C-Glycoside analogues of β-galactosylceramide with a simple ceramide substitute: Synthesis and binding to HIV-1 gp120

Article abstract of DOI:10.1016/j.bmc.2005.09.044

The synthesis and HIV-1 gp120 binding of C- and aza-C-glycoside analogues of β-galactosylceramide (GalCer) that contain a simple C-17 hydrocarbon chain as a ceramide substitute are described. Both compounds originate from stearic acid, and a carbohydrate-

Full text of DOI:10.1016/j.bmc.2005.09.044

Bioorganic & Medicinal Chemistry 14 (2006) 1182–1188
C-Glycoside analogues of b-galactosylceramide with a simple
ceramide substitute: Synthesis and binding to HIV-1 gp120
Line A. Augustin,a Jacques Fantinib,* and David R. Mootooa,*
aDepartment of Chemistry, Hunter College, 695 Park Avenue, New York, NY 10021, USA
bUniversite Paul Cezanne, Laboratoire de Biochimie et Physicochimie des Membranes Biologiques, INRA-UMR 1111,
Faculte des Sciences et Techniques de Saint-Jerome, 13397, Marseille Cedex 20, France
Received 15 August 2005; revised 15 September 2005; accepted 15 September 2005
Available online 10 October 2005
Abstract—The synthesis and HIV-1 gp120 binding of C- and aza-C-glycoside analogues of b-galactosylceramide (GalCer) that con-
tain a simple C-17 hydrocarbon chain as a ceramide substitute are described. Both compounds originate from stearic acid, and a
carbohydrate-derived thioacetal-alcohol, and their syntheses are potentially general for b-C-galactosides and their aza-C-partners.
They showed potent and specific affinity for gp120 in an assay based on the change of surface pressure when the glycolipid mon-
olayers were exposed to solutions of gp120. Interestingly, the aza-C-glycoside exhibited a significantly higher affinity than GalCer,
whereas the C-glycoside was as active as GalCer.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
It has been shown that interaction between the glyco-
sphingolipid GalCer 1 and gp120 is important for HIV
infectivity in CD4 negative cells (Fig. 1).1,2 GalCer has
also been proposed as a co-factor for gp120 attachment
in CD4 presenting cells.3 Consequently, GalCer ana-
logues have attracted interest as mechanistic probes for
the development of new HIV entry inhibitors. Struc-
ture–activity investigations have focused on variations
in the sugar and the fatty acid chain. Trends vary consid-
erably depending on the assay system used.4 It is generally
believed that specificity with respect to the galacto sugar
subunit is high and that the presence or absence of the fat-
ty acid residue or changes in its length or level of hydrox-
ylation can be tolerated.4–8 However, anomalies to these
trends have been reported. Recent studies with water-sol-
uble GalCer analogues suggest a much lower specificity
for the sugar,9 and investigations with analogues that
Figure 1.
were incorporated into monolayer, indicated high speci-
ficity for an a-hydroxylated fatty acid residue over the
sphingosine backbone have been less rigorous. Isolated
non-hydroxylated derivative.8 Structure–activity investi-
examples of analogues with ceramide substitutes of vary-
ing polarities have been reported to show good binding
gations with analogues containing modifications in the
affinity.9–12 Studies have also shown a nonlinear concen-
tration dependence of gp120 for GalCer, suggesting the
involvement of GalCer-rich microdomains.4,13 In view
of potential therapeutic applications, we were interested
Keywords: Galactosyl ceramide; C-Glycoside; Aza-C-glycoside gp120;
Oxocarbenium ion.
in GalCer analogues that were hydrolytically stable and
which contained simple replacements for the ceramide
*
Corresponding authors. Tel.: +1 212 772 4356; fax: +1 212 772 5332
(D.R.M); e-mail: dmootoo@hunter.cuny.edu
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.044

L. A. Augustin et al. / Bioorg. Med. Chem. 14 (2006) 1182–1188
1183
residue. Based on the observation that N-stearyl-1-
deoxynojirimycin 2 and related derivatives with simple
stearyl or stearoyl chains as ceramide substitutes exhibit-
ed gp120 affinity that was comparable to that of
GalCer,14 we set as initial goals, the synthesis of C-glyco-
side 3 and aza-C-glycoside 4, and the determination of
their binding to gp120.15
2. Synthesis
A synthetic strategy, in which 3 and 4 could be derived
from a central C1-substituted galactal precursor 5, was
envisaged (Scheme 1). Thus, stereoselective hydrobora-
tion of 5 was expected to provide the b-C-glycoside
framework, 3.16 Transformation of 5 to a diketone like
6 and stereoselective double reductive amination on 6
were plans for the b-aza-C-glycoside 4.17 Since C1-
substituted galactals 5 with aglycone substituents of vary-
ing complexities are available through our oxocarbenium
ion-enol ether cyclization methodology,16 this protocol
for 3 and 4 is a potentially general one for b-C-galacto-
sides and their corresponding aza-C-derivatives.
Scheme 2. Reagents: (a) stearic acid, DMAP, PhH; (b) Tebbe, py,
THF/toluene, 91% two steps; (c) MeOTf, DTBMP, CH2Cl2, 87%; (d)
BH3ÆMe2S, THF, 76%; (e) (i)—Bu4NF, THF; (ii)—HCl, MeOH, 87%
two steps.
ate diketone precursor. Dihydroxylation of 5 with
osmium tetroxide/NMNO proceeded with complete
a-selectivity to provide lactol 11 in 80% yield (Scheme
3). Selective acetylation of 11 and PCC oxidation of
the monoacetate 12 led to diketone 13 in 78% overall
yield from 11. The key double reductive amination reac-
tion was performed by treatment of 13 with ammonium
formate and NaCNBH3 in anhydrous methanol. The
b-aza-C-galactoside 14 was obtained as a single stereo-
isomer in 72% yield. The stereochemistry of 14 was
assigned on the basis of J values (J1,2 = 9.9, J2,3 = 7.7,
J3,4 = 5.1, and J4,5 = 2.2 Hz) and observation of 1%
NOE effects between H1, and H3 and H5, respectively.
