Synthesis of two glycolipid antigens of the causative agent of Lyme disease

Article abstract of DOI:10.1016/j.tet.2005.08.053

As a prelude to development of a human vaccine against Lyme disease, the first chemical synthesis of glycolipid antigens of Borrelia burkholderi is reported. First, cholesteryl β-d-galactopyranoside was synthesized and was converted to partially protected congeners having the HO-6 group of the galactose moiety unprotected. Treatment of these intermediates with palmitic and oleic acid, respectively, under dehydrative conditions followed by removal of the protecting groups afforded cholesteryl 6-O-palmitoyl/oleoyl-β-d- galactopyranosides that were identical to the glycolipids isolated from B. burkholderi.

Full text of DOI:10.1016/j.tet.2005.08.053

Tetrahedron 61 (2005) 10470–10481
Synthesis of two glycolipid antigens of the causative agent
of Lyme disease
*
¨
Vince Pozsgay, Joanna Kubler-Kielb, Bruce Coxon and Goran Ekborg
National Institute of Child Health and Human Development, National Institutes of Health, 31 Center Dr. MSC 2423 Bethesda,
MD 20892-2423, USA
Received 16 June 2005; revised 9 August 2005; accepted 11 August 2005
Available online 6 September 2005
Abstract—As a prelude to development of a human vaccine against Lyme disease, the first chemical synthesis of glycolipid antigens of
Borrelia burkholderi is reported. First, cholesteryl b-D-galactopyranoside was synthesized and was converted to partially protected
congeners having the HO-6 group of the galactose moiety unprotected. Treatment of these intermediates with palmitic and oleic acid,
respectively, under dehydrative conditions followed by removal of the protecting groups afforded cholesteryl 6-O-palmitoyl/oleoyl-b-D-
galactopyranosides that were identical to the glycolipids isolated from B. burkholderi.
Published by Elsevier Ltd.
1. Introduction
surface-exposed proteins are immunogenic9 and a recombi-
nant lipidated outer surface protein, referred to as OspA,
was the major component of a licensed vaccine10 LYMErix
(SmithKline Beecham) that was withdrawn from medical
use in 2002. Currently, there is no licensed vaccine to
prevent Lyme disease.
Lyme disease, a vector-borne infection caused by the tick-
transmitted spirochaete Borrelia burgdorferi, frequently
occurs in the United States, Europe, and North Asia.1,2 This
disease was identified in 1977 when an unusually large
number of children with arthritis-like symptoms in and
around the township of Lyme, Connecticut was identified.3
Since then, the Centers for Disease Control and Prevention
has reported a steadily increasing number of cases, reaching
more than 23,000 in 2002.4 Most cases occur in the age-
groups 5–14 and 50–59 years4 in northeastern, mid-Atlantic,
and north central states. Borrelia organisms enter the
circulatory system of the host via the bite of an infected
Ixodes tick and infect various organs, including the brain
and joints.5 Some patients develop chronic Lyme disease
that can be disabling6 when the infection persists for years.
Borrelia are considered Gram-negative bacteria but there is
no evidence for the presence of a characteristic lipopoly- or
lipooligosaccharide on their surface. Work in our labora-
tory11 has identified two major glycolipids (1, 2) in B.
burgdorferi that contain cholesteryl b-galactoside in which
O-6 of the galactose residue is substituted either by a
palmitoyl or an oleoyl group, respectively. Cholesteryl
glycosides occur in many plants12 and fungi13 but they are
rare in animals and bacteria. The bacteria Acholeplasma
spp.,14 Mycoplasma gallinarum,15 Spiroplasma citri,16
Borrelia hermsi,17 and Helicobacter pylori18 have been
reported to express cholesteryl glucoside but not galacto-
side. The presence of galactose in cholesteryl glycosides is,
therefore, a unique feature of B. burgdorferi. The native
glycolipids 1 and 2 differing in the fatty acid residue at O-6
of the galactose moiety could not be separated.11 Therefore,
the structural elucidation was performed on their mixture
estimated to contain 1 and 2 in an approx. 1:1 ratio. Because
of the difficulties in growing B. burgdorferi, glycolipids 1
and 2 could be obtained only in small quantities. This fact
combined with the difficulty to separate them, prompted us
to synthesize 1 and 2 in quantities and purity suitable for
immunization experiments. Availability of well-defined
compounds was expected to remove uncertainties that
B. burgdorferi belongs to the bacterial order Spirochaetales,
which is divided into two families, each divided into genera.
The first family, Spirochaetaceae includes four genera:
Spirochaeta, Cristaspira, Treponema, and Borrelia,
whereas the second family, Leptospiraceae encompasses
two genera: Leptospira and Leptonema.7 The pathogen’s
helically shaped, motile cells are surrounded by an outer cell
membrane, which together with flagella are assumed to play
a role in the host–parasite interaction.8 The bacterium’s
Keywords: Borrelia burkholderi; Cholesterol; Glycolipid; Lyme disease;
Vaccine.
Corresponding author. Tel.: C1 301 295 2266; fax: C1 301 295 1435;
e-mail: pozsgayv@mail.nih.gov
*
0040–4020/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.tet.2005.08.053

V. Pozsgay et al. / Tetrahedron 61 (2005) 10470–10481
10471
arose from the fact that the structural studies were carried
2. Results and discussion
out on a mixture. Here, we report chemical synthetic
approaches to glycolipids 1 and 2, and provide confirmatory
evidence for the native structures. We also describe a simple
method to eliminate the virtual couplings in the chloro-
form-d H NMR spectra of 1 and 2 and their synthetic
intermediates.
Two approaches were designed to the synthetic targets 1 and
2. The first involved the preparation of a 6-O-palmitoyl/
oleoyl-galactopyranosyl donor that was coupled to HO-3 of
cholesterol. Because of the limitations imposed upon the
synthetic scheme by the cholesterol’s own double bond, as
1
Scheme 1. Reagents and conditions: (a) 1.32 equiv of (CH3)3COCl, C5H5N, CHCl3, 23 8C, 24 h, 30%; (b) Ac2O, C5H5N, 23 8C, 4 h, quant.; (c) HBr/AcOH,
CH2Cl2, 0/23 8C, (d) 1.44 equiv of 5, 1.88 equiv of 2,6-di-tert-butyl-4-methylpyridine, 1.5 equiv of AgOTf, K40/0 8C, 1 h, 85%; (e) CH3ONa (excess),
CH3OH, CH2Cl2, 23 8C, 24 h, 94%.

