Modulation of innate immune responses with synthetic lipid a derivatives

Article abstract of DOI:10.1021/ja068922a

The lipid A moiety of lipopolysaccharides (LPS) initiates innate immune responses by interacting with Toll-like receptor 4 (TLR4), which results in the production of a wide range of cytokines. Derivatives of lipid A show potential for use as immuno-modula

Full text of DOI:10.1021/ja068922a

Published on Web 03/29/2007
Modulation of Innate Immune Responses with Synthetic Lipid
A Derivatives
Yanghui Zhang, Jidnyasa Gaekwad, Margreet A. Wolfert, and Geert-Jan Boons*
Contribution from the Complex Carbohydrate Research Center, The UniVersity of Georgia,
315 RiVerbend Road, Athens, Georgia 30602
Received December 13, 2006; E-mail: gjboons@ccrc.uga.edu
Abstract: The lipid A moiety of lipopolysaccharides (LPS) initiates innate immune responses by interacting
with Toll-like receptor 4 (TLR4), which results in the production of a wide range of cytokines. Derivatives
of lipid A show potential for use as immuno-modulators for the treatment of a wide range of diseases and
as adjuvants for vaccinations. Development to these ends requires a detailed knowledge of patterns of
cytokines induced by a wide range of derivatives. This information is difficult to obtain by using isolated
compounds due to structural heterogeneity and possible contaminations with other inflammatory compo-
nents. To address this problem, we have developed a synthetic approach that provides easy access to a
wide range of lipid A’s by employing a common disaccharide building block functionalized with a versatile
set of protecting groups. The strategy was employed for the preparation of lipid A’s derived from E. coli
and S. typhimurium. Mouse macrophages were exposed to the synthetic compounds and E. coli 055:B5
LPS, and the resulting supernatants were examined for tumor necrosis factor alpha (TNF-R), interferon
beta (IFN-â), interleukin 6 (IL-6), interferon-inducible protein 10 (IP-10), RANTES, and IL-1â. It was found
that for each compound, the potencies (EC50 values) for the various cytokines differed by as much as
100-fold. These differences did not follow a bias toward a MyD88- or TRIF-dependent response. Instead,
it was established that the observed differences in potencies of secreted TNF-R and IL-1â were due to
differences in the processing of respective pro-proteins. Examination of the efficacies (maximum responses)
of the various cytokines showed that each synthetic compound and E. coli 055:B5 LPS induced similar
efficacies for the production of IFN-â and IP-10. However, lipid A’s 1-4 gave lower efficacies for the
production of RANTES and IL-6 as compared to LPS. Collectively, the presented results demonstrate that
cytokine secretion induced by LPS and lipid A is complex, which can be exploited for the development of
immuno-modulating therapies.
Introduction
diseases including asthma, infections, and cancer. An important
concern of such therapies is, however, that over-activation of
The innate immune system is an evolutionarily ancient system
designed to detect the presence of microbial invaders and
activate protective reactions.1 It responds rapidly to compounds
that are integral parts of pathogens that are perceived as danger
signals by the host. Recognition of these molecular patterns is
mediated by sets of highly conserved receptors,2 whose activa-
tion results in acute inflammatory responses. These responses
include the production of a diverse set of cytokines and
chemokines, direct local attack against the invading pathogen,
and initiation of responses that activate and regulate the adaptive
component of the immune system.3-8
innate immunity may lead to the clinical symptoms of septic
shock.9,10 Thus, an important issue for the design of safe immune
modulators is a detailed knowledge of structure-activity
relationships to harness beneficial effects without causing
toxicity.
Lipopolysaccharides (LPS) are structural components of the
outer membrane of Gram-negative bacteria and offer great
promise for the development of immuno-modulators. LPS
consists of a hydrophobic domain known as lipid A, a non-
repeating core oligosaccharide, and a distal polysaccharide (or
O-antigen).11,12 The lipid A moiety of E. coli consists of a
hexaacylated bis-1,4′-phosphorylated glucosamine disaccharide,
which has (R)-3-hydroxymyristyl residues at C-2, C-2′, C-3,
and C-3′ (Figure 1). Furthermore, both of the primary (3)-
hydroxyacyl chains in the distal glucosamine moiety are
esterified with lauric and myristic acids, and the primary
Evidence is emerging that innate immune responses can be
exploited for therapeutic purposes such as the development of
adjuvants for vaccines and the treatment of a wide range of
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Modulation of Innate Immune Responses
A R T I C L E S
These structural differences probably account for the highly
variable in-vivo and in-vitro host responses to LPS.11,12,16-18
There is also some indication that structurally different lipid
A’s may differentially induce proinflammatory responses.19-22
For example, in one study, LPS from E. coli O55:B5 induced
the production of mediators (TNF-R, IL-1â, MCP-1, and
macrophage inflammatory protein 3alpha (MIP-3R)) arising
from the MyD88-dependent pathway, but caused less production
of mediators (IFN-â, nitric oxide, and IP-10) arising from the
TRIF-dependent pathway. In contrast, LPS from S. typhimurium
invoked strong production of mediators associated with the
TRIF-dependent pathway, but caused only minimal production
of TNF-R, IL-1â, MCP-1, and MIP-3R. Heterogeneity in the
structure of lipid A within a particular bacterial strain and
possible contamination with other inflammatory components of
the bacterial cell-wall complicate the use of either LPS or lipid
A isolated from bacteria to dissect the molecular mechanisms
responsible for the biological responses to specific lipid A
molecules.
Fortunately, homogeneous lipid A derivatives can be obtained
by chemical synthesis.17 The results of studies with small
numbers of synthetic analogues have shown that the number of
acyl chains and phosphate substitution are important for cytokine
production. These studies have not examined whether particular
structural modifications have different effects on the production
of particular cytokines and chemokines. The development of
safe immuno-modulators requires, however, such knowledge
because different mediators induce different biological effects.
To address this important issue, we have developed an efficient
synthetic approach whereby an advanced synthetic disaccharide
can easily be converted into lipid A analogues that differ in
phosphorylation and acylation pattern. This strategy has been
employed for the preparation of lipid A’s derived from E. coli
and S. typhimurium. Mouse macrophages were exposed to the
synthetic compounds and E. coli 055:B5 LPS, and the resulting
supernatants were examined for mouse TNF-R, IFN-â, IL-6,
IP-10, RANTES, and IL-1â. It has been found that particular
modifications had different effects on the potencies and effica-
cies of induction of the various cytokines. However, no bias
toward a MyD88- or TRIF-dependent response was observed.
Thus, for the first time, it has been shown that lipid A derivatives
can modulate innate immune responses in a complex manner.
Figure 1. Chemical structures of target lipid A’s 1-5.
hydroxyl at the C-6 position is linked to the polysaccharide
through a di-KDO carbohydrate moiety. It has been demon-
strated unequivocally that lipid A is the inflammation-inducing
moiety of LPS.13,14
Lipid A triggers innate immune responses through Toll-like
receptor 4 (TLR4), a member of the TLR family that participates
in pathogen recognition. Immediately distal to TLR4 activation
are two intracellular cascades that regulate signal transduction
processes, gene expression, and production of (pro)inflammatory
mediators.7 One of these cascades requires a specific intracellular
adaptor protein called MyD88, while the other cascade utilizes
the TRIF adaptor protein. The MyD88-dependent pathway leads
to up-regulation of cytokines and chemokines such as tumor
necrosis factor alpha (TNF-R), interleukin 1beta (IL-1â), IL-6,
and monocyte chemoattractant protein 1 (MCP-1), whereas the
TRIF-dependent pathway leads to the production of interferon-
beta (IFN-â), which in turn activates the STAT-1 pathway,
resulting in the production of mediators such as interferon-
inducible protein 10 (IP-10) and nitric oxide.15
Results and Discussion
Chemical Synthesis of Lipid A’s. To determine whether the
structure of lipid A can modulate innate immunological
responses, we have synthesized derivatives 1-5 (Figure 1) by
a highly convergent approach. Compound 1 is a prototypical
lipid A from E. coli and is hexa-substituted in an asymmetrical
fashion. Compound 2 is derived from compound 1, but several
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86.
Recent structural studies have demonstrated that the carbo-
hydrate backbone, degree of phosphorylation, and fatty acid
acylation patterns vary considerably among bacterial species.
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Eur. J. Immunol. 2001, 31, 2529-2538.
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A R T I C L E S
Zhang et al.
Scheme 1 a
of its acyl groups have been shortened. Compounds 3, 4, and 5
are hepta-acylated lipid A’s derived from S. typhimurium LPS
that differ in lipid length and phosphorylation pattern.23-26
Previously reported approaches for lipid A synthesis em-
ployed strategies whereby monosaccharides were functionalized
with lipids and phosphates, which were then used as glycosyl
donors and acceptors for disaccharide synthesis, which after
anomeric phosphorylation and deprotection provided target
compounds.17,27,28 Although this approach is attractive for one-
compound-at-a-time synthesis, detailed structure-activity re-
lationship studies require a synthetic approach that offers in a
straightforward manner a panel of lipid A’s. The strategy that
we have developed employs the advanced disaccharide inter-
mediate 13, which can selectively be modified with any lipid
at C-2, C-3, C-2′, and C-3′. A key feature of 13 is the use of
the allyloxycarbonate (Alloc), the anomeric tert-butyldimethyl
silyl ether (TBDMS), and the (9-fluorenylmethoxycarbamate
(Fmoc) and azido as a set of functional groups that in a
sequential manner can be deprotected or unmasked to allow
selective lipid modification at each position. It was envisaged
that disaccharide 13 could easily be prepared by a regio- and
stereoselective glycosylation of trichloroacetimidate 12 with
glycosyl acceptor 8. In this glycosylation, the higher glycosyl
accepting reactivity of the primary C-6 hydroxyl of 8 as
compared to its secondary C-3 hydroxyl, and the ability of the
Fmoc carbamate of 12 to control the â-anomeric configuration
by neighboring group participation,29 were exploited.
Glycosyl acceptor 8 and donor 12 could easily be prepared
from common intermediate 6 (Scheme 1). Thus, a regioselective
reductive opening of the benzylidene acetal of 6 using borane-
THF complex in the presence of the bulky Lewis acid Bu2-
BOTf gave glycosyl acceptor 8 as the only regio-isomer.
Alternatively, the C-3 hydroxyl of 630 could be protected by an
Alloc group by treatment with Alloc chloride in the presence
of N,N,N′,N′-tetramethylethylenediamine (TMEDA) in DCM to
give 7 in a yield of 88%. Regioselective reductive opening of
the benzylidene acetal of 7 using NaCNBH3 and HCl in diethyl
ether gave 9,31 which after phosphitylation with N,N-diethyl-
1,5-dihydro-2,3,4,-benzodioxaphosphepin-3-amine in the pres-
ence of 1H-tetrazole followed by in-situ oxidation with m-chlo-
roperoxybenzoic acid (mCPBA)32 provided the phosphotriester
10. Next, the azido function of 10 was reduced using activated
Zn in a mixture of acetic acid and DCM to give an amine, which
was immediately protected as an Fmoc carbamate by reaction
with 9-fluorenylmethyl chloroformate (FmocCl) in the presence
a Reagents and conditions: (a) BH3‚THF, Bu2OTf, THF; (b) AllocCl,
TMEDA, DCM; (c) NaCNBH3, 2 M HCl in diethyl ether, DCM; (d) N,N-
diethyl-1,5-dihyro-2,4,3-benzodioxaphosphepin-3-amine, tetrazole, DCM;
then mCPBA, -20 °C; (e) Zn/HOAc, DCM; then FmocCl, DIPEA, DCM;
(f) HF/pyridine, THF; then CNCCl3, NaH, THF; (g) TMSOTf, DCM,
-50 °C.
of N,N-diisopropylethylamine (DIPEA) in DCM to give fully
protected 11. Removal of the anomeric TBDMS ether of 11 by
treatment with HF in pyridine followed by conversion of the
resulting anomeric hydroxyl into a trichloroacetimidate by
reaction with trichloroacetonitrile in the presence of a catalytic
amount of NaH33 afforded glycosyl donor 12 in an overall yield
of 90%. A trimethylsilyl trifluoromethanesulfonate (TMSOTf)-
mediated glycosylation of the trichloroacetimidate 12 with
glycosyl acceptor 8 in dichloromethane gave the selectively
protected disaccharide 13 in a yield of 79% as only the
â-anomer. The alternative regioisomer resulting from glycosy-
lation of the C-3 hydroxyl or the trisaccharide arising from
glycosylation of both hydroxyls of 8 was not observed. The
acyloxy- and acyloxyacyl lipids 14-20 were prepared by a
reported procedure.34
(23) Kawasaki, K.; Ernst, R. K.; Miller, S. I. J. Biol. Chem. 2004, 279, 20044-
20048.
Having the advanced disaccharide 13 and lipids 14-20 at
hand, attention focused on the selective acylation of relevant
hydroxyls and amines (Scheme 2). Thus, removal of the Fmoc
protecting group of 13 using 1,8-diazabicyclo[5.4.0]undec-7-
ene (DBU) in DCM followed by acylation of the resulting amino
group with (R)-3-dodecanoyl-tetradecanoic acid (18) using 1,3-
dicyclohexylcarbodiimide (DCC) as the activation reagent gave
compound 21. Next, the C-3 hydroxyl of 21 was acylated with
(R)-3-benzyloxy-tetradecanoic acid (15) using DCC and 4-dim-
ethylaminopyridine (DMAP)35 to give 22 in a yield of 86%.
(24) Wick, M. J. Curr. Opin. Microbiol. 2004, 7, 51-57.
(25) Guo, L.; Lim, K. B.; Gunn, J. S.; Bainbridge, B.; Darveau, R. P.; Hackett,
M.; Miller, S. I. Science 1997, 276, 250-253.
(26) Kanegasaki, S.; Tanamoto, K.; Yasuda, T.; Homma, J. Y.; Matsuura, M.;
Nakatsuka, M.; Kumazawa, Y.; Yamamoto, A.; Shiba, T.; Kusumoto, S.;
Imoto, M.; Yoshimura, H.; Shimamoto, T. J. Biochem. 1986, 99, 1203-
1210.
(27) Kusumoto, S. Molecular Biochemistry and Cellular Biology; CRC Press:
Boca Raton, FL, 1992; Vol. 1, Bacterial Endotoxin Lipopolysaccharides;
Chapter 9, Chemical Synthesis of Lipid A, pp 81-105.
(28) Kusumoto, S.; Fukase, K.; Oikawa, M. In Endotoxin in Health and Disease;
Brade, H., Ed.; Marcel Dekker: New York, 1999; pp 243-256.
(29) Fukase, K.; Aoki, Y.; Kinoshita, I.; Suda, Y.; Kurosawa, M.; Zahringer,
U.; Rietschel, E. T.; Kusumoto, S. Tetrahedron Lett. 1995, 36, 8645-
8648.
(30) Lee, H. S.; Wolfert, M. A.; Zhang, Y. H.; Boons, G. J. ChemBioChem
2006, 7, 140-148.
(33) Patil, V. J. Tetrahedron Lett. 1996, 37, 1481-1484.
(34) Keegan, D. S.; Hagen, S. R.; Johnson, D. A. Tetrahedron: Asymmetry
1996, 7, 3559-3564.
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G. J. J. Am. Chem. Soc. 2003, 125, 6103-6112.
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31, 255-256.
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A R T I C L E S
Figure 2. Chemical structures of side products 25-27.
Scheme 2 a
a Reagents and conditions: (a) DBU, DCM; then (R)-3-dodecanoyloxy-tetradecanoic acid 18, DCC, DCM; (b) (R)-3-benzyloxy-tetradecanoic acid 15,
DCC, DMAP, DCM; (c) Pd(PPh3)4, HCO2H, n-buNH2, THF; (d) (R)-3-(p-methoxy)benzyloxy-tetradecanoic acid 17, DCC, DMAP, DCM; (e) DDQ, H2O,
DCM; (f) myristoyl chloride, pyridine, DMAP, DCM; (g) Zn/HOAc, DCM; then RCOOH, DCC, DCM; (h) HF/pyridine; (i) tetrabenzyl diphosphate, LiN(TMS)2,
THF, -78 °C; (j) H2 (50 psi), Pd-black, THF.
