Synthesis and structure-activity relationship study of isoglobotrihexosylceramide analogues

Article abstract of DOI:10.1021/jo701539k

(Chemical Equation Presented) Invariant natural killer T (iNKT) cells are innate T lymphocytes that express T cell receptors binding to exogenous and endogenous glycosphingolpid antigens presented by a nonpolymorphic, non-MHC antigen presenting molecule,

Full text of DOI:10.1021/jo701539k

Synthesis and Structure-Activity Relationship Study of
Isoglobotrihexosylceramide Analogues
Wenlan Chen,† Chengfeng Xia,‡ Jinhua Wang,‡ Prakash Thapa,§ Yusen Li,§ Janos Nadas,†
Wenpeng Zhang,‡ Dapeng Zhou,*,§ and Peng George Wang*,†,‡
Department of Chemistry, The Ohio State UniVersity, Columbus, Ohio 43210, Department of Biochemistry,
The Ohio State UniVersity, Columbus, Ohio 43210, Department of Melanoma Medical Oncology,
UniVersity of Texas M.D. Anderson Cancer Center, Houston, Texas 77054
dzhou@mdanderson.org; pgwang@chemistry.ohio-state.edu
ReceiVed July 13, 2007
Invariant natural killer T (iNKT) cells are innate T lymphocytes that express T cell receptors binding to
exogenous and endogenous glycosphingolpid antigens presented by a nonpolymorphic, non-MHC antigen
presenting molecule, CD1d. The endogenous glycosphingolipid metabolite, isoglobotrihexosylceramide
(iGb3), is the first known natural ligand for both human and mouse iNKT cells, whose activity has been
confirmed in a variety of iNKT cell clones generated by different investigators, representing the majority
of the iNKT cell population. The signaling pathway mediated by T cell receptor is largely influenced by
the structural variation of glycosphingolpid antigens, leading to multiple and varied biological functions
of iNKT cells. In order to investigate the structural requirements behind iGb3 triggered iNKT cell
activation, the structure-activity relationship (SAR) of iGb3 needs to be characterized. In this study,
iGb3 analogues containing 2′′′, 3′′′, 4′′′ and 6′′′ deoxy terminal galactose were synthesized for probing
the SAR between iGb3 and TCR. The biological assays on the synthetic iGb3 analogues were performed
with use of the murine iNKT cell hybridoma DN32.D3. The results showed that the 2′′′ and 3′′′ hydroxyl
groups of terminal galactose play more important roles for the recognition of iGb3 by TCR; while 4′′′
and 6′′′ hydroxyl groups were not as crucial for this recognition. These studies might help to understand
the general structural requirements for natural endogenous ligands recognized by iNKT cells.
Introduction
sented by the MHC class I-like CD1d protein are recognized
by natural killer T (NKT) cells.2 The major subset of CD1d
restricted NKT cells (also called invariant NKT cells) express
semi-invariant VR T cell receptor (VR14 in mouse and VR24
in human).3 iNKT cells play major roles as a bridging system
A group of specific peptide antigens bound to major histo-
compatibility complex (MHC) class II or I molecules are
recognized by CD4 and CD8 T cells, respectively.1 In contrast
to those peptide antigens, a variety of glycolipids antigens pre-
(1) (a) Krummel, M. F.; Davis, M. M. Curr. Opin. Immunol. 2002, 14,
66-74. (b) Bromley, S. K.; Burack, W. R.; Johnson, K. G.; Somersalo, K.;
Sims, T. N.; Sumen, C.; Davis, M. M.; Shaw, A. S.; Allen, P. M.; Dustin,
M. L. Annu. ReV. Immunol. 2001, 19, 375-396. (c) Gao, G. F.; Jakobsen,
B. K. Immunol. Today 2000, 21, 630-636.
* To whom correspondence should be addressed. P.G.W.: phone 614-292-
9884, fax 614-688-3106. D.Z.: phone 713-792-3134, fax 713-563-3424.
† Department of Chemistry, The Ohio State University.
‡ Department of Biochemistry, The Ohio State University.
§ University of Texas M.D. Anderson Cancer Center.
10.1021/jo701539k CCC: $37.00 © 2007 American Chemical Society
Published on Web 11/17/2007
9914
J. Org. Chem. 2007, 72, 9914-9923

Isoglobotrihexosylceramide Analogues
between innate and adaptive immunity.4 Like natural killer cells,
iNKT cells are activated in the first line of immune response
within 2 to 6 h upon stimulation to secret proinflammatory T
helper 1 (Th1) and immunomodulatory Th2 cytokines and
chemokines which allows iNKT cells to regulate a number of
disease states in vivo, including malignancy and infection, as
well as autoimmune diseases.5
but without the terminal galactose) could not stimulate mouse
VR14 iNKT TCR cells,11 and the isoglobotetrahexosylceramide
(iGb4) could not stimulate either.8
Although several CD1d/glycolipid structures have been
determined,12 the structure of the CD1d/glycolipid/TCR ternary
complex has become the most pressing target to completely
elucidate the SAR between the CD1d presented glycolipid and
TCR. This barrier was recently conquered by Borg et al.13 Most
surprisingly, the NKT TCR adopted an acute docking mode that
is directly contradicting to previous hypothetical models gener-
ated by molecular modeling.14 The NKT TCR bound ap-
proximately parallel to the long axis of the CD1d-antigen-
binding cleft, which is distinct from the “diagonal” footprints
observed for MHC class I restricted TCRs. More surprisingly,
comparison of unliganded NKT TCR to the NKT TCR in the
ternary structure did not show drastic change of conformation
upon ligand binding. Thus Borg et al. suggested a “lock-and-
key” instead of “induced fit” mode of NKT TCR binding. The
other striking finding is that only NKT TCRR is contacting the
R-GalCer antigen, while the CDR3â of NKT TCRâ is displaced
from the antigen binding groove. The 2′, 3′, and 4′ hydroxyl
groups of R-GalCer directly bind to CDR3R through hydrogen
bondings, thus leading to the recognition by TCR. While it is
not clear yet whether the terminal galactose of iGb3 directly
binds to CDR3R loop CD1d/R-GalCer/TCR studies showed
that the 3′ and 4′ hydroxyl groups of R-GalCer are adjacent to
a large positively charged pocket lined by the CDR3â of NKT
TCRâ. This positively charged pocket might also provide a
possibility to accommodate more bulky sugar head groups such
as the trisaccharide head of iGb3.
Ever since the first discovery of R-galactosylceramides (R-
GalCer),6 it has been verified that R-galactosyl lipids, e.g.,
KRN7000,7 can stimulate iNKT cells to produce cytokines like
interferon-γ (IFN-γ) and interleukin-4 (IL-4). Nevertheless,
R-GalCer is not the endogenous ligand for iNKT cells. In 2004,
Zhou et al. discovered that the lysosomal isoglobotrihexosyl-
ceramide (iGb3) was an endogenous glycolipid that could
stimulate both human and mouse iNKT cells.8 After Zhou et
al.’s initial discovery, iGb3 has been verified by five other
laboratories to stimulate a variety of mouse and human NKT
clones representing a majority of invariant NKT population.9
These include human NKT cell lines, and mouse hybridomas
VR14i-DOâ generated in by Gapin’s group,9e VR14i-24.8Aâ
that express the invariant T cell receptor R (TCRR) and the
TCRâ of the NKT clone 24.8.A originally from Brenner’s
group,9f and N38-2C11 and N38-3C generated by Hayakawa’s
group.9g Studies on iGb3 derivatives indicated that the terminal
galactose and its R(1,3) configuration might be crucial for
recognition.8,10 Further proof of this was that when the disac-
charide glycolipid Galâ1-4Glcâ1-1′ ceramide (same as iGb3
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The orientation and the fine structure of the terminal sugar
moiety might play a critical part for TCR binding, as NKT cells
recognize iGb3 (R1,3 linked terminal galactose), but not Gb3
(R1,4 linked terminal galactose).8 Therefore, the replacement
of the hydroxyl groups of terminal galactose with nonpolar
hydrogens has the possibility to eliminate potential hydrogen
bond interactions, consequently resulting in the SAR of these
positions (Figure 1).
(3) (a) Benglagha, K.; Bendelac, A. Semin Immunol. 2000, 12, 537-
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Results and Discussion
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The key component for the SAR study of iGb3 is the terminal
deoxy galatosyl moiety that is introduced in the form of the
deoxy galactosyl donor A. The retrosynthetic strategy of deoxy-
hydroxyl-iGb3 (dh-iGb3) is illustrated in Scheme 1. In this
strategy, phytosphingosine D was employed instead of the
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M. Science 1997, 278, 1626-1629.
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J. Org. Chem, Vol. 72, No. 26, 2007 9915

Chen et al.
FIGURE 1. The design of the iGb3 analogues for the SAR study.
SCHEME 1. Retrosynthetic Synthesis of dh-iGb3s
SCHEME 2. Synthesis of Terminal Deoxy Donors
original erythro-sphingosine for the synthesis to improve the
efficacy in stimulating iNKT cells,15 without interfering in the
study of the terminal sugar moiety. The deoxy trisaccharide
donor E can be arrived at by the glycosylation of the deoxy-
galactosyl donor A with the disaccharide acceptor B, which can
be prepared from the commercially available lactose. The
glycosylation between E and the phytosphingosine acceptor
(lipid) D followed by an amination reaction with the long chain
acid C yields the final product dh-iGb3.
The preparation of deoxy galactosyl donors 1-4 is shown in
Scheme 2. Peracetylation of the commercially available 2-deoxy
galactose 5 followed by the treatment of thiolphenol (PhSH) in
the presence of BF3-Et2O produced the 2-deoxy donor 1. The
3-deoxy donor 2 was obtained by deoxygenation of compound
716 through the formation of thiolcarbonate 8 followed by
treatment with Bu3SnH and azobisisobutyronitrile (AIBN).17 The
(16) Kanie, O.; Ito, Y.; Ogawa, T. Tetrahedron Lett. 1996, 37, 4551-
4554.
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195.
9916 J. Org. Chem., Vol. 72, No. 26, 2007

Isoglobotrihexosylceramide Analogues
SCHEME 3. Synthesis of dh-iGb3 Analogues
4-deoxy acetylated galactose 918 was treated with PhSH to give
the corresponding donor 3. The 6-deoxy donor 4 was prepared
according to the published protocol.19 Among these four deoxy
donors, the benzyl groups were employed as protecting groups
for compounds 2-4 instead of the acetyl group in donor 1. This
is to eliminate the neighboring group participating effect during
the preparation of 3′′′, 4′′′, and 6′′′ deoxy trisaccharides.
The synthesis of these four designed terminal deoxy iGb3
analogues as outlined in Scheme 3 yielded the final product in
about 8-10 steps. During the preparation of the deoxy trisac-
charide donors, the synthetic route for the 2′′′ deoxy trisaccharide
donor is slightly different from those for the 3′′′, 4′′′, and 6′′′
deoxy ones. It has been found that, among a variety of
promoters, the combination of triflic acid (TfOH) with N-
iodosuccinimide (NIS) at -20 °C provides a much better
R-selectivity for the glycosylations to provide R-galactosyl (R-
Gal) derivatives.20 The glycosylation between 2-deoxy donor 1
and disaccharide acceptor 109a proceeded smoothly to give
trisaccharide 11 by using TfOH as a promoter combining with
confirmed by the signal of â-1′′′-H at δ 5.28 ppm, which has a
small coupling constant with 2′′′-H. Hydrogenolysis of the only
benzyl group on 11 freed the anomeric hydroxyl group to
produce the trisaccharide 18, which was then converted into
the predominant R-configuration trichloroacetimidate donor 22
by using trichloroacetonitrile in the presence of 1,8-diazabicyclo-
[5.4.0]undec-7-ene (DBU).21
The preparation of trisaccharide donors 23-25 followed a
similiar procedure. Glycosylations of acceptor 10 with deoxy
galactosyl donors 2-4, under the same reaction conditions as
for 11, resulted in the R-Gal derivatives 12-14. All the benzyl
groups of compounds 12-14 were removed by hydrogenolysis,
and then the following acetylation yielded the fully ester-
protected trisaccharides 15-17. Regioselective deprotection of
the anomeric acetyl group with use of benzyl amine led to the
free anomeric hydroxyl trisaccharides 19-21,22 which were then
subjected to the stereoselective reaction with trichloroacetonitrile
to furnish R-trichloroacetimidate donors 23-25.
