Synthesis of cholesterol analogs having varying length alkyl side chains including cholesterol-23, 23, 24, 24, 25, 26, 26, 26, 27, 27, 27-d11 as probes of cholesterol's functions and properties

Article abstract of DOI:10.1016/j.chemphyslip.2013.08.003

Cholesterol-23, 23, 24, 24, 25, 26, 26, 26, 27, 27, 27-d₁₁ and nondeuterated long-chain analogs of cholesterol were prepared by alkylation of cyano-containing sterols with isopentyl-d₁₁ 4-methylbenzenesulfonate (1.0 equiv.) or with isoalkyl bromides, followed by reductive decyanation with excess potassium metal and a crown ether in toluene. The products are potentially useful probes of the role of the side-chain of cholesterol in the sterol's interactions with membrane lipids and proteins.

Full text of DOI:10.1016/j.chemphyslip.2013.08.003

Chemistry and Physics of Lipids 175–176 (2013) 99–104
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Short communication
Synthesis of cholesterol analogs having varying length alkyl side
chains including cholesterol-23, 23, 24, 24, 25, 26, 26, 26, 27, 27,
2
7-d as probes of cholesterol’s functions and properties
11
∗
Dong Jae Baek, Robert Bittman
Department of Chemistry and Biochemistry, Queens College of the City University of New York, Flushing, NY 11367-1597, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 July 2013
Received in revised form 8 August 2013
Accepted 12 August 2013
Available online 3 September 2013
Cholesterol-23, 23, 24, 24, 25, 26, 26, 26, 27, 27, 27-d11 and nondeuterated long-chain analogs
of cholesterol were prepared by alkylation of cyano-containing sterols with isopentyl-d11 4-
methylbenzenesulfonate (1.0 equiv.) or with isoalkyl bromides, followed by reductive decyanation with
excess potassium metal and a crown ether in toluene. The products are potentially useful probes of the
role of the side-chain of cholesterol in the sterol’s interactions with membrane lipids and proteins.
©
2013 Published by Elsevier Ireland Ltd.
Keywords:
Cholesterol synthesis
Cyano-containing sterol intermediates
Decyanation
Deuterium labeling
1
. Introduction
Our synthesis of cholesterol-d11 reported here represents the
first deuterium-labeled cholesterol with more than seven deu-
terium atoms. Visualization of the uptake and trafficking of
individual biomolecules in living cells with stable isotope labels (to
avoid the use of bulky fluorescent probes) is under intensive cur-
rent investigation by Raman spectroscopy, especially with lipids
and probes or metabolites added exogenously (for a recent review,
see Pezacki et al., 2011). Replacing C H bonds with C D bonds in
biomolecules shifts the stretching frequency to a spectral region in
which no other biomolecules have Raman peaks, giving distinctive,
narrow signals in minimally perturbed molecules. The symmetric
The role of the isooctyl side chain of cholesterol in cholesterol’s
ability to influence membrane properties and interactions with
phospholipids and proteins has been examined by using a variety
of biophysical methods (Chia et al., 1993; McMullen et al., 1995;
Vilchèze et al., 1996; Jaikishan and Slotte, 2011; Mattjus et al.,
1
995; Scheidt et al., 2013; for a review, see Bittman, 1997). Alter-
ation of the hydrophobic lengths of lipids and proteins affects both
lipid–lipid and lipid–protein interactions, thereby modifying the
organization of lateral domains in bilayer membranes. In addition
to the use of short side-chain and long side-chain analogs of choles-
terol to examine the hydrophobic mismatch effect in membranes,
deuterium has been introduced in the isooctyl chain of cholesterol
−
1
C
D stretching frequency (∼2097–2120 cm for CD groups and at
2
−
1
∼2194 cm for CD groups at the chain terminus) has been utilized
3
to monitor various lipids containing perdeuterated fatty acyl chains
such as palmitic acid-d31 and dipalmitoylphosphatidylcholine-
d62 (Li et al., 2005; Weeks and Huser, 2010) and the A ring of
cholesterol-d6 (Matthaus et al., 2012). Cholesterol-d11 may be
useful in imaging of side-chain deuterated cholesterol by coher-
ent anti-Stokes Raman scattering (CARS) and stimulated Raman
scattering (SRS) microscopy since it is expected to give higher
signal-to-noise ratios than the commercially available methyl-tail
deuterium-labeled (d6 and d7) cholesterol probes.
2
for use in H NMR and neutron diffraction studies to examine the
location of cholesterol in lipid model systems. For these studies,
“
methyl-tail” deuterated cholesterol analogs bearing 6 deuterium
atoms (at C26 and C27 in cholesterol) or 7 deuterium atoms (at
C25, C26, and C27) have been used frequently (e.g., Mihailescu
et al., 2011; Lütjohann et al., 1993). In this communication, we
report a new synthesis of alkyl side-chain analogs of cholesterol
and of cholesterol-d11, which bears deuterium atoms at C23, C23,
C24, C24, C25, C26, C26, C26, C27, C27, and C27. Our synthesis of
cholesterol bearing various isoalkyl chains is more practical than
the syntheses reported previously.
2. Results and discussion
2.1. Previous syntheses of sterols bearing a deuterated alkyl side
chain
∗ Corresponding author. Tel.: +1 718 997 3279; fax: +1 718 997 3349.
E-mail address: robert.bittman@qc.cuny.edu (R. Bittman).
Cholesterol-26, 26, 26, 27, 27, 27-d6 was synthesized
from pregnenolone via Wittig olefination reaction with
a
0
009-3084/$ – see front matter © 2013 Published by Elsevier Ireland Ltd.
http://dx.doi.org/10.1016/j.chemphyslip.2013.08.003

1
00
D.J. Baek, R. Bittman / Chemistry and Physics of Lipids 175–176 (2013) 99–104
Scheme 1. Known synthetic schemes for the preparation of (A) isoalkyl-chain analogs of cholesterol and (B) ent-cholesterol. Reagents and conditions: (a) iso-C7H15MgBr,
+
Li2CuCl4, THF; (b) H , MeOH, 52% (2 steps); (c) (i) iso-C5H11MgBr (30 equiv.), Li2CuCl4, THF; (ii) conc. HCl, MeOH, 55%; (d) 1-bromo-3-methylbutane (10 equiv.), LDA, THF,
8
2%; (e) (i) K, dicyclohexano-18-crown-6, toluene; (ii) 1 M HCl, THF, 83% (2 steps).