The latter two reactions exploited the finding that an amine can
selectively be acylated in the presence of a free hydroxyl using
DCC as the activator. The addition of DMAP provides, however,
a more reactive reagent and can acylate a less nucleophilic
hydroxyl. The removal of the Alloc protecting group of 22 could
easily be accomplished by treatment with Pd(PPh3)4;36 however,
the acylation of the resulting hydroxyl of 23 with (R)-
tetradecanoyltetradecanoic acid (19) using standard conditions
did not, unexpectedly, lead to the formation of 24. Instead,
compounds 25, 26, and 27 were identified (Figure 2). The
formation of these compounds can be rationalized by migration
of the phosphotriester to the C-3′ position and elimination of
the acyloxy chain of (R)-3-tetradecanoyl-tetradecanoic acid to
give tetradecanoic acid and tetradec-2-enoic acid. It is proposed
that compound 25 arises from acylation of the starting material
with tetradecanoic acid, whereas compounds 26 and 27 result
from phosphotriester migration followed by acylation with
tetradecanoic acid or 19, respectively. To circumvent these side
reactions, the (R)-3-tetadecanoyl-tetradecanoic ester was intro-
duced by a three-step procedure using (R)-3-(p-methoxy)-
benzyloxy-tetradecanoic acid (17) as the initial acylation reagent.
It was reasoned that the (p-methoxy)benzyl (PMB) ether of 17
would be less susceptible to elimination, and hence the formation
of the elimination product should be suppressed. Furthermore,
(36) Tsukamoto, Y.; Nakagawa, H.; Kajino, H.; Sato, K.; Tanaka, K.; Yanai,
T. Biosci., Biotechnol., Biochem. 1997, 61, 1650-1657.
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A R T I C L E S
Scheme 3 a
Zhang et al.
a Reagents and conditions: (a) (R)-3-benzyloxy-dodecanoic acid 14, DCC, DMAP, DCM; (b) Zn/HOAc, DCM; then RCOOH, DCC, DCM; (c) Pd(PPh3)4,
HCO2H, n-BuNH2, THF; then (R)-3-(p-methoxy)benzyloxy-dodecanoic acid 16, DCC, DMAP, DCM; (d) DDQ, H2O, DCM; (e) lauroyl chloride, pyridine,
DMAP, DCM; (f) HF/pyridine; then tetrabenzyl diphosphate, LiN(TMS)2, THF, -78 °C; (g) H2 (50 psi), Pd-black, THF; (h) HF/pyridine; then H2 (50 psi),
Pd-black, THF.
the higher reactivity of ether protected 17 may also suppress
phosphate migration. After installment of the (R)-3-(p-methoxy)-
benzyloxytetradecanoic ester and selective removal of the PMB
ether, the â-hydroxy functionality can be acylated to provide
the required compound. Thus, treatment of 23 with 17 in the
presence of DCC and DMAP to give 28 followed by removal
of the PMB ether using 2,3-dichloro-5,6-dicyano-1,4-benzo-
quinone (DDQ) in a mixture of DCM and water in the dark,
and acylation of the resulting â-hydroxyl of 29 with myristoyl
chloride in the presence of pyridine and DMAP, afforded 24.
Although the three-step procedure to convert 23 into 24 is more
laborious than direct acylation with an acyloxyacyl acid, it offers
an opportunity to devise a range of compounds that differ in
â-hydroxy acylation at the C-3′ position. Next, the azido
function of 24 was reduced with activated Zn in a mixture of
acetic acid and DCM, and the amine of the resulting compound
was reacted with 15 or 20 in the presence of DCC to give 30
and 31, respectively. Next, attention was focused on the
introduction of the anomeric phosphate and removal of the
permanent protecting groups. Thus, the anomeric TBS ether of
30 and 31 was removed by treatment with HF in pyridine,
conditions that do not affect the acyl and acyloxyacyl esters
and the phosphodiester, to give 32 and 33, respectively. These
derivatives were phosphorylated using tetrabenzyl diphosphate
in the presence of lithium bis(trimethyl)silylamide in THF at
-78 °C to give,37 after purification using Iatro beads, 34 and
35 as only R-anomers. Global deprotection of 34 and 35 by
catalytic hydrogenolysis over Pd-black gave the requisite lipid
A’s 1 and 3, respectively.
followed by reduction of the azido moiety and modification of
the corresponding amine. To study the orthogonality of the Alloc
and azido function, compounds 2, 4, and 5 were prepared by
an alternative sequence of reactions involving reduction of the
azido function and modification of the C-2 amine before
deprotection of Alloc group and acylation of the resulting C-3′
hydroxyl (Scheme 3). Thus, the C-3 hydroxyl of 21 was acylated
with 14 using DCC and DMAP to give 36 in a yield of 91%.
Next, the azido moiety of 36 was reduced with activated Zn in
a mixture of acetic acid and DCM without affecting the Alloc
group to provide an intermediate amine, which was immediately
acylated with (R)-3-benzyloxy-dodecanoic acid (14) or (R)-3-
tetradecanoyl-hexadecanoic acid (20), using DCC as the activat-
ing system, to give 37 and 38, respectively. Next, Pd(0)-
mediated removal of the Alloc group of 37 and 38, followed
by acylation of the resulting hydroxyl with (R)-3-(p-methoxy)-
benzyloxy-dodecanoic acid (16) in the presence of DCC/DMAP,
gave 39 and 40, which after treatment with DDQ to remove
the PMB ether were acylated with lauroyl chloride to give fully
acylated 43 and 44, respectively, in a good overall yield. Finally,
cleavage of the anomeric TBS ether of 43 and 44 was performed
under standard conditions to give intermediate lactols, which
were phosphorylated using tetrabenzyl diphosphate in the
presence of lithium bis(trimethyl)silylamide to give 45 and 46.
Deprotection of the latter compounds by catalytic hydrogenolysis
over Pd-black gave lipid A derivatives 2 and 4. Monophosphoryl
derivative 5 could easily be obtained by standard deprotection
of the intermediate lactol.
Biological Evaluation of Lipid A’s and LPS. Based on the
results of recent studies,5,7 it is clear that LPS-induced cellular
activation through TLR4 is complex as many signaling elements
are involved. However, it appears that there are two distinct
initiation points in the signaling process, one being a specific
Lipid A’s 1 and 3 were prepared by first removal of the Alloc
protecting group of 22 and acylation of the resulting hydroxyl
(37) Oikawa, M.; Shintaku, T.; Sekljic, H.; Fukase, K.; Kusumoto, S. Bull. Chem.
Soc. Jpn. 1999, 72, 1857-1867.
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Modulation of Innate Immune Responses
A R T I C L E S
Figure 3. TNF-R and IFN-â production by murine macrophages after stimulation with LPS and lipid A derivatives. Murine RAW γNO(-) cells were
incubated for 5.5 h with increasing concentrations of E. coli LPS or lipid A derivatives 1-5 as indicated. TNF-R (A) and IFN-â (B) in cell supernatants were
measured using ELISAs.
Table 1. EC50 Valuesa (nM) of E. coli LPS and Lipid A Derivatives
1, 2, and 4
intracellular adaptor protein called MyD88 and the other an
adaptor protein called TRIF, which operates independently of
MyD88. It is well-established that TNF-R secretion is a
prototypical measure for activation of the MyD88-dependent
pathway, whereas secretion of IFN-â is commonly used as an
indicator of TRIF-dependent cellular activation. There are some
indications that structurally different lipid A’s can differentially
utilize signal transduction pathways leading to complex patterns
of proinflammatory responses. Heterogeneity in lipid A of
particular bacterial strains as well as possible contamination with
other inflammatory components of the bacterial cell-wall
complicates the use of either LPS or lipid A isolated from
bacteria to dissect the molecular mechanisms responsible for
the biological responses to specific lipid A’s. To address these
issues, we have examined the well-defined compounds 1-5 and
E. coli LPS for the ability to initiate production of a wide range
of cytokines, including TNF-R, INF-â, IL-6, IP-10, RANTES,
and IL-1â. It was anticipated that analysis of potencies and
efficacies of the mediators would establish whether structural
differences in lipid A can modulate inflammatory responses.
Mouse macrophages (RAW 264.7 γNO(-) cells) were
exposed over a wide range of concentrations to compounds 1-5
and E. coli 055:B5 LPS. After 5.5 h, the supernatants were
harvested and examined for mouse TNF-R and IFN-â using a
commercial and in-house developed capture ELISA assay,
respectively. Potencies (EC50, concentration producing 50%
activity) and efficacies (maximal level of production) were
determined by fitting the dose-response curves to a logistic
equation using PRISM software. As can be seen in Figure 3,
the lipid A’s and E. coli 055:B5 displayed large differences in
potencies. Thus, lipid A’s 1, 2, and 4 and E. coli 055:B5 LPS
yielded clear dose response curves. S. typhimurium lipid A 3
gave only a partial response at the highest concentration tested,
whereas monophosphate 5 was inactive. Furthermore, the EC50
values for E. coli 055:B5 LPS were significantly smaller than
those of E. coli lipid 1 and 2 (Table 1). Probably, the higher
potency of LPS is due to its di-KDO moiety, which is attached
to the C-6′ position of lipid A. In this respect, recent studies38
have shown that meningococcal lipid A expressed by a strain
defect in KDO biosynthesis has significantly reduced bioactivity
as compared to KDO containing Meningococcal lipooligosac-
charides. It has also been shown that removal of the KDO
moieties by mild acidic treatment reduces cellular responses.35
E. coli LPS
lipid A 1
lipid A 2
lipid A 4
TNF-R
IFN-â
IL-6
0.016
(0.012-0.022)
0.038
(0.025-0.056)
0.063
21
(16-28)
124
(105-147)
157
4.1
(2.5-6.7)
16
(12-23)
14
60
(44-81)
234
(180-306)
462
(0.044-0.091)
0.030
(91-271)
44
(6-33)
12
(383-559)
156
IP-10
(0.019-0.046)
0.116
(37-52)
238
(201-281)
674
(10-16)
43
(36-51)
30
(120-204)
570
(478-681)
348
RANTES
IL-1â
(0.103-0.131)
1.74b
(1.59-1.91)
0.014
(0.008-0.025)
0.004
(622-728)
39
(32-47)
38
(26-35)
3.6
(1.4-9.4)
14
(284-428)
63
(48-84)
53
pro Il-1â
NF-κB
(0.003-0.005)
(30-48)
(9-20)
(42-67)
a Values of EC50 are reported as best-fit values and as minimum-
maximum range (best-fit value ( std error). b Plateau not reached; EC50
value is best-fit value according to Prism.
Further examination of the data revealed that the hexa-
acylated E. coli lipid A (1) is significantly more potent than
the hepta-acylated S. typhimurium lipid A (3). Shortening of
lipids, such as in compounds 2 and 4, resulted in higher
potencies (smaller EC50 values). In the case of the E. coli lipid
A’s (1 vs 2), the differences in EC50 values were relatively small,
whereas for the S. typhimurium lipid A’s (3 vs 4) an approximate
3 orders of magnitude of increase in potencies was observed.
Finally, a comparison of the EC50 values of TNF-R and IFN-â
for each compound indicated that the values of TNF-R are
slightly smaller than those of IFN-â (2-6-fold), indicating a
somewhat higher potency for TNF-R production. Previously, it
was observed that mice exposed to S. typhimurium LPS
provoked mainly cytokines associated with the TRIF-dependent
pathway.19 Interestingly, we have not observed such a bias. It
may be possible that such a bias may be due to contaminants,
or, alternatively, it may be due to lipid A derivatives that have
a different acylation pattern.
Having established the EC50 values of TNF-R and IFN-â
secretion by compounds 1-5 and E. coli 055:B5 LPS, attention
was focused on IL-6, IP-10, RANTES, and IL-1â responses.
Thus, the previously harvested supernatants were analyzed for
these cytokines using capture ELISA assays (Figure 4, Table
1). For IL-6, IP-10, and RANTES, a short incubation time of
5.5 h was sufficient for detection. To achieve significant IL-1â
secretion, the incubation had to be extended to 24 h. However,
(38) Zughaier, S. M.; Tzeng, Y. L.; Zimmer, S. M.; Datta, A.; Carlson, R. W.;
Stephens, D. S. Infect. Immun. 2004, 72, 371-380.
9
J. AM. CHEM. SOC. VOL. 129, NO. 16, 2007 5205

A R T I C L E S
Zhang et al.
Figure 4. Cytokine production by murine macrophages after stimulation with LPS and lipid A derivatives. Murine RAW γNO(-) cells were exposed to
increasing concentrations of E. coli LPS or lipid A derivatives 1-5 as indicated. Cytokine production was measured in supernatants after 5.5 h incubation
for IL-6 (A), IP-10 (B), and RANTES (C) or 24 h for IL-1â (D). The cell lysates were assayed for the presence of pro-IL-1â (E) after 5.5 h incubation.
analyzing cell lysates of the activated cells showed that after
5.5 h a significant quantity of IL-1â was present intracellularly.
IL-1â is expressed as a pro-protein (pro-IL-1â), which is cleaved
by caspase-1 into its active form (IL-1â), which is then secreted.
Indeed, analyzing IL-1â of the cell lysates by Western blotting
confirmed that it was present as a pro-protein (data not shown).
TNF-R is also produced as a pro-protein, which is proteolitically
cleaved by tumor necrosis factor-R converting enzyme (TA-
CE).39,40 Interestingly, after 5.5 h, no TNF-R could be detected
in the cell lysates, which indicates that proteolitic processing
and secretion is not the rate-limiting step. Furthermore, for each
of the synthetic compounds and LPS, EC50 values of secreted
TNF-R and intracellular pro-IL-1â were very similar. However,
EC50 values for secreted mature IL-1â were larger by as much
as 100-fold.
precursor protein and must be activated by stimulation with LPS
or other bacterial components.41,42 Although the mechanism of
LPS-mediated activation of caspase-1 is not well understood,
it has been shown that it is independent of TLR4 associated
adaptor proteins MyD88 and TRIF. Instead, experiments with
macrophages obtained from ACS-/- mice have implicated this
adaptor protein in LPS-mediated activation of caspase-1. Thus,
it appears that activation of caspase-1 is dependent on ACS,
whereas the expression of pro-IL-1â and pro-TNF-R is depend-
ent on MyD88. Furthermore, it has been suggested that ACS-
promoted caspase-1 activation constitutes the rate-limiting step
for IL-1â secretion. On the other hand, our results show that
processing of pro-TNF-R by TACE and subsequent secretion
are not rate-limiting steps. Thus, our results indicate that much
higher concentrations of lipid A or LPS are required for
caspase-1 activation than for pro-IL-1â expression.
TACE is constituently expressed in its active form. On the
other hand, caspase-1 is present in the cytoplasm as an inactive
(41) Yamamoto, M.; Yaginuma, K.; Tsutsui, H.; Sagara, J.; Guan, X.; Seki, E.;
Yasuda, K.; Akira, S.; Nakanishi, K.; Noda, T.; Taniguchi, S. Genes Cells
2004, 9, 1055-1067.
(39) Skotnicki, J. S.; Levin, J. I. Annu. Rep. Med. Chem. 2003, 38, 153-162.
(40) Duffy, M. J.; Lynn, D. J.; Lloyd, A. T.; O’Shea, C. M. Thromb. Haemostasis
2003, 89, 622-631.
(42) Ogura, Y.; Sutterwala, F. S.; Flavell, R. A. Cell 2006, 126, 659-662.
9
5206 J. AM. CHEM. SOC. VOL. 129, NO. 16, 2007

Modulation of Innate Immune Responses
A R T I C L E S
Table 2. Cytokine Top Valuesa (pg/mL) of Dose-Response
Curves of E. coli LPS, 1, 2, and 4
E. coli LPS
lipid A 1
lipid A 2
lipid A 4
TNF-R
IFN-â
IL-6
3118 ( 120
665 ( 38
451 ( 25
7439 ( 440
9367 ( 188
82b ( 2
3924 ( 179
724 ( 25
249 ( 29
6778 ( 214
5531 ( 214
92 ( 2
4223 ( 329
654 ( 37
299 ( 42
7207 ( 277
6851 ( 216
82 ( 2
4178 ( 250
710 ( 44
233 ( 12
7320 ( 415
5360 ( 263
86 ( 4
IP-10
RANTES
IL-1â
pro Il-1â
699 ( 73
565 ( 25
587 ( 87
610 ( 35
a Top values are reported as best-fit values ( std error. b Plateau not
reached; top value is best-fit value according to Prism.
production of IFN-â and IP-10. However, lipid A’s 1-4 gave
lower efficacies for the production of RANTES and IL-6 as
compared to LPS.
Figure 5. Response of HEK 293T cells expressing murine TLR4, MD2,
and CD14 to LPS and lipid A derivatives. Induction of NF-κB activation
was determined in triplicate cultures of HEK 293T cells stably transfected
with murine TLR4, MD2, and CD14 and transiently transfected with
pELAM-Luc, pRL-TK, and pcDNA3 plasmids. Forty-four hours post-
transfection, cells were treated with E. coli LPS or lipid A derivatives 1-5
at the indicated concentrations or were left untreated (control). Forty-eight
hours post-transfection, NF-κB activation was determined by firefly
luciferase activity relative to Renilla luciferase activity. In the transfection
experiment shown, human TNF-R (10 ng/mL) induced 12.1 ( 0.3-fold
activation of NF-κB.
