Synthesis of ent-25-hydroxycholesterol

Article abstract of DOI:10.1016/j.steroids.2006.01.007

25-Hydroxycholesterol (25-HC) appears to play a role in several important biological processes, including regulating cellular cholesterol levels and promoting apoptosis. However, in most cases the mechanisms by which 25-HC elicits its biological effects a

Full text of DOI:10.1016/j.steroids.2006.01.007

steroids 7 1 ( 2 0 0 6 ) 484–488
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Synthesis of ent-25-hydroxycholesterol
Emily J. Westover, Douglas F. Covey∗
Department of Molecular Biology and Pharmacology, Washington University School of Medicine,
660 S. Euclid, St. Louis, MO 63110, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
25-Hydroxycholesterol (25-HC) appears to play a role in several important biological pro-
cesses, including regulating cellular cholesterol levels and promoting apoptosis. However,
in most cases the mechanisms by which 25-HC elicits its biological effects are not known.
Insights into mechanisms of 25-HC action can be gained by studying the activity of its
enantiomer (ent-25-HC). ent-25-HC is physically and chemically identical to 25-HC; however,
25-HC and ent-25-HC can be distinguished in chiral environments, like a protein binding site.
In order to probe the mechanisms of 25-HC action, we have synthesized the enantiomer of
25-HC (ent-25-HC).
Received 15 November 2005
Accepted 24 January 2006
Published on line 6 March 2006
Keywords:
ent-25-Hydroxycholesterol
Enantiomer
© 2006 Elsevier Inc. All rights reserved.
Oxysterol
Abbreviations:
ABCA1, ATP-binding cassette,
subfamily A, member 1; ent-25-HC,
enantiomer of
25-hydroxycholesterol; 25-HC,
25-hydroxycholesterol; HMG CoA,
3-hydroxy-3-methylglutaryl
coenzyme A; LXR, liver X receptor;
SREBP, sterol regulatory element
binding protein
1.
Introduction
increasing storage of cholesterol [1,2]. Since that time, 25-HC
has been used in numerous studies of cellular cholesterol
25-Hydroxycholesterol (25-HC) has been implicated in
a
regulation despite some debate about the physiological rel-
evance of 25-HC. It is now known that 25-HC can be formed
from cholesterol by autooxidation or enzymatically by a
non-heme, iron-containing protein [3,4]. And some studies
report that significant levels of 25-HC are produced in cells
[3].
Experiments in cell culture have shown that 25-HC affects
cholesterol homeostasis at both the transcriptional and post-
translational levels. At the level of transcriptional control, 25-
HC decreases activation of the transcription factor sterol regu-
number of important biological processes, including cellu-
lar cholesterol homeostasis, atherosclerosis, and apoptosis.
Active research is underway to better characterize the in vivo
activities of 25-HC.
In the 1970’s in vitro studies showed that 25-HC was much
more potent than cholesterol at triggering cells to respond to
high sterol levels by decreasing 3-hydroxy-3-methylglutaryl
coenzyme A (HMG CoA) reductase activity and increasing
cholesterol esterification, thereby decreasing synthesis and
∗
Corresponding author at: Department of Molecular Biology and Pharmacology, Box 8103, St. Louis, MO 63110, United States.
Tel.: +1 314 362 1726; fax: +1 314 362 7058.
E-mail address: dcovey@wustl.edu (D.F. Covey).