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V. Pozsgay et al. / Tetrahedron 61 (2005) 10470–10481
well as the need for a ‘participating’ protecting group at O-2
to lead to the b anomeric stereochemistry, this scheme
required a per-O-acylated donor where the O-acyl groups at
HO-2, 3, and 4 were to be chemoselectively cleavable,
leaving the palmitoyl/oleoyl group at O-6 intact during
deprotection. For this purpose the levulinoyl and the
chloroacetyl groups were tested because they can be
selectively cleaved in the presence of alkanoyl and aroyl
groups. Unfortunately, this approach proved to be abortive
because attempted, selective cleavage of the protecting
groups at O-2, 3, and 4 caused extensive acyl migrations
leading to inseparable product mixtures. In the alternative
approach, cholesteryl galactopyranoside 8 was prepared first
as shown in Scheme 1, in which the 6-O-acyl moieties were
installed at a later stage. Thus, galactose was first treated
with pivaloyl chloride in pyridine containing 4-dimethyl-
aminopyridine, to afford a mixture of fully and partially
pivaloylated galactose derivatives. This protecting group
was chosen because it suppresses the formation of unwanted
orthoesters.19 Complete pivaloylation could not be achieved
even after extended reaction times at an elevated tempera-
ture. Previously, Kunz et al.20 reported that pivaloylation of
galactose with pivaloyl chloride in pyridine for 6 days
afforded penta-pivaloyl galactopyranose in 63% yield.20
Serendipitously, we found that 1,2,3,6-tetra-O-pivaloyl-b-
D-galactopyranose 3 could be isolated from the pivaloyl-
ation reaction mixture in pure form after only 1 day by
crystallization, in 30% yield. (Scheme 1) Because of the
inexpensive nature of the reagents, no attempt was made for
optimization. Compound 3 was converted to the fully
acylated derivative 4 by treatment with Ac2O in pyridine,
in a quantitative yield. Next, 4 was transformed into the
Scheme 2. Reagents and conditions: (a) 1.3 equiv of tert-butyl-diphenylsilyl chloride, C5H5N, 23 8C, 5 h, 96%; (b) (CH3O)2C(CH3)2 (excess), CSA (cat),
23 8C, 20 min, 91%; (c) 2.4 equiv of NaH, DMF, 1.4 equiv of All-Br, 0/23 8C, 92%; (d) Bu4NF (excess), THF, 23 8C, 2.5 h, 97%; (e) 3 equiv of palmitic acid,
6.4 equiv of DCC, MDAP, 23 8C, 6 h, 99%; (f) AcOH–MeOH–H2O, 70 8C, 6 h, 70%; (g) C34H38F6IrP3, THF, 23 8C, 24 h then AcOH/TFA, 23 8C, 24 h, 88%.

V. Pozsgay et al. / Tetrahedron 61 (2005) 10470–10481
10473
galactosyl donor 5 by treatment with hydrogen bromide.
Stereoselective glycosylation of cholesterol (6) with 5 under
promotion by silver triflate afforded the glycoside 7 in 85%
yield, without the formation of an orthoester.
propane in the presence of CSA (/9, 34%) in the hope that
subsequent O-acylations would take place preferentially or
exclusively at O-6. Unexpectedly, condensation of 9 with
palmitic acid in the presence of DCC afforded at least two
products that could not be separated and that are
conceivably the 2-O and the 6-O-acyl derivatives. This led
us to synthesize the highly protected intermediate 13 in a
series of protective-deprotective steps, as shown in Scheme
2. Thus, compound 8 was first converted to the 6-O-silyl
derivative 10 using tert-butyl-diphenylsilyl chloride in
pyridine in 96% yield, followed by acid catalyzed
transacetalization with 2,2-dimethoxypropane to afford the
alcohol 11 (91%). Treatment of 11 with allyl bromide and
NaH afforded the fully protected intermediate 12 (92%) as
expected. Next, reaction of compound 12 with tetrabuty-
lammonium fluoride provided the alcohol 13 (97%) from
which acylation with palmitic acid/DCC provided the fully
protected derivative 14 (99%). Compound 14 was depro-
tected in a three-step sequence involving acid-catalyzed
hydrolysis of the isopropylidene acetal in AcOH–MeOH–
H2O (/15, 70%), and allyl isomerization21,22 with
(1,5-cyclooctadiene)bis(methyldiphenylphosphine)iridium(I)
hexafluorophosphate in THF, followed by TFA hydrolysis
of the 2-O-(1-propylene) derivative (not isolated) afford-
ing23 1 in 88% yield.
Treatment of the glycoside 7 with sodium methoxide
afforded the tetraol 8 in 94% yield. The isolation of 8 in a
pure form was facilitated by the fact that 8, a crystalline
material, is insoluble in most organic solvents, whereas all
of the impurities are soluble. Next, we envisioned protection
schemes for the introduction of the acyl moieties at O-6 of
the galactose unit. Initially, we prepared the isopropylidene
derivative 9 by treating the tetraol 8 with 2,2-dimethoxy-
Scheme 3. Reagents and conditions: (a) 2 equiv of oleic acid, 5.9 equiv of DCC, DMAP, EtOAc, 23 8C, 2 h, 75%; (b) AcOH–MeOH–CH2Cl2, reflux, 6 h, 70%;
(c) C34H38F6IrP3, THF, 23 8C, 24 h then AcOH/TFA, 23 8C, 24 h.

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V. Pozsgay et al. / Tetrahedron 61 (2005) 10470–10481
Scheme 4. Reagents and conditions: (a) 6.6 equiv of NaH, DMF, 0 8C, 10 min, then 6 equiv of MBn–Br, 0 8C, 6 h, then Bu4NF (excess), THF, 23 8C, 6 h, 78%;
(b) 2.2 equiv of oleic acid, 6.8 equiv of DCC, DMAP, EtOAc, 23 8C, 6 h, 81%; (c) 1.3 equiv of CAN, CH2Cl2, H2O, 23 8C, 6 h, 93%; (d) AcOH–H2O, 65 8C,
4 h, 92%.
Figure 1. Partial 1H NMR spectra of cholesteryl 2-O-allyl-3,4-di-O-isopropylidene-b-D-galactopyranoside 13 at 500 MHz; (a) the spectrum of a solution in
CDCl3 shows ‘virtual coupling’ in the H-2 multiplet because of strong coupling of H-3 and H-4, and in the H-6 multiplet because of strong coupling of H-5 and
H-60; (b) no virtual coupling is observed in the spectrum of a solution of 13 in acetone-d6.

