One-pot syntheses of immunostimulatory glycolipids

Article abstract of DOI:10.1021/jo100366v

(Figure presented) Glycolipids containing α-linked galactosyl and glucosyl moieties have been shown to possess unique immunostimulatory activity creating a need for access to diverse and anomerically pure sources of these compounds for immunological studies. To meet this demand, glycosyl iodides were enlisted in the synthesis of these biologically relevant glycoconjugates. In the first-generation protocol, per-O-benzyl galactosyl iodide was efficiently coupled with activated sphingosine acceptors, but fully functionalized ceramides were found to be unreactive. To overcome this obstacle, per-O-trimethylsilyl glycosyl iodides were investigated and shown to undergo highly efficient coupling with ceramide and glycerol ester acceptors. Contrary to what has been observed with other donors, we detected little difference between the reactivity of glucosyl and galactosyl iodides. The trimethylsilyl protecting groups play a dual role in activating the donor toward nucleophilic attack while at the same time providing transient protection: the silyl groups are readily removed upon methanolysis. All reactions proceeded with complete acceptor regioselectivity, eliminating the need for additional protecting group manipulations, and the desired α-anomers were formed exclusively. This three-step, one-pot synthetic platform provides rapid access to an important class of immunostimulatory molecules including the first reported synthesis of the glucosyl analogue of the bacterial antigen BbGL-II.

Full text of DOI:10.1021/jo100366v

pubs.acs.org/joc
One-Pot Syntheses of Immunostimulatory Glycolipids
Matthew Schombs, Francine E. Park, Wenjun Du, Suvarn S. Kulkarni, and
Jacquelyn Gervay-Hague*
Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California 95616
gervay@chem.ucdavis.edu
Received February 26, 2010
Glycolipids containing R-linked galactosyl and glucosyl moieties have been shown to possess unique
immunostimulatory activity creating a need for access to diverse and anomerically pure sources of these
compounds for immunological studies. To meet this demand, glycosyl iodides were enlisted in the synthesis
of these biologically relevant glycoconjugates. In the first-generation protocol, per-O-benzyl galactosyl
iodide was efficiently coupled with activated sphingosine acceptors, but fully functionalized ceramides were
found to be unreactive. To overcome this obstacle, per-O-trimethylsilyl glycosyl iodides were investigated
and shown to undergo highly efficient coupling with ceramide and glycerol ester acceptors. Contrary to what
has been observed with other donors, we detected little difference between the reactivity of glucosyl and
galactosyl iodides. The trimethylsilyl protecting groups play a dual role in activating the donor toward
nucleophilic attack while at the same time providing transient protection: the silyl groups are readily removed
upon methanolysis. All reactions proceeded with complete acceptor regioselectivity, eliminating the need for
additional protecting group manipulations, and the desired R-anomers were formed exclusively. This three-
step, one-pot synthetic platform provides rapid access to an important class of immunostimulatory
molecules including the first reported synthesis of the glucosyl analogue of the bacterial antigen BbGL-II.
Introduction
In 1993, researchers in Japan reported the isolation and
bioactivity studies of the first known R-linked galactosylce-
ramides (R-GalCer).1 These glycolipids, also known as
agelasphins, were extracted from the marine sponge Agelas
mauritianus and possessed unique antitumor activity. Sub-
sequent structure-activity relationship (SAR) studies led
to the identification of the prototypical antigen, KRN7000
(Figure 1). The immunostimulatory effects of the agelas-
phins attracted considerable attention, as it was especially
curious that natural products derived from a marine sponge
FIGURE 1. Structures of immunostimulatory glycolipids.
could stimulate the immune system. Investigations aimed at
identifying other possible sources of R-linked glycolipids
commenced. One such study centered on Borrelia burgdorferi,
which is the causative agent of Lyme disease. Lyme is the most
common vector-borne disease in the United States and is a
multisystemic disorder that affects the skin, nervous system,
(1) Natori, T.; Koezuka, Y.; Higa, T. Tetrahedron Lett. 1993, 34 (35),
5591–5592.
DOI: 10.1021/jo100366v
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SCHEME 1. First-Generation Synthesis of KRN7000
heart, muscles, and joints.2 Its Gram-negative spirochete is
transmitted through the bite of infected ticks and contains the
R-linked glycolipid BbGL-II (Figure 1), which exhibits similar
antigenic activity to KRN7000. This discovery marked the
first example of an NKT cell antigen expressed by a human
pathogen and led to the hypothesis that the glycolipids
isolated from sponge were of bacterial origin.3-7
Subsequent biological investigations revealed that these
glycolipids interact with the lipid binding protein CD1d asso-
ciated with antigen presenting cells (APC).8,9 Once bound, the
carbohydrate headgroup of the glycolipid-CD1d complex is
presented to a T-cell receptor (TCR) on natural killer T-cells
(NKT) which, upon binding, initiates an immunological cas-
cade of events.10,11 This immune response includes the release
of cytokines mainly composed of the T helper 1 (Th1) cytokine
IFN-γ and the T helper 2 (Th2) cytokine IL-4.12,13 Th1 res-
ponses are important in controlling viral and bacterial infec-
tions, as well as tumor metastasis, whereas Th2 cytokine secre-
tion is associated with certain autoimmune diseases such as
type-1 diabetes and multiple sclerosis.14 However, simultane-
ous production of both Th1 and Th2 cytokines has an antago-
nizing effect resulting from reciprocal inhibition.15 Therefore,
research into the identification of analogues that elicit biased
cytokine production is of high interest for therapeutic use.16,17
Since a Th1 response requires prolonged CD1d-glycolipid-
TCR binding, the desired polarizing effects may be achieved
through modulation of ternary complex longevity.18 Attempts to
tune the stability of the CD1d-glycolipid-TCR construct have
focused primarily on modifications to the lipid portion of the
antigen. These alterations serve to attenuate complex stability by
shifting the spatial orientation of the carbohydrate. Recently it
has been shown that variation to the carbohydrate residue may
also bias cytokine production. And while it is known that
R-mannosyl ceramides (R-ManCer) exhibit no stimulatory acti-
vity, R-glucosyl ceramides (R-GlcCer) express similar activity
profiles as their R-GalCer epimers.11,19 As such, there is a need
for access to diverse and anomerically pure sources of R-linked
glycolipids to further probe the immunological effects of these
antigens. To meet this demand, we recently communicated
highly efficient syntheses of bioactive glycolipids with galactose
head groups.20,21 Herein we report a full account of those studies
and extension of the methodology to include glucosyl analogues.
