Synthesis of deuterium-labeled plant sterols and analysis of their side-chain mobility by solid state deuterium NMR

Article abstract of DOI:10.1021/jo960228y

The plant sterols sitosterol and stigmasterol exert very different effects on plant model membranes, the first one being a 'reinforcer' like cholesterol, the second one not. 25-²H-Stigmasterol has been synthesized by coupling of the 22-aldehyde derived from stigmasterol by ozonolysis, with the proper sulfone labeled in position 25. The configuration of the ethyl side chain at C-24 was controlled by separation of the diastereomers introduced via a chiral sulfoxide. This synthetic scheme allowed the introduction of a labeled side chain in plant sterols in eight steps for stigmasterol and nine for sitosterol (overall yield ca. 15%). Using both diastereomers, the 24-epimers of sitosterol (clionasterol) and stigmasterol (poriferasterol) have also been synthesized. Deuterium NMR on oriented lipid bilayers made of soybean phosphatidylcholine and containing these four labeled plant sterols clearly reveals the difference of orientation and mobility of the four side chains.

Full text of DOI:10.1021/jo960228y

4252
J . Org. Chem. 1996, 61, 4252-4257
Syn th esis of Deu ter iu m -La beled P la n t Ster ols a n d An a lysis of
Th eir Sid e-Ch a in Mobility by Solid Sta te Deu ter iu m NMR
Mary-Pierre Marsan,† William Warnock,‡ Isabelle Muller,† Yoichi Nakatani,‡
Guy Ourisson,‡ and Alain Milon*,†
Laboratoire de Pharmacologie et de Toxicologie Fondamentales, CNRS, 118 rte de Narbonne,
31062 Toulouse, France; Laboratoire de Chimie Organique des Substances Naturelles, Universite´ Louis
Pasteur, Centre de Neurochimie CNRS, 5 rue Blaise Pascal, 67084 Strasbourg, France
Received February 2, 1996X
The plant sterols sitosterol and stigmasterol exert very different effects on plant model membranes,
the first one being a “reinforcer” like cholesterol, the second one not. 25-2H-Stigmasterol has been
synthesized by coupling of the 22-aldehyde derived from stigmasterol by ozonolysis, with the proper
sulfone labeled in position 25. The configuration of the ethyl side chain at C-24 was controlled by
separation of the diastereomers introduced via a chiral sulfoxide. This synthetic scheme allowed
the introduction of a labeled side chain in plant sterols in eight steps for stigmasterol and nine for
sitosterol (overall yield ca. 15%). Using both diastereomers, the 24-epimers of sitosterol (clionasterol)
and stigmasterol (poriferasterol) have also been synthesized. Deuterium NMR on oriented lipid
bilayers made of soybean phosphatidylcholine and containing these four labeled plant sterols clearly
reveals the difference of orientation and mobility of the four side chains.
In animal cell membranes, cholesterol (cholest-5-en-
3â-ol) is the major sterol present. However, plant cell
membranes contain a complex mixture of sterols in which
sitosterol ((24R)-24-ethylcholest-5-en-3â-ol), stigmasterol
((24S)-24-ethylcholesta-5-(E)-22-dien-3â-ol), campesterol
((24R)-24-methylcholest-5-en-3â-ol) and 22,23-dihydro-
brassicasterol ((24S)-24-methylcholest-5-en-3â-ol) usually
predominate.1
The 24-ethylsterols (sitosterol and stigmasterol) have
the same R-configuration for the ethyl C-24 substituent,
and there is a mixture of both 24-R and 24-â epimers for
the 24-methylsterols. Moreover, stigmasterol differs from
sitosterol by a supplementary trans 22(23) double bond
(Figure 1). In addition to these sterols, poriferasterol and
clionasterol, respectively the C-24 epimers of stigmasterol
and sitosterol, are also present in certain algae, and
ergosterol is the major sterol in fungi.2
F igu r e 1. Structures of cholesterol (a), sitosterol (b), stig-
masterol (c), 24ê-methyl cholesterol (d), clionasterol (e),
poriferasterol (f).
These sterols comprise a 3â hydroxyl group, a quasi-
planar sterol skeleton, and a side chain, which are
essential requirements for reinforcing phospholipid-
sterol interactions in membranes.3 All these sterols are
indeed concentrated in the plasma membrane;4 they play
a structural role and act as regulators of some membrane
properties, but they are also precursors of effectors of a
variety of other biological functions. These sterols have
been incorporated into model membrane bilayers and
tested for their ability to regulate either membrane
fluidity as demonstrated by polarization fluorescence5
and deuterium NMR of properly labeled phospholipids,6
or water permeability.7 In particular, deuterium NMR
is a very efficient technique for the analysis of the
average orientation and of the dynamics of the n-alkyl
chains of deuterium-labeled phospholipids in the bio-
membranes.6-12 By using this technique with membrane
model systems prepared from soybean phosphatidylcho-
line (PC), a representative phospholipid of plant mem-
branes with a high content of unsaturated fatty acyl
chains,5 we have compared the effect of a variety of plant
sterols and analogues on the lipid dynamics and dem-
onstrated the surprising specificity of the sterol-lipid
interaction.6,7 Contrary to expectation, sitosterol and
* Author to whom correspondence should be addressed. Tel: 33
61.33.59.22, fax: 33 61.33.58.86, e-mail: milon@lptf.biotoul.fr
† Laboratoire de Pharmacologie et de Toxicologie Fundamentales.
‡ Laboratoire de Chimie Organique des Substances Naturelles.
X Abstract published in Advance ACS Abstracts, J une 1, 1996.
(1) Nes, W. R. Adv. Lipid. Res. 1977, 15, 233-324.
(2) Porter, J . W.; Surgeon, S. L.; Goodwin, T. W. Biosynth. Isoprenoid
Compds. 1981, 443-480.
(6) Krajewski-Bertrand, M. A.; Milon, A.; Hartmann, M. A. Chem.
Phys. Lipids 1992, 63, 235-241.
(7) Schuler, I.; Milon, A.; Nakatani, Y.; Ourisson, G.; Albrecht, A.