0039-128X/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2006.01.007

steroids 7 1 ( 2 0 0 6 ) 484–488
485
Fig. 1 – Structures of 25-hydroxycholesterol (1) and ent-25-hydroxycholesterol (2).
latory element binding protein (SREBP) by promoting its reten-
tion in the ER. This, in turn, leads to decreased production of
SREBP-responsive genes, like those encoding LDL receptors
and HMG CoA reductase. In addition, 25-HC is an agonist for
the liver X receptor (LXR) transcription factor, which increases
gene products encoding ATP binding cassette protein, sub-
family A, member 1 (ABCA1), a cholesterol efflux pump,
and cholesterol 7␣-hydroxylase, the rate-limiting enzyme
in bile salt synthesis. At the post-translational level, 25-HC
promotes the ubiquitination and subsequent degradation of
HMG CoA reductase. The fact that 25-HC affects all of these
systems has led some to hypothesize that 25-HC, or related
oxysterols, may serve as an indicator for high cholesterol in
cells [5], leading cells to increase storage and metabolism
while decreasing uptake and synthesis of cholesterol.
While 25-HC is more water-soluble than cholesterol
(log p25-HC = 7.6, log pcholesterol = 9.9),1 25-HC is quite hydropho-
bic and will partition into membranes. To date, only a few
biophysical studies have characterized the membrane behav-
ior of 25-HC. In monolayers, 25-HC does not condense other
lipids as cholesterol does [6,7]. Liposomes containing 25-HC
are more permeable to calcium, sodium and glucose [7,8]. In
red blood cell membranes, 25-HC also increases the suscep-
tibility of cholesterol to cholesterol oxidase [9]. These studies
clearly indicate that 25-HC may have important effects on cel-
lular membranes.
Except for LXR binding, the mechanisms by which 25-
HC mediates its many biological effects are not known.
Understanding the biological activities of 25-HC requires
considering its effects on both membranes and proteins.
This can be accomplished by using the enantiomer of 25-HC
(ent-25-HC). Fig. 1 shows 25-HC 1 and ent-25-HC 2. Enan-
tiomers have the same relative configuration and hence
physical properties. However, enantiomers have the opposite
absolute configuration and thus may be distinguished in
chiral environments, such as a protein binding site composed
of all l-amino acids (reviewed in [10]). Thus, ent-25-HC will
affect membranes identically to 25-HC, but may interact dif-
ferently with proteins. Substituting ent-25-HC for 25-HC may
provide important insights into the mechanisms of 25-HC
action.
2.
Experimental methods
2.1.
General methods
Solvents were either used as purchased or dried and purified
by standard methodology. All air- and/or moisture-sensitive
reactions were carried out under N2 or Ar using oven-dried
glassware and cooled under vacuum or N2. All extraction sol-
vents were dried with anhydrous Na2SO4. Flash chromatogra-
phy was performed using silica gel (32–63 ␮m) purchased from
Scientific Adsorbents (Atlanta, GA). Optical rotations were
determined on a Perkin-Elmer Model 341 polarimeter. Melt-
ing points were determined on a Kofler micro hot stage and
are uncorrected. IR spectra were recorded as films on an AgCl
plate with a Perkin-Elmer 1710 FT-IR spectrophotometer. NMR
spectra were recorded at ambient temperature in CDCl3 with
a 5 mm probe on a Varian Gemini 2000 operating at 300 MHz
(1H) or 75 MHz (13C). 1H and 13C NMR spectra were referenced
to CDCl3 (ı 7.27) and (ı 77.00), respectively. Elemental analyses
were performed by M-H-W Laboratories (Phoenix, AZ).