Table 1. Selected 1H chemical shiftsa of synthetic glycolipid antigens and their precursors
Compound
Galactose
H-4
Allyl
H-2
Cholesterol
H-3 H-6
Others
H-60
H-10
—
H-30
—
H-1
H-2
H-3
H-5
H-6
H-1
—
H-3
—
1b (at 300 K)
4.335
3.474
3.535 3.827
3.739
4.388
4.153
—
—
3.500
3.502
5.357
5.365
HO-2 4.051, HO-3 4.022, HO-4
3.674
Oleoyl H-9,10 5.371, 5.359
HO-2 4.059, HO-3 4.032, HO-4
3.678
2b
4.336
3.476
3.536 3.828
3.738
4.382
4.157
—
—
—
—
3
5.666
5.673
4.604
5.466
5.585
5.240
5.014 4.033
5.059 5.359
5.060 5.421
3.899
3.451
3.932
4.336
4.077
4.176
4.292
3.979
4.074
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Piv Me 1.205, 1.194, 1.182,
1.128
Piv Me 1.143, 1.126, 1.110,
1.110 Ac Me 1.658
Piv Me 1.180, 1.176, 1.112 Ac
Me 2.130
4c
7
—
—
3.478
5.326
8d
4.744
4.337
4.312
4.256
4.036
3.378
3.610
3.529
3.948 4.287
4.030 4.214
w3.577 4.049
4.065 4.237
3.898
3.863
3.519
3.851
4.145
3.758
3.916
3.956
4.107
3.733
3.876
3.916
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
3.810
3.553
3.556
3.553
5.401
5.345
5.344
5.352
9b,e
10
11
Ip Me 1.416, 1.275
t-Bu Me 1.050
Ip Me 1.507, 1.336, t-Bu Me
1.048
12
4.318
3.307
4.068 4.204
3.783
3.928
3.899
4.337
4.249
5.940
5.303
5.160
3.512
5.343
Ip Me 1.491, 1.324, t-Bu Me
1.043
Ip Me 1.441, 1.278
Ip Me 1.458, 1.296
13b
14b
15
4.435
4.440
4.395
4.439
3.207
3.228
3.393
3.228
4.078 4.220
4.124 4.221
3.575 3.895
4.123 4.221
3.853
4.078
3.635
4.076
3.745
4.360
4.328
4.358
3.714
4.201
4.295
4.203
4.306
4.304
4.454
4.304
4.216
4.213
4.176
4.214
5.909
5.905
5.935
5.906
5.307
5.307
5.295
5.307
5.100
5.106
5.201
5.107
3.546
3.508
3.524
3.510
5.361
5.373
5.355
5.366
16b
Oleoyl H-9,10 5.366 Ip Me
1.459, 1.297
Ip Me 1.332, 1.268, MBn CH2
4.772, 4.694, H-2,6 7.322, H-3,5
6.894, OMe 3.784
Ip Me 1.344, 1.283, MBn CH2
4.769, 4.692, H-2,6 7.319, H-3,5
6.896, OMe 3.785, Oleoyl H-9,
10 5.367
Ip Me 1.433, 1.291, Oleoyl H-9,
10 5.366
19b
4.479
4.490
3.268
3.283
4.117 4.210
4.158 4.197
3.856
4.073
3.740
4.351
3.709
4.192
—
—
—
—
—
—
—
—
—
—
3.578
3.539
5.364
5.367
20b
21b
4.351
3.386
4.075 4.216
4.094
4.367
4.210
—
—
—
—
—
3.511
5.366
a In ppm from internal Me4Si, in CDCl3 solution unless stated otherwise.
b In fresh acetone-d6.
c In 4:1 v/v benzene-d6/CDCl3.
d In 1:1 v/v methylsulfoxide-d6/pyridine-d5.
e At 318 K.

Table 2. Selected 13C chemical shiftsa of synthetic glycolipid antigens and their precursors
Compound
Galactose
C-4
Allyl
Cholesterol
C-5
Others
C-1
C-2
C-3
C-5
C-6
C-1
C-2
C-3
C-3
C-6
1b
2b
3
102.89
102.85
92.26
72.16
72.10
67.54
68.20
74.42
74.36
72.94
71.02
69.74
69.69
66.89
66.73
73.40
73.33
72.99
71.62
64.38
64.37
61.71
60.69
—
—
—
—
—
—
—
—
—
—
—
—
79.39
79.33
—
141.82
141.75
—
122.16
122.17
—
C]O 173.49
C]O 173.48, Oleoyl C-9,10 130.65, 130.53
Piv Me 27.11, 27.11, 27.08, 26.89
Piv Me 27.20, 27.09, 27.07, 26.92, Ac Me
20.00
4c
92.35
—
—
—
Piv C]O 177.20, 176.77, 176.36, 176.27
Ac C]O 169.68
7
100.19
68.88
70.97
67.08
70.69
61.47
—
—
—
80.13
140.29
122.10
Piv Me 27.20, 27.06, 26.96, Ac Me 20.68
Piv C]O 177.95, 177.27, 176.51, Ac C]O
169.97
8d
102.29
102.07
101.25
71.76
74.27b
72.22
74.43
80.65
73.68
69.25
74.71
68.83
75.92
74.77
74.44
61.50
62.38b
62.97
—
—
—
—
—
—
—
—
—
77.92
78.89
78.82
140.76
141.77
140.35
121.69
122.24
122.06
9b,e
10
Ip Me 28.53, 26.67, Ip OCO 109.87
TBDPS Ar 135.63, 135.55, 133.10, 132.94,
129.86, 129.85, 127.80, 127.79, t-Bu Me
26.77, t-Bu C-1 19.18
11
12
100.63
101.64
73.75
79.84
78.62
79.11
73.24
73.38
73.67
73.22
62.71
62.79
—
—
—
78.98
79.47
140.36
140.68
122.06
121.79
TBDPS Ar 135.63, 135.60, 133.42, 133.35,
129.69, 127.70, 127.63, Ip OCO 110.04, Ip Me
28.21, 26.31, t-Bu Me, 26.72, t-Bu C-1 19.20
TBDPS Ar 135.64, 135.60, 133.46, 133.38,
129.67, 127.69, 127.62, Ip OCO 109.75, Ip Me
28.03, 26.33, t-Bu Me 26.72, t-Bu C-1 19.20
Ip OCO 109.92, Ip Me 28.42, 26.64
C]O 173.39, Ip OCO 110.32, Ip Me 28.32,
26.60
72.88
135.14
117.03
13b
14b
101.99
102.08
81.11
80.84
80.09
80.12
74.74
74.69
74.44
71.50
62.17
64.00
72.93
72.92
136.85
136.74
115.93
116.06
79.14
79.79
141.56
141.57
122.35
122.35
15
16b
102.29
102.06
78.67
80.83
72.95
80.10
68.19
74.68
71.89
71.48
62.46
63.99
73.52
72.91
134.70
136.73
117.66
116.06
79.95
79.78
140.55
141.56
121.95
122.35
C]O 173.77
C]O 173.38, Oleoyl C-9,10 130.65, 130.50
Ip OCO, 110.31, Ip Me 28.31, 26.60
Ip Me 28.35, 26.66, Ip OCO 109.92
MBn CH2 73.64, C-1 132.04, C-2,6 130.21,
C-3,5 113.23, C-4 160.07, OMe 55.48
Ip Me 28.24, 26.63, Ip OCO 110.31, MBn CH2
73.63, C-1 131.92, C-2,6 130.26, C-3,5 114.25,
C-4 160.11, OMe 55.49
19b
101.99
102.11
80.78
80.48
80.13
80.13
74.72
74.66
74.43
71.48
62.18
64.00
—
—
—
—
—
—
79.09
79.77
141.57
141.59
122.37
122.38
20b
Oleoyl C-9,10 130.66, 130.51, C]O 173.39
Ip Me 28.44, 26.64, Ip OCO 110.19, Oleoyl
C-9,10, C]O 173.41
21b
102.02
73.97
80.60
74.54
71.66
64.07
—
—
—
79.40
141.66
122.25
a In ppm from internal Me4Si, in CDCl3 solution unless stated otherwise.
b In acetone-d6.
c In 4:1 v/v benzene-d6/CDCl3.
d In 1:1 v/v methylsulfoxide-d6/pyridine-d5.
e At 318 K.

