760 Acknowledgment. This work was supported by the National Institutes of Health in the form of a General Research Support Grant and by the Wisconsin Alumni Research Foundation. Thanks are
expressed to the University of Wisconsin Chemistry Department for use of the HA 100 nmr spectrometer and to Professor Pasupati Mukerjee for helpful discussions.
Reduction of A24of Lanosterol in the Biosynthesis of Cholesterol by Rat Liver Enzymes. 1. The Addition of a 24-Pro-$ Proton' J. B. Greig,'" K. R. Varma,2b and E. Caspi* Contribution from thc Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545. Received June 17, 1970 Abstract: The side chain of 17~i,20,24R-~H~-*~C~-cholesterol biosynthesized from 4R-(2-14C,4-3H)-mevalonic acid by rat liver enzymes was cleaved with an adrenal enzyme preparation and the 1,5-14C~-3R-3Hl-isocaproic acid encompassing carbons 22-27 of cholesterol was isolated. The 14C2,3Hl-iso~aproi~ acid was degraded, without disturbing the stereochemistry of the isotopic hydrogen, to 1S-3-14C,l-3H-isobutyl alcohol. The isobutyl alcohol was oxidized with NAD+ and yeast alcohol dehydrogenase (YADH) to 3-14C,1 -3H-isobutyraldehydewith complete retention of tritium, Since we proved that NAD+-YADH oxidation of isobutyl alcohol proceeds with the abstraction of the I-pro-R proton it follows that the isobutyl alcohol has the 1s stereochemistry. Hence the 14C,3Hcholesterol must have the 24R stereochemistry. This implies that a 24-pro-S proton adds to AZ4of lanosterol in the biosynthesis of cholesterol by rat liver enzymes. It was also demonstrated with the use of samples of l-3H1isobutyl alcohols that no significant isotope effects were detected in the NAD+-YADH oxidation of the alcohol to isobutyraldehyde by the procedures employed.
T
he biosynthetic transformation of lanosterol (1) to cholesterol (2) is a multistep process which involves among other transformations the reduction of the (2-24 double bond. In this paper we concern ourselves with the mode of reduction of this double bond in a rat liver enzyme system,4aab and particularly with the stereochemistry of addition of the hydrogen at (2-24. At the time that this work was undertaken it was already known5" that the biosynthesis of squalene, lanosterol, and cholesterol from exogenously supplied mevalonic acid (MVA) proceeds with the stereospecific retention of, respectively,'" six, five, and three 4-pro-R protons of MVA. Of the three protons retained in cholesterol the presence of the one at C-17 was proven e~perimentally.~" The other two were thought to be located at C-20jb and -24,4bwhich we later confirmed. The terminal methyls of lanosterol (and of cholesterol (2)) differ in that one originates from C-2 and the other from C-3' of MVA. From the work of Birch, et al.,'jait was inferred that the geometry at A Z 4of lanosterol is that shown in 1 (the heavy dots indicate carbons de(1) Work supported by grants from the National Institutes of Health (AM12156, HE10566, CAK316614), the National Science Foundation (GB8277), and the American Cancer Society (P-500H). (2) (a) Postdoctoral Fellow, 1966-1968; (b) Postdoctoral Fellow, 1966-1969. (3) For pertinent references see Ch. J. Sih and H. W. Whitlock, Annu. Rec. Biochem., 37,661 (1968); I. D. Frantz and G. J . Schroepfer, ibid., 36, 691 (1967); R . B. Clayton, Quart. Rea., Chem. Soc., 19, 168, 201 (1965). (4) (a) E. Caspi, I
