Synthesis, isolation and characterisation of β-sitosterol and β-sitosterol oxide derivatives

Article abstract of DOI:10.1039/b505069c

β-Sitosterol is the most prevalent plant cholesterol derivative (phytosterol) and can undergo similar oxidation to cholesterol, leading to β-sitosterol oxides. The biological impact of phytosterol oxides has only been evaluated in a phytosterol blend (usually of β-sitosterol, campesterol, stigmasterol and dihydrobrassicasterol). The lack of pure phytosterols, including β-sitosterol, hinders the collection of significant toxicity data on the individual β-sitosterol oxides. An efficient synthetic route to multi-gram quantities of pure β-sitosterol is described here, together with the first syntheses and characterisation of pure β-sitosterol oxides. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b505069c

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A R T I C L E
Synthesis, isolation and characterisation of b-sitosterol and
b-sitosterol oxide derivatives†
Florence O. McCarthy,*a Jay Chopra,a Alan Ford,a Sean A. Hogan,b Joe P. Kerry,b
Nora M. O’Brien,b Eileen Ryanb and Anita R. Maguirea
a Department of Chemistry and School of Pharmacy, Analytical and Biological Chemistry
Research Facility, University College Cork, Western Road, Cork, Ireland.
E-mail: f.mccarthy@ucc.ie; Fax: +353-21-427-4097; Tel: +353-21-490-3724
b Department of Food and Nutritional Sciences, University College Cork, Cork, Ireland
Received 12th April 2005, Accepted 14th June 2005
First published as an Advance Article on the web 13th July 2005
b-Sitosterol is the most prevalent plant cholesterol derivative (phytosterol) and can undergo similar oxidation to
cholesterol, leading to b-sitosterol oxides. The biological impact of phytosterol oxides has only been evaluated in a
phytosterol blend (usually of b-sitosterol, campesterol, stigmasterol and dihydrobrassicasterol). The lack of pure
phytosterols, including b-sitosterol, hinders the collection of significant toxicity data on the individual b-sitosterol
oxides. An efficient synthetic route to multi-gram quantities of pure b-sitosterol is described here, together with the
first syntheses and characterisation of pure b-sitosterol oxides.
the problem, there is a lack of pure commercially available phy-
tosterols hindering the development of individual phytosterol
oxides as standards.
Introduction
Background
A recent comment in the British Journal of Nutrition con-
cluded, “the development of accurate and sensitive methods
for qualitative and quantitative analyses of oxysterols and
oxyphytosterols in food, dietary products and biological samples
has become a new challenge.”13 As an initial step towards
developing a range of standard POP’s, the synthesis of pure
b-sitosterol and b-sitosterol oxidation products was undertaken.
The term phytosterols refers to sterols synthesized in plants, of
which the most prevalent are b-sitosterol 2 and campesterol 6
(comprising 95% of total sterols). Interest in phytosterols and b-
sitosterol has increased in recent years due to the incorporation
of phytosterol esters into foodstuffs, a function of which is
to lower cholesterol levels.1,2 Dietary intake of phytosterols is
projected to increase in Western countries as consumers respond
to health messages to increase vegetable oil consumption at the
expense of animal fats.3
b-Sitosterol is structurally similar to cholesterol with the addi-
tion of an ethyl substituent at the 24-position and, consequently,
it may undergo similar oxidation processes to cholesterol and
yield similar oxidation products. Cholesterol oxidation products
(COP’s) have well documented adverse effects, including a
harmful role in the development of atherosclerosis.4–6 The
consumption of dietary phytosterols in increased quantities has
lead to the possibility of increased levels of phytosterol oxides
in the blood.
Phytosterols and phytosterol oxidation products (POP’s) have
generally been assumed to absorb poorly from the diet and hence
decrease cholesterol uptake via disruption of cholesterol micelle
formation. Recently however, POP’s have been isolated from
the plasma of healthy human subjects.7 Discussion continues
as to whether these POP’s are absorbed or transformed in vivo
from the parent phytosterols.8 One paper which describes phy-
tosterolaemia (a disease of phytosterol storage as a dysfunctional
mutation of an active cellular sterol reexporter) would suggest
that phytosterols can passively enter the cellular lining of the
gut before their fate is determined.9 It appears that phytosterols
and POP’s can enter the bloodstream and this has a significant
impact on their respective toxicity profiles.
Toxicity data on POP’s is incomprehensive due to the fact
that b-sitosterol and campesterol have been difficult to purify
from natural sources and consequently POP’s are tested as part
of a phytosterol blend.9–12 The blend of phytosterol oxidation
products has inherent toxicity, however designation of toxicity to
individual POP’s from this blend is problematic. Compounding
Problematic synthesis of b-sitosterol
A synthetic route to b-sitosterol 2 is attractive as the natu-
ral extract usually contains some impurities (stigmasterol 1,
campesterol 6, dihydrobrassicasterol 7 etc.) in varying ratios,
which are virtually inseparable by chromatography (save for
HPLC which is arduous on a synthetically useful scale).14,15
b-Sitosterol has been synthesized previously starting from
stigmasterol 1 via simple hydrogenation of the side chain double
bond16. Prior to hydrogenation of the D22–23-double bond, the B-
ring alkene must be protected through an i-stigmasterol methyl
ether. Standard conditions for the subsequent hydrogenation
employ Pd/C as catalyst and ethyl acetate as solvent.16
Recently however, it has been reported that in the synthesis of
campesterol 6 (a compound similar in structure to b-sitosterol,
where the side chain ethyl group is replaced by a methyl group)
following similar methodology, the side chain is susceptible
to isomerisation during the hydrogenation of the D22–23-double
bond using Pd/CaCO3 as catalyst and ethanol as solvent
(Scheme 1).17 Critically, the stereochemistry is compromised
at C-24 during the hydrogenation step and a mixture of
products formed (4 and 5). This isomerisation had not been
considered previously in phytosterol synthesis. 13C NMR data
shows slight but distinct differences between the two isomers
synthesised (4 and 5), direct precursors of campesterol 6 and
dihydrobrassicasterol 7. It is suggested this isomerisation is
catalyst dependant in the case of campesterol. Spectral data
showed an appreciable amount of by-product 5 present, up to
10% when PtO2 was used as the catalyst and up to 25% when
Pd/CaCO3 was employed.17
The lack of 13C data in the literature regarding reactions of this
type leads us to our current investigation towards the synthesis
of pure b-sitosterol.