M.; Benveniste, P.; Hartmann, M. A. Proc. Natl. Acad. Sci. U.S.A..
1991, 88, 6926-6930.
(8) Davis, J . H. Biochim. Biophys. Acta 1983, 737, 117-171.
(9) Seelig, J . Quart. Rev. Biophys. 1977, 10, 353-411.
(10) Dufourc, E. J .; Parish, E. J .; Chitrakorn, S.; Smith, I. C. P.
Biochemistry 1984, 23, 6062-6071.
(11) Krajewski-Bertrand, M. A.; Milon, A.; Nakatani, Y.; Ourisson,
G. Biochim. Biophys. Acta 1992, 1105, 213-220.
(12) Le´onard, A.; Milon, A.; Krajewski-Bertrand, M. A.; Dufourc, E.
J . Bull. Magn. Reson. 1993, 15, 124-127.
(3) Demel, R. A.; De Kruyff, B. Biochim. Biophys. Acta 1976, 457,
109-132.
(4) Hartmann, M. A.; Benveniste, P. Methods Enzymol. 1987, 148,
632-650.
(5) Schuler, I.; Duportail, G.; Glasser, N.; Benveniste, P.; Hartmann,
M. A. Biochim. Biophys. Acta 1990, 1028, 82-88.
S0022-3263(96)00228-9 CCC: $12.00 © 1996 American Chemical Society

Synthesis of Deuterium-Labeled Plant Sterols
J . Org. Chem., Vol. 61, No. 13, 1996 4253
Sch em e 1. Syn th esis of th e Ch ir a l Syn th on a
a(a) n-BuLi, THF, -78 °C; (b) n-BuLi, C2H5CHO, THF, -78 °C;
(c) DBU, (CH3)2CHNO2; (d) separation of the diastereomers on
chromatography; (e) AIBN, Bu3SnD, benzene, 85 °C; (f) m-CPBA,
dichloromethane, 40 °C.
F igu r e 2. Retrosynthetic scheme.
stigmasterol had opposite effects: while sitosterol was
found to be very efficient in diminishing membrane
fluidity and permeability in a manner similar to choles-
terol in animal cell membranes, stigmasterol appeared
to be a poor membrane reinforcer.5-7
2H-Sitosterol derives from the i-methyl ether of deuter-
ated stigmasterol 9 by hydrogenation of the 22-double
bond, followed by recovery of the 3â-hydroxyl group and
the ∆5double bond. We have chosen the stereospecific
synthesis of a chiral deuterated synthon coupled with a
steroidal aldehyde, in order to obtain finally phytosterols
with both natural configurations of the asymmetric C-24
on the side chain.
Syn th esis of Syn th on 6. We wanted to synthesize
two diastereomers containing two chiral centers: an
asymmetric carbon bearing the ethyl group and a chiral
sulfoxide14 (Scheme 1); this would allow the separation
of the two diastereomers and the determination of their
configuration by X-ray crystallography.
The first step of the stereospecific synthesis of synthon
6 is the coupling of the commercial diastereomerically
pure (-)-(S)-menthyl p-toluenesulfinate 1 with dimethyl
methylphosphonate.15 This typical nucleophilic substitu-
tion takes place with complete inversion of configuration
at sulfur to give the (+)-(S)-sulfinyl phosphate 2.16 The
Horner-Wittig reaction15 of the lithio derivative of sul-
foxide 2 with propionaldehyde gives a mixture of E and
Z isomers of (R)-sulfoxide 3. Their structures were
determined by 1H NMR spectroscopy. The E- and Z-
sulfoxides 3 were used without separation in the follow-
ing reaction. The production of the deuterated sulfoxide
consists of a sequence of two steps: a Michael addition17
of 2-nitropropane on the R,â-unsaturation and a substitu-
tion of the nitro group to deuterium. The first step
catalyzed by DBU17 produced two diastereomers of the
nitro sulfoxide 4 (RR+RS) which could be separated by
In order to understand the molecular mechanism of
this specificity, we have now compared the orientation
and the dynamic behavior of the side chains of both
sterols in phospholipid bilayers. The dynamic properties
of the sterols themselves in membranes have been hardly
studied, except in the case of cholesterol itself, which can
be labeled at specific positions on rings A and B; this had
allowed the determination of its orientation and molec-
ular order parameter.10 However, since the difference
between stigmasterol and sitosterol is localized on the
side chain, it appeared of greater interest to analyze the
dynamics of the side chain, using specifically D-labeled
sterols. We have thus synthesized 25-2H-stigmasterol
and 25-2H-sitosterol as well as their epimers (25-2H-
poriferasterol and 25-2H-clionasterol) in order to measure
the order parameter of these sterols incorporated in
oriented bilayers.
The stereospecific introduction of the 24-R or the 24-S
ethyl group into a steroidal chain presents a significant
challenge. In 1970s, Sucrow and co-workers13 developed
an elegant stereoselective synthesis of (24R)- and (24S)-
ethylsterols by a Claisen rearrangement to generate a
specific chiral center on the steroidal side chain. How-
ever, this requires eight consecutive steps from the
starting i-methyl ether aldehyde to stigmasterol. We now
report a novel method controlling the chirality of the C-24
side chain, which was developed for the synthesis of
stigmasterol, but which should also be applicable to the
synthesis of other natural compounds. Our strategy is
illustrated in the retrosynthetic scheme (Figure 2). 25-
(14) Solladie´ G.; Carre-o, M. C. In Organosulfur Chemistry; Page,
P., Ed., Academic Press: New York, 1995; pp 1-47.
(15) Mikolajczyk, M.; Midura, W.; Grzejszczak, S.; Zatorski, A.;
Chefczynska, A. J . Org. Chem. 1978, 43, 473-478.
(16) Goldmann, S.; Hoffmann, R. W.; Maak, N.; Geueke, K. J . Chem.
Ber. 1980, 113, 831-844.
(13) Sucrow, W.; Caldeira, M.; Slokianka, P. P. Chem. Ber. 1973,
106, 2236-2245. Sucrow, W.; Slokianka, M.; Caldeira, P. P. Chem. Ber.