To improve the reactivity of the lipid acceptor, azido protected
phytosphingosine 26 served as the acceptor instead of the
ceramide.15,23 By doing extensive glycosylation experiments
1
NIS and 4 Å molecular sieves at -20 °C. From the H NMR
and 1H-1H COSY spectra of 11, the R configuration was
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J. Org. Chem, Vol. 72, No. 26, 2007 9917

Chen et al.
In a recent systemic mutagenesis study on the T cell receptor
of invariant NKT cells, Scott-Browne et al. proved that the
CDR3R, CDR1R, and CDR2â regions contain the “hot spots”
that might be involved in binding to iGb3 and R-GalCer.9e
Comparing their results to the CD1d/R-GalCer/TCR crystal
structure by Borg et al.,13 we may propose a model that 2′′′
and 3′′′ OH groups of the terminal R-Gal of iGb3 form hydrogen
bonds with CDR3R in a similar way as R-GalCer. In this model,
the second Gal of iGb3 might “clash” with the N30 of the
CDR1R side chain, as Scott-Browne et al.’s mutagenesis data
suggested that the mutation of N30 to A30 may create more
space that improve iGb3’s fitting to TCR, which is one
explanation for the biological assay that N30A mutation of
CDR1R surprisingly enhances the T cell hybridoma response
to iGb3 antigen; the second model for iGb3-TCR binding is
that the terminal R-Gal of iGb3 is accommodated to a “pocket”
created by CDR3â.9e
All these crystallographic and mutational analyses demon-
strated that the orientation of the terminal R-Gal of iGb3 is
significant for the recognition of TCR. The 2′′′ and 3′′′ hydroxyl
groups may be critical for this recognition due to the hydrogen
bond with CDR loops. On the other hand, the removal of 4′′′
or 6′′′ hydroxyl groups did not significantly diminish the activity.
This finding suggests that the modifications on the 4′′′ and 6′′′
positions of the terminal R-Gal will be more tolerated than
modifications on the 2′′′ and 3′′′ positions, and proper modifica-
tions would lead to further exploration of the activation of NKT
cells by CD1d/glycolipid.
FIGURE 2. Activation of NKT cells with iGb3 analogues. (Data were
the average of three independent experiments and analyzed by the SPPS
data program. An independent-sample t-test was used to measure the
statistic significance of the differences between HO-iGb3 and each
deoxy-iGb3 analogue at data points of 400 ng/mL. For the difference
between 2′′′-dh-iGb3 and HO-iGb3, as well as the difference between
3′′′-dh-iGb3 and HO-iGb3, the P value is lower than 0.001. For
the difference between 4′′′-dh-iGb3 and HO-iGb3, as well as the
difference between 6′′′-dh-iGb3 and HO-iGb3, the P value is lower
than 0.05.)
with a variety of promoters, the highly â-selective glycosylation
products with a fairly good yield were obtained when trimeth-
ylsilyl trifluoromethanesulfonate (TMSOTf) was used as catalyst
at a temperature of -30 °C.24 Glycosphingosines 27-30 were
obtained from the glycosylations between trisaccharide donors
22-25 and lipid acceptor 26 at -30 °C in the presence of 4 Å
molecular sieves and a catalytic amount of TMSOTf. The azido
groups on the lipid part were reduced by using triphenylphos-
phine in benzene with a trace amount of water at 60 °C. The
resulting intermediates were treated with cerotic acid and N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI)
in THF to produce the amides 31-34.25 Finally, the benzyl and
all the ester groups were removed by hydrogenolysis and saponi-
fication, respectively, to afford the four dh-iGb3s 35-38.
T cell stimulation assays with murine iNKT cell hybridomas
were performed as previously described.8 iGb3 and its analogues
were dissolved at 1 mg/mL in DMSO, and stored in -20 °C.
The iGb3 analogues in DMSO solution were examined by thin
layer chromatography before and after the bioassays to ensure
that the glycolipids were not degraded.
Conclusions
In summary, four iGb3 analogues containing terminal deoxy
galactose (compounds 35-38) were efficiently synthesized and
their activities against mouse NKT cells were investigated. This
strategy helped to reveal the SAR of the terminal R-Gal of iGb3.
The SAR study pointed out that 2′′′ and 3′′′ hydroxyl groups
should be crucial for the recognition of iGb3 by TCR. It was
also suggested that modifications of 4′′′ and 6′′′ positions of
iGb3 with other functional groups do not diminish their
stimulatory activity, and would lead to further investigation of
the recognition of CD1d/iGb3 by NKT cells. These studies
might help to understand the general structural requirements
for natural endogenous ligands recognized by NKT cells.
Experimental Section
Stimulatory activities of iGb3 analogues were studied through
the comparison between HO-iGb315 and compounds 35-38.
After removal of the 2′′′ or 3′′′ hydroxyl groups, iGb3 analogues
(compounds 35 and 36) showed much weaker stimulatory
activity compared to iGb3 with increased concentrations (Figure
2). At 400 ng/mL concentration, 2′′′-dh-iGb3 has a 4-fold
decrease, while 3′′′-dh-iGb3 has a 7-fold decrease. In contrast,
4′′′ and 6′′′ deoxy iGb3 analogues (compounds 37 and 38)
demonstrated much better stimulation as compared to the 2′′′
or 3′′′ deoxy ones. The 4′′′ and 6′′′ deoxy analogues had similar
stimulatory activities at the same concentrations. The bioassay
experiment was repeated three times giving the same trend each
time.
Phenyl 3,4,6-Tri-O-acetyl-2-deoxy-1-thio-D-galactopyranoside
(1). To a solution of acetylated compound 6 (0.84 g, 2.5 mmol) in
CH2Cl2 (15 mL) at 0 °C was added PhSH (0.31 mL, 3.0 mmol)
dropwise, followed by addition of BF3-Et2O (0.38 mL, 3.0 mmol).
The mixture was allowed to warm up to room temperature slowly.
After being stirred for 5 h, the reaction mixture was quenched by
saturated NaHCO3 solution (10 mL). The organic layer was washed
with water (15 mL) and brine (15 mL) and dried over anhydrous
Na2SO4. The concentrated residue was purified by flash chroma-
tography (4:1 hexanes/EtOAc) to give 2-deoxy donor 1 (R:â )
2:1; 0.80 g, 83%). 1H NMR (500 MHz, CDCl3) (R-isomer) δ 7.49-
7.47 (m, 2H), 7.31-7.24 (m, 3H), 5.76 (d, J ) 5.7 Hz, 1H), 5.38
(d, J ) 2.7 Hz, 1H), 5.27 (ddd, J ) 12.6, 5.0, 3.2 Hz, 1H), 4.69 (t,
J ) 6.4 Hz, 1H), 4.10-4.09 (m, 2H), 2.49 (ddd, J ) 13.0, 13.0,
5.9 Hz, 1H), 2.13 (s, 3H), 2.10-2.04 (m, 1H), 2.00 (s, 3H), 1.97
(s, 3H); 13C NMR (125 MHz, CDCl3) δ 170.6, 170.3, 170.0, 134.1,
131.7, 129.1, 127.6, 83.8, 67.7, 66.8 (2C), 62.5, 30.8, 20.9, 20.8,
20.7; HRMS calcd for C18H22O7SNa ([M + Na]+) 405.0984, found
405.0967.
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Isoglobotrihexosylceramide Analogues
Phenyl 2,4,6-Tri-O-benzyl-3-O-[(methylthio)thiocarbonyl]-1-
thio-â-D-galactopyranoside (8). To a solution of compound 7 (0.42
g, 0.775 mmol) in THF (10 mL) was added NaH (60 mg, 1.55
mmol). After 1 h, imidazole (2.64 mg, 0.039 mmol) was added
and the mixture was stirred for 30 min. CS2 (0.4 mL, 6.59 mmol)
was then added, followed by the addition of MeI (0.10 mL, 1.55
mmol) 10 min later. After 3 h, the reaction mixture was diluted
with CH2Cl2, then washed with saturated NaHCO3 solution and
water. The combined organic phase was dried over anhydrous Na2-
SO4 and concentrated. The residue was purified by flash chroma-
tography (10:1 hexanes/EtOAc) to give the product (0.41 g, 84%).
1H NMR (500 MHz, CDCl3) δ 7.56 (d, J ) 6.2 Hz, 2H), 7.33-
7.26 (m, 15H), 7.23-7.18 (m, 3H), 5.85 (dd, J ) 9.5, 2.8 Hz, 1H),
4.96 (d, J ) 10.3 Hz, 1H), 4.72 (d, J ) 9.7 Hz, 1H), 4.69 (d, J )
11.3 Hz, 1H), 4.63 (d, J ) 10.5 Hz, 1H), 4.49 (d, J ) 11.9 Hz,
1H), 4.45 (d, J ) 11.7 Hz, 1H), 4.43 (d, J ) 12.0 Hz, 1H), 4.28
(d, J ) 2.4 Hz, 1H), 4.12 (d, J ) 9.6 Hz, 1H), 3.76 (t, J ) 6.2 Hz,
1H), 3.68-3.60 (m, 2H), 2.56 (s, 3H); 13C NMR (125 MHz, CDCl3)
δ 215.4, 138.1, 137.8, 133.7, 131.7, 128.9, 128.4, 128.33, 128.30,
128.2, 128.0, 127.9, 127.8, 127.7, 127.3, 87.6, 85.8, 76.9, 75.5-
(2C), 74.8, 73.6(2C), 68.4, 19.3; HRMS calcd for C35H36O5S3Na
([M + Na]+) 655.1623, found 655.1620.
Phenyl 2,4,6-tri-O-benzyl-3-deoxy-1-thio-â-D-galactopyrano-
side (2). To a solution of 8 (0.38 g, 0.60 mmol) in anhydrous toluene
was added Bu3SnH (0.8 mL, 3.0 mmol) and AIBN (0.10 g, 0.60
mmol). The reaction mixture was refluxed for 1 h, then cooled,
filtered, and concentrated. The residue was purified by flash
chromatography (10:1, hexanes/EtOAc) to give 3-deoxy donor 2
(0.22 g, 70%). 1H NMR (400 MHz, CDCl3) δ 7.65-7.63 (m, 2H),
7.37-7.25 (m, 18H), 4.78 (d, J ) 9.5 Hz, 1H), 4.71 (d, J ) 11.5
Hz, 1H), 4.55 (d, J ) 11.4 Hz, 1H), 4.53 (d, J ) 11.1 Hz, 1H),
4.51 (d, J ) 12.0 Hz, 1H), 4.48 (d, J ) 11.6 Hz, 1H), 4.39 (d, J )
12.0 Hz, 1H), 3.81-3.74 (m, 5H), 2.48 (dt, J ) 13.7, 3.4 Hz, 1H),
1.57-1.50 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 138.4, 138.3,
138.2, 134.6, 131.6, 128.8, 128.5, 128.4, 128.3, 128.2, 127.9, 127.8,
127.75, 127.71, 127.6, 127.0, 89.4, 79.4, 73.6, 72.62, 72.60, 71.8,
71.1, 69.5, 34.1; HRMS calcd for C33H34O4SNa ([M + Na]+)
549.2076, found 549.2073.
