Minor sterols of marine invertebrates 37. Isolation of novel coprostanols and 4α-methyl sterols from the tunicate Ascidia nigra

The complex sterol mixture isolated from $\text{A, nigra}$ was found to contain a low level of Δ⁴-3-keto steroids, 5β-stanols and 4α-methyl sterols in addition to regular (4-demethyl) sterols. The following new marine sterols were isolated and identified using MS and 360 MHz NMR: 5β-cholest-22E-en-3β-ol, 24S-methyl-5β-cholest-22E-en-3β-ol, 24-methylene-5β-cholestan-3β-ol, both epimers at C-24 of 4α-methyl-24-ethyl-5α-cholest-22E-en-3β-ol, 4α, 22ξ, 23ξ-(or 24ξ-)trimethyl-5α-cholest-8(14)-en-3β-ol and (22S, 23S, 24S)-4α-24-dimethyl-22, 23-methylene-5α-cholestan-3β-ol. The latter sterol and 23-demethylgorqosterol have opposite configurations at C-22, C-23, and C-24; the Δ⁸⁽¹⁴⁾ sterol has an unprecedented side chain.     

Sterols of the cultured euglenid Eutreptia viridis: A novel Δ23-unsaturated sterol.

The sterol mixture isolated from the marine alga $\text{Eutreptia viridis}$ consists mainly of Δ^5,7-dienes which account for 80% of the free sterols. Eighteen different sterols were detected, including a novel sterol with the rare Δ²³-unsaturation, viz. 24-ethylcholesta-5,7,23Z-trien-3β-ol.     

Asymmetric reduction of steroidal 20-ketones: Chemical synthesis of corticosteroid derivatives containing and 20α, 21-diol and 17α, 20α, 21-triol side chains

A method is presented for the chemical synthesis of corticosteroid derivatives containing the 20α, 21-diol and 17α, 20α, 21-triol side chains. The ketol side chains of cortisol, corticosterone, 11-deoxycortisol, and 11-deoxycorticosterone were reduced at C-20 with sodium borohydride in a two-phase system consisting of aqueous calcium chloride and an organic phase of chloroform or ethyl acetate. Stereoselectivity of reduction was 92% α-oriented for cortisol and 79% α-oriented for 11-deoxycortisol at ?27°. The 20α-form diminished relative to the 20β-form with increasing temperature. For the 17-deoxy steroids, reduction to the 20α-form was 23% for 11-deoxycorticosterone and 41% for corticosterone. The $\text{20α}\text{20β}$ ratios of 17-deoxy steroids were unchanged between 0° and ?27°. Calcium ions increased the solubility of corticosteroids in the aqueous phase. We propose that calcium ions affect the stereochemistry of reduction by forming a bidentate complex with the side chains of 17α-hydroxy steroids, fixing them in an orientation favorable to 20α-reduction, and by altering the phase partition of the steroids.     

Bromine oxidation of 1,2-O-isopropylidene-α-d-Glucofuranose and sucrose

Sucrose and $\text{1,2-O-}\text{isopropylidene-α-}\text{d}\text{-glucofuranose}$ (1) were oxidised with bromine in aqueous solution at pH 7 and room temperature. The resulting keto derivatives were converted into their more-stable O-methyloximes, which were characterised by spectroscopic and chromatographic methods. Oxidation of 1 occurred at C-3 and C-5, with a preference for C-5. In the sucrose derivatives isolated after oxidation, those having a keto group in the glucopyranosyl moiety preponderated. The axial fructofuranosyl aglycon protects position 3 in the glucopyranosyl group and oxidation occurs only at C-2 and C-4. Small amounts of sucrose oxidised at C-3 in the fructofuranosyl moiety were also found.     

Preparation of 6 alpha- and 6 beta-carboxymethyl steroid conjugates and their use in radioimmunoassay for progesterone

A method was devised to utilize the Δ⁴-3-ketone structure of steroids to obtain a functional group at the 6 position which could be conjugated to proteins. The bis ethylene ketal of progesterone was formed and the resulting Δ⁵ double bond was subjected to epoxidation. The 5α,6α-epoxide formed was opened with allyl magnesium bromide after which the terminal carbon-carbon double bond was oxidized to provide a carboxyl group which could be coupled to the free amino groups of proteins.The 6α and 6β carboxymethyl progesterone derivatives formed by this method were coupled to bovine serum albumin (BSA) using tributyl amine and $\text{i}$-butyl chlorocarbonate. The 6α conjugate averaged 19 residues per molecule of BSA and the 6β, 24 residues per BSA. Antisera to the conjugates produced in rabbits showed very little cross-reaction with steroids which differed from progesterone at positions other than C-5 or C-6. There appeared to be very little difference between the specificities of the 6α and 6β antisera.     

