5,8-Epidioxysterols and related derivatives from a Chinese soft coral Sinularia flexibilis

Chromatographic separation of the methanolic extract of the marine soft coral, Sinularia flexibilis, resulted in the isolation and characterization of four new sterols, 5alpha,8alpha-epidioxygorgosta-6-en-3beta-ol (1), 5alpha,8alpha-epidioxygorgosta-6,9(11)-dien-3beta-ol (2), 22alpha,28-epidioxycholesta-5,23(E)-dien-3beta-ol (3) and its C-22 epimer (4), along with nine known sterols. The structures of the new compounds were determined on the basis of extensive spectroscopic data (IR, MS, 1H and 13C NMR, HMQC, HMBC, and NOESY) analyses.     

Analysis of sequence determinants of F1Fo-ATP synthase in the N-terminal region of alpha subunit for binding of delta subunit

The stator in F(1)F(o)-ATP synthase resists strain generated by rotor torque. In Escherichia coli, the b(2)delta subunit complex comprises the stator, bound to subunit a in F(o) and to the alpha(3)beta(3) hexagon of F(1). Previous work has shown that N-terminal residues of alpha subunit are involved in binding delta. A synthetic peptide consisting of the first 22 residues of alpha (alphaN1-22) binds specifically to isolated wild-type delta subunit with 1:1 stoichiometry and high affinity, accounting for a major portion of the binding energy between delta and F(1). Residues alpha6-18 are predicted by secondary structure algorithms and helical wheels to be alpha-helical and amphipathic, and a potential helix capping box occurs at residues alpha3-8. We introduced truncations, deletions, and mutations into alphaN1-22 peptide and examined their effects on binding to the delta subunit. The deletions and mutations were introduced also into the N-terminal region of the uncA (alpha subunit) gene to determine effects on cell growth in vivo and membrane ATP synthase activity in vitro. Effects seen in the peptides were well correlated with those seen in the uncA gene. The results show that, with the possible exception of residues close to the initial Met, all of the alphaN1-22 sequence is required for binding of delta to alpha. Within this sequence, an amphipathic helix seems important. Hydrophobic residues on the predicted nonpolar surface are important for delta binding, namely alphaIle-8, alphaLeu-11, alphaIle-12, alphaIle-16, and alphaPhe-19. Several or all of these residues probably make direct interaction with helices 1 and 5 of delta. The potential capping box sequence per se appeared less important. Impairment of alpha/delta binding brings about functional impairment due to reduced level of assembly of ATP synthase in cells.     

F1F0-ATP synthase. Binding of delta subunit to a 22-residue peptide mimicking the N-terminal region of alpha subunit

The stator in F(1)F(0)-ATP synthase resists strain generated by rotor torque. In Escherichia coli the b(2)delta subunit complex comprises the stator, bound to subunit a in F(0) and to alpha(3)beta(3) hexagon of F(1). Proteolysis and cross-linking had suggested that N-terminal residues of alpha subunit are involved in binding delta. Here we demonstrate that a synthetic peptide consisting of the first 22 residues of alpha ("alpha N1-22") binds specifically to isolated wild-type delta subunit with high affinity (K(d) = 130 nm), accounting for a major portion of the binding energy when delta-depleted F(1) and isolated delta bind together (K(d) = 1.4 nm). Stoichiometry of binding of alpha N1-22 to delta at saturation was 1/1, showing that in intact F(1)F(0)-ATP synthase only one of the three alpha subunits is involved in delta binding. When alpha N1-22 was incubated with delta subunits containing mutations in helices 1 or 5 on the F(1)-binding face of delta, peptide binding was impaired as was binding of delta-depleted F(1). Residues alpha 6-18 are predicted to be helical, and a potential helix capping box occurs at residues alpha 3-8. Circular dichroism measurements showed that alpha N1-22 had significant helical content. Hypothetically a helical region of residues alpha N1-22 packs with helices 1 and 5 on the F(1)-binding face of delta, forming the alpha/delta interface.     