Phenyl 2,3-Di-O-benzyl-6-O-acetyl-4-deoxy-1-thio-â-D-galac-
topyranoside (3). To a solution of compound 9 (0.73 g, 1.7 mmol)
in CH2Cl2 at 0 °C was added PhSH, followed by addition of BF3-
Et2O. The reaction mixture was stirred overnight. The reaction
mixture was quenched by adding saturated NaHCO3 solution. The
organic layer was washed with water and brine, then dried over
anhydrous Na2SO4. The concentrated residue was purified by flash
chromatography (8:1 hexanes/EtOAc) to give 4-deoxy donor 3 (0.70
g, 86%). 1H NMR (400 MHz, CDCl3) δ 7.51-7.50 (m, 2H), 7.43-
7.41 (m, 2H), 7.37-7.26 (m, 11H), 5.69 (d, J ) 5.2 Hz, 1H), 4.82
(d, J ) 11.6 Hz, 1H), 4.80 (d, J ) 11.9 Hz, 1H), 4.75 (d, J ) 11.9
Hz, 1H), 4.71 (d, J ) 11.6 Hz, 1H), 4.56-4.51 (m, 1H), 4.13 (dd,
J ) 11.8, 6.6 Hz, 1H), 4.06 (dd, J ) 11.7, 3.2 Hz, 1H), 3.89 (ddd,
J ) 12.6, 10.0, 5.1 Hz, 1H), 3.82 (dd, J ) 9.4, 5.1 Hz, 1H), 2.10
(ddd, J ) 12.6, 5.0, 2.2 Hz, 1H), 2.00 (s, 3H), 1.54 (dd, J ) 11.9,
11.8, 11.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 170.7, 138.6,
137.9, 134.4, 131.7, 128.9, 128.5, 128.4, 128.0, 127.9, 127.7, 127.6,
127.2, 87.3, 80.0, 75.2, 72.9, 72.5, 66.7, 65.8, 33.4, 20.8; HRMS
calcd for C28H30O5SNa ([M + Na]+) 501.1712, found 501.1713.
General Procedure for the Preparation of Trisaccharides 11-
14. Benzyl 3,4,6-Tri-O-acetyl-2-deoxy-R-D-galactopyranosyl-
(1,3)-2,4,6-tri-O-pivaloyl-â-D-galactopyranosyl-(1,4)-2,3,6-tri-O-
pivaloyl-â-D-glucopyranoside (11). A suspension of acceptor 10
(430 mg, 0.46 mmol), 2-deoxy donor 1 (160 mg, 0.42 mmol), and
4 Å molecular sieve (500 mg) in anhydrous CH2Cl2 (5 mL) was
stirred at room temperature for 30 min. After the solution was
cooled to -30 °C, N-iodosuccinamide (100 mg, 0.46 mmol)
followed by TfOH (8 µL, 0.10 mmol) was added. The resulting
mixture was stirred at -30 °C for 2 h, then was allowed to warm
slowly to 0 °C. The mixture was diluted with CH2Cl2 (5 mL) and
the molecular sieves were filtered. The filtrate was washed with
saturated aq NaHCO3 (5 mL), 10% Na2S2O3 (5 mL), and brine (5
mL). The combined organic phase was dried over anhydrous Na2-
SO4 and concentrated. The residue was purified by silica gel flash
chromatography (1:6 EtOAc/hexanes) to furnish trisaccharide 11
(360 mg, 72%) as white foam. 1H NMR (500 MHz, CDCl3) δ 7.35-
7.24 (m, 5H), 5.41 (d, J ) 2.6 Hz, 1H), 5.28 (m, 2H, 1′′′â-H),
5.18 (t, J ) 9.5 Hz, 1H), 5.09 (dd, J ) 9.3, 8.1 Hz, 1H), 4.96-
4.94 (m, 1H), 4.92 (dd, J ) 9.2, 8.0 Hz, 1H), 4.80 (d, J ) 11.9 Hz,
1H), 4.58 (d, J ) 8.2 Hz, 1H), 4.57 (d, J ) 8.2 Hz, 1H), 4.55 (d,
J ) 12.0 Hz, 1H), 4.48 (d, J ) 8.1 Hz, 1H), 4.27 (dd, J ) 11.9,
5.2 Hz, 1H), 4.12-4.03 (m, 5H), 3.92-3.87 (m, 2H), 3.85 (dd, J
) 10.5, 2.9 Hz, 1H), 3.57-3.54 (m, 1H), 2.10 (s, 3H), 2.05-2.00
(m, 1H), 2.04 (s, 3H), 1.84 (s, 3H), 1.55 (dd, J ) 12.9, 4.9 Hz,
1H), 1.25 (s, 9H), 1.23 (s, 9H), 1.22 (s, 9H), 1.21 (s, 9H), 1.18 (s,
9H), 1.10 (s, 9H); 13C NMR (125 MHz, CDCl3) δ 177.8, 177.6,
177.0, 176.7, 176.2, 170.3, 170.2, 169.8, 136.6, 128.4, 128.0, 127.9,
100.2, 99.4, 93.3, 73.7, 73.5, 72.1, 71.6, 71.5, 71.4, 70.5, 69.7, 67.0,
66.2, 65.8, 64.6, 62.2, 61.8, 61.5, 39.1, 38.9, 38.9, 38.8, 38.7, 38.6,
29.1, 27.3 (2C), 27.1 (2C), 20.7, 20.6 (2C), 14.2; HRMS calcd for
C61H92O24Na ([M + Na]+) 1231.5876, found 1231.5864.
Benzyl 2,4,6-Tri-O-benzyl-3-deoxy-R-D-galactopyranosyl-
(1,3)-2,4,6-tri-O-pivaloyl-â-D-galactopyranosyl-(1,4)-2,3,6-tri-O-
pivaloyl-â-D-glucopyranoside (12). Compound 12 (365 mg, 71%)
was obtained from acceptor 10 (390 mg, 0.42 mmol) and donor 2
1
(200 mg, 0.38 mmol). H NMR (500 MHz, CDCl3) δ 7.33-7.23
(m, 18H), 7.15-7.13 (m, 2H), 5.51 (d, J ) 2.8 Hz, 1H), 5.19 (t, J
) 9.5 Hz, 1H), 5.15 (dd, J ) 11.1, 8.2 Hz, 1H), 5.00 (d, J ) 2.7
Hz, 1H), 4.91 (dd, J ) 9.7, 8.0 Hz, 1H), 4.81 (d, J ) 11.9 Hz,
1H), 4.58-4.52 (m, 4H), 4.47 (s, 2H), 4.43 (d, J ) 12.1 Hz, 1H),
4.39-4.35 (m, 2H), 4.28-4.25 (m, 2H), 4.02 (d, J ) 6.5 Hz, 2H),
3.90 (t, J ) 7.1 Hz, 1H), 3.86 (d, J ) 9.6 Hz, 1H), 3.83-3.76 (m,
2H), 3.69 (t, J ) 6.5 Hz, 1H), 3.62 (s, 1H), 3.58-3.54 (m, 2H),
3.47 (dd, J ) 9.2, 5.9 Hz, 1H), 2.01 (ddd, J ) 13.0, 3.8, 3.8 Hz,
1H), 1.69 (ddd, J ) 13.0, 12.3, 2.3 Hz, 1H), 1.24 (s, 9H), 1.22 (s,
9H), 1.18 (s, 9H), 1.17 (s, 9H), 1.16 (s, 9H), 1.10 (s, 9H); 13C
NMR (125 MHz, CDCl3) δ 177.8, 177.7, 177.1, 177.0, 176.7, 175.9,
138.4, 138.3, 138.1, 136.5, 128.34, 128.32, 128.23, 128.20, 127.90,
127.88, 127.7, 127.57, 127.55, 127.51, 127.4, 100.3, 99.2, 95.2,
74.1, 73.7, 73.5, 73.1, 72.8, 71.9, 71.6, 71.3, 71.2, 71.0, 70.7, 70.4,
69.5, 68.4, 66.3, 61.9, 39.0, 38.8, 38.7, 38.66, 38.64, 38.58, 27.3,
27.2, 27.1, 26.3; HRMS calcd for C76H104O21Na ([M + Na]+)
1375.6968, found 1375.6975.
Benzyl 2,3-Di-O-benzyl-6-acetyl-4-deoxy-R-D-galactopyrano-
syl-(1,3)-2,4,6-tri-O-pivaloyl-â-D-galactopyranosyl-(1,4)-2,3,6-tri-
O-pivaloyl-â-D-glucopyranoside (13). Compound 13 (345 mg,
70%) was obtained from acceptor 10 (390 mg, 0.41 mmol) and
donor 3 (180 mg, 0.38 mmol). 1H NMR (500 MHz, CDCl3) δ 7.35-
7.24 (m, 15H), 5.54 (d, J ) 2.8 Hz, 1H), 5.19 (t, J ) 9.5 Hz, 1H),
5.09 (dd, J ) 10.2, 8.1 Hz, 1H), 4.93 (d, J ) 2.9 Hz, 1H), 4.90
(dd, J ) 9.6, 7.8 Hz, 1H), 4.83 (d, J ) 12.1 Hz, 1H), 4.80 (d, J )
12.0 Hz, 1H), 4.69 (d, J ) 11.5 Hz, 1H), 4.65 (d, J ) 12.0 Hz,
1H), 4.58-4.51 (m, 4H), 4.30 (d, J ) 8.3 Hz, 1H), 4.28 (dd, J )
11.8, 5.1 Hz, 1H), 4.01-4.00 (m, 4H), 3.92-3.88 (m, 1H), 3.87-
3.82 (m, 2H), 3.66 (dd, J ) 10.3, 3.2 Hz, 1H), 3.61 (t, J ) 6.4 Hz,
1H), 3.53 (ddd, J ) 9.6, 5.1, 1.8 Hz, 1H), 3.37 (dd, J ) 9.2, 3.2
Hz, 1H), 2.06 (s, 3H), 1.93-1.89 (m, 1H), 1.39 (ddd, J ) 12.3,
12.0, 12.0 Hz, 1H), 1.25 (s, 9H), 1.22 (s, 9H), 1.17 (s, 9H), 1.16
(s, 9H), 1.15 (s, 9H), 1.10 (s, 9H); 13C NMR (125 MHz, CDCl3) δ
177.8, 177.0, 176.9, 176.7, 175.8, 170.8, 138.7, 138.6, 136.6, 128.4,
128.37, 128.35, 128.0, 127.97, 127.95, 127.8, 127.7, 127.6, 127.5,
100.3, 99.4, 97.8, 80.5, 75.7, 74.9, 73.8, 73.6, 73.5, 72.6, 72.1, 71.6,
71.4, 70.6, 70.5, 67.1, 67.0, 65.8, 62.0, 39.0, 38.9, 38.8, 38.72,
38.71, 38.6, 33.1, 27.34, 27.32, 27.2, 27.14, 27.12, 27.0, 20.8;
HRMS calcd for C71H100O22Na ([M + Na]+) 1327.6604, found
1327.6620.
Benzyl 2,3,4-Tri-O-benzyl-6-deoxy-R-D-galactopyranosyl-
(1,3)-2,4,6-tri-O-pivaloyl-â-D-galactopyranosyl-(1,4)-2,3,6-tri-O-
pivaloyl-â-D-glucopyranoside (14). Compound 14 (300 mg, 63%)
was obtained from acceptor 10 (360 mg, 0.38 mmol) and donor 4
J. Org. Chem, Vol. 72, No. 26, 2007 9919

Chen et al.
1
(184 mg, 0.35 mmol). H NMR (500 MHz, CDCl3) δ 7.41-7.25
3.8 Hz, 1H), 4.41-4.36 (m, 4H), 4.35 (d, J ) 8.0 Hz, 1H), 4.07-
4.04 (m, 6H), 3.93-3.89 (m, 3H), 3.82-3.80 (m, 2H), 3.73-3.68
(m, 3H), 2.14 (s, 6H), 2.13 (s, 3H), 2.07 (s, 6H), 2.04 (s, 3H), 1.92
(s, 6H), 1.24-1.18 (m, 90H), 1.12-1.11 (m, 24H); 13C NMR (125
MHz, CDCl3) δ 177.8, 177.7, 177.2, 176.85, 176.83, 176.72,
176.71, 176.0, 175.9, 170.5, 170.2, 169.7, 168.8, 168.7, 100.3,
100.2, 95.1, 95.0, 91.7, 75.1, 74.9, 74.2, 73.0, 72.8, 72.2, 72.1, 71.3,
71.1, 70.9, 70.4, 70.0, 69.9, 69.8, 68.6, 67.9, 67.3, 67.2, 65.6, 65.3,
65.2, 61.8, 61.7, 61.6, 61.4, 61.3, 39.1, 39.05, 39.02, 39.0, 38.9,
38.8, 38.78, 38.75, 38.73, 38.6, 27.34, 27.31, 27.28, 27.24, 27.13,
27.09, 27.07, 27.0, 26.9, 21.1, 20.7, 20.6, 20.59, 20.57; HRMS calcd
for C56H88O25Na ([M + Na]+) 1183.5512, found 1183.5510.