Results and Discussion
(2) Orloski, K. A.; Hayes, E. B.; Campbell, G. L.; Dennis, D. T. MMWR
CDC Surveill. Summ. 2000, 49, 1–11.
Within the past decade, glycosyl iodide chemistry has
enjoyed a renaissance largely due to our ability to tame these
reactive donors through careful choice of protecting groups. In
the context of stereoselective sytheses of R-GalCer analogues,
we first reported the coupling of per-O-benzyl-protected galac-
tosyl iodide (1) with azido phytosphingosine (2, Scheme 1).20
TBAI was added to the reaction mixture to promote in situ
anomerizarion of the R-iodide to its more reactive β-anomer.
Nucleophilic addition of 2 to the reactive intermediate pro-
vided R-galactosyl azido phytosphingosine (3) exclusively. In
those investigations, we observed a correlation between yield
and the protecting groups appended to the nucleophile. For
example, when the secondary hydroxyls of 2 were protected
with benzoates (Bz), only a 30% yield was observed. Benzyl
(Bn) protecting groups, which are electron donating relative to
the benzoates, afforded increased product yield (67%), and
when p-methoxybenzyl (PMB) protecting groups were used,
essentially quantitative yields were achieved. Subsequent re-
duction of the azide and amidation of the resulting amine
followed by global deprotection afforded KRN7000 in argu-
ably the most efficient series of reactions reported prior to the
date of that publication.
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SCHEME 2. First-Generation Synthesis of Desoxy-KRN7000 (7)
These results cemented our understanding of the electro-
nic sensitivity of these reactions and the importance of
matching donor and acceptor reactivity. It is well-known
that per-O-benzylated glycosyl iodides are orders of magni-
tude more reactive than their corresponding per-O-acetyl-
ated analogues. This is evidenced by the fact that per-O-
acetylated glycosyl iodides can be easily purified by column
chromatography and even crystallized,22,23 whereas the ben-
zyl analogues are far more reactive and can only be isolated
with great care.24 Likewise, the more electron rich the
acceptor alcohol, the better suited it is to couple with a
donor. Conversely, more reactive donors are required for the
glycosidation of unreactive acceptors. The concept of match-
ing donor/acceptor reactivity changed our way of thinking
and resulted in a new direction for our research.
afford the KRN7000 analogue (7). In the case illustrated, the
double bond on the lipid was reduced during hydrogenolysis
although others have reported methods for selectively remov-
ing benzyl groups while leaving the double bond intact.25,27
Wishing to avoid these protecting group manipulations, we
turned our attention to the possibility of reacting glycosyl
iodides with fully functionalized ceramide acceptors (Figure 2).
Our ultimate goal was to be able to react fully functionalized
lipid acceptors with glycosyl iodide donors. We appreciated
the fact that glycosidation with fully functionalized ceramides
was notoriously difficult. For example, Sakai and co-workers
attempted a reaction between per-O-benzyl galactosyl fluoride
and a ceramide using stannous chloride/silver perchlorate
activation in which only a 23% yield of the desired R-product
was isolated.25 The main challenge with this strategy is the
diminished nucleophilicity of the ceramide primary alcohol
due to intramolecular hydrogen bonding with the amide
proton.26 We alleviated this problem in our first-generation
syntheses by masking the amide functionality as an azide;
however, this process involves many additional steps and
reduces the overall efficiency. As indicated in Scheme 2, start-
ing from commercially available sphingosine (4), four steps are
required to prepare the acceptor for glycosidation. First, the
amine is converted to an azide through the action of TfN3,
the primary alcohol is then protected with a trityl group, and
the secondary alcohol is reacted with p-methoxybenzyl chlo-
ride before removal of the trityl protecting group. Reaction of
azido sphingosine (5) with 1 is very efficient; however, after
glycosidation one must essentially reverse the reactions re-
quired to convert 4 to 5. First the azide is reduced, and then it is
amidated, and finally the protecting groups are removed to
FIGURE 2. Structures of ceramide- and glycerol-containing pro-
ducts and acceptors.
