Stereochemistry of reduction of the C-24,25 double bond in the conversion of desmosterol into cholesterol

Article abstract of DOI:10.1016/S0040-4039(02)02567-4

Feeding of the chemically prepared [24-¹³C, 24-²H]desmosterol to cell-free systems derived from rat liver and silkworm gut and to cultured cells of Oryza sativa followed by deuterium-decoupled ¹H, ¹³C shift correlation NMR analysis of the biosynthesized cholesterol revealed the stereospecific incorporation of hydrogen atoms from the re-face of the C-24 position of desmosterol.

Full text of DOI:10.1016/S0040-4039(02)02567-4

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 341–344
Stereochemistry of reduction of the C-24,25 double bond in the
conversion of desmosterol into cholesterol
Kyoko Takahashi,a Kenichiro Hashimoto,a Ayako Fujiyama,a Junko Yamada,a Noriko Kobayashi,a
Masuo Morisaki,a,* Sayaka Nakano,b Noriyuki Harab and Yoshinori Fujimotob
aKyoritsu College of Pharmacy, Shibakoen, Minato-ku, Tokyo 105-8512, Japan
bDepartment of Chemistry and Materials Science, Tokyo Institute of Technology, Meguro-ku, Tokyo 152-8551, Japan
Received 26 August 2002; accepted 8 November 2002
Abstract—Feeding of the chemically prepared [24-13C, 24-2H]desmosterol to cell-free systems derived from rat liver and silkworm
gut and to cultured cells of Oryza sativa followed by deuterium-decoupled 1H, 13C shift correlation NMR analysis of the
biosynthesized cholesterol revealed the stereospecific incorporation of hydrogen atoms from the re-face of the C-24 position of
desmosterol. © 2002 Elsevier Science Ltd. All rights reserved.
The final stage of sterol biosynthesis involves reduction
of D24 olefinic sterols such as desmosterol (1, R=H),
24-methyldesmosterol (1, R=Me) and 24-ethyldesmos-
terol (1, R=Et), which were established as the biosyn-
thetic precursors of cholesterol (2, R=H),
campesterol/dihydrobrassicasterol (2, R=Me) and
sitosterol (2, R=Et), respectively (Scheme 1).
dihydrobrassicasterol and sitosterol, respectively, by an
anti addition of hydrogen atoms on the respective D24
olefinic precursors.3 In this paper we have reexamined
the stereochemical course of the reduction of desmos-
terol (1, R=H) leading to cholesterol (2, R=H) in rat
liver, as well as in insects and higher plants. The results
clearly demonstrated that an anti addition uniformly
occurs, irrespective of the species examined, in contra-
diction to the dogma which has prevailed for 30 years.1
In regard to stereochemistry of the biohydrogenation
reaction catalyzed by rat liver 3b-hydroxysterol D24-
reductase (D24-sterol reductase) producing cholesterol, a
syn addition of hydrogen atoms on the 24-si face and
25-si face of the C-24,25 double bond was proposed,1
whereas an anti addition was reported for tigogenin
(and thence cholesterol) biosynthesis in Digitalis
lanata.2 We have recently demonstrated with cultured
cells of Oryza sativa that 24-methyldesmosterol and
24-ethyldesmosterol are converted into campesterol/
Because stereospecific hydrogen introduction on C-25
from the si-face during cholesterol biosynthesis was
amply demonstrated in various species,1,4 we have
presently focused on the stereochemical problem at
C-24, namely, whether the newly introduced hydrogen
on C-24 occupies the pro-R or the pro-S position in
cholesterol. This problem is difficult to solve, since C-24
of cholesterol is a prochiral carbon, which is located
Scheme 1. Reduction of D24-sterols (1) to yield naturally abundant sterols (2).
Keywords: steroids and sterols; cholesterol biosynthesis; desmosterol; stereochemistry; 3b-hydroxysterol D24-reductase.
* Corresponding author. Tel.: +81 3 3993 4841; fax: +81 3 3993 4847; e-mail: masuo-m@zephyr.dti.ne.jp
0040-4039/03/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)02567-4