Removal of the alcohol protecting groups in 14 under
standard conditions provided the desired b-aza-C-galac-
toside 4. The high stereoselectivity of the double reduc-
tive amination is consistent with the Stevens model of a
preferred axial attack by nucleophiles on six-membered
half-chair-like, iminium ions.21 That the galacto sub-
strate 13 shows the same sense of stereochemical bias
observed when related gluco-and manno-type diketones
were subjected to similar conditions supports this stereo-
chemistry model.22
The synthesis of 5 started with the DCC mediated ester-
ification of alcohol 718 and stearic acid to give ester 8
(Scheme 2). Tebbe olefination of 8 gave enol ether 9 in
91% overall yield from 7. Treatment of 9 with methyl tri-
flate in the presence of 2,6-di-tert-butyl-4-methylpyri-
dine (DTBMP) provided galactal 5 in 87% yield as a
single stereoisomer of the O-isopropylidene residue
and a single regioisomer of the alkene. The reaction is
presumed to proceed via attack of the enol ether on
the cyclic oxocarbenium derived from thioacetal 9. No
evidence was observed for isomers of 5 that contained
a trans-O-isopropylidene or an exocyclic alkene. These
selectivities are a likely consequence of torsional factors
associated with the 5,6 fused bicyclic framework.
Hydroboration of 5 provided the C-glycoside 10 as a
single diastereomer.19 The stereochemistry of 10 was
confirmed by the 1H COSY analysis of the derived
mono-acetate. The coupling constants (J1,2 = 9.2,
J2,3 = 7.5, J3,4 = 5.1, and J4,5 = 2.2 Hz) are in agreement
with those expected for the 3,4-O-isopropylidene
1b-galacto residue.20 Removal of the silyl ether and
acetonide protecting groups in 10 provided the desired
C-glycoside 3 in 87% overall yield from 10.
The first stage in the preparation of aza-C-glycoside 4
was the transformation of C1 galactal 5 to an appropri-
Scheme 3. Reagents: (a) OsO4 (cat), NMNO, acetone, 80%; (b) Ac2O,
EtOAc, DMAP 95%; (c) PCC, CH2Cl2, Celite, NaOAc, MS 4A, 82%;
(d) NH4HCO2, NaCNBH3, MeOH, 72%; (e) (i)—K2CO3, MeOH;
(ii)—Bu4NF, THF; (iii)—HCl, MeOH, then NaOMe 57%.
Scheme 1.

1184
L. A. Augustin et al. / Bioorg. Med. Chem. 14 (2006) 1182–1188
3. HIV-1 gp120 binding
cause the simple C-linked hydrocarbon chains in 3 and 4
are not expected to favor a well-defined conformation.
However, these results do not rule out the possibility
that the lipid chains in GalCer might be important for
clustering into microdomains, leading to multivalent
binding to gp120.4,13 In such a situation, substitution
of the ceramide residue with simple hydrocarbon chains
as in 3 and 4 might not have a pronounced effect on the
topography of a multivalent assembly, and as a conse-
quence, on binding affinity.25
The binding of 3 and 4 to gp120 was evaluated by mea-
suring the change in surface pressure (Dpi) versus initial
surface pressure (pi) at the air–water interface of the gly-
colipid monolayer, on exposure to an aqueous solution
of recombinant gp120 (10 nM).23 Increase in surface
pressure is associated with integration of gp120 into
the glycolipid monolayer. The critical pressure of inser-
tion, calculated by extrapolation for a null increase in
surface pressure, represents the pressure above which
lipid–lipid interactions are not disrupted by the protein,
and is often taken as a measure of binding affinity.
Together, a critical pressure greater than 10 mN/m and
a linear variation of Dp versus pi is generally interpreted
as an indication of specific binding.24 The critical pres-
sure of insertion was in the range of 24 mN/m for both
GalCer 1 and the C-glycoside 3, and 28.5 mN/m for the
aza-C-glycoside 4. These data suggest that gp120 exhib-
its similar binding to monolayers of 1 and 3, and, appre-
ciably higher affinity for the monolayer formed from 4
(Fig. 2).
That the interaction of gp120 with the aza analogue 4 is
stronger than for GalCer is in of itself noteworthy and
opens up new possibilities for mimetic design. This effect
could be related to aggregation factors in microdomain
formation, or to more intimate receptor contacts in the
individual sugar residues.26 In the absence of a wider set
of analogues additional speculation is premature. Paren-
thetically, the related aza sugar N-butyl-1-deoxynorjim-
icin has been shown to reduce syncytia formation.27 The
mechanism of action is believed to be a result of biosyn-
thetic alteration of the host cell receptor.28 The binding
behavior of 4 and 2 suggests that direct binding of Bu-
DNM to gp120 may be an alternative explanation for
the antiviral activity of Bu-DNM.
