Surprising unreactivity of cholesterol-5,6-epoxides towards nucleophiles

Article abstract of DOI:10.1194/jlr.M023689

We recently established that drugs used for the treatment and the prophylaxis of breast cancers, such as tamoxifen, were potent inhibitors of cholesterol-5,6-epoxide hydrolase (ChEH), which led to the accumulation of 5,6α-epoxy-cholesterol (5,6α-EC) and 5,6β-epoxy-cholesterol (5,6β-EC). This could be considered a paradox because epoxides are known as alkylating agents with putative carcinogenic properties. We report here that, as opposed to the carcinogen styrene-oxide, neither of the ECs reacted spontaneously with nucleophiles. Under catalytic conditions, 5,6β-EC remains unreactive whereas 5,6α-EC gives cholestan-3β,5 α-diol-6β-substituted compounds. These data showed that 5,6-ECs are stable epoxides and unreactive toward nucleophiles in the absence of a catalyst, which contrasts with the well-known reactivity of aromatic and aliphatic epoxides. These data rule out 5,6-EC acting as spontaneous alkylating agents. In addition, these data support the existence of a stereoselective metabolism of 5,6α-EC. Copyright

Full text of DOI:10.1194/jlr.M023689

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Surprising unreactivity of cholesterol-5,6-epoxides
towards nucleophiles
Michael R. Paillasse,*,† Nathalie Saffon,§ Heinz Gornitzka,** Sandrine Silvente-Poirot,*
Marc Poirot,1,* and Philippe de Medina*,†
INSERM UMR 1037-Cancer Research Center of Toulouse,* Université de Toulouse III, Institut Claudius
Regaud,* Toulouse, France; Affichem,† Toulouse, France; Institut de Chimie de Toulouse,§ Université Paul
Sabatier, Toulouse, France; and CNRS Laboratoire de Chimie de Coordination,** Toulouse, France
Abstract We recently established that drugs used for the
treatment and the prophylaxis of breast cancers, such as ta-
moxifen, were potent inhibitors of cholesterol-5,6-epoxide
hydrolase (ChEH), which led to the accumulation of 5,6␣-
epoxy-cholesterol (5,6␣-EC) and 5,6␤-epoxy-cholesterol
(5,6␤-EC). This could be considered a paradox because ep-
oxides are known as alkylating agents with putative carcino-
genic properties. We report here that, as opposed to the
carcinogen styrene-oxide, neither of the ECs reacted spon-
taneously with nucleophiles. Under catalytic conditions,
5,6␤-ECremainsunreactivewhereas5,6␣-ECgivescholestan-
3␤,5␣-diol-6␤-substituted compounds. These data showed
that 5,6-ECs are stable epoxides and unreactive toward nu-
cleophiles in the absence of a catalyst, which contrasts with
the well-known reactivity of aromatic and aliphatic epox-
ides. These data rule out 5,6-EC acting as spontaneous
alkylating agents. In addition, these data support the exis-
tenceofastereoselectivemetabolismof5,6␣-EC.—Paillasse,
M. R., N. Saffon, H. Gornitzka, S. Silvente-Poirot, M. Poirot,
and P. de Medina. Surprising unreactivity of cholesterol-5,6-
epoxides towards nucleophiles. J. Lipid Res. 2012. 53:
718–725.
thesis (2). We showed that ChEH was fully inhibited by
therapeutic doses of AEBS ligands, including drugs be-
longing to different pharmacological classes and widely
used nutritional compounds. These compounds include
tamoxifen (tam), one of the main drugs used for the first-
line and long-term hormone therapy of breast cancers;
raloxifene, which has been approved for the prevention of
osteoporosis (3); amiodarone, which is widely used for the
prevention and the treatment of anti-arrhythmia (4); trif-
luoroperazine, an antipsychotic drug used for the treat-
ment of schizophrenia (5); and the omega-3 fatty acid
docosahexaenoic acid, which is widely proposed as dietary
supplement with health benefits (2). Extensive knowledge
of the mechanism of action of all these molecules is re-
quired to prevent toxicity to patients submitted to long-
term treatment with such inhibitors of ChEH.
AEBS/ChEH ligands, such as tam and raloxifene, were
recently shown to induce cell differentiation and death of
breast cancer cells through a mechanism involving sterol
accumulation and autoxidation (6–8) leading to the pro-
duction of 5,6-EC, suggesting that 5,6-EC accumulation is
involved in their anticancer and chemopreventitive activ-
ity. This is contrary to the initial hypothesis that the bio-
logical function of ChEH was to detoxify 5,6-EC. This
hypothesis emerged because it was initially postulated that
5,6-EC could, like other chemicals bearing epoxide groups,
be potent alkylating substances and, hence, carcinogenic (9).
Supplementary key words SN2 • alkylation • ring opening • choles-
terol oxides
Cholesterol-5,6-epoxide hydrolase (ChEH; EC 3.3.2.11)
catalyzes the stereoselective hydration of cholesterol-5,6-
epoxide diastereoisomers (5,6-EC: 5,6␣-EC and 5,6␤-EC)
into cholestane-3␤,5␣,6␤-triol (CT) (1). We recently re-
ported the molecular identification of this enzyme and
established that ChEH activity was carried out by the mi-
crosomal antiestrogen binding site (AEBS), which consists
of two subunits: the 3␤-hydroxysterol-⌬8,⌬7-isomerase (EC
5.3.3.5) and the 3␤-hydroxysterol-⌬7-reductase (EC 1.3.1.21)
that are both involved in postlanosterol cholesterol biosyn-
Abbreviations: 2/3D, two/three dimensional; 5,6␣-EC, 5,6␣-epoxy-
cholesterol (common name) or 5,6␣-epoxy-5␣-cholestan-3␤-ol (system-
atic name); 5,6␤-EC, 5,6␤-epoxy-cholesterol (common name) or
5,6␤-epoxy-5␤-cholestan-3␤-ol (systematic name); AE, 2-aminoethanol;
AEBS, microsomal antiestrogen binding site; ChEH, cholesterol-5,
6-epoxide hydrolase; CM, culture medium; CT, cholestane-3␤,5␣,6␤-
triol; G, guanine; HAC, 5␣-hydroxy-6␤-[2-hydroxyethylamino]-cholestan-
3␤-ol; HSC, 5␣-hydroxy-6␤-[2-hydroxyethylsulfanyl]-cholestan-3␤-ol;
LXR, liver X receptor; ME, 2-mercaptoethanol; SN2, second-order
nucleophilic substitution; SO, styrene oxide; SULT2B1b, cholesterol
sulfotransferase.