1975, 108, 1101-1110. Sucrow, W.; Slokianka, M. Chem. Ber. 1975,
108, 3721-3729.
(17) Ono, N.; Kamimura, A.; Miyake, H.; Hamamoto, I.; Kaji, A. J .
Org. Chem. 1985, 50, 3692-3698.

4254 J . Org. Chem., Vol. 61, No. 13, 1996
Marsan et al.
Sch em e 2. Syn th esis of 25-2H-Stigm a ster ol a n d
25-2H-Sitoster ola
medium pressure chromatography. The Michael addition
of secondary nitro compounds to R- or â-substituted
alkenes or to R,â-unsaturated sulfoxides is rather dif-
ficult.17 Finally, we could resolve this problem by using
2-nitropropane as the solvent (as well as the reagent) and
by employing 2 equiv of DBU. The separation of the
diastereomers appeared to be easier at the level of the
nitro synthon than after its reduction by Bu3SnD. The
purity of each diastereomer was checked by 1H NMR. The
less polar diastereomer was crystallized, and its X-ray
analysis allowed us to deduce the configuration of the
asymmetric carbon, (S), from that of the sulfoxide (R).18
This analysis showed that the crystallized nitro sulfoxide
4a (R,S) has on the asymmetric carbon the correct
configuration required to obtain the natural 24-ethyl side
chain (Scheme 1). The replacement of the nitro group
by a deuterium in the nitrosulfoxides 4a (R,S) and 4b
(R,R) was performed using tributyltin deuteride in the
1
presence of AIBN at 80 °C.17 In addition to H NMR
analysis, the IR spectrum revealed the disappearance of
the nitro bands at 1537 and 1347 cm-1 and the appear-
ance of the two specific C-D stretching vibrations 2172
and 2110 cm-1
.
The deuterated sulfoxides 5a (R,S) and 5b (R,R) thus
obtained were then oxidized by m-CPBA to give the
deuterated sulfones 6a (S) an 6b (R). Two specific bands
for the sulfone group were present on the IR spectrum
at 1304 and 1146 cm-1. The products 6 contain now only
one asymmetric center: the chiral carbon bearing the
ethyl group with the (S) configuration in 6a will become
C-24 on the side chain of stigmasterol (configuration S)
and of sitosterol (configuration R).
a
(g) (1) n-BuLi, THF, -78 °C; (2) MeSO2Cl, THF, -10 °C; (3)
Na2HPO4, Na-Hg 6%, methanol/ethyl acetate, 4 °C; (h) H2, Pd-
C, methanol/ethyl acetate; (i) pTsOH, dioxane/water, 80 °C. The
same scheme was used with the other diastereomer 6b in order
to obtain 25-2H-clionasterol and 25-2H-poriferasterol. The yields
were (g) 81%, (h) 60%, (i) 80% for 12b, and (i) 60% for 10b.
Syn th esis of Sitoster ol, Stigm a ster ol, a n d Th eir
24 Ep im er s. These syntheses require coupling of the
deuterated chiral synthon 6a and the steroidal i-methyl
ether aldehyde 7 (Scheme 2), prepared from natural
stigmasterol according to the method described in the
literature.19 The coupling of the sulfone 6a with the
i-methyl ether aldehyde 7 was performed in the presence
of BuLi. To favor the formation of the 22-double bond
in the following procedure, the hydroxyl group is con-
verted in situ into a mesylate.20 The mixture of coupling
products is reduced with 6% sodium amalgam in presence
of disodium hydrogen phosphate according to J ulia’s
method.21 This method allowed the formation of the
trans olefin in the 25-deuterated i-methyl ether 9a . The
1H NMR reveals resonances around 5 ppm with J ) 15
Hz for the trans ethylenic protons on the lateral chain.
The presence of deuterium on the 25 position is confirmed
by the IR spectrum and by the comparison of the 1H NMR
of the deuterated stigmasterol and that of the commercial
stigmasterol: the specific doublets observed for the two
terminal methyls at C-26 and C-27 on the side chain of
stigmasterol are replaced by two broad singlets in the
deuterated compound because of the replacement of the
hydrogen in C-25 position by a deuterium. The third
confirmation of the structure is the observation of a
triplet at 32.1 ppm (J ) 19 Hz, C-25) in 13C-NMR. One
part of compound 9a was deprotected by treatment with
p-toluenesulfonic acid in aqueous dioxane to give the 25-
2H-stigmasterol 10a .22 The other part of 9a was submit-
ted to catalytic hydrogenation with palladium on carbon
(under control by GC as the i-methyl ether of stigmasterol
and sitosterol have very close Rf on TLC) to give the
deuterated i-methyl ether of sitosterol 11, which was
transformed to 25-2H-sitosterol 12a . The absence of
ethylenic protons in the 1H NMR spectrum of 11 confirms
the total hydrogenation of the ∆22 double bond to produce
sitosterol.
The diastereomers in position 24, 25-2H-poriferasterol,
10b, and 25-2H-clionasterol, 12b, have been also obtained
from the more polar oily diastereomer 4b (R,R) by the
same procedure as for 10a and 12a (yields indicated in
the legend of Scheme 2).
The approach presented here has allowed the synthesis
of stigmasterol and poriferasterol (respectively sitosterol
and clionasterol), deuterium labeled at C-25 in eight steps
(respectively nine steps) with an overall yield of about
15%. The configuration at C-24 (the ethyl chain) has
been controlled by the use of an optically pure synthon
which had been prepared from a chiral sulfoxide in five
steps including the separation of diastereomers by me-
dium pressure chromatography.
2H NMR An a lysis. Deuterated stigmasterol, sito-
sterol and their 24-epimers have been incorporated into
oriented bilayers. Four samples have been prepared in
conditions identical to those of previous studies.7 Samples
were composed of 16 mol % sterol, 10 mol % dimyris-
(18) Marsan, M. P.; Muller, I.; Milon, A.; Warnock, W.; Nakatani,
Y.; Ourisson, G.; J aud, J . J . Chem. Crystallogr. 1995, 25, 783-786.
(19) Hutchins, R. F. N.; Thompson, M. J .; Sovoboda, J . A. Steroids
1970, 15, 113-130.
