Syntheses of deuterium-labelled cholesteryl neoglycolipids

Article abstract of DOI:10.1002/jlcr.1961

Four deuterium-labelled neoglycolipids derived from cholesterol were synthesized for embedment into liposomes. Deuterium atoms were either incorporated by CH₂ replacement with a CD₂ group in the triethylene glycol spacer arm between the cholesteryl residue and the sugar moiety (products 2-4) or incorporated directly on the acetamido function in the sugar head (compound 5). Copyright

Full text of DOI:10.1002/jlcr.1961

Research Article
Received 21 September 2011,
Revised 18 November 2011,
Accepted 17 December 2011
Published online 25 January 2012 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1961
Syntheses of deuterium-labelled cholesteryl
neoglycolipids
a,b,c,d,e,f
Dominique Lafont,a,b,c,d,e,f Paul Boullanger,
Antoine Gambettaa,b,c,d,e,f
and
*
Four deuterium-labelled neoglycolipids derived from cholesterol were synthesized for embedment into liposomes.
Deuterium atoms were either incorporated by CH2 replacement with a CD2 group in the triethylene glycol spacer arm
between the cholesteryl residue and the sugar moiety (products 2–4) or incorporated directly on the acetamido function
in the sugar head (compound 5).
Keywords: neoglycolipids; deuterium; N-acetyl-D-glucosamine; glycosylation; hydrophobic anchor; isotopically labelled synthesis;
deuterium
the membrane surface being a major factor in the lectin accessi-
Introduction
bility, during recognition phenomenon with Wheat Germ
Deuterium-labelled glycolipids have been synthesized for more
Agglutinin (WGA). The most interesting results were obtained with
triethylene glycol and tetraethylene glycol spacer arms.14,15 The
than 25 years to study their behaviour in multicomponent model
membranes.1–5 Two different strategies have been developed
replacement of a methylene group of the spacer by a dideuterio-
for the replacement of selected hydrogen atoms by deuterium
methylene group at different places would give more informa-
in labelled glycolipids. In the first strategy, deuterium was incor-
tion about the glycolipid incorporation into the membrane.
The present paper describes the syntheses of analogues 2–5
porated into the lipidic moiety according to known methods,
which have been reviewed 15 years ago.6 Then, the labelled lipi-
of [8-(cholest-5-en-3b-yloxy)-3,6-dioxaoctyl] 2-acetamido-2-deoxy-
b-D-glucopyranoside (1)14 bearing deuterium atoms on the triethy-
dic part (most often an acid or an acyl chloride) was condensed
to a sugar derivative generally via an amide7,8 or an ester9–11
lene glycol spacer respectively at positions C-1 (compound 2), C-4
(compound 3) and C-7 (compound 4) or on the carbohydrate resi-
due on the acetamido function (compound 5; Figure 1).
Syntheses of glycosylation acceptors 9, 13 and 17 are
bond; deuterated alcohols have also been employed for the
preparation of ether bond.11 For example, an efficient and con-
venient synthesis of deuterium-labelled seminolipid isotopomers
was recently described by epoxide ring opening of (R)-glycidyl
2,3,4,6-tetra-O-benzyl-b-D-galactopyranoside: either the opening
was performed with labelled [16,16,16-D3]-hexadecanol under
acidic conditions (and the resulting hydroxyl group acylated
with hexadecanoic acid) or the epoxide was opened with
described in Scheme 1. Compound 9 was obtained in three steps
from cholesterol tosylate 6.17 Compound 6 was reacted with
diethylene glycol in refluxing dioxane, affording product 7 in
81% yield. Several methods were checked to realize the diethylene
chain elongation on derivative 7: the reaction with chloroacetic acid
hexadecanol (and then acylated with labelled [16,16,16-D3]-
in THF in the presence of sodium hydride as described by Abe
hexadecanoic acid).11 In the second strategy, the deuterium was
incorporated in the glycolipids on the saccharidic moieties on the
exocyclic hydroxymethyl group of terminal galactose, galac-
tosamine2,12 or glucosamine13 residues either by enzymatical or
chemical methods or by replacement of the N-acetyl group by an
N-trideuteroacetyl group.2,12
aLaboratoire de CO2-GLYCO, Institut de Chimie et Biochimie Moléculaires et
Supramoléculaires (ICBMS), 43 boulevard du 11 novembre 1918, Villeurbanne,
F-69622, France
bUMR5246, CNRS, Villeurbanne, F-69622, France
cUniversité de Lyon, Lyon, F-69622, France
dUniversité Lyon 1, Lyon, F-69622, France
eINSA Lyon, Villeurbanne, F-69622, France
fCPE Lyon, Villeurbanne, F-69616, France
The present paper deals with the syntheses of deuterium-
labelled analogues of N-acetyl-D-glucosamine glycosides of
cholesteryl oligoethylene glycol, which were used as anchors
into phosphatidylcholine bilayers.14–16
Results and discussion
Previous work in the laboratory has demonstrated that N-acetyl-
b-D-glucosaminyl glycosides of cholesteryl oligoethylene glycol
could be used as anchors into phosphatidylcholine liposomes,
*Correspondence to: Dominique Lafont, Laboratoire de CO2-GLYCO, Institut de
Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), 43 boulevard
du 11 novembre 1918, Villeurbanne F-69622, France.
the distance between the ligand (N-acetyl-D-glucosamine) and E-mail: lafont@univ-lyon1.fr
J. Label Compd. Radiopharm 2012, 55 88–95
Copyright © 2012 John Wiley & Sons, Ltd.

D. Lafont et al.
alcohol 15 was realized using di(chloroethyl)ether under phase
transfer conditions.19 Chloride 16 was recovered in 65% yield.
Two steps were necessary for the conversion of chloride 16 into
alcohol 17 by the intermediate of a formate22, which was
saponified under basic conditions.
Compounds 9, 13 and 17 were glycosylated with 1,2,3,4-
tetra-O-acetyl-2-allyloxycarbonylamino-2-deoxy-b-D-glucopyranose
(18)14,23,24 as donor (1.05 equiv.) in dichloromethane at
À20 ꢀC, the reaction being promoted by trimethylsilyl
trifluoromethanesulfonate; glycosides 19 (from 9), 20 (from
13) and 21 (from 17) were obtained in 75–79% yields
(Scheme 2). As expected, the cleavage of the tetrahydropyranyl
group occurred under the acidic conditions of the glycosylation
reaction. N-allyloxycarbonyl group cleavage for compounds
19–21 and for non-deuterated analogue 2214 was catalysed
by tetrakis(triplenylphosphine)palladium in the presence of
diethyl malonate in THF. The amino-free derivatives were
N-acetylated either with acetic anhydride for the deuterium-
labelled compounds or with hexadeuterated acetic anhydride
for the non-labelled derivative. Compounds 23–26 were finally
de-O-acetylated according to Zemplén method by using
catalytic sodium methylate in methanol, giving products 2–5
in good yields.
Figure 1. Structure of glycolipids 1–5.
et al.18 gave the expected acid in very low yields (30%
approximatively). However, under catalytic acidic conditions
(boron trifluoride etherate) at À20 ꢀC in dichloromethane,
alcohol 7 was reacted with ethyl diazoacetate, leading to ester
8 in 53% yield after purification. Increasing the temperature
gave more by-products and decreased the yield. Alcohol 9
was obtained by reduction of ester 8 with lithium aluminium
deuteride in THF. Similar reactions were applied for the
preparation of alcohol 10 (72%), ester 11 (56%) and alcohol
12 (75%). Condensation of alcohol 12 with pyranylated
2-chloroethylene glycol under phase transfer conditions19 in the
presence of powder sodium hydroxide and tetrabutylammonium
hydrogen sulfate afforded tetrahydropyranylated alcohol 13 in
80% yield. We did not try to deprotect the tetrahydropyranyl ether
before the glycosylation step, because cleavage must occur under
the glycosylation conditions. Reaction of cholesterol with ethyl
diazoacetate as described previously for 7 and 10 gave ester 14
(58% yield). This compound has formerly been synthesized in the
literature by using ethyl chloroacetate and benzene in the
presence of potassium.20,21 Ester 14 was reduced by lithium
aluminium deuteride (80% yield), and the chain elongation on
Experimental
General methods
Dichloromethane was washed twice with water, dried with CaCl2
and distilled from CaH2. THF and dioxane were distilled from
sodium–benzophenone. Methanol was distilled from magnesium.