† Electronic supplementary information (ESI) available: GCMS analysis
of TMS-ethers of b-sitosterol synthesised under various conditions. See
http://dx.doi.org/10.1039/b505069c
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 0 5 9 – 3 0 6 5
3 0 5 9
©

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Scheme 1 Synthesis of campesterol 6 and dihydrobrassicasterol 7 from common intermediate. Reagents and conditions: (i) H2, Pd/CaCO3 or PtO2
in EtOH or EtOH–EtOAc (5 : 3) (ii) p-TsOH, aq. dioxane, 80 ◦C.
The catalysts that effect hydrogenation are PtO2 and 5%
Pd/C. It can be seen from Table 1 that under conditions (a)
and following deprotection of the B-ring double bond, the b-
Results and discussion
Synthesis and isolation of b-sitosterol
sitosterol formed was of only 85.9% purity by GCMS. The use of
PtO2 improved the selectivity of hydrogenation in campesterol,
however little if any improvement was seen when used for b-
sitosterol, even on changing solvents (c and d). Other catalysts
used included Raney nickel and Wilkinson’s catalyst (e, f and g),
however no reaction was seen in these cases even when a large
excess of the catalyst and different solvents were used.19
Ethyl acetate was initially employed as solvent for the
hydrogenation due to solubility issues and literature precedent;
this however gave rise to a significant amount of an unidentified
isomer with characteristic alkene signals in the 13C NMR (a).
We believe that the unidentified isomer is formed by double
bond rearrangement on the side chain, catalysed by traces of
acetic acid in the ethyl acetate. Subsequent hydrogenation in the
presence of base yielded no reduced material (b).
The initial step in the synthesis of pure b-sitosterol is the
preparation of the stigmasterol tosylate 8 from the readily
available stigmasterol 1 (95% purity) using standard tosylation
conditions (Scheme 2).18
The tosylate was then treated with anhydrous MeOH and
pyridine in a solvolysis to give i-stigmasterol methyl ether 9
as an oily solid in 74% yield, as a mixture with stigmasterol
methyl ether 10 in a ratio of 5 : 1. This minor product has not
been previously identified as a side product from this reaction.
The mixture can be separated after repeated chromatography,
however the material was used unpurified and the minor product
removed by chromatography in the subsequent step.
Standard hydrogenation conditions for the conversion were
developed in 1963 and use Pd/C as catalyst and ethyl acetate
as solvent.16 As mentioned earlier, a recent publication has
identified isomerisation during hydrogenation in campesterol
synthesis under similar conditions.17 Therefore a range of
hydrogenation catalysts were screened and the yields and purities
obtained using these conditions are summarised in Table 1.
Ethanol was then used as solvent even though the starting
material 9 is only partially soluble in ethanol at rt. As the hy-
drogenation proceeded the organic material went into solution.
Following deprotection of the B-ring double bond, b-sitosterol 2
Scheme 2 Reagents and conditions: (i) p-TsCl, DMAP, pyridine (90%); (ii) MeOH (anhydrous), pyridine (74%); (iii) H2, catalyst and solvent as in
Table 1; (iv) p-TsOH, aq. dioxane, 80 ◦C (55% for 2 steps).
3 0 6 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 0 5 9 – 3 0 6 5

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Table 1 Summary of hydrogenation conditions employed in hydrogenation and deprotection (compound 9 to 2)
Reactiona
Catalyst
Pd/C 5%
PtO2
Scale
Yield (%)
Solvent
Purityb(%)
a
b
c
d
e
f
g
h
i
0.1 g
0.4 g
0.3 g
0.3 g
0.3 g
0.2 g
0.2 g
0.3 g
8.0 g
52
0
54
55
0
0
0
EtOAc
EtOAc/NaHCO3
EtOAc
EtOH
EtOAc
EtOAc
THF
EtOH
EtOH
85.9
—
<85
<85
—
—
—
Raney Ni
Wilkinson’s
Pd/C 5%
55
55
98.9
98.9
a All reactions were carried out at 50 psi. b Purity of compound 2 assessed by GCMS and NMR.
was obtained in good purity by NMR with no extra alkene
signals (h).
In the synthesis of the 7-keto derivative 13, the yield reported
by Wilson et al. for cholesterol derivatives was 65% using PCC
as the oxidant in benzene; however a slightly modified procedure
for b-sitosterol gave a yield of 79%.24
Initial synthesis of 7-b-hydroxysitosterol 14 was undertaken
with sodium borohydride and lithium aluminium hydride
with neither proving a very selective reduction (10–20%, 7-a-
hydroxysitosterol). The use of sodium borohydride and cerium
chloride heptahydrate however gave the 7-b-hydroxysitosterol
in excellent yield and selectivity (97 : 3). It was noted that the
two isomers are separable by flash chromatography on silica gel
using 40% ethyl acetate in hexane.
Whereas 1H NMR is of limited use in the synthesis of
phytosterols due to the number of alkane signals, 13C NMR
can be used to differentiate the stereochemistry at C-24.20–22 The
chemical shift at C-29 differs significantly between the R and S
epimers in b-sitosterol [(24-R)-b-sitosterol, d 12.0 and (24-S)-b-
sitosterol (clionasterol), d 12.3], which conclusively assigns our
structure as the correct epimer (24-R)-b-sitosterol.
GCMS analysis of the TMS-ethers of the b-sitosterol products
led to confirmation of the NMR data and assignment of purity
(Table 2).
The by-product formed by conditions (a) appears to be
an isomer of stigmasterol, which is slow to hydrogenate even
on using forcing conditions (high catalyst loading and long
reaction times). Using ethanol as solvent circumvents this
isomerisation, even though the substrate solubility is poor and
requires heating to dissolve. The remaining impurity appears to
be campesterol, which is residual from the stigmasterol starting
material.