Initial glycosylations between per-O-benzyl galactosyl iodide
(1) and ceramide (8) met with limited success, as only low
product yields were obtained. However, concurrent studies in
our lab utilizing per-O-trimethylsilyl galactosyl iodide were
more promising. We found this donor to be orders of magni-
tude more reactive than the per-O-benzyl analog. Per-O-TMS
glycosides are readily prepared on large scale, and the iodide
can be generated quantitatively upon reaction with iodotri-
methylsilane (TMSI).28 An additional benefit associated with
the use of silyl ether protecting groups is that they can be
readily removed by methanolysis upon completion of the
reaction, leaving the glycosidic linkage and olefin unaffected.
In this manner, the trimethylsilyl functionality serves as a
transient protecting group.
Our first attempts at developing this methodology focused
on galactosyl glycolipids.21 The syntheses of 13 - 16 began
with the addition of TMSI to a cooled solution of 12,
generating per-O-TMS galactosyl iodide in situ. The donor
(22) Fisher, E.; Fischer, H. Ber. 1910, 43, 2535.
(23) Mukhopadhyay, B.; Kartha, K. P. R.; Russell, D. A.; Field, R. A.
J. Org. Chem. 2004, 69 (22), 7758–7760.
(24) Caputo, R.; Kunz, H.; Mastroianni, D.; Palumbo, G.; Pedatella, S.;
Solla, F. Eur. J. Org. Chem. 1999, 1999 (11), 3147–3150.
(25) Sakai, T.; Ueno, H.; Natori, T.; Uchimura, A.; Motoki, K.;
Koezuka, Y. J. Med. Chem. 1998, 41, 650–652.
(27) Kinjo, Y.; Pei, B.; Bufali, S.; Raju, R.; Richardson, S. K.; Imamura,
M.; Fujio, M.; Wu, D.; Khurana, A.; Kawahara, K.; Wong, C.-H.; Howell,
A. R.; Seeberger, P. H.; Kronenberg, M. Chem. Biol. 2008, 15 (7), 654–664.
(28) Bhat, A.; Gervay-Hague, J. Org. Lett. 2001, 3 (13), 2081–2084.
(26) Polt, R.; Szabo, L.; Treiberg, J.; Li, Y.; Hruby, V. J. J. Am. Chem.
Soc. 1992, 114 (26), 10249–10258.
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TABLE 1. Second-Generation Synthesis of Biologically Relevant Glycolipids
entry
acceptor
solvent
conditions
product
R:β ratio (yield, %)
1
2
3
4
5
8
9
9
10
11
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
TBAI (3 equiv), rt, 48 h
TBAI (3 equiv), rt, 48 h
TBAI (3 equiv), μwave, 1.5 h
TBAI (4 equiv), rt, 24 h
TBAI (3 equiv), rt, 36 h
13
14
14
15
16
only R (77)
only R (30)
only R (67)
only R (81)
only R (72)
FIGURE 3. TBAI optimization study.
was then added to a mixture containing the acceptor (8-11),
TBAI, and N,N-diisopropylethylamine (DIPEA). After the
indicated time, the solvent was evaporated and the residue
was subjected to hydrolysis using acidic resin in methanol
affording the final products 13-16 (Table 1). Initially, the
glycosidation of acceptor 9 proved problematic and at room
temperature only low yields (30%, entry 2) were obtained.
However, to our delight we found that when the reaction was
conducted at 115 °C with 200 W of microwave irradiation,
the yield of 14 increased to 67% and the reaction time was
reduced from 48 h to 90 min (Table 1, entry 3).
We naturally thought that microwave irradiation would
also promote reactions with 8, but that proved not to be the
case. Instead, we observed trans-silylation of the acceptor as
evidenced by the disappearance of 8 by thin-layer chromato-
graphy (TLC) and its reappearance after methanolysis. In-
deed, we were able to confirm that trans-silylation had
occurred by isolating and characterizing a silyl-protected
acceptor. We hypothesized that TBAI could be promoting
desilylation of the sugar generating TMSI, which could in turn
react with the acceptor. In an attempt to reduce this process, we
explored the possibility of using fewer equiv of TBAI
(Figure 3). These studies indicated that at least 1.5 equiv of
TBAI is required for efficient conversion.29 We also attempted
microwave irradiation with reduced TBAI and again observed
primarily trans-silylation, indicating that room temperature
conditions are best for this system.
We next turned our attention to the syntheses of glucose-
containing bioactive glycolipids. While there are several pub-
lished methods for the synthesis of R-GalCer derivatives, there
are far fewer reports for the stereoselective synthesis of the
corresponding glucosyl analogues. The most commonly em-
ployed donor for the synthesis of 1,2-cis glucosides has been
2,3,4,6-tetra-O-benzyl glucopyranosyl fluoride.30 Typical pro-
tocols require the use of toxic reagents (stannous chloride and
silver perchlorate), result in R/β product mixtures, and proceed
without regioselectivity with respect to the acceptor.31 When
(29) We note that the authors of ref 27 experienced difficulty in removal of
TBAI during the synthesis of a galactosyl ceramide in which 3 equiv of TBAI
was employed and a yield was not reported. Reduction to 1.5 equiv may help
alleviate purification complications.
(30) Mukaiyama, T.; Murai, Y.; Shoda, S.-i. Chem. Lett. 1981, 431–432.