Dioxane, THF and CH2Cl2 were stored over 0.4nm molecular
sieves, and MeOH was stored over 0.3 nm molecular sieves. Melt-
ing points were determined on a Büchi apparatus and were uncor-
rected. Thin layer chromatography was performed on aluminium
sheets coated with silica gel 60 F254 (Merck Darmstadt, Germany).
Compounds were visualized by spraying the thin layer
Scheme 1. Reagents and conditions: (a) HO(CH2CH2O)2H, dioxane, reflux; (b) N2CH2COOEt, CH2Cl2, À20 ꢀC; (c) LiAlD4, THF; (d) HOCH2CH2OH, dioxane, reflux;
(e):ClCH2CH2OTHP, Bu4NHSO4, powder NaOH, 65 ꢀC; (f): (ClCH2CH2O)2O, Bu4NHSO4 powder NaOH, 65 ꢀC; (g) NaOCHO, Bu4NBr, DMSO, 115 ꢀC and then 12.5 N NaOH, rt.
J. Label Compd. Radiopharm 2012, 55 88–95
Copyright © 2012 John Wiley & Sons, Ltd.
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D. Lafont et al.
Scheme 2. Syntheses of glycosides 2–5, reagents and conditions: (a) TMSOTf, CH2Cl2, À20 ꢀC; (b) Pd(PPh3)4, CH2(COOMe)2, THF; (c) Ac2O, MeOH (for 19–21) or (CD3CO)2O
(for 22), MeOH; (d) MeONa (cat), MeOH.
chromatography plates with dilute 15% aq H2SO4, followed by 50.24 (C-9chol), 42.38 (C-13chol), 39.86 (C-12chol), 39.60 (C-24chol),
charring at 150 ꢀC for a few minutes. Column chromatography 39.05 (C-4chol), 37.28 (C-1chol), 36.91 (C-10chol), 36.28 (C-22chol),
was performed on silica gel Geduran Si 60 (Merck). Optical rota- 35.87 (C-20chol), 31.95 (C-7chol), 31.90 (C-8chol), 28.38 (C-2chol), 28.32
tions were recorded on a Perkin Elmer (Waltham, Massachussets, (C-16chol), 28.07 (C-25chol), 24.37 (C-15chol), 23.94 (C-23chol), 22.91
USA) 241 polarimeter in a 1 dm cell at 21 ꢀC. 1H NMR and 13C NMR (C-27chol), 22.66 (C-26chol), 21.15 (C-11chol), 19.45 (C-19chol),
spectra were recorded with a Bruker (Wissembourg, France) 18.81 (C-21chol), 11.94 (C-18chol).
AC-200 spectrometer working at 200 MHz and 50 MHz, respectively,
with Me4Si as the internal standard. Elemental analyses were per-
Ethyl 8-(cholest-5-en-3b-yloxy)-3,6-dioxaoctanoate (8)
formed by the ‘Laboratoire Central d’Analyses du CNRS’ (Vernaison,
France). ESI high resolution mass spectrometry measurements were
performed with a MAT 95 XL (Thermoquest Finnigan, Bremen,
Germany) electromagnetic mass spectrometer by the ‘Centre
Commun de Spectrometry de Masse’ (UMR 5246, Lyon, France).
Boron trifluoride etherate (15 mL, 0.012 mmol) was added to a
cold solution (À20 ꢀC) of alcohol 7 (0.687 g, 1.45 mmol) and ethyl
diazoacetate (0.18 mL, 1.71 mmol, 1.18 equiv.) in dry CH2Cl2
(10 mL). The solution was stirred overnight at À20 ꢀC and then
poured into a saturated aqueous NaHCO3 solution (30 mL), and
the product was extracted with CH2Cl2 (2 Â 15 mL). The organic
phase was dried (Na2SO4) and concentrated under diminished
5-(Cholest-5-en-3b-yloxy)-3-oxapentanol (7)
Product 7 was prepared as already described from cholesteryl pressure. The crude product was purified by column chromato-
tosylate 6 (4.00 g, 7.40 mmol) and diethylene glycol (25 mL, graphy (1:5 ethyl acetate–petroleum ether). Compound 8 was
0.263 mol) in dry dioxane (45 mL).14 Product 7 was obtained in recovered as an oily material in 53% yield: 0.428 g, Rf 0.23 (1:5
81% yield as a white solid: mp 52 ꢀC (lit.14 mp 51–52 ꢀC), Rf 0.40 ethyl acetate–petroleum ether), [a]D À10.1 (c 1.0, CHCl3); 1H
(1:1 ethyl acetate–petroleum ether), [a]D À29.2 (c 1.0, CHCl3); NMR (CDCl3): d 5.35 (bd, 1H, H-6chol), 4.23 (q, 2H, J 7.1 Hz,
1H NMR (CDCl3): d 5.34 (bd, 1H, H-6chol), 3.71–3.60 (m, 8H, CH3CH2OCO), 4.15 (s, 2H, OCH2COOEt), 3.75–3.63 (m, 8H,
4CH2O), 3.20 (m, 1H, H-3chol), 2.79 (bs, 1H, OH), 2.40–0.67 (m, 4OCH2), 3.18 (m, 1H, H-3chol), 1.28 (t, 3H, CH3CH2O), 2.40–0.67
43H, H cholesterol); 13C NMR (CDCl3): d 140.78 (C-5chol), 121.77 (m, 43H, H cholesterol); 13C NMR (CDCl3): d 170.50 (COOEt),
(C-6chol), 79.66 (C-3chol), 72.69, 70.82 (CH2OCH2CH2OH), 67.48 140.97 (C-5chol), 121.57 (C-6chol), 79.55 (C-3chol), 70.95, 70.92,
(CH2OChol), 61.83 (CH2OH), 56.94 (C-14chol), 56.25 (C-17chol), 70.74, 68.79 (CH2OCH2CH2OCH2COOEt), 67.35 (CH2OChol), 60.78
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Copyright © 2012 John Wiley & Sons, Ltd.
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D. Lafont et al.
(COOCH2CH3), 56.84 (C-14chol), 56.23 (C-17chol), 50.25 (C-9chol), À24.4 (c 1.0, CHCl3); 1H NMR (CDCl3): d 5.33 (bd, 1H, H-6chol),
42.37 (C-13chol), 39.85 (C-12chol), 39.57 (C-24chol), 39.12 (C-4chol), 4.23 (q, 2H, J 7.1 Hz, COOCH2CH3), 4.17 (s, 2H, OCH2COOEt),
37.30 (C-1chol), 36.91 (C-10chol), 36.25 (C-22chol), 35.84 (C-20chol), 3.74–3.66 (m, 4H, OCH2CH2O), 3.19 (m, 1H, H-3chol), 1.29 (t, 3H,
31.97 (C-7chol), 31.92 (C-8chol), 28.41 (C-2chol), 28.28 (C-16chol), 28.05 COOCH2CH3), 2.40–0.67 (m, 43H, H cholesterol); 13C NMR (CDCl3):
(C-25chol), 24.34 (C-15chol), 23.89 (C-23chol), 22.86 (C-27chol), 22.61 d 170.43 (COOEt), 140.80 (C-5chol), 121.52 (C-6chol), 79.48 (C-3chol),
(C-26chol), 21.12 (C-11chol), 19.42 (C-19chol), 18.78 (C-21chol), 71.17, 68.72 (CH2OCH2COOEt), 67.32 (CH2OChol), 60.66
14.26 (COOCH2CH3), 11.91 (C-18chol).
