Optimised conditions for the synthesis of 17O and 18O labelled cholesterol

Article abstract of DOI:10.1016/j.chemphyslip.2015.12.003

Conditions are described for the preparation of cholesterol with ¹⁷O and ¹⁸O labels from i-cholesteryl methyl ether using minimal amounts of isotopically enriched water. Optimum yields employed trifluoromethanesulfonic acid as catalyst in 1,4-dioxane at room temperature with 5 equivalents of water. An isotopic enrichment >90% of that of the water used for the reaction could be attained. Tetrafluoroboric acid could also be used as catalyst, at the expense of a lower overall reaction yield. Byproducts from the reaction included dicholesteryl ether, methyl cholesteryl ether, compounds formed by ether hydrolysis, and olefins arising from elimination reactions. Reactions in tetrahydrofuran yielded significant amounts of cholesteryl ethers formed by reaction with alcohols arising from hydrolysis of the solvent.

Full text of DOI:10.1016/j.chemphyslip.2015.12.003

Chemistry and Physics of Lipids 195 (2016) 58–62
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
17
18
Optimised conditions for the synthesis of O and O labelled
cholesterol
a
a
a
b
Celia de la Calle Arregui , Jonathan A. Purdie , Catherine A. Haslam , Robert V. Law ,
a,
John M. Sanderson *
a
Chemistry Department, Durham University, Durham, DH1 3LE, UK
Institute of Chemical Biology, Imperial College, Exhibition Road, London, SW7 2AZ, UK
b
A R T I C L E I N F O
A B S T R A C T
Conditions are described for the preparation of cholesterol with 17O and 18O labels from i-cholesteryl
methyl ether using minimal amounts of isotopically enriched water. Optimum yields employed
trifluoromethanesulfonic acid as catalyst in 1,4-dioxane at room temperature with 5 equivalents of water.
An isotopic enrichment >90% of that of the water used for the reaction could be attained. Tetrafluoroboric
acid could also be used as catalyst, at the expense of a lower overall reaction yield. Byproducts from the
reaction included dicholesteryl ether, methyl cholesteryl ether, compounds formed by ether hydrolysis,
and olefins arising from elimination reactions. Reactions in tetrahydrofuran yielded significant amounts
of cholesteryl ethers formed by reaction with alcohols arising from hydrolysis of the solvent.
ã 2016 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/).
Article history:
Received 28 October 2015
Received in revised form 30 November 2015
Accepted 17 December 2015
Available online 24 December 2015
Keywords:
Cholesterol
Synthesis
Oxygen isotope
1. Introduction
2001). It is therefore desirable to be able to label cholesterol with
isotopic labels that are less intrusive than bulky chromophores and
Cholesterol is one of the most important natural sterols because
of its role in modifying the properties of cell membranes (Róg and
Vattulainen, 2014) and the link between in vivo transport of
cholesterol in plasma lipoprotein fractions and disease states
related to dietary intake of cholesterol (Birner-Gruenberger et al.,
permit study by NMR or mass spectrometry methodologies.
17
18
Isotopes of oxygen ( O, O) are potentially valuable for this
17
purpose. The O isotope, with spin 5/2, is suitable for NMR studies,
particularly as it exhibits a wide chemical shift range of ꢀ2000
ppm (Lemaître et al., 2004; Klemperer, 1978). However, with a
natural abundance of 0.038% and low receptivity, isotopic
2014). Its presence modifies membrane fluidity (Fraenza et al.,
18
2014; Sanderson, 2012) and rigidity (Dimova, 2014), and in
enrichment is essential. The O isotope is not directly observable
13
mixtures of high and low melting lipids it is capable of inducing
phase separation to form cholesterol-rich liquid ordered phases in
the presence of more fluid disordered phases (Róg and Vattulainen,
by NMR, but does induce isotopic shifts in C NMR spectra (Risley
and Van Etten, 1979) that can be used analytically (Risley and Van
Etten, 1989). Both isotopes also have utility as isotopic markers for
mass spectrometry studies (Ye et al., 2009; Zyakun, 2011). A
convenient route to cholesterol analogues has been described in
the literature, involving acid-catalyzed reaction of the commer-
cially available i-cholesteryl methyl ether (1) with water
(Scheme 1).