The a-epoxide 16 was generated through mCPBA oxidation
of b-sitosterol yielding a mixture of a- and b-epoxide in a 6 :
1 ratio which could not be separated by chromatography on a
small scale.10
Triol 17 is synthesised in a one-pot procedure featuring a three
step synthesis generating performic acid in situ.
Conclusion
Hydrogenation method (h) was scaled up to 8 g with excellent
reproducibility of purity and yield (i). This material was used in
the synthesis of phytosterol oxides as outlined in Scheme 3.
As mentioned earlier, the hydrogenation step was key to the
synthesis of pure b-sitosterol. By employing ethanol as the
solvent we have eliminated the unidentified impurity that was
produced using the standard conditions.
Synthesis of b-sitosterol oxides
This synthesis of b-sitosterol 2 (98.9% purity) has lead to the
synthesis of standards for toxicity testing in b-sitosterol oxides.
The synthesis of b-sitosterol oxides proceeds in a facile manner
as per the literature precedent for cholesterol and provides for the
first time standard samples of b-sitosterol oxides. The toxicity
of these compounds has been evaluated, with the 7-keto-b-
sitosterol and 7-b-hydroxy-b-sitosterol being the most toxic to
U937, Caco2 and HepG2 cell lines in a similar fashion to their
corresponding cholesterol counterparts. This toxicology is the
subject of another publication.25
The initial targets in the synthesis of b-sitosterol oxides were
chosen to form a comparative study with the most toxic COP’s
and our strategy for the synthesis of phytosterol oxides was based
on a variety of methods used previously to synthesise cholesterol
oxidation products. Starting from b-sitosterol, the acetate 12 was
produced using standard acetylation conditions (Scheme 3).23
The synthesis of the b-epoxide 15 was carried out using the
procedure described by Wilson et al., using copper sulfate,
potassium permanganate and t-butanol in DCM, followed by
cleavage of the acetate.24 The yield of this reaction seems to be
variable and modest when compared with Wilson and coworkers
reports of yields in the region of 70%. The b-epoxide was
obtained in 93% purity; the other 7% being the a-epoxide. The
Experimental
Materials and methods
1
isomers are easily distinguished by H NMR (H-6 comes at d
Reagents were purchased from the Aldrich chemical com-
2.90 for the a-epoxide and d 3.05 for the b-isomer).
pany (Poole, Dorset, UK), including b-sitosterol (>40%) and
Table 2 GCMS analysis of hydrogenation reaction products†
Conditions
Retention/min
Peak area
M+
Product (as TMS-ether)
Commercial sample ∼40% purity
9.8
11.1
11.7
13.2
11.1
11.6
13.2
11.1
13.2
0.4%
31.6%
13.0%
55.0%
1.1%
13.0%
85.9%
1.1%
471
473
485
487
473
485
487
473
487
Brassicasterol/crinosterol or isomer
Campesterol or isomer
Stigmasterol or isomer
b-Sitosterol
Campesterol or isomer
Stigmasterol or isomer
b-Sitosterol
a
h
Campesterol or isomer
b-Sitosterol
98.9%
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 0 5 9 – 3 0 6 5
3 0 6 1

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Scheme 3 Reagents and conditions: (i) Ac2O, pyridine (88%); (ii) CuSO4/KMnO4, t-BuOH, H2O (54%), followed by K2CO3, MeOH (71%); (iii)
CrO3, dimethylpyrazole, DCM, −20 ◦C to 5 ◦C (79%), followed by K2CO3, MeOH (91%); (iv) CeCl3·7H2O, NaBH4, (87%); (v) mCPBA, DCM, 0 ◦C
(70%); (vi) HCOOH, 80 ◦C, 10 min, followed by H2O2, followed by MeOH, NaOH (49% for 3 steps).
stigmasterol (95%). mCPBA was purified by washing with
phosphate buffer, followed by filtration and drying. Melting
points are uncorrected. Impurities could be identified on spectra
run on a 500 MHz NMR spectrometer and are detailed.
All spectra were recorded at 20 ◦C in deuterated chloroform
(CDCl3) unless otherwise stated, using tetramethylsilane (TMS)
as internal standard. Chemical shifts are expressed in parts per
million (ppm) and coupling constants in Hertz (Hz). Infrared
spectra were measured as potassium bromide (KBr) discs for
solids or thin films on sodium chloride plates for oils. Thin
layer chromatography (TLC) was carried out on precoated
silica gel plates (Merck 60 PF254). Column chromatography
was performed using Merck silica gel 60 and the fractions are
reported in the order in which they eluted unless otherwise
stated. Visualisation of compounds on TLC plates was achieved
by UV (254 nm) light detection and vanillin or potassium
permanganate staining.
(0.6 mL) and chlorotrimethylsilane (0.3 mL). The reaction
mixture was stirred for 12 h at rt. Water was then added
(20 mL) and the sample was extracted into hexane (30 mL).
The organic layer was washed with water (20 mL), saturated aq.
sodium chloride (2 × 20 mL) and dried over magnesium sulfate.
Solvent was then evaporated and the sample stored in an inert
atmosphere until ready for GCMS analysis.