(COOCH2CH3), 56.73 (C-14chol), 56.14 (C-17chol), 50.14 (C-9chol),
Anal. calc. for C35H60O5 (560.83): C, 74.95; H, 10.78. Found: C, 42.27 (C-13chol), 39.75 (C-12chol), 39.48 (C-24chol), 38.99
74.80; H, 10.92.
(C-4chol), 37.18 (C-1chol), 36.80 (C-10chol), 36.15 (C-22chol),
35.75 (C-20chol), 31.89 (C-7chol), 31.85 (C-8chol), 28.27 (C-2chol),
28.19 (C-16chol), 27.95 (C-25chol), 24.24 (C-15chol), 23.80
8-(Cholest-5-en-3b-yloxy)-1-[2H2]-3,6-dioxaoctan-1-ol (9)
A solution of ester 8 (0.400 g, 0.71 mmol) in THF (2 mL) was (C-23chol), 22.77 (C-27chol), 22.52 (C-26chol), 21.03 (C-11chol), 19.32
slowly added during 15 min. under argon to a suspension of (C-19chol), 18.68 (C-21chol), 14.17 (COOCH2CH3), 11.81 (C-18chol).
lithium aluminium deuteride (0.070 g, 1.67 mmol) in THF (3 mL).
Anal. calc. for C33H56O4 (516.78): C, 76.69; H, 10.92. Found: C,
Stirring was maintained overnight, and the excess of lithium 76.37; H, 10.78.
aluminium deuteride was destroyed by careful addition of a
5-(Cholest-5-en-3b-yloxy)-1-[2H2]-3-oxapentan-1-ol (12)
15% NaOH aqueous solution. Then, the mixture was acidified
to pH 5–6, and THF was evaporated in vaccuo. The product was Compound 12 was obtained as described previously from
extracted with diethyl ether (4 Â 30 mL), and the organic phase compound 9, starting from ester 11 (0.600 g, 1.56 mmol) and
was washed with water (20 mL) and dried. After concentration, lithium aluminium deuteride (0.130 g, 3.12 mmol). Pure
the residue was purified by column chromatography by using compound 12 was recovered as an oily material in 75% yield
pure ethyl acetate as eluent. Pure compound 9 was recovered after purification by column chromatography (3:2 ethyl
in 62% yield as an amorphous solid: 0.235 g, Rf 0.45 (ethyl acetate–petroleum ether): 0.422 g, Rf 0.52 (1:1 ethyl acetate–
1
1
acetate), [a]D À23.9 (c 1.0, CHCl3); H NMR (CDCl3): d 5.35 (bd, petroleum ether), [a]D À28.4 (c 1.0, CHCl3); H NMR (CDCl3): d
1H, H-6chol), 3.67–3.61 (m, 10H, 5CH2O), 3.19 (m, 1H, H-3chol), 5.36 (bd, 1H, H-6chol), 3.68–3.62 (m, 6H, 3CH2O), 3.18 (m, 1H,
2.40–0.67 (m, 43H, H cholesterol); 13C NMR (CDCl3): d 140.88 H-3chol), 2.40–0.67 (m, 43H, H cholesterol); 13C NMR (CDCl3): d
(C-5chol), 121.58 (C-6chol), 79.54 (C-3chol), 72.54, 70.88, 70.67, 70.39 140.80 (C-5chol), 121.71 (C-6chol), 79.46 (C-3chol), 72.48, 70.84
(CH2OCH2CH2OCH2), 67.28 (CH2OChol), 60.95 (q, J 21.2 Hz, CD2), (CH2OCH2), 67.45 (CH2OChol), 61.18 (q, J 21.0 Hz, CD2), 56.83
56.81 (C-14chol), 56.24 (C-17chol), 50.22 (C-9chol), 42.35 (C-13chol), (C-14chol), 56.23 (C-17chol), 50.23 (C-9chol), 42.36 (C-13chol),
39.84 (C-12chol), 39.56 (C-24chol), 39.06 (C-4chol), 37.28 (C-1chol), 36.87 39.83 (C-12chol), 39.56 (C-24chol), 39.03 (C-4chol), 37.25 (C-1chol),
(C-10chol), 36.26 (C-22chol), 35.85 (C-20chol), 31.97 (C-7chol), 31.93 36.97 (C-10chol), 36.24 (C-22chol), 35.82 (C-20chol), 31.98
(C-8chol), 28.35 (C-2chol), 28.29 (C-16chol), 28.03 (C-25chol), 24.35 (C-7chol), 31.93 (C-8chol), 28.375 (C-2chol), 28.26 (C-16chol), 28.03
(C-15chol), 23.92 (C-23chol), 22.88 (C-27chol), 22.63 (C-26chol), (C-25chol), 24.32 (C-15chol), 23.68 (C-23chol), 22.85 (C-27chol),
21.02 (C-11chol), 19.41 (C-19chol), 18.79 (C-21chol), 11.91 (C-18chol).
22.60 (C-26chol), 21.11 (C-11chol), 19.41 (C-19chol), 18.76 (C-21chol),
Anal. calc. for C33H56D2O4,H2O (538.46): C, 73.55; H, 11.58. 11.89 (C-18chol).
Found: C, 73.27; H, 11.18.
Anal. calc. for C31H52D2O3,0.5H2O (485.77): C, 76.64; H, 11.00.
Found: C, 76.94; H, 11.19.
2-(Cholest-5-en-3b-yloxy)-ethanol (10)
8-(Cholest-5-en-3b-yloxy)-4-[2H2]-1-tetrahydropyranyl-3,6-diox-
aoctan-1-ol (13)
Product 10 was prepared from cholesteryl tosylate 6 (5.90 g,
10.9 mmol) and ethylene glycol (40 mL, 0.717 mol) in dry dioxane
(60 mL) as already described.14 Product 10 was obtained in 72% A mixture of compound 12 (0.360 g, 0.756 mmol), tetrahydro-
yield as a white solid: mp 96–97 ꢀC (lit.14 mp 96–97 ꢀC), Rf 0.62 pyranylated 2-chloroethanol (0.520 g, 3.50 mmol, 4.63 equiv.),
(1:1 ethyl acetate–petroleum ether), [a]D À30.1 (c 1.0, CHCl3); tetrabutylammonium hydrogen sulfate (0.167 g, 0.49 mmol)
1H NMR (CDCl3): d 5.35 (bs, 1H, H-6chol), 3.75–3.71 (m, 2H, and powder sodium hydroxide (0.152 g, 3.80 mmol) was
CH2OChol), 3.60–3.56 (m, 2H, CH2OH), 3.20 (3, 1H, H-3chol), stirred at 65 ꢀC for 2 h. After cooling to room temperature,
2.40–0.67 (m, 43H, H cholesterol); 13C NMR (CDCl3): d 140.38 the mixture was poured into water, and the product was
(C-5chol), 121.84 (C-6chol), 80.55 (C-3chol), 69.13 (CH2OChol), extracted with dichloromethane (2 Â 50 mL). The organic
62.14 (CH2OH), 56.87 (C-14chol), 56.28 (C-17chol), 50.27 (C-9chol), phase was washed with water (20 mL), dried (Na2SO4) and
42.41 (C-13chol), 39.88 (C-12chol), 39.62 (C-24chol), 39.20 (C-4chol), concentrated to afford a crude product, which was purified
37.28 (C-1chol), 39.95 (C-10chol), 36.30 (C-22chol), 35.90 by column chromatography by using 1:2 ethyl acetate–
(C-20chol), 32.07 (C-7chol), 31.95 (C-8chol), 28.50 (C-2chol), petroleum ether as eluent. Product 13 was recovered in a pure
28.34 (C-16chol), 28.11 (C-25chol), 24.39 (C-15chol), 23.51 form as an oily material in 80% yield: 0.357 g, Rf 0.53 (1:2 ethyl
(C-23chol), 22.92 (C-27chol), 22.67 (C-26chol), 21.18 (C-11chol), acetate–petroleum ether), [a]D À20.9 (c 1.0, CHCl3); 1H NMR
19.47 (C-19chol), 18.82 (C-21chol), 11.96 (C-18chol).