V. Pozsgay et al. / Tetrahedron 61 (2005) 10470–10481
10477
An initial approach to the synthesis of the oleoyl glycolipid
2 was based on the method just described for the preparation
of 1. (Scheme 3.) Thus, the alcohol 13 was acylated with
oleic acid/DCC to afford compound 16 in a near quantitative
yield, from which acid-catalyzed removal of the iso-
propylidene group provided the diol 17 in an uneventful
transformation. Removal of the allyl group by isomerization
with the iridium reagent, followed by hydrolysis as
described for 1 gave a chromatographically homogeneous
material (18) that showed the expected C-5 and C-6 13C
NMR resonances for the double bond of the cholesterol unit
(140 and 122 ppm, respectively) but lacked the character-
istic olefinic carbon resonances of the oleoyl residue (18:1)
at w130 ppm. Instead, a number of low intensity signals
appeared at w130 ppm, hinting at extensive migration of
the double bond. In order to gain insight into the extent of
isomerization, compound 18 was subjected to trans-
esterification with methanol followed by reaction with
2-amino-2-methylpropanol as described.24 GC–MS of the
resulting oxazoline derivatives revealed unsaturations
mainly at C-7, C-8, and C-15. Thus, while the iridium
reagent preserved the location of the cholesterol double
bond, it caused extensive double bond migration in the
oleoyl residue, necessitating an alternative approach
depicted in Scheme 4. Alcohol 11 was first treated with
4-methoxybenzyl bromide/NaH followed by removal of the
silyl protecting group with tetrabutylammonium fluoride in
THF to afford compound 19 in 78% combined yield. Next,
treatment of the alcohol 19 with oleic acid/DCC led to the
desired oleoyl ester 20, from which removal of the
4-methoxybenzyl (/21) and isopropylidene groups using
standard procedures successfully afforded the oleoyl
glycolipid 2 in 86% combined yield.
(large J/d) of H-3 and H-4 of the Gal residue, which led to
second order effects in the H-2 and H-5 multiplets in the
form of additional lines in the multiplets that complicated
the first order analysis of the spectra. (Fig. 1) Rather than
resorting to iterative spectral simulations, the virtual
coupling problem was overcome by the use of solvent
shifts in acetone-d6 to increase the dispersion of the spectra.
Selected 1H and 13C chemical shifts of the synthetic
glycolipids 1 and 2 and their precursors are shown in
Tables 1 and 2, respectively, and selected 1H coupling
constants in Table 3.
The measured values of the 1H–1H coupling constant 3J1,2
Z
1
7.5–8.4 Hz and JC-1,H-1Z155.2–166.3 Hz indicated that
except for 5, all of the galactopyranose precursors,
glycolipid intermediates, and target glycolipids had the b
anomeric configuration, as required for duplicative syn-
thesis of the natural glycolipids. For the pivaloyl esters 3
1
and 4, the observed values JC-1,H-1Z165.7–166.3 Hz
(Table 3) are 5–10 Hz greater than the range for many
b-D-galactopyranosides, due to the electronegativity of the
acyloxy group at C-1.26
As usual, TOCSY experiments did not work well for
compounds containing undistorted galactopyranose resi-
dues, because of the small J4,5 values, which inhibited
transfer of magnetization from H-4 through to H-5, H-6, and
H-60. However, in compounds 9, 11–14, 16, and 19–21 the
attachment of the 3,4-O-isopropylidene ring flattened the
chair form of the galactopyranose ring somewhat, causing
J4,5 to be larger (w2 Hz, Table 3), with a resulting
improvement in TOCSY transfer. Nevertheless, in the end,
most of the 1H assignments could be made by simple COSY
experiments, and the 13C assignments by HSQC based on
the 1H assignments. The positions of the various sub-
stituents on the Gal ring were indicated by HMBC
connectivities.
For solutions of members of this series of glycolipids in
1
CDCl3, virtual coupling25 in their H NMR spectra was
extremely common. This was often due to strong coupling
Table 3. Selected 1H coupling constants (Hz)a of synthetic glycolipid antigens and their precursors
Compound
Galactose
Allyl
0
0
0
0
J1 ,2 J1,3
0
0
0
0
J1 ,3 J1 ,3 J2,3
0
0
J3,3
J1,2
J2,3
9.5
9.5
10.3
10.2
10.4
9.6
J3.4
3.5
3.5
3.4
3.6
3.6
3.4
5.6
J4,5
J5,6
J5,6
J6,6
JC-1,H-1
J1,1
J1,2
J1,3
J2,3
1b (at 300 K)
7.5
7.5
8.3
8.4
8.0
7.6
8.1
7.5
8.3
8.2
8.1
8.1
7.7
8.1
8.1
8.1
8.1
1.1 8.3
1.1 8.2
1.1 6.2
1.2 6.9
1.1 7.0
1.1e 6.1
2.2 6.5
4.1
4.2
6.7
7.0
6.6
6.3
5.8
5.5
6.3
6.4
5.9
3.5
6.9
3.6
5.8
3.6
3.7
11.4 57.4
11.3 56.1
11.5 165.7
11.1 166.3
11.2 158.5
11.0 156.3e
11.1 156.1
10.5 160.8
10.0 155.9
10.0 155.2
11.1 156.8
11.6 157.2
11.4 157.8
11.5 157.0
11.1 156.8
11.5 157.0
11.5 156.8
2b
3
4c
7
8d
9b,f
10
7.1
9.4 w2.0 w0.9 6.2
11
7.5
7.1
7.1
7.0
9.5
7.0
7.1
6.9
7.1
5.4
5.5
5.6
5.6
3.3
5.6
5.6
5.6
5.6
2.2 7.0
2.1 6.9
2.1 6.5
2.1 8.7
1.2 6.3
2.1 8.6
2.1 6.6
2.1 8.7
2.2 8.6
12
12.7 5.5
13.4 5.9
13.4 5.0
12.6 5.5
13.4 5.1
6.0
5.5
5.5
6.3
5.5
1.6
1.7
1.8
1.5
1.8
1.4
1.6
1.6
1.3
1.5
1.5
1.6
1.6
1.4
1.7
1.3
1.5
1.5
1.2
1.4
17.2 10.4 1.9
17.3 10.5 2.0
17.3 10.5 2.0
17.2 10.3 1.4
17.3 10.5 2.1
13b
14b
15
16b
19b,g
20b,g
21b
a Measured at 500 MHz, for CDCl3 solutions unless stated otherwise.
b In fresh acetone-d6, J2,HO-2Z3.5, J3,HO-3Z5.0, J4,HO-4Z4.0, J5,HO-4Z0.9.
c In 4:1 v/v benzene-d6/CDCl3.
d In 1:1 v/v methylsulfoxide-d6/pyridine-d5.
e In pyridine-d5.
f
At 318 K.
g MBn J(CH2)Z11.5 Hz.