4. Discussion
That 3 and 4 bind HIV-1 gp120 with comparable or
even higher affinity than GalCer suggests that it may
be possible to develop GalCer analogues with simple
ceramide substitutes as potent inhibitors of GalCer-
gp120 binding. This data is consistent with observations
on related, stearyl, and stearoyl monosaccharide deriva-
tives with single lipid chains,14 but appear to disagree
with other studies that point to the critical importance
of the fatty acid residue in the ceramide moiety. Thus,
in the identical monolayer assay, the GalCer analogue
without the a-hydroxy group in the acyl chain was com-
pletely inactive8 and in a solid-phase assay psychosine
(without the acyl chain) is inactive.6 The unusual behav-
ior of the single chain analogues 3 and 4 also appears to
be in conflict with the notion that the polar head region
of the ceramide is preorganized for binding to gp120, be-
5. Conclusion
In summary, novel analogues of GalCer with a simpli-
fied hydrocarbon chain as an aglycone substitute were
prepared and shown, in a monolayer binding assay with
gp120, to have similar properties as GalCer. A closer
examination of the molecular basis of these effects using
complementary binding assays is underway and will be
reported in due course. From a synthetic standpoint,
the preparation of 3 and 4 provides a potentially general
protocol for accessing C- and aza-C-b-galactoside pairs
from a common precursor.
6. Experimental
6.1. General synthesis
Solvents were purified by standard procedures or used
from commercial sources as appropriate. Petroleum
ether refers to the fraction of petroleum ether boiling be-
tween 40 and 60 °C. Ether refers to diethyl ether. Unless
otherwise stated thin-layer chromatography (TLC) was
done on 0.25 mm thick precoated Silica Gel 60 (HF-
254, Whatman) aluminum sheets and flash chromatog-
raphy (FCC) was performed using Silica Gel 60 (32–63
mesh, Scientific Adsorbents). Elution for FCC usually
employed a stepwise solvent polarity gradient, correlat-
ed with TLC mobility. Chromatograms were observed
under UV (short and long wavelength) light and/or were
visualized by heating plates that were dipped in a solu-
tion of ammonium (VI) molybdate tetrahydrate
(12.5 g) and cerium (IV) sulfate tetrahydrate (5.0 g) in
10% aqueous sulfuric acid (500 mL). Optical rotations
([a]D) were recorded using a Rudolph Autopol III polar-
imeter at 589 nm (sodium D-line). NMR spectra were
1 (open circles, dotted line) 3 (full triangles, solid line)
4 (full circles, dashed line)
Figure 2. Binding of gp120 to glycolipid analogues.

L. A. Augustin et al. / Bioorg. Med. Chem. 14 (2006) 1182–1188
1185
recorded using GE QE 300, JEOL 400 or Bruker Ultra
Shield instruments. Unless otherwise stated spectra were
recorded in CDCl3 with residual CHCl3 as internal stan-
dard (dH 7.26, dC 77.0). Chemical shifts are quoted in
ppm relative to tetramethylsilane (dH 0.00) and coupling
constants (J) are given in Hertz. First order approxima-
tions are employed throughout. High resolution mass
spectrometry (HRMS) was performed on Micromass
70-SE-4F or Micromass Q-Tof Ultima instruments at
the Mass Spectrometry Laboratory of the University
of Illinois, Urbana-Champaign.
34.0, 37.6, 62.2, 74.7, 80.6, 82.2, 85.5, 111.9, 128.7,
129.4, 130.2, 132.4, 133.9, 135.2, 135.2, 163.3.
6.4. (1S)-1,5-Anhydro-2-deoxy-1-C-heptadecyl-3,4-O-
isopropylidene-6-O-tert-butyldiphenylsilyl-D-lyxo-hex-2-
enitol 6
A mixture of TIA–enol ether 9 (1.39 g, 1.80 mmol), 2,6-
di-tert-butyl-4-methylpyridine (4.40 g, 21.5 mmol), and
˚
freshly activated, powdered 4 A molecular sieves
(2.00 g) in anhydrous CH2Cl2 (200 mL) was stirred for
15 min at rt under an argon atmosphere. The mixture
was cooled to 0 °C. Methyl triflate (2.05 mL, 18.1 mmol)
was then introduced and the reaction mixture was
warmed to rt, and maintained at this temperature for
an additional 24 h, at which time triethylamine
(2.60 mL) was added. The mixture was diluted with
ether, washed with saturated aqueous NaHCO3 and
brine, dried (Na2SO4), filtered, and evaporated under re-
duced pressure. The residue was purified by FCC over
basic alumina (Brockmann I, 150 mesh) to give glycal
6.2. 1,2-O-Isopropylidene-3-O-octadecanoyl-4-O-tert-
butyldiphenylsilyl-D-threo-S-phenyl-mono-thiohemiacetal
8
DCC (600 mg, mmol) was added at 0 °C to a solution of
TIA–alcohol 718 (1.00 g, 1.97 mmol), stearic acid
(840 mg, 2.95 mmol), and DMAP (25 mg, 0.21 mmol)
in anhydrous benzene (50 mL). The reaction was
warmed to rt and stirred for 2 h. The mixture was dilut-
ed with ether and filtered through Celite. The filtrate was
washed with 0.1 N aqueous HCl and brine, dried
(Na2SO4), filtered, and evaporated under reduced pres-
sure. The residue was purified by FCC to give ester 8
6 (1.19 mg, 87%): clear oil; Rf = 0.55 (2% ethyl ace-
25
D
tate:petroleum ether); ½aꢀ þ 18 (c 1.2, CHCl3); 1H
NMR (500 MHz, C6D6) d 0.92 (t, J = 7.0 Hz, 3H),
1.21 (s, 9H), 1.35 (m, 28H), 1.44 (s, 3H), 1.46 (s, 3H),
2.11 (m, 2H), 4.00 (t, J = 6.7 Hz, 1H), 4.17 (q,
J = 6.5 Hz, 2H), 4.25 (m, 2H), 4.57 (dd, J = 3.0,
7.0 Hz, 1H), 4.72 (d, J = 8.3 Hz, 1H), 7.24–7.81 (m,
10H). 13C NMR (125 MHz, C6D6): d 14.9, 23.7, 27.6,
28.4, 29.2, 29.7, 30.7, 30.8, 31.2, 32.4, 32.9, 34.6, 38.1,
41.5, 64.7, 70.9, 72.7, 76.8, 98.7, 110.7, 127.8, 128.1,
128.9, 130.6, 136.6, 136.7, 157.3. HRMS (FAB) calcd
for C42H65O4Si (MꢁH): 661.4652. Found: 661.4649.