4
-methylpentyl-d -triphenylphosphonium bromide (Holm and
2.3. Coupling reactions via LDA treatment of cyano-bearing sterol
intermediates
6
Crossland, 1996). Cholesterol-25, 26, 26, 26, 27, 27, 27-d7 was
prepared by addition of isopropyl-d7-magnesium bromide to
a steroid bearing a formyl group at C-23 (6␤-methoxy-3␣,5-
cyclo-5␣-cholan-24-al) (Chu and Li, 2000). Deuterioalkyl groups
have also been introduced by nucleophilic displacement reactions
of sterol derivatives with ␣-lithiated deuterioalkyl phenylsul-
fones (Kirk et al., 1983; Ciuffreda et al., 2003). Many of
these synthetic strategies proceeded in low overall yields. The
present work describes the synthesis of such compounds more
expeditiously.
Scheme 2 shows the preparation of iso-C12, iso-C13, and iso-
C14 from THP-protected alcohol 1 in nine steps and overall yields
of ∼39%. Alcohol 1 was prepared from commercially available
bisnorcholenic acid as previously reported (Li et al., 2006). The
reaction steps in Scheme 2 are easily controlled. It is notewor-
thy that alkylation of the ␣-carbanion generated by LDA treatment
of sterol nitrile 2 with the silyl ether of an alkyl bromide (12,
13, and 14; step c in Scheme 2) required only 1 equiv. of the
halide. Reaction of the ␣-carbanion of isocapronitrile with mesy-
lates 6, 7, and 8 afforded the diastereomeric isoalkyl sterols 9,
2.2. Previous synthetic approaches to side-chain analogs of
1
0, and 11 in ∼80% yield. The use of isocapronitrile in this syn-
cholesterol and installation of the side chain into ent-cholesterol
thetic strategy is a convenient route to isoalkyl chain analogs,
since many isoalkyl halides are not available commercially. Finally,
decyanation with potassium and dicyclohexano-18-crown-6, fol-
lowed by quenching of the excess potassium with methanol, and
hydrolysis of the protected alcohol afforded iso-C12, iso-C13, and
iso-C14.
Scheme 1A shows the synthetic route we and others employed
previously to prepare isoalkyl-chain modified cholesterol analogs.
The copper-catalyzed Grignard reaction of a hydroxyl-protected
sterol-bearing tosylate (1) with an alkyl halide affords the prod-
ucts, such as cholesterol (iso-C8) and its homologue, iso-C10, but
the reaction gives low yields, requires careful drying to secure the
generation of the Grignard reagent, and consumes a large excess (as
much as 30 equiv.) of the halide compound (Morisaki et al., 1980;
Slotte et al., 1994).
2.4. Preparation of cholesterol-d11 (17)
Primary nitriles undergo ␣-alkylation reactions via formation
of ␣-carbanion intermediates, which undergo substitution reac-
tions with alkylating agents (Rojas et al., 2007). Reductive cleavage
of a carbon-cyano bond has been achieved by using a variety
of reductive methodologies with hydride ion or hydrogen atom
sources, affording a carbon–hydrogen bond in moderate to good
yield (Savoia et al., 1980; Mattalia et al., 2006; Patra et al., 2013).
The use of a crown ether and a benzyl radical anion generated in
a one-electron transfer reaction from potassium metal in toluene
was shown to be effective for reductive cyanation of primary,
secondary, and tertiary nitriles (Ohsawa et al., 1985). Scheme 1B
shows a key step in the synthesis of the unnatural enantiomer
of cholesterol, ent-cholesterol, in which the isooctyl chain was
installed via alkylation of a nitrile-bearing aliphatic chain with
We used nitrile 2 as the starting material for the synthesis
of the target product 17, as depicted in Scheme 3. Alkyl-
ation of 2 with 1 equiv. of tosylate 18, which was prepared
from commercially available 3-methyl-1-butanol-d11, afforded 15
(
epimeric at C-22) in 89% yield. We used tosylate 18 (1 equiv.)
for alkylation rather than the corresponding bromide (as in
Scheme 2) because 1-bromo-3-methylbutane is a volatile liq-
◦
uid (bp, 121 C). Reductive decyanation with potassium metal
and dicyclohexano-18-crown-6 in toluene, followed by acid-
mediated hydrolysis of the tetrahydropyranyl ether, afforded
target product 17. Thus, cholesterol-d11 was prepared in only
four steps and in an overall yield of 71% from compound
2
.
The synthetic strategy described here has the potential of being
1
0 equiv. of an alkyl bromide, followed by reductive decyanation
readily scaled up to prepare gram quantities of cholesterol-d11 and
isoalkyl analogs of cholesterol with shorter or longer chains than
the iso-C8 chain of natural cholesterol.
with potassium and a crown ether in toluene (Rychnovsky and
Mickus, 1992).

D.J. Baek, R. Bittman / Chemistry and Physics of Lipids 175–176 (2013) 99–104
101
Scheme 2. Synthesis of non-deuterated sterol analogs with varying isoalkyl side-chain lengths. Reagents and conditions: (a) MsCl (3 equiv.), Et3N, CH2Cl2; (b) KCN, DMSO, 82%
◦
for 2 steps; (c) 12, 13, or 14, LDA, THF, −78 C, 90% for 3, 92% for 4, 88% for 5; (d) K, dicyclohexano-18-crown-6, toluene; (e) TBAF, THF, overall yields (2 steps) 74% for 6, 76% for
◦
7
, 77% for 8; (f) MsCl, Et3N, CH2Cl2; (g) isocapronitrile (1 equiv.), LDA, THF, −78 C, overall yields (2 steps) 81% for 9, 78% for 10, 79% for 11; (h) K, dicyclohexano-18-crown-6,
toluene; (i) 1 M HCl, THF, overall yields (2 steps) 88% for iso-C12, 84% for iso-C13, and 85% for isoC14; (j) TIPSCl (1 equiv.), imidazole (2 equiv.), CH2Cl2, 85% for 12, 88% for
3, 79% for 14.