Six new ergosterols from the marine-derived fungus Rhizopus sp

Six new ergosterols, including 3beta-hydroxyl-(22E, 24R)-ergosta-5,8,22-trien-7,15-dione (1), 3beta-hydroxyl-(22E, 24R)-ergosta-5,8,14,22-tetraen-7-one (2), 3beta,15beta-dihydroxyl-(22E, 24R)-ergosta-5,8(14),22-trien-7-one (3), 3beta,15alpha-dihydroxyl-(22E, 24R)-ergosta-5,8(14),22-trien-7-one (4), 3beta-hydroxyl-(22E, 24R)-ergosta-5,8(14),22-trien-7,15-dione (5), and 5alpha,8alpha-epidioxy-23,24(R)-dimethylcholesta-6,9(11),22-trien-3beta-ol (6), have been isolated from the marine-derived fungus Rhizopus sp., along with four known ones (7-10). The structures of the new compounds were determined on the basis of extensive spectroscopic data. All compounds were evaluated for their cytotoxic activities on P388, A549, HL-60, and BEL-7420 cell lines by the MTT and SRB methods.     

Alternative pathways of sterol synthesis in yeast. Use of C(27) sterol tracers to study aberrant double-bond migrations and evaluate their relative importance

Yeast produce traces of aberrant sterols by minor alternative pathways, which can become significant when normal metabolism is blocked by inhibitors or mutations. We studied sterols generated in the absence of the delta(8)-delta(7) isomerase (Erg2p) or delta(5) desaturase (Erg3p) by incubating three mutant strains of Saccharomyces cerevisiae with 5 alpha-cholest-8-en-3beta-ol, 8-dehydrocholesterol (delta(5,8) sterol), or isodehydrocholesterol (delta(6,8) sterol), together with the corresponding 3 alpha-3H isotopomer. Nine different incubations gave altogether 16 sterol metabolites, including seven delta(22E) sterols formed by action of the yeast C-22 desaturase (Erg5p). These products were separated by silver-ion high performance liquid chromatography (Ag(+)-HPLC) and identified by gas chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy, and radio-Ag(+)-HPLC. When delta(8)-delta(7) isomerization was blocked, exogenous delta(8) sterol underwent desaturation to delta(5,8), delta(6,8), and delta(8,14) sterols. Formation of delta(5,8) sterol was strongly favored over delta(6,8) sterol, but both pathways are essentially dormant under normal conditions of sterol synthesis. The delta(5,8) sterol was metabolically almost inert except for delta(22) desaturation, whereas the delta(6,8) sterol was readily converted to delta(5,7), delta(5,7,9(11)), and delta(7,9(11)) sterols. The combined results indicate aberrant metabolic pathways similar to those in mammalian systems. However, delta(5,7) sterol undergoes only slight isomerization or desaturation in yeast, an observation that accounts for the lower levels of delta(5,8) and delta(5,7,9(11)) sterols in wild-type yeast compared to Smith-Lemli-Opitz individuals.     

土壤真菌 Aspergillus fumigatus固体发酵产物的化学成分及抗氧化活性研究

为研究来源于哀牢山国家级自然保护区河谷的土壤真菌Aspergillus fumigatus固体发酵产物的化学成分及抗氧化活性研究,采用正相硅胶柱层析、反相硅胶柱层析和Sephadex LH-20分离纯化,借助核磁共振波谱等方法鉴定化合物结构,从中共得到13个化合物,分别鉴定为rubrofusarin B(1)、rubrofusarin A(2)、carbonarone A(3)、aspernigrin A(4)、flavasperone(5)、(22 E,24 R)-5α,8α-过氧麦角甾-6,22-二烯-3β-醇(6)、(22 E,24 R)-5α,8α-过氧麦角甾-6,9(11),22-三烯-3β-醇(7)、ourosperone A(8)、(22 E,24 R)-麦角甾-5,7,22-三烯-3β-醇(9)、stigmast-1,5-dien-3β-ol(10)、fonsecinones A(11)、asperpyrone C(12)、asperpyrones B(13)。其中,化合物1~5和7~13为从该菌种中首次分离。抗氧化活性结果显示,化合物8对DPPH(IC₅₀=3.453 mg/mL)、ABTS+(IC₅₀=0.155 mg/mL)、OH(IC₅₀=0.019 mg/mL)自由基都有一定的清除效果。     