(CDCl3): d 5.34 (bd, 1H, H-6chol), 4.63 (dd, 1H, OCHTHP), 3.95–3.45
(m, 12H, 5CH2O, OCH2THP), 3.18 (m, 1H, H-3chol), 2.40–0.67 (m, 49H,
3CH2THP, H cholesterol); 13C NMR (CDCl3): d 140.97 (C-5chol), 121.50
Ethyl 5-(cholest-5-en-3b-yloxy)-3-oxapentanoate (11)
Boron trifluoride etherate (12 mL, 0.010 mmol) was added to a (C-6chol), 79.48 (C-3chol), 70.92, 70.53, 70.46 (CH2OCH2CD2OCH2),
cold solution (À20 ꢀC) of alcohol 10 (0.430 g, 1.00 mmol) and 69.86 (q, J 21.4 Hz, CD2), 67.32 (CH2OChol), 66.65 (CH2OTHP), 62.04
ethyl diazoacetate (0.11 mL, 1.10 mmol, 1.10 equiv.) in dry CH2Cl2 (OCH2THP), 56.78 (C-14chol), 56.20 (C-17chol), 50.20 (C-9chol), 42.31
(10 mL). The reaction was treated as described previously, and (C-13chol), 39.80 (C-12chol), 39.52 (C-24chol), 39.08 (C-4chol), 37.26
pure compound 11 was recovered as an oily material in 56% (C-1chol), 36.83 (C-10chol), 36.21 (C-22chol), 35.79 (C-20chol), 31.94
yield: 0.289 g, Rf 0.48 (1:5 ethyl acetate–petroleum ether), [a]D (C-7chol), 31.89 (C-8chol), 30.55 (CH2THP), 28.36 (C-2chol), 28.22
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D. Lafont et al.
(C-16chol), 27.98 (C-25chol), 25.47 (CH2THP), 24.29 (C-15chol), 23.86 organic phase was washed with water (2 Â 20 mL) and dried
(C-23chol), 22.83 (C-27chol), 22.58 (C-26chol), 21.08 (C-11chol), 19.44 (C- (Na2SO4). The crude residue obtained after concentration
19chol), 19.39 (CH2THP), 18.74 (C-21chol), 11.86 (C-18chol).
under diminished pressure was purified by column chromatogra-
Anal. calc. for C38H64D2O5 (604.94): C, 75.45; H, 11.33. Found: C, phy (1:3 ethyl acetate–petroleum ether). Pure product 16 was
75.28; H, 11.27.
recovered as an oily material in 65% yield: 0.817 g, Rf 0.55 (1:3 ethyl
acetate–petroleum ether), [a]D À21.2 (c 1.0, CHCl3); 1H NMR
(CDCl3): d 5.35 (bd, 1H, H-6chol), 3.79 (t, 2H, CH2Cl), 3.64–3.54
Ethyl 2-(cholest-5-en-3b-yloxy)-ethanoate (14)
Boron trifluoride etherate (64 mL, 0.52 mmol) was added to a cold (m, 8H, OCH2CH2OCH2CH2OChol), 3.18 (m, 1H, H-3chol), 2.40–0.67
solution (À20 ꢀC) of cholesterol (2.00 g, 5.17 mmol) and ethyl dia- (m, 43H, H cholesterol); 13C NMR (CDCl3): d 140.93 (C-5chol),
zoacetate (0.60 mL, 5.70 mmol, 1.10 equiv.) in dry CH2Cl2 (10 mL). 121.53 (C-6chol), 79.48 (C-3chol), 71.37, 70.75, 70.70.71 (4 CH2O),
The solution was stirred overnight at À20 ꢀC and then poured 70.23 (q, J 21.0 Hz, CD2), 67.16 (CH2OChol), 56.77 (C-14chol), 56.16
into a saturated aqueous NaHCO3 solution (30 mL), and the (C-17chol), 50.18 (C-9chol), 42.67 (CH2Cl), 42.31 (C-13chol), 39.78
product was extracted with CH2Cl2 (2 Â 25 mL). The organic (C-12chol), 39.51 (C-24chol), 39.07 (C-4chol), 37.24 (C-1chol),
phase was dried (Na2SO4) and concentrated under diminished 36.86 (C-10chol), 36.19 (C-22chol), 35.78 (C-20chol), 31.94
pressure. The crude product was purified by column chromato- (C-7chol), 31.89 (C-8chol), 28.35 (C-2chol), 28.23 (C-16chol), 28.00
graphy (1:10 ethyl acetate–petroleum ether). Compound 14 (C-25chol), 24.29 (C-15chol), 23.83 (C-23chol), 22.82 (C-27chol),
was recovered as an amorphous solid in 58% yield: 1.12 g, Rf 22.57 (C-26chol), 21.07 (C-11chol), 19.37 (C-19chol), 18.72 (C-21chol),
0.60 (1:10 ethyl acetate–petroleum ether), [a]D À30.9 (c 1.0, 11.99 (C-18chol).
CHCl3); 1H NMR (CDCl3): d 5.34 (bd, 1H, H-6chol), 4.16 (q, 2H, J
Anal. calc. for C33H55ClD2O3 (539.25): C, 73.49; H, 11.02. Found:
7.1 Hz, CH3CH2OCO), 4.11 (s, 2H, OCH2COOEt), 3.19 (m, 1H, C, 72.91; H, 10.44.
H-3chol), 2.40–0.67 (m, 43H, H cholesterol), 1.28 (t, 3H, CH3CH2O);
8-(Cholest-5-en-3b-yloxy)-7-[2H2]-3,6-dioxaoctan-1-ol (17)
13C NMR (CDCl3): d 170.86 (COOEt), 140.53 (C-5chol), 121.94
(C-6chol), 80.035 (C-3chol), 65.76 (CH2OChol), 60.78 (COOCH2CH3),
56.80 (C-14chol), 56.22 (C-17chol), 50.21 (C-9chol), 42.35 (C-13chol), 39.80
(C-12chol), 39.567 (C-24chol), 38.75 (C-4chol), 37.19 (C-1chol), 36.84
(C-10chol), 36.24 (C-22chol), 35.82 (C-20chol), 31.92 (C-7chol),
31.92 (C-8chol), 28.26 (C-2chol), 28.12 (C-16chol), 28.03 (C-25chol),
24.32 (C-15chol), 23.88 (C-23chol), 22.85 (C-27chol), 22.60
(C-26chol), 21.11 (C-11chol), 19.38 (C-19chol), 18.76 (C-21chol),
14.25 (COOCH2CH3), 11.89 (C-18chol).
A mixture of compound 16 (0.800 g, 1.48 mmol), sodium formate
(0.250 g, 3.68 mmol) and tetrabutylammonium bromide (0.030 g,
0.09 mmol) in DMSO was stirred at 115 ꢀC for 24 h. After cooling
to room temperature, 12.5 M sodium hydroxide (0.23 mL,
2.95 mmol) was added, and stirring was maintained overnight
at room temperature. The crude product was obtained after
addition of water (10 mL), extraction with dichloromethane
(2 Â 30 mL), drying (Na2SO4) and concentration of the organic
phase. Purification was realized by column chromatography
(2:1 ethyl acetate–petroleum ether). Compound 17 was obtained
as an oily material in 55% yield: 0.425 g, Rf 0.53 (2:1 ethyl acet-
Anal. calc. for C31H52O3 (472.73): C, 78.76; H, 11.09. Found: C,
78.43; H, 11.25.