1
Scheme 3. Synthesis of cholesterol-d11 (17). Reagents and conditions: (a) 18, LDA, THF, 89%; (b) K, dicyclohexano-18-crown-6, toluene, 92%; (c) 1 M HCl, THF, 87%; (d) TsCl,
DMAP, Et3N, CH2Cl2, 98%.
3
. Experimental procedures
(m, 2H), 3.47–3.57 (m, 2H), 3.89–3.95 (m, 1H), 4.70–4.72 (m, 1H),
.34–5.35 (m, 1H); 13C NMR (100 MHz, CDCl ) ı 11.9, 14.7, 19.3,
5
3
3.1. General synthetic methods
19.4, 20.0, 20.1, 21.0, 21.8, 24.2, 24.8, 25.5, 28.1, 31.3, 31.8, 33.6,
6.5, 36.7, 36.8, 37.2, 38.7, 39.4, 40.2, 42.7, 50.0, 54.7, 54.8, 56.5,
3
For a description of the general methods, see Byun and Bittman
2012).
62.9, 63.0, 71.7, 76.0, 96.9, 97.0, 119.1, 121.3, 121.4; ESI-HRMS
(
(M+H)+ m/z calcd. for C28H43D43NO2 425.3294, found 425.3289.
3.2. 22-Cyanochol-5-en-3ˇ-ol THP ether (2)
3.3. Synthesis of compounds 3, 4, and 5
A solution of alcohol 1 (300 mg, 0.72 mmol), which was prepared
After a solution of 2 (100 mg, 0.23 mmol) in THF (4 mL) was
◦
from bisnorcholenic acid by a known procedure (Li et al., 2006),
cooled to −78 C, 0.63 mL (0.94 mmol) of LDA (1.5 M solution in
◦
and NEt3 (1.0 mL, 7.2 mmol) in CH Cl2 (10 mL) was cooled to 0 C.
cyclohexane) was added dropwise, followed by for 30 min. The
halide-containing compound (12, 13, or 14) (1.0 equiv.) was added,
and stirring was continued for another 4 h. The reaction was
2
Methanesulfonyl chloride (0.28 mL, 3.6 mmol) was added drop-
wise. After the addition was complete, the mixture was stirred at
room temperature overnight. The solution was washed with water,
dried, and concentrated. To a solution of the resulting mesylate in
DMSO (10 mL) was added potassium cyanide (234 mg, 3.6 mmol).
quenched with aqueous NH Cl solution. The layers were separated,
4
and the aqueous layer was extracted with EtOAc. The combined
organic layers were dried (Na SO ), filtered, and concentrated
2
4
◦
The mixture was heated at 100 C for 1 d, and then was diluted
under reduced pressure. The product was purified by chromatogra-
phy (hexane/EtOAc 10:1) to give compounds 3, 4, and 5 as colorless
oils.
with EtOAc and water. The organic layer was separated, washed
with brine, dried (Na SO ), and concentrated. The residue was puri-
2
4
fied by chromatography (hexane/EtOAc 8:1) to give 251 mg (82%) of
Compound 3: 90%; 1H NMR (400 MHz, CDCl ) ı 0.69 (s, 3H),
3
◦
1
nitrile 2 as a white solid: mp 180–182 C; H NMR (400 MHz, CDCl )
0.97–1.11 (m, 30H), 1.22–1.30 (m, 4H), 1.41–1.57 (m, 15H),
1.63–1.75 (m, 3H), 1.83–2.00 (m, 6H), 2.17–2.22 (m, 1H), 2.33–2.37
(m, 1H), 2.60–2.65 (m, 1H), 3.47–3.56 (m, 2H), 3.65–3.71 (m, 2H),
3
ı 0.70 (s, 3H), 1.01 (s, 3H), 1.02–1.65 (m, 19H), 1.17 (d, J = 6.6 Hz, 3H),
.78–1.87 (m, 4H), 1.95–1.99 (m, 2H), 2.20–2.29 (m, 2H), 2.32–2.39
1

1
02
D.J. Baek, R. Bittman / Chemistry and Physics of Lipids 175–176 (2013) 99–104
3
(
.89–3.93 (m, 1H), 4.70–4.72 (m, 1H), 5.33–5.36 (m, 1H); 13C NMR
100 MHz, CDCl ) ı 11.7, 11.8, 11.9, 12.3, 12.5, 13.4, 14.1, 14.5,
5.5, 17.1, 17.7, 18.0, 18.8, 19.4, 20.1, 20.7, 20.1, 22.7, 23.7, 24.1,
4.2, 24.8, 25.3, 25.5, 27.6, 28.0, 28.1, 29.1, 29.7, 29.8, 30.7, 31.3,
1.6, 31.8, 31.9, 32.4, 32.7, 34.5, 34.7, 36.1, 36.5, 36.7, 36.8, 37.2,
7.4, 38.1, 38.8, 39.5, 40.2, 42.4, 42.5, 43.8, 49.9, 50.0, 52.4, 54.1,
35.9, 36.7, 36.8, 37.2, 37.4, 38.8, 39.8, 40.3, 42.3, 50.1, 50.2, 56.1,
56.7, 62.8, 62.9, 76.0, 96.8, 96.9, 121.5, 121.6, 140.8, 141.0, ESI-
HRMS (M+H)+ m/z calcd. for C32H55O3 487.4151, found 487.4143.