短角湿生冷水花化学成分的研究

为研究短角湿生冷水花(Pilea aquarum subsp.brevicornuta)化学成分。实验采用色谱法从中分离得到7个化合物,包括5个五环三萜、一个甾醇和一个木脂素。利用波谱学方法鉴定了它们的结构,分别鉴定为pilearbornol(1)、rubiarbonone D(2)、camarolide(3)、表齐墩果酸(4)、齐墩果酮酸(5)、5,8-epidioxy-(3β,5α,8α,22E)-ergosta-6,9(11),22-trien-3-ol(6)和桉脂素(7)。其中,化合物1和2属于乔木萜烷型(arborane)三萜,化合物1为新化合物,化合物2的绝对构型首次通过X-射线单晶衍射得到了确定。所有化合物均为首次从该植物中分离得到,这也是首次从荨麻科植物中分离得到乔木萜烷型三萜化合物。     

T cell receptor gamma gene status of human alpha/beta+ and gamma/delta+ T cell clones: absence of V9JP rearrangements in alpha/beta+ clones is not a result of a lack of rearrangements involving more 5' J gamma segments

T cell receptor (TCR) gamma gene rearrangements were examined in panels of human T cell clones expressing TCR alpha/beta or gamma/delta heterodimers. Over half of the alpha/beta+ clones had both chromosomes rearranged to C gamma 2 but this was the case for only 20% of the gamma/delta+ clones. While more than half of the gamma/delta+ clones showed a V9JP rearrangement, this configuration was absent from all 49 alpha/beta+ clones analysed. However, this was not a result of all rearrangements being to the more 3' J gamma genes as 11 alpha/beta+ clones had rearrangement(s) to JP1, the most 5' J gamma gene segment. Both alpha/beta+ and gamma/delta+ clones showed a similar pattern of V gamma gene usage in rearrangements to J gamma 1 or J gamma 2 with a lower proportion of the more 3' genes being rearranged to J gamma 2 than for the more 5' genes. Several alpha/beta+ and several gamma/delta+ clones had noncoordinate patterns of rearrangement involving both C gamma 1 and C gamma 2. Eleven out of fourteen CD8+ clones tested had both chromosomes rearranged to C gamma 2 whereas all clones derived from CD4-8- cells and having unconventional phenotypes (CD4-8- or CD4+8+) had at least one C gamma 1 rearrangement. Twelve out of twenty-seven CD4+ clones also had this pattern, suggesting that CD4-8+ clones had a tendency to utilize more 3' J gamma gene segments than CD4+ clones. There was some evidence for interdonor variation in the proportions of TCR gamma rearrangements to C gamma 1 or C gamma 2 in alpha/beta+ clones as well as gamma/delta+ clones. The results illustrate the unique nature of the V9JP rearrangement in gamma/delta+ clones and the possible use of a sequential mechanism of TCR gamma gene rearrangements during T cell differentiation is discussed.     

Cholestane rhamnosides from the bulbs of Ornithogalum saundersiae.