(1.40 g, 92%): colorless oil; Rf = 0.60 (10% ethyl acetate:
25
D
petroleum ether); ½aꢀ ꢁ 39:9 (c 5.00, CHCl3); 1H NMR
(500 MHz) d 0.88 (t, J = 6.9 Hz, 3H), 1.03 (s, 9H), 1.25
(m, 28H), 1.44 (s, 3H), 1.48 (s, 3H), 1.58 (m, 2H), 2.29
(m, 2H), 4.34 (dd, J = 3.6, 6.6 Hz, 1H), 5.25 (m, 2H),
7.20–7.70 (m, 15H). 13C NMR (125 MHz) d 14.7, 23.3,
25.5, 26.8, 27.3, 27.8, 29.9, 30.0 (two peaks), 30.2,
30.3, 32.5, 34.9, 62.9, 72.0, 79.7, 85.9, 112.3, 128.2,
128.3, 129.6, 130.3, 130.3, 132.9, 133.6, 134.3, 136.2.
HRMS (ESI) calcd for C47H70O5SSiNa (M+Na):
797.4572. Found: 797.4611.
6.5. (1S)-1,5-Anhydro-1-C-heptadecyl-3,4-O-isopropylid-
ene-6-O-tert-butyldiphenylsilyl-D-galactitol 10
BH3ÆMe2S (1.09 mL of a 1 M solution in CH2Cl2,
1.09 mmol) was added at 0 °C to a solution of glycal 6
(180 mg, 0.27 mmol) in anhydrous THF, under an atmo-
sphere of argon. The mixture was warmed to rt and stir-
red for an additional 1 h at this temperature. The solution
was then re-cooled to 0 °C, and a mixture of 3 N NaOH
(2 mL) and 30% aqueous H2O2 (2 mL) was carefully add-
ed. After stirring at this temperature for an additional
30 min, the mixture was diluted with ether and washed
with saturated aqueous NaHCO3 and brine, dried
(Na2SO4), filtered, and evaporated under reduced pres-
sure. FCC of the residue provided 10 (185 mg, 76%): clear
oil; Rf = 0.30 (15% ethyl acetate: petroleum ether);
6.3. 1,2-O-Isopropylidene-3-O-(2-nondecen-2-yl)-4-O-
tert-butyldiphenylsilyl-D-threo-S-phenyl-mono-thiohemi-
acetal 9
Tebbe reagent in toluene (7.84 mL of a 0.5 M solution,
3.91 mmol) was added at ꢁ78 °C, dropwise under an
atmosphere of argon, to a solution of ester 8 (1.40 g,
1.80 mmol) in a mixture of pyridine (0.2 mL), anhy-
drous toluene (18 mL), and THF (6 mL). The reaction
mixture was warmed to 0 °C, stirred at this temperature
for 1 h, and then for an additional 15 min at rt. The mix-
ture was then slowly poured into 1 N aqueous NaOH at
0 °C. The resulting suspension was extracted with ether,
and the organic phase was washed with brine, dried
(Na2SO4), filtered, and concentrated under reduced
pressure. The residue was purified by FCC on basic alu-
mina (Brockmann I, 150 mesh) to give the enol ether 9
(1.39 g, 99%): colorless oil; Rf = 0.65 (2% ethyl acetate:
25
1
½aꢀ þ 5:1 (c 1.7, CHCl3); H NMR (400 MHz) d 0.89
D
(t, J = 7.0 Hz, 3H), 1.07 (s, 9H), 1.25 (m, 30H), 1.36 (s,
3H), 1.51 (s, 3H), 1.54 (m, partly buried under singlet at
1.51, 1H), 1.83 (m, 1H), 2.81 (br s, 1H, D2O ex.), 3.04
(br t, J = 7.0 Hz, 1H), 3.41 (dd, J = 7.4, 9.9 Hz, 1H),
3.80 (dt, J = 2.2, 8.1 Hz, 1H), 3.92 (m, 2H), 3.98 (dd,
J = 5.5, 7.0 Hz, 1H), 4.30 (dd, J = 2.2, 5.5 Hz, 1H),
7.39–7.72 (m, 10H). 13C NMR (75 MHz): 14.4, 19.5,
23.0, 25.6, 26.7, 27.1, 28.6, 29.6, 29.7, 30.0, 31.8, 32.2,
63.2, 74.0, 76.8, 78.4, 79.5, 109.8, 127.7, 127.8, 127.9,
129.7, 130.0, 133.6, 135.7, 135.8. HRMS (FAB) calcd
for C38H59O4Si (M–C4H9): 623.4132. Found: 623.4133.