2.1.1. (3˛, 8˛, 9ˇ, 10˛, 13˛, 14ˇ, 17˛, 20S)-Chol-5-
en-24-oic acid, methyl ester (5)
The methyl ester of (3␣, 8␣, 9␤, 10␣, 13␣, 14␤, 17␣, 20S)-3-[[(1,1-
dimethylethyl)dimethylsilyl]oxy]chol-5-en-24-oic acid 3a was
prepared as described previously [11]. This synthesis produces
a small amount (<10%) of the corresponding methyl ester of
ent-cholan-24-oic acid 3b. This mixture of steroids, 3a and 3b,
(270 mg, 0.54 mmol) was dissolved in Et2O (15 mL). A mixture
of HOAc (1.5 mL), NaOAc (16 mg) and Br2 (0.06 mL) was then
added to the steroid solution. After stirring the mixture for
3 h, water (10 mL) was added. The mixture was extracted with
EtOAc (3 × 20 mL) and washed with NaHCO3 (2 × 20 mL) and
brine (2 × 20 mL). The organic extracts were dried, filtered and
concentrated in vacuo to give an orange oil. The oil was imme-
diately applied to a column packed with silica (15% EtOAc in
hexanes). The dibromosteroid 4a was easily separated from
the saturated 4b. The dibromosteroid 4a product was recov-
ered as a clear, colorless oil (160 mg, 55%) which was not stable
and was used immediately after purification. 1H NMR ı 4.84
(1H, m), 4.44 (1H, m), 3.67 (3H, s, OMe-H), 2.69–1.47 (17H, m),
1.45 (3H, s, 19-H), 1.41–1.07 (8H, m), 0.93 (3H, d, J = 6.32 Hz, 21-
H), 0.71 (3H, s, 18-H); 13C NMR ı 174.89, 89.85, 69.31, 56.27, 55.91,
55.30, 51.66, 47.53, 45.89, 42.88, 42.06, 39.73, 37.34, 36.87, 35.48,
31.20, 31.11, 30.97, 30.33, 28.19, 24.15, 21.45, 20.50, 18.40, 12.34.
The dibromosteroid 4a (130 mg, 0.24 mmol) was dissolved
in HOAc (10 mL). Zinc dust (0.5 g) was added and the mixture
Enantiomeric steroids including ent-25-HC are not natu-
rally available and must be chemically synthesized. We here
report the first synthesis of ent-25-HC.
1
Calculated using Advanced Chemistry Development
(ACD/Labs) Software V8.14 for Solaris.

486
steroids 7 1 ( 2 0 0 6 ) 484–488
dried, filtered and concentrated in vacuo to give a white solid
(130 mg, 80%) which was used without further purification.
1H NMR ı 7.79 (2H, d, J = 9.2 Hz), 7.35 (2H, d, J = 8.0 Hz), 5.32
(1H, m, 6-H), 4.00 (2H, dt, J = 6.59, 2.47 Hz, 24-H), 3.48 (1H, m,
3-H), 2.45 (3H, s, Ph-CH3), 2.38–1.00 (28H, m), 0.99 (3H, s, 19-H),
0.89 (9H, s, C(CH3)3), 0.64 (3H, s, 18H), 0.06 (6H, s, Si(CH3)2);
13C NMR ı 144.58, 141.51, 133.25, 129.76, 127.85, 121.06, 72.58,
71.20, 56.69, 55.75, 50.10, 42.77, 42.27, 39.71, 37.34, 36.52, 35.15,
32.03, 31.84 (×2), 31.39, 28.09, 25.91 (5 × C), 25.48, 24.19, 21.61,
20.99, 19.38, 18.41, 18.23, 11.78, −4.63 (2 × C).
was stirred at rt for 1.5 h. Ether (10 mL) was added and the zinc
was removed by filtration through Celite. The organic phase
was washed with water (3 × 20 mL), NaHCO3 (2 × 20 mL), and
brine (2 × 20 mL). The organic layer was dried, filtered and con-
centrated to yield a white solid 5 (80 mg, 90%); mp 141–142.5 ◦C;
[˛]2D4 = +39.0 (c = 1.0, CHCl3); IR 2936, 1740, 1438, 1375, 1194,
1173, 1058, 734 cm−1 1H NMR ı 5.32 (1H, m, 6-H), 3.64 (3H, s,
;
OMe-H) 3.49 (1H, m, 3-H), 2.37–1.00 (26H, m), 0.98 (3H, s, 19-
H), 0.90 (3H, d, J = 6.32 Hz, 21-H), 0.65 (3H, s, 18-H); 13C NMR
ı 174.90, 140.86, 121.62, 71.63, 56.62, 55.66, 51.38, 49.97, 42.23,
42.14, 39.61, 37.13, 36.34, 35.22, 31.73 (2 × C), 31.47, 30.91, 30.86,
27.95, 24.10, 20.91, 19.22, 18.15, 11.68.