3
1
2
3
3
5
1
6
◦
1
Compound 8: 77%; mp 124–125 C; H NMR (400 MHz, CDCl3)
ı 0.67 (s, 3H), 0.90–0.94 (m, 4H), 0.97–1.11 (m, 8H), 1.14–1.33
(m, 12H), 1.42–1.56 (m, 11H), 1.69–1.74 (m, 2H), 1.78–1.86 (m,
4H), 1.94–2.01 (m, 2H), 2.17–2.36 (m, 2H), 3.46–3.54 (m, 2H), 3.62
(t, J = 6.6 Hz, 2H), 3.89–3.94 (m, 1H), 4.71–4.72 (m, 1H), 5.32–5.36
(m, 1H); 13C NMR (100 MHz, CDCl3) ı 11.9, 14.1, 18.7, 19.4, 20.0,
20.1, 21.1, 22.7, 24.3, 25.5, 25.6, 25.8, 26.0, 28.0, 28.2, 29.3, 29.4,
29.5, 29.7, 30.1, 31.3, 31.9, 32.8, 35.7, 35.9, 36.8, 37.2, 37.5, 38.8,
39.8, 40.2, 42.3, 50.2, 53.8, 56.1, 56.8, 62.8, 62.9, 63.0, 76.0, 96.8,
97.0, 121.5, 121.6, 140.9, 141.1; ESI-HRMS (M+Na)+ m/z calcd. for
C33H56O3Na 523.4127, found 523.4123.
6.5, 56.7, 62.9, 63.0, 63.1, 76.0, 96.9, 97.0, 121.1, 121.3, 121.4,
+
22.9, 140.9, 141.1; ESI-HRMS (M+H) m/z calcd. for C41H72NO Si
3
54.5281, found 654.5277.
1
Compound 4: 92%; H NMR (400 MHz, CDCl ) ı 0.69 (s, 3H),
3
1
.00–1.13 (m, 30H), 1.15–1.30 (m, 4H), 1.40–1.48 (m, 7H), 1.50–1.58
(m, 10H), 1.63–1.75 (m, 3H), 1.80–2.01 (m, 6H), 2.16–2.38 (m,
2
3
H), 2.60–2.64 (m, 1H), 3.47–3.56 (m, 2H), 3.69 (t, J = 6.3 Hz, 2H),
.89–3.94 (m, 1H), 4.70–4.72 (m, 1H), 5.33–5.36 (m, 1H); 13C NMR
(
100 MHz, CDCl ) ı 11.8, 11.9, 14.6, 18.0, 18.7, 18.9, 19.4, 20.1, 21.0,
3
2
3
3
7
3.9, 24.1, 25.5, 25.6, 27.6, 28.0, 28.1, 29.7, 29.8, 30.9, 31.3, 31.8,
1.9, 32.7, 32.8, 35.9, 36.4, 36.7, 36.8, 37.2, 37.4, 38.0, 38.2, 38.8,
9.5, 40.2, 42.4, 42.5, 43.7, 49.9, 50.0, 54.2, 56.5, 62.9, 63.2, 63.3,
3.5. Synthesis of compounds 9, 10, and 11
To a solution of 6, 7 or 8 (0.32, 0.42, or 0.25 mmol, respectively)
and NEt3 (10 equiv.) in CH2Cl2 (3 mL) was added methanesulfonyl
chloride (3 equiv.). After being stirred at room temperature for
3 h, the reaction mixture was evaporated under reduced pressure,
diluted with water, and the product was extracted with EtOAc. The
extract was washed with brine, dried, and evaporated. After a solu-
6.0, 96.9, 97.0, 121.2, 121.3, 121.4, 122.9, 140.9, 141.0, 141.1;
+
ESI-HRMS (M+H) m/z calcd. for C42H74NO Si 668.5438, found
3
6
68.5437.
Compound 5: 88%; 1H NMR (400 MHz, CDCl ) ı 0.69 (s, 3H),
3
1
1
.00–1.11 (m, 30H), 1.16–1.1.30 (m, 9H), 1.35–1.46 (m, 4H),
.48–1.57 (m, 10H), 1.61–1.75 (m, 3H), 1.83–2.00 (m, 6H), 2.20–2.43
◦
tion of isocapronitrile (1 equiv.) in THF (3 mL) was cooled to −78 C,
(
3
(
m, 2H), 2.59–2.64 (m, 1H), 3.45–3.57 (m, 2H), 3.64–3.69 (m, 2H),
.89–3.94 (m, 1H), 4.70–4.72 (m, 1H), 5.33–5.36 (m, 1H); 13C NMR
100 MHz, CDCl ) ı 11.8, 11.9, 12.0, 12.6, 14.6, 17.7, 18.0, 18.8, 18.9,
LDA (1.5 M solution in cyclohexane, 4 equiv.) was added dropwise,
and the resulting solution was stirred for 30 min. Mesylated com-
pound (1 equiv.) was added, and stirring was continued for another
4 h. The reaction was quenched with aqueous NH4Cl solution. The
layers were separated, and the aqueous layer was extracted with
EtOAc. The combined organic layers were dried (Na2SO4), filtered,
and concentrated under reduced pressure. The product was puri-
fied by chromatography (hexane/EtOAc 6:1) to give compounds 9,
3
1
2
3
9
9.4, 20.1, 21.0, 23.8, 24.1, 25.5, 25.6, 25.7, 27.8, 28.1, 29.0, 29.3,
9.7, 30.9, 31.3, 31.8, 31.9, 32.9, 33.0, 36.5, 36.7, 36.8, 37.2, 37.4,
8.3, 38.8, 39.5, 40.3, 42.4, 50.0, 54.2, 56.5, 63.0, 63.3, 63.4, 76.0,
+
7.0, 97.1, 121.2, 121.4, 122.8, 141.1; ESI-HRMS (M+H) m/z calcd
for C43H76NO Si 682.5594, found 682.5591.
3
1
0, and 11 as waxy solids.