Intramolecular catalysis. VII: The nature of side chain shielding of the 12alpha-hydroxyl group of steroids

A series of 12α-hydroxy steroids with varying side chains was prepared, and their 24-hour acetylation yields were compared, l2α-Hydroxy-5β-pregnan-20-one ($\text{lb}$) was prepared from 3α, 12α-diacetoxy-5β~pregnan-20-one ($\text{2}$) and also by side chain degradation of 12α-acetoxy-5β-cholanoic acid ($\text{5d}$). 21-Benzyl-5β-pregnan-12α-ol ($\text{1g}$) was synthesized by hydrogenation of the 21-benzylidine derivative of ketone $\text{1b}$. 23-Pheny1-5β-norcholan-12α-ol ($\text{1k}$) was obtained by the Grignard reaction of 2-phenyl-ethylmagnesium bromide and ketone $\text{1b}$, dehydration, hydrogenation and hydride reduction; a similar sequence produced 20-methyl-5β-pregnan-12α-ol ($\text{lm}$). The acetylation results (Table 11) imply that branching at C-20 may be more significant for 12α-hydroxyl reactivity than side chain length or type. An additional compound with an unbranched side chain, 21-nor-5β-cholan-12α-ol ($\text{14}$), was synthesized by a Grignard reaction on the 21-bromo intermediate $\text{11b}$. Acetylation rates determined by glc indicate (Table 111) That compounds with unbranched side chains have 12α-hydroxyl groups about ten times as reactive as their analogs with 20-methyl groups.     

Conversion of sterols and triterpenes by mycobacteria. II. Transformation of 7-oxygenated sterols into androstane derivatives via a 7-deoxygenation

The bioconversion of 7-oxygenated sterols by Mycobacterium aurum was studied in a preliminary investigation of the microbial conversion of wool wax. 7-Oxocholesterol was found to be transformed mainly into 3,17-dioxygenated androstane derivatives. 7 xi-Hydroxylated sterols were formed in an initial reduction step, and the C-7 hydroxyl group was then eliminated in a dehydration reaction. This was thought to take place during the isomerisation of cholest-4-en-3-one to cholest-5-en-3-one. Deuterium labelling experiments showed that this elimination proceeded faster for the C-7 alpha isomer, although it was not stereospecific. The C-7 alpha and C-7 beta-hydroxy isomers were weakly interconverted via the 7-oxo derivatives. Cholest-4-en-3-one, cholest-1,4-dien-3-one and cholest-4,6-dien-3-one all lost their side chains following a hydrogenation/dehydrogenation reaction. The resulting 3,17-dioxoandrostene or 3,17-androstadiene derivatives were mainly hydrogenated into 5 alpha-androstane-3,17-dione and 5 alpha-androstane-3 beta-ol-17-one. Elimination of the 3 beta-hydroxyl groups giving cholesta-3,5-dien-7-one, and subsequent microbial degradation of the side chain was not observed to any significant extent. The convergence of the bioconversion pathways of cholesterol and the 7-oxygenated cholesterols enabled crude, partially auto-oxidised cholesterol to be used as a substrate for the production of 3,17-dioxygenated androstane derivatives by M. aurum.     

Rifamycin derivatives: specific inhibitors of nucleic acid polymerases

Rifampicin and three rifamycin SV derivatives with different lipophilic side chains were tested as inhibitors of a number of purified enzymes including the α and αβ forms of RNA-directed DNA polymerase of avian myeloblastosis virus (AMV). $\text{AF}\text{ABDMP}$ (2,5-dimethyl-4-N-benzyl demethyl rifampicin), $\text{AF}\text{013}$ (O-n-octyloxime of 3-formyl rifamycin SV) and C-27 (rifamycin SV with a dicyclohexylalkyl substituted piperidyl ring at the 3-position) at concentrations less than 20 to 40 μg/ml completely inhibited the RNA- and DNA-directed DNA polymerase and RNase H activities of both AMV enzymes. Rifampicin was inactive at 100 μg/ml. When used against a variety of non-polymerizing enzymes such as alkaline phosphatase, glutamate-oxaloacetate transaminase, DNase I, and RNase A, these derivatives were inactive at drug concentrations between 100 and 200 μg/ml. Polynucleotide phosphorylase was inhibited slightly by all three derivatives. These results support the idea that rifamycin SV derivatives with appropriate 3-substituted side-chains are specific inhibitors of nucleic acid polymerizing enzymes.     