25
1
petroleum ether); ½aꢀ ꢁ 57:0 (c 1.4, CHCl3); H NMR
(500 MHz, C6D6) dD0.92 (t, J = 7.0 Hz, 3H), 1.18 (s,
9H), 1.33 (m, 30H), 1.52 (s, 3H), 1.53 (s, 3H), 2.16 (t,
J = 7.9 Hz, 2H), 3.95 (m, 2H), 4.15 (m, 2H), 4.52 (m,
1H), 4.77 (dd, J = 1.8, 7.0 Hz, 1H), 5.81 (d, J = 7.0 Hz,
1H), 7.10–7.80 (m, 15H). 13C NMR (125 MHz, C6D6)
d 14.6, 23.3, 27.2, 30.0, 30.3, 30.8, 32.0, 32.5, 33.3,

1186
L. A. Augustin et al. / Bioorg. Med. Chem. 14 (2006) 1182–1188
6.6. (1S)-1,5-Anhydro-1-C-heptadecyl-D-galactitol 3
reaction mixture was stirred at rt for 5 min, then
quenched with methanol (0.5 mL) and evaporated under
reduced pressure. The residue was purified by FCC to
give 12 (91 mg, 95%): clear oil; Rf = 0.85 (20% ethyl ace-
TBAF (0.85 mL of 1 M solution) was added at rt to a
solution of 11 (190 mg, 0.279 mmol) in anhydrous
THF (2 mL) under nitrogen. The reaction mixture was
stirred for 4 h and then concentrated under reduced
pressure. FCC of the residue provided a homogeneous
material (127 mg): Rf = 0.13 (20% ethyl acetate:petro-
leum ether). This material was dissolved in methanol
(2 mL) and treated with an approximately 1 N solution
of HCl in ether (0.1 mL) at rt for 10 min. The mixture
was then neutralized with a solution of NaOMe in meth-
anol, diluted with brine, and extracted with ether. The
organic extract was dried (Na2SO4), filtered, and evapo-
rated in vacuo. FCC of the residue provided 3 (100 mg,
87 %): clear oil; Rf = 0.23 (10% methanol: chloroform);
1H NMR (500 MHz, CD3OD) d 0.90 (t, J = 7.0 Hz,
3H), 1.29 (m, 30H), 1.59 (m, 1H), 1.84 (m, 1H), 3.06
(t, J = 7.9 Hz, 1H), 3.38 (m, 3H), 3.69 (m, 2H), 3.87
(m, 1H); 13C NMR (125 MHz, CD3OD) d 14.6, 23.8,
26.8, 30.6, 30.8, 30.9, 33.0, 33.2, 62.9, 71.0, 72.9, 76.7,
80.2, 81.7. HRMS (FAB) calcd for C23H47O4 (M+H):
403.3424. Found: 403.3423.
1
tate:petroleum ether); H NMR (300 MHz) d 0.86 (t,
J = 7.0 Hz, 3H), 1.10 (s, 9H), 1.25 (s, 30H), 1.32 (s,
3H), 1.53 (s, 3H), 2.10 (m, 2H), 2.12 (s, 3H), 3.85, 3.96
(both m, 1H ea), 4.32 (m, 3H), 4.95 (d, J = 7.50 Hz,
1H), 7.40–7.60 (m, 10H); 13C NMR (75 MHz) d 19.6,
21.4, 22.1, 23.0, 26.9, 27.0, 27.1, 27.2, 28.0, 29.7, 29.8,
30.0, 30.2, 32.2, 38.3, 63.2, 69.2, 73.4, 73.7, 75.3, 76.8,
97.9, 109.7, 127.7, 127.8, 127.9, 128.0, 128.1, 129.7,
129.8, 130.0, 133.1, 133.7, 133.8, 135.8, 170.3.
HRMS(FAB) calcd for C44H71O5SiN: 722.5180. Found:
722.5183.
6.9. (3R,4S,5R)-3,4-O-Isopropylidene-1-tert-butyldiphe-
nylsilyloxy-2,6-dioxotricosan-5-yl acetate 13
To a mixture of PCC (147 mg, 0.136 mmol), Celite
(147 mg), florisil (15 mg), sodium acetate (56 mg,
˚
0.68 mmol), freshly activated 4 A molecular sieves
(200 mg), and CH2Cl2 (3 mL), under an argon atmo-
sphere, was added a solution of 12 (100 mg, 0.136 mmol)
in CH2Cl2 (2 mL). The reaction mixture was stirred at rt
for 3 h and then filtered through a bed of Celite. The fil-
trate was concentrated under reduced pressure, and the
residue was purified by FCC to give 13 (82 mg, 82%):
colorless oil; Rf = 0.80 (10% ethyl acetate:petroleum
6.7. (3R,4R,5S,6R)-2-Heptadecyl-tetrahydro-4,5-O-
isopropylidene-6-tert-butyldiphenylsilyloxy-methyl-2H-
pyran-2,3-diol 11
N-Methylmorpholine-N-oxide (0.57 mL, 60 wt% in
H2O, 3.32 mmol) and osmium tetroxide (2.08 mL 2.5
wt% in tert-butanol, 0.17 mmol) were added to a solu-
tion of 6 (1.1 g, 1.66 mmol) in acetone (20 mL). The
reaction mixture was stirred at rt for 0.5 h, at which time
a solution of 1 N aqueous sodium bisulfite (0.57 mL,
1 N) was added and the mixture was stirred for addi-
tional 0.5 h. Most of the solvent was evaporated under
reduced pressure, and the residue was diluted with water
and extracted with ethyl acetate. The combined organic
phase was dried (Na2SO4), filtered, and evaporated in
vacuo. FCC of the residue gave mixture 11 (0.93 g,
80%): colorless oil; Rf = 0.10 (10% ethyl acetate:petro-
1
ether); H NMR (400 MHz) d 0.86 (t, J = 7.0 Hz, 3H),
1.10 (s, 9H), 1.25 (br s, 30H), 1.27, 1.5 (both s, each
3H) 2.08 (s, 3H), 2.42 (m, 2H), 4.40 (ABq,
J = 17.0 Hz, Dd = 0.15 ppm, 1H), 4.66 (d, J = 2.2 Hz,
1H), 4.95 (dd, J = 2.2, 8.4 Hz, 1H), 5.17 (d, J = 8.4 Hz,
1H), 7.40–7.60 (m, 10H); 13C NMR (125 MHz) d 14.7,
19.8, 20.9, 23.3, 23.4, 24.9, 26.7, 27.3, 29.6, 29.9, 30.0,
30.2, 30.3, 32.5, 39.4, 69.1, 77.1, 80.1, 111.2, 128.3,
128.5, 130.2, 130.6, 132.9, 133.3, 135.4, 136.0, 136.2,
171.2, 206.0, 207.2. HRMS(FAB) calcd for C44H68O7Si
(M+Na): 759.4629. Found: 759.4632.