(20) Hsiao, C-N.; Shechter, H. Tetrahedron Lett. 1982, 23, 1963-
1966.
(22) Burger, A.; Colobert, F.; Hetru, C.; Luu, B. Tetrahedron Lett.
1988, 44, 1141-1152.
(21) J ulia, M.; Paris, J . M. Tetrahedron Lett. 1973, 49, 4833-4836.

Synthesis of Deuterium-Labeled Plant Sterols
J . Org. Chem., Vol. 61, No. 13, 1996 4255
splittings that we observe, the unsaturated side chain
appears to be more rigid than the saturated one; however,
a smaller quadrupolar splitting may be obtained even
with a more rigid molecule, if the average orientation of
the CD bond is different. Therefore, an exact interpreta-
tion of these data in terms of a model for the molecular
motions of the sterols in a lipid bilayer would require
more extensive NMR experiments to be performed; in
particular, the behavior of the tetracyclic ring system
may be analyzed by labeling on ring A and B.10 T1
measurements have to be performed in order to extract
correlation times for the movements responsible for the
averaging of the quadrupolar interaction. Molecular
dynamics simulations of the four sterols may indicate
preferential orientation of each side chain, the number
of accessible conformers, and the amplitude of fluctuation
around each of them. These simulations may be per-
formed either in vacuum or in a force field describing the
cooperative van der Waals interactions with the sur-
rounding lipids. All these experiments are now being
performed and will be published later.
One obvious and unexpected result is that the 24-
diastereomers of sitosterol and stigmasterol display an
even stronger difference in their side-chain mobility than
the natural ones. We had not determined previously the
influence of these unnatural epimers on water perme-
ability and on the fatty acid order parameters of lipid
bilayers. This needs now to be performed to understand
better the specificity of plant sterols-plant lipids interac-
tions.
Exp er im en ta l Section
Ch em ica ls. Gen er a l P r oced u r e. All reactions were run
under argon. All solvents and reagents used were reagent
grade and used as obtained except as noted below. THF was
distilled from LiAlH4, dry benzene and dry ethyl acetate were
obtained by distillation from CaH2, CH2Cl2 was distilled from
P2O5, and dry methanol was obtained by distillation from
sodium. 2-Nitropropane was purified by distillation from
MgSO4, and pyridine from KOH. Dioxane was used without
further purification. All the reactions were followed by TLC,
on plates of silica gel 60 F254 (Merck). “Extractive workup”
refers to extraction of the material into the indicated solvent;
the organic layer was dried over anhydrous Na2SO4, or MgSO4,
and the solvent was removed under reduced pressure. Chro-
matography was performed for all reaction products on silica
gel 60 (Merck), 230-400 mesh. 1H and 13C NMR spectra were
recorded in CDCl3 at 500 MHz and 125 MHz, respectively;
significant chemical shifts are reported in ppm and coupling
constants (J values) are given in hertz (Hz).
F igu r e 3. Proton-decoupled deuterium NMR spectra of 90°-
oriented multibilayers consisting of 10 mol % DMPC/73 mol
% SPC/1 mol% BHT/16 mol % sterol; stigmasterol (A), sito-
sterol (B), poriferasterol (C), clionasterol (D). ∆νQ, the qua-
drupolar splitting of the C-D bond, is characteristic of the
amplitude of motion and average orientation of this bond.
Spectrum C was acquired under proton decoupling in order
to resolve the small doublet coupling.
toylphosphatidylcholine (DMPC), 1 mol % butylated
hydroxytoluene (BHT) as an antioxidant, and 73 mol %
soybean phosphatidylcholine (SPC).
2
In Figure 3 are shown the H NMR spectra recorded
for 25-2H-stigmasterol (3A), for 25-2H-sitosterol (3B), for
25-2H-poriferasterol (3C), and for 25-2H-clionasterol (3D).
In each case, one doublet is observed, from which the
deuterium quadrupolar splitting is measured: 7.3 kHz
for sitosterol, 11.7 kHz for stigmasterol, 0.44 kHz for
poriferasterol, and 17.4 kHz for clionasterol. This split-
ting is proportional to the order parameter of the C-D
bond: SCD ) 3 cos2 θ - 1 /2, where θ is the angle between
the C-D bond and the bilayer normal and where 3 cos2
θ - 1 denotes a time and ensemble average on all the
possible orientations of the C-D bond.
(+)-(S)-(Dim eth ylp h osp h or yl)m eth yl p-Tolyl Su lfoxid e
(2). This compound was prepared from (-)-(S)-menthyl p-tolyl
sulfinate according to the procedure described in the litera-
ture.15
(E)- a n d (Z)-(R)-(p -t olylsu lfin yl)b u t en es 3. (+)-(S)-
(Dimethylphosphoryl)methyl p-tolyl sulfoxide (3 g , 11.4 mmol)
was dissolved in 34 mL of THF and cooled to -78 °C; a solution
of n-butyllithium (8.1 mL, 13 mmol) in hexane was then added.
After 30 min at -78 °C, a solution of propionaldehyde (661
mg, 11.4 mmol) in THF (26 mL) was added to this mixture.
Stirring at this temperature was continued for 30 min. After
having returned to room temperature, the solution was washed
with saturated NH4Cl (3 × 30 mL) and then purified by
chromatography, eluting with ether/cyclohexane (8:2), to give
These data show immediately that the four sterols
behave very differently when they are incorporated into
a lipid bilayer. A simple, intuitive, analysis of the data
fails to explain why stigmasterol is a less efficient sterol
than sitosterol, since one would expect to correlate a good
sterol efficiency with a high rigidity of the molecule. The
double bond of stigmasterol is expected to increase the
side chain mobility of the sterol. Indeed, although the
double bond itself is rigid, the dihedral angles on each
side of it have much more rotational freedom than in the
case of a saturated chain. According to the quadrupole
1.47 g (66%) of a mixture of E and Z isomers : [R]25 ) -168
D
(c 1.88, CHCl3); IR (NaCl) 3069, 2968, 1618, 1454, 1083, 1038,
1
810 cm-1; H NMR (200 MHz) δ 1.04 (t, J ) 7.3 Hz, 3H) and
1.11 (t, J ) 7.6 Hz, 3H), 2.25 (m, 2H) and 2.58 (m, 2H), 6.19
(m, 2H (Z), 1H (E)), 6.62 (dt, 1H (E)), 7.29 and 7.47 (AB q, ∆ν
) 36.8 Hz, J ) 8.2 Hz, 4H); 13C NMR (CDCl3, 200 MHz) δ
13.5, 21.2, 22.6, 123.8, 129.8, 134.1, 136.1, 140.9, 141.1, 142.2,
143.0.