This work was supported by the Institut National de la Santé et de la Recherche
Médicale, the Conseil Regional Midi-Pyrénées, and the Ministère Français de la
Recherche et de l’Enseignement Supérieur through the GenHomme. S.S.-P. is in
charge of research at the Centre National de la Recherche Scientifique.
1 To whom correspondence should be addressed.
e-mail: marc.poirot@inserm.fr
The online version of this article (available at http://www.jlr.org)
contains supplementary data in the form of one figure.
Manuscript received 19 December 2011 and in revised form 26 January 2012.
Published, JLR Papers in Press, January 29, 2012
DOI 10.1194/jlr.M023689
Copyright © 2012 by the American Society for Biochemistry and Molecular Biology, Inc.
718
Journal of Lipid Research Volume 53, 2012
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Silica Gel 60-F254 250 ␮m precoated plates and visualized by
ChEH is ubiquitously expressed in healthy and tumoral mam-
malian tissues, and its inhibition by AEBS ligands could lead
to the accumulation of 5,6-EC throughout patients’ bodies.
Epoxides are three-member strained rings leading in
general or normally to electrophilic compounds with reac-
tivity toward nucleophiles to give ␤-substituted alcohols in
a regio- and stereoselective way in SN2 conditions (10–12).
This reactivity makes chemicals bearing an epoxide group,
such as benzo(a)pyrene diol epoxide or styrene oxide
(SO), potent alkylating agents producing covalent adducts
with proteins and nucleic acids, which contributes to their
mutagenicity and carcinogenicity in mammals (13, 14).
5,6-EC exist as ␣- and ␤-diastereoisomers (5,6␣-EC,
5,6␤-EC), depending on the orientation of the oxirane
group with regard to the plane defined by the sterol back-
bone (1). 5,6-EC are known as major autoxidation prod-
ucts of cholesterol (15), and 5,6␣-EC has been reported to
be produced stereoselectively by a cytochrome p450 in the
bovine adrenal cortex (16). 5,6-EC diastereoisomers are
rapidly metabolized in mammals through different routes.
They are extensively transformed into CT by ChEH, and
they can be esterified with fatty acids acyl ester by acyl-
coA:cholesterol acyl transferases (ACAT1 and ACAT2)
(17) into fatty acyl-cholesteryl esters, and with sulfate by
cholesterol sulfotransferase (SULT2B1b) into the 3␤-
sulfate of 5,6-EC (18). The 3␤-sulfate of 5,6␣-EC has been
shown to antagonize the liver X receptor (LXR) signaling
pathway (19, 20), and recently 5,6␣-EC was found to be a
direct modulator of LXR, suggesting a physiological role
for this 5,6-EC diastereoisomer (21). Interestingly 5,6␣-EC
has been shown to be a better substrate than 5,6␤-EC for
ACATs and SULT2B1b and the sole 5,6-EC substrate for
glutathione transferase B (22, 23). The stereoselectivity of
these enzymes and the receptors that are targets of 5,6␣-EC
suggest structural and possibly conformational differences
in both diastereoisomers. Because of the presence of a pu-
tative reactive epoxide group, 5,6-EC were initially sus-
pected to be carcinogenic and involved in the etiology of
cutaneous cancers (24, 25), although 5,6-EC were shown
to be nontumorigenic in rodents (26) and the reactivity of
5,6-EC has never been clearly addressed.
warming after spraying either with 50% methanolic sulfuric acid
or 0.2% ethanolic ninhydrin. Atmospheric pressure column
chromatography on Merck-60 70-200 mesh silica was used for all
1
purifications. H, 13C, COSY, HSBC, and HSQC NMR spectra
were run at either 250 or 300 MHz using Bruker AC-250 or AC-
300 spectrometers, and NOESY experiments were done using
500 MHz and mass spectrometry analysis on a quadrupolar Ner-
mag R10-10 by either electrospray or chemical ionization. All RX
data for all structures represented in this paper were collected at
low temperatures using an oil-coated, shock-cooled crystal on a
Bruker-AXS CCD 1000 diffractometer with MoK␣ radiation (␭ =
0.71073 Å). The structures were solved by direct methods (27),
and all nonhydrogen atoms were refined anisotropically using
the least-squares method on F2 (SHELXL-97, Program for Crystal
Structure Refinement, G. M. Sheldrick, University of Göttingen,
1997). Cambridge Crystallographic Data Centre (CCDC) 851178
(5,6␣-EC • 1/2 methanol); CCDC 851179 (5,6␤-EC • methanol);
CCDC 851180 (HSC); and CCDC 851181 (HAC) contain the
supplementary crystallographic data for this work. These data
can be obtained free of charge via www.ccdc.cam.ac.uk/conts/
retrieving.html (or from the CCDC, 12 Union Road, Cambridge
CB2 1EZ, UK; e-mail: deposit@ccdc.cam.ac.uk).