2-(Cholest-5-en-3b-yloxy)-1-[2H2]-ethano1 (15)
1
ate–petroleum ether), [a]D À21.9 (c 1.0, CHCl3); H NMR (CDCl3):
Compound 15 was prepared as described previously for 8 from
ester 14 (1.70 g, 3.60 mmol) and lithium aluminium deuteride
(0.300 g, 7.20 mmol). The crude reaction product was purified
by column chromatography by using 1:1 ethyl acetate–petro-
leum ether as eluent. Pure compound 15 was recovered in
82% yield as a solid: 1.28 g, Rf 0.60 (1:1 ethyl acetate–
d 5.34 (bd, 1H, H-6chol), 3.70–3.63 (m, 10H, 5CH2O), 3.18 (m, 1H,
H-3chol), 2.40–0.67 (m, 43H, H cholesterol); 13C NMR (CDCl3): d
140.85 (C-5chol), 121.54 (C-6chol), 79.51 (C-3chol), 72.56, 70.53,
70.34 (CH2OCH2CH2O), 70.00 (q, J 21.2 Hz, CD2), 67.07 (CH2OChol),
61.68 (CH2OH), 56.74 (C-14chol), 56.15 (C-17chol), 50.17 (C-9chol),
42.33 (C-13chol), 39.77 (C-12chol), 39.50 (C-24chol), 38.98
(C-4chol), 37.20 (C-1chol), 36.83 (C-10chol), 36.18 (C-22chol),
35.76 (C-20chol), 31.91 (C-7chol), 31.87 (C-8chol), 28.28 (C-2chol),
28.20 (C-16chol), 27.97 (C-25chol), 24.26 (C-15chol), 23.82
(C-23chol), 22.79 (C-27chol), 22.54 (C-26chol), 21.05 (C-11chol), 19.35
(C-19chol), 18.70 (C-21chol), 11.84 (C-18chol).
1
petroleum ether), mp 98 ꢀC, [a]D À29.1 (c 1.0, CHCl3); H NMR
(CDCl3): d 5.33 (bd, 1H, H-6chol), 3.57 (s, 2H, CH2OChol), 3.18
(m, 1H, H-3chol), 2.40À0.67 (m, 43H, H cholesterol); 13C NMR
(CDCl3): d 140.64 (C-5chol), 121.70 (C-6chol), 79.44 (C-3chol),
68.95 (CH2OChol), 61.28 (q, J 21.1 Hz, CD2), 56.76 (C-14chol),
56.19 (C-17chol), 50.18 (C-9chol), 42.30 (C-13chol), 39.79
(C-12chol), 39.52 (C-24chol), 39.08 (C-4chol), 37.19 (C-1chol),
36.83 (C-10chol), 36.20 (C-22chol), 35.79 (C-20chol), 31.91
(C-7chol), 31.88 (C-8chol), 28.38 (C-2chol), 28.32 (C-16chol),
27.99 (C-25chol), 24.28 (C-15chol), 23.86 (C-23chol), 22.80 (C-27chol),
22.55 (C-26chol), 21.07 (C-11chol), 19.35 (C-19chol), 18.79
(C-21chol), 11.85 (C-18chol).
Anal. calc. for C33H56D2O4,0.75H2O (534.61): C, 74.14; H, 11.59.
Found: C, 74.14; H, 11.21.
General method for the glycosylation of deuterated
cholesteryl derivatives
Trimethylsilyl trifluoromethanesulfonate (1.05 equiv.) was added
Anal. calc. for C29H48D2O2,0.5H2O (441.73): C, 78.85; H, 12.09. at À20 ꢀC under argon to a mixture of 1,3,4,6-tetra-O-acetyl-
Found: C, 78.91; H, 11.84.
2-allyloxycarbonylamino-2-deoxy-b-D-glucopyranose (18)23,24
(1–1.05 equiv) and alcohol 9 (or 17) or tetrahydropyranylated
alcohol 13 (1 equiv.) in dry alcohol-free CH2Cl2 (15 mL/mmol).
1-Chloro-8-(cholest-5-en-3b-yloxy)-7-[2H2]-3,6-dioxaoctane (16)
A mixture of compound 15 (1.00 g, 2.26 mmol), 2-chloroethyl The mixture was stirred for 16 h at À20 ꢀC and then poured into
ether (1.60 mL, 13.65 mmol), powder sodium hydroxide a saturated aqueous NaHCO3 solution (30 mL); the aqueous
(0.550 g, 13.75 mmol) and tetrabutylammonium hydrogen phase was extracted with CH2Cl2 (2 Â 20 mL), and the organic
sulfate (0.550 g, 1.62 mmol) was heated for 1 h at 65 ꢀC with phase was dried and concentrated under diminished pressure.
vigorous stirring. After cooling to room temperature, the The crude product was purified by column chromatography (2:1
mixture was diluted with dichloromethane (80 mL), and the EtOAc–petroleum ether) to afford the pure glycosides.
www.jlcr.org
Copyright © 2012 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 88–95

D. Lafont et al.
[8-(Cholest-5-en-3b-yloxy)-1-[2H2]-3,6-dioxaoctyl] 3,4,6-tri-O-acetyl-2- 0.70 (2:1 ethyl acetate–petroleum ether), [a]D À17.2 (c 1.0, CHCl3);
allyloxy-carbonylamino-2-deoxy-b-D-glucopyranoside (19)
1H NMR (CDCl3): d 5.89 (m, 1H, CH), 5.75 (m, 1H, NH), 5.34 (bd, 1H,
H-6chol), 5.24–5.09 (m, 3H, CH2=, H-3), 5.05 (dd, 1H, J3,4 9.3 Hz, J4,5
9.7 Hz, H-4), 4.79 (d, 1H, J1,2 8.4 Hz, H-1), 4.56 (m, 2H, allyl CH2),
4.27 (dd, 1H, J5,6a 4.7 Hz, J6a,6b 12.3 Hz, H-6a), 4.12 (dd, 1H, J5,6b
2.0 Hz, H-6b), 3.95–3.60 (m, 12H, 5CH2O, H-2, H-5), 3.18 (m, 1H,
H-3chol), 2.17, 2.08, 2.01 (3s, 9H, 3CH3COO), 2.40–0.67 (m, 43H,
H cholesterol); 13C NMR (CDCl3): d 170.76, 170.51, 169.48
(CH3COO), 156.25 (NHCOO), 140.91 (C-5chol), 132.88 (CH2=CH),
121.60 (C-6chol), 117.26 (CH2=), 101.90 (C-1), 79.48 (C-3chol), 73.01
(C-3), 71.68 (C-5), 70.66, 70.57, 70.42 (CH2CH2OCH2CH2OCH2), 70.03
(q, J 21.2 Hz, CD2), 68.81 (C-4), 67.32 (CH2OChol), 65.50 (allyl CH2),
62.21 (C-6), 56.78 (C-14chol), 56.16 (C-17chol), 55.97 (C-2), 50.20
(C-9chol), 42.32 (C-13chol), 39.72 (C-12chol), 39.52 (C-24chol),
39.08 (C-4chol), 37.23 (C-1chol), 36.8775 (C-10chol), 36.20
(C-22chol), 35.78 (C-20chol), 31.95 (C-7chol), 31.90 (C-8chol),
28.36 (C-2chol), 28.24 (C-16chol), 28.00 (C-25chol), 24.29 (C-15chol),
23.83 (C-23chol), 22.83 (C-27chol), 22.57 (C-26chol), 21.08
(C-11chol), 20.78, 20.72, 20.65 (CH3COO), 19.40 (C-19chol),
18.73 (C-21chol), 11.87 (C-18chol).