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V. Pozsgay et al. / Tetrahedron 61 (2005) 10470–10481
The chemical synthesis of the palmitoyl (1) and oleoyl (2)
glycolipids as separate entities afforded us the opportunity
to test a previous interpretation11 of the 13C NMR spectrum
of a mixture of two major and several minor glycolipids
termed ‘Glycolipid I’, isolated from B. burgdorferi. It was
proposed11 that the doubling of the C-4, C-5, and C-6
signals of Gal and the C-3 resonance of the cholesteryl
moiety that was observed for glycolipid I was due to its
isolation as a chromatographically inseparable mixture of
mainly 1 and 2, even though the points of difference in the
fatty acid residues of these molecules are quite remote from
the nuclei showing the doubling of signals. When, in the
current work we recorded the 13C spectrum of a 1:1 mixture
of synthetic 1 and 2, this spectrum was almost identical with
that of glycolipid I, thus confirming our previous
interpretation.11 This was true as long as care was taken
with concentration, because 1 and 2 showed an unexpected
sensitivity to concentration of the galactose C-2 resonance,
possibly due to molecular aggregation, amounting to
changes in the 13C chemical shifts as large as 1 ppm.
Molecular aggregation may produce this effect. Hydroxyl
proton coupled 1H NMR spectra were obtained for 1 and 2
by use of freshly opened vials of acetone-d6 solvent (see OH
chemical shifts and coupling constants in Tables 1 and 3,
respectively). Otherwise, the use of acetone-d6 that had been
500 MHz by using 32,768 point data sets, a 308 pulse (2.2–
2.4 ms), and a pulse recycle time of 6 s. An optimum
concentration for obtaining good resolution and sensitivity
was 5–6 mg of glycolipid intermediate in 0.5 mL of solvent.
The resolution of the spectra was enhanced by Gaussian
multiplication of the free induction decay, using a line-
broadening of K1 to K3 Hz and a Gaussian truncation
fraction of 0.3, together with linear prediction to 65,536
points, or linear prediction to 131,072 points with zero-
filling to 262,144 points. 13C NMR spectra were acquired at
126 MHz by using 65,536 point data sets, a 908 pulse
(14 ms), and a pulse recycle time of 1.5 s. Resolution was
enhanced by exponential multiplication with a line-
broadening of K0.2 Hz, together with forward, complex
1
linear prediction to 131,072 points. H and 13C chemical
shifts were referenced to internal tetramethylsilane (0 ppm).
1H-coupled 13C NMR spectra were acquired with the
nuclear Overhauser effect (NOE) by use of gated, WALTZ-
16 irradiation at the 1H frequency. These spectra were used
to measure 1JC-1,H-1. All 2D NMR spectra were acquired by
field gradient-selected methods. Forward linear prediction
to larger data sizes was used to improve the resolution of 2D
spectra, where necessary. 2D COSY and HMBC NMR
spectra were obtained by using 2048 (t2)!512 (t1) point
data sets, zero-filled to 2048 (F2)!2048 (F1) points. For 2D
COSY and TOCSY, the spectral width was 5.48 kHz in each
dimension, and for COSY, the read pulse was 308 (2.2–
2.4 ms). For 2D HSQC and HMBC, the 1H and 13C spectral
widths were 5.48 kHz (F2) and 25.1 kHz (F1), respectively.
2D TOCSY NMR spectra were acquired by use of 16,384
(t2)!512 (t1) point data sets, zero-filled to 32,768 (F2)!
2048 (F1) points. High-resolution 2D HSQC spectra were
acquired in 2048!512 point data sets, linear predicted to
4096 points in both dimensions.
1
exposed to the atmosphere a number of times yielded H
NMR spectra in which a major proportion of the hydroxyl
proton signals was absent due to decoupling of the OH
protons by chemical exchange. In these spectra, the OH
proton coupled species appeared only as a minor component
amounting to 14% for 1 at 310 K, and 15% for 2 at 300 K.
3. Conclusion
In summary, we have presented successful syntheses of
O-lipidated galactosyl cholesterols 1 and 2 that can now be
used individually for detailed biological studies. Current
work is directed towards synthesizing an experimental
vaccine against Lyme disease. This vaccine will consist of
glycolipid 1 carrying a reactive end group at the alkyl chain
through which it will be covalently attached to protein
carriers27 to form immunogenic lipoglycoproteins.
The mass spectra were recorded at the Laboratory of
Bioorganic Chemistry, NIDDK, NIH, Bethesda, MD. The
fast atom bombardment (FAB) mass spectra were obtained
using 6 keV Xe atoms to ionize samples from dithiothreitol/
dithioerythritol, 3-nitrobenzyl alcohol, or glycerol as
the matrix. The purity of all new compounds was
determined to be O95% by 1H NMR spectroscopy.
Elemental analyses were performed by Atlantic Microlab,
Inc., Norcross, GA.
4. Experimental
4.1.1. 1,2,3,6-Tetra-O-trimethylacetyl-b-D-galacto-
pyranose (3). To a stirred mixture of anhydrous CHCl3
(80 mL), C5H5N (45 mL), and trimethylacetyl chloride
(45 mL, 365 mmol) was added D-galactose (10 g, 55 mmol,
0.75 equiv) in portions. After 24 h at room temperature,
TLC (3:1 hexane/EtOAc) indicated the formation of several
products. Their ratios did not change significantly after the
addition of more trimethylacetyl chloride or heating for
another 24 h. The mixture was concentrated under reduced
pressure and the residue so obtained was triturated with
diethyl ether. The solids were isolated by filtration and were
discarded. To the filtrate was added isopropyl ether and the
mixture was left standing at room temperature for 3 h.
Filtration followed by washing with isopropyl ether afforded
3 (8.5 g, 30%) as a crystalline material: [a]D C15 (c 0.8,
CHCl3). For NMR data, see Tables 1–3. HRMS m/z Calcd
for C26H44O10Li 523.3105, found 523.3095.
4.1. General methods
All chemicals were commercial grade and were used
without purification. Solvents for chromatography were
distilled prior to use. Anhydrous solvents were obtained
from Aldrich. Column chromatography was performed on
silica gel 60 (0.040–0.063 mm). Melting points were taken
on a Meltemp capillary melting point apparatus and are
uncorrected. Optical rotations were measured at 23 8C with
a Perkin-Elmer Type 341 polarimeter.
NMR spectroscopy. NMR spectra were acquired at 300 K by
use of a Bruker DRX-500 NMR spectrometer equipped with
a 5 mm TXI HCN cryoprobe with z-gradient coil, a Silicon
Graphics O2 workstation, and Bruker XWINNMR software,
version 3.5, patch level 6. 1H NMR spectra were acquired at

