Chemical Synthesis of Δ7,24-[3α-3H]Cholestadien-3β-ol and Its Conversion to Cholesterol in the Rat

A7J4-[3~aH]Cholestadien-3P-01 was synthesized by catalytic hydrogenation of A5*7*24 [3cu 3H]cholestatrien-3/3 01. The elemental composition was proved correct by high resolution mass spectrometry. The mass spectrum of A7~24-[3~-3H]cholestadien-3/I-01 was compared with the spectra of Asand A’-cholesten-3P-ol and A5024-cholestadien3/3-01. The infrared spectrum of synthetic A7s24-[3~-3H]cholestadien-3p-ol is compatible with that of the biological sterol. Their metabolic behavior is also similar.

A sterol with the probable structure of A7j24-eholestadien-3fi-01 has been isolated by Frantz and Mobberley (1) from the livers, and by Clayton et al. (2) and Frantz et al. (3) from the skins of MER-29..treated rats. The conversion of the biosynthetic A7*24-cholestadien-3fi-ol to cholesterol and intermediates has been studied by Frantz et al. (3) in vivo and Dempsey (4) in &TO. This paper describes the chemical synthesis of A7fZ4-[3a-3H]cholestadien-3/3-ol and its conversion to cholesterol and some intermediates in a cell-free preparation of rat liver.
The silicic acid-Super Cel chromatography has previously been described by Clayton et a.Z. (2). The silver nitrate chromatography was done according to the method of Zacchei (5) or Paliokas et al. (6) and Lee et al. (7). Melting points were determined in sealed evacuated capillary tubes in a Thomas-Hoover melting point apparatus.
Radioactivity was measured with a Packard Tri-Carb scintillation spectrometer. Ultraviolet spectra were recorded on a Beckman model DU spectrometer.
Infrared spectra were recorded on a PerkinElmer 521 spectrometer equipped with a reflecting beam condenser unit.
To elute the A5,1,24 compound, 60 7' hexane-40% atmospheric pressure for 3.5 hours. Under these conditions benzene was used; it was followed by a small amount of more 10 to 15% of the 24,25 double bond was also hydrogenated, The sterol mixture was separated on a silver nitrate column (5) using benzene as eluent.
The sterol has no specific absorption in the 220-to 300-nm region.
The molecular ion mass measured with a high resolution mass spectrometer was 384.3385 compared to the calculated value of 384.3394 for C&TH~~O.
Infrared Specfrometyy-The infrared spectrum of synthetic A7*2"-[301~3H]cho1estadien-3~-ol is compared with the spectrum of A7-cholesten-3P-ol in Fig. 1. In the 1300 to 1400 cm-l region, assigned to methyl and methylene bending vibrations, the spectrum of the A7J4-sterol, compared to the saturated side chain analog, shows the characteristic decrease in the intensity of the 1363 cm-l band compared to its higher frequency companion at 1378 cm-l (10). In the 800 to 900 cm-l region, assigned to the out-of-plane bending vibrations of hydrogen atoms attached to doubly bonded carbon, an increase in the intensity of the 825 cm-1 band compared to the 843 cm-1 band is noticeable with the introduction of the 24 double bond. This may be related to a Iow intensity band observed in Az4-cholesten-30-01 at 824 cm-l (10). The infrared spectrum of the synthetic A7%terol is compatible with that of biological sterol (3).
Mass Spectrometry-The mass spectra of A5-and A?-cholesten-3/3-01 and A5J4-and A7T24-(3a-3H]cholest,adien-3fi-ol are shown in Fig. 2. According to Wyllie and Djerassi (ll), a diagnostically important cleavage is associated with the presence of the 24 double bond in the sterol side chain. The intense peak at m/e 271, corresponding to the loss of the side chain together with 2 hydrogen atoms from the steroid nucleus, appears in the As,nr-and A7J4-sterols, but. is absent in the corresponding saturated side chain sterols.
The triplet of peaks at m/e 299, 300, and 301, also assumed characteristic of the A24 unsaturation, is present in the A7J4-sterol, although of lower intensity than in the Asaz4 compound It has been shown in several instances that the position of the nuclear double bond has some influence on the fragmentation pattern induced by the side chain double bond. The m/e 271 ion, or its equivalent at m/e 343 in the trimethylsilyl ethers, is present in both A5 and A7 analogs of the 24-methylene and 24ethylidene sterols, the m/e 343 peak being the base peak in the A7 compounds (12). The double bond in position 8,9, however, has a definite inhibiting effect on the double hydrogen transfer in the 17-20 side chain fragmentation of zymosterol (13). The base peak at m/e 69 of A 7~24-cholestadien-3/3-ol corresponds to t,he allylic cleavage of the 22-23 bond (14). In sterol acetates this base peak is characteristic for 24 unsaturation.
The saturated side chain sterols have their base peak at m/e 43. In trimethylsilyl derivatives the m/e 69 ion is of no diagnostic value (13). In the free sterols, Fig. 2, the m/e 69 fragment is of considerably higher relative intensity in the A7,24-sterol than in its A5 analog, and may be characteristic of the A7,24 configuration. More information about the fragmentation patterns of free sterols is needed before any definite conclusions can be drawn.
Biological The incubation was carried out at 37' under oxygen for 20 min in a Dubnoff shaker. The incubation mixture was saponified to insure the absence of any sterol esters formed during the incubation.
The chromatographic separation of the sterols is shown in Fig. 3, the composition of the sterol mixture is given in Table I. The relatively small amounts of A7 and A5v7 intermediates compared to the A5.7J4 and A6sz4 compounds indicate the preference of the unsaturated side chain pathway in cholesterol synthesis under the conditions used.