To the dry steroidal tosylate (130 mg, 0.21 mmol) was added
NaCN (15 mg, 0.31 mmol). After evacuating and filling the flask
with N2, anhydrous DMSO (5 mL) was added and the mixture
was heated to 90 ◦C for 3.5 h. The reaction was then cooled
and added to a mixture of ice and saturated NaCl (20 mL). The
organic layer was extracted with EtOAc (3 × 20 mL), washed
with brine (2 × 20 mL), and dried with sodium sulfate. After fil-
tration and concentration, the crude material was applied to
a column packed with silica gel (15% EtOAc in hexanes). The
desired product was recovered as a white solid and recrys-
tallized from Et2O/hexanes to obtain clear, colorless crystals
of the carbonitrile 7 (80 mg, 80%); mp 196–198 ◦C; [˛]D24 = +32.3
(c = 1.0, CHCl3); IR 2929, 2253, 1256, 1094, 837, 775; 1H NMR ı 5.32
(1H, s, 6-H), 3.48 (1H, m, 3-H), 2.34–1.03 (27H, m), 1.00 (3H, s, 19-
H), 0.94 (3H, d, J = 6.59 Hz, 21-H), 0.89 (9H, s, C(CH3)3), 0.68 (3H,
s, 18-H), 0.06 (6H, s, Si(CH3)2); 13C NMR ı 141.50, 121.06, 119.82,
72.57, 56.71, 55.72, 50.10, 42.77, 42.33, 39.74, 37.34, 36.52, 35.20,
35.00, 32.04, 31.84 (2 × C), 28.17, 25.91 (3 × C), 24.19, 22.16, 21.01,
19.38, 18.49, 18.21, 17.51, 11.81, −4.62 (2 × C). Anal. Calcd. for
2.1.2. (3˛, 8˛, 9ˇ, 10˛, 13˛, 14ˇ, 17˛, 20S)-
3-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]chol-5-en-24-ol (6)
4-(Dimethylamino)pyridine (DMAP) (70 mg, 0.52 mmol), t-
butyldimethylsilyl chloride (80 mg, 0.52 mmol), CH2Cl2 (3 mL),
THF (1 mL) and distilled triethylamine (0.75 mL) were added
to alcohol 5 (100 mg, 0.26 mmol). The mixture was stirred
overnight at rt. Aqueous NH4Cl (7 mL) was added. After stirring
for 10 min, the mixture was extracted with CH2Cl2 (3 × 10 mL).
The combined organic extracts were dried and concentrated.
The residue was passed through a silica plug using CH2Cl2
as the eluent and concentrated to yield the protected steroid
as a white solid (130 mg, 99%); mp 135.5–137.5 ◦C; [˛]D24 = +25.2
(c = 0.88, CHCl3); IR 2929, 2895, 2853, 1744, 1636, 1253, 1100, 837,
777 cm−1 1H NMR ı 5.31 (1H, m, 6-H), 3.65 (3H, s, OMe-H), 3.47
;
(1H, m, 3-H), 2.35–1.02 (25H, m), 0.99 (3H, s, 19-H), 0.92 (3H, d,
J = 6.04 Hz, 21-H), 0.88 (9H, s, C(CH3)3), 0.66 (3H, s, 18-H), 0.04
(6H, s, Si(CH3)2); 13C NMR ı 174.84, 141.56, 121.15, 72.55, 56.66,
56.39, 55.66, 51.37, 50.05, 42.70, 42.25, 39.64, 37.25, 36.43, 35.24,
31.94, 31.74, 30.88, 27.98, 25.80 (4 × C), 24.11, 20.89, 19.27, 18.15,
18.10, 11.68, −4.78 (2 × C).