1
leum ether); H NMR (500 MHz) d 0.91 (t, J = 7.0 Hz,
6.10. (1S)-2-O-Acetyl-1,5-dideoxy-1-C-heptadecyl-3,4-O-
isopropylidene-1,5-imino-D-galactitol 4 14
3H), 1.08 (s, ca. 3H), 1.10 (s. 6H), 1.35 (s. ca 1H), 1.38
(s, 2H), 1.50 (s, ca. 2H), 1.52 (s, ca. 1H), 1.25–1.70 (m,
30H), 1.74 (m, ca. 1.33 H), 2.19 (d, J = 6.0 Hz, ca.
0.67 H, D2O ex), 2.57 (dt, J = 7.5, 17.5 Hz, ca. 0.33H),
2.68 (dt, J = 7.0, 17.5 Hz, ca. 0.33H), 3.41 (br s, ca.
0.33H, D2O ex), 3.59 (t, J = 6.0 Hz, ca. 0.67 H), 3.75–
4.00 (m, ca. 2.33H), 4.18–4.40 (m, ca. 2.33 H), 4.35
(m, ca. 0.33H), 4.51 (m, ca. 0.33H), 7.40–7.80 (m,
10H). 13C NMR (125 MHz) d 14.4, 19.4, 22.39, 22.9,
25.2, 26.3, 27.7, 28.3, 29.6, 29.7, 32.1, 38.7, 63.2, 65.3,
69.0, 69.6, 72.7, 73.3, 76.3, 98.1, 109.4, 127.8, 128.0,
129.8, 130.1, 133.7, 135.7, 135.9. HRMS (FAB) calcd
for C42H68O6Si (M+Na): 719.4684. Found: 719.4683.
Sodium cyanoborohydride (75 mg, 1.1 mmol) was add-
ed to a mixture of 13 (250 mg, 0.34 mmol), ammonium
˚
formate (38.0 mg, 0.6 mmol), and 4 A powdered molec-
ular sieves (100 mg) in anhydrous methanol (10 mL),
under an argon atmosphere. The reaction mixture was
stirred for 30 min at rt and then filtered through a bed
of Celite. The filter cake was washed with ether and
the filtrate was concentrated under reduced pressure.
The residue was dissolved in ether (20 mL) and washed
with saturated aqueous NaHCO3 and brine, dried
(Na2SO4), filtered, and concentrated under reduced
pressure. FCC of the residue to give 14 (176.3 mg,
72%): clear oil; Rf = 0.45 (15% ethyl acetate:petroleum
6.8. (3R,4S,5S,6R)-2-Heptadecyl-tetrahydro-2-hydroxy-
4,5-O-isopropylidene-6-tert-butyl-diphenylsilyloxymeth-
yl-2H-pyran-3-yl acetate 12
1
ether); H NMR (300 MHz) d 0.86 (br t, J = 7.0 Hz,
3H), 1.10 (s, 9H), 1.25 (s, 30H), 1.33 (s, 3H), 1.62 (s,
3H), 1.20–1.40 (m, partially buried under singlets at
1.33 and 1.62 ppm, 2H), 2.45 (t, J = 9.3 Hz, 1H), 3.11
(m, 1H), 3.82 (m, 2H), 3.98 (m, 1H), 4.18 (m, 1H),
4.83 (dd, J = 7.8, 9.9 Hz, 1H), 7.40–7.60 (m, 10H); 13C
To a solution of 11 (90 mg, 1.3 mmol) and DMAP
(15.9 mg, 0.13 mmol) in ethyl acetate (10 mL) was added
acetic anhydride (0.16 mL, 1.56 mmol) dropwise. The

L. A. Augustin et al. / Bioorg. Med. Chem. 14 (2006) 1182–1188
1187
NMR (75 MHz) d 14.4, 19.5, 21.4, 23.0, 25.9, 26.8, 26.9,
27.1, 28.1, 29.6, 29.9, 30.0, 31.6, 32.2, 57.3, 57.8, 64.4,
74.1, 76.5, 78.7, 86.9, 109.9, 127.8, 129.8, 133.5, 133.6,
134.9, 135.7, 170.3. HRMS(FAB) calcd for C44H71O5S-
iN (M+H): 722.5183. Found: 722.5179.
surface pressure (pi) of the monolayer. The data were
analyzed with the Filmware 2.5 program (Kibron,
Inc.). The results are expressed as the variations of Dp
as a function of pi for compounds 1, 3, and 4. The accu-
racy of the system under our experimental conditions
was 0.25 mN/m for surface pressure.