(31) Morita, M.; Natori, T.; Akimoto, K.; Osawa, T.; Fukushima, H.;
Koezuka, Y. Bioorg. Med. Chem. Lett. 1995, 5 (7), 699–704.
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TABLE 2. Examples from Competition Studies
SCHEME 3. Competition Experiment between Per-O-TMS Galactosyl and Glucosyl Iodides
applied to the glucosidation of unprotected ceramides, mixtures
of both mono and diglycosylated products are formed. Other
donors show similar reactivities. For example, Fan and co-
workers recently utilized 2,3,4,6-tetra-O-benzyl thiophenyl glu-
copyranoside in the NIS/TfOH promoted glycosidation of a
serine-based ceramide. This protocol afforded GlcCer in 46%
yield with 2:1 R/β selectivity.32 The reactivity, stereoselectivity,
and overall reaction efficiencies are typically reduced with
glucosyl donors relative to galactosyl donors. As illustrated by
competition studies comparing the reactivity of thioether and
trichloroacetimidate donors (Table 2), the relative reactivity
ratios were reported to be 6.4:1, 4:1, and 5:1 respectively.33-38 A
number of theories have been proposed to account for these
observations including stereoelectronic effects and torsional
strain.34,35,38-42
glycosyl iodides. We were hopeful that the synergistic effects
of the TMS groups and the reactive nature of the glycosyl
iodide would be sufficient to overcome the relative electron
deficiency typically associated with glucosyl donors, result-
ing in a similar reactivity profile to their galactosyl counter-
parts. As a first step in our quest to extend this protocol, we
initiated a competition study to quantify the relative reacti-
vity of per-O-TMS Gal:Glc iodide donors (Scheme 3). Equal
amounts of per-O-TMS-galactoside (12) and per-O-TMS-
glucoside (17) were mixed and TMSI was added to the
reaction to generate the respective glycosyl iodide donors
in situ.28 Once iodide formation was complete, as evidenced
by TLC, the solution was cannulated into a flask containing
TBAI, DIPEA, and (S)-(þ)-1,2-isopropylidine glycerol (18),
which was chosen as an acceptor because of its relevance to
target glucoside 24. After 36 h, the mixture was subjected to
acidic resin-mediated global deprotection followed by acet-
ylation of the resulting free hydroxyls, which allowed for
complete characterization of the products. In agreement
with our previous reports and based upon 1H and 13C
nuclear magnetic resonance (NMR) experiments, only the
R-linked glycosides were formed.20,21 The Gal:Glc product
ratio was found to be 1.6:1, which is much closer than is
usually observed with the aforementioned donors. In order
to verify the results of the competition experiment, both
reactions were conducted separately. In the individual ex-
periments, 19 was obtained in 90% yield and 20 in 79% yield
validating the competition experiment.
The key to extending our methodology to glucosyl systems
hinged upon the unique reactivity of per-O-trimethylsilyl
(32) Fan, G.-T.; Pan, Y.-s.; Lu, K.-C.; Cheng, Y.-P.; Lin, W.-C.; Lin, S.;
Lin, C.-H.; Wong, C.-H.; Fang, J.-M.; Lin, C.-C. Tetrahedron 2005, 61 (7),
1855–1862.
(33) Bulow, A.; Meyer, T.; Olszewski, T. K.; Bols, M. Eur. J. Org. Chem.
2004, 2, 323–329.
(34) Namchuk, M. N.; McCarter, J. D.; Becalski, A.; Andrews, T.;
Withers, S. G. J. Am. Chem. Soc. 2000, 122, 1270–1277.
(35) Zhang, Z.; Ollmann, I. R.; Ye, X. S.; Wischnat, R.; Baasov, T.;
Wong, C. H. J. Am. Chem. Soc. 1999, 121 (4), 734–753.
(36) Lahmann, M.; Oscarson, S. Can. J. Chem. 2002, 80, 889–893.
(37) Hadd, M. J.; Gervay-Hague, J. J. Org. Chem. 1997, 62, 6961–6967.
(38) Jensen, H. H.; Bols, M. Acc. Chem. Res. 2006, 39 (4), 259–265.
(39) Crich, D.; de la Mora, M.; Vinod, A. U. J. Org. Chem. 2003, 68 (21),
8142–8148.
Next, we focused on the synthesis of the glucosyl analo-
gues 22-24. We began with the glucosidation of ceramide
acceptor 8. As described previously, per-O-TMS glucosyl
iodide was transferred into a flask containing the acceptor,
(40) Edward, J. T. Chem. Ind. (London) 1955, 1102–1104.
(41) Khan, S. H.; O’Neill, R. A. Modern Methods in Carbohydrate
Synthesis; Harwood Academic Publishers: Chichester, U.K., 1996; p 558.
(42) Pedersen, C. M.; Nordstrom, L. U.; Bols, M. J. Am. Chem. Soc. 2007,
129 (29), 9222–9235.