Our results show that the relative quantities of secreted
cytokines depend on the nature and concentration of the
employed lipid A. This information is of critical importance
for the development of lipid A’s as immune modulators. For
example, at a relative low dose of LPS or lipid A no IL-1â will
be produced. This cytokine is important for the induction of
IFN-γ, which in turn is important for biasing an adaptive
immune response toward a T helper-1 (Th1) phenotype.
To obtain further support that the EC50 values of secreted
TNF-R protein are not affected by transcriptional, translational,
or protein processing processes, dose response curves for the
activation of the transcription factor NF-κB were determined
for each compound, and the results were compared to similar
data for secretion of TNF-R protein. Thus, compounds 1-5,
and E. coli LPS, were exposed at a range of concentrations to
HEK 293T cells stably transfected with human TLR4/MD2/
CD14 and transiently transfected with a plasmid containing the
reporter gene pELAM-Luc (NF-κB-dependent firefly luciferase
reporter vector) and a plasmid containing the control gene pRL-
TK (Renilla luciferase control reporter vector). As a negative
control, wild-type HEK 293T cells transiently transfected with
plasmids containing the reporter gene pELAM-Luc and control
gene pRL-TK were used. After an incubation time of 4 h, the
activity was measured using a commercial dual-luciferase assay.
As can be seen in Figure 5 and Table 1, the EC50 values for
NF-κB activation for each compound are very similar to those
of TNF-R protein production, demonstrating that transcription,
translation, and protein processing do not impact the dose
responses. However, the EC50 values for secreted IL-1â protein
are at least 2 orders of magnitude larger, demonstrating that
down-stream processes control the dose response of this
cytokine. Collectively, our data indicate that a difference in the
processing of pro-TNF-R and pro-IL-1â is responsible for the
observed differences in EC50 values, which represents a novel
mechanism for modulating innate immune responses.
Differences in EC50 values were observed for the other
cytokines. For example, for each compound, the EC50 value for
RANTES secretion was approximately 10-fold larger than that
of TNF-R. Differential responses were also observed for S.
typhimurium lipid 3, which at the highest concentration
tested induced the production of TNF-R, IFN-â, and IP-10,
whereas no formation of IL-6, RANTES, and IL-1â could be
measured.
Examination of the efficacies (maximum responses) of the
various cytokines also provided unexpected structure-activity
relationships (Table 2). For example, each synthetic compound
and E. coli 055:B5 LPS induced similar efficacies for the
Conclusions
The results of previous studies have shown that the number
of acyl chains and phosphates of lipid A are important
determinants for potencies of cytokine production. These reports,
however, have described the inductions of only one mediator
such as TNF-R or IL-6 protein. We have determined, for the
first time, the potencies and efficacies of a wide range of (pro)-
inflammatory mediators induced by a number of well-defined
lipid As. This undertaking required the development of a new
synthetic approach that allowed for the convenient synthesis of
a panel of lipid A’s. The synthetic approach uses a highly
functionalized disaccharide building block that is selectively
protected with an Alloc, Fmoc, and anomeric TBDMS group
and an azido function, which in a sequential manner can be
deprotected or unmasked allowing selective lipid modifications
at each position of the disaccharide backbone. The strategy was
employed for the preparation of lipid A’s derived from E. coli
and S. typhimurium. Cellular activation studies with the synthetic
compounds and LPS revealed a number of novel structure-
activity relationships. For example, it was found that hepta-
acylated S. typhimurium lipid A gave much lower activities than
hexa-acylated E. coli lipid A. Furthermore, shortening of lipids,
such as in compounds 2 and 4, resulted in higher potencies. In
the case of the E. coli lipid A’s (1 vs 2), the differences in
EC50 values were relatively small, whereas for the S. typhimu-
rium lipid A’s (3 vs 4), an approximately 3 orders of magnitude
increase in potencies was observed. LPS gave much higher
potencies than the synthetic lipid A’s, which is probably due to
its di-KDO moiety. It has been shown, for the first time, that
cellular activation with a particular compound can give EC50
values for various mediators that differ as much as 100-fold.
The differences in responses did not follow a bias toward a
MyD88- or TRIF-dependent response. For example, for each
compound, potencies and efficacies for the induction of TNF-R
and IFN-â, which are the prototypical cytokines for the MyD88-
or TRIF-dependent pathway, respectively, differed only margin-
ally. On the other hand, large differences were observed between
9
J. AM. CHEM. SOC. VOL. 129, NO. 16, 2007 5207

A R T I C L E S
Zhang et al.
solution of compound 6 (3.0 g, 7.37 mmol) and TMEDA (666 µL,
4.42 mmol) in DCM (30 mL) was added dropwise allyl chloroformate
(1.00 mL, 8.85 mmol). The reaction mixture was stirred at room
temperature for 10 h, and then diluted with DCM (50 mL) and washed
with saturated aqueous NaHCO3 (2 × 100 mL) and brine (2 × 50 mL).
The organic phase was dried (MgSO4) and filtered. Next, the filtrate
was concentrated in vacuo. The residue was purified by silica gel
column chromatography (hexane/ethyl acetate, 25/1, v/v) to give 7 as
the efficacies of secreted TNF-R and IL-1â, which both depend
on the MyD88 pathway. Both cytokines are expressed as pro-
proteins, which are processed to the active form by the proteases
TACE and caspase-1, respectively. The rate-limiting step for
the secretion of IL-1â is the activation of caspase-1, whereas
for TNF-R it is the expression of the pro-protein. Surprisingly,
our results indicate that LPS-mediated activation of MyD88
resulting in the production of pro-Il-1â and pro-TNF-R requires
a much lower concentration of LPS or lipid A than does ACS-
mediated activation of caspase-1. As a result, the EC50 values
for secreted IL-1â and TNF-R differ significantly. Differences
in potencies were also observed for the production of other
cytokines. For example, S. typhimurium lipid 3 induced the
secretion of TNF-R, IFN-â, and IP-10 at the highest concentra-
tion tested, whereas no formation of IL-6, RANTES, and IL-
1â could be measured. Further studies are required to uncover
the origin of the differences of these responses. Examination
of the efficacies (maximum responses) of the various cytokines
also provided unexpected structure-activity relationships. For
example, each synthetic compound and E. coli 055:B5 LPS
induced similar efficacies for the production of IFN-â and IP-
10. However, lipid A’s 1-4 gave lower efficacies for the
production of RANTES and IL-6 as compared to LPS.
Collectively, the results presented in this paper demonstrate
that cytokine secretion induced by LPS and lipid A is complex.
In particular, the relative quantities of secreted cytokines depend
on the nature of the compounds and employed concentration
of initiator. This information is critical for the development of
lipid A’s as immune modulators. Future examination of the
utilization of signaling transduction and processing pathways
of pro-proteins to the active form by different compounds at
different concentrations may provide further insight into the
underlying mechanism of immune modulation.
a colorless oil (3.20 g, 88%). Rf ) 0.57 (hexane/ethyl acetate, 5/1, v/v).
1
[R]25 ) -36.8° (c ) 1.0, CHCl3). H NMR (300 MHz, CDCl3): δ
D
7.39-7.33 (m, 5H, aromatic), 5.97-5.88 (m, 1H, OCH2CHdCH2), 5.47
(s, 1H, >CHPh), 5.33 (d, J ) 17.4 Hz, OCH2CHdCH2), 5.22 (d, J )
17.4 Hz, OCH2CHdCH2), 4.81 (t, J2,3 ) J3,4 ) 9.9 Hz, H-3), 4.71 (d,
1H, J1,2 ) 7.5 Hz, H-1), 4.65 (d, 2H, J ) 5.4 Hz, OCH2CHdCH2),
4.28 (d, 1H, J5,6a ) 5.1 Hz, J6a,6b ) 10.5 Hz, H-6a), 3.77 (dd, 1H, J5,6b
) J6a,6b ) 10.5 Hz, H-6b), 3.67 (d, 1H, J3,4 ) J4,5 ) 9.0 Hz, H-4),
3.50-3.40 (m, 2H, H-3, H-5), 0.92 (s, 9H, SiC(CH3)3), 0.16 (s, 3H,
Si(CH3)), 0.14 (s, 3H, Si(CH3)). 13C NMR (75 MHz, CDCl3): δ 154.10
(CdO), 136.72-126.15 (aromatic), 131.13 (OCH2CHdCH2), 119.03
(OCH2CHdCH2), 101.51 (>CHPh), 97.69 (C-1), 78.55 (C-4), 75.18
(C-3), 68.90 (OCH2CHdCH2), 68.41 (C-6), 66.95 (C-5), 66.33 (C-2),
25.48 (SiC(CH3)3), 17.86 (SiC(CH3)3), -4.46 (Si(CH3)2), -5.23 (Si-
(CH3)2). HR MS (m/z) calcd for C23H33N3O7Si [M + Na]+, 514.1985;
found, 514.1907.
tert-Butyldimethylsilyl 2-Azido-4-O-benzyl-2-deoxy-â-D-glucopy-
ranoside (8). Compound 6 (1.32 g, 3.49 mmol) was dissolved in a
solution of BH3 (1 M) in THF (17.5 mL). After the mixture was stirred
at 0 °C for 5 min, dibutylboron triflate (1 M in DCM, 3.49 mL) was
added dropwise, and the reaction mixture was stirred at 0 °C for another
1 h. Subsequently, triethylamine (0.5 mL) and methanol (∼0.5 mL)
were added until the evolution of H2 gas had ceased. The solvents were
evaporated in vacuo, and the residue was coevaporated with methanol
(3 × 50 mL). The residue was purified by silica gel column
chromatography (hexane/ethyl acetate, 8/1, v/v) to give 8 as a colorless
oil (1.21 g, 85%). Rf ) 0.40 (hexane/ethyl acetate, 3/1, v/v). [R]25
)
D
1
+0.9° (c ) 1.0, CHCl3). H NMR (300 MHz, CDCl3): δ 7.32-7.31
(m, 5H, aromatic), 4.81 (d, 1H, J2 ) 11.4 Hz, CH2aPh), 4.70 (d, 1H, J2
) 11.4 Hz, CH2bPh), 4.55 (d, 1H, J1,2 ) 7.5 Hz, H-1), 3.84 (dd, 1H,
J5,6a ) 2.4 Hz, J6a,6b ) 12.0 Hz, H-6a), 3.70 (dd, 1H, J5,6b ) 1.5 Hz,
J6a,6b ) 12.0 Hz, H-6b), 3.49-3.43 (m, 2H, H-3, H-4), 3.33 (broad,
1H, H-5), 3.22-3.17 (m, 1H, H-2), 0.92 (s, 9H, SiC(CH3)3), 0.14 (s,
6H, Si(CH3)2). 13C NMR (75 MHz, CDCl3): δ 137.89-128.11
(aromatic), 96.98 (C-1), 77.17 (C-3 or C-4), 75.22 (C-5), 74.88
(C-3 or C-4), 74.75 (CH2Ph), 68.69 (C-2), 61.97 (C-6), 25.56 (SiC-
(CH3)3), 17.91 (SiC(CH3)3), -4.27 (Si(CH3)2), -5.16 (Si(CH3)2). HR
MS (m/z) calcd for C19H31N3O5Si [M + Na]+, 432.1931; found,
432.1988.
Experimental Section
General Synthetic Methods. Column chromatography was per-
formed on silica gel 60 (EM Science, 70-230 mesh). Reactions were
monitored by thin-layer chromatography (TLC) on Kieselgel 60 F254
(EM Science), and the compounds were detected by examination under
UV light and by charring with 10% sulfuric acid in MeOH. Solvents
were removed under reduced pressure at <40 °C. CH2Cl2 was distilled
from NaH and stored over molecular sieves (3 Å). THF was distilled
from sodium directly prior to the application. MeOH was dried by
refluxing with magnesium methoxide and then was distilled and stored
under argon. Pyridine was dried by refluxing with CaH2 and then was
distilled and stored over molecular sieves (3 Å). Molecular sieves (3
and 4 Å), used for reactions, were crushed and activated in vacuo at
390 °C during 8 h in the first instance and then for 2-3 h at 390 °C
directly prior to application. Optical rotations were measured with a
Jasco model P-1020 polarimeter. 1H NMR and 13C NMR spectra
were recorded with Varian spectrometers (models Inova500 and
Inova600) equipped with Sun workstations. 1H NMR spectra were
recorded in CDCl3 and referenced to residual CHCl3 at 7.24 ppm,
and 13C NMR spectra were referenced to the central peak of
CDCl3 at 77.0 ppm. Assignments were made by standard gCOSY and
gHSQC. High-resolution mass spectra were obtained on a Bruker
model Ultraflex MALDI-TOF mass spectrometer. Signals marked with
a subscript L symbol belong to the biantennary lipids, whereas
signals marked with a subscript L′ symbol belong to their side chain.
Signals marked with a subscript S symbol belong to the monoantennary
lipids.
tert-Butyldimethylsilyl 3-O-Allyloxycarbonyl-2-azido-6-O-benzyl-
2-deoxy-â-D-glucopyranoside (9). A suspension of compound 7 (3.20
g, 6.52 mmol) and molecular sieves (4 Å, 500 mg) in THF (50 mL)
was stirred at room temperature for 1 h, and then NaCNBH3 (2.46 g,
39.0 mmol) was added. A solution of HCl (2 M in diethyl ether) was
added dropwise to this reaction mixture until the mixture became acidic
(∼5 mL, pH ) 5). After being stirred another 0.5 h, the reaction mixture
was quenched with solid NaHCO3, diluted with diethyl ether (100 mL),
and washed with saturated aqueous NaHCO3 (2 × 100 mL) and brine
(2 × 50 mL). The organic phase was dried (MgSO4) and filtered. Next,
the filtrate was concentrated in vacuo. The residue was purified by
silica gel column chromatography (hexane/ethyl acetate, 7/1, v/v) to
give 9 as a colorless oil (3.20 g, 88%). Rf ) 0.42 (hexane/ethyl acetate,
4/1, v/v). [R]25 ) -6.2° (c ) 1.0, CHCl3). 1H NMR (300 MHz,
D
CDCl3): δ 7.39-7.34 (m, 5H, aromatic), 6.02-5.89 (m, 1H, OCH2CHd
CH2), 5.39 (d, 1H, J ) 17.4 Hz, OCH2CHdCH2), 5.30 (d, 1H, J )
10.5 Hz, OCH2CHdCH2), 4.70-4.58 (m, 5H, H-1, H-3, OCH2CHd
CH2, CH2Ph), 3.79-3.70 (m, 3H, H-4, H-6a, H-6b), 3.52-3.46 (m,
1H, H-5), 3.37 (dd, 1H, J1,2 ) 8.4 Hz, J2.3 ) 9.6 Hz, H-2), 0.94 (s, 9H,
tert-Butyldimethylsilyl 3-O-Allyloxycarbonyl-2-azido-4,6-O-ben-
zyldidine-2-deoxy-â-D-glucopyranoside (7). To a cooled (0 °C)
9
5208 J. AM. CHEM. SOC. VOL. 129, NO. 16, 2007

Modulation of Innate Immune Responses
A R T I C L E S
SiC(CH3)3), 0.17 (s, 6H, Si(CH3)2). 13C NMR (75 MHz, CDCl3): δ
154.81 (CdO), 137.59-127.32 (aromatic), 131.07 (OCH2CHdCH2),
118.97 (OCH2CHdCH2), 96.92 (C-1), 78.79 (C-3), 74.25 (C-5), 73.44
(CH2Ph), 69.89, 69.55, 68.84 (C-4, C-6, OCH2CHdCH2), 65.84 (C-
2), 25.42 (SiC(CH3)3), 17.75 (SiC(CH3)3), -4.50 (Si(CH3)2), -5.40
(Si(CH3)2). HR MS (m/z) calcd for C23H35N3O7Si [M + Na]+, 516.2142;
found, 516.2197.
Si(CH3)2), 0.10 (s, 3H, Si(CH3)2). 13C NMR (75 MHz, CDCl3): δ
155.52 (CdO), 154.80 (CdO), 143.71-119.94 (aromatic), 131.34
(OCH2CHdCH2), 118.85 (OCH2CHdCH2), 95.41 (C-1), 74.77 (C-4),
73.85 (C-5), 73.47 (CH2Ph), 68.94, 68.57, 68.50, 68.42 (C-3, C-6,
OCH2CHdCH2, OC6H4(CH2O)P), 68.50 (OC6H4(CH2O)P), 67.12
(CO2CH2CH, Fmoc), 58.69 (C-2), 47.04 (CO2CH2CH, Fmoc), 25.52
(SiC(CH3)3), 17.88 (SiC(CH3)3), -4.26 (Si(CH3)2), -5.38 (Si(CH3)2).