Phytochemical examination of the bulbs of Ornithogalum saundersiae yielded six cholestane rhamnosides, two of which had previously been isolated from the same plant material. However, detailed spectroscopic analysis of the aglycone led us to revise the configuration of the C-11 hydroxyl group of the latter two and reassign their structures as (22S)-cholest-5-ene-3 beta,11 alpha,16 beta,22-tetrol 16-O-alpha-L-rhamnopyranoside and (22S)-cholesta-5,24-diene-3 beta,11 alpha,16 beta,22-tetrol 16-O-alpha-L-rhamnopyranoside, respectively. The other four are new naturally occurring constituents and their structures were determined to be (22S)-cholest-5-ene-3 beta,11 alpha,16 beta,22-tetrol 16-O-(2,3-di-O-acetyl-alpha-L-rhamnopyranoside), (22S)-cholest-5-ene-3 beta,11 alpha,16 beta,22-tetrol 16-O-{2-O-acetyl-3-O-(3,4,5-trimethoxybenzoyl)-alpha-L-rhamnopyran oside}, (22S)-cholest-5-ene-3 beta,11 alpha,16 beta,22-tetrol 16-O-{2-O-acetyl-3-O-(p-methoxybenzoyl)-alpha-L-rhamnopyranoside} and (22S)-cholesta-5,24-diene-3 beta,11 alpha,16 beta,22-tetrol 16-O-(2,3-di-O-acetyl-alpha-L-rhamnopyranoside), respectively. The isolated compounds were evaluated for their cytostatic activity against leukemia HL-60 cells.     

A computer investigation of intramolecular bond ordering: unsaturated chains of natural lipids

Computer simulations (by the Monte Carlo method) of unperturbed linear hydrocarbon chains of 18-22 carbon atoms with methylene-interrupted cis-double bonds (18:0, 20:0, 22:0, 18:1delta11cis, 18:2delta9, 12cis, 18:3delta9, 12, 15cis, 20:3delta5, 8, 11cis, 20:4delta5, 8, 11, 14cis, 20:5delta5, 8, 11, 14, 17cis, 22:6delta4, 7, 10, 13, 16, 19cis), typical components of natural lipids, at a temperature of 298 K have been carried out. The conformations generated with continuous variation of all single C-C bond rotation angles within the (0, 360 degrees) range have been considered. The energy of nonbonded interactions and torsion and electrostatic terms have been taken into account. The intramolecular bond order parameters S(CC) and S(CH) about the axes along inertia tensor eigenvectors and bond orientation distributions rho(theta) with respect to the maximum molecule span axis (theta is the angle between the bond and the axis) have been calculated. The relation of the bond orientation distributions rho(theta) to the order parameters S are analyzed in terms of angles thetamax (a "geometric" factor, rho(thetamax) = max) and widths deltatheta of the distributions (factor of "fluctuations"). The results indicate that fluctuation factors depend on both the segment chemical structure and location in the chain; fluctuations increase from the centre of the chain towards the terminals, all things being equal. The two deltatheta values of C-H bonds flanking the cis-double bond are smaller than that obtained for adjacent CH2 groups by a factor of 1.5-2. Defining these properties is a necessary step to gaining a more complete understanding of polyunsaturated lipid hydrocarbon chains significance. The mean molecule magnitudes of ?S(CH)? decrease when unsaturation increases. The cis-double bond parameters S(CC) are found to be higher than those of adjacent single C-C bonds: the parameter S(CC) odd-even effect in the polyunsaturated molecules of such structure changes the "sign" between double bonds. The order parameter profiles -S(CH) of cis-18:1 and cis-18:2 obtained from the simulations (at the portion which corresponds to the double bonds location) are in qualitative agreement with experimental data on bilayers in the liquid-crystal phase. This has made possible the quantitative prognosis of the ordering properties of experimentally uninvestigated unsaturated lipids.     

Studies of the oxysterol inhibition of tumor cell growth

The oxysterols 3 beta-hydroxy-5 alpha-cholest-8-en-11-one, 3 beta-hydroxy-5 alpha-cholest-8-en-7-one, 3 beta-hydroxy-5 alpha-cholest-8(14)-en-7-one, 3 beta-hydroxy-4,4'-dimethylcholest-5-ene-7 one, 4,4'-dimethylcholest-5-ene-3 beta, 7 alpha-diol, 4,4'-dimethylcholest-5-ene-3 beta, 7 beta-diol, lanost-8-ene-3 beta, 25-diol, 25-hydroxylanost-8-en-3-one, 9 alpha, 11 alpha-epoxy-5 alpha-cholest-7-en-3 beta-ol, 3 beta-hydroxycholest-5 alpha-en-22-one, and 3 beta-hydroxycholest-5-en-22-one oxime were evaluated with respect to their ability to inhibit cell growth. All of the sterols were found to possess cytotoxicity when incubated with hepatoma (HTC) and lymphoma (RDM-4) cells in culture at 10-30 microM concentrations.     