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TABLE 3. Results of the Glucosylation Study
TBAI, and DIPEA. After 48 h, the solvent was removed and
the residue was reconstituted in methanol where it was
subjected to acidic resin mediated deprotection, affording
the desired final product after flash column chromatogra-
phy. Once again, the reaction progressed in a completely
stereoselective fashion, providing 22 in 52% yield from
starting material to final product (Table 3, entry 1). The
reaction between 17 and glyceride acceptor 10 proceeded in a
similar manner, resulting in the first reported synthesis of the
glucosyl analogue of the immunological antigen BbGL-II
(24) in 58% overall yield (Table 3, entry 3). The anomeric
configuration of the products was determined using 1H and
13C NMR, and complete assignment was possible using a
combination of 1D and 2D homonuclear and heteronuclear
NMR experiments. The glucose H-H (3J1-2) coupling con-
stants were all less than J = 4 Hz as expected for a 1,2-cis
linkage. The presence of only one anomeric carbon, between
96 and 100 ppm, is in agreement with the literature and
further indicated the desired R-linkage.
As was the case with our galactose study, initial attempts to
incorporate acceptor 9 at room temperature were unsuccessful
as none of the desired product (23) was recovered. The
decreased reactivity is attributed to micelle formation.43-45
Various reaction conditions were explored including changes
in the solvent, temperature and stoichiometry without success.
The only thing we have found thus far to promote this reaction
is microwave irradiation. Over the three steps, a 32% yield of 23
was obtained when the mixture was subjected to 200 W and
115 °C for 1.5 h in a sealed microwave vessel (Table 3, entry 2).
Although we do not completely understand the mode of action,
we have postulated that the microwave conditions increase
acceptor accessibility by disrupting ordered lipid arrangement.
anomerization of R-glycosyl iodides to the corresponding
β-linked reactive intermediates. Our first-generation protocol
involved the glycosidation of protected sphingosine or phyto-
sphingosine acceptors with per-O-benzyl galactosyl iodide.
While this process proceeded with complete R-selectivity, it
was only efficient when the acceptor was outfitted with electron-
releasing protecting groups. These findings led to the hypothesis
that electron-rich glycosyl donors may undergo reaction with
fully functionalized ceramide acceptors. Subsequent studies
revealed that per-O-trimethylsilyl ethers are more reactive
than the per-O-benzyl counterpart and are indeed suffi-
ciently reactive to glycosidate ceramides. An extra added
benefit of the trimethylsilyl protecting group is the ready
removal upon methanolysis providing a one-pot method for
the syntheses of bioactive R-linked galactosyl ceramides and
glycerides. Encouraged by these results, we extended the
methodology to encompass traditionally less reactive glucosyl
donors, but contrary to our expectation, per-O-TMS glucosyl
iodide was nearly as reactive as the galactosyl iodide. This
methodology provides selectivity for primary alcohols over
allylic or secondary alcohols, and no detectable amounts of
either the β-glycosides or over glycosylated products were
observed. Furthermore, this technology allows for the presence
of other functional groups such as esters, amides, and alkenes.
This three-step, one-pot protocol is economical and provides
rapid access to an important class of immunostimulatory mole-
cules including the first reported synthesis of the glucosyl
analogue of the bacterial antigen BbGL-II. We are currently
exploring the scope of this methodology for the synthesis of
biologically relevant oligosaccharides and glycoconjugates.
Experimental Section
1,2-Di-O-acetyl-3-O-(2,3,4,6-tetra-O-r-D-galactopyranosyl)-
(S)-glycerol (19). A solution of per-O-trimethylsilyl-D-galactopyra-
noside (12, 1.31 g, 2.42 mmol) in CH2Cl2 (3 mL) was cooled to 0 °C
followed by the addition of TMSI (532 mg, 2.66 mmol). The
reaction mixture was stirred for 20 min at 0 °C then quenched by
addition of anhydrous benzene (10 mL) followed by evaporation
under reduced pressure to afford the glucosyl iodide as a viscous
yellow oil. The iodide was next dissolved in CH2Cl2 (3.0 mL) and
kept under an argon atmosphere. In a separate flask, TBAI (1.34 g,
3.63 mmol), (S)-(þ)-1,2-isopropylidine glycerol (18, 107 mg,
0.81 mmol), and DIPEA (469 mg, 3.63 mmol) were dissolved in
CH2Cl2 (5.0 mL), and the mixture was stirred at rt under argon.
Conclusions
We have developed a robust platform for the synthesis of
biologically relevant glycolipids based on reacting transiently
protected glycosyl iodides with fully functionalized and un-
protected acceptors. TBAI was utilized to promote in situ
(43) Nakano, M.; Inoue, R.; Koda, M.; Baba, T.; Matsunaga, H.; Natori,
T.; Handa, T. Langmuir 2000, 16 (18), 7156–7161.
(44) Karttunen, M.; Haataja, M. P.; Saily, M.; Vattulainen, I.; Holopainen,
J. M. Langmuir 2009, 25 (8), 4595–600.
(45) Van Veldhoven, P. P.; Bishop, W. R.; Yurivich, D. A.; Bell, R. M.
Biochem. Mol. Biol. Int. 1995, 36 (1), 21–30.
4896 J. Org. Chem. Vol. 75, No. 15, 2010

Schombs et al.