Investigations on purine and pyrimidine bases stacking associations in aqueous solutions by the fluorescence quenching method: II. Heteroassociation between 2-aminopurine and thymidine

Heteroassociation between A and B compounds in liquid solution was considered. Provided that concentration of A molecules is low, a geneial equation describing fluorescence quantum yield and lifetime of compound A as a function of B molecules concentration was derived. The heteroassociation between 2-aminopurine and thymidine in aqueous solutions was examined within the range of temperatures 0 to 90° C. The equilibrium constants of the first step of association, namely heterodimer formation, were determined and its thermodynamic parameters (ΔH = ?2.76 $\text{kcal}\text{mol}$, ΔS = ?5.9 e.u.) were calculated. The observed changes of the stacking rate constants with temperature confirm the two-step mechanism of the reaction. The activation energy (~2.7 $\text{kcal}\text{mol}$) and the encounter distance (~10.7 A) are only slightly larger than in the case of 2-aminopurine autoassociation, most probably because of a stronger solvation of thymidine molecules.     

Minor and trace sterols from marine invertebrates 29. (22E)-ergosta-5,22,25-trien-3β-ol and (22E,24R)-24,26-dimethylcholesta-5,22,25(27)-trien-3β-ol. Two new marine sterols from the sponge Pseudaxinella lunacharta

Two new marine sterols have been isolated from the sponge $\text{Pseudaxinella lunaecharta}$, and are shown to contain two double bonds in the side chains. The structures have been deduced by spectroscopic methods and confirmed by transformation of one of them to a known compound.     

Contribution of microsomal cytochrome b5 electron transport system coupled with Δ5-3β-hydroxysteroid dehydrogenase to androgen synthesis from progesterone

Cytochromes P-450 and $\text{b}$₅ were observed in the microsomal fraction of interstitial tissue of rat testes. Microsomal cytochrome $\text{b}$₅ was reduced by the NADH coupled with the activities of Δ⁵-3β-hydroxysteroid dehydrogenase with Δ⁵-Δ⁴ isomerase through conversion of pregnenolone to progesterone. Activities of NADPH-supported 17α-hydroxylase and C-17-C-20 lyase which converted progesterone to androstenedione were stimulated by either the presence of NADH or the oxidative reaction by the dehydrogenase upon Δ⁵-3β-hydroxysteroids. Androstenedione production enhanced by the reaction of the dehydrogenase was decreased by addition of the antibody against NADH-cytochrome $\text{b}$₅ reductase which was purified from rat hepatic microsomes, suggesting the active participation of cytochrome $\text{b}$₅ in the androgen synthesis.     

Influence of sterols on ion transport through lipid bilayer membranes

Charge-pulse relaxation experiments with the negatively charged lipophilic ions, dipicrylamine and tetraphenylborate, (as well as with the positively charged carrier system Rb⁺-valinomycin) have been carried out in order to study the influence of sterols on the ion transport through the lipid bilayer membrane. The mol fraction of the sterols (cholesterol, epicholesterol, ergosterol, stigmasterol, dihydrocholsterol, epicoprostanol and cholesterololeate) as referred to total lipid was varied in a wide range (mol fractions 0–0.8).The monoolein/sterol or dioleoylphosphatidylcholine/sterol mixtures were dissolved in $\text{n}$-hexadecane in order to minimize effects of the sterol on the membrane thickness.Cholesterol had a strong influence on the transport of the lipophilic ions. Its incorporation into monoolein membranes increased the rate constant ${}_{\mathrm{<mtext>i</mtext>}}$ of translocation up to 8-fold, but incorporation into phosphatidylcholine membranes had virtually no influence on $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$. The other sterols with one hydroxy group and cholesterololeate had no influence on the rate constant or the partition coefficient β. The results are discussed on the basis of a possible change of dipole potential of the membrane caused by cholesterol and its derivatives.In the case of valinomycin-mediated Rb⁺ transport only cholesterol had a strong influence on transport properties. The rate constants of association ($\text{k}{}_{\mathrm{<mtext>R</mtext>}}$) as well as the rate constants of translocation of the complex ($\text{k}{}_{\mathrm{<mtext>MS</mtext>}}$) and of the free carrier ($\text{k}{}_{\mathrm{<mtext>S</mtext>}}$) were reduced by incorporation of cholesterol up to eight-fold. The decrease of $\text{k}{}_{\mathrm{<mtext>S</mtext>}}$ and $\text{k}{}_{\mathrm{<mtext>MS</mtext>}}$ are possibly caused by a decrease of membrane fluidity, whereas the decrease of $\text{k}{}_{\mathrm{<mtext>R</mtext>}}$ may be due to an increase of surface potential. The different action of cholesterol on the two transport systems is discussed under the assumption that the adsorption plane of the lipophilic ion is located more towards the aqueous side and that of the ion-carrier complexes more towards the hydrocarbon side of the dipole layer.     