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K. Takahashi et al. / Tetrahedron Letters 44 (2003) 341–344
four carbons apart from the nearest chiral carbon
(C-20). The strategy of feeding of [24-13C,24-2H]des-
24-alcohol (5). Oxidation of 5 with tetra-n-propylam-
monium perruthenate/N-methylmorpholine N-oxide,
Wittig olefination and desilylation produced [24-13C,
24-2H]desmosterol (6) in good yield. Incubation of this
substrate 6 with the 24,000×g supernatant fraction
of rat liver homogenate and the 1500×g supernatant
fraction prepared from the guts of silkworm larvae
(Bombyx mori ), and with the cultured cells of O.
sativawascarriedoutasdescribedpreviously.4 Sterolfrac-
tions were separated by silica gel chromatography and
cholesterols were further purified by HPLC. The
cholesterol samples were analyzed with deuterium-
1
mosterol (6) followed by deuterium-decoupled H, 13C
shift correlation NMR analysis5 of the biosynthesized
cholesterol appears promising, as evidenced from our
recent investigation on stereochemical details occurring
at the C-28 methylene group during sitosterol biosyn-
thesis.6
The required substrate [24-13C, 24-2H]desmosterol (6)
was prepared as shown in Scheme 2.7 Horner–
Emmons reaction of the 3-t-butyldimethylsilyl(TBS)-
oxy-22-al (3) derived from bisnorcholenic acid with
[1-13C]triethyl phosphonoacetate (Aldrich) gave the
a,b-unsaturated ester 4 which on catalytic hydrogena-
tion with PtO2 in ethanol followed by reduction with
lithium aluminum deuteride afforded [24-13C,24-2H2]-
1
decoupled H, 13C shift correlation NMR (Fig. 1). The
approximate conversion yield of cholesterol from
desmosterol was estimated to be 80% in rat liver, 50%
in B. mori and 0.9% (based on desmosterol added to
the culture medium) in O. sativa.
Scheme 2. Synthesis of [24-13C, 24-2H]desmosterol (6).
1
Figure 1. Deuterium-decoupled HMQC spectra of cholesterol (500 MHz for H/125 MHz for 13C, in CDCl3). (A) Non-labeled
cholesterol; (B) (24S)-[24-2H]cholesterol (13); (C) (24R)-[24-2H] cholesterol (14); (D–F) biosynthesized cholesterol from [24-13C,
24-2H]desmosterol (6) on incubation with rat liver homogenate (D), with cultured cells of O. sativa (E) and with silkworm gut
homogenate (F).

K. Takahashi et al. / Tetrahedron Letters 44 (2003) 341–344
343
Scheme 3. Synthesis of (24S)-[24-2H]cholesterol (13) and (24R)-[24-2H]cholesterol (14).
Scheme 4. Conversion of [24-13C, 24-2H]desmosterol (6) into (24S)-[24-13C, 24-2H]cholesterol (15).
(24-C)/l 1.08 (24-H) (Fig. 1 B) and l 39.1 (24-C)/l 1.12
(24-H) (Fig. 1 C), respectively. These shifted values are
consistent with the expected a-isotope shift (13C NMR)
and b-isotope shift (1H NMR).5 Thus, Figure 1 A, B
and C allowed definitive stereochemical assignment that
the pro-R and pro-S hydrogens at C-24 appear upfield
and downfield, respectively. Figure 1 D, E and F show
spectra of the cholesterol biosynthesized in rat liver, O.
sativa and silkworm gut, respectively. Almost single and
intense peaks were observed at l 1.08, indicating that
these peaks are due to the pro-R hydrogen at C-24. It is
concluded that the biohydrogenation of [24-13C,24-
2H]desmosterol (6) furnishes (24S)-[24-13C,24-2H]chol-
esterol (15) (Scheme 4), implying that introduc-
tion of hydrogen into C-24 of desmosterol occurs from
the re-face.11 Since 25-si face addition was well estab-
lished,1,4 the present work demonstrates that addition
of hydrogen on the 24(25)-double bond of desmos-
terol takes place in an anti fashion from the re-face
of C-24 and the si-face of C-25, as opposed to a syn
addition proposed by Caspi and co-workers 30 years
ago.1
In order to aid unequivocal assignment of the chemical
shifts of the pro-R and pro-S hydrogens at C-24 of
cholesterol, stereochemically defined [24-pro-S-2H] and
[24-pro-R-2H] cholesterols (13 and 14), were synthe-
sized (Scheme 3). Grignard reaction of the 3-OTBS-24-
al (7) derived from cholenic acid gave a diastereomeric
mixture of (24R)- and (24S)-24-alcohol, (8a) and (8b),
whose benzoates (9a and 9b) were prepared and sepa-
rated by HPLC (TOSOH TSK-GEL, SILICA-60, 7.8×
300 mm, n-hexane-dichloromethane 3:1, 6.5 ml/min).
The less mobile benzoate (9a) and the more mobile
isomer (9b) were derivatized to 3-OTBS-24-ol (8a
and 8b), 3,24-dibenzoates(10a and 10b), (R)- and
(S)-2-methoxy-2-phenyl-2-(trifluoromethyl)acetic acid
(MTPA) esters (11aR/S and 11bR/S), and methanesul-
fonates (12a and 12b). The configurations at the C-24
position of these compounds were determined by 13C
NMR of 3-OTBS-24-ol (8) and 3,24-dibenzoates(10)
compared with our previously reported ones,8 and
further confirmed by application of the advanced
Mosher’s method9 for MTPA esters (11).10 Reductive
substitution of (24R)- and (24S)-mesylate (12a and 12b)
with sodium borodeuteride in HMPA followed by desi-
lylation afforded (24S)-[24-2H]cholesterol (13) and
(24R)-[24-2H]cholesterol (14), respectively.
References
The {1H}{2H}HMQC spectra of 13 and 14 are included
in Figure 1. It can be seen that non-labeled cholesterol
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(24-H) (Fig. 1 A), whereas the corresponding peak of
24-deuterated samples (13) and (14) appeared at l 39.1
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