Stigmasterol tosylate (8)
A solution of stigmasterol 1 (25.010 g, 57 mmol, 95% pure), 4-
DMAP (0.7 g, 10 mol%) and tosyl chloride (23.0 g, 120 mmol)
in pyridine (250 mL) was stirred at rt for 6 h. The solution
was poured into 10% aq. sodium bicarbonate (1000 mL) and
the precipitate was filtered, washed with water, dried and
recrystallised from acetone to give the corresponding tosylate 8
as white needles (29.003 g, 90%): mp 142–144 ◦C (from acetone)
(found: C, 76.27; H, 9.60. Calc. for C36H54O3S: C, 76.26; H,
9.67%). mmax/cm−1 2956, 2867, 1716, 1598, 1464, 1354, 1192,
1172, 1098, 965, 943, 892, 871, 817, 666, 555; dH 0.55–2.36 (43H,
m), 2.44 (3H, s, Ts–CH3), 4.12–4.39 (1H, m, 3a-H), 5.01 (1H, dd,
J 8.4, 15.2, 22-H or 23-H), 5.16 (1H, dd, J 8.5, 15.2, 22-H or 23-
H), 5.30 (1H,d, J 5.2, 6-H), 7.39 (2H, d, J 8.3, ArH), 7.72 (2H,
d, J 8.3, ArH); dC (75.5 MHz) 12.41 (CH3), 12.65 (CH3), 19.37
(CH3), 19.54 (CH3), 21.36 (CH2), 21.48 (CH3), 21.60 (CH3),
22.03 (CH3), 24.71 (CH2), 25.80 (CH2), 29.02 (CH2), 29.27
(CH2), 32.13 (CH), 32.23 (CH), 32.26 (CH2), 36.75 (quaternary
C), 37.27 (CH2), 39.25 (CH2), 39.94 (CH2), 40.87 (CH), 42.57
(quaternary C), 50.31 (CH), 51.62 (CH), 56.29 (CH), 57.13
(CH), 82.77 (CH), 123.90 (CH), 128.03 (CH, 2 × ArCH), 129.70
(CH), 130.13 (CH, 2 × ArCH), 135.08 (quaternary C), 138.64
(CH), 139.25 (quaternary C), 144.79 (quaternary C).
GCMS analysis
GCMS analysis was effected on a Varian CP 3800 gas chromato-
graph coupled to a Varian Saturn 2000 mass spectrometer. The
column used was a Chrompack WCOT fused silica CP-Sil 8 CB
low bleed (30 m × 0.25 mm id, film thickness 0.25 lm).
The protocol used in GCMS runs was: column oven 275 ◦C for
30 min, injector 350 ◦C (split mode), detector 300 ◦C, flow 30psi
He. Mass spectroscopy setup was in EI, scan range 40 to 650 m/z,
(transfer line 260 ◦C, trap 200 ◦C, manifold 100 ◦C). A Combi
Pal (CTC Analytics AG, CH-4222 Zwingen, Switzerland) auto-
sampler was used for injections and chromatographic data
processing was undertaken with Saturn GC/MS Workstation
Version 5.51 software.
Representative procedure for preparation of TMS-ethers for
GCMS samples
i-Stigmasterol methyl ether (9)
The tosylate 8 (14.303 g, 25.2 mmol) and pyridine (6.1 mL, 3 eq.)
were dissolved in anhydrous methanol and refluxed for 6 h.
The solution was evaporated and extracted into ethyl acetate
b-Sitosterol (155 mg, 0.37 mmol) and pyridine (0.9 mL)
were added under nitrogen, followed by hexamethyldisilizane
3 0 6 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 0 5 9 – 3 0 6 5

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(400 mL) and washed with water (2 × 300 mL). The organic layer
was washed with saturated aq. sodium chloride (2 × 200 mL)
and dried over magnesium sulfate. The residue was then purified
by chromatography on silica gel using 5% ethyl acetate in hexane
to give the i-stigmasterol methyl ether 9 (8.211 g, 74%) as an oily
solid as a mixture (5 : 1) with stigmasterol methyl ether 10. An
analytical sample of 9 and 10 was provided by repeated chro-
matography, however this mixture was used unpurified in the
next step. i-Stigmasterol methyl ether 9: clear oil; dH 0.39–0.49
(1H, m), 0.61–2.09 (43H, m), 2.77 (1H, t, J 3.0, CHOMe), 3.32
(3H, s, OCH3), 5.01 (1H, dd, J 8.5, J 15.2), 5.15 (1H, dd, J 8.5, J
15.2); dC (75.5 MHz) 12.26 (CH3), 12.44 (CH3), 13.08 (CH2),
19.07 (CH3), 19.29 (CH3), 21.10 (CH3), 21.22 (CH3), 21.48
(CH), 22.70 (CH2), 24.27 (CH2), 24.97 (CH2), 25.42 (CH2), 29.02
(CH2), 30.49 (CH), 31.89 (CH), 33.36 (CH2), 35.09 (CH2), 35.27
(quaternary C), 40.20 (CH2), 40.55 (CH), 42.68 (quaternary C),
43.40 (quaternary C), 48.08 (CH), 51.26 (CH), 56.13 (CH), 56.56
(CH3), 56.65 (CH), 82.43 (CH), 129.22 (CH), 138.33 (CH).
Stigmasterol methyl ether 10 (white needles): mp 116–117 ◦C
(from EtOAc–hexane); dH 0.70–2.41 (43H, m), 3.04 (1H, m, 3a-
H), 3.35 (3H, s, OCH3), 5.01 (1H, dd, J 8.3, 15.3, 22-H or 23-H),
5.15 (1H, dd, J 8.5, 15.3, 22-H or 23-H), 5.35 (1H,d, J 5.1, 6-H);
dC (75.5 MHz) 12.44 (CH3), 12.66 (CH3), 19.38 (CH3), 19.77
(CH3), 21.45 (CH2), 21.51 (CH3), 21.63 (CH3), 24.76 (CH2),
25.81 (CH2), 28.41 (CH2), 29.35 (CH2), 32.28 (CH), 32.28 (CH),
32.32 (CH2), 37.29 (quaternary C), 37.58 (CH2), 39.08 (CH2),
40.08 (CH2), 40.93 (CH), 42.60 (quaternary C), 50.60 (CH),
51.64 (CH), 56.00 (CH3), 56.33 (CH), 57.27 (CH), 80.74 (CH),
121.99 (CH), 129.64 (CH), 138.74 (CH), 141.25 (quaternary C).
b-Sitosterol acetate (12)
Acetic anhydride and pyridine were combined and b-sitosterol 2
(820 mg, 2.0 mmol) was added slowly. The reaction mixture was
stirred at rt for 24 h. The reaction mixture was then poured into
water (200 mL) and extracted with ethyl acetate (4 × 50 ml).