4256 J . Org. Chem., Vol. 61, No. 13, 1996
Marsan et al.
(R,S)- a n d (R,R)-1-(p-tolylsu lfin yl)-2-eth yl-3-n itr o-3-
m eth ylbu ta n es (4a , 4b). DBU (2.7 mL,18 mmol) was added
to a solution of (R)-p-tolyl sulfinyl butene (1.4 g, 7.2 mmol) in
2-nitropropane (3.26 mL, 36 mmol). The mixture was stirred
in darkness at room temperature. After 28 h, 40 mL of ether
and 20 mL of water were added. The organic phase was
washed with 2 N HCl (2 × 40 mL) and then with water (2 ×
40 mL). The mixture of the pure diastereomeric nitro sulfox-
ides, (R,S) and (R,R), obtained after the chromatography
(ether/cyclohexane (8:2)) represented 1.48 g (73%). The sepa-
ration of the diastereomers by medium pressure chromatog-
raphy (30 g of Lichroprep silicagel 60 (Merck, 5-10 µm),
column pressure, 8 bars) gave 710 mg of the pure (R,S)
diastereomer (less polar) and 601 mg of the pure (R,R)
diastereomer (more polar).
19.5, 22.3, 24.0, 28.6 (t, J ) 20 Hz), 41.1, 58.1, 128.7, 130.5,
137.8, 145.1.
(R) sulfone: [R]25 ) -5 (c 1.21, CHCl3); IR (NaCl) 3020,
D
2960, 2171 (ν C-D), 2108 (ν C-D), 1463, 1304 (νas SO2), 1146
1
(νss SO2), 1083, 818 cm-1; H NMR (200 MHz) δ 0.76 (s, 6H),
0.82 (t, J ) 7.4 Hz, 3H), 1.45 (m, 2H), 1.74 (m, 1H), 2.45 (s,
3H), 2.86 and 3.04 (AB qd, ∆ν ) 45 Hz, 2J ) 14.4 Hz, 3J ) 6.3
and 4.4 Hz, 2H), 7.35 (d, J ) 7.9 Hz, 2H) and 7.79 (d, J ) 7.9
Hz, 2H); 13C NMR (CDCl3, 500 MHz) δ 11.8, 18.3, 22.1, 23.8,
28.5 (t, J ) 20 Hz), 41.0, 58.0, 128.5, 130.5, 137.3, 145.2. Anal.
Calcd for C14H21DSO2: C, 65.89; H, 8.23; S, 12.56. Fd: C,
64.80; H, 8.73; S, 12.12.
(S)- an d (R)-6â-Meth oxy-3r,5-cyclo-25-deu ter iostigm ast-
22(E)-en es 9a , 9b. The deuterated (S) sulfone 6a (140 mg,
0.56 mmol) [or the deuterated (R) sulfone 6b: 70 mg, 0.29
mmol] was dissolved in 6 mL [or 3.4 mL] of dry THF.
A
The (R,S) diastereomer 4a was crystalline: mp 89-95 °C;
D
[R]25 ) +255 (c 0.57, CHCl3); IR (NaCl) 3020, 2917, 1537 (νas
solution of nBuLi in hexane (360 µL, 0.57 mmol) [or 200 µL,
0.31 mmol] was added to the mixture cooled at -78 °C. After
10 min, 190 mg (0.55 mmol) [or 101 mg, 0.29 mmol] of the
i-methyl ether aldehyde 7 in 6 mL [or 3.5 mL] of dry THF was
added dropwise under stirring. The mixture was kept at -78
°C during 4 h, and then, after returning to room temperature
(1 h), methanesulfonyl chloride (51 µL, 0.66 mmol) [or 27 µL,
0.35 mmol] in THF (340 µL) [180 µL] was added at -10 °C.
After a reflux at 80 °C for 25 min, THF was removed and the
residue was extracted with ether. The organic layer was
washed with aqueous saturated NaHCO3, dried, and evapo-
rated to yield an oily product. This mixture of diastereomers
8 (273 mg of (S)) [and 202 mg of (R)] was dissolved in
methanol/ethyl acetate (2:1, 5.9 mL) [or 7.9 mL], and 257 mg
(1.82 mmol) [or 191 mg, 1.3 mmol] of Na2HPO4 was added.
Under stirring at room temperature, then at 4 °C, freshly
prepared 6% sodium amalgam (3.1 g) [or 2.4 g] was added and
the stirring continued for 5 h. Water was added and the
extraction was performed with dichloromethane (6 × 35 mL).
Chromatography of the crude product [cyclohexane/ethyl
acetate (97:3)] yielded as colorless oils the (S) diastereomer
9a (131 mg, 76%) and the (R) diastereomer 9b [100 mg, 81%].
(S) diastereomer 9a : [R]25D ) +30 (c 0.71, CHCl3); IR (NaCl)
NO2), 1450, 1406, 1345 (νss NO2), 1086, 1038 (νs SO), 805 cm-1
;
1H NMR (200 MHz) δ 0.99 (t, J ) 6 Hz, 3H), 1.48 (m, 1H),
1.55 (s, 3H), 1.58 (m, 1H), 1.62 (s, 3H), 2.43 (s, 3H), 2.51 (m,
1H), 2.55 (m, 1H), 2.90 (dd, J ) 10 Hz, J ) 2 Hz, 1H), 7.35
and 7.55 (ABq, ∆ν ) 40 Hz, J ) 8 Hz, 4H); 13C NMR (CDCl3,
500 MHz) δ 13.2, 22.2, 24.1, 24.5, 25.8, 44.3, 61.2, 91.8, 124.7,
130.8, 141.7, 142.7.