Procedure for comparing epoxide reactivity with
2-mercaptoethanol, 2-aminoethanol, and guanine
Styrene oxide, 5,6␣-EC, or 5,6␤-EC (0.1 mmol) were dissolved
in ethanol (5 ml). Four equivalents of the test nucleophile
[2-mercaptoethanol (ME), 2-aminoethanol (AE), or guanine
(G)] were added and the mixture stirred at the appropriate tem-
perature. After 48 h, the mixture was concentrated in vacuo, dis-
solved in EtOAc, washed with water, brined, dried over MgSO4,
and the solvent was removed. The oil obtained was purified by
column chromatography, and fractions containing adducts were
pooled. For the reaction with G, the same experimental condi-
tions were used except for the work-up; i.e., the concentrated
mixture was diluted in butanol and extracted with 1 M aqueous
HCl. Aqueous layers were pooled, neutralized, and extracted
with butanol. The organic layer was dried over MgSO4, and the
solvent was removed. The reaction was monitored by TLC
(EtOAc) and revealed with concentrated sulfuric acid and heat-
ing at 180°C for 1 min for 5,6␣-EC and 5,6␤-EC and UV detection
at 254 nm for SO. The disappearance of the spot corresponding
to the 5,6-EC and SO was used to measure the occurrence of the
reaction of addition of nucleophiles to epoxides to give more
polar compounds that did not migrate under these conditions.
The objective of the present study was to evaluate the reac-
tivity of 5,6-EC toward nucleophiles under SN2 conditions
that could putatively give the compounds shown in Fig. 1. We
showed that 5,6-EC were neither reactive at ambient room
temperature nor at physiological temperature (37°C). We es-
tablished the first structural analysis of 5,6-EC. We further es-
tablished that 5,6␣-EC was the only diastereoisomer that can
undergo a ring opening reaction leading to the formation of
cholestan-3␤,5␣-diol-6␤-substituted compounds through a
trans-diaxial mechanism under catalytic conditions.
Procedure for comparing epoxide reactivity in cell
culture medium
Cholestan-5,6-epoxy-3␤-ol. One microliter of an ethanolic
solution of [14C]5,6␣-EC or [14C]5,6␤-EC was added to 1 ml of
DMEM culture medium (CM) supplemented with 10% FCS to
give a final concentration of 60 µM (1 µCi/condition). The mix-
ture was stirred for 48 h at 37°C. At the indicated time (1 h, 48 h),
a 30 µl aliquot was applied to silica gel 60, 20 × 20 cm. TLC plates
(Fluka, Germany) that had previously been heated for 1 h at
100°C and were developed using EtOAc. Radioactive 5,6-EC were
visualized using a Storm 840 apparatus (Molecular Dynamics)
and quantified by densitometry with Imagequant software (Mo-
lecular Dynamics). Retention factors (Rf) were determined for
each spot on the TLC plates as the ratio of the distance of migra-
tion of the eluate from the origin and the distance of the solvent
from the origin. 5,6-EC have a Rf = 0.67 (2).
MATERIALS AND METHODS
Unless otherwise noted, all reagents were purchased from
Sigma or Steraloids and checked for purity. [14C]5,6␣-EC or
[14C]5,6␤-EC was synthesized exactly as previously described (2).
All reactions were carried out under a positive pressure of argon.
Reactions were analyzed by thin-layer chromatography on Merck
Cholesterol-5,6-epoxides are not spontaneous alkylating epoxides
719

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Fig. 1. (A) Putative products of the addition of
5,6␣-EC and 5,6␤-EC to nucleophiles. The four rings
of the steroid backbone are lettered A, B, C and D,
and carbons are numbered on 5,6␣-EC. Carbon 5 and
6 are numbered in red on 5,6␤-EC. (B) Solid-state
structure of HAC and HSC as determined by X-ray
analysis. Oxygen atoms are in red, carbon atoms are
in gray, and sulfur atoms are in yellow. (C) 5,6␣-EC
gives a single product of addition with nucleophiles
only under catalytic conditions, whereas 5,6␤-EC re-
mains unreactive.
Styrene oxide. Styrene oxide was added to 10 ml of DMEM
culture medium supplemented with 10% FCS to a final concen-
tration of 60 µM. The mixture was stirred either for 1 min or
48 h at 37°C. After the indicated time, total lipids were extracted
using the Blight and Dyer methodology (28). The organics were
concentrated to dryness in vacuo, dissolved in 50 µl ethanol,
and submitted to TLC (elutant 1:1 hexane:diethyl ether) to vi-
sualize the disappearance of the SO spot after 48 h reaction
(UV, 254 nm).
127.01, 65.55, 61.04, 52.01, 32.96; HRMS m/z = 198.0722 (calcu-
lated for C29H52SO3 198.0715).
2-(2-Hydroxy-ethylamino)-1-phenyl-ethanol. TLC Rf [metha-
nol] = 0.37; NMR ␦H (300 MHz, MeOD) 7.44–7.27 (5H, m), 4.85
(1H, t, J 6.6 Hz), 3.72 (1H, dd, J 5.2, 1.2 Hz), 3.35–3.33 (2H, m),
2.91–2.84 (4H, m); NMR ␦C (75 MHz, MeOD) 142.91, 128.11,
127.38, 125.71, 71.62, 59.62, 56.18, 50.55; HRMS m/z = 181.1098
(calculated for C29H52NO3 181.1103).
Procedure for quantifying cholesterol-5,6-epoxide
reactivity with ME and AE
2-(7-Guanidyl)-1-phenyl-ethanol. TLC Rf [28% aqueous
NH3/methanol 9:1] = 0.71; NMR ␦H (300 MHz, MeOD) 8.38
(1H, s), 7.45–7.21 (5H, m), 4.91 (1H, t, J 7.3 Hz), 4.09 (2H, d,
J 7.2 Hz); NMR ␦C (75 MHz, MeOD) 155.91, 153.77, 151.12,
143.24, 138.87, 128.01, 127.19, 124.71, 107.65, 74.62, 52.62;
HRMS m/z = 271.1077 (calculated for C29H52NO3 271.1069).