2014; Simons and Vaz, 2004). The distribution of cholesterol
within plasma membranes has consequently been subject to a
great deal of investigation using a range of spectroscopic and
analytical techniques. The membrane activity of cholesterol is
intrinsically linked to its hydrocarbon structure. Minor perturba-
tions to the structure of cholesterol, such as the addition of a
chromophore to facilitate luminescence studies, frequently modify
the behaviour of the sterol in an unpredictable manner. For
example, the addition of an 7-nitro-2,1,3-benzoxadiazol-4-yl
This reaction has been known for some time and generally gives
good yields of cholesterol (2) when water is present in large excess
(Chu and Li, 2000; Riegel et al., 1946; Steele and Mosettig, 1963;
Smith et al., 1993; Nicotra et al., 1981). It has been used for the
18
(
NBD) group in the aliphatic hydrocarbon region produces a
preparation of O-cholesterol (Wong et al.,1995). However, for the
preparation of isotopically enriched cholesterol, particularly with a
fluorescent analogue that preferentially partitions into the liquid
disordered phase rather than the liquid ordered phase (Loura et al.,
17
O isotope, the use of a large excess of water is undesirable given
the high cost of water enriched with oxygen isotopes
17
(
e.g. O enriched water typically costs >$1000 per ml). Further-
more, in order to maximize isotopic enrichment, it is desirable to
either use an acid catalyst without exchangeable oxygen, or if the
*
Corresponding author. Fax: +44 1913844737.
E-mail address: j.m.sanderson@durham.ac.uk (J.M. Sanderson).
http://dx.doi.org/10.1016/j.chemphyslip.2015.12.003
009-3084/ã 2016 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
0

C. de la Calle Arregui et al. / Chemistry and Physics of Lipids 195 (2016) 58–62
59
H+
H O
2
HO
2
OMe
1
Scheme 1. Acid-catalyzed reaction of i-cholesteryl methyl ether (1) with water to
form cholesterol (2).
use of such a catalyst is unavoidable, minimize the amount of
catalyst required. In this communication, we investigate the ability
of different acids to catalyse the reaction, and establish conditions
that use minimal amounts of water and catalyst.
2. Results and discussion
2.1. Choice of acid catalyst
Initial studies were conducted using 1 and non-enriched water
in 1,4-dioxane using a selection of acid catalysts (Table 1). This
solvent was chosen because of its miscibility with water and its
good solvation properties for cholesterol. In addition, as an aprotic
solvent it was not expected to participate in the reaction. All acid
catalysts were investigated at a ratio of 5 mol% with respect to 1.
Fig. 1. Byproducts from hydrolysis reactions of 1 in 1,4-dioxane.
temperature and the ratios of trifluoromethanesulfonic acid
catalyst and water to ether 1. These outcomes of these experiments
are summarised in Table 2 and Fig. 2. The best yield of cholesterol
was found with 0.05 equivalents of acid and 5 equivalents of water
at 20 C (entry 8). Increasing the number of equivalents of water
beyond 5 did not lead to significant benefits, either in overall yield
The acids investigated covered a range of pK
a
values in organic
solvents, including the superacids trifluoromethanesulfonic acid
ꢁ
(
(
pK
pK
a
= À11.4 in dichloroethane) and tetrafluoroboric acid
= À10.3 in dichloroethane) (Kütt et al., 2011). From the data
a
(
entry 11) or in the ratio of cholesterol to other products (entry 9).
it is apparent that trifluoromethanesulfonic acid produced by some
margin the greatest yield of cholesterol, at the expense of having
exchangeable oxygen. Tetrafluoroboric acid, on the other hand,
gave a poor yield of cholesterol but, being a hydracid, did offer the
potential for preparing cholesterol with an isotopic enrichment
matching that of the water used for its preparation. It should be
noted though, that in the reaction utilizing this acid, a significant
quantity of 1 remained unreacted after 5 h, raising the possibility
that a longer reaction time would increase the yield of cholesterol.