Two new polyhydroxysteroids from the gorgonian Isis hippuris

Two new polyhydroxysteroids isihippurols A (1) and B (2) were isolated from the MeOH extract of the gorgonian Isis hippuris in addition to nine known steroids namely gorgosterol, 3alpha-acetoxy-24-methyl 11beta,18; 18,20beta; 22,25-triepoxy-5alpha-furostane, hippurin-1, 22-epi-hippuristanol, 22-epi-hippurin-1, 3-acetyl-2-deacetyl-22-epi-hippurin-1, 2-deacetylhippurin-1, 3beta, 7alpha, 11alpha-trihydroxygorgost-5-ene-12beta-acetate and 2-deacetyl-22-epi-hippurin-1. The structures of 1 and 2 have been determined as 1alpha, 3beta, 5alpha, 6beta, 11alpha-pentahydroxygorgosta-12beta-monoacetate and 1alpha, 3beta, 11alpha-trihydroxygorgosta-5alpha, 6alpha-epoxy-12beta-monoacetate respectively by spectral analysis and chemical correlation.     

Metabolism of eicosapentaenoic acid by aorta: formation of a novel 13-hydroxylated prostaglandin

We have investigated the metabolism by fetal calf aorta of eicosapentaenoic acid (20:5) and docosahexaenoic acid (22:6), two polyunsaturated fatty acids found in high concentrations in marine oils. The major product formed from 20:5 by particulate fractions from fetal calf aorta is delta 17-6-oxoprostaglandin F1 alpha. In addition, we detected a novel isomer of delta 17-6-oxoprostaglandin F1 alpha, in which a hydroxyl group is present in the 13-position instead of the 15-position. Eicosapentaenoic acid is also converted to 12-hydroxy-5,8,10,14-heptadecatetraenoic acid as well as to five monohydroxy isomers with hydroxyl groups present in the 11, 12, 14, 15, and 18 positions. Although 20:5 was metabolized at about one-third the rate of arachidonic acid (20:4), greater amounts of monohydroxy fatty acids, the major one being the 11-hydroxy metabolite, were formed from 20:5. Unlike 20:5, 22:6 was not metabolized to any detectable products by fetal calf aorta, but both of these polyunsaturated fatty acids inhibited the oxygenation of 20:4 by cyclooxygenase from aorta with IC50 values of 4.1 microM (22:6) and 15 microM (20:5). These results suggest that 20:5 has a high affinity for cyclooxygenase, but that the intermediate 11-oxygenated intermediate has a lower affinity than the corresponding intermediate from 20:4, resulting in a greater loss of substrate after a single oxygenation. The formation of oxygenation products from both 20:4 and 20:5 was inhibited by 13-hydroperoxy-9,11-octadecadienoic acid (13hp-18:2). The IC50 values for inhibition of cyclooxygenase products by 13hp-18:2 were about twice as high as those for inhibition of prostacyclin synthase products. Consequently, there was little diversion of prostaglandin endoperoxides to other prostaglandins in the presence of 13hp-18:2.     