The (R,R) diastereomer 4b was oily: [R]25 ) +106 (c 0.76,
D
CHCl3); IR (NaCl) 3020, 2969, 1537 (νas NO2), 1461, 1400, 1347
(νss NO2), 1086, 1044 (νs SO), 810 cm-1; 1H NMR (200 MHz) δ
1.07 (t, J ) 7.3 Hz, 3H), 1.28 (m, 1H), 1.44 (s, 3 H), 1.50 (m,
1H), 1.58 (s, 3H), 2.38 (m, 1H), 2.43 (s, 3H), 2.79 (dd, J ) 4.3
Hz, 2H), 7.34 and 7.55 (ABq, ∆ν ) 42 Hz, J ) 8.3 Hz, 4H); 13
C
NMR (CDCl3, 500 MHz) δ 13.1, 22.1, 24.4, 24.8, 44.5, 60.6,
91.9, 125.0, 130.8, 141.5, 142.9. Anal. Calcd for C14H21NSO3:
C, 59.4; H, 7.41; N, 4.94; S, 11.32. Fd: C, 59.58; H, 7.50; N,
4.86; S, 11.59.
(R,S)- a n d (R,R)-1-(p-tolylsu lfin yl)-2-eth yl-3-d eu ter io-
3-m eth ylbu ta n es (5a , 5b). To a solution of (R,S)-4a (290 mg,
1.02 mmol) [or 120 mg, 0.42 mmol of (R,R)-4b] in dry benzene
(14 mL) [or 5.5 mL] were added 84.1 mg (0.51 mmol) [or 35
mg, 0.21 mmol] of AIBN and then 1.38 mL (4.9 mmol) [or 534
µL, 2.1 mmol] of tributyltin deuteride. The mixture was
heated at 85 °C for 6 h and cooled slowly to room temperature.
Chromatography, eluting with ether/cyclohexane (7:3), gave
196 mg (80%) of deuterated (R,S) sulfoxide 5a and 88.2 mg
(87%) of deuterated (R,R) sulfoxide 5b, both as a white solids.
3050, 2930, 2141 (ν C-D), 1595, 1451, 1376, 1093, 1018 cm-1
;
1H NMR (200 MHz) δ 0.41 (dd, J ) 8.2 Hz, J ) 5.3 Hz, 1H),
0.63 (t, J ) 5 Hz, 1H), 0.71 (s, 3H), 0.77 (s, 3H), 0.79 (t, J )
7.04 Hz, 3H), 0.82 (s, 3H), 0.99 (d, J ) 5.3 Hz, 3H), 2.75 (t, J
) 2.6 Hz, 1H), 3.31 (s, 3H), 5.0 and 5.12 (AB qd, ∆ν ) 24 Hz,
2J ) 15 Hz, 3J ) 8.5 Hz and 7.9 Hz, 2H); 13C NMR (CDCl3,
500 MHz) δ 12.9, 13.1, 13.8, 19.6, 20.0, 21.7, 21.9, 22.2, 23.5,
25.0, 25.7, 26.1, 29.7, 31.2, 32.1 (t, J ) 19.2 Hz), 34.1, 35.8,
36.0, 40.9, 41.2, 43.4, 44.1, 48.8, 51.8, 56.8, 57.3, 83.1, 130.0,
139.1.
(R,S) Diastereomer: mp 50.3-51.4 °C; [R]25D ) +186 (c 0.6,
CHCl3); IR (NaCl) 3018, 2917, 1445, 1043, 815; 1H NMR (200
MHz) δ 0.76 (s, 3H), 0.84 (s, 3H), 0.88 (t, J ) 7.6 Hz, 3H), 2.40
2
3
(s, 3H), 2.63 and 2.77 (AB qd, ∆ν ) 28 Hz, J ) 12.9 Hz, J )
6.2 Hz), 7.31 and 7.53 (AB q, ∆ν ) 44 Hz, J ) 8.5 Hz, 4H); 13
C
(R) diastereomer 9b: [R]25D ) +30 (c 1.15, CHCl3); IR (NaCl)
3060, 2932, 2857, 2141 (ν C-D), 1638, 1606, 1451, 1376, 1093,
NMR (CDCl3, 500 MHz) δ 12.4, 19.1, 19.3, 22.1, 24.0, 29.0 (t,
J ) 19.3Hz), 41.3, 61.5, 125.0, 130.6, 142.3.
1
1024 cm-1; H NMR (200 MHz) δ 0.41 (dd, J ) 7.9 Hz, J ) 5
(R,R) diastereomer: mp 63-65 °C; IR (NaCl) 3020, 2958,
2172 (ν C-D), 2110 (ν C-D), 1461, 1083, 1036 (νs SO), 808
cm-1; 1H NMR (200 MHz) δ 0.78 (s, 3H), 0.86 (s, 3H), 0.9 (t, J
) 7.4 Hz, 3H), 1.44 (m, 3H), 2.42 (s, 3H), 2.66 and 2.79 (AB
qd, ∆ν ) 26.1 Hz, 2J ) 13.1 Hz, 3J ) 6.2 Hz, 2H), 7.32 and
7.55 (AB q, ∆ν ) 46 Hz, J ) 8.1 Hz, 4H).
(R)- a n d (S)-1-(p -t olylsu lfon yl)-2-et h yl-3-d eu t er io-3-
m eth ylbu ta n es (6a , 6b). A 163 mg (0.68 mmol) amount of
(R,S)-5a [or 82 mg, 0.32 mmol of (R,R)-5b] was dissolved in
Hz, 1H), 0.63 (t, J ) 4.7 Hz, 1H), 0.72 (s, 3H), 0.77 (s, 3H),
0.80 (t, J ) 7.3 Hz, 3H), 0.82 (s, 3H), 1.00 (d, J ) 6.5 Hz, 3H),
1.01 (s, 3H), 2.75 (t, J ) 3 Hz, 1H), 3.31 (s, 3H), 5.00 and 5.14
(AB qd, ∆ν ) 28 Hz, 2J ) 15.3 Hz, 3J ) 8.5 Hz and 7.9 Hz,
2H); 13C NMR (CDCl3, 500 MHz) δ 13.1, 13.8, 19.5, 20.0, 21.5,
21.9, 22.2, 23.5, 24.9, 25.7, 26.1, 29.6, 31.2, 32.0 (t, J ) 20 Hz),
34.0, 35.8, 36.0, 40.9, 41.2, 43.4, 44.1, 48.8, 51.8, 56.8, 57.3,
83.1, 129.9, 139.0.