Comparative responses of the yeast mutant strain GL7 to lanosterol,cycloartenol, and cyclolaudenol

Under anaerobic growth conditions the isomeric 4,4′,14-trimethylcholestane derivatives lanosterol and, more efficiently, cycloartenol satisfy the sterol requirement of the yeast sterol auxotroph $\text{Saccharomyces cerevisiae}$ strain GL7. Aerobic mutant growth is supported only by cycloartenol and not by lanosterol, suggesting different structural requirements for aerobic and anaerobic cells. It is proposed that the non-planar conformation imposed by the 9,19-cyclopropane ring of cycloartenol moderates the adverse membrane effects of the nuclear methyl groups at C-4 and C-14. Under both aerobic and anaerobic conditions cyclolaudenol, a C-24-methyl derivative of cycloartenol, is a significantly more effective sterol source for strain GL7 than cycloartenol. This result is in keeping with the predominance of C-24-methyl sterols (ergosterol) in wild-type yeast.     

Investigations on purine and pyrimidine bases stacking associations in aqueous solutions by the fluorescence quenching method: I. Autoassociation of 2-aminopurine

A general equation was derived, describing fluorescence quantum yield and lifetime of an autoassociating compound in liquid solutions. The autoassociation of 2-aminopurine in aqueous solution was examined within the range from 0 to 90°C. The compound seemed to associate cooperatively. The thermodynamic parameters of polymerization change with temperature, so that its free enthalpy ΔG = ?0.0797 T² + 45.4 T ?7893. The dimerization enthalpy and entropy are approximately temperature-independent (ΔH₂ = ?4.17 $\text{kcal}\text{mol}$, ΔS₂ = ?10.9 e.u.), although the function: ΔG₂ = ?0.0308 T² + 30.3 T - 7213 fits experimental points better. The observed dependences can be explained by the increasing role of the hydrophobic effect with temperature and size of the aggregates. The association rate constants were determined, and a two-step reaction mechanism was demonstrated. The first step is diffusion-controlled. The second is characterized by an activation energy of ~2 $\text{kcal}\text{mol}$ and an encounter distance of ~8.3 Å.     

An intramolecular C-2 → C-1 hydrogen transfer during the acid-catalyzed conversion of d-xylose to d-threo-pentulose (d-xylulose)

Aldose-ketose isomerases are known to catalyze a partial and sometimes complete intramolecular hydrogen transfer between C-1 of the ketose and C-2 of the aldose. It was recently shown (Feather, M. S., and Harris, D. W. (1975) J. Amer. Chem. Soc.97, 178–181) that the same type of transfer occurs during the acid-catalyzed interconversion of d-fructose, d-glucose, and d-mannose. A similar transfer is demonstrated herein for the conversion of d-xylose to d-xylulose in acid solution. d-[2-³H]xylose was isomerized in aqueous sulfuric acid and the resulting d-[³H]xylulose was isolated in 6% yield. The ketose had 18.3% the activity of the starting aldose. Chemical degradation showed that all the carbon-bound tritium of the d-[³H]xylulose was located at C-1, thus indicating a C-2 → C-1 intramolecular hydrogen transfer. During the reaction, less than 1.2% of the total radiochemical activity was found in the solvent, and, the unreacted d-[2-³H]xylose was recovered, having an activity nearly the same as the starting material. The differences in activity, therefore, of the d-[2-³H]xylose and the d-[1-³H]xylulose are due to an isotope effect ($\text{K}{}_{\mathrm{<mtext>H</mtext>}}\text{K}{}_{\mathrm{<mtext>T</mtext>}}$) which is indicated to be 5.4. The data are discussed in terms of currently accepted models for isomerase mechanisms.     

Quantitative analysis of the binding of melittin to planar lipid bilayers allowing for the discrete-charge effect

The interaction of melittin with lecithin bilayers was studied using the resulting surface potentials at the bilayer/water interfaces to monitor the association. Melittin added to the aqueous phase binds strongly to the interface but remains localized on that side of the bilayer to which it is added. The analysis of the binding curves reveals the inadequacy of the Gouy-Chapman theory for the fixed-charge surface potential in describing the electrostatic potential experienced by the adsorbed molecules. Calculations based on the Stern equation, modified for a discrete charge distribution, give a good fit to the experimental data. The thermodynamic analysis revealed different binding energies, Δ$\text{G}$°, at 10 and 100 mM ionic strength (?7.85 and ?8.26 kcal/mol, respectively). Binding saturates at an area of 650 Ų per melittin molecule. A change in the surface dipole potential corresponding to ?1.1 debye/?_a ($\text{?}{}_{\mathrm{a}}\text{= dielectric constant of the adsorption region}$) had to be postulated. The Debye-Hückel length for a charge bound to the membrane/solution interface was found to be about one-third smaller than in bulk solution.     