Structural characterization of the interaction of the delta and alpha subunits of the Escherichia coli F1F0-ATP synthase by NMR spectroscopy

A critical point of interaction between F(1) and F(0) in the bacterial F(1)F(0)-ATP synthase is formed by the alpha and delta subunits. Previous work has shown that the N-terminal domain (residues 3-105) of the delta subunit forms a 6 alpha-helix bundle [Wilkens, S., Dunn, S. D., Chandler, J., Dahlquist, F. W., and Capaldi, R. A. (1997) Nat. Struct. Biol. 4, 198-201] and that the majority of the binding energy between delta and F(1) is provided by the interaction between the N-terminal 22 residues of the alpha- and N-terminal domain of the delta subunit [Weber, J., Muharemagic, A., Wilke-Mounts, S., and Senior, A. E. (2003) J. Biol. Chem. 278, 13623-13626]. We have now analyzed a 1:1 complex of the delta-subunit N-terminal domain and a peptide comprising the N-terminal 22 residues of the alpha subunit by heteronuclear protein NMR spectroscopy. A comparison of the chemical-shift values of delta-subunit residues with and without alpha N-terminal peptide bound indicates that the binding interface on the N-terminal domain of the delta subunit is formed by alpha helices I and V. NOE cross-peak patterns in 2D (12)C/(12)C-filtered NOESY spectra of the (13)C-labeled delta-subunit N-terminal domain in complex with unlabeled peptide verify that residues 8-18 in the alpha-subunit N-terminal peptide are folded as an alpha helix when bound to delta N-terminal domain. On the basis of intermolecular contacts observed in (12)C/(13)C-filtered NOESY experiments, we describe structural details of the interaction of the delta-subunit N-terminal domain with the alpha-subunit N-terminal alpha helix.     

Measurement of urinary steroid profile in patients with adrenal tumor as a screening method for carcinoma

Results of measurement of urinary steroid metabolite profile using gas chromatographic analysis in eight patients with adrenocortical tumors, i.e. 3 adenomas with Cushing's Syndrome, one adenoma with virilization, one adenoma without clinical manifestations, one carcinoma with Cushing's syndrome and virilization, one carcinoma with Cushing's syndrome and feminization, and one carcinoma without endocrinological symptoms, are reported. A unique pattern dominated by 5 beta and 11 beta-hydroxy steroid metabolites was confirmed in five patients with Cushing's syndrome consisting of three cases with adenomas and two with carcinomas. Excessive 3 alpha, 17 alpha, 21-trihydroxy-5 beta-pregnan-20-one (tetrahydro-11-deoxycortisol, THS) and delta 5-pregnene-3 beta, 11 alpha, 20 alpha-triol (delta 5-pregnenetriol) values were found in all three carcinomas including a nonfunctional carcinoma. These findings would strongly suggest the tumor to be a carcinoma, although excessive excretion of THS and delta 5-pregnenetriol was detected in one patient with a large adenoma associated with virilization. One patient with carcinoma was responsive to ACTH stimulation while the remainder show almost no response to exogenous ACTH. Urinary steroid profiling using gas chromatographic analysis, especially the values for THS and delta 5-pregnenetriol, appears to be a useful method to use in detecting these steroid metabolic characteristics in patients with adrenocortical carcinoma.     

Novel function for intestinal intraepithelial lymphocytes. Murine CD3+, gamma/delta TCR+ T cells produce IFN-gamma and IL-5.

Intestinal intraepithelial lymphocytes (IEL) from mice are greater than 80% CD3+ T cells and could be separated into four subsets according to expression of CD4 and CD8. In our studies designed to assess the functions of IEL, namely, cytokine production, it was important to initially characterize the various subsets of T cells that reside in IEL. The major subset was CD4-, CD8+ (75% of CD3+ T cells), which contained approximately 45 to 65% gamma/delta TCR+ and 35 to 45% alpha/beta TCR+ T cells. Approximately 7.5% of IEL T cells were CD4-, CD8- (double negative) and gamma/delta+ population. On the other hand, CD4+, CD8+ (double positive) and CD4+, CD8- fractions represented 10% and 7.5% of CD3+ T cells, respectively, which were all alpha/beta TCR+. Inasmuch as CD3+, CD4-, CD8+ T cells are a major subset of IEL which contain both gamma/delta TCR or alpha/beta TCR-bearing cells, the present study was focused on the capability of this subset of IEL T cells to produce the cytokines IFN-gamma and IL-5. Both gamma/delta TCR+ and alpha/beta TCR+ IEL spontaneously produced IFN-gamma and IL-5, although higher frequencies of cytokine spot-forming cells were associated with the alpha/beta TCR+ subset. Approximately 30% of CD8+, gamma/delta TCR+ cells produced both cytokines, whereas approximately 90% of alpha/beta TCR+ T cells produced either IFN-gamma or IL-5. Both gamma/delta TCR+ and alpha/beta TCR+ IEL possessed large quantities of cytokine-specific mRNA, clearly showing that these IEL were programmed for cytokine production. When IEL were activated with anti-gamma/delta or anti-CD8 antibodies, higher numbers of IFN-gamma and IL-5 spot-forming cells were noted. The present study has provided direct evidence that a major function of IEL involves cytokine production, and this is the first evidence that gamma/delta TCR+ cells in IEL possess the capability of producing both IL-5 and IFN-gamma.     