3,4,6-Tri-O-acetyl-2-deoxy-R-D-galactopyranosyl-(1,3)-2,4,6-
tri-O-pivaloyl-â-D-galactopyranosyl-(1,4)-2,3,6-tri-O-pivaloyl-D-
glucopyranose (18). Palladium hydroxide (150 mg, 20 wt %) was
added to a solution of trisaccharide 11 (350 mg, 0.29 mmol) in
MeOH/EtOAc (4 mL) and the reaction mixture was stirred under
hydrogen balloon pressure for 12 h. The reaction mixture was then
filtered through a celite pad and concentrated in vacuo to give crude
product. Purification on flash chromatography (3:1 hexanes/EtOAc)
(m, 20H), 5.54 (d, J ) 2.5 Hz, 1H), 5.18 (t, J ) 9.6 Hz, 1H), 5.07
(dd, J ) 10.1, 8.3 Hz, 1H), 4.93-4.90 (m, 2H), 4.88-4.87 (m,
1H), 4.85 (s, 2H), 4.80 (d, J ) 11.9 Hz, 1H), 4.71 (d, J ) 11.8 Hz,
1H), 4.61 (t, J ) 10.8 Hz, 2H), 4.58-4.52 (m, 3H), 4.30-4.27
(m, 2H), 3.98-3.93 (m, 3H), 3.86 (dd, J ) 10.1, 2.2 Hz, 1H), 3.85
(t, J ) 9.4 Hz, 1H), 3.79 (d, J ) 6.5 Hz, 1H), 3.58-3.51 (m, 4H),
1.26 (s, 9H), 1.21 (s, 9H), 1.17 (s, 9H), 1.15 (s, 18H), 1.10 (s, 9H),
1.04 (d, J ) 6.3 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 177.9,
177.8, 177.0, 176.8, 176.7, 175.9, 139.1, 138.7, 138.6, 136.6,
128.44, 128.41, 128.37, 128.33, 128.2, 128.0, 127.97, 127.95,
127.62, 127.61, 127.4, 99.4, 99.1, 79.3, 77.9, 74.8, 74.8, 73.8, 73.5,
73.4, 72.1, 71.7, 71.4, 70.5, 68.0, 67.9, 62.04, 62.01, 39.0, 38.9,
38.8, 38.7, 38.6, 27.3, 27.26, 27.21, 27.15, 27.11, 16.7; HRMS calcd
for C76H104O21Na ([M + Na]+) 1375.6968, found 1375.6915.
General Procedure for the Preparation of Trisaccharides 15-
17. Acetyl 2,4,6-Tri-O-acetyl-3-deoxy-R-D-galactopyranosyl-
(1,3)-2,4,6-tri-O-pivaloyl-â-D-galactopyranosyl-(1,4)-2,3,6-tri-O-
pivaloyl-D-glucopyranoside (15). Palladium hydroxide (200 mg,
20 wt %) was added to a solution of trisaccharide 12 (340 mg,
0.25 mmol) in MeOH/EtOAc (2 mL) and the mixture was shaken
under 50 psi H2 for 12 h. The palladium hydroxide was then filtered
through a celite pad and concentrated in vacuo to give crude alcohol
product. The resulting alcohol was then acetylated to give R,â
1
gave compound 18 (310 mg, 96%). H NMR (500 MHz, CDCl3)
δ 5.51 (t, J ) 9.8 Hz, 1H), 5.41 (d, J ) 2.6 Hz, 1H), 5.35 (d, J )
3.7 Hz, 1H), 5.28-5.26 (m, 2H), 5.27 (d, J ) 9.0 Hz, 1H), 5.23
(d, J ) 8.9 Hz, 1H), 5.12-5.08 (m, 1H), 5.00-4.96 (m, 1H), 4.73
(d, J ) 7.8 Hz, 1H), 4.70 (s, 1H), 4.70-4.69 (m, 1H), 4.68 (d, J
) 3.7 Hz, 1H), 4.54-4.50 (m, 1H), 4.53 (d, J ) 8.2 Hz, 1H), 4.49
(d, J ) 7.9 Hz, 1H), 4.35-4.29 (m, 2H), 4.14-4.03 (m, 10H),
3.93 (t, J ) 9.6 Hz, 1H), 3.91 (t, J ) 9.7 Hz, 1H), 3.88-3.83 (m,
4H), 3.59-3.57 (m, 1H), 2.09 (s, 6H), 2.07-2.02 (m, 2H), 2.03
(s, 3H), 2.02 (s, 3H), 1.93 (s, 6H), 1.64-1.59 (m, 1H), 1.56 (dd, J
) 12.7, 4.5 Hz, 1H), 1.24 (s, 18H), 1.234 (s, 9H), 1.232 (s, 9H),
1.227 (s, 9H), 1.22 (s, 9H), 1.217 (s, 9H), 1.212 (s, 9H), 1.208 (s,
9H), 1.202 (s, 9H), 1.17 (s, 18H); 13C NMR (125 MHz, CDCl3) δ
178.7, 177.8, 177.7, 177.6, 176.8, 176.7, 176.3, 176.2, 170.3 (2C),
170.2, 169.8, 100.0, 95.8, 93.3, 89.9, 73.8, 73.1, 72.0, 71.7, 71.5,
69.6, 68.9, 68.4, 67.0, 66.2, 65.8, 64.6, 62.1 (2C), 61.6, 61.5 (2C),
39.1, 39.0, 38.99, 38.96, 38.9, 38.8, 38.77, 38.68, 29.7, 29.1, 27.37,
27.33, 27.29, 27.26, 27.14, 27.09, 27.0, 20.7, 20.65, 20.61; HRMS
calcd for C54H86O24Na ([M + Na]+) 1141.5407, found 1141.5409.
3,4,6-Tri-O-acetyl-2-deoxy-R-D-galactopyranosyl-(1,3)-2,4,6-
tri-O-pivaloyl-â-D-galactopyranosyl-(1,4)-2,3,6-tri-O-pivaloyl-R-
D-glucopyranosyl-(1,1)-trichloroacetimide (22). Compound 18
(140 mg, 0.125 mmol) was dissolved in anhydrous CH2Cl2 (6 mL).
Then CCl3CN (0.13 mL, 1.25 mmol) and DBU (18 uL, 0.125 mmol)
were added, respectively. The reaction mixture was stirred at room
temperature for 4 h. The solvent was removed in vacuo and the
residue was purified by silica gel flash chromatography (1:4 EtOAc/
1
mixture 15 (220 mg, 2 steps yield 75%). H NMR (500 MHz,
CDCl3) (R and â mixture; R:â ) 1:2.7) δ 6.24 (d, J ) 3.8 Hz,
1H), 5.68 (d, J ) 8.2 Hz, 1H), 5.47-5.43 (m, 2H), 5.22 (t, J ) 9.5
Hz, 1H), 5.15-5.10 (m, 4H), 5.05 (d, J ) 3.0 Hz, 2H), 5.00 (s,
4H), 4.93 (t, J ) 8.3 Hz, 1H), 4.85 (dd, J ) 10.2, 3.9 Hz, 1H),
4.46-4.40 (m, 4H), 4.28 (dd, J ) 12.2, 4.4 Hz, 2H), 4.11-4.05
(m, 6H), 4.00-3.95 (m, 4H), 3.92-3.88 (m, 2H), 3.84-3.78 (m,
4H), 3.68-3.66 (m, 1H), 2.10 (s, 3H), 2.06 (s, 6H), 2.02-2.00
(m, 15H), 1.96-1.95 (m, 2H), 1.90-1.84 (m, 2H), 1.21-1.16 (m,
90H), 1.09-1.08 (m, 18H); 13C NMR (125 MHz, CDCl3) δ 177.8,
177.7, 177.64, 177.58, 177.1, 176.9, 176.86, 176.83, 176.70, 175.95,
175.91, 170.4, 170.1, 169.9, 168.8, 168.7, 100.2, 100.1, 93.9, 93.8,
91.6, 88.6, 74.2, 72.9, 71.9, 71.2, 70.4, 70.1, 67.3, 67.2, 66.4, 65.7,
61.9, 61.5, 61.4, 39.0, 38.9, 38.88, 38.76, 38.72, 38.69, 38.66, 27.4,
27.3, 27.29, 27.26, 27.2, 27.1, 27.0, 26.95, 26.91, 21.2, 20.9, 20.63,
20.62; HRMS calcd for C56H88O25Na ([M + Na]+) 1183.5512,
found 1183.5524.
Acetyl 2,3,6-Tri-O-acetyl-4-deoxy-R-D-galactopyranosyl-(1,3)-
2,4,6-tri-O-pivaloyl-â-D-galactopyranosyl-(1,4)-2,3,6-tri-O-piv-
aloyl-D-glucopyranoside (16). Compound 16 (220 mg, 2 steps yield
82%) was obtained from trisaccharide 13 (300 mg, 0.23 mmol).
1H NMR (500 MHz, CDCl3) (R and â mixture; R:â ) 1:1.7) δ
6.27 (d, J ) 3.8 Hz, 1H), 5.69 (d, J ) 8.2 Hz, 1H), 5.49-5.43 (m,
1H), 5.41-5.39 (m, 2H), 5.24 (t, J ) 9.3 Hz, 1H), 5.18-5.12 (m,
4H), 4.97-4.93 (m, 5H), 4.87 (dd, J ) 10.1, 3.9 Hz, 1H), 4.43-
4.35 (m, 6H), 4.14-4.00 (m, 10H), 3.93-3.89 (m, 3H), 3.82-
3.77 (m, 4H), 3.68-3.66 (m, 1H), 2.16 (s, 2H), 2.06 (s, 12H), 2.04
(s, 6H), 1.97 (s, 6H), 1.38-1.27 (m, 2H), 1.22-1.18 (m, 90H),
1.11 (s, 9H), 1.10 (s, 9H); 13C NMR (125 MHz, CDCl3) δ 177.8,
177.7, 177.66, 177.61, 177.1, 176.8, 176.79, 176.73, 176.6, 176.0,
175.9, 170.5, 170.1, 169.9, 168.8, 168.6, 100.2, 100.1, 94.4, 94.3,
91.6, 88.5, 74.2, 73.6, 73.5, 72.9, 72.7, 72.0, 71.9, 71.3, 71.1, 70.6,
70.3, 69.7, 69.6, 68.6, 67.6, 65.8, 65.7, 65.2, 65.1, 61.6, 61.5, 61.3,
61.1, 39.0, 38.9, 38.8, 38.7, 38.69, 38.68, 38.65, 38.62, 32.4, 27.3,
27.2, 27.18, 27.15, 27.12, 27.05, 27.03, 26.9, 26.8, 21.1, 20.9, 20.7,
20.67, 20.6; HRMS calcd for C56H88O25Na ([M + Na]+) 1183.5512,
found 1183.5490.
1
hexanes) to afford compound 22 (140 mg, 89%). H NMR (500
MHz, CDCl3) δ 8.62 (s, 1H), 6.44 (d, J ) 2.6 Hz, 1H), 5.56 (td,
J ) 9.8, 1.1 Hz, 1H), 5.39 (s, 1H), 5.25 (s, 2H), 5.07 (t, J ) 9.3
Hz, 1H), 4.94 (ddd, J ) 10.2, 3.7, 1.4 Hz, 1H), 4.90 (m, 1H), 4.47
(d, J ) 8.1 Hz, 1H), 4.44 (d, J ) 12.1 Hz, 1H), 4.32 (dd, J ) 12.2,
4.2 Hz, 1H), 4.13-4.11 (m, 1H), 4.10-4.08 (m, 2H), 4.06 (d, J )
9.1 Hz, 1H), 4.02-4.01 (m, 2H), 3.97 (td, J ) 9.7, 1.1 Hz, 1H),
3.86 (d, J ) 6.7 Hz, 1H), 3.84-3.82 (m, 1H), 2.08 (s, 3H), 2.02-
2.01 (m, 1H), 2.00 (s, 3H), 1.91 (s, 3H), 1.52 (dd, J ) 12.7, 4.4
Hz, 1H), 1.20-1.18 (m, 36H), 1.17 (s, 9H), 1.10 (s, 9H); 13C NMR
(125 MHz, CDCl3) δ 177.7, 177.6, 177.54, 177.51, 176.7, 176.2,
170.3, 170.2, 169.8, 160.5, 100.1, 93.3, 92.54, 92.49, 90.8, 72.7,
71.9, 71.8, 71.5, 71.4, 70.1, 69.7, 68.7, 67.0, 66.2, 65.8, 64.5, 62.2,
61.4, 39.1, 38.94, 38.91, 38.8, 38.73, 38.69, 29.0, 27.3, 27.29, 27.24,
27.2, 27.16, 27.12, 27.09, 27.0, 26.9, 20.8, 20.7, 20.6; HRMS calcd
for C56H86Cl3NO24Na ([M + Na]+) 1284.4503, found 1284.4457.