Compound 9: 81%; 1H NMR (400 MHz, CDCl ) ı 0.67 (s, 3H),
3
3.4. Synthesis of compounds 6, 7, and 8
0.86–0.96 (m, 12H), 0.98–1.02 (m, 6H), 1.03–1.11 (m, 4H), 1.15–1.28
m, 8H), 1.33–1.38 (m, 4H), 1.43–1.63 (m, 9H), 1.68–1.71 (m, 2H),
(
To 70 mg of compound 3, 4, or 5 in 4 mL of toluene was added
excess (>6 equiv.) potassium metal and dicyclohexyl-18-crown-6
1.2 equiv.) at room temperature. After 8 h, methanol was added
1.77–1.86 (m, 4H), 1.93–2.04 (m, 2H), 2.18–2.37 (m, 2H), 3.47–3.53
(m, 2H), 3.91–3.93 (m, 1H), 4.72–4.73 (m, 1H), 5.33–5.36 (m, 1H);
13
(
C NMR (100 MHz, CDCl ) ı 11.9, 14.1, 14.2, 18.7, 19.4, 20.0,
3
carefully to quench the excess potassium. The mixture was diluted
with EtOAc, and the organic layer was separated, washed with
20.1, 21.1, 21.3, 21.5, 21.7, 22.3, 22.4, 22.7, 22.9, 24.2, 24.3, 25.5,
25.8, 26.1, 26.2, 27.2, 28.0, 28.3, 29.4, 29.7, 30.0, 30.9, 31.1, 31.3,
31.9, 32.7, 35.7, 35.8, 36.8, 37.2, 37.4, 37.5, 38.8, 39.8, 40.3, 41.3,
42.3, 42.8, 49.7, 50.2, 56.0, 56.8, 60.4, 62.8, 62.9, 64.3, 76.0, 96.8,
97.0, 121.5, 121.6, 122.5, 141.1; ESI-HRMS (M+H)+ m/z calcd. for
brine, dried (Na SO ), and concentrated under reduced pressure.
2
4
To a solution of the residue in THF (4 mL) was added 3 equiv. of
TBAF (tetra-n-butylammonium fluoride, 1 M solution in THF). After
being stirred at room temperature for 12 h, the reaction mixture
was diluted with water, and the product was extracted with EtOAc.
The extract was washed with brine, dried, and evaporated. Purifica-
tion by silica gel chromatography, eluting with hexane/EtOAc (2:1),
gave compounds (6, 7, and 8) as white solids.
C37H62NO2 552.4781, found 552.4765.
1
Compound 10: 78%; H NMR (400 MHz, CDCl ) ı 0.67 (s, 3H),
3
0.87–0.96 (m, 12H), 0.98–1.02 (m, 6H), 1.04–1.12 (m, 4H), 1.15–1.29
(m, 8H), 1.31–1.38 (m, 4H), 1.40–1.62 (m, 11H), 1.64–1.79 (m, 2H),
1.81–1.88 (m, 4H), 1.95–2.05 (m, 2H), 2.15–2.38 (m, 2H), 3.47–3.55
(m, 2H), 3.91–3.93 (m, 1H), 4.72–4.74 (m, 1H), 5.35–5.36 (m, 1H);
◦
1
Compound 6: 74%; mp 118–120 C; H NMR (400 MHz, CDCl ) ı
3
13
0.67 (s, 3H), 0.90–0.98 (m, 4H), 1.01–1.12 (m, 8H), 1.15–1.38 (m,
12H), 1.42–1.62 (m, 7H), 1.71–1.75 (m, 2H), 1.83–1.86 (m, 4H),
1.92–2.01 (m, 2H), 2.17–2.36 (m, 2H), 3.47–3.52 (m, 2H), 3.62 (t,
C NMR (100 MHz, CDCl ) ı 11.9, 14.1, 17.2, 18.7, 19.4, 20.1, 21.1,
3
22.4, 22.7, 24.3, 25.5, 25.8, 25.9, 26.0, 27.0, 28.0, 28.3, 28.7, 29.4,
29.5, 29.7, 30.0, 31.1, 31.3, 31.9, 32.7, 35.7, 35.8, 36.8, 37.2, 37.5,
38.8, 39.8, 40.3, 42.3, 43.6, 45.2, 50.2, 56.1, 56.8, 60.4, 62.8, 62.9,
64.3, 76.0, 96.8, 97.0, 121.5, 121.6, 122.4, 141.1; ESI-HRMS (M+Na)+
J = 6.6 Hz, 2H), 3.89–3.93 (m, 1H), 4.70–4.72 (m, 1H), 5.33–5.36 (m,
1
3
1
2
3
6
H); C NMR (100 MHz, CDCl ) ı 11.9, 14.1, 18.7, 19.4, 20.0, 20.1,
3
1.1, 24.3, 25.5, 25.9, 26.2, 28.0, 28.3, 29.7, 31.3, 31.9, 32.2, 32.9,
5.7, 35.9, 36.8, 37.2, 37.4, 38.8, 39.8, 40.2, 42.3, 50.1, 50.2, 56.1,
2.8, 62.9, 63.0, 76.0, 96.8, 96.9, 121.5, 121.6, 140.9, 141.0; ESI-
HRMS (M+H) m/z calcd. for C31H53
Compound 7: 76%; mp 142–144 C; H NMR (400 MHz, CDCl3)
ı 0.67 (s, 3H), 0.90–0.95 (m, 4H), 1.00–1.11 (m, 8H), 1.14–1.1.32
m/z calcd. for C38H63NO Na 588.4756, found 588.4791.
2
1
Compound 11: 79%; H NMR (400 MHz, CDCl ) ı 0.67 (s, 3H),
3
0.87–0.97 (m, 12H), 0.98–1.03 (m, 6H), 1.04–1.19 (m, 4H), 1.21–1.40
(m, 12H), 1.43–1.62 (m, 13H), 1.66–1.74 (m, 2H), 1.77–1.89 (m, 4H),
1.91–2.05 (m, 2H), 2.17–2.37 (m, 2H), 3.47–3.55 (m, 2H), 3.89–3.94
(m, 1H), 4.71–4.72 (m, 1H), 5.33–5.36 (m, 1H); 13C NMR (100 MHz,
+
O
3
473.3995, found 473.3991.