The organic layer was washed with water (3 × 50 mL), saturated
aq. sodium chloride (2 × 50 mL), dried over magnesium sulfate
and solvent concentrated under a reduced pressure to give an
off-white solid 12, which was used without further purification
(801 mg, 88%): mp 118–119 ◦C (white needles from EtOAc–
hexane) (found: C, 81.50; H, 11.32. Calc. for C31H52O2: C, 81.52;
H, 11.48%). mmax/cm−1 2938, 1731, 1467, 1368, 1251, 1039; dH
0.61–2.41 (50H, m), 4.55–4.66 (1H, m, 3a-H), 5.38 (1H, bd,
J 4.4, H-6); dC (125.8 MHz) 11.86 (CH3), 11.99 (CH3), 18.80
(CH3), 19.06 (CH3), 19.31 (CH3), 19.83 (CH3), 21.04 (CH2),
21.42 (CH3), 23.08 (CH2), 24.30 (CH2), 26.11 (CH2), 27.79
(CH2), 28.25 (CH2), 29.17 (CH), 31.88 (CH), 31.91 (CH), 33.96
(CH2), 36.16 (CH2), 36.60 (quaternary C), 37.01 (CH2), 38.14
(CH2), 39.74 (CH2), 42.32 (quaternary C), 45.86 (CH), 50.05
(CH), 56.06 (CH), 56.70 (CH), 73.98 (CH), 122.33 (CH), 139.66
(quaternary C), 170.49 (quaternary C). The following peaks were
distinguishable for the minor (approx. 1%) impurity; dC 12.25,
12.32, 18.90, 18.99, 19.22, 19.45, 19.60, 21.18, 23.00, 23.03,
24.21, 26.39, 26.57, 28.02, 28.96, 28.98, 31.74, 33.41, 35.76,
36.29, 36.68, 36.91, 39.84, 42.37, 46.00, 46.08, 49.91, 50.18,
55.74, 55.91, 56.19.
7-Keto-b-sitosterol (13)
Chromium trioxide (985 mg, 9.8 mmol) was suspended in
dry dichloromethane (40 mL) and stirred for 30 min at
−25 ◦C. Dimethylpyrazole (947 mg, 9.8 mmol) was added in one
portion and the reaction mixture stirred for 30 min at −20 ◦C.
b-Sitosterol acetate 12 (300 mg, 0.65 mmol) was added and
the mixture stirred at −20 ◦C allowing to warm to 5 ◦C over
2.5 h. Ethyl acetate (200 mL) was then added and the brown
suspension filtered through celite. The filtrate was concentrated
under a reduced pressure to give a brown residue. This residue
was purified by chromatography on silica gel using hexane–
ethyl acetate (100 : 0 to 75 : 25), y◦ielding 7-keto-b-sitosterol
acetate (241 mg, 79%): mp 151–153 C (needles from EtOAc–
hexane) (found: C, 78.52; H, 10.47. Calc. for C31H50O3: C, 79.1;
H, 10.71%). mmax/cm−1 2959, 2873, 1731, 1673, 1466, 1375, 1264,
1044; dH 0.59–2.60 (48H, m), 4.61–4.79 (1H, m, 3a-H), 5.79 (1H,
bs, H-6); dC (75.5 MHz) 11.97 (2 × CH3), 17.26 (CH3), 18.91
(CH3), 19.02 (CH3), 19.80 (CH3), 21.16 (CH2), 21.28 (CH3),
23.02 (CH2), 26.04 (CH2), 26.31 (CH2), 27.35 (CH2), 28.56
(CH2), 29.08 (CH), 33.91 (CH2), 35.99 (CH2), 36.09 (CH), 37.74
(CH2), 38.31 (quaternary C), 38.63 (CH2), 43.10 (quaternary
C), 45.41 (CH), 45.79 (CH), 49.78 (CH), 49.93 (CH), 54.65
(CH), 72.21 (CH), 126.71 (CH), 163.86 (quaternary C), 170.31
(quaternary C), 202.02 (quaternary C).
22,23-Dihydro-i-stigmasterol methyl ether (11) and b-sitosterol
(2)
i-Stigmasterol methyl ether 9 was dissolved in the appropriate
solvent and the catalyst (5% by wt.) was added (see Table 1).
The resulting suspension was shaken at rt for 16 h. Reaction
completion was then confirmed by 1H NMR. The reaction
mixture was then filtered through celite and concentrated to give
the hydrogenated methyl ether 11. A solution of 11 (6.820 g,
15.84 mmol), TsOH (0.3 g, 1.58 mmol) in aqueous dioxane
◦
(132 mL of dioxane and 15 mL of water) was heated at 80 C
for 3 h before the evaporation of the dioxane. The residue was
taken up in CHCl3 and the organic layer was washed with water
(2 × 50 mL), saturated aq. sodium chloride (2 × 50 mL), dried
using anhydrous magnesium sulfate, then concentrated to give
the crude product, which was then purified by chromatography
on silica gel using 20% ethyl acetate in hexane to give b-
sitosterol 2 as a white solid in the various purities and yields
outlined in Table 1. b-Sitosterol (98.9%): mp 130–134 ◦C (needles
from EtOAc–hexane) (found: C, 82.47; H, 12.08. Calc. for
C29H50O·(1 H2O): C, 82.20; H, 12.13%). mmax/cm−1 3434, 2937,
2
1466, 1382, 1054; dH 0.59–2.34 (48H, m), 3.41–3.59 (1H, m, 3a-
H), 5.35 (1H, bd, J 5.1, H-6); dC (125.8 MHz) 11.99 (CH3,
C-29), 12.18 (CH3), 18.80 (CH3), 19.06 (CH3), 19.40 (CH3),
19.83 (CH3), 21.10 (CH2), 23.31 (CH2), 24.13 (CH2), 26.11
(CH2), 28.26 (CH2), 29.18 (CH), 31.66 (CH2), 31.92 (CH2 and
overlapping CH), 33.96 (CH2), 36.16 (CH), 36.52 (quaternary
C), 37.28 (CH2), 39.79 (CH2), 42.30 (CH2), 42.33 (quaternary C),
45.85 (CH), 50.15 (CH), 56.08 (CH), 56.78 (CH), 71.80 (CH),
121.70 (CH), 140.77 (quaternary C). The following peaks were
distinguishable for the minor (approx. 1%) impurity; dC 12.24,
12.33, 18.84, 18.99, 19.45, 19.61, 21.18, 23.00, 23.03, 24.21,
24.40, 26.57, 28.04, 29.03, 29.31, 33.40, 33.93, 35.77, 36.28,
39.90, 42.46, 50.18, 56.22.