[14C]5,6␣-EC or [14C]5,6␤-EC (1 µmol) were dissolved in a so-
lution of the considered nucleophile (4 equivalents) in ethanol
(500 µl) containing LiClO4 (4 equivalents), if needed. The mix-
ture was stirred at the appropriate temperature for 48 h, and a
25 µl aliquot was applied to silica gel 60 and analyzed as described
above. The transformation rate was determined by quantification
of the spot comigrating with nonlabeled HSC or HAC compared
with the total radioactivity of the lane.
Synthesis of HSC
To a stirred solution of 56␣-EC (400 ␮mol) in ethanol (5 ml)
were added 2 M sodium hydroxide (800 ␮mol, 2 equivalents)
and then ME (1.6 mmol, 4 equivalents). After refluxing for 24 h,
the mixture was cooled and diluted with ethyl acetate (10 ml) and
washed sequentially with water and brine. The organic layer was dried
over MgSO4, and the solvent was removed under reduced pressure.
The crude product was purified by column chromatography
2-(2-Hydroxy-ethylsulfanyl)-2-phenyl-ethanol. TLC Rf [EtOAc] =
0.38; NMR ␦H (300 MHz, MeOD) 7.42–7.24 (5H, m), 4.07–4.00
(2H, m), 3.73–3.69 (1H, m), 3.58 (2H, t, J 6.7 Hz), 2.59–2.54
(2H, m); NMR ␦C (75 MHz, MeOD) 143.72, 128.12, 128.02,
720
Journal of Lipid Research Volume 53, 2012

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(0–10% methanol in ethyl acetate) to obtain the product of inter-
Z = 4, T = 173(2) K. 28,335 reflections (4,710 independent, Rint =
est as a white solid (90 ␮mol, 23%). To obtain crystals suitable for
X-ray analysis, HSC was dissolved in hot acetone, and water was
added drop-wise until supersaturation. The solution was then al-
lowed to cool at room temperature overnight for optimal nucle-
ation and crystallization. TLC Rf [EtOAc] = 0.25; NMR ␦H (300
MHz, DMSO-d6) 3.88–3.80 (1H, m), 3.43–3.40 (2H, m), 2.65–2.60
(1H, m), 2.40–2.35 (1H, m), 2.29 (1H, m), 1.03 (3H, s), 0.89 (3H,
d, J 3.9 Hz), 0.86 (3H, d, J 1.5 Hz), 0.81 (3H, d, J 1.5 Hz), 0.63
(3H, s); NMR ␦C (75 MHz, DMSO-d6) 75.56, 66.43, 64.97, 60.86,
56.21, 56.12, 52.15, 45.45, 42.72, 42.51, 39.45, 38.46, 36.06, 35.68,
32.83, 31.36, 30.80, 30.43, 28.29, 27.81, 24.35, 23.64, 23.11, 22.83,
21.16, 18.95, 17.35, 12.47; HRMS m/z = 481.3701 (calculated for
C29H52SO3 481.3710).
0.0541) were collected. Largest electron density residue: 0.179 e Å-3,
R1 [for I > 2␴(I)] = 0.0360 and wR2 = 0.0787 (all data).
Molecular modeling
The structure analysis and comparison between the three-di-
mensional (3D) structures obtained by X-ray analysis were done
exactly as described previously with other compounds (30, 31).
Superimposition of the structures of 5,6␣-EC and 5,6␤-EC was
done by superimposing carbons of rings C and D of the steroid
backbone (C8, C9, C14, and C17). Van der Waals volume analy-
ses were done using the steroid core of 5,6-EC, excluding the
isooctane side chain of superimposed 5,6-EC. The percentage
corresponding to the difference in the van der Waals volumes of
superimposed 5,6-EC was calculated by subtracting the van der
Waals volumes of 5,6␤-EC to 5,6␣-EC and the ratio of this value
with the van der Waals volume of 5,6␣-EC. The total energy of
5,6␣-EC, 5,6␤-EC, and HAC was calculated using the structure
obtained by X-ray analysis and the CVFF force field (InsightII,
version 2000.1; Accelrys, San Diego, CA). The total energy of 5␤-
hydroxy-6␣-[2-hydroxyethylamino]cholestan-3␤-ol was calculated
using the Discover calculation engine with the CVFF force field
(InsightII). The Build module was used for the creation of the
5␤-hydroxy-6␣-[2-hydroxyethylamino]cholestan-3␤-ol molecule,
using the X-ray structure of 5,6␤-EC as a starting point. The C5-O
bond was deleted, and the O replaced by the N of the aminoetha-
nol moiety. H6 was replaced by a hydroxyl group, and a new pro-
ton was added on C6 to complete the molecule. The resulting
conformation was then minimized using 250 steps of the steepest
descent method, followed by the conjugated gradient method
until the root-mean-square gradient was less than 0.001 kcal/
mol/Å. The CVFF force-field was used together with a distant-
dependent dielectric term (␧ = r) and a 20 Å nonbonded cut-off
distance. The final energy of the system was compared with that
of the solid-state structure of HAC. The conformations of the
molecules were compared using the Search Compare module.