V. Pozsgay et al. / Tetrahedron 61 (2005) 10470–10481
10479
4.1.2. 4-O-Acetyl-1,2,3,6-tetra-O-(trimethylacetyl)-b-D-
galactopyranose (4). Compound 3 was treated with Ac2O
and C5H5N at room temperature followed by removal of the
volatiles to afford 4 as a crystalline material in a quantitative
yield: [a]D C9 (c 1.2, CHCl3). For NMR data, see Tables
1–3. HRMS m/z Calcd for C28H46O11Li 565.3230, found
565.3200.
pyridine. After 5 h, MeOH was added (2 mL) followed by
concentration under reduced pressure. Column chromato-
graphic purification of the residue afforded 10 (11.93 g,
96%) as a syrup: [a]D K44 (c 0.4, CHCl3). For NMR data,
see Tables 1–3. HRMS m/z Calcd for C49H74O6LiSi
793.5420, found 793.5415.
4.1.8. Cholesteryl 6-O-(tert-butyl-diphenylsilyl)-3,4-O-
isopropylidene-b-D-galactopyranoside (11). To a stirred
solution of triol 10 (10.4 g (18.9 mmol) in 2,2-dimethoxy-
propane (100 mL) was added at room temperature
camphorsulfonic acid (0.5 g). After 20 min TLC (4:1
hexane/EtOAc) indicated that no starting material remained.
To the solution was added triethylamine (2 mL). The
solution was concentrated and the residue purified by
column chromatography using a gradient of hexane/EtOAc
10:1–5:1 to obtain 11 (10.0 g, 91%) as a syrup: [a]D K26 (c
0.6, CHCl3). For NMR data, see Tables 1–3. HRMS m/z
Calcd for C48H86O11LiSi 873.6071, found 873.6099.
4.1.3. 4-O-Acetyl-2,3,6-tri-O-(trimethylacetyl)-a-D-
galactopyranosyl bromide (5). To a solution of compound
4 (8.5 g, 15 mmol) in anhydrous CH2Cl2 (50 mL) at 0 8C
was added a 30% solution of hydrogen bromide in
acetic acid (15 mL). The solution was allowed to
reach room temperature. After 1 h, the solution was
extracted with ice-water (5!100 mL) followed by drying
(Na2SO4), filtration, and concentration to afford a colorless
syrup that was used in the next step without further
purification.
4.1.4. Cholesteryl 4-O-acetyl-2,3,6-tri-O-(trimethyl-
acetyl)-b-D-galactopyranoside (7). To a stirred solution
of compound 5 (2.0 g, 3.73 mmol), cholesterol (6, 1.0 g,
2.58 mmol), and di-tert-butyl-4-methylpyridine (1.0 g,
4.87 mmol) in anhydrous CH2Cl2 (10 mL) was added at
K40 8C CF3SO3Ag (2.0 g, 3.89 mmol). The mixture was
allowed to reach 0 8C then was treated with Bu4NBr
followed by aq NaHCO3. The solids were removed by
filtration and the organic layer was concentrated under
vacuum. The residue was stirred in MeOH overnight. The
solids were collected by filtration and were washed with
cold MeOH to afford pure 7 (1.85 g, 85%) as a crystalline
material: [a]D K18 (c 0.5, CHCl3). For NMR data, see
Tables 1–3. HRMS m/z Calcd for C50H82O10Li 849.6071,
found 849.6068.
4.1.9. Cholesteryl 2-O-allyl-6-O-(tert-butyl-diphenyl-
silyl)-3,4-O-isopropylidene-b-D-galactopyranoside (12).
To a stirred solution of 11 (384 mg, 0.46 mmol) in DMF
(2 mL) was added at 0 8C NaH (45 mg of a 60% suspension
in oil). After 20 min, allyl bromide (56 mL, 0.65 mmol) was
added. The solution was allowed to reach room temperature
and was stirred for a further period of 45 min. The mixture
was cooled to 0 8C and was treated with CH3OH (1 mL)
followed by CH3CO2H (0.5 mL).The mixture was concen-
trated and the residue was equilibrated between CHCl3 and
H2O. Concentration of the organic layer followed by
column chromatographic purification of the residue using
10:1 hexane/EtOAc afforded 12 (370 mg, 92%) as a syrup:
[a]D K18 (c 0.4, CHCl3). For NMR data, see Tables 1–3.
HRMS m/z Calcd for C48H86O11LiSi 873.6071, found
873.6099.
4.1.5. Cholesteryl b-D-galactopyranoside (8). A stirred
mixture of compound 7 (1.8 g, 2.1 mmol), CH2Cl2 (5 mL),
and CH3OH (5 mL) was treated with an excess of CH3ONa.
After 24 h, the solids were isolated by filtration and were
washed with CH3OH to afford 8 (1.10 g, 94%) as a
crystalline material: [a]D K58 (c 0.4, C5H5N). For NMR
data, see Tables 1–3. Anal. Calcd for C33H56O7: C, 72.22;
H, 10.29. Found: C, 71.83; H, 10.29.
4.1.10. Cholesteryl 2-O-allyl-3,4-O-isopropylidene-b-D-
galactopyranoside (13). To a solution of compound 12
(2.9 g, 3.6 mmol) in anhydrous THF (20 mL) was added at
room temperature a 1 M solution of tetrabutylammonium
fluoride in tetrahydrofuran (1.2 mL) under argon. After 2
1⁄2 h, the solution was concentrated. Silica gel column
chromatographic purification of the residue using a gradient
of 10:1–3:1 hexane/EtOAc afforded 13 (2.05 g, 97%) as a
syrup: [a]D K21 (c 1.0, CHCl3). For NMR data, see Tables
1–3. HRMS m/z Calcd for C39H64O6Li 635.4866, found
635.4863.
4.1.6. Cholesteryl 3,4-O-isopropylidene-b-D-galacto-
pyranoside (9). To a solution of 8 (1.1 g, 2.0 mmol) in
DMF (5 mL) was added 2,2-dimethoxypropane (5 mL) and
camphorsulfonic acid (100 mg). Thin-layer chromato-
graphy (5:1 hexanes/EtOAc) revealed the initial formation
of two products. After 48 h, the faster-moving component
had almost completely disappeared. Neutralization
(NaHCO3) followed by extractive work-up (CHCl3/H2O)
afforded an amorphous residue that was purified by silica
gel column chromatography using 8:1 hexanes/EtOAc as
the eluant to afford 9 (365 mg, 34%): [a]D K24 (c 0.8,
CHCl3). For NMR data, see Tables 1–3. MS m/z Calcd for
C36H60O6Li 593.4533, found 595.4550.
4.1.11. Cholesteryl 2-O-allyl-3,4-O-isopropylidene-6-O-
palmitoyl-b-D-galactopyranoside (14). A stirred solution
of 13 (300 mg, 0.38 mmol), palmitic acid (300 mg,
1.17 mmol),
dicyclohexyl
carbodiimide
(0.5 g,
2.42 mmol), and 4-dimethylaminopyridine (100 mg) in
EtOAc (6 mL) was kept at room temperature for 6 h.
MeOH (2 mL) was added and the mixture was filtered. The
solids were discarded and the filtrated concentrated. Silica
gel column chromatographic purification of the residue
(100:2 CHCl3/MeOH) afforded 14 (410 mg, 99%) as a
syrup: [a]D K20 (c 0.7, CHCl3). For NMR data, see Tables
1–3. Anal. Calcd for C55H94O7: C, 76.16; H, 10.92. Found:
C, 75.95; H, 10.49.
4.1.7. Cholesteryl 6-O-(tert-butyl-diphenylsilyl)-b-D-
galactopyranoside (10). To a stirred solution of tetraol 9
(8.2 g, 14.9 mmol) in C5H5N (50 mL) were added at room
temperature tert-butyl-diphenylsilyl chloride (8.2 mL,
19.2 mmol) and a catalytic amount of 4-dimethylamino-