HR MS (m/z) calcd for C46H54NO12PSi [M + Na]+, 894.3051; found,
894.3937.
tert-Butyldimethylsilyl 3-O-Allyloxycarbonyl-2-azido-6-O-benzyl-
2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-
3yl)-â-D-glucopyranoside (10). To a solution of compound 9 (1.30 g,
2.50 mmol) and 1H-tetrazole (3 wt %, 10.0 mmol) in DCM (30 mL)
was added N,N-diethyl-1,5-dihydro-3H-2,4,3-benzodioxaphosphepin-
3-amine (1.20 g, 1.05 mmol). After the reaction mixture was stirred at
room temperature for 15 min, it was cooled (-20 °C), stirred for another
10 min, and then mCPBA (3.40 g, 50-55 wt %, 10.0 mmol) was added.
The reaction mixture was stirred at -20 °C for 20 min, and then
quenched by the addition of saturated aqueous NaHCO3 (40 mL) and
diluted with DCM (30 mL). The organic phase was washed with
saturated aqueous NaHCO3 (2 × 60 mL) and brine (2 × 40 mL), dried
(MgSO4), and filtered. Next, the filtrate was concentrated in vacuo.
The residue was purified by silica gel column chromatography (hexane/
ethyl acetate, 5/1-3/1, v/v) to give 10 as a pale yellow oil (1.48 g,
3-O-Allyloxycarbonyl-6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-
oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-3yl)-2-(9-fluorenylmethox-
ycarbonylamino)-D-glucopyranosyl Trichloroacetimidate (12). HF/
pyridine (1 mL) was added dropwise to a stirred solution of 11 (1.37
g, 1.58 mmol) in THF (10 mL). The reaction mixture was stirred at
room temperature for 12 h, after which it was diluted with ethyl acetate
(40 mL), and then washed with saturated aqueous NaHCO3 (2 × 40
mL) and brine (2 × 40 mL), successively. The organic phase was dried
(MgSO4) and filtered. Next, the filtrate was concentrated in vacuo. The
residue was purified by silica gel column chromatography (hexane/
ethyl acetate, 3/2, v/v) to give a lactol as a pale yellow oil (1.02 g,
98%). HR MS (m/z) calcd for C40H40NO12P [M + Na]+, 780.2186;
found, 780.2379. The lactol (1.02 g, 1.35 mmol) thus obtained was
dissolved in DCM (20 mL), and trichloroacetonitrile (10 mL) and NaH
(5 mg) were added, successively. The reaction mixture was stirred at
room temperature for 30 min, after which another portion of NaH (5
mg) was added. After the suspension was stirred for another 20 min,
the solids were removed by filtration, and the filtrate was concentrated
in vacuo. The residue was purified by silica gel column chromatography
(hexane/ethyl acetate, 1/1, v/v) to give 12 as a colorless solid (1.14 g,
92%).
89%). Rf ) 0.40 (hexane/ethyl acetate, 1/1, v/v). [R]25 ) -10.3° (c
D
1
) 1.0, CHCl3). H NMR (300 MHz, CDCl3): δ 7.35-7.15 (m, 9H,
aromatic), 5.98-5.85 (m, 1H, OCH2CHdCH2), 5.65 (d, 1H, J ) 1.2
Hz, J ) 17.4 Hz, OCH2CHdCH2), 5.50 (d, 1H, J ) 1.2 Hz, J ) 10.5
Hz, OCH2CHdCH2), 5.18-5.01 (m, 4H, C6H4(CH2O)P), 3.81 (dd, 1H,
J2,3 ) 10.5 Hz, J3,4 ) 9.3 Hz, H-3), 4.64-4.52 (m, 6H, H-1, H-4, OCH2-
CHdCH2, CH2Ph), 3.82 (d, 1H, J6a,6b ) 9.0 Hz, H-6a), 3.72-3.61 (m,
2H, H-5, H-6b), 3.41 (dd, 1H, J1,2 ) 7.4 Hz, J2,3 ) 10.5 Hz, H-2),
0.92 (s, 9H, SiC(CH3)3), 0.16 (s, 3H, Si(CH3)), 0.15 (s, 3H, Si(CH3)).
13C NMR (75 MHz, CDCl3): δ 154.38 (CdO), 138.02-127.56
(aromatic), 131.33 (OCH2CHdCH2), 118.99 (OCH2CHdCH2), 97.13
(C-1), 76.77 (C-3), 74.27 (C-4), 74.08 (C-5), 73.50 (CH2Ph), 69.06
(OCH2CHdCH2), 68.74 (C-6), 68.55 (OC6H4(CH2O)P), 68.50 (OC6H4-
(CH2O)P), 65.97 (C-2), 25.53 (SiC(CH3)3), 17.92 (SiC(CH3)3), -4.35
(Si(CH3)2), -5.28 (Si(CH3)2). HR MS (m/z) calcd for C31H42N3O10PSi
[M + Na]+, 698.2275; found, 698.2315.
tert-Butyldimethylsilyl 3-O-Allyloxycarbonyl-6-O-benzyl-2-deoxy-
4-O-(1,5-dihydro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-3yl)-2-
(9-fluorenylmethoxycarbonylamino)-â-D-glucopyranoside (11). Ace-
tic acid (300 µL, 5.20 mmol) was added dropwise to a stirred suspension
of 10 (1.40 g, 2.08 mmol) and zinc powder (676 mg, 10.4 mmol) in
DCM (15 mL). The reaction mixture was stirred at room temperature
for 2 h, after which it was diluted with ethyl acetate (50 mL). The
solids were removed by filtration and washed with ethyl acetate (2 ×
10 mL). The combined filtrates were washed with saturated aqueous
NaHCO3 (2 × 40 mL) and brine (2 × 40 mL). The organic phase was
dried (MgSO4), filtered, and the filtrate was concentrated in vacuo to
afford a crude amine as a pale yellow oil. Rf ) 0.21 (hexane/ethyl
acetate, 1/1, v/v). FmocCl (645 mg, 2.50 mmol) was added to a stirred
solution of the crude amine and DIPEA (435 µL, 2.50 mmol) in DCM
(15 mL) at 0 °C. The reaction mixture was stirred at room temperature
for 5 h, after which it was diluted with DCM (40 mL) and washed
with brine (2 × 50 mL). The organic phase was dried (MgSO4) and
filtered. Next, the filtrate was concentrated in vacuo. The residue was
purified by silica gel column chromatography (hexane/ethyl acetate,
4/1-2/1, v/v) to afford 11 as a colorless solid (1.45 g, 80% over two
steps). Rf ) 0.54 (hexane/ethyl acetate, 1/1, v/v). [R]25D ) -3.9° (c )
1.0, CDCl3). 1H NMR (500 MHz, CDCl3): δ 7.78-7.20 (m, 17H,
aromatic), 5.92-5.82 (m, 1H, OCH2CHdCH2), 5.42 (broad, 1H, H-3),
5.31 (d, 1H, J ) 17.6 Hz, OCH2CHdCH2), 5.20-5.07 (m, 6H, H-1,
OCH2CHdCH2, C6H4(CH2O)P), 4.67-4.56 (m, 5H, H-4, OCH2CHd
CH2, CH2Ph), 4.41-4.23 (m, 3H, COOCH2, Fmoc, COOCH2CH,
Fmoc), 3.89-3.87 (broad, 1H, H-6a), 3.76-3.74 (broad, 2H, H-5,
H-6b), 3.49-3.47 (m, 1H, H-2), 0.88 (s, 4H, SiC(CH3)3), 0.14 (s, 3H,
tert-Butyldimethylsilyl 6-O-[3-O-Allyloxycarbonyl-6-O-benzyl-2-
deoxy-4-O-(1,5-dihydro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-
3yl)-2-(9-fluorenylmethoxycarbonylamino)-â-D-glucopyranosyl]-2-
azido-4-O-benzyl-2-deoxy-â-D-glucopyranoside (13). A suspension
of trichloroacetimidate 12 (1.04 g, 1.21 mmol), acceptor 8 (740 mg,
1.82 mmol), and molecular sieves (4 Å, 500 mg) in DCM (20 mL)
was stirred at room temperature for 1 h. The mixture was cooled
(-60 °C), and then TMSOTf (18 µL, 0.09 mmol) was added. After
the reaction mixture was stirred for 15 min, it was quenched with solid
NaHCO3. The solids were removed by filtration, and the filtrate was
washed with saturated aqueous NaHCO3 (2 × 50 mL) and brine (2 ×
40 mL). The organic phase was dried (MgSO4) and filtered. Next, the
filtrate was concentrated in vacuo. The residue was purified by silica
gel column chromatography (hexane/ethyl acetate, 2/1, v/v) to give 13
as a colorless solid (1.09 g, 79%). Rf ) 0.37 (DCM/methanol, 50/1,
v/v). [R]26 ) -3.8 (c ) 1.0, CHCl3). 1H NMR (600 MHz, CD3-
D
COCD3): δ 7.84-7.20 (m, 22H, aromatic), 6.98 (d, 1H, JNH′,2′ ) 9.0
Hz, NH′), 5.83 (m, 1H, OCH2CHdCH2), 5.41 (t, 1H, J2′,3′ ) J3′,4′
)
9,6 Hz, H-3′), 5.29-5.21 (m, 3H, OCH2CHdCH2, C6H4(CH2O)2P),
5.13-5.03 (m, 3H, H-1′, OCH2CHdCH2, C6H4(CH2O)2P), 4.96-4.91
(m, 2H, CH2aPh, C6H4(CH2O)2P), 4.73-4.45 (m, 7H, H-1, H-4′, CH2b-
Ph, CH2Ph, OCH2CHdCH2), 4.24-4.13 (m, 4H, H-6, CO2CH2, Fmoc,
CO2CH2CH, Fmoc), 3.93-3.79 (m, 4H, H-5′, H-6a, H-6′a, H-6′b), 3.69
(m, 1H, H-2′), 3.54 (broad, 3H, H-3, H-4, H-5), 3.19 (dd, 1H, J1,2
)
7.8 Hz, J2,3 ) 9,0 Hz, H-2), 0.92 (s, 9H, SiC(CH3)3), 0.17 (s, 6H, Si-
(CH3)2). 13C NMR (75 MHz, CD3COCD3): δ 156.39 (CdO), 155.28
(CdO), 144.96-120.56 (aromatic, OCH2CHdCH2), 118.41 (OCH2-
CHdCH2), 101.14 (C-1′), 97.33 (C-1), 78.54, 77.87, 75.72, 75.25-
74.42 (m), 73.83, 70.35, 69.52, 69.04-68.73 (m), 67.91, 67.12, 57.03
(C-2′), 47.64 (CO2CH2, Fmoc), 25.83 (SiC(CH3)3), 18.27 (SiC(CH3)3),
-3.85 (Si(CH3)2), -5.21 (Si(CH3)2). HR MS (m/z) calcd for C59H69N4O16-
PSi [M + Na]+, 1171.4113; found, 1171.4256.
tert-Butyldimethylsilyl 6-O-{3-O-Allyloxycarbonyl-6-O-benzyl-2-
deoxy-4-O-(1,5-dihydro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-
3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-â-D-glucopyra-
nosyl}-2-azido-4-O-benzyl-2-deoxy-â-D-glucopyranoside (21). DBU
9
J. AM. CHEM. SOC. VOL. 129, NO. 16, 2007 5209

A R T I C L E S
Zhang et al.
(200 µL) was added dropwise to a solution of 13 (730 mg, 0.637 mmol)
in DCM (10 mL). The reaction mixture was stirred at room temperature
for 1 h, after which it was concentrated in vacuo. The residue was
purified by silica gel column chromatography (DCM/methanol, 100/
1-100/3, v/v) to afford the free amine as a colorless syrup (567 mg,
96%). Rf ) 0.32 (DCM/methanol, 50/1, v/v). 1H NMR (500 MHz,
CDCl3): δ 7.39-7.18 (m, 14H, aromatic), 5.96-5.88 (m, 1H,
OCH2CHdCH2), 5.38 (d, 1H, J ) 17.0 Hz, OCH2CHdCH2), 5.25 (d,
1H, J ) 11.0 Hz, OCH2CHdCH2), 5.21-5.06 (m, 4H, C6H4(CH2O)2P),
4.85 (t, 1H, J2′,3′ ) J3′,4′ ) 9.5 Hz, H-3′), 4.79 (d, 1H, J ) 11.0 Hz,
CH2aPh), 4.67 (d, 1H, J ) 11.0 Hz, CH2bPh), 4.63-4.55 (m, 5H, H-4′,
2 × CH2Ph, OCH2CHdCH2), 4.52 (d, 1H, J1,2 ) 7.5 Hz, H-1), 4.22
(d, 1H, J1′,2′ ) 8.0 Hz, H-1′), 4.14 (d, 1H, J6a,6b ) 10.5 Hz, H-6a), 3.87
(d, 1H, J6′a,6′b ) 10.5 Hz, H-6′a), 3.73-3.69 (m, 1H, H-6′b), 3.67-
3.65 (m, 1H, H-5′), 3.62-3.59 (m, 1H, H-6b), 3.55-3.52 (m, 1H, H-5),
3.46 (t, 1H, J2,3 ) J3,4 ) 9,5 Hz, H-3), 3.32 (t, 1H, J3,4 ) J4,5 ) 9.0
5.34 (d, 1H, J ) 17.5 Hz, OCH2CHdCH2), 5.22 (d, 1H, J ) 10.0 Hz,
OCH2CHdCH2), 5.08-4.95 (m, 7H, H-1, H-3, H-3L, C6H4(CH2O)2P),
4.61-4.44 (m, 10H, H-1, H-4′, 3 × CH2Ph, OCH2CHdCH2), 3.96 (d,
1H, J6′a,6′b ) 10.5 Hz, H-6′a), 3.88-3.85 (m, 1H, H-3S), 3.80 (d, 1H,
J6a,6b ) 9.5 Hz, H-6a), 3.72-3.66 (m, 3H, H-5′, H-6b, H-6′b), 3.55-
3.52 (m, 2H, H-4, H-5), 3.47-3.41 (m, 1H, H-2′), 3.27 (dd, 1H, J1,2
)
7.5 Hz, J2,3 ) 10.0 Hz, H-2), 2.56 (dd, 1H, J2Sa,2Sb ) 16.0 Hz, J2Sa,3S
) 7.0 Hz, H-2Sa), 2.43 (dd, 1H, J2Sa,2Sb ) 16.0 Hz, J2Sb,3S ) 7.0 Hz,
H-2Sb), 2.35 (dd, 1H, J2La,2Lb ) 15.0 Hz, J2La,3L ) 6.0 Hz, H-2La), 2.30-
2.20 (m, 3H, H-2L′, H-2L′b), 1.59-1.52 (m, 6H, H-4L, H-4S, H-3L′),
1.23 (broad, 52H, 26 × CH2, lipid), 0.90 (s, 9H, SiC(CH3)3), 0.88-
0.84 (m, 9H, 3 × CH3, lipid), 0.12 (s, 6H, Si(CH3)2). 13C NMR (75
MHz, CDCl3): δ 173.40 (CdO), 170.55 (CdO), 169.94 (CdO), 154.42
(CdO), 138.46-127.35 (aromatic, OCH2CHdCH2), 118.74 (OCH2-
CHdCH2), 99.29 (C-1′), 96.96 (C-1), 75.89, 75.62, 74.75, 74.28, 74.02,
73.67, 73.41, 71.34, 70.89, 68.87-67.85 (m), 66.48, -4.18 (Si(CH3)2),
-5.38 (Si(CH3)2). HR MS (m/z) calcd for C91H139N4O19PSi [M + Na]+,
1673.9438; found, 1674.1754.
Hz, H-4), 3.22 (t, 1H, J1,2 ) J2,3 ) 9.0 Hz, H-2), 2.93 (t, 1H, J1′,2′
)
8.0, J2′,3′ ) 10.0 Hz, H-2′), 0.94 (s, 9H, SiC(CH3)3), 0.19 (s, 6H, Si-
(CH3)2). HR MS (m/z) calcd for C44H59N4O14PSi [M + Na]+, 949.3432;
found, 949.4922. DCC (202 mg, 0.979 mmol) was added to a stirred
solution of (R)-3-dodecanoyl-tetradecanoic acid 18 (313 mg, 0.734
mmol) in DCM (10 mL). After the reaction mixture was stirred for 10
min, the amine (567 mg, 0.612 mmol) in DCM (4 mL) was added,
and stirring was continued for another 12 h. The insoluble materials
were removed by filtration, and the residue was washed with DCM (2
× 2 mL). The combined filtrates were concentrated in vacuo, and the
residue was purified by silica gel column chromatography (hexane/
ethyl acetate, 2/1, v/v) to give 21 as a white solid (760 mg, 93%). Rf
tert-Butyldimethylsilyl 6-O-{6-O-Benzyl-2-deoxy-4-O-(1,5-dihy-
dro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dode-
canoyloxy-tetradecanoylamino]-3-O-[(R)-3-(p-methoxy)benzyloxy-
tetradecanoyl]-â-D-glucopyranosyl}-2-azido-4-O-benzyl-3-O-[(R)-3-
benzyloxy-tetradecanoyl]-2-deoxy-â-D-glucopyranoside
(28).