Synthesis of HAC
To a stirred solution of 5,6␣-EC (400 ␮mol) in Ethanol (5 ml)
were added LiClO4 (1.6 mmol, 4 equivalents) and then etha-
nolamine (1.6 mmol, 4 equivalents). The mixture was refluxed
for 5 days, and after cooling was diluted with ethyl acetate (10 ml)
and washed sequentially with water and brine. The organic layer
was dried over MgSO4, and the solvent was removed under re-
duced pressure. The crude material was purified by column chro-
matography (0–10% methanol in ethyl acetate) to obtain the
final product as a light-yellow solid (101 ␮mol, 25%). To obtain
crystals suitable for X-ray analysis, HAC was dissolved in hot etha-
nol, and water was added drop-wise until supersaturation. The
solution was then allowed to cool at room temperature overnight
for optimal nucleation and crystallization. TLC Rf [EtOAc/meth-
anol 3:1] = 0.42; NMR ␦H (300 MHz, DMSO-d6) 3.95–3.84 (1H, m),
3.78–3.67 (2H, m), 3.18–2.96 (3H, m), 1.03 (3H, s), 0.93 (3H, d,
J 6.0 Hz), 0.87 (3H, d, J 1.2 Hz), 0.85 (3H, d, J 1.2 Hz), 0.68 (3H, s);
NMR ␦C (75 MHz, DMSO-d6) 73.94, 65.77, 62.93, 56.16, 56.10,
55.90, 52.03, 50.43, 44.62, 42.94, 40.09, 38.01, 36.13, 35.73, 32.57,
31.24, 29.67, 28.32, 27.89, 27.33, 23.92, 23.73, 23.16, 22.89, 21.11,
19.92, 18.90, 16.34, 12.62; HRMS m/z = 464.4146 [M+H]+, calcu-
lated for C29H53NO3: 464.4104; [␣]D = Ϫ4.6°/mol/dm.
RESULTS
Crystallographic data
To perform X-ray crystallographic analysis, commercial 5,6␣-EC
and 5,6␤-EC were recrystallized in hot methanol.
5,6␣- and 5,6␤-EC are unreactive toward nucleophiles in
noncatalytic conditions, in contrast to SO
In the first set of experiments, we evaluated the reactivity
of 5,6␣- and 5,6␤-EC toward nucleophiles and putative
products of addition (Fig. 1A). AE, ME, G, and a complex
cell CM were used as nucleophiles. AE contains a primary
amine, ME contains a thiol group, and G contains two nu-
cleophilic centers with an aromatic primary amine and a
secondary aromatic amine. DMEM, the CM used for rou-
tine eukaryotic cell growth, contained about 13 mmol/l nu-
cleophiles (mainly amino acids). The reactivity of the 5,6-EC
was studied in protic solvents in which both reactants were
soluble or directly without addition of solvent when testing
CM. Thin layer chromatography was used to monitor the
transformations of the 5,6-EC in conditions enabling their
separation from putative transformation products. In the
case of CM, we used [14C]5,6-EC and followed up their
transformation by TLC and autoradiography. Table 1 shows
that 5,6␣-EC and 5,6␤-EC did not react with nucleophiles
after 48 h at room temperature or even at physiological
temperature (37°C). Under the same conditions, the carci-
nogenic compound SO reacted with the tested nucleophiles.
These data established that 5,6␣-EC and 5,6␤-EC are stable
epoxide-bearing chemicals that are unreactive toward
5,6␣-EC • 1/2 methanol. C27.5H48O2.5, M = 418.66, mono-
clinic, P21, a = 12.406(1) Å, b = 6.043(1) Å, c = 35.224(2) Å, ␤ =
94.042(3)°, V = 2634.1(2) Å3, Z = 4, T = 193(2) K. 20,471 reflec-
tions (7,379 independent, Rint = 0.0722) were collected. Largest
electron density residue: 0.161 e Å-3, R1 [for I > 2␴(I)] = 0.0628
and wR2 = 0.1664 (all data) with R1 = ⌺||Fo|-|Fc||/⌺|Fo| and wR2 =
(⌺w (Fo2-Fc2)2/⌺w(Fo2)2)0.5
.
5,6␤-EC • methanol. C28H50O3, M = 434.68, monoclinic, P21,
a = 12.393(1) Å, b = 7.247(1) Å, c = 15.007(1) Å, ␤ = 95.534(2)°, V =
1341.42(7) Å3, Z = 2, T = 193(2) K. 25587 reflections (5399 indepen-
dent, Rint = 0.0583) were collected. Largest electron density residue:
0.293 e Å-3, R1 (for I>2␴(I)) = 0.0546 and wR2 = 0.1476 (all data).
HSC. C29H52O3S, M = 480.77, orthorhombic, P212121, a =
7.688(1) Å, b = 11.080(1) Å, c = 33.401(1) Å, V = 2845.4(2) Å3,
Z = 4, T = 193(2) K. 25,229 reflections (5,769 independent, Rint
=
0.0301) were collected. Largest electron density residue: 0.270 e Å-3,
R1 [for I > 2␴(I)] = 0.0372 and wR2 = 0.0955 (all data). The abso-
lute structure parameter refined to 0.00(7) (29).
HAC. C29H53NO3, M = 463.72, orthorhombic, P212121, a =
7.691(1) Å, b = 10.794(1) Å, c = 33.676(1) Å, V = 2795.7(2) Å3,
Cholesterol-5,6-epoxides are not spontaneous alkylating epoxides
721

Supplemental Material can be found at:
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.html
nucleophiles under our experimental conditions. This dif-
fers strongly from SO, which has alkylating properties that
have been linked to carcinogenicity (32).
TABLE 2. Measurement of the reactivity of 5,6␣-EC and 5,6␤-EC
toward nucleophiles in catalytic conditions
Compound Solvent Temperature Catalyst Nucleophile Reaction Yield
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
RT
RT
RT
—
—
—
—
—
—
—
—
LiCl04
LiCl04
LiCl04
LiCl04
LiCl04
LiCl04
LiCl04
LiCl04
EA
ME
EA
ME
EA
ME
EA
ME
EA
ME
EA
ME
EA
ME
EA
ME
NR
NR
NR
NR
+
—
—
—
5,6␣-EC
5,6␤-EC
5,6␣-EC
5,6␤-EC
5,6␣-EC
5,6␤-EC
5,6␣-EC
5,6␤-EC
5,6␣-EC is the sole 5,6-EC diastereoisomer that reacts
toward nucleophiles under catalytic conditions
RT
—
Next, we tested whether [14C]5,6␣-EC and [14C]5,6␤-EC
could react with AE and ME under catalytic conditions.