Compound 19 was obtained in 79% yield as described pre-
viously from alcohol 9 (0.520 g, 1.00 mmol) and donor 18
(0.452 mmol, 1.05 mmol): 0.705 g, Rf 0.70 (2:1 ethyl acetate–
petroleum ether), [a]D À17.0 (c 1.0, CHCl3); 1H NMR (CDCl3): d
5.89 (m, 1H, CH), 5.78 (m, 1H, NH), 5.34 (bd, 1H, H-6chol), 5.30–5.13
(m, 3H, CH2=, H-3), 5.10 (dd, 1H, J3,4 9.3 Hz, J4,5 9.9 Hz, H-4), 4.80
(d, 1H, J1,2 8.5 Hz, H-1), 4.57 (m, 2H, allyl CH2), 4.27 (dd, 1H, J5,6a
4.6 Hz, J6a,6b 12.2 Hz, H-6a), 4.12 (dd, 1H, J5,6b 2.2 Hz, H-6b),
3.95–3.55 (m, 12H, 5CH2O, H-2, H-5), 3.18 (m, 1H, H-3chol),
2.16, 2.08, 2.01 (3s, 9H, 3CH3COO), 2.40–0.67 (m, 43H, H cholesterol);
13C NMR (CDCl3): d 170.74, 170.49, 169.46 (CH3COO), 156.23
(NHCOO), 140.90 (C-5chol), 132.93 (CH2=CH), 121.58 (C-6chol),
117.25 (CH2=), 101.87 (C-1), 79.43 (C-3chol), 73.01 (C-3), 71.68
(C-5), 70.66, 70.57 (CH2OCH2CH2OCH2), 70.03 (q, J 21.2 Hz,
CD2), 68.82 (C-4), 67.33 (CH2OChol), 65.49 (allyl CH2), 62.21
(C-6), 56.78 (C-14chol), 56.16 (C-17chol), 55.96 (C-2), 50.21 (C-9chol),
42.32 (C-13chol), 39.75 (C-12chol), 39.52 (C-24chol), 39.08
(C-4chol), 37.24 (C-1chol), 36.87 (C-10chol), 36.19 (C-22chol),
35.78 (C-20chol), 31.95 (C-7chol), 31.90 (C-8chol), 28.37 (C-2chol),
28.22 (C-16chol), 28.01 (C-25chol), 24.29 (C-15chol), 23.82
(C-23chol), 22.83 (C-27chol), 22.58 (C-26chol), 21.08 (C-11chol),
20.78, 20.72, 20.65 (CH3COO), 19.40 (C-19chol), 18.73 (C-21chol),
11.87 (C-18chol).
Anal. calc. for C49H77D2NO13 (892.145): C, 65.96; H, 9.15; N 1.57.
Found: C, 65.82; H, 9.07; N: 1.53.
General procedure for the preparation of compounds 23–26
Tris(dibenzylideneacetone)dipalladium (0.016 g, 0.0168 mmol)
and PPh3 (0.050 g, 0.190 mmol) were reacted for 10 min in dry
oxygen-free THF (2 mL) under argon. The solution was added
to a solution of the N-allyloxycarbonyl derivatives 19–22
(0.40 mmol) and diethyl malonate (0.80 mL) in dry THF (2–3 mL),
and the mixture was stirred for 16 h under argon. After concen-
tration, the residue was eluted on a short column of silica gel to
separate the free amino derivative, which was acetylated with
Ac2O (1.5 equiv.) in dry MeOH (4 mL) (from 19 to 21) or with
(CD3CO)2O in MeOH (from 22). The pure peracetylated deriva-
tives 23–26 were obtained after concentration and purification
of the residue by column chromatography by using 5:1 ethyl
acetate–acetone mixture as eluent.
Anal. calc. for C49H77D2NO13 (892.145): C, 65.96; H, 9.15; N 1.57.
Found: C, 65.71; H, 9.05; N: 1.78.
[8-(Cholest-5-en-3b-yloxy)-4-[2H2]-3,6-dioxaoctyl] 3,4,6-tri-O-acetyl-2-
allyloxy-carbonylamino-2-deoxy-b-D-glucopyranoside (20)
Compound 20 was obtained in 75% yield as described pre-
viously from tetrahydropyranylated derivative 13 (0.362 g,
0.60mmol) and donor 18 (0.265 g, 0.62 mmol): 0.401 g, mp 110 ꢀC
(petroleum ether), Rf 0.70 (2:1 ethyl acetate–petroleum ether), [a]D
À19.2 (c 1.0, CHCl3); 1H NMR (CDCl3): d 5.89 (m, 1H, CH), 5.78
(m, 1H, NH), 5.34 (bd, 1H, H-6chol), 5.24–5.09 (m, 3H, CH2=, H-3),
5.05 (dd, 1H, J3,4 9.3 Hz, J4,5 9.8 Hz, H-4), 4.79 (d, 1H, J1,2 8.5 Hz,
H-1), 4.57 (m, 2H, allyl CH2), 4.27 (dd, 1H, J5,6a 4.7 Hz, J6a,6b
12.2 Hz, H-6a), 4.11 (dd, 1H, J5,6b 2.0 Hz, H-6b), 3.95–3.60
(m, 12H, 5CH2O, H-2, H-5), 3.19 (m, 1H, H-3chol), 2.16, 2.08,
2.01 (3s, 9H, 3CH3COO), 2.40–0.67 (m, 43H, H cholesterol); 13C
NMR (CDCl3): d 170.55, 170.28, 169.35 (CH3COO), 156.16
(NHCOO), 140.73 (C-5chol), 132.93 (CH2=CH), 121.46 (C-6chol),
117.06 (CH2=), 101.62 (C-1), 79.32 (C-3chol), 72.92 (C-3), 71.52
(C-5), 70.96, 70.67, 70.42 (CH2CH2OCD2CH2OCH2), 69.83 (q, J
21.2 Hz, CD2), 68.82 (C-4), 67.32 (CH2OChol), 65.30 (allyl CH2),
62.14 (C-6), 56.68 (C-14chol), 56.09 (C-17chol), 55.81 (C-2), 50.10
(C-9chol), 42.23 (C-13chol), 39.71 (C-12chol), 39.44 (C-24chol),
39.00 (C-4chol), 37.16 (C-1chol), 36.75 (C-10chol), 36.12 (C-22chol),
35.72 (C-20chol), 31.85 (C-7chol), 31.81 (C-8chol), 28.27 (C-2chol), 28.17
(C-16chol), 27.90 (C-25chol), 24.22 (C-15chol), 23.77 (C-23chol), 22.78
(C-27chol), 22.53 (C-26chol), 21.01 (C-11chol), 20.69, 20.60, 20.53
(CH3COO), 19.31 (C-19chol), 18.68 (C-21chol), 11.81 (C-18chol).
[8-(Cholest-5-en-3b-yloxy)-1-[2H2]-3,6-dioxaoctyl] 2-acetamido-3,4,6-
tri-O-acetyl-2-deoxy-b-D-glucopyranoside (23)
Compound 23 was obtained in 80% yield as described pre-
viously from glycoside 19 (0.357 g, 0.40 mmol): 0.271 g, Rf 0.55
(5:1 ethyl acetate–acetone), mp 126 ꢀC, [a]D À26.6 (c 1.0, CHCl3);
1H NMR (CDCl3): d 6.70 (d, 1H, J2,NH 9.3 Hz, NH), 5.31 (bd, 1H,
H-6chol), 5.11–5.02 (m, 2H, H-3, H-4), 4.79 (d, 1H, J1,2 8.6 Hz, H-1),
4.25 (dd, 1H, J5,6a 4.6 Hz, J6a,6b 12.2 Hz, H-6a), 4.10 (dd, 1H, J5,6b
2.2 Hz, H-6b), 4.09–4.04 (m, 1H, H-2), 3.80–5.55 (m, 11H,
5CH2O, H-5), 3.15 (m, 1H, H-3chol), 2.07, 1.99, 1.99, 1.97 (4s,
12H, 4CH3CO), 2.34–0.66 (m, 43H, H cholesterol); 13C NMR
(CDCl3): d 170.92, 170.84, 170.82, 169.47 (CH3CO), 140.87
(C-5chol), 121.88 (C-6chol), 102.13 (C-1), 79.60 (C-3chol), 73.56
(C-3), 71.84 (C-5), 71.76, 71.04, 70.87, 70.83 (CH2OCH2-
CH2OCH2), 68.85 (C-4), 67.45 (CH2OChol), 62.34 (C-6), 56.89
(C-14chol), 56.28 (C-17chol), 53.96 (C-2), 50.33 (C-9chol), 42.45
(C-13chol), 39.90 (C-12chol), 39.63 (C-24chol), 39.29 (C-4chol),
37.33 (C-1chol), 36.99 (C-10chol), 36.27 (C-22chol), 35.91 (C-20chol),
32.06 (C-7chol), 32.02 (C-8chol), 28.43 (C-2chol), 28.36 (C-16chol), 28.14
Anal. calc. for C49H77D2NO13 (892.145): C, 65.96; H, 9.15; N 1.57.