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V. Pozsgay et al. / Tetrahedron 61 (2005) 10470–10481
4.1.12. Cholesteryl 2-O-allyl-6-O-palmitoyl-b-D-galacto-
pyranoside (15). To a solution of 14 (400 mg, 0.46 mmol)
in CH2Cl2 (2 mL) was added a solution of AcOH in MeOH
(80%, 25 mL) followed by H2O (2 mL). The solution was
stirred at 70 8C for 6 h. Removal of the volatiles under
reduced pressure afforded a syrup, which was purified by
silica gel column chromatography using 10:1 CHCl3/MeOH
as the eluant to afford 15 (270 mg, 70%) as a syrup: [a]D
K12 (c 0.1, CHCl3). For NMR data, see Tables 1–3. MS m/z
Calcd for C52H90O7Na 849.66, found 849.60. Anal. Calcd
for C52H90O7: C, 75.50; H, 10.97. Found: C, 74.47; H,
10.91.
of 11 (10.0 g, 12.1 mmol) in dry DMF (50 mL) at 0 8C was
added NaH (2.2 g of a 60% suspension in oil). After 10 min,
freshly prepared 4-methoxybenzyl bromide (15 mL) was
added dropwise and the mixture was stirred for 6 h followed
by the usual work-up and silica gel column chromatography
using 4:1 hexanes/EtOAc as the eluant to afford a syrup.
This was dissolved in dry THF (100 mL) and the solution so
obtained was treated with a solution of tetrabutylammonium
fluoride (25 mL of a 1 M solution in THF) at 23 8C. After
6 h, the solution was concentrated and the residue purified
by silica gel column chromatography using a 4:1/1:1
hexanes/EtOAc gradient as the eluant to afford 19 (6.7 g,
78%) as a solid: [a]D C22 (c 0.6, CHCl3). For NMR data,
see Tables 1–3. MS m/z Calcd for C44H68O7Na 731.49,
found 732.08.
4.1.13. Cholesteryl 6-O-palmitoyl-b-D-galactopyranoside
(1). To a solution of 15 (240 mg, 0.29 mmol) in THF (5 mL)
at room temperature was added a solution of (1,5-
cyclooctadiene)bis(methyldiphenylphosphine)iridium(I)
hexafluorophosphate21 (40 mg, 0.28 mmol) in THF (5 mL)
through which H2 was bubbled for 15 min before addition.
After 24 h, the solution was concentrated and the residue
was dissolved in AcOH (5 mL) followed by addition of
TFA (1 mL) at room temperature. After 6 h, the solution
was concentrated and the residue was purified by silica gel
column chromatography using 20:1 CHCl3/MeOH as the
eluant to afford 1 (200 mg, 88%) as an amorphous
substance: [a]D K29 (c 0.5, CHCl3). For NMR data, see
Tables 1–3. MS m/z Calcd for C49H86O7Na 809.63, found
809.68. Anal. Calcd for C49H86O7$MeOH: C, 73.30; H,
11.07. Found: C, 73.49; H, 11.00.
4.1.18. Cholesteryl 3,4-O-isopropylidene-2-O-(4-
methoxybenzyl)-6-O-oleoyl-b-D-galactopyranoside (20).
A stirred solution of 19 (1.1 g, 1.41 mmol), oleic acid
(890 mg, 3.1 mmol), dicyclohexyl carbodiimide (2.0 g,
9.68 mmol), and 4-dimethylaminopyridine (100 mg) in
EtOAc (6 mL) was kept at room temperature for 6 h.
MeOH (4 mL) was added and the mixture was filtered. The
solids were discarded and the filtrate was concentrated.
Silica gel column chromatographic purification of the
residue (100:2 CHCl3/MeOH) afforded 20 (1.2 g, 81%) as
a waxy solid: [a]D C17 (c 1.0, CHCl3). For NMR data, see
Tables 1–3. Anal. Calcd for C49H86O7: C, 76.50; H, 10.35.
Found: C, 76.53; H, 10.51.
4.1.14. Cholesteryl 2-O-allyl-3,4-O-isopropylidene-6-O-
oleoyl-b-D-galactopyranoside (16). A stirred solution of