◦
1
(
1
m, 12H), 1.42–1.58 (m, 9H), 1.68–1.73 (m, 2H), 1.78–1.87 (m, 4H),
.92–2.01 (m, 2H), 2.18–2.36 (m, 2H), 3.45–3.54 (m, 2H), 3.62 (t,
J = 6.6 Hz, 2H), 3.87–3.94 (m, 1H), 4.70–4.72 (m, 1H), 5.33–5.36 (m,
CDCl ) ı 11.9, 14.2, 18.7, 19.4, 20.0, 20.2, 21.1, 21.6, 22.4, 22.7, 22.9,
3
23.3, 24.3, 25.5, 25.8, 26.0, 26.2, 27.2, 27.5, 28.0, 28.3, 28.7, 29.2,
29.3, 29.4, 29.7, 29.8, 30.0, 30.3, 31.3, 31.9, 32.7, 34.3, 35.6, 35.8,
36.8, 37.2, 37.5, 38.5, 38.8, 39.3, 39.9, 41.5, 42.4, 50.2, 56.1, 56.8,
60.6, 62.8, 62.9, 76.1, 96.8, 97.0, 121.5, 121.6, 122.5, 140.9, 141.1;
13
1
2
H); C NMR (100 MHz, CDCl ) ı 11.9, 18.7, 19.4, 20.0, 21.0, 24.3,
3
5.5, 25.8, 26.1, 28.0, 28.2, 29.7, 29.9, 31.2, 31.3, 31.9, 32.8, 35.7,

D.J. Baek, R. Bittman / Chemistry and Physics of Lipids 175–176 (2013) 99–104
103
ESI-HRMS (M+Na)+ m/z calcd. for C39H65NO Na 602.4913, found
28.3, 29.7, 30.0, 30.2, 31.9, 35.8, 36.0, 36.5, 37.3, 39.1, 39.8, 42.3,
50.1, 56.1, 56.8, 71.8, 121.7, 140.8; ESI-HRMS (M+H) m/z calcd. for
2
+
6
02.4910.
C33H59O 471.4566, found 471.4559.
3.6. Synthesis of compounds 12, 13, and 14
3.8. Isopentyl-d11 4-methylbenzenesulfonate (18)
To a solution of 100 mg of alcohol (4-bromo-1-butanol, 5-
bromo-1-pentanol, or 6-bromo-1-hexanol) in 5 mL of CH Cl were
To a solution of 3-methyl-1-butanol-d11 (100 mg, 1.0 mmol)
2
2
added 2 equiv. of imidazole and 1.0 equiv. of TIPSCl (triisopropy-
lsilyl chloride). After being stirred at room temperature for 4 h,
the reaction mixture was diluted with water, and the product was
extracted with EtOAc. The extract was washed with brine, dried,
and evaporated. The product was purified by chromatography (hex-
ane/EtOAc 20:1) to give compounds 12, 13, and 14 as colorless
oils.
in CH2Cl2 (3 mL) were added 0.7 mL (5.0 mmol) of NEt3, 12 mg
(0.10 mmol) of DMAP, and 384 mg (2.0 mmol) of p-toluenesulfonyl
chloride. After being stirred at room temperature for 5 h, the reac-
tion mixture was diluted with EtOAc and water. The organic layer
was separated, washed with brine, dried (Na2SO4), and concen-
trated. The residue was purified by chromatography (hexane/EtOAc
10:1) to give 250 mg (98%) of tosylate 18 as a colorless oil. 1H NMR
(400 MHz, CDCl3) ı 2.45 (s, 3H), 7.34 (d, J = 8.0 Hz, 2H), 7.79 (d,
J = 8.3 Hz, 2H); 13C NMR (100 MHz, CDCl3) ı 20.6, 20.8, 21.0, 21.1,
21.3, 21.6, 23.1, 23.3, 23.5, 35.8, 36.0, 36.2, 36.4, 36.5, 68.0, 68.3,
68.5, 68.7, 69.0, 127.9, 129.8, 133.2, 144.7; ESI-HRMS (M+H)+ m/z
calcd. for C12H7D11O3S 253.1656, found 253.1667.
Compound 12: 85%; 1H NMR (400 MHz, CDCl ) ı 1.03–1.10 (m,
3
2
1H), 1.68 (quin, J = 6.1 Hz, 2H), 1.98 (quin, J = 7.0 Hz, 2H), 3.46 (t,
13
J = 6.8 Hz, 2H), 3.72 (t, J = 6.1 Hz, 2H); C NMR (100 MHz, CDCl ) ı
1
3
+
1.9, 18.0, 29.6, 31.5, 34.0, 62.4; ESI-HRMS (M+H) m/z calcd. for
C13H30BrOSi 309.1249, found 309.1248.
1
Compound 13: 88%; H NMR (400 MHz, CDCl ) ı 1.05–1.11 (m,
3
2
2
2
3
1H), 1.49–1.58 (m, 4H), 1.89 (t, J = 7.6 Hz, 2H), 3.41 (t, J = 6.9 Hz,
3.9. 22-Cyanochol-23, 23, 24, 24, 25, 26, 26, 26, 27, 27,
27-d11-5-en-3ˇ-ol THP ether (15)
H), 3.69 (t, J = 6.1 Hz, 2H); 13C NMR (100 MHz, CDCl ) ı 12.0, 18.0,
3
+
4.6, 32.1, 32.7, 33.8; ESI-HRMS (M+H) m/z calcd. for C14H32BrOSi
23.1406, found 323.1401.
After a solution of 2 (220 mg, 0.52 mmol) in THF (8 mL) was
Compound 14: 79%: 1H NMR (400 MHz, CDCl ) ı 1.00–1.12
cooled to −78 C, 1.4 mL (2.07 mmol) of LDA (1.5 M solution in
◦
3
(m, 21H), 1.33–1.50 (m, 4H), 1.55 (quin, J = 6.8 Hz, 2H), 1.87 (quin,
cyclohexane) was added dropwise. The reaction mixture was
stirred for 30 min. Deuterated tosylate 18 (131 mg, 0.52 mmo1) was
added, and stirring was continued for another 4 h. The reaction was
quenched with aqueous NH4Cl solution. The layers were separated,
and the aqueous layer was extracted with EtOAc. The combined
organic layers were dried (Na2SO4), filtered, and concentrated
under reduced pressure. The product was purified by chromatogra-
phy (hexane/EtOAc 3:1) to give 235 mg (89%) of alkylated product
13
J = 7.2 Hz, 2H), 3.41 (t, J = 6.9 Hz, 2H), 3.68 (t, J = 6.4 Hz, 2H); C NMR
(
100 MHz, CDCl ) ı 12.0, 18.0, 25.1, 28.0, 32.8, 32.9, 33.9, 63.2; ESI-
3
+
HRMS (M+H) m/z calcd for C15H34BrOSi 337.1562, found 337.1557.