In all cases, unreacted 1 was present, alongside a number of
byproducts, the structures of which are given in Fig. 1.
Although in the latter case the reaction had not gone to completion
within 5 h, the major product after this time was alcohol 5 (full
details are in the ESI). Increasing the number of equivalents of acid
catalyst led to a significant increase in the formation of byproducts,
particularly methyl ether 4, which formed >20% of the products
when 1 equivalent of acid was used. Decreasing the amount of acid
ꢁ
below 0.05 equivalents gave incomplete reactions after 5 h at 20 C,
but in all cases cholesterol was not the major product after this
time and would not have given cholesterol yields greater than
entry 8 had they been allowed to go to completion.
When the mole fraction of cholesterol in the products is
analysed in relation to the ratio of acid to water (Fig. 2) in the
reaction, the conditions corresponding to entries 7 and 8 in Table 2,
with an acid/water ratio of 0.01, are found to be optimal for
increasing the proportion of cholesterol in the mixture. At lower
acid to water ratios, the predominant byproduct is the alcohol 3
resulting from hydrolysis of ether 1. At higher ratios, the major
byproduct becomes methyl ether 4.
Some of these byproducts, including dicholesteryl ether 3 and
cholesteryl methyl ether 4 could be accounted for by reaction of 1
with other nucleophiles formed during the progress of the
reaction. Alcohol 5 was formed by a competing nucleophilic
displacement of methanol from 1. The other products, 6 and 7,
could be accounted for by elimination reactions of either
cholesterol, 3 or 4 (for 6) and 1 or 5 (for 7).
On the basis of the optimal conditions, and assuming complete
2
.2. Optimization of reaction conditions
16
exchange between the
O isotope of the catalyst and the
18
O
isotope of the enriched water, the maximum isotopic
On the basis of this preliminary work, further experiments were
enrichment of cholesterol theoretically attainable (not allowing
conducted to investigate the effects on cholesterol yield of
Table 1
a
The influence of acid catalyst on the formation of cholesterol from 1 in the presence of water.
Acid
CF SO
p-TsOH
CH SO
Yield cholesterol (%)b
Byproductsc
3
3
H
73 (61)
trace
1
18 (16)
7 (6)
3 (9%), 4 (9%), 5 (trace), 6 (trace), other (9%) + 1 (trace)
3,4, 5 (all trace) + 1 (>99%)
3 (trace), 4 (2%), 5 (2%), 7 (1%), other (1%) + 1 (93%)
3 (1%), 4 (3%), 5 (12%), 6 (1%), 7 (trace), other (10%) + 1 (55%)
3 (1%), 4 (2%), 5 (9%), 6 (trace), 7 (1%), other (1%) + 1 (79%)
3
3
H
HBF
HCl
4
a
ꢁ
Conditions in all cases: 5 eq. water, 0.05 eq. acid, 1,4-dioxane, 80 C, 5 h.
b
c
Yields are calculated from analysis of crude NMR spectra. Figures in parentheses indicate isolated yields.
Yields are calculated from analysis of crude NMR spectra. Trace products were detectable by thin layer chromatography but not 1H NMR. The relative yields of ‘other’
products were determined on the basis of 3-H signals or pairs of olefinic signals that could not be attributed to any of 1–7.

6
0
C. de la Calle Arregui et al. / Chemistry and Physics of Lipids 195 (2016) 58–62
+ 5 eq. 18OH
at 80 C for 40 h.
ꢁ
2
. 1 + 0.05 eq. HBF
4 2
These gave isolated yields of 62% and 40% respectively.