JOCFeatured Article
The glycosyl iodide solution was transferred dropwise, via cannula,
to the acceptor flask. Once transferred, the reaction mixture was
stirred at rt for 36 h. Next, the solvent was removed, and the mixture
was reconstituted in MeOH (15 mL) and stirred with Dowex
50WX8-200 ion-exchange resin (1 g) at rt for 4 h. The resin was
then removed by filtration, and the solvent was removed in vacuo to
afford a brown oil. The crude mixture was acetylated using
standard conditions followed by purification using flash column
chromatography (CH2Cl2/acetone =97:3, Rf = 0.24) to afford 19
as a viscous oil (367 mg, 90%): 1H NMR (600 MHz, CDCl3) δ 1.98
(s, 3H), 2.04 (s, 3H), 2.060 (s, 3H), 2.064 (s, 3H), 2.09 (s, 3H), 2.13 (s,
3H), 3.61-3.64 (dd, J = 11.4, 5.4 Hz, 1H, H-1a), 3.81-3.83 (dd,
J= 11.4, 4.2 Hz, 1H, H-1b), 4.08 (d, J= 6.6 Hz, 2H, H-60a, H-60b),
4.13-4.16 (dd, J= 11.4, 5.4 Hz, 1H, H-3a), 4.20 (t, J= 6.6 Hz, 1H,
H-50), 4.30-4.33 (dd, J = 12.0, 4.2 Hz, 1H, H-3b), 5.08-5.10 (dd,
J = 10.2, 3.6 Hz, 1H, H-20), 5.12 (d, J = 3.6 Hz, 1H, H-10), 5.17-
5.20 (p, 1H, H-2), 5.30-5.32 (dd, J = 10.8, 3.0 Hz, 1H, H-30), 5.45
(appd, J= 3.0 Hz, 1H, H-40);13CNMR(150MHz, CDCl3) δ20.7,
20.78, 20.81, 21.1, 61.8, 62.3, 66.6, 66.7, 67.6, 68.0, 68.1, 70.2, 96.7,
170.1, 170.30, 170.33, 170.5, 170.59, 170.61; ESI-MS calcd for
C21H30O14 [M þ Na]þ = 529.15, found 529.30.
cannula, to the acceptor flask. Once transferred, the reaction
mixture was stirred at rt for 48 h. Next, the solvent was removed,
and the mixture was reconstituted in MeOH (10 mL) and stirred
with Dowex 50WX8-200 ion-exchange resin (0.5 g) at rt for 4 h.
The resin was then removed by filtration, and the solvent was
removed in vacuo to afford a brown oil which was purified using
flash column chromatography (CHCl3/MeOH = 95:5, Rf =
0.36) to afford 24 as a white foam (36 mg, 58%): [R]25D þ34.4
(c = 1.0, CH2Cl2); 1H NMR (600 MHz, CDCl3) δ 0.87 (t, J =
6.6 Hz, 7H), 1.25-1.29 (m, 54H), 1.60 (bs, 4H), 2.01 (q, J =
12.0, 6.0 Hz, 4H), 2.31 (dt, J = 7.2, 4.2 Hz, 4H), 3.49 (app d, J =
13.2 Hz, 1H, H-20), 3.56-3.60 (m, 3H, H-3a, H-40, H-50), 3.72
(bt, 1H, H-30), 3.82 (bs, 3H, H-3b, H-60a, H-60b), 4.15 (dd, J =
11.4, 6.0 Hz, 1H, H-1a), 4.38 (d, J = 9.6 Hz, 1H, H-1b), 4.86
(app s, 1H, H-10), 5.25 (bt, 1H, H-2), 5.34 (dt, J = 10.8, 6.0 Hz,
2H, HCdCH); 13C NMR (150 MHz, CDCl3) δ 14.2, 22.8, 22.9,
25.0, 27.35, 27.38, 29.27, 29.34, 29.41, 29.48, 29.49, 29.53, 29.69,
29.71, 29.83, 29.84, 29.88, 29.91, 29.92, 32.06, 32.08, 34.3, 34.4,
61.7, 62.7, 66.3, 69.9, 72.0, 72.2, 74.3, 99.3, 129.8, 130.2, 173.4,
173.9; ESI-HRMS calcd for C43H80O10 [M - H]- = 755.5668,
found 755.5665.