Bilayers of dipalmitoyl-3-sn-phosphatidylcholine: Conformational differences between the fatty acyl chains

$\text{Dipalmitoyl}\text{-3-sn-}\text{phosphatidylcholine}$ is specifically deuterated at the C-2 position of the fatty acyl chains. Using deuterium magnetic resonance it is then possible to probe the chain conformation in the vicinity the polar head groups. Three separate quadrupole splittings are observed for bilayers of 1,2[2′-²H₂] $\text{palmitoyl}\text{-3-sn-}\text{phosphatidylcholine}$, indicating that the two chains behave differently. The synthesis of phosphatidylcholines each deuterated in only one chain allows the assignment of the three resonances. It is concluded that the beginnings of the two chains have orientations parallel and perpendicular to the bilayer normal. The data further suggest the possibility of two long-lived conformations of the glycerol constituent.     

The artifactual nature of fluoride inhibition of reverse transcriptase and associated ribonuclease H

Surprisingly, the $\text{sn}$-1 configuration of 1-0-hexadecyl-2-acetyl-glycerylphosphorylcholine showed significant activity, 3.22 × 10^?9 M, when compared to the $\text{sn}$-3 enantiomer, 2.92 × 10^?10 M and a racemic mixture with a value of 7.2 × 10^?10 M. A methoxy substitution at the C-1 or C-2 position of octadecyl glycerylphosphorylcholine gave a derivative with high biological activity for stimulating serotonin release from rabbit platelets. A 1-0-dodecyl-2-methoxy analogue showed very low activity; also, a comparable series of 0-benzyl derivatives were inactive. Examination of 1-0-hexadecyl, 1-0-octadecyl- or 1-0-dodecyl-2-acetyl-$\text{sn}$-glyceryl-3-phosphorylcholine showed that the hexadecyl compound had three times the biological activity of the octadecyl and five times that of the dodecyl.     

Irreversible inactivation of mammalian Δ5-3β-hydroxysteroid dehydrogenases by 5, 10-secosteroids. Enzymatic oxidation of allenic alcohols to the corresponding allenic ketones

Novel 4,5-allenic 3β-hydroxy-5,10-secosteroids have been synthesized by sodium borohydride reduction of the corresponding conjugated allenic 3-oxo-5, 10-secosteroids. The secosteroid allenic alcohols are substrates for bovine adrenal and human placental Δ⁵-3β-hydroxysteroid dehydrogenases, and the resulting electrophilic conjugated allenic ketones are shown to inactivate these dehydrogenases in a time-dependent manner. Inactivated enzyme did not recover activity after filtration through Sephadex G-25. In contrast, the secosteroid allenic alcohols were not oxidized at C-3 by the bacterial 3β(and 17β)-hydroxysteroid dehydrogenase from $\text{P. testosteroni}$, nor did the corresponding allenic ketones inactivate this enzyme when incubated directly.     

Biosynthesis of Δ5.23 sterols in etiolated coleoptiles from Zea mays

In addition to the previously found ergosta-5, E-23-dien-3β-ol and 5α-ergosta-7, E-23-dien-3β-ol, the following Δ23 sterols have been identified in etiolated maize coleoptiles: cyclosadol, 4α, 14α-dimethyl-5α-ergosta-8, E-23-dien-3β-ol, 4α, 14α-dimethyl-9β, 19-cyclo-5α-ergosta-8, E-23-dien-3β-ol and 4α-methyl-5α-ergosta-7, E-23-dien-3β-ol. The incubation of maize coleoptile microsomes in the presence of cycloartenol and of [¹⁴C-methyl]S-adenosyl methionine gave a mixture of labelled 24-methylene cycloartanol and cyclosadol. No trace of cyclolaudenol could be detected in these conditions. It is suggested that Δ²³ sterols are products of the C-24 methyltransferase reaction and they probably do not arise from a Δ²⁴ → Δ²³ isomerization occurring at a later stage of the biosynthesis. The Δ¹³-sterols may play an intermediary role in the biosynthesis of 24-methyl sterols in this plant material.