When the reaction mixture was heated by refluxing in eth-
anol (79°C), 5,6␣-EC reacted with AE or ME with a 2% and
7% yield, respectively, whereas no reaction was observed
with 5,6␤-EC under the same conditions (Table 2). The
addition of a Lewis acid (LiClO4) did not catalyze the reac-
tion at room temperature, but at 79°C there was a 25 and
53% yield of the products of transformation of 5,6␣-EC
with AE and ME respectively, showing a 12.5 and 7.5-fold
increase in the reaction yield. Even in these conditions
5,6␤-EC was unreactive (Table 2). These results highlight a
difference in the reactivity of both diastereoisomers under
catalytic conditions and established that both 5,6␣-EC and
5,6␤-EC are not reactive under noncatalytic conditions.
79°C
79°C
79°C
79°C
RT
RT
RT
2%
7%
—
—
—
—
—
—
+
NR
NR
NR
NR
NR
NR
+
+
NR
NR
RT
79°C
79°C
79°C
79°C
25%
53%
—
—
[14C]5,6-EC were incubated at a concentration of 0.8 mg/ml for
48 h in the absence or presence of AE or ME at room temperature, or
refluxed in ethanol (79°C) in the absence or presence of LiClO4.
+, reaction; AE, 2-aminoethanol; ME, 2-mercaptoethanol; NR, no
reaction, RT, room temperature.
5,6␣-EC gives 6␤-substituted-5␣-cholestan-3␤,5␣-diols by
reaction with AE or ME under catalytic conditions
We next analyzed the structure of the products obtained
through the ring opening of the oxirane group of 5,6␣-EC
following addition of AE or ME under catalytic conditions.
The 3D solid-state structures of the products of addition of
5,6␣-EC with EA, 5␣-hydroxy-6␤-[2-hydroxyethylamino]
cholestan-3␤-ol (HAC), and with ME, 5␣-hydroxy-6␤-[2-
hydroxyethylsulfanyl]cholestan-3␤-ol (HSC) (Fig. 1B),
were determined by X-ray diffraction analysis. Both HAC
and HSC are 6␤-substituted-5␣-cholestan-3␤,5␣-diols, indi-
cating a trans-diaxial opening of the epoxide ring with AE
and ME, reflecting an SN2 mechanism resulting from the
regioselective attack of the nucleophile at C6 (Fig. 1C).
Both HAC and HSC had an extended conformation with
the four rings of the steroid backbone in a chair conforma-
tion with trans junctions in the A, B, C, and D rings. Analy-
ses of the structure of HSC by NMR showed that spatial
H-H interactions were estimated between protons from
C19 and protons from C28 (from the hydroxyethyl-sulfanyl
moiety) and between protons from C19 and protons from
C6. Only an interaction between protons from C19 and
one proton from C28 could be observed, which is in ac-
cordance with the 5␣-hydroxy-6␤-substituted products
(supplementary Fig. I).
TABLE 1. Evaluation of the alkylating properties 5,6␣-EC, 5,
6␤-EC, and SO
Compound
Solvent
Temperature
Nucleophile
Reaction
Ethanol
Ethanol
Ethanol
Ethanol
CM
Ethanol
Ethanol
Ethanol
Ethanol
CM
Ethanol
Ethanol
Ethanol
Ethanol
CM
Ethanol
Ethanol
Ethanol
Ethanol
CM
RT
RT
RT
RT
RT
37°C
37°C
37°C
37°C
37°C
RT
RT
RT
RT
RT
37°C
37°C
37°C
37°C
37°C
RT
—
AE
ME
G
—
—
AE
ME
G
—
—
AE
ME
G
—
—
AE
ME
G
—
—
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
+
5,6␣-EC
5,6␤-EC
SO
Ethanol
Ethanol
Ethanol
Ethanol
C.M.
RT
RT
RT
RT
AE
ME
G
+
+
+
—
Analysis of the structure of 5,6-EC diastereoisomers
Ethanol
Ethanol
Ethanol
Ethanol
CM
37°C
37°C
37°C
37°C
37°C
—
NR
+
+
+
+
AE
ME
G
To determine whether the difference in the reactivity
of 5,6-EC diastereoisomers was due to a difference in
their conformation, we carried out structural analyses.
Common two-dimensional (2D) representation of rings
A and B of 5,6-EC epimers is presented in Fig. 2A (33,
34). 5,6-EC diastereoisomers were crystallized and their
structures are shown in Fig. 2B. Rings A, C, and D are
identical in both 5,6-EC and are in a chair conforma-
tion. Ring B of both 5,6-EC is twisted with a different
distortion at the junction of rings A and B. In 5,6␣-EC,
—
Compounds were incubated at a concentration of 8 mg/ml for
48 h in the absence or presence of AE, ME, or G at a concentration
0.32 mol/l or in complete growth CM for eukaryote cells at the
indicated temperatures. The reaction was followed as described in
Materials and Methods.
+, reaction; AE, 2-aminoethanol; CM, culture medium; G, guanine;
ME, 2-mercaptoethanol; NR, no reaction, RT, room temperature; SO,
styrene oxide.
722
Journal of Lipid Research Volume 53, 2012

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TABLE 3. Comparison of torsion angles of 5,6-EC diastereoisomers
in the solid state and in solution
3
Compound
Torsion Angle
Measured Angle JH-H
Calculated Angle
Solid state
Ϫ143
Ϫ158.32
172.76
Ϫ171
88
Ϫ28
Ϫ149
Ϫ32
Ϫ108
Ϫ159.10
171.95
Ϫ142
61
Ϫ57
Ϫ176
Ϫ58
Hz
Solution
C3-C4-C5-C6
C4-C5-C6-C7
5,6␣-EC
C2-C1-C10-C9
C1-C10-C9-C8
H6-C6-C7-H7␣
H6-C6-C7-H7␤
H7␣-C7-C8-H8
H7␤-C7-C8-H8
C3-C4-C5-C6
0
4.5
9.45
6.0
90
Ϫ41
Ϫ148
Ϫ42
5,6␤-EC
C4-C5-C6-C7
C2-C1-C10-C9
C1-C10-C9-C8
H6-C6-C7-H7␣
H6-C6-C7-H7␤
H7␣-C7-C8-H8
H7␤-C7-C8-H8
0.6
3.45
10.95
3.45
78
Ϫ63
Ϫ159
Ϫ56
For solid-state data, angles were measured using the measure tool
from InsightII software (Accelrys, Inc., CA). 3JH-H were determined from
1H-NMR analysis of the molecule in MeOD, and angles were calculated
using the online interactive data table. When 3JH-H corresponded to two
or more possible angles, the closest one (between Ϫ90 and +90 degrees
from the solid-state angle) was chosen.