A
suspension of 7-keto-b-sitosterol acetate (239 mg,
0.51 mmol) in methanol (25 mL) was stirred at rt for 5 min.
Potassium carbonate (77 mg, 0.56 mmol) dissolved in water
(5 mL) was added to the suspension and the mixture stirred
at rt over 24 h. The reaction mixture was then partitioned
between water (100 mL) and ethyl acetate (50 mL). Washed
with water (2 × 100 mL) and saturated aq. sodium chloride (2 ×
100 mL), dried over magnesium sulfate and the solution was then
concentrated under a reduced pressure to yield the crude product
as a white solid (240 mg), which was purified by chromatography
on silica gel eluting with ethyl acetate–hexane (0 : 100 to 100 : 0)
to yield the pure product 13 as a white solid (199 mg, 91%): mp
◦
119–121 C (from EtOAc–hexane) (found: C, 79.50; H, 11.20.
GCMS analysis of TMS-ether. Retention time 11.1 min:
1.1% TMS-campesterol or isomer; m/z (EIMS) 473 (21%), 383
(100), 344 (62).
Calc. for C29H48O2·(1 H2O): C, 79.58; H, 11.28%). mmax/cm−1
2
3535, 3338, 2937, 2871, 1673, 1658, 1464, 1385, 1066; dH 0.59–
2.60 (46H, m), 3.55–3.77 (1H, m, 3a-H), 5.73 (1H, bs, H-6); dC
(125.8 MHz) 12.03 (2 × CH3), 17.37 (CH3), 18.99 (CH3), 19.11
(CH3), 19.86 (CH3), 21.28 (CH2), 23.11 (CH2), 26.15 (CH2),
Retention time 13.2 min: 98.9% TMS-sitosterol; m/z (EIMS)
487 (M+, 48%), 397 (100), 358 (52), 256 (23), 130 (40).
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 0 5 9 – 3 0 6 5
3 0 6 3

View Article Online
26.39 (CH2), 28.61 (CH2), 29.19 (CH), 31.21 (CH2), 33.99 (CH2),
36.13 (CH), 36.42 (CH2), 38.36 (CH2), 38.75 (quaternary C),
41.89 (CH2), 43.16 (quaternary C), 45.48 (CH), 45.87 (CH),
49.99 (CH), 50.02 (CH), 54.76 (CH), 70.50 (CH), 126.09 (CH),
165.46 (quaternary C), 202.49 (quaternary C).
The following peaks were distinguishable for the minor
(approx. 1%) impurity; dC 12.29, 12.37, 14.25, 15.59, 18.87,
19.04, 19.27, 19.49, 19.64, 21.28, 21.34, 23.03, 23.06, 26.30,
26.13, 28.39, 28.98, 29.00, 30.92, 32.91, 33.72, 33.73, 35.12,
35.51, 36.24, 39.74, 43.21, 46.05, 46.09, 52.01, 54.35, 54.72,
60.49, 63.11, 68.24, 68.70.
(CH2), 22.02 (CH2), 23.16 (CH2), 24.98 (CH2), 26.18 (CH2),
27.14 (CH2), 28.09 (CH), 28.70 (CH), 31.43 (CH2), 32.84 (CH2),
33.99 (quaternary C), 35.06 (CH), 35.64 (CH2), 36.97 (CH2),
38.73 (CH2), 41.24 (quaternary C), 44.77 (CH), 49.94 (CH),
55.04 (CH), 55.14 (CH), 61.51 (quaternary C), 62.59 (CH), 70.33
(CH), 169.56 (quaternary C).
A suspension of b-sitosterol-5,6-b-epoxide acetate (62 mg,
0.13 mmol) in methanol (14 mL) was stirred for 5 min at rt.
Sodium carbonate (30 mg, 0.28 mmol) was added and the
mixture stirred at rt for 16 h. The reaction mixture was then
concentrated under a reduced pressure to give the crude epoxide
as a white solid. The product was purified by chromatography on
silica gel using hexane–ethyl acetate (100 : 0 to 80 : 20) to give the
epoxide 15 (40 mg, 71%) in a 93 : 7 mixture of the b-epoxide : a-
epoxide as a white solid: mmax/cm−1 3424, 2937, 1465, 1382, 1063;
dH 0.55–2.10 (48H, m), 3.05 (1H, d, J 2.2, H-6), 3.60–3.78 (1H,
m, 3a-H); dC (75.5 MHz) 11.76 (CH3), 11.97 (CH3), 17.05 (CH3),
18.72 (CH3), 19.01 (CH3), 19.82 (CH3), 21.99 (CH2), 23.04
(CH2), 24.20 (CH2), 26.02 (CH2), 28.17 (CH2), 29.11 (CH), 29.77
(CH), 31.04 (CH2), 32.60 (quaternary C), 33.87 (CH2), 34.84
(quaternary C), 36.08 (CH), 37.23 (CH2), 39.82 (CH2), 42.22
(CH2), 42.29 (CH2), 45.78 (CH), 51.32 (CH), 56.09 (CH), 56.22
(CH), 62.94 (quaternary C), 63.74 (CH), 69.45 (CH). Charac-
teristic signals for the a-epoxide were also seen (see below).
GCMS analysis of TMS-ether. Retention time 24.2 min:
TMS-7-keto-b-sitosterol; m/z (EIMS) 501 (M+, 100%), 414 (34),
396 (62).