General Procedure for the Preparation of Trisaccharide
Donors 23-25. 2,4,6-Tri-O-acetyl-3-deoxy-R-D-galactopyrano-
syl-(1,3)-2,4,6-tri-O-pivaloyl-â-D-galactopyranosyl-(1,4)-2,3,6-tri-
O-pivaloyl-R-D-glucopyranosyl-(1,1)-trichloroacetimide (23). Ben-
zylamine (0.2 mL, 1.8 mmol) was added to a solution of compound
15 (0.21 g, 0.18 mmol) in THF (10 mL). After being stirred for 24
h at room temperature, the reaction mixture was concentrated in
Acetyl 2,3,4-Tri-O-acetyl-6-deoxy-R-D-galactopyranosyl-(1,3)-
2,4,6-tri-O-pivaloyl-â-D-galactopyranosyl-(1,4)-2,3,6-tri-O-piv-
aloyl-D-glucopyranoside (17). Compound 17 (146 mg, 2 steps yield
85%) was obtained from trisaccharide 14 (200 mg, 0.15 mmol).
1H NMR (500 MHz, CDCl3) (R and â mixture; R:â ) 1:2.1) δ
6.28 (d, J ) 3.9 Hz, 1H), 5.70 (d, J ) 8.2 Hz, 1H), 5.50-5.46 (m,
1H), 5.41(d, J ) 2.6 Hz, 1H), 5.39(d, J ) 2.3 Hz, 1H), 5.29-5.21
(m, 6H), 5.18-5.13 (m, 4H), 5.04 (t, J ) 3.4 Hz, 1H), 5.01 (t, J )
3.3 Hz, 1H), 4.96 (dd, J ) 9.6, 8.4 Hz, 1H), 4.88 (dd, J ) 10.1,
9920 J. Org. Chem., Vol. 72, No. 26, 2007

Isoglobotrihexosylceramide Analogues
vacuo. The residue was diluted with CH2Cl2 (10 mL), then washed
with 1 N HCl (5 mL), saturated aq NaHCO3 (5 mL), and brine (5
mL). The combined organic phase was dried over anhydrous Na2-
SO4 and concentrated. The above crude residue was dissolved in
anhydrous CH2Cl2 (10 mL). Then CCl3CN (0.2 mL, 2.0 mmol)
and DBU (30 uL, 0.20 mmol) were added, respectively. After the
solution was stirred at room temperature for 2 h, the solvent was
removed in vacuo and the residue was purified by silica gel flash
chromatography (1:4 EtOAc/hexanes) to afford compound 23 (190
being dried over anhydrous Na2SO4 and concentrated, the concen-
trated residue was purified by silica gel flash chromatography (1:4
EtOAc/hexanes) to provide 27 (130 mg, 67%) as clear oil. 1H NMR
(500 MHz, CDCl3) δ 7.33-7.26 (m, 10H), 5.66 (d, J ) 5.6 Hz,
1H), 5.28 (s, 2H), 5.17 (t, J ) 9.6 Hz, 1H), 5.09 (dd, J ) 10.1, 8.3
Hz, 1H), 4.98-4.94 (m, 1H), 4.88 (dd, J ) 9.6, 8.3 Hz, 1H), 4.64
(d, J ) 11.4 Hz, 1H), 4.59 (d, J ) 11.4 Hz, 1H), 4.56 (d, J ) 11.6
Hz, 1H), 4.52 (d, J ) 11.3 Hz, 1H), 4.51-4.46 (m, 2H), 4.43 (d,
J ) 7.9 Hz, 1H), 4.26 (dd, J ) 11.7, 5.0 Hz, 1H), 4.14-4.03 (m,
2H), 4.00 (dd, J ) 10.0, 7.3 Hz, 1H), 3.92-3.86 (m, 2H), 3.84
(dd, J ) 10.4, 2.8 Hz, 1H), 3.78 (dd, J ) 10.3, 2.8 Hz, 1H), 3.70-
3.67 (m, 1H), 3.58-3.57 (m, 2H), 3.52-3.50 (m, 1H), 2.11 (s,
3H), 2.04 (s, 3H), 2.03-2.00 (m, 1H), 1.94 (s, 3H), 1.66-1.47
(m, 3H), 1.55 (dd, J ) 12.9, 4.4 Hz, 1H), 1.25-1.20 (m, 26H),
1.23 (s, 9H), 1.22 (s, 9H), 1.20 (s, 9H), 1.19 (s, 9H), 1.18 (s, 9H),
1.14 (s, 9H), 0.88 (t, J ) 7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3)
δ 177.8, 177.6, 177.57, 177.0, 176.6, 176.3, 170.3, 170.2, 169.8,
138.4, 138.0, 128.4, 128.36, 128.1, 128.0, 127.9, 127.8, 127.7,
127.6, 100.8, 100.1, 93.3, 79.3, 79.2, 73.8, 73.5, 73.4, 72.1, 72.0,
71.6, 71.5, 71.4, 69.7, 69.5, 67.0, 66.2, 65.8, 64.5, 62.2, 61.9, 61.7,
61.4, 39.4, 39.2, 39.1, 39.0, 38.9, 38.74, 38.72, 38.67, 31.9, 30.0,
29.73, 29.69, 29.66, 29.6, 29.57, 29.4, 29.1, 27.3, 27.2, 27.14, 27.11,
27.08, 25.8, 25.4, 22.7, 20.7, 20.64, 20.62, 14.1; HRMS calcd for
C86H133N3O26Na ([M + Na]+) 1646.9075, found 1646.9060.
2,4,6-Tri-O-acetyl-3-deoxy-R-D-galactopyranosyl-(1,3)-2,4,6-
tri-O-pivaloyl-â-D-galactopyranosyl-(1,4)-2,3,6-tri-O-pivaloyl-â-
D-glucopyranosyl-(1,1)-(2S,3S,4R)-2-azido-3,4-benzyloxyoctade-
cane (28). Compound 28 (148 mg, 61%) was obtained from donor
23 (184 mg, 0.15 mmol) and acceptor 26 (83 mg, 0.16 mmol). 1H
NMR (500 MHz, CDCl3) δ 7.34-7.25 (m, 10H), 5.46 (d, J ) 2.6
Hz, 1H), 5.19 (t, J ) 9.6 Hz, 1H), 5.13 (dd, J ) 10.1, 8.1 Hz, 1H),
5.07 (d, J ) 2.9 Hz, 1H), 5.04-5.00 (m, 2H), 4.86 (dd, J ) 9.6,
7.9 Hz, 1H), 4.64 (d, J ) 11.2 Hz, 1H), 4.58 (d, J ) 11.2 Hz, 1H),
4.55 (d, J ) 11.6 Hz, 1H), 4.50-4.46 (m, 2H), 4.43 (d, J ) 7.8
Hz, 2H), 4.25 (dd, J ) 12.0, 5.2 Hz, 1H), 4.10-4.07 (m, 3H),
4.03-3.97 (m, 3H), 3.88 (t, J ) 9.6 Hz, 1H), 3.85 (t, J ) 7.2 Hz,
1H), 3.82 (dd, J ) 10.3, 3.1 Hz, 1H), 3.77 (dd, J ) 10.4, 2.8 Hz,
1H), 3.68 (m, 1H), 3.58-3.55 (m, 2H), 3.51 (ddd, J ) 9.7, 5.3,
2.0 Hz, 1H), 2.09 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 2.00 (ddd, J
) 13.0, 4.2, 4.2 Hz, 1H), 1.91 (ddd, J ) 12.7, 10.5, 3.0 Hz, 1H),
1.66-1.58 (m, 1H), 1.52-1.46 (m, 1H), 1.25-1.23 (m, 24H), 1.22
(s, 9H), 1.190 (s, 9H), 1.194 (s, 9H), 1.180 (s, 9H), 1.179 (s, 9H),
1.14 (s, 9H), 0.88 (t, J ) 7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3)
δ 177.8, 177.7, 177.0, 176.9, 176.5, 175.9, 170.4, 170.1, 169.9,
138.3, 137.9, 128.3, 127.9, 127.8, 127.7, 127.6, 100.7, 100.2, 93.9,
79.2, 79.1, 74.4, 73.7, 73.5, 73.4, 71.9, 71.8, 71.5, 71.4, 70.3, 69.4,
67.4, 67.3, 66.4, 65.7, 61.9, 61.8, 61.4, 39.0, 38.84, 38.82, 38.7,
38.6, 31.9, 29.7, 29.65, 29.61, 29.56, 29.52, 29.3, 27.4, 27.3, 27.2,
27.1, 27.08, 27.06, 27.03, 25.3, 22.6, 21.2, 20.9, 20.6, 14.1; HRMS
calcd for C86H133N3O26Na ([M + Na]+) 1646.9075, found 1646.9021.
1
mg, 2 steps yield 83%). H NMR (500 MHz, CDCl3) δ 8.63 (s,
1H), 6.46 (d, J ) 3.7 Hz, 1H), 5.57 (t, J ) 9.8 Hz, 1H), 5.46 (d,
J ) 2.2 Hz, 1H), 5.16 (dd, J ) 10.3, 7.9 Hz, 1H), 5.08 (d, J ) 3.0
Hz, 1H), 5.05-5.01 (m, 2H), 4.96 (dd, J ) 10.2, 3.7 Hz, 1H),
4.47 (d, J ) 7.9 Hz, 1H), 4.45 (dd, J ) 12.3, 2.0 Hz, 1H), 4.34
(dd, J ) 12.3, 4.7 Hz, 1H), 4.15-4.05 (m, 4H), 4.02-3.95 (m,
3H), 3.86-3.82 (m, 2H), 2.08 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H),
2.00 (ddd, J ) 13.5, 4.2, 4.2 Hz, 1H), 1.89 (ddd, J ) 13.5, 12.0,
3.1 Hz, 1H), 1.22 (s, 9H), 1.21 (s, 9H), 1.20 (s, 9H), 1.19 (s, 9H),
1.15 (s, 9H), 1.12 (s, 9H); 13C NMR (125 MHz, CDCl3) δ 177.7,
177.6, 177.5, 176.9, 176.7, 175.9, 170.4, 170.1, 169.9, 160.5, 100.2,
93.8, 92.5, 90.8, 74.1, 72.8, 71.9, 71.5, 70.2, 70.0, 68.7, 67.3, 66.4,
65.6, 61.9, 61.5, 61.3, 60.4, 39.0, 38.9, 38.8, 38.79, 38.7, 38.6, 27.4,
27.3, 27.2, 27.07, 27.04, 26.99, 21.2, 21.0, 20.9, 20.6, 14.2. (This
compound is not stable enough to obtain the HRMS spectra.)