C31H53NOSi: C, 76.95; H, 11.04; N, 2.89. Obsd. C, 77.12; H, 11.26;
N, 2.63. A small amount (<10 mg) of the 24-alcohol 6 was also
recovered.
The protected methyl ester (130 mg, 0.26 mmol) was dis-
solved in Et2O (8 mL) and added dropwise into a flask con-
taining Et2O (8 mL) and LAH (60 mg, 1.56 mmol) at 0 ◦C. After
30 min, reduction of the ester was complete. The reaction
was quenched by addition of water (0.06 mL), followed by 15%
NaOH (0.06 mL). After stirring for 30 min, additional water
(0.18 mL) was added. The solvent was then decanted from the
white aluminum salts, which were washed several times with
Et2O. Removal of solvent in vacuo produced a white solid, the
alcohol 6 (120 mg, 98%); mp 164–165 ◦C; [˛]D24 = +28.4 (c = 1.0,
2.1.4. (3˛, 8˛, 9ˇ, 10˛, 13˛, 14ˇ, 17˛,
20S)-27-Norcholest-5-en-25-one (8)
THF (6 mL) and MeLi (1.25 mL of 1.6 M solution in Et2O, 2 mmol)
were introduced to a dry flask. The mixture turned cloudy
upon cooling to 0 ◦C. The steroidal nitrile 7 (80 mg, 0.2 mmol)
was then dissolved in THF (4 mL) and slowly added to the
MeLi solution. The flask was rinsed with additional THF
(3 mL) and added to the reaction. The reaction was stirred
at 0 ◦C for 20 min, then at rt for 2.5 h. The reaction was
monitored by TLC (10% EtOAc in hexanes), visualized with
2,4-dinitrophenylhydrazine because the nitrile 7 and methyl
ketone 8 have identical Rf values. Next, H2SO4 in dioxane
(3 mL of 3 M solution in 6 mL dioxane) was added to the reac-
tion and heated to 65 ◦C for 1.5 h. After cooling, the layers
were separated. The aqueous layer was neutralized with 10%
NaOH and extracted with EtOAc (3 × 10 mL). The combined
organic layers were washed with brine, dried, filtered, and con-
centrated. The resultant white solid was dissolved in CH2Cl2
and applied to a column of silica gel in 25% EtOAc in hex-
anes. The desired product 8 was recovered as a white solid
(60 mg, 90%) and recrystallized from MeOH as thin white crys-
tals; mp 116–118 ◦C; [˛]D24 = +44.0 (c = 0.6, CHCl3); IR 3435, 2938,
1712, 1375, 1045; 1H NMR ı 5.35 (1H, m, 6-H), 3.53 (1H, m, 3-
H), 2.42–2.23 (4H, m), 2.14 (3H, s, 26-H), 2.09–1.04 (24H, m),
1.01 (3H, s, 19-H), 0.94 (3H, d, J = 6.59 Hz, 21-H), 0.67 (3H, s,
18-H); 13C NMR ı 209.45, 140.75, 121.64, 71.76, 56.71, 55.76,
50.07, 44.30, 42.30, 42.26, 39.71, 37.22, 36.47, 35.58, 35.41, 31.86
CHCl3); IR 3370, 2937, 2857, 1253, 1082, 839, 774 cm−1 1H NMR
;
ı 5.32 (1H, m, 6-H), 3.61 (1H, OH), 3.48 (1H, m, 3-H), 2.28–1.03
(27H, m), 1.00 (3H, m, 19-H), 0.94 (3H, d, J = 6.31 Hz, 21-H), 0.89
(9H, s, C(CH3)3), 0.68 (3H, s, 18-H), 0.06 (6H, s, Si(CH3)2); 13C
NMR ı 141.54, 121.13, 72.63, 63.58, 56.77, 55.95, 50.16, 42.79,
42.32, 39.76, 37.35, 36.55, 35.55, 32.06, 31.87, 31.81 (2 × C), 29.37,
28.22, 25.92 (3 × C), 24.25, 21.04, 19.41, 18.67, 18.24, 11.84, −4.62
(2 × C).