7-b-Hydroxy-b-sitosterol (14)
A suspension of 7-keto-b-sitosterol 13 (119 mg, 0.28 mmol) and
cerium chloride heptahydrate (156 mg, 0.42 mmol) in methanol
(5 mL) was stirred at rt for 10 min. Sodium borohydride (12 mg,
0.3 mmol) was added to the suspension and the mixture stirred
at rt over 3 h. The reaction was worked up by partitioning
between water (30 mL) and ethyl acetate (30 mL). The organic
layer was washed with water (2 × 20 mL) and saturated aq.
sodium chloride (2 × 20 mL), dried over magnesium sulfate and
concentrated under a reduced pressure to yield the crude product
(104 mg, 87%, b : a-ratio 97 : 3 from NMR) as white solid.
The crude product was purified by chromatography on silica
gel eluting with hexane–ethyl acetate (60 : 40), the first fraction
yielding the b-alcohol 14 as a single stereoisomer (50 mg, 41%)
and the second fraction as a mixture of isomers (28 mg, 23%): mp
GCMS analysis of TMS-ether. Retention time 17.4 min:
93% TMS-b-sitosterol-5,6-b-epoxide; m/z (EIMS) 503 (M+,
55%), 488 (48), 485 (52), 474 (73), 413 (100), 385 (79).
Retention time 17.9 min: 7% TMS-b-sitosterol-5,6-a-epoxide;
m/z (EIMS) 503 (M+, 51%), 488 (29), 485 (26), 413 (100), 385
(91).
◦
137–139 C (from EtOAc–hexane) (found: C, 79.30; H, 11.43.
b-Sitosterol-5,6-a-epoxide (16)
Calc. for C29H50O2·(1 H2O): C, 79.21; H, 11.69%). mmax/cm−1
2
3400, 2959, 2871, 1465, 1384, 1056; dH 0.70–2.32 (47H, m), 3.51–
3.58 (1H, m, 3a-H), 3.85 (1H, bd, J = 2.6, H-7), 5.29 (1H, bs, H-
6); dC (75.5 MHz) 12.21 (CH3), 12.37 (CH3), 19.22 (CH3), 19.41
(CH3), 19.55 (CH3), 20.22 (CH3), 21.46 (CH2), 23.44 (CH2),
26.46 (CH2), 26.78 (CH2), 28.95 (CH2), 29.51 (CH), 31.94 (CH2),
34.35 (CH2), 36.49 (CH), 36.82 (quaternary C), 37.32 (CH2),
39.93 (CH2), 41.28 (CH), 42.10 (CH2), 43.31 (quaternary C),
46.21 (CH), 48.64 (CH), 55.74 (CH), 56.33 (CH), 71.81 (CH),
73.75 (CH), 125.82 (CH), 143.87 (quaternary C). The following
peaks from the 1H NMR of the mixture could be attributed to
the 7-a-hydroxy-b-sitosterol: dH 3.63–3.69 (1H, m, 3a-H), 5.69
(1H, bs, H-6).
A solution of mCPBA (100%, 125 mg, 0.7 mmol) in dichloro-
methane (10 mL) was added dropwise to a stirred ice-cold
solution of b-sitosterol 2 (250 mg, 0.6 mmol) in dichloromethane
(30 mL). The resulting mixture was stirred for 2 h at rt. The
reaction mixture was then washed with 10% aq. sodium
hydrogen sulfite solution (2 × 50 mL), 5% aq. sodium thiosulfate
solution (2 × 50 mL), saturated aq. sodium bicarbonate (2 ×
100 mL) and saturated aq. sodium chloride (2 × 100 mL).
The dichloromethane extracts were then dried over magne-
sium sulfate and concentrated under a reduced pressure to
produce a white solid (180 mg, 70%). NMR analysis proved
this to be a mixture of the a- and b-epoxides in a ratio of 6 : 1,
which could not be separated using chromatography. a-
Epoxide 16: mmax/cm−1 3431, 2959, 2869, 1467, 1377, 1064;
dH 0.61–2.07 (48H, m), 2.90 (s, 1H, H-6), 3.85–3.95 (1H, m, 3a-
H); dC (75.5 MHz) 10.84 (CH3), 10.95 (CH3), 14.90 (CH3), 17.68
(CH3), 18.00 (CH3), 18.81 (CH3), 19.62 (CH2), 22.02 (CH2),
23.04 (CH2), 25.04 (CH2), 27.08 (CH2), 27.79 (CH2), 28.09 (CH),
28.86 (CH), 30.02 (CH2), 31.38 (CH2), 32.86 (C quaternary),
33.83 (CH2), 35.11 (CH), 38.37 (CH2), 38.83 (CH2), 41.31 (C
quaternary), 41.52 (CH), 44.78 (CH), 54.75 (CH), 55.83 (CH),
58.32 (CH), 64.75 (C quaternary), 67.66 (CH). Characteristic
b-epoxide peaks also present.
GCMS analysis of TMS-ether. Retention time 13.9 min: 3%
TMS-7-a-hydroxy-b-sitosterol; m/z (EIMS) 574 (M+ − 1, 1%),
485 (M+ − 90, 100%).
Retention time 15.4 min: 97% TMS-7-b-hydroxy-b-sitosterol;
m/z (EIMS) 485 (M+ − 90, 100%).
b-Sitosterol-5,6-b-epoxide (15)
Copper sulfate pentahydrate (1.008 g) and potassium perman-
ganate (2.280 g) were ground together into a fine powder with
a mortar and pestle to which water (0.5 mL) was added. The
resulting paste was transferred to a flask containing the acetate
12 (221 mg, 0.48 mmol) in dichloromethane (30 mL). Tert-
butanol (0.3 mL) was added and the reaction mixture was
refluxed for 15 min before cooling to rt. The reaction mixture
was then stirred for a further 16 h at rt. The reaction mixture
was then filtered through silica gel plug column eluting with
dichloromethane. The product rich layer was then dried over
magnesium sulfate and concentrated under a reduced pressure
to yield the crude protected epoxide (123 mg, 54%) as a white
solid. This crude product was used directly in the next step:
mmax/cm−1 2959, 2868, 1732, 1468, 1376, 1265, 1242, 1044; dH
0.57–2.26 (50H, m), 3.00 (1H, d, J 2.1, H-6), 4.64–4.75 (1H, m,
3a-H); dC (75.5 MHz) 10.73 (CH3), 10.95 (CH3), 16.02 (CH3),
17.75 (CH3), 17.99 (CH3), 18.80 (CH3), 20.31 (CH3), 20.90
GCMS analysis of TMS-ether. Retention time 17.4 min:
14% TMS-b-sitosterol-5,6-b-epoxide; m/z (EIMS) 503 (M+,
59%), 488 (44), 485 (49), 474 (67), 413 (100), 385 (77).