13 (205 mg, 0.33 mmol), oleic acid (200 mL, 180 mg,
4.1.19. Cholesteryl 3,4-O-isopropylidene-6-O-oleoyl-b-D-
galactopyranoside (21). To a stirred mixture of 20 (1.1 g,
1.13 mmol), CH2Cl2 (10 mL), and H2O (0.4 mL) was added
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (333 mg,
1.47 mmol). After 6 h, the reaction mixture treated with
aq NaHCO3 followed by extractive work-up (CHCl3/H2O).
Silica gel column chromatographic purification of the
residue, using 6:1 hexanes/EtOAc as the eluant afforded
21 (1.0 g, 93%) as an amorphous solid: [a]D K17 (c 0.6,
CHCl3). For NMR data, see Tables 1–3. HRMS m/z Calcd
for C54H92O7Li 859.7032, found 859.7003.
0.63 mmol),
dicyclohexyl
carbodiimide
(0.4 g,
1.94 mmol), and 4-dimethylaminopyridine (100 mg) in
EtOAc (5 mL) was kept at room temperature for 2 h.
MeOH (1 mL) was added and the mixture was filtered. The
solids were discarded and the filtrated concentrated. Silica
gel column chromatographic purification of the residue (8:1
hexanes/EtOAc) afforded 16 (220 mg, 75%) as a syrup: [a]D
K23 (c 0.2, CHCl3). For NMR data, see Tables 1–3. HRMS
m/z Calcd for C57H96O7Li 899.7297, found 899.7361.
4.1.20. Cholesteryl 6-O-oleoyl-b-D-galactopyranoside
(2). A solution of 21 (250 mg, 0.29 mmol) in AcOH
(15 mL) and H2O (1.3 mL) was stirred at 65 8C until TLC
(1:1 hexanes/EtOAc) indicated disappearance of 21
(approx. 4 h). Removal of the volatiles under reduced
pressure afforded a semisolid that was purified by silica gel
column chromatography using a 1:1/1:3 hexanes/EtOAc
gradient as the eluant to afford glycolipids 2 (220 mg, 92%)
as a waxy solid: [a]D K29 (c 0.5, CHCl3). For NMR data,
see Tables 1–3. MS m/z Calcd for C51H88O7Na 835.64,
found 836.41. Anal. Calcd for C51H88O7$1⁄2 MeOH: C,
74.59; H, 10.91. Found: C, 74.36; H, 10.99.
4.1.15. Cholesteryl 2-O-allyl-6-O-oleoyl-b-D-galacto-
pyranoside (17). To a solution of 16 (200 mg, 0.22 mmol)
in CH2Cl2 (2 mL) was added a solution of AcOH in MeOH
(80%, 25 mL) followed by H2O (2 mL). The solution was
stirred under reflux for 6 h. Removal of the volatiles under
reduced pressure afforded a syrup, which was purified by
silica gel column chromatography using 10:1 CHCl3/MeOH
as the eluant to afford 17 (133 mg, 70%) as a syrup: [a]D
K10 (c 0.3, CHCl3). For NMR data, see Tables 1–3. Anal.
Calcd for C54H92O7: C, 76.01; H, 10.87. Found: C, 75.86; H,
11.06.
4.1.16. Cholesteryl 6-O-octadecenoyl-b-D-galactopyrano-
sides (18a,b,c,d). Compound 17 was treated as described for
the preparation of 1. The product was purified by silica gel
column chromatography using 20:1 EtOAc/MeOH as the
eluant to afford 18a–d, as determined by GC–MS.
References and notes
1. Steere, A. C.; Grodzicki, R. L.; Kornblatt, A. N.; Craft, J. E.;
Barbour, A. G.; Burgdorfer, W.; Schmid, G. P.; Johnson, E.;
Malawista, S. E. N. Engl. J. Med. 1983, 308, 733–740.
4.1.17. Cholesteryl 3,4-O-isopropylidene-2-O-(4-methoxy-
benzyl)-b-D-galactopyranoside (19). To a stirred solution

V. Pozsgay et al. / Tetrahedron 61 (2005) 10470–10481
10481
2. Steere, A. C. N. Engl. J. Med. 2001, 345, 115–125.
3. Steere, A. C.; Malawista, S. E.; Snydman, D. R.; Shope, R. E.;
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