Tetrakis(triphenylphosphine)palladium (29.0 mg, 0.0255 mmol)
was added to a solution of 22 (210 mg, 0.127 mmol), n-BuNH2 (25.0
µL, 0.255 mmol), and HCOOH (10.0 µL, 0.255 mmol) in THF (5 mL).
After the reaction mixture was stirred at room temperature for 20 min,
it was diluted with DCM (20 mL), and washed successively with water
(20 mL), saturated aqueous NaHCO3 (2 × 20 mL), and brine (2 × 20
mL). The organic phase was dried (MgSO4) and filtered. Next, the
filtrate was concentrated in vacuo. The residue was purified by silica
gel column chromatography (hexane/ethyl acetate, 4/3, v/v) to give
compound 23. A solution of (R)-3-(p-methoxy)benzyloxy-tetradecanoic
acid 17 (69 mg, 0.191 mmol) and DCC (52 mg, 0.254 mmol) in DCM
(4 mL) was stirred at room temperature for 10 min, and then the
intermediate 23 in DCM (1 mL) and DMAP (7 mg, 0.060 mmol) were
added. The reaction mixture was stirred for another 10 h, after which
the solids were removed by filtration and washed with DCM (2 × 2
mL). The combined filtrates were concentrated in vacuo. The residue
was purified by silica gel column chromatography (hexane/ethyl acetate,
) 0.68 (hexane/ethyl acetate, 1/1, v/v). [R]27 ) -3.0° (c ) 1.0,
D
CHCl3). 1H NMR (600 MHz, CDCl3): δ 7.33-7.14 (m, 14H, aromatic),
5.92 (d, 1H, JNH′,2′ ) 7.8 Hz, NH′), 5.91-5.85 (m, 1H, OCH2CHd
CH2), 5.46 (t, 1H, J2′,3′ ) J3′,4′ ) 9.6 Hz, H-3′), 5.34 (d, 1H, J ) 16.8
Hz, OCH2CHdCH2), 5.21 (d, 1H, J ) 10.2 Hz, OCH2CHdCH2), 5.09-
5.04 (m, 4H, C6H4(CH2O)2P), 4.99 (d, 1H, J1′,2′ ) 8.4 Hz, H-1′), 5.00-
4.96 (m, 1H, H-3L), 4.73 (d, 1H, J2 ) 12.0 Hz, CH2aPh), 4.63 (d, 1H,
J2 ) 12.0 Hz, CH2bPh), 4.59-4.48 (m, 6H, H-1, H-4′, CH2Ph, OCH2-
CHdCH2), 4.00 (d, 1H, J6′a,6′b ) 10.2 Hz, H-6′a), 3.82 (d, 1H, J6a,6b
)
10.2 Hz, H-6a), 3.73-3.67 (m, 3H, H-5′, H-6b, H-6′b), 3.49-3.36 (m,
4H, H-2′, H-3, H-4, H-5), 3.18 (t, 1H, J1,2 ) J2,3 ) 8.4 Hz, H-2), 2.33
(s, 1H, OH), 2.37 (dd, 1H, J2La,2Lb ) 14.4 Hz, J2La,3L ) 6.0 Hz, H-2La),
2.29-2.22 (m, 3H, H-2L′, H-2Lb), 1.61-1.53 (m, 4H, H-4L, H-3L′), 1.23
(broad, 34H, 17 × CH2, lipid), 0.90 (s, 9H, SiC(CH3)3), 0.85-0.78
(m, 6H, 2 × CH3, lipid), 0.13 (s, 6H, Si(CH3)2). 13C NMR (75 MHz,
CDCl3): δ 173.70 (CdO), 170.00 (CdO), 154.59 (CdO), 138.08-
127.57 (aromatic, OCH2CHdCH2), 118.84 (OCH2CHdCH2), 99.30 (C-
1′), 96.95 (C-1), 77.65, 77.21, 76.05, 75.04, 74.41, 74.32, 73.78, 71.13,
68.95-67.93 (m), 55.91 (C-2′), -4.02 (Si(CH3)2), -5.26 (Si(CH3)2).
HR MS (m/z) calcd for C70H107N4O17PSi [M + Na]+, 1357.7036; found,
1357.8037.
4/1, v/v) to afford 28 as a white solid (182 mg, 75%). Rf ) 0.46 (hexane/
1
ethyl acetate, 2/1, v/v). [R]26 ) -2.8° (c ) 1.0, CHCl3). H NMR
D
(500 MHz, CDCl3): δ 7.38-6.79 (m, 23H, aromatic), 5.73 (d, 1H,
JNH′, 2′ ) 7.5 Hz, NH′), 5.57 (t, 1H, J2′,3′ ) J3′,4′ ) 9.5 Hz, H-3′), 5.07-
4.87 (m, 6H, H-1, H-3, C6H4(CH2O)2P), 4.66-4.47 (m, 11H, H-1, H-4′,
H-3L, 3 × CH2Ph, CH2PhOCH3), 3.98 (d, 1H, J6a,6b ) 11.0 Hz, H-6a),
3.91-3.69 (m, 9H, H-5′, H-6b, H-6′a, H-6′b, 2 × H-3S, CH3OPh),
3.55-3.52 (m, 2H, H-4, 5), 3.47-3.41 (m, 1H, H-2′), 3.38-3.31 (m,
2H, H-2, H-2′), 2.67-2.07 (m, 8H, H-2L, 2 × H-2S, H-2L′), 1.62-1.59
(m, 8H, H-4L, 2 × H-4S, H-3L′), 1.27 (broad, 70H, 35 × CH2, lipid),
0.93 (s, 9H, SiC(CH3)3), 0.92-0.87 (m, 12H, 4 × CH3, lipid), 0.16 (s,
6H, Si(CH3)2). 13C NMR (75 MHz, CDCl3): δ 173.65 (CdO), 171.18
(CdO), 170.63 (CdO), 169.87 (CdO), 159.17-113.78 (aromatic),
99.77 (C-1′), 97.06 (C-1), 75.95, 75.71, 75.26, 74.89, 74.43, 74.09,
73.97, 73.75, 73.53, 72.07, 71.48, 71.07, 70.66, 68.90-68.13 (m), 66.54
(C-2), 56.22 (C-2′), 55.17 (CH3OC6H5), -4.08 (Si(CH3)2), -5.31 (Si-
(CH3)2). HR MS (m/z) calcd for C109H169N4O20PSi [M + Na]+,
1936.1735; found, 1936.2613.
tert-Butyldimethylsilyl 6-O-{3-O-Allyloxycarbonyl-6-O-benzyl-2-
deoxy-4-O-(1,5-dihydro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-
3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-â-D-glucopyra-
nosyl}-2-azido-4-O-benzyl-3-O-[(R)-3-benzyloxy-tetradecanoyl]-2-
deoxy-â-D-glucopyranoside (22). A reaction mixture of (R)-3-benzyloxy-
tetradecanoic acid 15 (100 mg, 0.293 mmol) and DCC (93 mg, 0.450
mmol) in DCM (5 mL) was stirred at room temperature for 10 min,
and then disaccharide 21 (300 mg, 0.225 mmol) in DCM (3 mL) and
DMAP (11 mg, 0.090 mmol) were added. The reaction mixture was
stirred at room temperature for 14 h, after which the solids were
removed by filtration, and the residue was washed with DCM (2 × 1
mL). The combined filtrates were concentrated in vacuo, and the residue
was purified by silica gel column chromatography (hexane/ethyl acetate,
tert-Butyldimethylsilyl 6-O-{6-O-Benzyl-2-deoxy-4-O-(1,5-dihy-
dro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dode-
canoyloxy-tetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxy-tet-
radecanoyl]-â-D-glucopyranosyl}-2-azido-4-O-benzyl-3-O-[(R)-3-
benzyloxy-tetradecanoyl]-2-deoxy-â-D-glucopyranoside (24). DDQ
(36 mg, 0.158 mmol) was added to a stirred solution of 15 (200 mg,
0.105 mmol) in a mixture of DCM and H2O (4 mL, 10/1, v/v). The
reaction mixture was stirred at room temperature for 1 h, after which
4/1, v/v) to give 22 as a white solid (319 mg, 86%). Rf ) 0.41 (hexane/
1
ethyl acetate, 2/1, v/v). [R]26 ) -2.8° (c ) 1.0, CHCl3). H NMR
D
(500 MHz, CDCl3): δ 7.33-7.15 (m, 19H, aromatic), 5.94-5.85 (m,
2H, NH, OCH2CHdCH2), 5.45 (t, 1H, J2′,3′ ) J3′,4′ ) 9.5 Hz, H-3′),
9
5210 J. AM. CHEM. SOC. VOL. 129, NO. 16, 2007

Modulation of Innate Immune Responses
A R T I C L E S
it was diluted with DCM. The mixture was washed with brine (20 mL),
dried (MgSO4), and concentrated in vacuo. The residue was purified
by silica gel column chromatography (hexane/ethyl acetate, 3/1, v/v)
to give the alcohol 29 as a colorless syrup (170 mg, 90%). Rf ) 0.50
(hexane/ethyl acetate, 5/3, v/v). HR MS (m/z) calcd for C101H161N4O19-
PSi [M + Na]+, 1816.1160; found, 1816.3214. Lauroyl chloride (128
µL, 0.475 mmol) was added to a solution of the alcohol 29 (170 mg,
0.095 mmol), pyridine (60 µL, 0.760 mmol), and DMAP (12 mg, 0.095
mmol) in DCM (4 mL). After the reaction mixture was stirred at room
temperature for 12 h, it was diluted with DCM and washed with
saturated aqueous NaHCO3 (2 × 20 mL) and brine (2 × 20 mL). The
organic phase was dried (MgSO4) and filtered. Next, the filtrate was
concentrated in vacuo. The residue was purified by silica gel column
chromatography (hexane/ethyl acetate, 4/1, v/v) to afford 24 as a white
H-6b, H-6′b, H-3S), 3.57 (t, 1H, J3,4 ) J4,5 ) 9.0 Hz, H-4), 3.53-3.50
(m, 1H, H-5), 3.43-3.38 (m, 1H, H-2′), 2.66-2.22 (m, 12H, 2 × H-2L,
2 × H-2S, 2 × H-2L′), 1.71-1.45 (m, 12H, 2 × H-4L, 2 × H-4S, 2 ×
H-3L′), 1.26 (broad, 108H, 54 × CH2, lipid), 0.91-0.88 (m, 18H, 6 ×
CH3, lipid), 0.86 (s, 9H, SiC(CH3)3), 0.10 (s, 3H, SiCH3), 0.05 (s, 3H,
SiCH3). 13C NMR (75 MHz, CDCl3): δ 178.19 (CdO), 173.68 (Cd
O), 173.55 (CdO), 171.45 (CdO), 170.87 (CdO), 170.10 (CdO),
138.62-127.42 (aromatic), 99.48 (C-1′), 96.25 (C-1), 76.13, 75.85,
75.44, 74.76, 74.38, 74.10, 72.61, 71.34, 70.62, 70.53, 70.29, 68.94,
68.88-68.22 (m), 56.48 (C-2), 56.04 (C-2′), -3.72 (Si(CH3)2), -5.05
(Si(CH3)2). HR MS (m/z) calcd for C136H221N2O22PSi [M + Na]+,
2316.5641; found, 2316.9641.
tert-Butyldimethylsilyl 6-O-{6-O-Benzyl-2-deoxy-4-O-(1,5-dihy-
dro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dode-
canoyloxy-tetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxy-tet-
radecanoyl]-â-D-glucopyranosyl}-4-O-benzyl-3-O-[(R)-3-benzyloxy-
tetradecanoyl]-2-deoxy-2-[( R)-3-hexadecanoyloxy-
tetradecanoylamino]-â-D-glucopyranoside (31). The free amine obtained
above (56.0 mg, 0.028 mmol) was acylated in a manner similar to the
synthesis of 30 with (R)-3-(hexadecanoyl)oxy-tetradecanoic acid 20 (27
mg, 0.057 mmol) to yield 31 as a white solid (47 mg, 68%). Rf ) 0.48
(hexane/ethyl acetate, 5/2, v/v). [R]25D ) -0.87° (c ) 1.0, CHCl3). 1H
NMR (500 MHz, CDCl3): δ 7.39-7.21 (m, 19H, aromatic), 6.20 (d,
1H, JNH′,2′ ) 7.5 Hz, NH′), 5.76 (d, 1H, JNH,2 ) 9.0 Hz, NH), 5.58 (t,
1H, J2′,3′ ) J3′,4′ ) 9.5 Hz, H-3′), 5.29-5.26 (m, 1H, H-3L), 5.15-4.97
(m, 8H, H-1′, H-3, 2 × H-3L, C6H4(CH2O)2P), 4.72 (d, 1H, J1,2 ) 8.0
Hz, H-1), 4.64-4.44 (m, 7H, H-4, H-3 × CH2Ph), 4.02 (d, 1H, J6a,6b
) 10.5 Hz, H-6a), 3.87-3.81 (m, 3H, H-2, H-6′a, H-3S), 3.74-3.69
(m, 3H, H-5′, H-6′b, H-6b), 3.59-3.58 (m, 2H, H-4, H-5), 3.44-3.39
(m, 1H, H-2), 2.64-2.22 (m, 14H, 3 × H-2L, H-2S, 3 × H-2L′), 1.60
(broad, 14H, 3 × H-4L, H-4S, 3 × H-3L), 1.26 (broad, 132H, 66 ×
CH2, lipid), 0.90-0.87 (m, 30H, 7 × CH3, lipid, SiC(CH3)3), 0.12 (s,
3H, SiCH
solid (162 mg, 85%). Rf ) 0.46 (hexane/ethyl acetate, 5/2, v/v). [R]26
D
1
) -2.8° (c ) 1.0, CHCl3). H NMR (500 MHz, CDCl3): δ 7.39-
7.22 (m, 19H, aromatic), 6.26 (d, 1H, JNH′, )7.5 Hz, NH′), 5.58 (t,
2′
1H, J2′,3′ ) J3′,4′ ) 9.5 Hz, H-3′), 5.32-5.27 (m, 1H, H-3L), 5.16-4.99
(m, 7H, H-1′, 3, H-3L, C6H4(CH2O)2P), 4.66-4.49 (m, 8H, H-1, 4′, 3
× CH2Ph), 4.03 (d, 1H, J6a,6b )10.5 Hz, H-6a), 3.93-3.88 (m, 1H,
H-3S), 3.82-3.74 (m, 3H, H-5′, H-6b, H-6′a), 3.70 (dd, 1H, J5′,6′b
)
5.0 Hz, J6′a,6′b ) 10.5 Hz, H-6′b), 3.62-3.55 (m, 2H, H-4, H-5), 3.48
(m, 1H, H-2′), 3.33 (dd, 1H, J1,2 ) 8.0 Hz, J2,3 ) 10.5 Hz, H-2), 2.70-
2.22 (m, 10H, 2 × H-2L, H-2S, 2 × H-2L′), 1.61-1.51 (m, 10H, 2 ×
H-4L, H-4S, 2 × H-3L′), 1.26 (broad, 108H, 54 × CH2, lipid), 0.95 (s,
9H, SiC(CH3)3), 0.92-0.90 (m, 15H, 5 × CH3, lipid), 0.19 (s, 3H,
SiCH3), 0.18 (s, 3H, SiCH3). 13C NMR (75 MHz, CDCl3): δ 173.65
(CdO), 173.60 (CdO), 170.62 (CdO), 170.14 (CdO), 170.10 (Cd
O), 138.53-127.41 (aromatic), 99.64 (C-1′), 97.05 (C-1), 75.93, 75.70,
75.43, 74.06, 73.73, 73.50, 72.60, 71.46, 70.52, 70.29, 68.82-68.24
(m), 66.54 (C-2), 56.34 (C-2′), -4.12 (Si(CH3)2), -5.32 (Si(CH3)2).
HR MS (m/z) calcd for C115H187N4O20PSi [M + Na]+, 2026.3143; found,
2026.6381.