3.7. Synthesis of iso-C12, iso-C13, and iso-C14
To a solution of cyano-sterol 9, 10, or 11 (0.030 mmol) in 4 mL
◦
1
of toluene were added excess potassium metal and 1.2 equiv. of
dicyclohexyl-18-crown-6. After 8 h, methanol was added to quench
the excess potassium. The mixture was diluted with EtOAc, the
organic layer was separated, washed with brine, dried (Na SO ),
15 as a white solid: mp 189–190 C; H NMR (400 MHz, CDCl3)
ı 0.69 (s, 3H), 0.98 (s, 3H), 1.07 (d, J = 6.5 Hz, 3H), 1.09–1.75 (m,
19H), 1.83–2.05 (m, 6H), 2.17–2.37 (m, 2H), 2.56 (d, J = 2.6 Hz, 1H),
3.47–3.55 (m, 2H), 3.90–3.93 (m, 1H), 4.70–471 (m, 1H), 5.33–5.35
(m, 1H); 13C NMR (100 MHz, CDCl3) ı 12.0, 14.6, 19.4, 20.2, 21.0,
21.2, 21.4, 21.5, 21.6, 21.7, 22.1, 22.2, 22.3, 22.4, 24.1, 25.5, 26.4,
26.6, 26.8, 27.7, 28.0, 28.1, 29.7, 31.3, 31.8, 31.9, 36.2, 26.4, 36.6,
36.7, 36.8, 37.2, 37.4, 38.0, 38.2, 38.3, 38.8, 39.5, 40.2, 42.4, 42.5,
49.9, 50.0, 52.4, 54.2, 56.5, 56.6, 63.0, 67.1, 76.0, 97.0, 97.1, 121.3,
2
4
and concentrated. The residue was dissolved in THF (3 mL), and
M HCl (0.5 mL) was added. The mixture was stirred at room tem-
1
perature for 24 h. The solution was concentrated, and the residue
was purified by chromatography (hexane/EtOAc 3:1) to give the
desired cholesterol analogs as white solids.
Compound iso-C12: 88%; mp 112–114 C; 1H NMR (400 MHz,
◦
121.4, 140.9, 141.1; ESI-HRMS (M+H) m/z calcd. for C33H42D11NO2
+
CDCl ) ı 0.68 (s, 3H), 0.87 (d, J = 6.6 Hz, 6H), 0.91 (d, J = 6.6 Hz,
506.4756, found 506.4768.
3
3
H), 0.93–1.01 (m, 5H), 1.05–1.22 (m, 8H), 1.23–1.40 (m, 12H),
.43–1.59 (m, 6H), 1.80–1.88 (m, 4H), 1.94–2.02 (m, 2H), 2.23–2.29
1
3.10. Chol-23, 23, 24, 24, 25, 26, 26, 26, 27, 27,
27-d11-5-en-3ˇ-ol THP ether (16)
(
m, 2H), 3.52 (tt, J = 4.8, 11.6 Hz, 1H), 5.35 (d, J = 5.2 Hz, 1H); 13
C
NMR (100 MHz, CDCl ) ı 11.9, 18.7, 19.4, 21.1, 22.7, 24.3, 26.1, 27.5,
3
2
3
8.0, 28.2, 29.8, 30.0, 30.2, 31.7, 31.9, 35.8, 36.0, 36.5, 37.3, 39.1,
9.8, 42.3, 50.1, 56.1, 56.8, 71.8, 121.8, 140.8; ESI-HRMS (M+H)
To 200 mg (0.39 mmol) of 22-cyanocholesterol THP ether 15
in 6 mL of toluene were added 100 mg (2.56 mmol) of potassium
metal and 294 mg (0.79 mmol) of dicyclohexyl-18-crown-6. After
8 h, methanol was added carefully to quench the excess potassium.
The mixture was diluted with EtOAc, and the organic layer was
separated, washed with brine, dried (Na2SO4), and concentrated.
The residue was purified by chromatography (hexane/EtOAc 20:1)
+
m/z calcd. for C31H55O 443.4253, found 443.4233.
Compound iso-C13: 84%; mp 111–112 C; 1H NMR (400 MHz,
◦
CDCl ) ı 0.68 (s, 3H), 0.86 (d, J = 6.6 Hz, 6H), 0.90 (d, J = 6.6 Hz,
3
3
H), 0.93–0.1.01 (m, 5H), 1.03–1.19 (m, 8H), 1.22–1.40 (m, 12H),
.45–1.61 (m, 8H), 1.80–1.87 (m, 4H), 1.95–2.05 (m, 2H), 2.23–2.29
1
(
(
m, 2H), 3.53 (tt, J = 4.5, 11.5 Hz, 1H), 5.35 (d, J = 5.2 Hz, 1H); 13C NMR
◦
1
to give 175 mg (92%) of 16 as a white solid: mp 148–150 C; H
100 MHz, CDCl ) ı 11.9, 18.7, 19.4, 21.1, 22.7, 24.3, 26.1, 27.5, 28.0,
NMR (400 MHz, CDCl3) ı 0.67 (s, 3H), 0.91 (d, J = 6.5 Hz, 3H), 1.01
(s, 3H), 0.94–1.74 (m, 21H), 1.80–1.86 (m, 4H), 1.94–2.02 (m, 2H),
2.17–2.37 (m, 2H), 3.46–3.57 (m, 2H), 3.91–3.94 (m, 1H), 4.70–4.72
(m, 1H), 5.33–5.36 (m, 1H); 13C NMR (100 MHz, CDCl3) ı 11.9, 18.7,
19.4, 20.0, 20.1, 21.1, 21.2, 21.4, 21.6, 21.8, 24.3, 25.5, 26.7, 26.8,
27.0, 28.0, 28.3, 29.7, 31.3, 31.9, 32.0, 35.4, 35.5, 35.7, 35.8, 36.0,
36.8, 36.9, 37.2, 37.4, 37.8, 38.0, 38.2, 38.4, 38.5, 38.8, 39.8, 40.3,
42.3, 50.2, 56.2, 56.8, 62.9, 76.0, 96.8, 97.0, 121.5, 121.6, 140.9,
141.1; ESI-HRMS (M+H)+ m/z calcd. for C32H43D11O2 481.4803,
found 481.4818.