Found: C, 65.68; H, 9.00; N: 1.69.
[8-(Cholest-5-en-3b-yloxy)-7-[2H2]-3,6-dioxaoctyl] 3,4,6-tri-O-acetyl-2-
allyloxy-carbonylamino-2-deoxy-b-D-glucopyranoside (21)
Compound 21 was obtained in 75% yield as described pre- (C-25chol), 24.41 (C-15chol), 23.93 (C-23chol), 23.23 (NHCOCH3), 22.96
viously from alcohol 17 (0.362 g, 0.60 mmol) and donor 18 (C-27chol), 22.70 (C-26chol), 21.18 (C-11chol), 20.91, 20.87, 20.78
(0.265 g, 0.62 mmol): 0.401 g, mp 109 ꢀC (petroleum ether), Rf (CH3COO), 19.52 (C-19chol), 18.85 (C-21chol), 12.00 (C-18chol).
J. Label Compd. Radiopharm 2012, 55 88–95
Copyright © 2012 John Wiley & Sons, Ltd.
www.jlcr.org

D. Lafont et al.
Anal. calc. for C47H75D2NO12 (850.105): C, 66.40; H, 9.36; N 1.65. 2.0 Hz, H-6b), 4.07–4.03 (m, 1H, H-2), 3.85–3.55 (m, 13H, 6CH2O,
Found: C, 66.35; H, 9.17; N: 1.64.
H-5), 3.15 (m, 1H, H-3chol), 2.05, 1.97, 1.97 (3s, 9H, 3CH3CO),
2.35–0.65 (m, 43H, H cholesterol); 13C NMR (CDCl3): d 170.82,
170.82, 170.69, 169.40 (CH3CO), 140.78 (C-5chol), 121.79
(C-6chol), 102.07 (C-1), 79.52 (C-3chol), 73.48 (C-3), 71.75 (C-5),
[8-(Cholest-5-en-3b-yloxy)-4-[2H2]-3,6-dioxaoctyl] 2-acetamido-3,4,6-
tri-O-acetyl-2-deoxy-b-D-glucopyranoside (24)
Compound 24 was obtained in 75% yield as described pre- 71.75, 70.95, 70.77, 70.72, 68.72 (CH2OCH2CH2OCH2CH2),
viously from glycoside 20 (0.350 g, 0.39 mmol): 0.250 g, Rf 0.55 68.81 (C-4), 67.35 (CH2OChol), 62.28 (C-6), 56.81 (C-14chol), 56.20
(5:1 ethyl acetate–acetone), mp 127 ꢀC, [a]D À28.4 (c 1.0, CHCl3); (C-17chol), 53.86 (C-2), 50.25 (C-9chol), 42.37 (C-13chol), 39.82
1H NMR (CDCl3): d 6.73 (d, 1H, J2,NH 9.2 Hz, NH), 5.33 (bd, 1H, (C-12chol), 39.57 (C-24chol), 39.21 (C-4chol), 37.26 (C-1chol),
H-6chol), 5.12–5.03 (m, 2H, H-3, H-4), 4.80 (d, 1H, J1,2 8.6 Hz, H-1), 36.91 (C-10chol), 36.25 (C-22chol), 35.84 (C-20chol), 31.98 (C-7chol),
4.25 (dd, 1H, J5,6a 4.6 Hz, J6a,6b 12.2Hz, H-6a), 4.11 (dd, 1H, J5,6b 31.94 (C-8chol), 28.35 (C-2chol), 28.29 (C-16chol), 28.06
2.2 Hz, H-6b), 4.10–4.07 (m, 1H, H-2), 3.91–3.58 (m, 11H, (C-25chol), 24.34 (C-15chol), 23.88 (C-23chol), 22.90 (C-27chol),
5CH2O, H-5), 3.17 (m, 1H, H-3chol), 2.08, 2.00, 2.00, 1.97 (4s, 22.64 (C-26chol), 21.14 (C-11chol), 20.83, 20.79, 20.70
12H, 4CH3CO), 2.40–0.67 (m, 43H, H cholesterol); 13C NMR (CDCl3): (CH3COO), 19.45 (C-19chol), 18.79 (C-21chol), 11.93 (C-18chol).
d 170.99, 170.99, 170.89, 169.52 (CH3CO), 140.68 (C-5chol), 121.93
Anal. calc. for C47H74D3NO12 (851.13): C, 66.32; H, 9.47; N 1.65.
(C-6chol), 102.22 (C-1), 79.66 (C-3chol), 73.60 (C-3), 71.89 (C-5), Found: C, 66.05; H, 9.29; N: 1.68.
71.84, 70.89, 70.79, 68.88 (OCH2CH2CH2OCD2CH2OCH2), 68.88
(C-4), 67.49 (CH2OChol), 62.37 (C-6), 56.93 (C-14chol), 56.32
(C-17chol), 53.91 (C-2), 50.37 (C-9chol), 42.49 (C-13chol), 39.94
(C-12chol), 39.69 (C-24chol), 39.32 (C-4chol), 37.37 (C-1chol),
37.03 (C-10chol), 36.36 (C-22chol), 35.95 (C-20chol), 32.10
(C-7chol), 32.05 (C-8chol), 28.46 (C-2chol), 28.40 (C-16chol), 28.19
(C-25chol), 24.46 (C-15chol), 23.99 (C-23chol), 23.28 (NHCOCH3),
23.00 (C-27chol), 22.74 (C-26chol), 21.25 (C-11chol), 20.97, 20.92,
20.83 (CH3COO), 19.57 (C-19chol), 18.90 (C-21chol), 12.04
(C-18chol).
General procedure for the cleavage of the O-deacetylation of
compounds 23–26
A solution of compounds 23–26 (0.200 g, 0.23 mmol) in MeOH
(25 mL) containing a chip of sodium was stirred overnight at
room temperature. After neutralization with Amberlyst IR 120
[H+] and filtration, the solvent was evaporated under diminished
pressure to afford products 2–5.
[8-(Cholest-5-en-3b-yloxy)-1-[2H2]-3,6-dioxaoctyl] 2-acetamido-2-deoxy-
b-D-glucopyranoside (2)
Anal. calc. for C47H75D2NO12 (850.105): C, 66.40; H, 9.36; N 1.65.
Found: C, 66.17; H, 9.21; N: 1.66.
Product 2 was obtained in 92% yield as described previously
from glycoside 23 (0.200 g, 0.23 mmol): 0.156 g, Rf 0.62
(67:25:8 ethyl acetate–ethanol–water), amorphous solid, [a]D
À45.1 (c 1.0, CHCl3); 1H NMR (C5D5N): d 8.82 (d, 1H, J2,NH
8.5 Hz, NH), 5.43 (bd, 1H, H-6chol), 5.10 (d, 1H, J1,2 8.4 Hz, H-1),
4.55 (ddd, 1H, J2,3 10.1 Hz, H-2), 4.54 (dd, 1H, J5,6a 2.5 Hz, J6a,6b
12.1 Hz, H-6a), 4.39–4.33 (m, 2H, H-3, H-6b), 4.21 (dd, 1H, J3,4
8.6 Hz, J4,5 9.5 Hz, H-4), 3.91 (ddd, 1H, J5,6b 5.6 Hz H-5), 3.72–3.70
(m, 10H, 5OCH2), 3.28 (m, 1H, H-3chol), 2.15 (s, 3H, CH3CO),
2.58–0.67 (m, 43H, H cholesterol).