Following purification, the isotopic enrichment of the cholesterol
13
was determined by C NMR spectroscopy (Fig. 4).
18
The inclusion of the O induced a readily detectable isotopic
13
shift of the 3-C C resonance of 17 Hz. Isotopic enrichment was
13
16
determined by the relative peak areas for the C signals of the O-
18
and O-isotopomers to be 90.1 Æ1.9 atom % with trifluorometha-
nesulfonic acid as catalyst, and 90.9 Æ1.9 atom % with tetrafluor-
oboric acid as catalyst. These isotopic enrichments are lower than
the theoretical maximum achievable of 92.9 Æ 0.04 atom % when
consideration is given to the exchangeable atoms of the catalysts
and the isotopic enrichment of the water. The differences are
accounted for by residual water in the solvent and acid catalysts.
-
3.20
-2.64
-2.08
-1.52
-0.96
-0.40
log1 ([CF SO H] / [H O])
0
3
3
2
Fig. 2. Mole fraction of cholesterol in the steroid products of reactions in 1,4-
dioxane with CF SO H as catalyst in response to changes in the ratio of acid to water.
17
Using the same procedure, O-cholesterol was also prepared using
3
3
17
2
OH .
for residual water in the catalyst and solvent) would be 97.1% of
that of the original water, which is acceptable.
3
. Conclusions
Tetrahydrofuran (THF) was investigated as an alternative
solvent to 1,4-dioxane and good yields of cholesterol could be
obtained in this solvent. However, in all cases, products arising via
lysis of the solvent were significant byproducts, with compounds
From the data presented above, we conclude that 1,4-dioxane is
the best solvent for the preparation of isotopically enriched
cholesterol. For maximum recovery of cholesterol where a small
17
18
dilution of the O or O isotope is acceptable, trifluorometha-
nesulfonic acid is the optimum choice of acid catalyst. In order to
obtain an acceptable yield of product, it should not be necessary to
use more than 5 equivalents of enriched water relative to
i-cholesteryl methyl ether.
8
–10 (Fig. 3) typically forming 20–50% of the reaction products.
Further experiments were not conducted in THF and we would
not recommend using this solvent as first choice for reactions that
are not conducted in the presence of a large excess of water. For
reactions in 1,4-dioxane, products equivalent to those in Fig. 3 were
present in some cases (as judged by NMR), but were not isolated in
sufficient quantity to characterise and always represented less
than 3% of the reaction products.
4
. Experimental procedures
4
.1. General methods
2.3. Synthesis of labelled cholesterol
Dry solvents were sourced from Fisher Scientific (Lough-
borough, UK; THF) and Sigma–Aldrich (Dorset, UK; 1,4-dioxane;
ꢂ0.003% H O manufacturers specification, measured as 0.007% by
Karl–Fischer titration at the time of use). Cholesterol,
i-cholesteryl methyl ether (95%), 4 M HCl in dioxane, tetrafluor-
oboric acid diethyl ether complex and the rest of the acids were
obtained from Sigma–Aldrich (Dorset, UK). Isotopically enriched
Having determined suitable reaction conditions for the
preparation of cholesterol labelled with oxygen isotopes, the final
2
a
step was to verify that yields and isotopic enrichment were as
1
8
expected. Reactions were therefore performed with 1 and OH
95.7 atom%
2
18
(
O) in two sets of conditions, with either
trifluoromethanesulfonic acid or tetrafluoroboric acid as catalyst.
The latter was included to examine the maximum achievable
isotopic enrichment achievable:
water was obtained from Cortecnet (Voisins-Le-Bretonneux,
18
France). Isotopic enrichments were 95.7 atom % for OH
measurement, lot number 139808A-P) and 41.1% for
2
(our
17
OH
2
H + 5 eq. 18OH
ꢁ
(manufacturers specification, lot number 1040171A-P). Stock
solutions of acids in THF or 1,4-dioxane were prepared immedi-
ately before use. Purification was performed by flash column
chromatography using a silica gel support (230–400 mesh, 40–
1
. 1 + 0.05 eq. CF
3
SO
3
2
at 20 C for 5 h; and
60 mm) from Sigma–Aldrich (UK). NMR data were collected on a
Table 2
Formation of cholesterol from 1 using trifluoromethanesulfonic acid as catalyst in
,4-dioxane.