Fig. 2. (A) Commonly used 2D representation of rings A and B
of 5,6␣-EC and 5,6␤-EC. (B) Solid-state structures of 5,6␣-EC and
5,6␤-EC as established by X-ray analysis and 2D representation of
rings A and B of both diastereoisomers. (C) Superimposition of
5,6␣-EC (orange) and 5,6␤-EC (blue). Superposition was done us-
ing the Search Compare module of Insight II (Accelrys Inc., CA).
(D) The van der Waals volume of 5,6␣-EC and 5,6␤-EC (excluding
the isodecane side chain) are 241.84 Å3 and 242.16 Å3, respectively.
The difference between the van der Waals volumes of 5,6-EC (de-
picted in yellow) is 22.19 Å3, showing that 5,6␣-EC and 5,6␤-EC
have 87% of their van der Waals volume in common and 13%
difference.
and B (supplementary Fig. I). In 5,6␣-EC, the hydrogen
H6 carried by the C6 and oriented toward the ␤-side of
5,6␣-EC gave NOESY signals showing the interactions
between the hydrogens from C8 and C19 (H8 and H19)
(Fig. 3), which highlighted a similar conformation to
that observed in the solid state (Fig. 2B). In 5,6␤-EC,
the hydrogen H6 is oriented toward the ␣-side of
5,6␤-EC and interacts with the hydrogen H3 from C3
but not with H8 and H19, which is consistent with the
bent conformation observed in the solid state (Fig. 2B).
These data constitute the first experimental evidence of
the existence of conformational differences between
5,6␣-EC and 5,6␤-EC. The calculation of the dihedral
angles deduced from the measured coupling constants
are given in Table 3. We observed less difference in the
conformations of 5,6-EC in solution than that observed
in the solid state, showing that the bending of rings at
the A,B junction was also less in solution than in the
solid state. Energy calculations on the solid state and
the energy deduced from dihedral angle measurements
in the liquid state showed that 5,6␤-EC had a surpris-
ingly lower energy state than 5,6␣-EC. This results in a
greater energy barrier for the transformation of 5,6␤-EC
under SN2 conditions (Fig. 3B, C). These results could
explain a decrease, but not the lack of reactivity, of the
5,6␤-EC toward nucleophiles if an attack occurred at
C6: the optimal angle of attack for the opening of an
epoxide is in the plane of the oxirane ring at 165° com-
pared with the C-O bond to be opened (35). According
to the rule of Furst and Plattner, the ring opening of an
oxirane is mainly driven by the stability of the product
of the reaction, which has to be in a trans-diaxial con-
figuration (36). The calculated energy for 5␤-Hydroxy-
6␣-[2-hydroxyethylamino]cholestan-3␤-ol is 96 kcal/
mol, establishing an increase of 11 kcal/mol compared
the angle C1-C10-C9 is 110.91° and the angle C4-C5-C6
is 120.23°, whereas in 5,6␤-EC these angles were 106.68°
and 119.21°, respectively. The conformational differ-
ences are highlighted through the superposition of
5,6-EC (Fig. 2C). We measured a displacement of 0.68 Å
of the oxygen from the hydroxyl in C3 and a displace-
ment of 0.83 Å of the C19 methyl groups. The van der
Waals volume of the steroid backbone of 5,6-EC was
241.84 Å3 for 5,6␣-EC and 242.16 Å3 for 5,6␤-EC. The
difference in the van der Waals volume after superposi-
tion of 5,6-EC was 22.19 Å3, showing that 5,6␣-EC and
5,6␤-EC have 87% of their van der Waals volume in com-
mon; however, there was a 13% difference at the level of
ring A of the methyl C19 and of the epoxide ring from
5,6␣-EC (Fig. 2D). The calculation of the total energy of
the 5,6-EC showed that 5,6␤-EC is in a lower energy state
and thus more stable than 5,6␣-EC (⌬E = 15 kcal/mol).
Measurement of dihedral angles at the junction of rings
A and B showed different distortions around C7 and
C10 (Table 3) between the 5,6-EC, but there were no
significant differences in the length of C-O bonds and
of the O-C6-C5 angles between the diastereoisomers.
We then examined the conformations of 5,6-EC in solu-
tion through NOESY NMR experiments to study poten-
tial interactions between hydrogen atoms from rings A
Cholesterol-5,6-epoxides are not spontaneous alkylating epoxides
723

Supplemental Material can be found at:
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.html
Fig. 3. Determination of 5,6␣-EC and 5,6␤-EC confor-
mation in solution using 1D and 2D-NMR. (A) NOE inter-
actions of proton on carbon 6 (H6) for 5,6␣-EC (left) and
5,6␤-EC (right). Plain arrows represent spatial interactions
effectively found, and dashed arrows represent interac-
tions not found. (B) 3D (left) and Newman’s representa-
tions (center and right) of ring B of 5,6␣-EC. Newman
representations are made along the C6-C7 (center) or
C8-C7 (right) bond. They show that the carbons of the B
ring are in a synperiplanar conformation. (C) 3D (left)
and Newman’s representations (center and right) of the B
ring of 5,6␤-EC. Newman representations are made along
the C6-C7 (center) or C8-C7 (right) bond.
positioning of the O of the hydroxyl on C3 and of the C19
methyl, in addition to differences due to the presence of
the oxirane ring of the ␣ and the ␤ sides of the 5,6-EC.