3), 0.10 (s, 3H, SiCH3). 13C NMR (75 MHz, CDCl3): δ 173.68
(CdO), 173.63 (CdO), 173.57 (CdO), 171.54 (CdO), 170.15 (Cd
O), 170.10 (CdO), 169.17 (CdO), 138.52-127.46 (aromatic), 99.45
(C-1′), 96.16 (C-1), 76.00, 75.40, 74.92, 74.45, 74.14, 73.50, 72.58,
71.26, 70.84, 70.53, 70.28, 68.89-68.33 (m), 56.40 (C-2 or 2′), 56.35
(C-2 or 2′), -3.83 (Si(CH3)2), -5.13 (Si(CH3)2). HR MS (m/z) calcd
for C145H245N2O23PSi [M + Na]+, 2464.7468; found, 2465.0632.
tert-Butyldimethylsilyl 6-O-{6-O-Benzyl-2-deoxy-4-O-(1,5-dihy-
dro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dode-
canoyloxy-tetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxy-tet-
radecanoyl]-â-D-glucopyranosyl}-4-O-benzyl-3-O-[(R)-3-benzyloxy-
tetradecanoyl]-2-[(R)-3-benzyloxy-tetradecanoylamino]-2-deoxy-â-
D-glucopyranoside (30). A suspension of 16 (100 mg, 0.05 mmol),
zinc (33.0 mg, 0.50 mmol), and acetic acid (18 µL, 0.30 mmol) in
DCM (4 mL) was stirred at room temperature for 12 h, after which it
was diluted with ethyl acetate (25 mL). The solids were removed by
filtration and washed with ethyl acetate (2 × 3 mL), and the combined
filtrates were washed with saturated aqueous NaHCO3 (2 × 20 mL)
and brine (2 × 20 mL). The organic phase was dried (MgSO4) and
filtered. Next, the filtrate was concentrated in vacuo. The residue was
purified by silica gel column chromatography (hexane/ethyl acetate,
2.5/1, v/v) to afford the amine as a pale yellow syrup (94 mg, 95%).
Rf ) 0.29 (hexane/ethyl acetate, 5/2, v/v). HR MS (m/z) calcd for
6-O-{6-O-Benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ5-3H-2,4,3-
benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoy-
lamino]-3-O-[(R)-3-tetradecanoyloxy-tetradecanoyl]-â-D-glucopyr-
anosyl}-4-O-benzyl-3-O-[(R)-3-benzyloxy-tetradecanoyl]-2-[(R)-3-
benzyloxy-tetradecanoylamino]-2-deoxy-r-D-glucopyranose (32). HF/
pyridine (50 µL) was added dropwise to a stirred solution of 30 (20.0
mg, 0.0087 mmol) in THF (3 mL). The reaction mixture was stirred at
room temperature for 5 h, after which it was diluted with ethyl acetate
(15 mL), and washed with saturated aqueous NaHCO3 (2 × 25 mL)
and brine (2 × 20 mL). The organic phase was dried (MgSO4) and
filtered. Next, the filtrate was concentrated in vacuo. The residue was
purified by silica gel column chromatography (hexane/ethyl acetate,
3/1-4/3, v/v) to give 32 as a white solid (16.0 mg, 84%). Rf ) 0.38
(hexane/ethyl acetate, 1/1, v/v). 1H NMR (500 MHz, CDCl3): δ 7.39-
7.19 (m, 24H, aromatic), 6.36 (d, 1H, JNH′, 2′ ) 7.0 Hz, NH′), 6.28 (d,
1H, JNH,2 ) 9.5 Hz, NH), 5.52 (d, 1H, J1′,2′ ) 9.0 Hz, H-1′), 5.51 (t,
1H, J2′,3′ ) J3′,4′ ) 9.5 Hz, H-3′), 5.41 (t, 1H, J2,3 ) J3,4 ) 10.0 Hz,
H-3), 5.27-5.25 (m, 1H, H-3L), 5.15-4.96 (m, 6H, H-1, H-3L, C6H4-
(CH2O)2P), 4.64-4.43 (m, 9H, H-4′, 4 × CH2Ph), 4.23-4.19 (m, 1H,
H-2), 4.13-4.09 (m, 1H, H-5), 3.94-3.82 (m, 4H, H-6a, H-6′a, 2 ×
H-3S), 3.76-3.69 (m, 3H, H-5′, H-6′a, H-6b), 3.36-3.33 (m, 2H, H-2′,
H-4), 2.69-2.27 (m, 12H, 2 × H-2L, 2 × H-2S, 2 × H-2L′), 1.58 (broad,
12H, 2 × H-4L, 2 × H-4S, 2 × H-3L′), 1.26 (broad, 108H, 54 × CH2,
lipid), 0.91-0.81 (m, 18H, 6 × CH3, lipid). HR MS (m/z) calcd for
C
115H189N2O20PSi [M + Na]+, 2000.3238; found, 2000.6035. DCC (12
mg, 0.06 mmol) was added to a stirred solution of (R)-3-benzyloxy-
tetradecanoic acid 15 (10.0 mg, 0.03 mmol) in DCM (1.5 mL). After
the reaction mixture was stirred for 10 min, the amine (30.0 mg, 0.015
mmol) in DCM (1 mL) and DMAP (1.0 mg, 0.0075 mmol) were added,
and stirring was continued for another 12 h. The insoluble materials
were removed by filtration, and the residue was washed with DCM (2
× 1 mL). The combined filtrates were concentrated in vacuo, and the
residue was purified by preparative silica gel TLC chromatography
(hexane/ethyl acetate, 3.5/1, v/v) to give 30 as a white solid (22.0 mg,
64%). Rf ) 0.54 (hexane/ethyl acetate, 2/1, v/v). [R]26D ) -2.6° (c )
1.0, CHCl3). 1H NMR (500 MHz, CDCl3): δ 7.38-7.19 (m, 24H,
aromatic), 6.21 (d, 1H, JNH′,2′ ) 7.0 Hz, NH′), 6.15 (d, 1H, JNH,2 ) 9.5
Hz, NH), 5.59 (t, 1H, J2′,3′ ) J3′,4′ ) 9.5 Hz, H-3′), 5.31-5.26 (m, 1H,
H-3L), 5.15-4.97 (m, 7H, H-1′, H-3, H-3L, C6H4(CH2O)2P), 4.65-
4.44 (m, 10H, H-1, H-4′, 4 × CH2Ph), 4.01 (d, 1H, J6a,6b ) 9.5 Hz,
H-6a), 3.90-3.82 (m, 3H, H-2, H-6′a, H-3S), 3.76-3.69 (m, 4H, H-5′,
C
130H207N2O22PSi [M + Na]+, 2202.4776; found, 2202.8279.
6-O-{6-O-Benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3λ5-3H-2,4,3-
benzodioxaphosphepin-3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoy-
9
J. AM. CHEM. SOC. VOL. 129, NO. 16, 2007 5211

A R T I C L E S
Zhang et al.
lamino]-3-O-[(R)-3-tetradecanoyloxy-tetradecanoyl]-â-D-glucopyr-
anosyl}-4-O-benzyl-3-O-[(R)-3-benzyloxy-tetradecanoyl]-2-deoxy-2-
[(R)-3-hexadecanoyloxy-tetradecanoylamino]-r-D-glucopyranose (33).
31 (39.0 mg, 0.016 mmol) was deprotected in a manner similar to the
synthesis of 32 with HF/pyridine (100 mL) in THF (5 mL) to yield 33
as a white solid (33.0 mg, 89%). Rf ) 0.52 (hexane/ethyl acetate, 4/3,
35 (9.0 mg, 0.0035 mmol) was deprotected in a manner similar to the
synthesis of 1 to provide 3 as a colorless film (5.4 mg, 75%). 1H NMR
(600 MHz, CDCl3): δ 5.11 (broad, 1H, H-1), 4.87-4.82 (m, 5H, H-3,
H-3′, 3 × H-3L), 4.40 (d, 1H, J1′,2′ ) 8.4 Hz, H-1′), 3.92-3.88 (m, 1H,
H-4′), 3.85-2.83 (m, 1H, H-2), 3.77 (broad, 1H, H-5), 3.71-3.62 (m,
3H, H-3S), 3.53-3.43 (m, 3H, H-2′), 3.18 (t, J3,4 ) J4,5 ) 9.6 Hz, H-4),
3.10-3.07 (m, 1H, H-5′), 2.34-1.96 (m, 14H, 3 × H-2L, H-2S, 3 ×
H-2L′), 1.23 (broad, 14H, 3 × H-4L, H-4S, 3 × H-3L), 0.99 (broad,
132H, 66 × CH2, lipid), 0.57-0.55 (m, 21H, 7 × CH3, lipid). HR MS
(m/z) (negative) calcd for C110H208N2O26P2, 2035.4491; found, 2034.4668
[M - H], 2035.4692 [M].
1
v/v). H NMR (500 MHz, CDCl3): δ 7.40-7.17 (m, 19H, aromatic),
6.41 (d, 1H, JNH′,2′ ) 6.5 Hz, NH′), 5.95 (d, 1H, JNH,2 ) 9.0 Hz, NH),
5.56 (d, 1H, J1′,2′ ) 8.5 Hz, H-1′), 5.51 (t, 1H, J2′,3′ ) J3′,4′ ) 10.0 Hz,
H-3′), 5.39 (t, 1H, J2,3 ) J3,4 ) 10.0 Hz, H-3), 5.29-5.26 (m, 1H,
H-3L), 5.15-4.95 (m, 7H, H-1, 2 × H-3L, C6H4(CH2O)2P), 4.65-4.42
(m, 7H, H-4′, 3 × CH2Ph), 4.17-4.08 (m, 2H, H-2, H-5), 3.92 (d, 1H,
J6a,6b ) 12.0 Hz, H-6a), 3.91-3.82 (m, 2H, H-6′a, H-3S), 3.76-3.69
(m, 3H, H-5′, H-6b, H-6′b), 3.36-3.30 (m, 2H, H-2′, H-4), 2.69-2.27
(m, 14H, 3 × H-2L, H-2S, 3 × H-2L′), 1.59 (broad, 14H, 3 × H-4L,
H-4S × 2, 3 × H-3L), 1.26 (broad, 132H, 66 × CH2, lipid), 0.90-0.88
(m, 21H, 7 × CH3, lipid). HR MS (m/z) calcd for C139H231N2O23PSi
[M + Na]+, 2350.6603; found, 2350.8623.
Bis(benzyloxy)phosphoryl 6-O-{6-O-Benzyl-2-deoxy-4-O-(1,5-di-
hydro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dode-
canoyloxy-tetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxy-tet-
radecanoyl]-â-D-glucopyranosyl}-4-O-benzyl-3-O-[(R)-3-benzyloxy-
tetradecanoyl]-2-[(R)-3-benzyloxy-tetradecanoylamino]-2-deoxy-r-
D-glucopyranose (34). To a cooled (-78 °C) solution of 32 (16.0 mg,
0.0073 mmol) and tetrabenzyl diphosphate (16.0 mg, 0.029 mmol) in
THF (4 mL) was added dropwise lithium bis(trimethylsilyl)amide in
THF (1.0 M, 30 µL, 0.03 mmol). The reaction mixture was stirred for
1 h, and then allowed to warm to -20 °C. After the reaction mixture
was stirred at -20 °C for 1 h, it was quenched with saturated aqueous
NaHCO3 (10 mL) and extracted with ethyl acetate (15 mL). The organic
phase was washed with brine (2 × 15 mL), dried (MgSO4), and
concentrated in vacuo. The residue was purified by Iatro beads column
chromatography (hexane/ethyl acetate, 5/1-3/1-4/3, v/v) to give 34
as a pale yellow oil (12.0 mg, 67%).
tert-Butyldimethylsilyl 6-O-{3-O-Allyloxycarbonyl-6-O-benzyl-2-
deoxy-4-O-(1,5-dihydro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-
3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-â-D-glucopyra-
nosyl}-2-azido-4-O-benzyl-3-O-[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-
â-D-glucopyranoside (36). A solution of (R)-3-benzyloxy-dodecanoic
acid 14 (86 mg, 0.281 mmol) and DCC (78 mg, 0.376 mmol) in DCM
(5 mL) was stirred at room temperature for 10 min, and then
disaccharide 21 (250 mg, 0.188 mmol) in DCM (2 mL) and DMAP
(11 mg, 0.094 mmol) were added. The reaction mixture was stirred
for another 14 h, after which the solids were removed by filtration,
and the residue was washed with DCM (2 × 1 mL). The combined
filtrates were concentrated in vacuo, and the residue was purified by
silica gel column chromatography (hexane/ethyl acetate, 4/1, v/v) to
give 36 as a white solid (277 mg, 91%). Rf ) 0.41 (hexane/ethyl acetate,
2/1, v/v). [R]26 ) -3.0° (c ) 1.0, CHCl3). 1H NMR (600 MHz,
D
CDCl3): δ 7.34-7.15 (m, 19H, aromatic), 6.01 (d, 1H, JNH′,2 ) 7.2
Hz, NH′), 5.92-6.86 (m, 1H, OCH2CHdCH2), 5.46 (t, 1H, J2′,3′
)
J3′,4′ ) 9.6 Hz, H-3′), 5.34 (d, 1H, J ) 16.8 Hz, OCH2CHdCH2), 5.22
(d, 1H, J ) 10.8 Hz, OCH2CHdCH2), 5.08-4.97 (m, 7H, H-1′, H-3,
H-3L, C6H4(CH2O)2P), 4.61-4.45 (m, 10H, H-1, H-4′, 3 × CH2Ph,
OCH2CHdCH2), 3.97 (d, 1H, J6′a,6′b ) 10.5 Hz, H-6′a), 3.88-3.86 (m,
1H, H-3S), 3.81 (d, 1H, J6a,6b ) 9.5 Hz, H-6a), 3.73-3.68 (m, 3H,
H-5′, H-6b, H-6′b), 3.56-3.46 (m, 3H, H-2′, H-4, H-5), 3.28 (dd, 1H,
J1,2 ) 7.8 Hz, J2,3 ) 10.2 Hz, H-2), 2.56 (dd, 1H, J2Sa,2Sb ) 15.6 Hz,
J2Sa,3S ) 7.2 Hz, H-2Sa), 2.44 (dd, 1H, J2Sa,2Sb ) 15.6 Hz, J2Sb, S3 ) 6.0
Hz, H-2Sb), 2.36 (dd, 1H, J2La,2Lb ) 15.0 Hz, J2La,3 L ) 6.0 Hz, H-2La),
2.30-2.21 (m, 3H, H-2L′, H-2L′b), 1.57-1.53 (m, 6H, H-4L, H-4S, H-3L′),
1.24 (broad, 48H, 24 × CH2, lipid), 0.90 (s, 9H, SiC(CH3)3), 0.89-
0.85 (m, 9H, 3 × CH3, lipid), 0.13 (s, 6H, Si(CH3)2). 13C NMR (75
MHz, CDCl3): δ 173.54 (CdO), 170.64 (CdO), 170.03 (CdO), 154.49
Bis(benzyloxy)phosphoryl 6-O-{6-O-Benzyl-2-deoxy-4-O-(1,5-di-
hydro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dode-
canoyloxy-tetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxy-tet-
radecanoyl]-â-D-glucopyranosyl}-4-O-benzyl-3-O-[(R)-3-benzyloxy-
tetradecanoyl]-2-deoxy-2-[( R)-3-hexadecanoyloxy-
tetradecanoylamino]-r-D-glucopyranose (35). The phosphorylation
of 33 (12 mg, 0.0052 mmol) was performed in a manner similar to
that for 34 to give 35 as a white solid (9.0 mg, 68%).
(CdO), 138.52-127.43 (aromatic, OCH
2CHdCH2), 118.84 (OCH2
-
CHdCH2), 99.34 (C-1′), 97.02 (C-1), 76.00, 75.70, 74.33, 74.08, 73.73,
73.48, 71.46, 71.01, 68.95-67.99 (m), 66.55, -4.13 (Si(CH3)2), -5.32
(Si(CH3)2). HR MS (m/z) calcd for C89H135N4O19PSi [M + Na],
1645.9125; found, 1646.2435.