2.1.3. (3˛, 8˛, 9ˇ, 10˛, 13˛, 14ˇ, 17˛, 20S)-3-[[(1,1-Dime-
thylethyl)dimethylsilyl]oxy]chol-5-en-24-carbonitrile (7)
To the alcohol
6
(120 mg, 0.25 mmol), p-toluenesu-
lfonylchloride (100 mg, 0.5 mmol) and pyridine (5 mL) were
added. The mixture was kept at 4 ◦C for 2 days. The reaction
was then poured into ice/water (10 mL). The white solid was
filtered, solubilized with EtOAc, and washed with both 0.5 N
HCl (1 × 10 mL) and water (2 × 5 mL). The organic layer was

steroids 7 1 ( 2 0 0 6 ) 484–488
487
(2 × C), 31.62, 29.84, 28.17, 24.22, 21.04, 20.35, 19.37, 18.58, 11.81.
m, 6-H), 3.53 (1H, m, 3-H), 2.29–1.24 (25H, m), 1.22 (6H, 26 and
27-H), 1.17–1.05 (4H, m), 1.02 (3H, s, 19-H), 0.93 (3H, d, J = 6.59 Hz,
21-H), 0.69 (3H, s, 18-H); 13C NMR ı 140.76, 121.70, 71.79, 71.11,
56.75, 56.05, 50.10, 44.41, 42.28 (×2), 39.76, 37.23, 36.49, 36.44,
35.72, 31.88 (×2), 31.64, 29.35, 29.18, 28.23, 24.26, 21.06, 20.74,
19.38, 18.66, 11.85. Anal. Calcd. for C27H46O2: C, 80.54; H, 11.51.
Obsd. C, 80.70; H, 11.52.
Anal. Calcd. for C26
10.65.
H42O2: C, 80.77; H, 10.95. Obsd. C, 80.45; H,
2.1.5. (3˛, 8˛, 9ˇ, 10˛, 13˛, 14ˇ, 17˛,
20S)-25-Hydroxycholesterol (2)
The steroid methyl ketone 8 (55 mg, 0.15 mmol) and THF (5 mL)
were added to a dry flask. The mixture was cooled to 0 ◦C. Then
MeLi was added dropwise (4.7 mL of 1.6 M solution in Et2O,
7.5 mmol). The mixture was stirred on ice for 30 min and at
rt for an additional 90 min. Then water (5 mL) was added cau-
tiously to the reaction mixture. The layers were separated and
the aqueous phase was extracted with EtOAc (3 × 10 mL). The
combined organic layers were washed with brine (2 × 10 mL),
dried, filtered, and concentrated. The resultant white solid
was applied to a silica gel column (30% EtOAc in hexanes)
to separate the alcohol product 2 from the methyl ketone
starting material. Approximately 25% of the methyl ketone
8 was recovered. The desired product 2 was recovered as a
white solid (35 mg, 60%); mp 174–175 ◦C; [˛]D24 = +37.7 (c = 0.44,
CHCl3); IR 3306, 2935, 1465, 1377, 1058, 734; 1H NMR ı 5.35 (1H,
3.
Results and discussion
The strategy we used for preparing ent-25-HC was simi-
lar to that used to make ent-desmosterol [11]; namely, we
prepared the ent-steroid nucleus and then added the side
chain piecewise. This strategy required the production of the
steroidal methyl ester 3a (Scheme 1). However, as described
in [11], the method used to make 3a also produces a small
amount (<10%) of the corresponding saturated stanol 3b.