(2S,3R,4E)-1-O-(r-D-Glucopyranosyl)-2-(N-octadecanosyl-
amino)octadec-4-ene-1,3-diol (22). A solution of per-O-tri-
methylsilyl-D-glucopyranoside (17, 162 mg, 0.3 mmol) in CH2-
Cl2 (3 mL) was cooled to 0 °C followed by the addition of TMSI
(56 mg, 0.3 mmol). The reaction mixture was stirred for 45 min
at 0 °C and then quenched by addition of anhydrous benzene
(10 mL) followed by evaporation under reduced pressure to
afford the glucosyl iodide as a viscous yellow oil. The iodide was
next dissolved in CH2Cl2 (2.0 mL) and kept under an argon
atmosphere. In a separate flask, TBAI (222 mg, 0.6 mmol), 8 (56
mg, 0.1 mmol), and DIPEA (77 mg, 0.6 mmol) were dissolved in
CH2Cl2 (3.0 mL) and stirred at rt under argon. The glycosyl
iodide solution was transferred dropwise, via cannula, to the
acceptor flask. Once transferred, the reaction mixture was
stirred at rt for 48 h. Next, the solvent was removed, and the
mixture was reconstituted in MeOH (10 mL) and stirred with
Dowex 50WX8-200 ion-exchange resin (0.5 g) at rt for 4 h. The
resin was then removed by filtration, and the solvent was
removed in vacuo to afford a brown oil which was purified
using flash column chromatography (CH2Cl2/MeOH = 90:10,
Rf = 0.36). The product (22) was obtained as a white foam
(37 mg, 52%): [R]25D -31.6 (c = 1.0, CH2Cl2); 1H NMR (600
MHz, CDCl3/CD3OD) δ 0.69 (t, J = 7.2 Hz, 8H), 1.07-1.11, m,
66H), 1.16-1.18 (m, 4H), 1.39-1.41 (m, 2H), 1.84 (q, J = 7.2
Hz, 2H), 2.01 (t, J = 7.2 Hz, 2H), 3.17 (p, J = 1.4 Hz, 2H), 3.19
(t, J = 9.6 Hz, 1H, H-40), 3.26 (dd, J = 9.6, 4.2 Hz, 1H, H-20),
3.36 (dq, J = 4.2, 2.4 Hz, 1H, H-50), 3.47 (t, J = 9.0 Hz, 1H,
H-30), 3.53 (dd, J = 4.2, 1.2 Hz, 1H, H-1a), 3.54 (app t, J = 4.8,
3.0 Hz, 1H, H-60a), 3.58 (app t, J = 4.2, 3.0 Hz, 1H, H-60b), 3.61
(t, J = 3.0 Hz, 1H, H-1b), 3.77 (p, J = 3.6 Hz, 1H, H-2), 3.91
(app t, J = 6.6 Hz, 1H, H-3), 4.63 (d, J = 3.6 Hz, 1H, H-10), 5.26
(dd, J = 15.0, 7.2 Hz, 1H, HC=CH), 5.54 (dt, J = 15.0, 7.2 Hz,
1H, HCdCH); 13C NMR (150 MHz, CDCl3/CD3OD) δ 13.8,
22.5, 25.8, 29.10, 29.17, 29.23, 29.28, 29.39, 29.46, 29.47, 29.51,
31.7, 32.2, 36.3, 53.4, 61.3, 67.2, 70.0, 71.86, 71.88, 73.6,
99.3, 128.9, 134.1, 174.5; ESI-HRMS calcd for C42H81NO8
[M þ H]þ = 728.6035, found 728.6031.
1,2-Di-O-acetyl-3-O-(2,3,4,6-tetra-O-r-D-glucopyranosyl)-
(S)-glycerol (20).Asolutionofper-O-trimethylsilyl-D-glucopyrano-
side (17, 1.31 g, 2.42 mmol) in CH2Cl2 (3 mL) was cooled to 0 °C
followed by the addition of TMSI (532 mg, 2.66 mmol). The
reaction mixture was stirred for 20 min at 0 °C then quenched by
addition of anhydrous benzene (10 mL) followed by evaporation
under reduced pressure to afford the glucosyl iodide as a viscous
yellow oil. The iodide was next dissolved in CH2Cl2 (3.0 mL) and
kept under an argon atmosphere. In a separate flask, TBAI (1.34 g,
3.63 mmol), (S)-(þ)-1,2-isopropylidine glycerol (18, 107 mg, 0.81
mmol), and DIPEA (469 mg, 3.63 mmol) were dissolved in CH2Cl2
(5.0 mL), and the mixture was stirred at rt under argon. The
glycosyl iodide solution was transferred dropwise, via cannula, to
the acceptor flask. Once transferred, the reaction mixture was
stirred at rt for 36 h. Next, the solvent was removed, and the
mixture was reconstituted in MeOH (15 mL) and stirred with
Dowex 50WX8-200 ion-exchange resin (1 g) at rt for 4 h. The resin
was then removed by filtration, and the solvent was removed in
vacuo to afford a brown oil. The crude mixture was acetylated using
standard conditions followed by purification using flash column
chromatography (CH2Cl2/acetone = 97:3, Rf = 0.24) to afford 20
as a viscous oil (323 mg, 79%): 1H NMR (600 MHz, CDCl3) δ 1.99
(s, 3H), 2.02 (s, 3H), 2.05 (s, 3H), 2.06 (s, 3H), 2.079 (s, 3H), 2.083 (s,
3H), 3.62-3.65 (dd, J = 11.4, 5.2 Hz, 1H, H-1a), 3.80-3.83 (dd,
J = 10.8, 4.2 Hz, 1H, H-1b), 3.97-4.00 (dq, J = 10.2, 2.4 Hz, 1H,
H-50), 4.07-4.09 (dd, J = 12.6, 2.4 Hz, 1H, H-60a), 4.14-4.17 (dd,
J = 12.0, 6.0 Hz, 1H, H-3a), 4.23-4.26 (dd, J = 12.6, 4.8 Hz, 1H,
H-60b), 4.30-4.33 (dd, J = 12.0, 4.8 Hz, 1H, H-3b), 4.82-4.85 (dd,
J= 10.2, 4.2 Hz, 1H, H-20), 5.04 (app t, J= 10.2, 9.6 Hz, 1H, H-40),
5.09 (d, J = 4.2 Hz, 1H, H-10), 5.17-5.20 (p, 1H, H-2), 5.44 (app t,
J = 10.2, 9.6 Hz, 1H, H-30); 13C NMR (150 MHz, CDCl3) δ 20.70,
20.73, 20.8, 21.0, 61.9, 62.3, 66.7, 67.7, 68.6, 70.1, 70.2, 70.8, 96.3,
169.7, 170.2, 170.3, 170.4, 170.6, 170.7; ESI-MS calc for C21H30O14
[M þ Na]þ = 529.15, found 529.30.