Retention time 17.9 min: 86% TMS-b-sitosterol-5,6-a-
epoxide; m/z (EIMS) 503 (M+, 53%), 488 (27), 485 (25), 413
(100), 394 (93).
b-Sitosterol-3,5,6-triol (17)
A solution of formic acid (95%, 3 mL) was added to b-sitosterol
2 (149 mg, 0.36 mmol) and this suspension was stirred for
10 min at 80 ◦C (turns a dark colour and an oily layer forms
on top of the formic acid). The reaction mixture was then
cooled to 25 ◦C, forming a solid from the oil, and hydrogen
3 0 6 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 0 5 9 – 3 0 6 5

View Article Online
peroxide was then added (1 mL). The suspension was stirred
at 25 ◦C for 16 h. Boiling water (50 mL) was then added to
the suspension and extracted using dichloromethane (100 mL).
The dichloromethane layer was washed with saturated aq.
sodium chloride (2 × 50 mL), dried over magnesium sulfate
and the solvent evaporated yielding a white solid used without
purification.
2 A. Berger, P. J. H. Jones and S. S. Abumweis, Lipids Health Dis.,
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This solid was dissolved in methanol (20 mL) and sodium
hydroxide (25 mg) added. The suspension was stirred for 3 h
at rt. The reaction was partitioned between water (50 mL) and
ethyl acetate (50 mL). The organic layer was washed with water
(2 × 50 mL) and saturated aq. sodium chloride (2 × 50 mL),
dried over magnesium sulfate and concentrated under a reduced
pressure to yield the crude product (130 mg) as white solid,
which was purified by column chromatography eluting with
ethyl acetate to yield the triol 17 as a white solid (79 mg, 49%):
mmax/cm−1 3436, 2956, 2870, 1466, 1386, 1293, 1044; dH (d6-
DMSO) 0.47–1.95 (46H, m), 2.56 (s, 1H), 3.35 (broad s, 1H),
3.68 (s, 1H, H-6), 3.83–3.89 (1H, m, 3a-H), 4.22 (1H, d, J =
5.5 Hz), 4.44 (1H, d, J = 4.0 Hz); dC (75.5 MHz) 12.11 (CH3),
12.27 (CH3), 16.62 (CH3), 18.93 (CH3), 19.27 (CH3), 20.06
(CH3), 21.09 (CH2), 22.94 (CH2), 24.25 (CH2), 25.87 (CH2),
28.26 (CH2), 29.03 (CH), 30.35 (CH), 31.44 (CH2), 32.36 (CH2),
33.70 (CH2), 34.83 (CH2), 35.98 (CH), 38.11 (C quaternary),
40.13 (CH2), 41.24 (CH2), 42.61 (C quaternary), 44.89 (CH),
45.47 (CH), 56.02 (CH), 56.15 (CH), 66.10 (CH), 74.48 (CH),
74.64 (C quaternary).
10 L. Maguire, M. Konoplyannikov, A. Ford, A. R. Maguire and
N. M. O’Brien, Br. J. Nutr., 2003, 90, 1. This reference describes
the evaluation of epoxide (16) prepared by the mCPBA oxidation of
commercial b-sitosterol (∼60% purity).
11 J. Plat, H. Brzezinka, D. Lutjohann, R. P. Mensink and K. Von
Bergmann, J. Lipid Res., 2001, 42, 2030.
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13 D. Lutjohann, Br. J. Nutr., 2004, 91, 3.
14 A. Grandgirard, L. Martine, P. Joffre and O. Berdeaux, J. Chro-
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15 C. Johannes and R. L. Lorenz, Anal. Biochem., 2004, 325, 107.
16 J. A. Steele and E. Mosettig, J. Org. Chem., 1963, 28, 571.
17 V. A. Khripach, V. N. Zhabinskii, O. V. Konstantinova, N. B.
Khripach, A. P. Antonchick and B. Schneider, Steroids, 2002, 67,
597.
18 S. K. Chung, M. G. Kang and H. I. Kang, Tetrahedron, 1998, 54,
15899.
19 A. Jourdant, E. Gonzalez-Zamora and J. Zhu, J. Org. Chem., 2002,
67, 3163.
20 H. Nagano, M. Matsuda and T. Yajima, J. Chem. Soc., Perkin Trans.
1, 2001, 174.
21 S. Seo, A. Uomori, Y. Yoshimura, K. Takeda, H. Seto, Y. Ebizuka,
H. Noguchi and U. Sankawa, J. Chem. Soc., Perkin Trans. 1, 1988,
2407.
22 Y. Fujimoto, N. Sato, K. Iwai, H. Hamada, J. Yamada and M.
Morisaki, Chem. Commun., 1997, 681.
23 J. N. Carroll, F. D. Pinkerton, X. Su, N. Gerst, W. K. Wilson and
G. J. Schroepfer, Jr., Chem. Phys. Lipids, 1998, 94, 209.
24 W. K. Wilson, S. Li, J. Pang and G. J. Schroepfer, Jr., Chem. Phys.
Lipids, 1999, 99, 33.
GCMS analysis of TMS-ether. Retention time 20.5 min:
TMS-b-sitosterol-3,5,6-triol; m/z (EIMS) 575 (M+ − 90, 18%),
560 (27), 546 (24), 485 (100), 432 (70)
Acknowledgements
The authors would like to thank Enterprise Ireland for funding
this research.
References
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O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 0 5 9 – 3 0 6 5
3 0 6 5