Sterol 3a and stanol 3b cannot be separated by chromatog-
raphy. In the earlier work, the small amount of stanol was
removed when the side chain was complete, containing only
Scheme 1 – (a) (i) Br2, HOAc, NaOAc, (ii) chromatography, 55%; (b) Zn, HOAc, 90%; (c) TBDMSCI, DMAP, MeCI2, THF, TEA, 99%;
(d) LiAIH, Et2O, 0 ◦C, 98%; (e) (i) p-TsCI, pyr, 4 ◦C, (ii) NaCN, DMSO, 90 ◦C, 80%; (f) (i) MeLi, THF, 0 ◦C, (ii) H2SO4, dioxane, 65 ◦C,
90%; (g) MeLi, THF, 60%.

488
steroids 7 1 ( 2 0 0 6 ) 484–488
hydrocarbons, e.g. ent-TBDMS-desmosterol or ent-TBDMS-
cholesterol. We predicted that such a separation would not
be possible in this work because the side chain would contain
some functionality, i.e. the 25-hydroxyl group. Therefore,
we sought to remove the saturated material by a different
method.
We reacted esters 3a and 3b with Br2 in HOAc. These
conditions removed the silyl protecting group from the 3-
hydroxyl group in both compounds. Otherwise, ester 4b did
not react, while 3a was converted to the 5,6-dibromide 4a.
The dibromide 4a and saturated 4b were easily separated by
chromatography. Prolonged storage (several days) of the dibro-
mide resulted in significant degradation of the product. Thus,
after separation, the dibromide 4a was immediately debromi-
nated by treatment with Zn in HOAc to give the desired methyl
ester of ent-chol-5-en-3-ol 5, uncontaminated by the saturated
steroid.
r e f e r e n c e s
[1] Brown MS, Goldstein JL. Suppression of
3-hydroxy-3-methylglutaryl coenzyme A reductase activity
and inhibition of growth of human fibroblasts by
7-ketocholesterol. J Biol Chem 1974;249(22):7306–14.
[2] Kandutsch AA, Chen HW. Inhibition of cell growth by
oxygenated derivatives of cholesterol. J Biol Chem
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[3] Johnson KA, Morrow CJ, Knight GD, Scallen TJ. In vivo
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cholesterol after a single meal, dietary cholesterol
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cloning of mouse and human cholesterol 25-hydroxylases,
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The alcohol 5 was easily reprotected and the methyl ester in
the side chain was reduced to give the alcohol 6. This primary
alcohol was converted to the tosylate, which was then dis-
placed by NaCN to give the carbonitrile 7. Methylation followed
by acidic workup afforded the methyl ketone 8. Subsequent
methylation provided the tertiary alcohol in the desired prod-
uct, ent-25-HC 2.
[5] Kandutsch AA, Chen HW, Heiniger H-J. Biological activity
of some oxygenated sterols. Science 1978;201:498–501.
[6] Kauffman JM, Westerman PW, Carey MC, Goldman M.
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exhibit interfacial properties similar to cholesterol. J Lipid
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[7] Theunissen JJ, Jackson RL, Kempen HJ, Demel RA.
Membrane properties of oxysterols. Interfacial orientation,
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[8] Holmes RP. 25-Hydroxysterols increase the permeability of
liposomes to Ca2+ and other cations. Biochim Biophys
Acta 1984;792(3):371–5.
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The overall yield of ent-25-HC from the methyl
ester
testosterone the yield was 6%. Efforts are currently
underway to use ent-25-hydroxycholesterol as tool to
3 was 21% and from the steroidal precursor ent-
a
understand the mechanisms by which 25-hydroxycholesterol
affects cellular cholesterol homeostasis and cholesterol
transport.
[10] Westover EJ, Covey DF. The enantiomer of cholesterol. J
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Acknowledgements
The authors gratefully acknowledge funding from the NIH
Training Grant T32 HL07275 (EJW) and GM47969 (DFC).