2,3,6-Tri-O-acetyl-4-deoxy-R-D-galactopyranosyl-(1,3)-2,4,6-
tri-O-pivaloyl-â-D-galactopyranosyl-(1,4)-2,3,6-tri-O-pivaloyl-R-
D-glucopyranosyl-(1,1)-trichloroacetimide (24). Compound 24
(160 mg, 2 steps yield 70%) was obtained from 16 (210 mg, 0.18
1
mmol). H NMR (500 MHz, CDCl3) δ 8.63 (s, 1H), 6.47 (d, J )
3.6 Hz, 1H), 5.58 (t, J ) 9.8 Hz, 1H), 5.40 (d, J ) 2.4 Hz, 1H),
5.19 (d, J ) 2.8 Hz, 1H), 5.16 (dd, J ) 10.5, 8.2 Hz, 1H), 4.99-
4.93 (m, 3H), 4.44 (d, J ) 7.9 Hz, 1H), 4.41 (d, J ) 3.2 Hz, 2H),
4.15-4.03 (m, 4H), 3.96 (t, J ) 9.7 Hz, 3H), 3.83-3.79 (m, 2H),
2.19-2.15 (m, 1H), 2.07 (s, 6H), 1.97 (s, 3H), 1.56 (ddd, J ) 12.3,
11.7, 11.7 Hz, 1H), 1.23 (s, 9H), 1.22 (s, 9H), 1.200 (s, 9H), 1.198
(s, 9H), 1.19 (s, 9H), 1.12 (s, 9H); 13C NMR (125 MHz, CDCl3) δ
177.8, 177.7, 177.5, 176.7, 176.6, 176.0, 171.2, 170.6, 170.2, 170.0,
160.6, 100.4, 94.3, 92.6, 90.8, 73.5, 72.9, 72.0, 71.5, 70.7, 70.1,
69.7, 68.7, 67.7, 65.8, 65.2, 61.5, 61.4, 60.4, 39.0, 38.9, 38.8, 38.73,
38.71, 32.5, 30.6, 27.3, 27.2, 27.12, 27.11, 27.09, 27.03, 21.1, 21.0,
20.9, 20.7, 14.2. (This compound is not stable enough to obtain
the HRMS.)
2,3,4-Tri-O-acetyl-6-deoxy-R-D-galactopyranosyl-(1,3)-2,4,6-
tri-O-pivaloyl-â-D-galactopyranosyl-(1,4)-2,3,6-tri-O-pivaloyl-R-
D-glucopyranosyl-(1,1)-trichloroacetimide (25). Compound 25
(210 mg, 2 steps yield 80%) was obtained from 17 (240 mg, 0.21
1
mmol). H NMR (500 MHz, CDCl3) δ 8.64 (s, 1H), 6.48 (d, J )
3.7 Hz, 1H), 5.59 (t, J ) 9.8 Hz, 1H), 5.40 (d, J ) 2.8 Hz, 1H),
5.24 (dd, J ) 10.9, 3.4 Hz, 1H), 5.23 (d, J ) 3.2 Hz, 1H), 5.19-
5.15 (m, 2H), 5.03 (dd, J ) 10.9, 3.2 Hz, 1H), 4.97 (dd, J ) 10.3,
3.7 Hz, 1H), 4.43 (dd, J ) 12.3, 4.5 Hz, 1H), 4.40 (d, J ) 8.1 Hz,
1H), 4.36 (dd, J ) 12.2, 1.6 Hz, 1H), 4.17-4.14 (m, 1H), 4.13-
4.04 (m, 3H), 3.96 (t, J ) 9.7 Hz, 1H), 3.82 (t, J ) 6.7 Hz, 1H),
3.73 (dd, J ) 10.3, 3.0 Hz, 1H), 2.14 (s, 3H), 2.07 (s, 3H), 1.92 (s,
3H), 1.23 (s, 18H), 1.20 (s, 27H), 1.13-1.12 (m, 12H). (This
compound is not stable enough to obtain the 13C NMR and HRMS
spectra.)
2,3,6-Tri-O-acetyl-4-deoxy-R-D-hexopyranosyl-(1,3)-2,4,6-tri-
O-pivaloyl-â-D-galactopyranosyl-(1,4)-2,3,6-tri-O-pivaloyl-â-D-
glucopyranosyl-(1,1)-(2S,3S,4R)-2-azido-3,4-dibenzyloxyoctade-
cane (29). Compound 29 (130 mg, 57%) was obtained from donor
24 (180 mg, 0.14 mmol) and acceptor 26 (82 mg, 0.16 mmol). 1H
NMR (500 MHz, CDCl3) δ 7.35-7.26 (m, 10H), 5.40 (d, J ) 2.4
Hz, 1H), 5.21-5.17 (m, 2H), 5.14 (dd, J ) 10.2, 8.0 Hz, 1H),
4.97-4.95 (m, 2H), 4.87 (dd, J ) 9.6, 7.9 Hz, 1H), 4.65 (d, J )
11.3 Hz, 1H), 4.59 (d, J ) 11.2 Hz, 1H), 4.56 (d, J ) 11.5 Hz,
1H), 4.50 (d, J ) 11.5 Hz, 1H), 4.45-4.43 (m, 2H), 4.40 (d, J )
7.9 Hz, 1H), 4.31 (dd, J ) 12.1, 4.9 Hz, 1H), 4.14-4.11 (m, 2H),
4.09-4.06 (m, 2H), 4.05-4.01 (m, 1H), 4.00 (d, J ) 10.3, 7.3 Hz,
1H), 3.88 (t, J ) 9.5 Hz, 1H), 3.82 (t, J ) 7.2 Hz, 1H), 3.79 (dd,
J ) 10.3, 3.1 Hz, 2H), 3.69 (m, 1H), 3.59-3.55 (m, 2H), 3.52
(ddd, J ) 9.8, 4.9, 2.0 Hz, 1H), 2.18-2.15 (m, 1H), 2.08 (s, 3H),
2.07 (s, 3H), 1.98 (s, 3H), 1.68-1.47 (m, 3H), 1.39-1.36 (m, 1H),
1.25-1.23 (m, 23H), 1.23 (s, 18H), 1.20 (s, 9H), 1.19 (s, 9H), 1.18
(s, 9H), 1.14 (s, 9H), 0.88 (t, J ) 6.9 Hz, 3H); 13C NMR (125
MHz, CDCl3) δ 177.8, 177.7, 177.0, 176.8, 176.6, 176.0, 170.6,
170.1, 170.0, 138.4, 138.0, 128.4, 128.0, 127.8, 127.7, 127.6, 100.8,
General Procedure for the Preparation of Glycosphingosines
27-30. 3,4,6-Tri-O-acetyl-2-deoxy-R-D-galactopyranosyl-(1,3)-
2,4,6-tri-O-pivaloyl-â-D-galactopyranosyl-(1,4)-2,3,6-tri-O-piv-
aloyl-â-D-glucopyranosyl-(1,1)-(2S,3S,4R)-2-azido-3,4-dibenzyloxy-
octadecane (27). A suspension of trisaccharide donor 22 (160 mg,
0.12 mmol), sphingosine acceptor 26 (75 mg, 0.15 mmol), and 4
Å molecular sieve (500 mg) in anhydrous CH2Cl2 (5 mL) was
stirred at room temperature for 30 min. After the mixture was cooled
to -20 °C, TMSOTf (5 uL, 0.024 mmol) was added. The resulting
mixture was stirred at -20 °C for 2 h, and then diluted with CH2-
Cl2 (5 mL). The resulting mixture was quenched by saturated
NaHCO3 solution (5 mL) and filtered through a celite pad. The
organic layer was separated and washed with brine (5 mL). After
J. Org. Chem, Vol. 72, No. 26, 2007 9921

Chen et al.
100.3, 94.5, 79.3, 79.2, 73.8, 73.7, 73.5, 73.4, 72.0, 71.9, 71.6, 71.4,
70.8, 69.9, 69.5, 67.7, 65.9, 65.3, 65.2, 61.9, 61.8, 61.6, 39.0, 38.9,
38.8, 38.7, 38.6, 32.4, 31.9, 30.0, 29.7, 29.66, 29.61, 29.6, 29.4,
27.4, 27.3, 27.23, 27.21, 27.12, 27.10, 25.4, 22.7, 21.1, 20.9, 20.7,
14.1; HRMS calcd for C86H133N3O26Na ([M + Na]+) 1646.9075,
found 1646.9073.
2,4,6-Tri-O-acetyl-3-deoxy-R-D-galactopyranosyl-(1,3)-2,4,6-
tri-O-pivaloyl-â-D-galactopyranosyl-(1,4)-2,3,6-tri-O-pivaloyl-â-
D-glucopyranosyl-(1,1)-(2S,3S,4R)-2-hexacosanoylamino-3,4-
dibenzyloxyoctadecane (32). Compound 32 (71 mg, 45%) was
obtained from 28 (136 mg, 0.08 mmol). 1H NMR (500 MHz,
CDCl3) δ 7.35-7.24 (m, 10H), 5.65 (d, J ) 8.2 Hz, 1H), 5.45 (d,
J ) 2.4 Hz, 1H), 5.20 (t, J ) 9.5 Hz, 1H), 5.13 (dd, J ) 9.6, 7.9
Hz, 1H), 5.07 (d, J ) 2.8 Hz, 1H), 5.04-5.00 (m, 2H), 4.84 (dd,
J ) 9.6, 7.9 Hz, 1H), 4.77 (d, J ) 11.3 Hz, 1H), 4.59 (d, J ) 11.7
Hz, 1H), 4.54 (d, J ) 11.3 Hz, 1H), 4.48 (d, J ) 11.7 Hz, 1H),
4.43 (d, J ) 7.8 Hz, 1H), 4.40 (d, J ) 7.9 Hz, 1H), 4.33 (dd, J )
12.1, 1.8 Hz, 1H), 4.27 (dd, J ) 12.1, 4.8 Hz, 1H), 4.15-4.13 (m,
2H), 4.11-4.05 (m, 3H), 4.02-3.96 (m, 2H), 3.87 (t, J ) 9.5 Hz,
1H), 3.84-3.77 (m, 3H), 3.61-3.60 (m, 1H), 3.53-3.49 (m, 1H),
3.45-3.43 (m, 1H), 2.08 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 2.02-
1.98 (m, 2H), 1.96-1.88 (m, 2H), 1.66-1.58 (m, 3H), 1.51-1.43
(m, 3H), 1.24-1.23 (m, 66H), 1.22 (s, 9H), 1.19 (s, 9H), 1.18 (s,
9H), 1.17 (s, 9H), 1.15 (s, 9H), 1.13 (s, 9H), 0.87 (t, J ) 6.9 Hz,
6H); 13C NMR (125 MHz, CDCl3) δ 177.7, 177.5, 176.9, 176.88,
176.83, 175.9, 172.4, 170.4, 170.1, 169.9, 138.7, 138.6, 128.3,
128.2, 127.7, 127.6, 127.55, 127.51, 101.0, 100.1, 93.9, 79.9, 78.4,
74.4, 73.7, 73.5, 73.3, 71.9, 71.8, 71.5, 71.2, 70.2, 68.8, 67.4, 67.2,
66.4, 65.6, 61.9, 61.8, 61.4, 49.8, 39.0, 38.8, 38.84, 38.78, 38.76,
38.68, 38.64, 36.7, 31.9, 29.8, 29.7, 29.69, 29.67, 29.62, 29.4, 29.34,
29.32, 27.4, 27.3, 27.2, 27.1, 27.08, 27.04, 26.1, 25.6, 22.7, 21.2,
20.9, 20.6, 14.1; HRMS calcd for C112H185NO27Na ([M + Na]+)
1999.3032, found 1999.3079.