1-O-Palmitoyl-2-O-oleoyl-3-O-r-D-glucopyranosyl-sn-glycer-
ol (Glc-BbGL-II, 24). A solution of per-O-trimethylsilyl-D-glu-
copyranoside (17, 136 mg, 0.25 mmol) in CH2Cl2 (3 mL) was
cooled to 0 °C followed by the addition of TMSI (56 mg, 0.28
mmol). The reaction was stirred for 45 min at 0 °C then
quenched by addition of anhydrous benzene (10 mL) followed
by evaporation under reduced pressure to afford the glucosyl
iodide as a viscous yellow oil. The iodide was next dissolved in
CH2Cl2 (2.0 mL) and kept under under an argon atmosphere. In
a separate flask, TBAI (186 mg, 0.50 mmol), 10 (50 mg, 0.08
mmol), and DIPEA (65 mg, 0.50 mmol) were dissolved in
CH2Cl2 (3.0 mL), and the mixture was stirred at rt under argon.
The glycosyl iodide solution was transferred dropwise, via
(2S,3S,4R)-1-O-(r-D-Glucopyranosyl)-2-(N-octadecanosyl-
amino)octadecane-1,3,4-triol (23). The microwave-assisted one-
pot glycosylation began with the cooling of a solution of per-O-
trimethylsilyl-D-glucopyranoside (17, 82 mg, 0.15 mmol) in
CH2Cl2 (3 mL) to 0 °C followed by the addition of TMSI
(30 mg, 0.15 mmol). The reaction mixture was stirred for 45
min at 0 °C then quenched by addition of anhydrous benzene
(10 mL) followed by evaporation under reduced pressure to
afford the glucosyl iodide as a viscous yellow oil. The iodide was
next dissolved in CH2Cl2 (2.0 mL) and kept under an argon
J. Org. Chem. Vol. 75, No. 15, 2010 4897

Schombs et al.
JOCFeatured Article
atmosphere. Next, TBAI (167 mg, 0.45 mmol), 9 (30 mg, 0.05
mmol), and DIPEA (60 mg, 0.45 mmol) were added to a 10 mL
microwave vessel containing CH2Cl2 (3.0 mL) and stirred at rt
under argon. The glycosyl iodide solution was transferred, via
cannula, to the sealed microwave reaction vessel, and the vessel
was placed into a microwave reactor. The reaction was con-
ducted at 115 °C for 90 min at 200 W. The reaction mixture was
transferred to a round-bottomed flask, and the solvent was
removed affording a brown viscous oil which was reconstituted
in MeOH (10 mL) and stirred with Dowex 50WX8-200 ion-
exchange resin (0.5 g) at rt for 4 h. The resin was then removed
by filtration, and the solvent was removed in vacuo to afford a
brown oil, which was purified using flash column chromato-
graphy as described above to afford 23 as a white foam (12 mg,
32%): [R]25D -36.8 (c = 1.0, CH2Cl2/MeOH, 93:7); 1H NMR
(600 MHz, C5D5N) δ 0.98 (t, J = 6.6 Hz, 6H), 1.33-1.39 (m,
50H), 1.42-1.53 (m, 6H), 1.75 (m, 1H), 1.91 (m, 2H), 2.37 (m,
1H), 2.52 (t, J = 7.2 Hz, 2H), 4.24 (dd, J = 9.6, 3.6 Hz, 1H,
H-20), 4.32 (t, J = 9.6 Hz, 1H, H-40), 4.40-4.50 (m, 4H, H-1a,
H-60a, H-4, H-3), 4.52-4.54 (m, 1H, H-50), 4.58 (dd, J = 11.4,
1.8 Hz, 1H, H-60b), 4.67 (t, J = 9.0 Hz, 1H, H-30), 4.83
(dd, J = 10.8, 6.0 Hz, 1H, H-1b), 5.37 (m, 1H, H-2), 5.69 (d,
J = 4.2 Hz, 1H, H-10), 8.47 (d, J = 8.4 Hz, 1H, N-H); 13C NMR
(150 MHz, C5D5N) δ14.8, 23.4, 26.85, 26.94, 30.1, 30.2, 30.3,
30.35, 30.41, 30.47, 30.49, 30.50, 30.51, 30.6, 30.9, 32.6, 34.9,
37.3, 51.8, 63.2, 68.6, 72.4, 72.8, 74.0, 75.1, 75.9, 77.2, 101.5,
173.7; ESI-HRMS calc for C42H83NO9 [M þ H]þ = 746.6141,
found 746.6143.
Acknowledgment. This work is supported by the National
Institutes of Health, NIH Grant No. R01GM090262. NSF
CRIF program (CHE-9808183), NSF Grant No. OSTI 97-
24412, and NIH Grant No. RR11973 provided funding for
the NMR spectrometers used on this project. HRMS sam-
ples were analyzed by Dr. William Jewell at the UC Davis,
Campus Mass Spectrometry Facilities.
Supporting Information Available: 1H, 13C, 2D COSY, and
HETCOR NMR spectral data. This material is available free of
charge via the Internet at http://pubs.acs.org.
4898 J. Org. Chem. Vol. 75, No. 15, 2010