[8-(Cholest-5-en-3b-yloxy)-7-[2H2]-3,6-dioxaoctyl] 2-acetamido-3,4,6-tri-
O-acetyl-2-deoxy-b-D-glucopyranoside (25)
Compound 25 was obtained in 78% yield as described pre-
viously from glycoside 21 (0.350 g, 0.39 mmol): 0.260 g, Rf 0.55
(5:1 ethyl acetate–acetone), mp 126 ꢀC, [a]D À28.7 (c 1.0, CHCl3);
1H NMR (CDCl3): d 6.74 (d, 1H, J2,NH 9.2 Hz, NH), 5.32 (bd, 1H,
H-6chol), 5.12–5.03 (m, 2H, H-3, H-4), 4.79 (d, 1H, J1,2 8.6 Hz, H-1),
4.25 (dd, 1H, J5,6a 4.5 Hz, J6a,6b 12.2 Hz, H-6a), 4.10 (dd, 1H, J5,6b
2.1 Hz, H-6b), 4.10–4.07 (m, 1H, H-2), 3.92–3.58 (m, 11H, 5CH2O,
H-5), 3.16 (m, 1H, H-3chol), 2.07, 1.99, 1.99, 1.97 (4s, 12H, 4CH3CO),
2.36–0.66 (m, 43H, H cholesterol); 13C NMR (CDCl3): d 170.98,
170.90, 170.87, 169.51 (CH3CO), 140.89 (C-5chol), 121.93 (C-6chol),
101.18 (C-1), 79.66 (C-3chol), 73.60 (C-3), 71.88 (C-5), 71.88,
70.84, 70.80 (CH2OCH2CH2), 68.86 (C-4), 68.86 (OCH2), 67.34
(CH2OChol), 62.36 (C-6), 56.92 (C-14chol), 56.31 (C-17chol), 54.01
(C-2), 50.35 (C-9chol), 42.48 (C-13chol), 39.93 (C-12chol), 39.67
(C-24chol), 39.29 (C-4chol), 37.36 (C-1chol), 37.02 (C-10chol), 36.34
(C-22chol), 35.94 (C-20chol), 32.09 (C-7chol), 32.04 (C-8chol), 28.45
(C-2chol), 28.29 (C-16chol), 28.17 (C-25chol), 24.45 (C-15chol), 23.98
(C-23chol), 23.27 (NHCOCH3), 22.99 (C-27chol), 22.73 (C-26chol),
21.24 (C-11chol), 20.95, 20.91, 20.78 (CH3COO), 19.56 (C-19chol),
18.88 (C-21chol), 12.03 (C-18chol).
HRMS (ESI) calc. for MNa: C41H69D2NNaO9, 746.5152. Found:
746.5154.
[8-(Cholest-5-en-3b-yloxy)-4-[2H2]-3,6-dioxaoctyl] 2-acetamido-2-deoxy-
b-D-glucopyranoside (3)
Compound 3 was obtained in 91% yield as described pre-
viously from glycoside 24 (0.200 g, 0.23 mmol): 0.152 g, Rf
0.62 (67:25:8 ethyl acetate–ethanol–water), amorphous solid,
1
[a]D À45.2 (c 1.0, CHCl3); H NMR (C5D5N): d 8.81 (d, 1H, J2,NH
8.2 Hz, NH), 5.52 (bd, 1H, H-6chol), 5.08 (d, 1H, J1,2 8.3 Hz, H-1),
4.54–4.50 (m, 2H, H-2, H-6a), 4.37–4.32 (m, 2H, H-3, H-6b), 4.20
(dd, 1H, J3,4 8.8 Hz, J4,5 9.2 Hz, H-4), 4.14 (m, 1H, CH2CHAHBOC-1),
3.92–3.84 (m, 2H, H-5, CH2CHAHBOC-1), 3.73–3.70 (m, 8H, 4OCH2),
3.28 (m, 1H, H-3chol), 2.15 (s, 3H, CH3CO), 2.56–0.68 (m, 43H, H
cholesterol).
Anal. calc. for C47H75D2NO12 (850.105): C, 66.40; H, 9.36; N 1.65.
Found: C, 66.64; H, 9.20; N: 1.68.
HRMS (ESI) calc. for MNa: C41H69D2NNaO9, 746.5152. Found:
746.5149.
[8-(Cholest-5-en-3b-yloxy)-3,6-dioxaoctyl] 3,4,6-tri-O-acetyl-2-deoxy-2-
trideuterioacetamido-b-D-glucopyranoside (26)
[8-(Cholest-5-en-3b-yloxy)-7-[2H2]-3,6-dioxaoctyl] 2-acetamido-deoxy-
b-D-glucopyranoside (4)
Compound 26 was obtained in 81% yield as described pre-
viously from glycoside 22 (0.350 g, 0.39 mmol): 0.270 g, Rf 0.39
(6:1 ethyl acetate–acetone), mp 127 ꢀC, [a]D À29.5 (c 1.0, CHCl3); Compound 4 was obtained in 90% yield as described previously
1H NMR (CDCl3): d 6.78 (d, 1H, J2,NH 9.3 Hz, NH), 5.31 (bd, 1H, from glycoside 25 (0.200 g, 0.23 mmol): 0.150 g, Rf 0.62 (67:25:8
H-6chol), 5.11–5.01 (m, 2H, H-3, H-4), 4.79 (d, 1H, J1,2 8.6 Hz, H-1), ethyl acetate–ethanol–water), amorphous solid, [a]D À45.5
1
4.24 (dd, 1H, J5,6a 4.5 Hz, J6a,6b 12.2 Hz, H-6a), 4.08 (dd, 1H, J5,6b (c 1.0, CHCl3); H NMR (C5D5N): d 8.82 (d, 1H, J2,NH 8.4 Hz, NH),
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J. Label Compd. Radiopharm 2012, 55 88–95

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5.42 (bd, 1H, H-6chol), 5.09 (d, 1H, J1,2 8.4 Hz, H-1), 4.55 (ddd, 1H,
J2,3 9.9 Hz, H-2), 4.54 (m, 1H, H-6a), 4.40–4.33 (m, 2H, H-3, H-6b),
4.21 (dd, 1H, J3,4 8.8 Hz, J4,5 9.2 Hz, H-4), 4.15 (m, 1H, CH2CHAHBOC-1),
3.88 (ddd, 1H, J5,6a 2.4 Hz, J5,6b 5.4 Hz, H-5), 3.86 (m, 1H,
CH2CHAHBOC-1), 3.75–3.67 (m, 8H, 4OCH2), 3.28 (m, 1H, H-3chol),
2.15 (s, 3H, CH3CO), 2.58–0.67 (m, 43H, H cholesterol).
HRMS (ESI) calc. for MNa: C41H69D2NNaO9, 746.5152. Found:
746.5155.
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[8-(Cholest-5-en-3b-yloxy)-3,6-dioxaoctyl] 2-deoxy-2-
trideuteroacetamido-b-D-glucopyranoside (5)
Compound 5 was obtained in 90% yield as described previously
from glycoside 26 (0.200 g, 0.23 mmol): 0.150 g, Rf 0.62 (67:25:8
ethyl acetate–ethanol–water), amorphous solid, [a]D À45.2 (c 1.0,
CHCl3); 8.82 (d, 1H, J2,NH 8.4 Hz, NH), 5.42 (bd, 1H, H-6chol),
5.10 (d, 1H, J1,2 8.4 Hz, H-1), 4.59–4.53 (m, 2H, H-2, H-6a),
4.40–4.33 (m, 2H, H-3, H-6b), 4.21 (dd, 1H, J3,4 8.9 Hz, J4,5
9.3 Hz, H-4), 4.15 (m, 1H, CH2CHAHBOC-1), 3.90 (m, 1H, H-5),
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HRMS (ESI) calc. for MNa: C41H68D3NNaO9, 747.5215. Found:
747.5217.
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Conflict of Interest
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Chem. 1996, 33, 1385–1387.
The authors did not report any conflict of interest.
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J. Label Compd. Radiopharm 2012, 55 88–95
Copyright © 2012 John Wiley & Sons, Ltd.
www.jlcr.org