2,3,6-Tri-O-acetyl-4-deoxy-R-D-galactopyranosyl-(1,3)-2,4,6-
tri-O-pivaloyl-â-D-galactopyranosyl-(1,4)-2,3,6-tri-O-pivaloyl-â-
D-glucopyranosyl-(1,1)-(2S,3S,4R)-2-hexacosanoylamino-3,4-
dibenzyloxyoctadecane (33). Compound 33 (80 mg, 51%) was
obtained from 29 (130 mg, 0.08 mmol). 1H NMR (500 MHz,
CDCl3) δ 7.34-7.25 (m, 10H), 5.66 (d, J ) 8.4 Hz, 1H), 5.39 (d,
J ) 2.2 Hz, 1H), 5.20 (t, J ) 9.5 Hz, 1H), 5.19 (s, 1H), 5.13 (dd,
J ) 10.1, 8.1 Hz, 1H), 4.98 (br, 2H), 4.85 (dd, J ) 9.6, 8.0 Hz,
1H), 4.78 (d, J ) 11.2 Hz, 1H), 4.60 (d, J ) 11.7 Hz, 1H), 4.54
(d, J ) 11.2 Hz, 1H), 4.49 (d, J ) 11.7 Hz, 1H), 4.44 (d, J ) 7.8
Hz, 1H), 4.37 (d, J ) 7.8 Hz, 1H), 4.35 (dd, J ) 12.1, 4.2 Hz,
1H), 4.27 (dd, J ) 12.1, 1.4 Hz, 1H), 4.18-4.00 (m, 7H), 3.87 (t,
J ) 9.6 Hz, 1H), 3.81-3.76 (m, 3H), 3.60 (dd, J ) 9.6, 3.2 Hz,
1H), 3.51 (m, 1H), 3.44 (dt, J ) 8.4, 2.7 Hz, 1H), 2.16 (m, 1H),
2.08 (s, 3H), 2.07 (s, 3H), 1.98 (s, 3H), 1.67-1.45 (m, 10H), 1.25-
1.24 (m, 65H), 1.23 (s, 9H), 1.22 (s, 9H), 1.19 (s, 9H), 1.18 (s,
9H), 1.15 (s, 9H), 1.14 (s, 9H), 0.87 (t, J ) 6.9 Hz, 6H); 13C NMR
(125 MHz, CDCl3) δ 177.8, 177.6, 177.0, 176.8, 176.7, 176.0,
172.4, 170.6, 170.1, 170.0, 138.7, 138.6, 128.3, 128.2, 127.8, 127.6,
127.57, 127.53, 101.1, 100.2, 94.4, 80.0, 78.3, 73.7, 73.4, 73.3,
71.9, 71.6, 70.7, 67.6, 65.8, 65.1, 65.0, 61.5, 49.7, 39.0, 38.9, 38.8,
38.77, 38.68, 38.65, 32.4, 31.9, 29.8, 29.7, 29.68, 29.63, 29.5, 29.4,
29.35, 29.33, 27.3, 27.2, 27.16, 27.15, 27.08, 27.06, 26.1, 25.6,
22.7, 20.7, 14.1; HRMS calcd for C112H185NO27Na ([M + Na]+)
1999.3032, found 1999.3038.
2,3,4-Tri-O-acetyl-6-deoxy-R-D-galactopyranosyl-(1,3)-2,4,6-
tri-O-pivaloyl-â-D-galactopyranosyl-(1,4)-2,3,6-tri-O-pivaloyl-â-
D-glucopyranosyl-(1,1)-(2S,3S,4R)-2-azido-3,4-dibenzyloxyocta-
decane (30). Compound 30 (120 mg, 72%) was obtained from
donor 25 (130 mg, 0.10 mmol) and acceptor 26 (59 mg, 0.11 mmol).
1H NMR (500 MHz, CDCl3) δ 7.35-7.26 (m, 10H), 5.40 (d, J )
2.6 Hz, 1H), 5.24-5.20 (m, 3H), 5.16-5.12 (m, 2H), 5.03 (dd, J
) 11.0, 3.2 Hz, 1H), 4.88 (dd, J ) 9.6, 7.9 Hz, 1H), 4.64 (d, J )
11.3 Hz, 1H), 4.59 (d, J ) 11.2 Hz, 1H), 4.56 (d, J ) 11.6 Hz,
1H), 4.50 (d, J ) 11.5 Hz, 1H), 4.44 (d, J ) 7.9 Hz, 1H), 4.40
(dd, J ) 12.0, 1.6 Hz, 1H), 4.37 (d, J ) 8.0 Hz, 1H), 4.32 (dd, J
) 12.1, 4.9 Hz, 1H), 4.08-4.06 (m, 3H), 4.01 (dd, J ) 10.4, 7.3
Hz, 1H), 3.88 (t, J ) 9.5 Hz, 1H), 3.83 (t, J ) 6.5 Hz, 1H), 3.79
(dd, J ) 10.4, 3.0 Hz, 1H), 3.72 (dd, J ) 10.3, 3.1 Hz, 1H), 3.70-
3.67 (m, 1H), 3.59-3.56 (m, 2H), 3.52 (ddd, J ) 9.8, 4.7, 2.1 Hz,
1H), 2.14 (s, 3H), 2.07 (s, 3H), 1.92 (s, 3H), 1.68-1.47 (m, 3H),
1.39-1.36 (m, 1H), 1.26-1.24 (m, 22H), 1.23 (s, 18H), 1.19 (s,
18H), 1.18 (s, 9H), 1.14 (s, 9H), 1.12 (d, J ) 6.2 Hz, 3H), 0.88 (t,
J ) 6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 177.9, 177.8,
177.0, 176.8, 176.6, 176.0, 170.6, 170.2, 169.8, 138.4, 138.0, 128.4,
128.0, 127.8, 127.7, 127.6, 100.8, 100.2, 95.1, 79.3, 79.2, 75.0,
73.8, 73.5, 73.4, 72.1, 72.0, 71.6, 71.3, 70.9, 68.0, 67.3, 65.6, 65.2,
61.9, 61.6, 39.1, 39.0, 38.9, 38.7, 38.6, 31.9, 30.0, 29.7, 29.69,
29.66, 29.62, 29.5, 29.4, 27.4, 27.3, 27.28, 27.22, 27.16, 27.13,
27.11, 27.08, 27.05, 25.4, 22.7, 21.1, 20.6, 20.5, 14.1; HRMS calcd
for C86H133N3O26Na ([M + Na]+) 1646.9075, found 1646.9076.
General Procedure for the Preparation of Glycoceramides
31-34. 3,4,6-Tri-O-acetyl-2-deoxy-R-D-galactopyranosyl-(1,3)-
2,4,6-tri-O-pivaloyl-â-D-galactopyranosyl-(1,4)-2,3,6-tri-O-piv-
aloyl-â-D-glucopyranosyl-(1,1)-(2S,3S,4R)-2-hexacosanoylamino-
3,4-dibenzyloxyoctadecane (31). Triphenylphosphine (36 mg, 0.14
mmol) was added to a solution of azide 27 (114 mg, 0.07 mmol)
in benzene (10 mL) and water (0.1 mL). The reaction mixture was
stirred at 50 °C for 8 h. The solvent was evaporated under reduced
pressure and azetroped with benzene (2 × 10 mL), then the residue
was dissolved in anhydrous THF (5 mL) and treated with cerotic
acid (40 mg, 0.10 mmol) and EDCI (19 mg, 0.10 mmol). After the
solution was stirred for 10 h at room temperature, the solvent was
evaporated and the residue was partitioned between CH2Cl2 (10
mL) and water (5 mL). The organic layer was separated and dried
over anhydrous Na2SO4. After being concentrated, the residue was
purified by silica gel flash chromatography (1:5 EtOAc/hexanes)
1
to provide 31 (69 mg, 2 steps yield 50%). H NMR (500 MHz,
CDCl3) δ 7.34-7.33 (m, 4H), 7.30-7.27 (m, 6H), 5.65 (d, J )
8.1 Hz, 1H), 5.41 (d, J ) 2.4 Hz, 1H), 5.28 (s, 2H), 5.19 (t, J )
9.5 Hz, 1H), 5.08 (dd, J ) 10.2, 8.2 Hz, 1H), 4.95 (ddd, J ) 12.4,
4.7, 2.8 Hz, 1H), 4.85 (dd, J ) 9.6, 7.9 Hz, 1H), 4.78 (d, J ) 11.2
Hz, 1H), 4.60 (d, J ) 11.7 Hz, 1H), 4.54 (d, J ) 11.2 Hz, 1H),
4.49 (d, J ) 11.7 Hz, 1H), 4.44 (d, J ) 7.8 Hz, 2H), 4.35 (dd, J )
12.2, 1.7 Hz, 1H), 4.28 (dd, J ) 12.1, 4.6 Hz, 1H), 4.17-4.13 (m,
2H), 4.12-4.09 (m, 3H), 4.06-4.01 (m, 2H), 3.89 (t, J ) 9.5 Hz,
1H), 3.86-3.81 (m, 2H), 3.78 (dd, J ) 7.1, 2.2 Hz, 1H), 3.62-
3.59 (m, 1H), 3.52-3.49 (m, 1H), 3.45-3.43 (m, 1H), 2.11 (s,
3H), 2.04 (s, 3H), 2.03-1.91 (m, 2H), 1.94 (s, 3H), 1.65-1.58
(m, 5H), 1.55 (m, 1H), 1.53-1.43 (m, 4H), 1.25-1.24 (m, 64H),
1.23 (s, 9H), 1.22 (s, 9H), 1.20 (s, 9H), 1.19 (s, 9H), 1.15 (s, 9H),
1.13 (s, 9H), 0.88 (t, J ) 6.9 Hz, 6H); 13C NMR (125 MHz, CDCl3)
δ 177.8, 177.6, 177.5, 177.0, 176.9, 176.3, 172.4, 170.4, 170.3,
169.8, 138.7, 138.6, 128.4, 128.3, 127.8, 127.7, 127.6, 127.5, 101.1,
100.0, 93.3, 80.0, 78.4, 73.7, 73.6, 73.2, 72.0, 71.9, 71.6, 71.4, 71.3,
69.6, 68.9, 67.0, 66.1, 65.8, 64.4, 62.1, 61.7, 61.4, 49.8, 39.1, 39.0,
38.8, 38.7, 38.6, 36.8, 31.9, 29.75, 29.72, 29.7, 29.6, 29.44, 29.40,
27.3, 27.2, 27.1, 27.0, 22.7, 20.8, 20.7, 20.6, 14.1; HRMS calcd
for C112H185NO27Na ([M + Na]+) 1999.3032, found 1999.2994.
2,3,4-Tri-O-acetyl-6-deoxy-R-D-galactopyranosyl-(1,3)-2,4,6-
tri-O-pivaloyl-â-D-galactopyranosyl-(1,4)-2,3,6-tri-O-pivaloyl-â-
D-glucopyranosyl-(1,1)-(2S,3S,4R)-2-hexacosanoylamino-3,4-
dibenzyloxyoctadecane (34). Compound 34 (80 mg, 58%) was
obtained from compound 30 (120 mg, 0.07 mmol). 1H NMR (500
MHz, CDCl3) δ 7.33-7.25 (m, 10H), 5.64 (d, J ) 8.3 Hz, 1H),
5.38 (d, J ) 2.6 Hz, 1H), 5.23-5.19 (m, 3H), 5.15-5.12 (m, 2H),
5.02 (dd, J ) 10.9, 3.2 Hz, 1H), 4.85 (dd, J ) 9.5, 7.9 Hz, 1H),
4.77 (d, J ) 11.2 Hz, 1H), 4.59 (d, J ) 11.7 Hz, 1H), 4.54 (d, J )
11.2 Hz, 1H), 4.49 (d, J ) 11.7 Hz, 1H), 4.44 (d, J ) 7.8 Hz, 1H),
4.36-4.33 (m, 2H), 4.25 (dd, J ) 11.7, 1.6 Hz, 1H), 4.17-4.12
(m, 2H), 4.09-4.05 (m, 3H), 3.87 (t, J ) 9.5 Hz, 1H), 3.80-3.78
(m, 2H), 3.70 (dd, J ) 10.2, 3.0 Hz, 1H), 3.60 (dd, J ) 8.6, 2.6
Hz, 1H), 3.52 (ddd, J ) 9.6, 4.3, 2.1 Hz, 1H), 3.44 (dt, J ) 8.1,
2.7 Hz, 1H), 2.13 (m, 3H), 2.06 (s, 3H), 1.92 (s, 3H), 1.72-1.45
(m, 8H), 1.25-1.24 (m, 66H), 1.23 (s, 9H), 1.22 (s, 9H), 1.18 (s,
18H), 1.15 (s, 9H), 1.14 (s, 9H), 1.11 (d, J ) 6.5 Hz, 3H), 0.88-
0.85 (m, 6H); 13C NMR (125 MHz, CDCl3) δ 177.8, 177.7, 177.0,
9922 J. Org. Chem., Vol. 72, No. 26, 2007

Isoglobotrihexosylceramide Analogues
176.9, 176.7, 175.9, 172.4, 170.6, 170.2, 169.7, 138.7, 138.6,
128.35, 128.31, 127.8, 127.7, 127.6, 127.5, 101.1, 100.2, 80.0, 78.5,
74.8, 73.7, 73.5, 73.3, 72.1, 72.0, 71.6, 71.2, 70.9, 69.9, 68.9, 67.9,
67.2, 65.5, 65.2, 61.9, 61.6, 49.8, 39.0, 38.9, 38.83, 38.80, 38.7,
38.6, 36.7, 36.6, 31.9, 29.8, 29.7, 29.69, 29.65, 29.5, 29.4, 29.39,
29.35, 27.3, 27.2, 27.18, 27.13, 27.0, 26.1, 25.6, 24.7, 23.4, 22.7,
21.1, 20.6, 20.5, 15.7, 14.1; HRMS calcd for C112H185NO27Na ([M
+ Na]+) 1999.3032, found 1999.2950.