6.11. (1S)-1,5-Dideoxy-1-C-heptadecyl-1,5-imino-D-
galactitol 4
Acknowledgments
To a solution of 14 (150 mg, 0.21 mmol) in anhydrous
methanol (5 mL) was added potassium carbonate
(21 mg, 0.21 mmol). The reaction mixture was stirred
at rt for 0.5 h and then evaporated in vacuo. The crude
material was dissolved in THF (5 mL) and treated with
n-Bu4NF (0.4 mL, 1 M in THF) for 1 h at rt. The mix-
ture was then diluted with water and extracted with
ether. The organic phase was washed with brine, dried
(Na2SO4), filtered, and concentrated under reduced
pressure. FCC of the residue afforded the derived diol
(64 mg, 72%): clear oil; Rf = 0.1 (40% ethyl acetate:pe-
This investigation was supported by Grants R01
GM57865 and SCORE S06 GM60654 from the Nation-
al Institute of General Medical Sciences of the National
Institutes of Health (NIH). ꢀResearch Centers in Minor-
ity Institutionsꢁ award RR-03037 from the National
Center for Research Resources of the NIH, which sup-
ports the infrastructure and instrumentation of the
Chemistry Department at Hunter College, is also
acknowledged.
1
troleum ether); H NMR (300 MHz, CDCl3) d 0.86 (br
t, J = 7.0 Hz, 3H), 1.25 (br s, 32H), 1.32 (s, 3H), 1.52
(s, 3H), 2.42 (m, 1H), 3.11 (m, 1H), 3.32 (dd, J = 7.8,
9.9 Hz, 1H), 3.82 (m, 2H), 3.98 (m, 1H), 4.18 (m, 1H).
Supplementary data
Supplementary data associated with this article can be
found in the online version at doi:10.1016/
j.bmc.2005.09.044.
An approximately 1 N solution of HCl in ether (0.1 mL)
was added at rt to a solution of the material from the
previous step (60 mg, 0.14 mmol) and in anhydrous
methanol (5 mL). The reaction was maintained at this
temperature and monitored by TLC. When the starting
material had completely disappeared, solid NaOMe was
carefully added to a pH of 7. The solution was then con-
centrated in vacuo and the residue as purified by FCC to
give 4 (44 mg, 80%): clear oil; Rf = 0.1 (20% metha-
nol:ethyl acetate). 1H NMR (500 MHz, CD3OD) d
0.90 (br t, J = 7.0 Hz, 3H), 1.30 (m, 28H), 1.50 (m,
2H), 1.62 (m, 1H), 1.94 (m, 1H), 2.00 (s, 1H, –NH),
2.78 (m, 1H), 3.15 (t, J = 6.5 Hz, 1H), 3.42 (dd,
J = 3.0, 8.5 Hz, 1H), 3.62 (t, J = 10.0 Hz, 1H), 3.78 (m,
2H), 4.00 (br s, 1H); 13C NMR (75 MHz, CD3OD) d
14.6, 23.9, 26.8, 30.6, 30.7, 30.9, 31.1, 32.0, 32.2, 60.9,
61.2, 61.5, 68.9, 71.7, 75.6. HRMS(FAB) calcd for
C23H48NO4 (M+H): 402.3583. Found: 402.3584.
References and notes
1. Harouse, J. M.; Bhat, S.; Spitalnik, S. L.; Laughlin, M.;
Stefano, K.; Silberberg, D. H.; Gonzalez-Scarano, F.
Science 1991, 252, 320–323.
2. Fantini, J.; Cook, D. G.; Nathanson, N.; Spitalnik, S. L.;
Gonzalez-Scarano, F. Proc. Natl. Acad. Sci. U.S.A. 1993,
90, 2700–2704.
3. Fotopoulos, G.; Harari, A.; Michetti, P.; Trono, D.;
Pantaleo, G.; Kraehenbuhi, J.-P. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 9410–9414.
4. For a discussion of inherent limitations with different
assays: Conboy, J. C.; McReynolds, K. D.; Gervay-
Hague, J.; Saavedra, S. S. J. Am. Chem. Soc. 2002, 124,
968–977.
5. Bhat, S.; Spitalnik, S. L.; Gonzalez-Scarano, F.; Silber-
berg, D. H. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 7131–
7134.
6.12. Surface pressure measurements
6. Long, D.; Berson, J. F.; Cook, D. G.; Doms, R. W.
J. Virol. 1994, 68, 5890–5898.
7. McAlarney, T.; Apostolski, S.; Lederman, S.; Latov, N.
J. Neurosci. Res. 1994, 37, 453–460.
The surface pressure was measured with a fully auto-
mated microtensiometer (lTROUGH SX; Kibron,
Inc., Helsinki, Finland). The apparatus allowed the
real-time recording of the kinetics of interaction of a sol-
uble ligand with the monomolecular film, using a set of
specially designed Teflon troughs. All experiments were
carried out in a controlled atmosphere at 20 1 °C.
Monomolecular films of 1, 3, and 4 were spread on pure
water subphases (volume of 800 lL) from hexane–chlo-
roform–ethanol solution as described previously.21 After
spreading of the film, 5 min was allowed for solvent
evaporation. To measure the interaction of gp120 with
GalCer and its derivatives, recombinant gp120 (IIIB iso-
late, 10 nM) was injected in the subphase with a 10 lL
Hamilton syringe, and pressure increases produced were
recorded until reaching a stable value (Dp). The experi-
ment was repeated at different values of the initial
´
´
8. Hammache, D.; Pieroni, G.; Yahi, N.; Delezay, O.; Koch,
N.; Lafont, H.; Tamalet, C.; Fantini, J. J. Biol. Chem.
1998, 273, 7967–7971.
