in medium containing campesterol. 24-Methyl-23-dehydrocholesterol was not detected when the nematodes were propagated in medium containing 22-dihydrobrassicasterol or 24-methylenecholesterol. This may be a result of the greater efficiency of dealkylation of the latter two sterols. This is the first report of the natural occurrence of this sterol in a non-photosynthetic organism, and the first report in organisms that dealkylate 24-alkylsterols.     

FadD19 of Rhodococcus rhodochrous DSM43269, a steroid-coenzyme A ligase essential for degradation of C-24 branched sterol side chains

The actinobacterial cholesterol catabolic gene cluster contains a subset of genes that encode β-oxidation enzymes with a putative role in sterol side chain degradation. We investigated the physiological roles of several genes, i.e., fadD17, fadD19, fadE26, fadE27, and ro04690DSM43269, by gene inactivation studies in mutant strain RG32 of Rhodococcus rhodochrous DSM43269. Mutant strain RG32 is devoid of 3-ketosteroid 9α-hydroxylase (KSH) activity and was constructed following the identification, cloning, and sequential inactivation of five kshA gene homologs in strain DSM43269. We show that mutant strain RG32 is fully blocked in steroid ring degradation but capable of selective sterol side chain degradation. Except for RG32ΔfadD19, none of the mutants constructed in RG32 revealed an aberrant phenotype on sterol side chain degradation compared to parent strain RG32. Deletion of fadD19 in strain RG32 completely blocked side chain degradation of C-24 branched sterols but interestingly not that of cholesterol. The additional inactivation of fadD17 in mutant RG32ΔfadD19 also did not affect cholesterol side chain degradation. Heterologously expressed FadD19DSM43269 nevertheless was active toward steroid-C26-oic acid substrates. Our data identified FadD19 as a steroid-coenzyme A (CoA) ligase with an essential in vivo role in the degradation of the side chains of C-24 branched-chain sterols. This paper reports the identification and characterization of a CoA ligase with an in vivo role in sterol side chain degradation. The high similarity (67%) between the FadD19(DSM43269) and FadD19H37Rv enzymes further suggests that FadD19H37Rv has an in vivo role in sterol metabolism of Mycobacterium tuberculosis H37Rv.     

The effect of altered sterol composition on cytochrome oxidase and S-adenosylmethionine: delta 24 sterol methyltransferase enzymes of yeast mitochondria

Arrhenius kinetics of two mitochondrial enzymes, cytochrome oxidase and S-adenosylmethionine: Δ 24 sterol methyltransferase were analyzed in wild-type and sterol mutant strains of yeast. Temperature effects on the enzymes isolated from the ergosterol producing wild-type and nystatin resistant mutants (major sterol Δ⁸(9), 22 ergostadiene-3-β-ol) were compared. Transition temperatures were lower in both mutant strains compared to wild-type. Lipid analysis shows a relationship between sterol content and the temperature dependent transition phases.     

Structural requirements for transformation of substrates by the S-adenosyl-L-methionine:delta 24(25)-sterol methyltransferase. Inhibition by analogs of the transition state coordinate.

Microsomes from sunflower seedlings were used to investigate the transition state coordinate for the C-24 methylation reaction that mediates phytosterol biosynthesis. They were then used to study structurally related cationic and uncharged compounds of the natural sterol substrate, which were designed to interfere with the reaction progress. The hypothetical reaction course is described to proceed through an Sn2 formation of an activated complex involving the initial production of a covalent structure with a dative bond (methyl from AdoMet attacks si-face of the 24,25-double bond of the sterol) and the secondary production of a series of high energy intermediates, the stabilization of which determines the final C-24 methylated product. Derivatives of lanosterol and cholesterol with a methyl, hydrogen, oxygen, or bromine atom introduced into the side chain and/or at C-3 in place of the natural nucleophile were studied as inhibitors that interfere with the formation of the hypothetical tertiary isopropylcarbinyl cation intermediate in the conversion of cycloartenal to 24(28)-methylene cycloartanol. The data indicate the most potent inhibitor is a sterol with an aziridine group attached to C-24(25), which mimics the bridged C-24(25) carbenium ion generated in the transition state, and the methyltransferase possesses two strategic sites: one that recognizes the proximal end of the sterol acting as a proton donor and the other that recognizes the distal end that acts as a proton acceptor. The best fit (binding/catalysis) involves a flat sterol (including substrate and inhibitor) with intact unsubstituted nucleophilic centers at C-3 and C-24 and a freely rotating side chain that can assume the pseudocyclic conformation.