NMR (500 MHz, pyridine-d5) δ 8.54 (d, J ) 8.8 Hz, 1H), 7.60 (s,
1H), 7.37 (d, J ) 5.2 Hz, 1H), 6.72 (d, J ) 3.9 Hz, 1H), 6.67 (m,
1H), 6.51 (d, J ) 6.2 Hz, 1H), 6.44-6.42 (m, 2H), 6.13 (s, 1H),
5.89 (d, J ) 7.1 Hz, 1H), 5.84 (s, 1H), 5.60 (d, J ) 3.5 Hz, 1H),
5.14-5.09 (m 1H), 4.94 (m, 1H), 4.87 (d, J ) 8.0 Hz, 2H), 4.77
(dd, J ) 10.6, 5.4 Hz, 1H), 4.57 (br, 2H), 4.52-4.47 (m, 1H),
4.43 (br, 3H), 4.38-4.35 (m, 2H), 4.33-4.29 (m, 1H), 4.23-4.17
(m, 4H), 4.06-3.99 (m, 5H), 3.81-3.79 (m, 1H), 3.59 (d, J ) 5.1
Hz, 1H), 2.48-2.44 (m, 1H), 2.42 (t, J ) 7.5 Hz, 2H), 2.25-2.19
(m, 1H), 2.05 (ddd, J ) 12.2, 12.0, 12.0 Hz, 1H), 1.96-1.90 (m,
2H), 1.84-1.77 (m, 2H), 1.68 (m, 1H), 1.31-1.24 (m, 67H), 0.88-
0.84 (m, 6H); 13C NMR (125 MHz, pyridine-d5) δ 173.4, 105.5,
105.3, 97.8, 82.1, 80.2, 76.7, 76.5, 75.9, 75.6, 74.7, 72.7, 70.7, 70.5,
68.5, 66.0, 65.5, 62.0, 61.8, 52.2, 37.2, 36.9, 33.6, 32.2, 30.4, 30.2,
30.1, 30.0, 29.96, 29.94, 29.86, 29.78, 29.63, 26.7, 26.4, 23.0, 14.3;
HRMS calcd for C62H119NO18Na ([M + Na]+) 1188.8325, found
1188.8330.
General Procedure for the Preparation of iGb3 analogues
35-38. 2-Deoxy-R-D-galactopyranosyl-(1,3)-â-D-galactopyrano-
syl-(1,4)-â-D-glucopyranosyl-(1,1)-(2S,3S,4R)-2-hexacosanoy-
laminooctadecen-3,4-diol (2-dh-iGb3) (35). A suspension of
glycoceramide 31 (46 mg, 0.025 mmol) and Pd(OH)2/C (30 mg,
20 wt %) in MeOH (3 mL) was shaken for 4 h under H2 atmosphere
(50 psi). The catalytic Pd(OH)2/C was filtered off through a celite
pad and the filtrate was concentrated. Then the residue was
dissolved in anhydrous MeOH (5 mL) and freshly prepared NaOMe
(4 mg, 0.07 mmol) was added. The resulting mixture was heated
to reflux for 24 h. After the solution was cooled to room
temperature, the precipitate was collected by centrifuge. The
precipitate was washed with MeOH (2 × 2 mL) and dissolved in
pyridine. The insoluble impurities were removed by centrifuge and
the clear solution was concentrated to give 2-dh-iGb3 35 (12 mg,
6-Deoxy-R-D-galactopyranosyl-(1,3)-â-D-galactopyranosyl-
(1,4)-â-D-glucopyranosyl-(1,1)-(2S,3S,4R)-2-hexacosanoylami-
nooctadecen-3,4-diol (6-dh-iGb3) (38). Compound 38 (23 mg,
1
50%) was obtained from compound 34 (80 mg, 0.04 mmol). H
NMR (500 MHz, pyridine-d5) δ 8.49 (d, J ) 8.7 Hz, 1H), 7.26 (d,
J ) 5.1 Hz, 1H), 6.61 (s, 1H), 6.48 (br, 1H), 6.45 (d, J ) 5.2 Hz,
1H), 6.39 (s, 1H), 6.16 (s, 1H), 6.13 (s, 1H), 5.82 (d, J ) 7.0 Hz,
1H), 5.57 (d, J ) 2.8 Hz, 1H), 5.14-5.11 (m 1H), 5.06 (d, J ) 7.9
Hz, 1H), 4.88 (d, J ) 7.9 Hz, 2H), 4.77 (dd, J ) 10.6, 5.4 Hz,
1H), 4.65-4.62 (m, 1H), 4.57 (s, 1H), 4.52-4.51 (m, 2H), 4.45
(m, 3H), 4.38-4.36 (m, 3H), 4.22 (d, J ) 8.7 Hz, 3H), 4.12 (s,
1H), 4.06 (t, J ) 5.9 Hz, 1H), 4.01 (t, J ) 7.9 Hz, 1H), 3.82-3.80
(m, 1H), 3.61 (s, 1H), 2.43 (t, J ) 7.3 Hz, 2H), 2.26-2.20 (m,
1H), 1.95-1.93 (m, 2H), 1.85-1.79 (m, 2H), 1.71-1.68 (m, 1H),
1.58 (d, J ) 6.4 Hz, 3H), 1.31-1.27 (m, 68H), 0.89-0.87 (m,
6H); 13C NMR (125 MHz, pyridine-d5) δ 173.8, 105.8, 105.6, 98.0,
82.4, 80.4, 77.0, 76.9, 76.8, 76.2, 75.0, 73.8, 73.0, 72.0, 71.0, 70.9,
70.4, 68.1, 66.3, 62.4, 62.2, 52.5, 37.3, 33.9, 32.5, 30.7, 30.5, 30.4,
30.38, 30.31, 30.28, 30.21, 30.1, 30.0, 29.97, 27.0, 26.7, 23.3, 17.6,
14.6; HRMS calcd for C62H119NO18Na ([M + Na]+) 1188.8325,
found 1188.8322.
Bioassay Experiment. Stimulatory activities of glycolipids were
assayed by a murine iNKT stimulation system as described,8 using
bone marrow-derived mouse DC as APC and a murine iNKT
hybridoma, DN32D3, as responder cells. DN32D3 is a NKT cell
hybridoma that secrets IL2 upon recognition of glycolipids presented
by dendritic cells. iGb3 and its analogues (35-38) were dissolved
at 1 mg/mL in DMSO and stored at -20 °C. In this experiment,
they were further diluted in cell culture medium at indicated
concentrations and pulsed to 100 000 mouse (C57BL6) bone
marrow derived dendritic cells for 8 h, after which the dendritic
cells were washed with culture medium and mixed with 50 000
DN32.D3 hybridoma cells. Then the resulted mixture was co-
cultured for 24 h, and the released IL2 was measured by CTLL
assay.
1
41%) as white powder. H NMR (500 MHz, pyridine-d5) δ 8.51
(d, J ) 8.8 Hz, 1H), 7.54 (s, 1H), 7.32 (d, J ) 4.5 Hz, 1H), 6.62
(s, 1H), 6.46-6.44 (m, 2H), 6.41-6.38 (m, 2H), 6.15 (s, 1H), 6.11-
6.09 (s, 2H), 5.84 (d, J ) 6.7 Hz, 1H), 5.67 (s, 1H), 5.10-5.05 (m
2H), 4.91 (t, J ) 5.9 Hz, 2H), 4.86 (d, J ) 7.7 Hz, 1H), 4.76 (dd,
J ) 10.5, 5.7 Hz, 1H), 4.62 (m, 2H), 4.58-4.54 (m, 1H), 4.49 (s,
1H), 4.43-4.40 (m, 6H), 4.36-4.30 (m, 4H), 4.23-4.17 (m, 3H),
4.00-3.97 (m, 1H), 3.80 (br, 1H), 2.51 (td, J ) 12.1, 3.1 Hz, 1H),
2.42 (t, J ) 7.4 Hz, 2H), 2.23-2.19 (m, 2H), 1.95-1.89 (m, 2H),
1.85-1.77 (m, 2H), 1.73-1.63 (m, 1H), 1.32-1.25 (m, 65H),
0.88-0.85 (m, 6H); 13C NMR (125 MHz, pyridine-d5) δ 173.6,
105.7, 105.3, 94.6, 82.1, 77.9, 77.0, 76.7, 76.5, 75.9, 74.7, 72.7,
72.5, 70.7, 70.5, 69.7, 66.2, 65.3, 63.0, 62.1, 62.0, 52.2, 36.9, 34.1,
33.6, 32.2, 30.4, 30.2, 30.1, 30.0, 29.99, 29.97, 29.89, 29.8, 29.67,
29.65, 26.7, 26.4, 23.0, 14.3; HRMS calcd for C62H119NO18Na ([M
+ Na]+) 1188.8325, found 1188.8257.
3-Deoxy-R-D-galactopyranosyl-(1,3)-â-D-galactopyranosyl-
(1,4)-â-D-glucopyranosyl-(1,1)-(2S,3S,4R)-2-hexacosanoylami-
nooctadecen-3,4-diol (3-dh-iGb3) (36). Compound 36 (20 mg,
1
62%) was obtained from compound 32 (55 mg, 0.028 mmol). H
NMR (500 MHz, pyridine-d5) δ 8.45 (d, J ) 8.7 Hz, 1H), 7.54
(m, 1H), 7.30 (d, J ) 4.9 Hz, 1H), 6.59 (m, 1H), 6.48 (m, 1H),
6.44-6.40 (m, 2H), 6.37-6.36 (m 2H), 6.10 (s, 1H), 5.80 (d, J )
7.0 Hz, 1H), 5.75 (s, 1H), 5.55 (d, J ) 2.7 Hz, 1H), 5.14-5.09 (m
1H), 5.04 (d, J ) 7.8 Hz, 1H), 4.89-4.87 (m, 2H), 4.76 (dd, J )
10.4, 5.2 Hz, 1H), 4.56-4.52 (m, 3H), 4.43-4.40 (m, 4H), 4.38-
4.33 (m, 4H), 4.31-4.25 (m, 2H), 4.23-4.18 (m, 2H), 4.02-4.00
(m, 2H), 3.83-3.81 (m, 1H), 2.43-2.40 (m, 4H), 2.21 (m, 1H),
1.93-1.91 (m, 2H), 1.82-1.78 (m, 2H), 1.68 (m, 1H), 1.31-1.25
(m, 67H), 0.88-0.85 (m, 6H); 13C NMR (125 MHz, pyridine-d5)
δ 173.4, 105.5, 105.3, 97.0, 82.1, 79.9, 76.7, 76.6, 76.5, 75.9, 74.7,
72.7, 71.9, 70.7, 70.6, 67.1, 66.1, 64.5, 62.8, 62.0, 61.8, 52.2, 36.9,
36.1, 33.6, 32.2, 32.1, 30.4, 30.2, 30.1, 30.0, 29.96, 29.93, 29.85,
Acknowledgment. This project was supported by The Ohio
State University (PGW) and the M. D. Anderson Cancer Center
(DZ).
29.78, 29.65, 29.63, 26.7, 26.4, 23.0, 14.3; HRMS calcd for C62H119
-
Supporting Information Available: 1H and 13C NMR spectra
of all new compounds 1-3, 8, 11-18, 22-25, 27-38 and 1H-1H
COSY of compound 11. This material is available free of charge
via the Internet at http://pubs.acs.org.
NO18Na ([M + Na]+) 1188.8325, found 1188.8328.
4-Deoxy-R-D-galactopyranosyl-(1,3)-â-D-galactopyranosyl-
(1,4)-â-D-glucopyranosyl-(1,1)-(2S,3S,4R)-2-hexacosanoylami-
nooctadecen-3,4-diol (4-dh-iGb3) (37). Compound 37 (16 mg,
1
69%) was obtained from compound 33 (39 mg, 0.020 mmol). H
JO701539K
J. Org. Chem, Vol. 72, No. 26, 2007 9923