6-O-{2-Deoxy-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-3-O-
[(R)-3-tetradecanoyloxy-tetradecanoyl]-â-D-glucopyranosyl}-2-deoxy-
3-O-[(R)-3-hydroxy-tetradecanoyl]-2-[(R)-3-hydroxy-tetradecanoy-
lamino]-r-D-glucopyranose 1,4′-Bisphosphate (1). A mixture of 34
(12.0 mg, 0.0049 mmol) and Pd-black (15.0 mg) in anhydrous THF (5
mL) was shaken under an atmosphere of H2 (50 psi) at room
temperature for 30 h, after which it was neutralized with triethylamine
(10 µL), the catalyst was removed by filtration, and the residue was
washed with THF (2 × 1 mL). The combined filtrates were concentrated
in vacuo to afford 1 as a colorless film (6.3 mg, 72%). 1H NMR (600
MHz, CDCl3): δ 5.19 (broad, 1H, H-1), 4.87-4.83 (m, 4H, H-3, H-3′,
2 × H-3L), 4.43 (d, 1H, J1′,2′ ) 8.4 Hz, H-1′), 3.93-3.89 (m, 1H, H-4′),
3.87-3.85 (m, 1H, H-2), 3.74 (broad, 1H, H-5), 3.70 (d, 1H, J6a,6b or
J6′a,6′b ) 11.4 Hz, H-6a or 6′a), 3.65 (broad, 1-H, H-3S), 3.57-3.48
(m, 4H, H-6a or 6′a, 6b, 6′b, H-3S), 3.21 (t, J3,4 ) J4,5 ) 9.6 Hz, H-4),
3.14-3.11 (m, 1H, H-5′), 2.37-1.96 (m, 12H, 2 × H-2L, 2 × H-2S, 2
× H-2L′), 1.27 (broad, 12H, 2 × H-4L, 2 × H-4S, 2 × H-3L), 0.94
(broad, 108H, 54 × CH2, lipid), 0.56-0.54 (m, 18H, 6 × CH3, lipid).
HR MS (m/z) (negative) calcd for C94H178N2O25P2, 1797.2194; found,
1796.5488 [M - H], 1797.5510 [M].
tert-Butyldimethylsilyl 6-O-{3-O-Allyloxycarbonyl-6-O-benzyl-2-
deoxy-4-O-(1,5-dihydro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-
3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-â-D-glucopyra-
nosyl}-4-O-benzyl-3-O-[(R)-3-benzyloxy-dodecanoyl]-2-[(R)-3-
benzyloxy-tetradecanoyl]-2-deoxy-â-D-glucopyranoside (37). A
suspension of 36 (180 mg, 0.111 mmol), zinc (72 mg, 1.11 mmol),
and acetic acid (25 µL, 0.444 mmol) in DCM (5 mL) was stirred at
room temperature for 12 h, after which it was diluted with ethyl acetate,
the solids were removed by filtration, and the residue was washed with
ethyl acetate (2 × 2 mL). The combined filtrates were washed with
saturated aqueous NaHCO3 (2 × 15 mL) and brine (2 × 15 mL). The
organic phase was dried (MgSO4) and filtered. Next, the filtrate was
concentrated in vacuo. The residue was purified by silica gel column
chromatography (hexane/ethyl acetate, 2.5/1, v/v) to afford the amine
as a pale yellow syrup (160 mg, 90%). Rf ) 0.35 (hexane/ethyl acetate,
2/1, v/v). HR MS (m/z) calcd for C89H137N2O19PSi [M + Na]+,
1619.9220; found, 1620.1069. DCC (34 mg, 0.169 mmol) was added
to a stirred solution of (R)-3-benzyloxy-tetradecanoic acid 15 (47 mg,
0.141 mmol) in DCM (1.5 mL). After the mixture was stirred for 10
min, the amine (150 mg, 0.094 mmol) in DCM (1 mL) was added.
6-O-{2-Deoxy-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-3-O-
[(R)-3-tetradecanoyloxy-tetradecanoyl]-â-D-glucopyranosyl}-2-deoxy-
2-[(R)-3-hexadecanoyl-tetradecanoylamino]-3-O-[(R)-3-hydroxy-
tetradecanoyl]-r-D-glucopyranose 1,4′-Bisphosphate (3). Compound
9
5212 J. AM. CHEM. SOC. VOL. 129, NO. 16, 2007

Modulation of Innate Immune Responses
A R T I C L E S
The reaction mixture was stirred at room temperature for 10 h, after
which the insoluble materials were removed by filtration, and the residue
was washed with DCM (2 × 1 mL). The combined filtrates were
concentrated in vacuo, and the residue was purified by preparative silica
gel TLC chromatography (hexane/ethyl acetate, 5/1, v/v) to give 37 as
Next, the filtrate was concentrated in vacuo. The residue was purified
by silica gel column chromatography (hexane/ethyl acetate, 4/3, v/v)
to give the alcohol intermediate. A solution of (R)-3-(p-methoxy)-
benzyloxy-dodecanoic acid 16 (16.5 mg, 0.049 mmol) and DCC (13.6
mg, 0.066 mmol) in DCM (5 mL) was stirred at room temperature for
10 min, after which the alcohol intermediate in DCM (1 mL) and
DMAP (7 mg, 0.060 mmol) were added. The reaction mixture was
stirred at room temperature for 5 h, after which the solids were removed
by filtration and washed with DCM (2 × 2 mL). The combined filtrates
were concentrated in vacuo, and the residue was purified by preparative
silica gel TLC (hexane/ethyl acetate, 3/1, v/v) to afford 39 as a white
a white solid (153 mg, 85%). Rf ) 0.34 (hexane/ethyl acetate, 3/2,
1
v/v). [R]26 ) -2.3° (c ) 1.0, CHCl3). H NMR (500 MHz, CDCl3):
D
δ 7.38-7.19 (m, 24H, aromatic), 6.15 (d, 1H, JNH, ) 9.0 Hz, NH),
2
5.97-5.89 (m, 2H, NH′, OCH2CHdCH2), 5.57 (t, 1H, J2′,3′ ) J3′,4′
)
9.5 Hz, H-3′), 5.38 (d, 1H, J ) 17.5 Hz, OCH2CHdCH2), 5.26 (d, 1H,
J ) 10.5 Hz, OCH2CHdCH2), 5.15-5.02 (m, 7H, H-1′, H-3, H-3L,
C6H4(CH2O)2P), 4.67-4.44 (m, 10H, H-1, H-4′, 4 × CH2Ph), 4.01 (d,
1H, J6a,6b ) 11.5 Hz, H-6a), 3.90-3.81 (m, 3H, H-2, H-6′a, H-3S),
solid (47 mg, 75%). Rf ) 0.29 (hexane/ethyl acetate, 5/2, v/v). [R]26
D
1
) -4.5° (c ) 1.0, CHCl3). H NMR (600 MHz, CDCl3): δ 7.38-
3.76-3.67 (m, 4H, H-5′, H-6b, H-6′b, H-3S), 3.57 (t, 1H, J3,4 ) J4,5
)
6.72 (m, 28H, aromatic), 6.11 (d, 1H, JNH, ) 9.0 Hz, NH), 5.74 (d,
2
9.0 Hz, H-4), 3.53-3.50 (m, 1H, H-5), 3.45-3.40 (m, 1H, H-2′), 2.61-
2.25 (m, 8H, H-2L, 2 × H-2S, H-2L′), 1.61-1.44 (m, 8H, H-4L, 2 ×
H-4S, H-3L′), 1.27 (broad, 66H, 33 × CH2, lipid), 0.91-0.86 (m, 21H,
4 × CH3, lipid, SiC(CH3)3), 0.09 (s, 3H, SiCH3), 0.04 (s, 3H, SiCH3).
13C NMR (75 MHz, CDCl3): δ 173.49 (CdO), 171.43 (CdO), 170.82
(CdO), 170.10 (CdO), 154.45 (CdO), 138.54-127.44 (aromatic,
OCH2CHdCH2), 118.79 (OCH2CHdCH2), 98.94 (C-1′), 96.27 (C-1),
76.07, 75.89, 75.77, 75.41, 74.89, 74.63, 74.18, 73.78, 73.66, 71.32,
70.95, 70.56, 68.93-68.24 (m), 56.18 (C-2 or 2′), 55.96 (C-2 or 2′),
-3.74 (Si(CH3)2), -5.11 (Si(CH3)2). HR MS (m/z) calcd for
1H, JNH′, ) 7.8 Hz, NH′), 5.59 (t, 1H, J2′,3′ ) J3′,4′ ) 9.6 Hz, H-3′),
2′
5.10-5.06 (m, 2H, H-1′, H-3), 5.00-4.85 (m, 5H, H-3L, C6H4-
(CH2O)2P), 4.61 (t, 1H, J3′,4′ ) J4′,5′ ) 9.0 Hz, H-4′), 4.57-4.41 (m,
11H, H-1, 4 × CH2Ph, CH2PhOCH3), 3.97 (d, 1H, J6a,6b ) 10.8 Hz,
H-6a), 3.88-3.81 (m, 4H, H-2, H-6′a, 2 × H-3S), 3.71-3.68 (m, 7H,
H-5′, H-6b, H-6′b, H-3S, CH3OPh), 3.55 (t, 1H, J3,4 ) J4,5 ) 9.0 Hz,
H-4), 3.47 (broad, 1H, H-5), 3.30-3.26 (m, 1H, H-2′), 2.64-1.69 (m,
10H, H-2L, 3 × H-2S, H-2L′), 1.67-1.41 (m, 10H, H-4L, 3 × H-4S,
H-3L′), 1.24 (broad, 80H, 40 × CH2, lipid), 0.87-0.81 (m, 24H, 5 ×
CH3, lipid, SiC(CH3)3), 0.06 (s, 3H, SiCH3), 0.01 (s, 3H, SiCH3). HR
MS (m/z) calcd for C126H195N2O22PSi [M + Na]+, 2170.3606; found,
2170.4929.
C
110H169N2O21PSi [M + Na]+, 1936.1622; found, 1936.2714.
tert-Butyldimethylsilyl 6-O-{3-O-Allyloxycarbonyl-6-O-benzyl-2-
deoxy-4-O-(1,5-dihydro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-
3yl)-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-â-D-glucopyra-
nosyl}-4-O-benzyl-3-O-[(R)-3-benzyloxy-dodecanoyl]-2-deoxy-2-
[(R)-3-hexadecanoyl-tetradecanoyl]-â-D-glucopyranoside (38). In a
manner similar to the synthesis of 37, the free amine (99 mg, 0.062
mmol) synthesized by reduction of 36 was acylated with (R)-3-
hexadecanoyl-tetradecanoic acid 20 (45 mg, 0.093 mmol), using DCC
(26 mg, 0.124 mmol) as activating agents, to yield 38 as a white solid
tert-Butyldimethylsilyl 6-O-{6-O-Benzyl-2-deoxy-4-O-(1,5-dihy-
dro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dode-
canoyloxy-tetradecanoylamino]-3-O-[(R)-3-(p-methoxy)benzyloxy-
dodecanoylamino]-â-D-glucopyranosyl}-4-O-benzyl-3-O-[(R)-3-
benzyloxy-dodecanoyl]-2-deoxy-2-[(R)-3-hexadecanoyloxy-
tetradecanoyl]-â-D-glucopyranoside (40). In a manner similar to that
described for the synthesis of 39, the Alloc group of 38 (72 mg, 0.035
mmol) in THF (6 mL) was removed with tetrakis(triphenylphosphine)-
palladium (12 mg, 0.011 mmol) in the presence of n-BuNH2 (6.9 µL,
0.07 mmol) and HCOOH (2.6 µL, 0.07 mmol). After purification by
silica gel column chromatography (hexane/ethyl acetate, 4/3, v/v), the
resulting intermediate was acylated with (R)-3-(p-methoxy)benzyloxy-
docanoic acid 16 (18 mg, 0.052 mmol) in DCM (5 mL), using DCC
(15 mg, 0.07 mmol) and DMAP (2.5 mg, 0.02 mmol) as activating
agents. Purification by preparative silica gel TLC (hexane/ethyl acetate,
3/1, v/v) afforded 40 as a white solid (49 mg, 61%). Rf ) 0.30 (hexane/
ethyl acetate, 5/2, v/v). [R]25D ) -6.0° (c 1.0, CHCl3). 1H NMR (500
MHz, CDCl3): δ 7.39-6.73 (m, 23H, aromatic), 5.80-5.79 (broad,
2H, NH, NH′), 5.64 (t, 1H, J2′,3′ ) J3′,4′ ) 9.5 Hz, H-3′), 5.16-5.11
(m, 2H, H-1′, H-3), 5.06-4.84 (m, 6H, 2 × H-3L, C6H4(CH2O)2P),
4.71 (d, 1H, J1,2 ) 8.0 Hz, H-1), 4.67-4.44 (m, 9H, H-4′, 4 × CH2-
Ph), 4.01 (d, 1H, J6a,6b ) 10.5 Hz, H-6a), 3.88-3.79 (m, 4H, H-2, 6′a,
2 × H-3S), 3.74-3.69 (6, 3H, H-5′, H-6b, H-6′b, CH3OPh), 3.61-
3.58 (m, 2H, H-4, H-5), 3.30-3.25 (m, 1H, H-2′), 2.65-2.01 (m, 12H,
2 × H-2L, 2 × H-2S, 2 × H-2L′), 1.61-1.50 (m, 12H, 2 × H-4L, 2 ×
H-4S, 2 × H-3L′), 1.25 (broad, 102H, 51 × CH2, lipid), 0.88-0.84 (m,
27H, 6 × CH3, lipid, SiC(CH3)3), 0.08 (s, 3H, SiCH3), 0.06 (s, 3H,
SiCH3). 13C NMR (75 MHz, CDCl3): δ 173.63 (CdO), 171.62 (Cd
O), 171.14 (CdO), 169.88 (CdO), 169.14 (CdO), 159.20-113.77
(aromatic), 99.61 (C-1′), 96.17 (C-1), 75.98, 75.39, 75.23, 74.91, 74.42,
74.15, 73.92, 73.51, 72.02, 71.26, 71.05, 70.81, 70.63, 68.95, 68.52-
68.18 (m), 56.32 (C-2 or 2′), 55.17 (CH3OPh), -3.83 (Si(CH3)2), -5.13
(Si(CH3)2). HR MS (m/z) calcd for C135H219N2O23PSi [M + Na],
2318.5433; found, 2318.7700.
(103 mg, 81%). Rf ) 0.52 (hexane/ethyl acetate, 2/1, v/v). [R]26
)
D
1
-5.3° (c ) 1.0, CHCl3). H NMR (600 MHz, CDCl3): δ 7.36-7.17
(m, 19H, aromatic), 5.98 (d, 1H, JNH′,2′ ) 7.2 Hz, NH′), 5.93-5.87
(m, 1H, OCH2CHdCH2), 5.76 (d, 1H, JNH,2 ) 9.0 Hz, NH), 5.56 (t,
1H, J2′,3′ ) J3′,4′ ) 9.0 Hz, H-3′), 5.36 (d, 1H, J ) 17.4 Hz, OCH2-
CHdCH2), 5.23 (d, 1H, J ) 10.2 Hz, OCH2CHdCH2), 5.14-4.99 (m,
8H, H-1′, 3, 2 × H-3L, C6H4(CH2O)2P), 4.70 (d, 1H, J1,2 ) 7.8 Hz,
H-1), 4.62-4.42 (m, 7H, H-4′, 3 × CH2Ph), 3.99 (d, 1H, J6a,6b ) 11.4
Hz, H-6a), 3.84-3.78 (m, 3H, H-2, H-6′a, H-3S), 3.74-3.67 (m, 3H,
H-5, H-5′, H-6′b), 3.58-3.55 (m, 2H, H-4, H-6b), 3.41-3.37 (m, 1H,
H-2′), 2.54-2.19 (m, 10H, 2 × H-2L, H-2S, 2 × H-2L′), 1.59-1.50
(m, 10H, 2 × H-4L, H-4S, 2 × H-3L′), 1.23 (broad, 90H, 45 × CH2,
lipid), 0.88-0.84 (m, 24H, 5 × CH3, lipid, SiC(CH3)3), 0.09 (s, 3H,
SiCH3), 0.06 (s, 3H, SiCH3). 13C NMR (75 MHz, CDCl3): δ 173.60
(CdO), 173.43 (CdO), 171.57 (CdO), 170.04 (CdO), 169.14 (Cd
O), 154.42 (CdO), 138.47-127.43 (aromatic, OCH2CHdCH2), 118.72
(OCH2CHdCH2), 99.09 (C-1′), 96.05 (C-1), 75.96, 75.36, 74.95, 74.80,
74.26, 74.10, 73.77, 73.69, 71.21, 70.91, 70.76, 68.87-67.98 (m), 56.32,
56.03 (C-2′), -3.91 (Si(CH3)2), -5.20 (Si(CH3)2). HR MS (m/z) calcd
for C110H169N2O21PSi [M + Na]+, 2084.3450; found, 2084.6633.
tert-Butyldimethylsilyl 6-O-{6-O-Benzyl-2-deoxy-4-O-(1,5-dihy-
dro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-3yl)-2-[(R)-3-dode-
canoyloxy-tetradecanoylamino]-3-O-[(R)-3-(p-methoxy)benzyloxy-
dodecanoylamino]-â-D-glucopyranosyl}-4-O-benzyl-3-O-[(R)-3-
benzyloxy-dodecanoyl]-2-[(R)-3-benzyloxy-tetradecanoyl]-2-deoxy-
â-D-glucopyranoside (39). Tetrakis(triphenylphosphine)palladium (6.6
mg, 0.006 mmol) was added to a solution of 37 (55 mg, 0.029 mmol),
n-BuNH2 (5.7 µL, 0.058 mmol), and HCOOH (2.2 µL, 0.058 mmol)
in THF (5 mL). After the reaction mixture was stirred at room
temperature for 20 min, it was diluted with DCM (15 mL), and washed
with water (10 mL), saturated aqueous NaHCO3 (2 × 10 mL), and
brine (2 × 10 mL). The organic phase was dried (MgSO4) and filtered.
tert-Butyldimethylsilyl 6-O-{6-O-Benzyl-2-deoxy-4-O-(1,5-dihy-
dro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-3yl)-3-O-[(R)-3-dode-
canoyloxy-dodecanoyl]-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-
â-D-glucopyranosyl}-4-O-benzyl-3-O-[(R)-3-benzyloxy-dodecanoyl]-
2-[(R)-3-benzyloxy-tetradecanoylamino]-2-deoxy-â-D-
glucopyranoside (43). DDQ (5 mg, 0.0223 mmol) was added to a
9
J. AM. CHEM. SOC. VOL. 129, NO. 16, 2007 5213

A R T I C L E S
Zhang et al.
stirred solution of 39 (32 mg, 0.0149 mmol) in a mixture of DCM and
H2O (3 mL, 10/1, v/v). After the reaction mixture was stirred at room
temperature for 1 h, it was diluted with DCM (10 mL) and washed
with brine (10 mL). The organic phase was dried (MgSO4) and filtered.