9. McReynolds, K. D.; Hadd, M. J.; Gervay-Hague, J.
Bioconjugate Chem. 1999, 10, 1021–1031; McReynolds, K.
D.; Bhat, A.; Conboy, J. C.; Saavedra, S. S.; Gervay-
Hague, J. Biorg. Med. Chem. 2002, 10, 625–637.
10. Bertozzi, C. R.; Cook, D. G.; Kobertz, W. R.; Gonzalez-
Scarano, F.; Bednarski, M. D. J. Am. Chem. Soc. 1992,
114, 10639–10641.
´
´
11. Fantini, J.; Hammache, D.; Delezay, O.; Yahi, N.; Andre-
Barres, C.; Rico-Lattes, I.; Lattes, A. J. Biol. Chem. 1997,
`
´
272, 7245–7252; Rico-Lattes, I.; Gouzy, M.-F.; Andre-
Barres, ; Guidetti, B.; Lattes, A. New J. Chem. 1998, 6,
`
451–457.

1188
L. A. Augustin et al. / Bioorg. Med. Chem. 14 (2006) 1182–1188
12. Villard, R.; Hammache, D.; Delapierre, G.; Fotiadu, F.;
Buono, G.; Fantini, J. ChemBioChem 2002, 3, 517–525.
13. Hammache, D.; Yahi, N.; Maresca, M.; Pieroni, G.;
Fantini, J. J. Virol. 1999, 73, 5244–5248.
22. Baxter, E. W.; Reitz, A. B. J. Org. Chem. 1994, 59, 3175–
3185; Martin, O. R.; Saavedra, O. M. J. Org. Chem. 1996,
61, 6987–6993.
23. Mahfoud, R.; Mylvaganam, R.; Lingwood, C. A.; Fantini,
J. J. Lipid Res. 2002, 43, 1670.
24. Briggs, M. S.; Gierasch, L. M.; Zlotnick, A.; Lear, J. D.;
DeGrado, W. F. Science 1985, 228, 1096–1099; Baten-
burg, A. M.; Demel, R. A.; Verkleijg, A. J.; de Kruijff, B.
Biochemistry 1988, 27, 5678–5685.
25. Kensinger, R. D.; Catalone, B. J.; Krebs, F. C.; Wigdahl,
B.; Schengrund, C. Antimicrob. Agents Chemother. 2004,
1614–1623.
26. An aromatic Phe residue in the V3 loop of HIV-1 gp120
is directly involved in GalCer binding, so that the
aromatic phenyl cycle stacks onto the sugar cycle. Taieb,
N.; Yahi, N.; Fantini, J. Adv. Drug Deliv. Rev. 2004, 56,
779–794, An interesting speculation is that the proton-
ated azasugar analogue could engage in a favorable NH–
p interaction with the aromatic ring, leading to increased
binding of 4 to gp120 (relative to 1 and 3). Alternatively,
it is conceivable that the electrostatic repulsion between
the protonated amine in adjacent molecules of 4 could
reduce the overall lipid–lipid attraction in monolayers
formed from 4 relative to monolayers from the neutral
analogues 1 and 3. This effect could lead to a monolayer
of 4 that is intrinsically more ꢀpenetrableꢁ (i.e., with a
greater critical pressure of insertion).
14. Weber, K. T.; Hammache, D.; Fantini, J.; Ganem, B.
Bioorg. Med. Chem. Lett. 2000, 10, 1011–1014.
15. For seminal work on C-glycoside analogues of GalCer, see
Ref. 10. Other examples of C-glycolipids: Yang, G.;
Franck, R. W.; Bittman, R.; Samadder, P.; Arthur, G.
Org. Lett. 2001, 3, 197–200; Yang, G.; Franck, R. W.;
Byun, H.-S.; Bittman, R.; Samadder, P.; Arthur, G. Org.
Lett. 1999, 1, 2149–2151; Yang, G.; Schmieg, J.; Tsuji, M.;
Franck, R. W. Angew. Chem. Int. Ed. Engl. 2004, 43,
3818–3822; Dondoni, A.; Perrone, D.; Turturici, E. J. Org.
Chem. 1999, 64, 5557–5564; Cipolla, L.; Nicotra, F.;
Vismara, E.; Guerrini, M. Tetrahedron 1997, 53, 6163–
6170.
16. Khan, N.; Cheng, X.; Mootoo, D. R. J. Am. Chem. Soc.
1999, 121, 4918–4919.
17. Cheng, X.; Kumaran, G.; Mootoo, D. R. Chem. Commun.
2001, 811–812.
18. Alcohol 7 is available in five steps from commercially
available D-lyxose in an overall yield of approximately
60%. Khan, N.; Cheng, X.; Mootoo, D. R. J. Org. Chem.
2000, 65, 2544–2547.
19. For related examples of stereoselective hydroboration of
Cl-substituted galactals: Calimente, D.; Postema, M. H.
D. J. Org. Chem. 1999, 64, 1770–1771; Schmidt, R. R.;
Dietrich, H. Liebigs. Ann. Chem. 1994, 975–981.
20. Barili, P. L.; Catelani, G.; DꢁAndrea, F.; Mastrorilli, E.
J. Carbohydr. Chem. 1997, 16, 1001–1010.
27. Karpas, A.; Fleet, G. W.; Dwek, R. A.; Petursson, S.;
Namgoong, S. K.; Ramsden, N. G.; Jacob, G. S.; Radem-
acher, T. W. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 9229.
28. Karlsson, G. B.; Butters, T. B.; Dwek, R. A.; Platt, F. M.
J. Biol. Chem. 1993, 268, 570.
21. Stevens, R. V. Acc. Chem. Res. 1984, 17, 289–296.