1
13
Bruker Avance-400 (at 400 MHz for H; 100.6 MHz for C) or
1
13
1
Varian VNMRS (at 700 MHz for H; 176 MHz for C). NMR spectra
were obtained in CDCl and are reported in ppm using residual
CHCl at 7.26 ppm as the internal reference. C NMR spectra were
referenced to solvent as internal reference (77.23 ppm for CDCl ).
ꢁ
d
3
Entry
T ( C) Equiv.
Equiv. waterb Yield cholesterol (%)c
x
chol
13
Ha
3
3 3
CF SO
3
1
2
3
4
5
6
7
8
9
20
20
20
80
20
20
80
20
20
20
20
0.005
0.005
0.01
0.01
0.01
0.05
0.05
0.05
0.05
1
2
5
2
2
5
2.5
5
5
20
5
9
4
0.37
0.35
0.47
0.52
0.50
0.58
0.73
0.76
0.27
0.59
0.57
Atmospheric solids analysis probe (ASAP) mass spectrometry was
performed on a Xevo QToF mass spectrometer (Waters Ltd,
Wilmslow, UK) equipped with an Agilent 7890 GC gas
30
52
21
19
73
76
7
1
0
59
57
11
1
20
a
Equivalents relative to 1.
Yields are calculated from analysis of NMR spectra of the crude reaction
b
products.
c
Cholesterol mole fraction in the steroid products (i.e. excluding 1). Errors are
Æ7%.
Fig. 3. Major products from reactions of 1 in THF.

C. de la Calle Arregui et al. / Chemistry and Physics of Lipids 195 (2016) 58–62
61
Conflicts of interest
All third-party financial support for the work in the submitted
manuscript: nothing to disclose.
All financial relationships with any entities that could be
viewed as relevant to the general area of the submitted
manuscript: nothing to disclose.
All sources of revenue with relevance to the submitted work
who made payments to you, or to your institution on your behalf,
in the 36 months prior to submission: nothing to disclose.
Any other interactions with the sponsor of outside of the
submitted work should also be reported: nothing to disclose.
Any relevant patents or copyrights (planned, pending, or
issued): nothing to disclose.
Any other relationships or affiliations that may be perceived by
readers to have influenced, or give the appearance of potentially
influencing, what you wrote in the submitted work: nothing to
disclose.
Fig. 4. The region of the 13C NMR spectrum (176 MHz, CDCl
) corresponding to the
3
18
3
-carbon of O-cholesterol prepared by Method 1.
chromatography apparatus (Agilent Technologies UK Ltd, Stock-
ꢁ
port, UK) at 450 C. Full details are presented in the supporting
Acknowledgements
information.
We thank the EPSRC (EP/J017566/1) for support and Dr. Darren
R. Gröcke for assistance with water isotopic enrichment analyses.
4.2. Optimised experimental procedure in 1,4-dioxane
A 12.5 mM solution of the acid in dry 1,4-dioxane (1 ml,
12 mmol) was added to a solution of 1 (0.10 g, 0.25 mmol), and
Appendix A. Supplementary data
water (22
m
l, 1.2 mmol) in 1,4-dioxane (4 ml) and the solution
ꢁ
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
chemphyslip.2015.12.003.
stirred for 5 h at 20 C. After concentrating to ꢀ1 ml in vacuo,
CH
water (2 Â10 ml). The organic solution was dried (MgSO
and concentrated in vacuo. Purification was by flash column
chromatography on silica using a gradient of CH Cl /hexane, 1:1 to
CH Cl
The procedure when using HBF
2
Cl
2
(20 ml) was added and the organic solution washed with
4
), filtered
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