Fatty acid structural requirements for activity of arachidonoyl-CoA synthetase

We have examined the fatty acid substrate specificity of arachidonoyl-CoA synthetase from human platelet membranes. A variety of positional isomers and chain-length analogs of arachidonic acid [20:4(5, 8, 11, 14)] were synthesized, and assayed for their ability to inhibit arachidonoyl-CoA formation or to serve as substrates for the synthetase. The chain-length specificity of the synthetase for delta 8,11,14 trienoic fatty acids was C19 greater than C18 = C20 much greater than C21 greater C22. Inhibition activity by positional isomers of arachidonate was 20:4(5, 8, 11, 14) approximately equal to 20:4(6, 9, 12, 15) = 20:4(7, 10, 13, 16) much greater than 20:4(4, 7, 10, 13), however, Vmax for arachidonate was greater than that for 20:4(6, 9, 12, 15). The enzyme apparently "counts" double bonds from the carboxyl terminus. As counted from the methyl terminus we found that several n-6,-9,-12 fatty acids were ineffective as inhibitors [18:3(6, 9, 12); 19:4)4, 7, 10, 13); 21:3(9, 12, 15)], whereas all methylene-interrupted tri- and tetraenoic fatty acids which contained delta 8 and delta 11 double bonds were potent inhibitors. The delta 11 double bond was best associated with optimal inhibition: 20:3(5, 11, 14) had a lower Ki than 20:3(5, 8, 14). 13-Methyl-20:3(8, 11, 14) did not inhibit the enzyme. Partially purified enzyme from calf brain, depleted of nonspecific long-chain acyl-CoA synthetase, exhibited the same fatty acid specificity as crude platelet enzyme.     

Inhibitors of sterol synthesis. Spectral characterization of derivatives of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one

Described herein are the chemical syntheses of a number of deuterated derivatives of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one. These include the [2,2,3 alpha,4,4,7,7,9 alpha,16,16-2H10]-, [7 alpha,9 alpha,16,16-2H4]-, [7,7,9 alpha,16,16-2H5]-, and [2,2,3 alpha,4,4-2H5]-analogs of the delta 8(14)-15-ketosterol. Also included are the syntheses of the 3 beta-acetate derivatives of the latter three deuterated analogs and of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one, and 5 alpha-cholest-8(14)-en-3 alpha-ol-15-one. Low resolution mass spectral data on these compounds and on 5 alpha-cholest-8(14)-en-15-one, 5 alpha-cholest-8(14)-en-3 beta-ol-15-one, 5 alpha-cholest-8(14)-en-3 alpha-ol-15-one, 3 beta-benzoyloxy-5 alpha-cholest-8(14)-en-15-one, and the trimethylsilyl ethers of the free sterols have been presented. The results of these studies, supplemented with high resolution mass spectral data on five of these compounds, have been used to evaluate the electron impact mass spectral fragmentation of the delta 8(14)-15-ketosterols and their derivatives. Also presented herein are the results of 1H, 2H, and 13C nuclear magnetic resonance studies of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one and its derivatives.