3
2
4
8.3, 29.8, 30.0, 30.2, 31.7, 31.9, 35.8, 36.0, 36.5, 37.3, 39.1, 39.8,
2.3, 50.1, 56.1, 56.8, 71.8, 121.8, 140.8; ESI-HRMS (M+H) m/z calcd
+
for C32H57O 457.4409, found 457.4385.
Compound iso-C14: 85%; mp 108–110 C; 1H NMR (400 MHz,
◦
CDCl ) ı 0.68 (s, 3H), 0.86 (d, J = 6.6 Hz, 6H), 0.90 (d, J = 6.6 Hz,
3
3
H), 0.92–0.1.01 (m, 5H), 1.03–1.19 (m, 8H), 1.21–1.36 (m, 12H),
.40–1.59 (m, 10H), 1.78–1.87 (m, 4H), 1.94–2.05 (m, 2H), 2.19–2.32
1
(
m, 2H), 3.53 (tt, J = 4.6, 11.1 Hz, 1H), 5.35 (d, J = 5.2 Hz, 1H); 13
C
NMR (100 MHz, CDCl ) ı 11.9, 18.7, 19.4, 21.1, 22.7, 24.3, 27.5, 28.0,
3

1
04
D.J. Baek, R. Bittman / Chemistry and Physics of Lipids 175–176 (2013) 99–104
3.11. Cholesterol-23, 23, 24, 24, 25, 26, 26, 26, 27, 27, 27-d11 (17)
Li, Z., Mintzer, E., Bittman, R., 2006. First synthesis of free cholesterol-BODIPY con-
jugates. J. Org. Chem. 71, 1718–1721.
Lütjohann, D., Meese, C.O., Crouse 3rd, J.R., von Bergmann, K., 1993. Evaluation of
deuterated cholesterol and deuterated sitostanol for measurement of choles-
terol absorption in humans. J. Lipid Res. 34, 1039–1046.
Mattalia, J.-M., Marchi-Delapierre, C., Hazimah, H., Chanon, M., 2006. The reductive
decyanation reaction: chemical methods and synthetic applications. ARKIVOC,
To a solution of ether 16 (50 mg, 0.10 mmol) in THF (2 mL) was
added 1 M HCl (0.5 mL). The mixture was stirred at room tem-
perature for 2 d. The solution was concentrated, and the residue
was purified by chromatography (hexane/EtOAc 3:1) to give 36 mg
90–118.
◦
23
◦
(
(
(
3
(
2
2
3
7
3
87%) of product 17 as a white solid: mp 138–139 C; [˛] −41.3
Matthaus, C., Krafft, C., Dietzek, B., Brehm, B.R., Lorkowski, S., Popp, J., 2012. Non-
invasive imaging of intracellular lipid metabolism in macrophages by Raman
microscopy in combination with stable isotopic labeling. Anal. Chem. 84,
D
1
c 0.75, CHCl ); H NMR (400 MHz, CDCl ) ı 0.68 (s, 3H), 0.91
3
3
d, J = 6.5 Hz, 3H), 1.01 (s, 3H), 0.93–1.61 (m, 16H), 1.78–1.87 (m,
H), 1.94–2.05 (m, 2H), 2.19–2.32 (m, 2H), 3.48–3.56 (m, 1H), 5.35
8549–8556.
Mattjus, P., Bittman, R., Vilchèze, C., Slotte, J.P., 1995. Lateral domain formation in
cholesterol/phospholipid monolayers as affected by the sterol side chain con-
formation. Biochim. Biophys. Acta 1240, 237–247.
McMullen, T.P., Vilchèze, C., McElhaney, R.N., Bittman, R., 1995. Differential scan-
ning calorimetric study of the effect of sterol side chain length and structure on
dipalmitoylphosphatidylcholine thermotropic phase behavior. Biophys. J. 69,
169–176.
Mihailescu, M., Soubias, O., Worcester, D., White, S.H., Gawrisch, K., 2011. Struc-
ture and dynamics of cholesterol-containing polyunsaturated lipid membranes
studied by neutron diffraction and NMR. J. Membr. Biol. 239, 63–71.
Morisaki, M., Shibata, M., Duque, C., Imamura, N., Ikekawa, N., 1980. Studies on
steroids LXIII. Synthesis of cholesterol analogs with a modified side chain. Chem.
Pharm. Bull. 28, 606–611.
1
3
d, J = 5.2 Hz, 1H); C NMR (100 MHz, CDCl ) ı 11.9, 18.7, 19.4,
3
1.1, 21.3, 21.4, 21.7, 21.9, 22.5, 22.7, 22.8, 23.0, 23.2, 23.3, 24.3,
6.6, 26.8, 27.0, 28.3, 31.7, 31.9, 35.4, 35.5, 35.7, 35.8, 36.0, 37.3,
7.8, 38.0, 38.2, 38.4, 38.5, 39.8, 42.4, 50.1, 53.4, 56.1, 56.2, 56.8,
+
1.8, 121.7, 140.8; ESI-HRMS (M+H) m/z calcd. for C27H35D11
O
97.4228, found 397.4242.
Acknowledgment
We thank the Finnish Medical Foundation for financial support.
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