25-Deu ter iostigm a ster ol (10a ). A solution of 9a (40 mg,
94 µmol) in dioxane/water (7:3, 2.6 mL) was stirred at 80 °C
for 5 h in presence of a catalytic amount of p-toluenesulfonic
acid. The mixture was poured into aqueous saturated NaCl
(10 mL), and the crude product was extracted three times with
10 mL of ether. Column chromatography, eluting with cyclo-
hexane/ethyl acetate (8:2), afforded the pure deuterated stig-
masterol 10a as a white solid (28.5 mg, 75%): mp ) 164.2-
dry CH2Cl2 (8.2 mL) [or 4.1 mL] and cooled to -15 °C.
A
solution of m-CPBA (470 mg, 2.7 mmol) [or 237 mg, 1.37 mmol]
in dry CH2Cl2 (41 mL) [or 20 mL] was added under stirring at
this temperature. After returning to room temperature, the
mixture was heated at 40 °C for 2.5 h, cooled, washed (3 × 20
mL saturated NaHCO3), dried over anhydrous MgSO4, and
chromatographed, eluting with ether/cyclohexane (2:8) to give
the pure deuterated (S) sulfone 6a (140 mg, 81%) and the (R)
sulfone 6b (70.2 mg, 80%) as oils.
166 °C; [R]25 ) -50 (c 0.57, CHCl3); IR (NaCl) 3348, 2942,
D
2857, 2141 (ν C-D), 1659, 1638, 1446, 1376, 1045 cm-1
;
1H
(S) Sulfone: [R]25 ) +4 (c 1.27, CHCl3); IR (NaCl) 3020,
NMR (500 MHz) δ 0.74 (s, 3H), 0.82 (s, 3H), 0.84 (t, J ) 7.4
Hz, 3H), 0.88 (s, 3H), 1.05 (s, 3H), 1.06 (d, J ) 6.6 Hz, 3H),
1.74 (m, 1H), 1.88 (m, 2H), 2.27 (m, 1H), 2.32 (m, 1H), 3.57
(m, 1H), 5.06 and 5.19 (AB qd, ∆ν ) 65 Hz, 2J ) 15.2 Hz, 3J )
8.7 Hz and 9.1 Hz, 2H), 5.39 (m, 1H); 13C NMR (CDCl3, 500
MHz) δ 12.7, 12.9, 19.5, 20.1, 21.7, 21.8, 21.9, 25.1, 26.1, 29.6,
D
2953, 1456, 1301 (νas SO2), 1141 (νss SO2), 1082, 815 cm-1; 1
H
NMR (200 MHz) δ 0.75 (s, 6H), 0.82 (t, J ) 7.4 Hz, 3H), 1.25
2
(m, 1H), 1.42 (m, 2H), 2.46 (s, 3H), 2.86 and 3.04 (AB q, J )
3
13.6 Hz, J ) 1.3 Hz, 2H), 7.36 (d, J ) 8.3 Hz, 2H) and 7.80
(d, J ) 8.4 Hz, 2H); 13C NMR (CDCl3, 500 MHz) δ 12.1, 18.6,

Synthesis of Deuterium-Labeled Plant Sterols
J . Org. Chem., Vol. 61, No. 13, 1996 4257
32.1 (t, J ) 18.9 Hz), 32.4, 32.6, 37.2, 37.9, 40.4, 41.2, 42.9,
43.0, 50.8, 51.8, 56.6, 57.6, 72.5, 122.4, 129.9, 139.0, 141.4.
25-Deu ter iop or ifer a ster ol (10b). With 35 mg of deuter-
ated i-methyl ether 9b, 20 mg (60%) of pure deuterated
saturated NaCl solution (20 mL). The crude product was
extracted three times with ether and chromatographed [cy-
clohexane/ethylacetate (8:2)] to give a white solid (34 mg,
95%): [R]25D ) -30 (c 1, CHCl3); mp 131.0-133.4 °C; 1H NMR
(500 MHz) δ 0.72 (s, 3H), 0.84 (s, 3H), 0.87 (s, 3H), 0.88 (t, J
) 7.5 Hz, 3H), 0.96 (d, J ) 6.5 Hz, 3H), 1.05 (s, 3H), 1.89 (m,
1H), 2.28 (m, 1H), 2.33 (m, 1H), 3.57 (m, 1H), 5.39 (m, 1H);
13C NMR (CDCl3, 500 MHz) δ 12.6, 12.7, 19.5, 19.6, 20.1, 20.4,
21.8, 23.8, 25.0, 26.7, 28.9, 29.3 (t, J ) 19.7 Hz), 30.4, 32.4,
32.6, 32.6, 34.6, 36.8, 37.2, 37.9, 45.5, 43.0, 46.4, 50.8, 56.7,
57.5, 72.5, 122.4, 141.5.
poriferasterol 10b was obtained: mp ) 154.0-155.0 °C; [R]25
D
) -42 (c 0.50, CHCl3); IR (NaCl) 3362, 2928, 2854, 2135, 1665,
1
1613, 1449, 1375, 1047, 962 cm-1; H NMR (500 MHz) δ 0.70
(s, 3H), 0.79 (s, 3H), 0.81 (t, J ) 7 Hz, 3H), 0.83 (s, 3H), 1.01
(s, 3H), 1.02 (d, J ) 6.5 Hz, 3H), 1.99 (m, 1H), 2.05 (m, 1H),
2.23 (m, 1H), 2.30 (m, 1H), 3.53 (m, 1H), 5.02 and 5.16 (AB
qd, ∆ν ) 70 Hz, 2J ) 15 Hz, 3J ) 8.5 Hz and 9.5 Hz, 2H), 5.35
(dd, J ) 3 Hz, J ) 2.5 Hz, 1H); 13C NMR (CDCl3, 500 MHz) δ
11.9, 12.3, 18.7, 19.3, 20.7, 21.0, 21.1, 24.2, 25.3, 28.7, 29.6,
30.8, 31.2 (t, J ) 20 Hz), 31.6, 36.4, 37.2, 39.6, 40.3, 42.1, 42.2,
50.1, 51.0, 55.8, 56.8, 71.7, 121.6, 129.2, 138.1, 140.6.