These elements could represent structural motifs respon-
sible for discrimination by enzymes and receptor targets of
the 5,6-EC.
with 5␣-hydroxy-6␤-[2-hydroxyethylamino]cholestan-
3␤-ol (85 kcal/mol). Taken together, the lower energy
of 5,6␤-EC combined with the higher energy of its prod-
uct of aminolysis could explain the lack of reactivity of
this diastereoisomer in our conditions.
Although cholesterol was shown to have antioxidant
properties because of the sensitivity of its double bond to
oxidation (38), we showed here that the oxidation prod-
uct at ⌬5, such as in 5,6-epoxides, is also a possible route
for stereoselective transformations. We show that 5,6-EC
have a restricted possibility of transformation from one di-
astereoisomer to obtain one product through a SN2 mech-
anism, allowing a stereoselectivity in the reactants as well
as in the products of the reaction as only one (5,6␣-EC) of
the two 5,6-EC diastereoisomers gave a reaction and only
one out four possible theoretical products was obtained
through a SN2 mechanism.
This delineates an attractive mechanism for the stereo-
selective transformation of 5,6-EC. These data suggest
that if an enzyme could catalyze such stereoselective bi-
otransformations, it would give 6␤-substituted-5␣-cholestan-
3␤,5␣-diol. This hypothesis is supported by the fact that
5,6␣-EC was shown to be selectively transformed by gluta-
thionetogive3␤,5␣-dihydroxycholestan-6␤-yl-S-glutathione
(22, 23) and to produce 5␣-hydroxy-6␤-[2-(1H-imidazol-
4-yl)ethylamino] cholestan-3␤-ol (dendrogenin A) in mam-
malian tissues (de Medina et al., unpublished data). In a
previous study, we reported that the products of the cata-
lytic aminolysis of 5,6␣-EC with polyamines such as sper-
midine and spermine had potent neurodifferentiation
properties (39), showing that 5,6␣-EC could control the
production of other putative metabolites and thus justify-
ing the existence of a metabolic pathway centered on
5,6␣-EC. The fact that the ⌬5 double bond of cholesterol
drives a stereospecific process of transformation supports
previous studies underlining the importance of the ⌬5
double bond of cholesterol. These studies established that
the ⌬5 was required for proliferation (40), whereas genetic
CONCLUSIONS
In this work, we report that under normal SN2 condi-
tions, 5,6-EC were not reactive toward nucleophiles. This
is in contrast to SO, which was reactive in the tested con-
ditions and which is well-known as a weak, directly muta-
genic and carcinogenic substance. The reactivity of SO is
due to its alkylating properties on nucleophiles found in
biomolecules (37). We demonstrated that the oxirane
group present on the C5-C6 position of each diastereo-
isomer of 5,6-EC is exceptionally stable toward nucleo-
philes, including guanine, showing that 5,6-EC are not
spontaneous alkylating substances. This ruled out a di-
rect mutagenic and carcinogenicity potency of 5,6-EC in
mammals. We next established that 5,6␣-EC can react at
C6 with nucleophiles only under catalytic conditions and
through a trans-diaxial ring opening, leading to 6␤-
substituted-5␣-cholestan-3␤,5␣-diols. In that case the hy-
droxyl at C5 is in an anti position with regard to the C19
methyl. In the case of 5,6␤-EC, no reaction occurred be-
cause i) the product of trans-diaxial opening at C6 would
have given an energetically unfavorable constrained
structure with the hydroxyl at C5 in the syn position with
regard to the C19 methyl, and ii) no attack occurred at
C5 for the same reason reported above for 5,6␣-EC.
Structure analyses of the 5,6-EC diasteroisomers showed
a less pronounced distortion than expected and usually
depicted between rings A and B in 5,6␤-EC, in which the
A-B ring junction is cis (33, 34).
The main difference in the structure of the rigid steroid
backbone of 5,6-EC diastereoisomers occurred at the level
of the ring A-B junction and showed differences in the
724
Journal of Lipid Research Volume 53, 2012

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macrophage cholesterol efflux through hydroxymethylglutaryl-
CoA reductase inhibition: a role for RhoA in ABCA1-mediated
cholesterol efflux. J. Biol. Chem. 280: 22212–22221.
studies have shown that the null mutation on the gene en-
coding lathosterol oxidase, which catalyses the formation
of ⌬5 to give cholesterol from lathosterol, led to a malfor-
mation syndrome known as lathosterolosis associated with
pre- or postnatal death (41).
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725

Supplemental Material can be found at:
http://www.jlr.org/content/suppl/2012/01/29/jlr.M023689.DC1
.html
ERRATA
In the article “Surprising unreactivity of cholesterol-5,6-epoxides towards nucleophiles” J. Lipid Res. 2012. 53: 718–725,
Figure 3 was erroneously repeated as Figure 1 in the print version. The final online version has been corrected. The cor-
rect Figure 1 appears below. The Journal regrets this error.
Fig. 1. (A) Putative products of the addition of
5,6␣-EC and 5,6␤-EC to nucleophiles. The four rings
of the steroid backbone are lettered A, B, C and D,
and carbons are numbered on 5,6␣-EC. Carbon 5 and
6 are numbered in red on 5,6␤-EC. (B) Solid-state
structure of HAC and HSC as determined by X-ray
analysis. Oxygen atoms are in red, carbon atoms are
in gray, and sulfur atoms are in yellow. (C) 5,6␣-EC
gives a single product of addition with nucleophiles
only under catalytic conditions, whereas 5,6␤-EC re-
mains unreactive.
DOI 10.1194/jlr.M023689ERR
Journal of Lipid Research Volume 53, 2012
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