The Reversibility of the Isomerization of the Δ⁸ to Δ⁷ Bond in Cholesterol Biosynthesis

Abstract

The reversibility of the isomerization of the A s to A7 bond in the biosynthesis of cholesterol has been re-examined. For this purpose [4-'4C,9a-3H,]cholest-7-en-3~-ol has been incubated with the 10000 x g soluble fraction of rat liver homogenate both under anaerobic and aerobic conditions. Almost complete exchange of the 9a hydrogen with the medium has been observed under anaerobic conditions, whereas under aerobic conditions 34 % exchange with the medium has been calculated from the 3H/14C ratio found in cholesterol isolated after incubation. The stereospecificity of the A7 to As isomerization has been established by incubating in analogous conditions [4-14C,7-3Hl]cholest-7-en-3P-01. The synthesis of each labelled substrate is described.Cholesterol biosynthesis involves the isomerization of the double bond at position 8 of lanosterol to the 7 position. The reaction may occur at any step of the biosynthesis after the removal of the methyl group at C-32 [l]. The isomerization implies the stereospecific removal of the 7P hydrogen and the addition of a solvent hydrogen at the 9a positionThe possible reversibility of this reaction has been considered and different authors [l ,4] have presented evidence that no detectable amounts of cholest-8-en-3P-01 are formed by incubating cholest-7-en-3~-01 with a microsomal preparation of rat liver. On the other hand it has been recently demonstrated that tritium from the medium is incorporated at the 9a position of cholest-7-en-3/3-01, when this sterol is incubated either with microsomes or with the 10000 x g soluble fraction of rat liver homogenate under anaerobic conditions [5]. On the basis of these results it has been concluded that the biological conversion of cholest-8-en-3P-01 to cholest-7-en-38-01 is a reversible process. However under physiological conditions cholest-7-en-3/l-ol is efficiently converted to cholesterol and it does not accumulate in the same way as under the anaerobic conditions utilized in the above-mentioned experiments. It was of interest therefore to consider the reversibility of the isomerization under aerobic conditions. For this purpose [4-14C,9a-3H1]cholest-7-en3p-01 was synthesized and incubated with the 10 000 x g soluble fraction of rat liver homogenate under aerobic ~3 1 .conditions. The decreased 3H/14C ratio found in cholesterol derived from the incubations demonstrated that isomerization from cholest-7-en-3P-01 to cholest-8-en-3P-01 takes place also during the biological conversion of the former to cholesterol. The stereospecificity of the reaction is also demonstrated under both aerobic an anaerobic conditions. MATERIALS AND METHODS MaterialsTritiated water and tritiated sodium boron hydride were obtained from Amersham (U.K.) ; N,O-bis(trimethylsily1)-trifluoroacetamide from Pierce Chemicals; osmium tetroxide from Merck ; cholesta-5,7-dien-38-01 acetate from Fluka A.G. Radioactive Thin-Layer Chromatography ScanningThis was carried out using a Packard radioactivechromatogram scanning system, model 7201 and silica gel H F layers (0.25 mm ...

Studies on the 14α‐Demethylation Mechanism in Cholesterol Biosynthesis

“…Despite the demonstrated sequence of lanosterol metabolism [30] indicating that formation of the 8,14-diene system and reduction of the A 1 4 double bond precedes the elimination of the methyl groups at position 4, the results indicate an active conversion of 3P-hydroxy5a-[7-3H]cholest-7-ene-14a-carbaldehyde to cholesterol by the 10000 x g supernatant fraction of rat liver homogenate [14], similar to that previously described for 5a-cholest-7-en-3~-01 [31]. In addition, 3HCOOH was found to be released by incubation of 3~-hydroxy-Sa-[32-3H]cholest-7-ene-14a-carbaldehyde with liver microsomes (Table 1).…”

“…Therefore the catabolism of the 7a-oxygenated intermediate was studied in the presence of soluble proteins by following the aerobic incubation of the aldehyde with microsomes with an anaerobic incubation in the presence of the 1OOOOxg supernatant of rat liver homogenate. The anaerobic conditions were chosen to avoid both the introduction of the A 5 double bond in 5a-cholest-7-en-38-01 possibly formed [31,32] and the conversion of the residual 14a-carbaldehyde. The 10000 x g supernatant proteins were added in order to provide both fresh microsomal proteins and sterol carrier proteins which might be required for the metabolism of the A s ('4)7a-0-substituted compound.…”

Conversion of methyl ketones into terminal acetylenes and (E)-trisubstituted olefins of terpenoid origin

“…4-(2,6,6-Trimethyl-l-cyclohexyl)-2-butanone (5). The following procedure is based on that reported by Ashby, Lin, and Kovar.6 To cuprous iodide (38.1 g, 200 mmol) placed in a three-necked flask were added 400 mL of dry THF and a THF solution of LiAlH4 (1.35 M, 37 mL, 50 mmol) at 0 °C.…”

“…As the isolation of 2 is not usually required, such a sequence would amount to a two-step but highly stereoselective alternative to conventional carbonyl olefination reactions. In order to demonstrate the practicality of such a procedure, we decided to synthesize monocyclofarnesol (3)5 from ¡8-ionone (4) (see Scheme II). Unfortunately, however, our attempts to convert dihydro-/3-ionone (5), obtained in quantitative yield by reducing 4 with LiAlH4 and Cul,6 into the required intermediate 6 by various known procedures7 were disappointing. Even the best of those examined, which was developed by Craig and Moyle,7f…”

Stereochemical course of the catalytic reduction and of the acidic isomerization of 14.beta. steroids. Synthesis of .DELTA.8-14.beta. and 8.alpha., 9.alpha., 14.beta. steroids

“…The structural proof is additionally unsatisfactory because the 60-MHz spectrum of 5 is reported1 as 4.62-3.58 (m, 6), 2.57-1.66 (m, 6), and 1.45-0.78 (m, 4). Such a spectrum is inconsistent with a structure (5) for which only five protons in the region 4.62-3.58 corresponding to CHOH and CH2OH would be anticipated.…”