(S)- a n d (R)-6â-Met h oxy-3r,5-cyclo-25-d eu t er iosit o-
ster ol (11a , 11b). To a solution of (E)-6-methoxy-3, 5-cyclo-
25-deuteriostigmast-22-ene (9a ) (37 mg, 87 µmol) [or 9b: 29
mg, 68 µmol] in methanol/ethyl acetate (1:1, 3.8 mL) was added
7.5 mg [or 5.8 mg] of activated Pd-C. The mixture was stirred
at room temperature in a hydrogen atmosphere during 2 h.
After filtration on Celite and washing with ethyl acetate, the
filtrate was evaporated in vacuo. The crude product was
chromatographed on silica gel [cyclohexane/ethyl acetate (98:
2)] to give 28 mg of 11a (75%) and 18 mg (60%) of 11b as pure
colorless oils.
25-Deu ter ioclion a ster ol 12b. With 17 mg of deuterated
methyl ether 11b, by the above procedure, 13 mg (80%) of pure
deuterated clionasterol was obtained: [R]25 ) -42 (c 0.56,
D
CHCl3); mp 137.0-138.5 °C; IR (NaCl) 3380, 2932, 2868, 2141,
1643, 1456, 1376, 1104, 1045 cm-1; 1H NMR (500 MHz) δ 0.72
(s, 3H), 0.84 (s, 3H), 0.86 (s, 3H), 0.89 (t, J ) 7.5 Hz, 3H), 0.96
(d, J ) 6.5 Hz, 3H), 1.05 (s, 3H), 1.88 (m; 1H), 2.27 (m, 1H),
2.33 (m, 1H), 3.56 (m, 1H), 5.39 (m, 1H); 13C NMR (CDCl3,
500 MHz) δ 12.5, 13.0, 19.5, 20.0, 20.2, 21.8, 23.7, 25.0, 27.0,
28.9, 30.4, 32.4, 32.6, 32.6, 34.6, 37.0, 37.2, 37.9, 40.5, 43.0,
46.6, 50.8, 54.1, 56.7, 57.4, 72.5, 122.4, 141.4.
2H NMR Exp er im en ts. Lipid mixtures were prepared
with one of the sterols (2 mg, 16 mol %), DMPC (2 mg, 10 mol
%) (Sygena, Inc., Liestal), SPC (16 mg, 73 mol %) (Sigma), and
1 mol % BHT (Sigma) in 1.4 mL of 2-propanol and deposited
onto 40-50 circular coverslips (diameter 4.5 mm, Polylabo).
The plates were placed under vacuum overnight and stacked
into tubes (diameter 7 mm). The lipids were hydrated with
2H-depleted water (Aldrich) at 37 °C overnight, and then 18
µL of this water was added to the tube. The stacks of bilayers
thus obtained were oriented with their normal at 90° with
respect to the magnetic field.
Proton decoupled 2H-NMR spectra were acquired at 30 °C
in a 7 mm coil doubly tuned for deuterium (76.77 MHz) and
proton with a Bruker AMX 500 spectrometer equiped for solid
state NMR. Quadrupolar echos8 were obtained by using two
90° pulses of 3.5 µs, width separated by 30 µs, the sweepwidth
was 250 kHz, proton decoupling was performed during acqui-
sition (decoupling power 15 W), the repetition rate was 1 s to
allow for thermal dissipation of the decouping power, and the
acquisition time was 4 ms.
(R) Diastereomer 11a : [R]25D ) +50 (c 1.4, CHCl3); IR (NaCl)
2934, 2868, 2141, 1633, 1451, 1381, 1098, 1029 cm-1; 1H NMR
(500 MHz) δ 0.43 (dd, J ) 8 Hz, J ) 5.1 Hz, 1H), 0.64 (t, J )
4.9 Hz, 1H), 0.71 (s, 3H), 0.80 (s, 3H), 0.82 (s, 3H), 0.84 (t, J
) 7.4 Hz, 3H), 0.91 (d, J ) 6.5 Hz, 3H), 1.02 (s, 3H), 2.77 (t, J
) 2.7 Hz, 1H), 3.32 (s, 3H); 13C NMR (CDCl3, 500 MHz) δ 12.7,
13.0, 13.8, 19.5, 19.6, 20.0, 20.4, 22.2, 23.5, 23.8, 24.9, 25.7,
26.8, 29.0, 29.3 (t, J ) 18.8 Hz), 31.2, 34.1, 34.6, 35.8, 36.0,
36.9, 41.0, 43.5, 44.1, 46.4, 48.7, 57.0, 57.2, 83.2.
(S) Diastereomer 11b: IR (NaCl) 2933, 2888, 2141, 1643,
1
1456, 1376, 1093, 1018 cm-1; H NMR (500 MHz) δ 0.44 (dd,
J ) 8 Hz, J ) 5.1 Hz, 1H), 0.66 (t, J ) 5.1 Hz, 1H), 0.73 (s,
3H), 0.81 (s, 3H), 0.83 (s, 3H), 0.86 (t, J ) 7.4 Hz, 3H), 0.93 (d,
J ) 6.5 Hz, 3H), 1.03 (s, 3H), 2.78 (t, J ) 2.8 Hz, 1H), 3.34 (s,
3H).
25-Deu ter iositoster ol (12a ). A catalytic quantity of p-
toluenesulfonic acid was added to a solution of 11a (37 mg,
86.2 µmol) in dioxane/water (7:3, 2.6 mL). After stirring at
80 °C during 1.5 h, the mixture was poured into an aqueous
J O960228Y