Next, the filtrate was concentrated in vacuo. The residue was purified
by silica gel column chromatography (hexane/ethyl acetate, 3/1, v/v)
to give free alcohol 41 as a colorless syrup (29 mg, 96%). Rf ) 0.36
(hexane/ethyl acetate, 2/1, v/v). 1H NMR (500 MHz, CDCl3): δ 7.39-
7.18 (m, 24H, aromatic), 6.30 (d, 1H, JNH′, 2′ ) 7.5 Hz, NH′), 6.16 (d,
1H, JNH,2 ) 9.0 Hz, NH), 5.60 (t, 1H, J2′,3′ ) J3′,4′ ) 10.0 Hz, H-3′),
5.15-4.98 (m, 7H, H-1′, H-3, H-3L, C6H4(CH2O)2P), 4.68-4.63 (m,
1H, H-4′), 4.58-4.44 (m, 9H, H-1, 4 × CH2Ph), 4.07 (broad, 1H, H-3S),
4.01 (d, 1H, J6a,6b ) 10.0 Hz, H-6a), 3.87-3.82 (m, 3H, H-2, H-6′a,
× CH2, lipid), 0.87-0.84 (m, 27H, 6 × CH3, lipid, SiC(CH3)3), 0.08
(s, 3H, SiCH3), 0.06 (s, 3H, SiCH3). HR MS (m/z) calcd for
C
127H211N2O22PSi [M + Na]+, 2198.4858; found, 2198.7722. In a
manner similar to the synthesis of 43, alcohol 42 (28 mg, 0.013 mmol)
was acylated with lauroyl chloride (50 µL) in the presence of pyridine
(100 µL) and DMAP (1.6 mg, 0.013 mmol) in DCM (2 mL).
Purification by silica gel column chromatography (toluene/ethyl acetate,
10/1-6/1, v/v) afforded 44 as a pale yellow oil (28.5 mg, 94%). Rf )
0.52 (hexane/ethyl acetate, 2/1, v/v). [R]26D ) -1.7° (c ) 1.0, CHCl3).
1H NMR (500 MHz, CDCl3): δ 7.34-7.16 (m, 19H, aromatic), 6.14
(d, 1H, JNH′,2′ ) 8.0 Hz, NH′), 5.73 (d, 1H, JNH,2 ) 9.5 Hz, NH), 5.57
(t, 1H, J2′,3′ ) J3′,4′ ) 9.5 Hz, H-3′), 5.29-5.27 (m, 1H, H-3L), 5.15-
4.99 (m, 8H, H-1′, 3, 2 × H-3L, C6H4(CH2O)2P), 4.73 (d, 1H, J1,2
)
7.5 Hz, H-1), 4.65-4.40 (m, 7H, H-4′, 3 × CH2Ph), 4.02 (d, 1H, J6a,6b
) 10.5 Hz, H-6a), 3.88-3.79 (m, 3H, H-2, H-6′a, H-3S), 3.75-3.69
(m, 3H, H-5′, H-6′b, H-6b), 3.62-3.59 (m, 2H, H-4, H-5), 3.46-3.41
(m, 1H, H-2), 2.68-2.23 (m, 14H, 3 × H-2L, H-2S, 3 × H-2L′), 1.63-
1.61 (m, 14H, 3 × H-4L, H-4S, 3 × H-3L), 1.27 (broad, 120H, 60 ×
CH2, lipid), 0.91-0.88 (m, 30H, 7 × CH3, lipid, SiC(CH3)3), 0.13 (s,
3H, SiCH3), 0.10 (s, 3H, SiCH3). 13C NMR (75 MHz, CDCl3): δ 173.67
(CdO), 173.62 (CdO), 173.55 (CdO), 171.62 (CdO), 170.13 (Cd
O), 170.10 (CdO), 169.15 (CdO), 138.52-127.48 (aromatic), 99.57
(C-1′), 96.15 (C-1), 76.00, 75.40, 74.91, 74.45, 74.14, 73.50, 72.56,
71.26, 70.83, 70.54, 70.27, 68.89-68.33 (m), 56.36 (C-2 or 2′), -3.84
(Si(CH3)2), -5.13 (Si(CH3)2). HR MS (m/z) calcd for C139H233N2O23-
PSi [M + Na]+, 2380.6529; found, 2380.8301.
H-3S), 3.73-3.71 (m, 4H, H-5′, H-6b, H-6′b, H-3S), 3.59 (t, 1H, J3,4
)
J4,5 ) 9.0 Hz, H-4), 3.53-3.50 (m, 1H, H-2′, H-5), 2.64-2.23 (m,
10H, H-2L, 3 × H-2S, H-2L′), 1.69-1.46 (m, 10H, H-4L, 3 × H-4S,
H-3L′), 1.26 (broad, 80H, 40 × CH2, lipid), 0.91-0.84 (m, 24H, 5 ×
CH3, lipid, SiC(CH3)3), 0.09 (s, 3H, SiCH3), 0.04 (s, 3H, SiCH3). HR
MS (m/z) calcd for C118H187N2O21PSi [M + Na]+, 2050.3031; found,
2050.5063. Lauroyl chloride (50 µL) was added to a solution of alcohol
41 (27 mg, 0.0133 mmol), pyridine (100 µL), and DMAP (1.2 mg,
0.01 mmol) in DCM (2 mL). After the reaction mixture was stirred at
room temperature for 12 h, it was diluted with DCM (15 mL) and
washed with saturated aqueous NaHCO3 (2 × 10 mL) and brine (2 ×
10 mL). The organic phase was dried (MgSO4) and filtered. Next, the
filtrate was concentrated in vacuo. The residue was purified by
preparative silica gel TLC (toluene/ethyl acetate, 5/1, v/v) to afford 43
Bis(benzyloxy)phosphoryl 6-O-{6-O-Benzyl-2-deoxy-4-O-(1,5-di-
hydro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-3yl)-3-O-[(R)-3-
dodecanoyloxy-dodecanoyl]-2-[(R)-3-dodecanoyloxy-tetradecanoy-
lamino]-â-D-glucopyranosyl}-4-O-benzyl-3-O-[(R)-3-benzyloxy-
dodecanoyl]-2-[(R)-3-benzyloxy-tetradecanoylamino]-2-deoxy-r-D-
glucopyranose (45). Compound 43 (16 mg, 0.72 µmol) was deprotected
in a manner similar to the synthesis of 32 with HF/pyridine (50 µL) in
THF (3 mL) to yield the intermediate lactol as a white solid (13 mg,
86%). Rf ) 0.35 (hexane/ethyl acetate, 1/1, v/v). 1H NMR (500 MHz,
as a white solid (25 mg, 86%). Rf ) 0.56 (hexane/ethyl acetate, 2/1,
1
v/v). [R]26 ) -2.9° (c ) 1.0, CHCl3). H NMR (500 MHz, CDCl3):
D
δ 7.38-7.21 (m, 24H, aromatic), 6.19 (d, 1H, JNH′, 2′ ) 7.5 Hz, NH′),
6.17 (d, 1H, JNH, 2 ) 9.0 Hz, NH), 5.59 (t, 1H, J2′,3′ ) J3′,4′ ) 9.5 Hz,
H-3′), 5.30-5.27 (m, 1H, H-3L), 5.15-4.98 (m, 7H, H-1′, H-3, H-3L,
C6H4(CH2O)2P), 4.65-4.42 (m, 10H, H-1, H-4′, 4 × CH2Ph), 4.01 (d,
1H, J6a,6b ) 9.5 Hz, H-6a), 3.91-3.82 (m, 3H, H-2, H-6′a, H-3S), 3.75-
3.69 (m, 4H, H-5′, H-6b, H-6′b, H-3S), 3.58 (t, 1H, J3,4 ) J4,5 ) 9.0
Hz, H-4), 3.53-3.50 (m, 1H, H-5), 3.43-3.38 (m, 1H, H-2′), 2.65-
2.22 (m, 12H, 2 × H-2L, 2 × H-2S, 2 × H-2L′), 1.66-1.52 (m, 12H,
2 × H-4L, 2 × H-4S, 2 × H-3L′), 1.27 (broad, 96H, 48 × CH2, lipid),
0.91-0.88 (m, 18H, 6 × CH3, lipid), 0.86 (s, 9H, SiC(CH3)3), 0.09 (s,
3H, SiCH3), 0.05 (s, 3H, SiCH3). 13C NMR (75 MHz, CDCl3): δ 178.18
(CdO), 173.66 (CdO), 173.54 (CdO), 171.45 (CdO), 170.89 (Cd
O), 170.12 (CdO), 138.64-127.45 (aromatic), 99.52 (C-1′), 96.26 (C-
1), 76.15, 75.88, 75.44, 74.78, 74.39, 74.10, 72.65, 71.36, 70.62, 70.54,
70.29, 68.96, 68.89-68.22 (m), 56.50 (C-2), 56.06 (C-2′), -3.77 (Si-
(CH3)2), -5.09 (Si(CH3)2). HR MS (m/z) calcd for C130H209N2O22PSi
[M + Na]+, 2232.4702; found, 2232.8787.
tert-Butyldimethylsilyl 6-O-{6-O-Benzyl-2-deoxy-4-O-(1,5-dihy-
dro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-3yl)-3-O-[(R)-3-dode-
canoyloxy-dodecanoyl]-2-[(R)-3-dodecanoyloxy-tetradecanoylamino]-
â-D-glucopyranosyl}-4-O-benzyl-3-O-[(R)-3-benzyloxy-dodecanoyl]-
2-deoxy-2-[(R)-3-hexadecanoyloxy-tetradecanoylamino]-â-D-
glucopyranoside (44). The PMB group of 40 (41 mg, 0.018 mmol)
was removed in a manner similar to the synthesis of 41 with DDQ
(6.1 mg, 0.158 mmol) in a mixture of DCM and H2O (5 mL, 10/1,
v/v). Purification by silica gel column chromatography (hexane/ethyl
acetate, 3/1, v/v) gave free alcohol 42 as a colorless syrup (32 mg,
83%). Rf ) 0.39 (hexane/ethyl acetate, 2/1, v/v). 1H NMR (600 MHz,
CDCl3): δ 7.34-7.15 (m, 24H, aromatic), 6.26 (d, 1H, JNH′,2′ ) 7.2
CDCl3): δ 7.40-7.18 (m, 24H, aromatic), 6.37 (d, 1H, JNH′, ) 7.5
2′
Hz, NH′), 6.26 (d, 1H, JNH,2 ) 9.5 Hz, NH), 5.55 (d, 1H, J1′,2′ ) 8.0
Hz, H-1′), 5.52 (t, 1H, J2′,3′ ) J3′,4′ ) 9.5 Hz, H-3′), 5.42 (t, 1H, J2,3
)
J3,4 ) 10.0 Hz, H-3), 5.28-5.24 (m, 1H, H-3L), 5.15-4.96 (m, 6H,
H-1, H-3L, C6H4(CH2O)2P), 4.65-4.43 (m, 9H, H-4′, 4 × CH2Ph),
4.24-4.19 (m, 1H, H-2), 4.13-4.09 (m, 1H, H-5), 3.94-3.82 (m, 4H,
H-6a, H-6′a, 2 × H-3S), 3.77-3.68 (m, 3H, H-5′, H-6b, H-6′b), 3.37-
3.31 (m, 2H, H-2′, H-4), 2.69-2.27 (m, 12H, 2 × H-2L, 2 × H-2S, 2
× H-2L′), 1.59 (broad, 12H, 2 × H-4L, 2 × H-4S, 2 × H-3L′), 1.26
(broad, 80H, 40 × CH2, lipid), 0.91-0.88 (m, 18H, 6 × CH3, lipid).
HR MS (m/z) calcd for C124H195N2O22PSi [M + Na]+, 2118.3837; found,
2118.6284. The anomeric hydroxyl of the resulting lactol (16.0 mg,
0.0073 mmol) was phosphorylated in a manner similar to the synthesis
of 34 to afford 45 as a white solid (11.0 mg, 72%).
Bis(benzyloxy)phosphoryl 6-O-{6-O-Benzyl-2-deoxy-4-O-(1,5-di-
hydro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-3yl)-3-O-[(R)-3-
dodecanoyloxy-dodecanoyl]-2-[(R)-3-dodecanoyloxy-tetradecanoy-
lamino]-â-D-glucopyranosyl}-4-O-benzyl-3-O-[(R)-3-benzyloxy-
dodecanoyl]-2-deoxy-2-[(R)-3-hexadecanoyloxy-tetradecanoylamino]-
r-D-glucopyranose (46). Compound 44 (24 mg, 0.010 mmol) was
deprotected in a manner similar to the synthesis of 32 with HF/pyridine
(100 µL) in THF (3 mL) to yield the intermediate lactol as a white
solid (22 mg, 97%). Rf ) 0.52 (hexane/ethyl acetate, 1/1, v/v). 1H NMR
(600 MHz, CDCl3): δ 7.39-7.15 (m, 19H, aromatic), 6.33 (d, 1H,
JNH′,2′ ) 7.2 Hz, NH′), 5.89 (d, 1H, JNH,2 ) 9.0 Hz, NH), 5.55 (d, 1H,
J1′,2′ ) 8.4 Hz, H-1′), 5.48 (t, 1H, J2′,3′ ) J3′,4′ ) 9.6 Hz, H-3′), 5.36 (t,
1H, J2,3 ) J3,4 ) 9.6 Hz, H-3), 5.26-5.22 (m, 1H, H-3L), 5.11-4.88
(m, 7H, H-1, 2 × H-3L, C6H4(CH2O)2P), 4.62-4.40 (m, 7H, H-4′, 3 ×
CH2Ph), 4.14-4.05 (m, 2H, H-2, H-5), 3.89 (d, 1H, J6a,6b ) 12.6 Hz,
H-6a), 3.84-3.79 (m, 2H, H-6′a, H-3S), 3.74-3.67 (m, 3H, H-5′, H-6b,
H6′b), 3.31-3.28 (m, 2H, H-2′, H-4), 2.66-2.23 (m, 14H, 3 × H-2L,
Hz, NH), 5.71 (d, 1H, JNH,2 ) 9.0 Hz, NH), 5.55 (t, 1H, J2′,3′ ) J3′,4′
)
9.6 Hz, H-3′), 5.13-4.95 (m, 8H, H-1′, H-3, 2 × H-3L, C6H4(CH2O)2P),
4.71 (d, 1H, J1,2 ) 7.8 Hz), 4.65-4.59 (m, 1H, H-4′), 4.55-4.10 (m,
6H, 3 × CH2Ph), 4.04 (broad, 1H, H-3S), 3.99 (d, 1H, J6a,6b ) 10.2
Hz, H-6a), 3.82-3.76 (m, 3H, H-2, H-6′a, H-3S), 3.73-3.68 (m, 3H,
H-5′, H-6b, H-6′b), 3.60-3.54 (m, 2H, H-4, H-5), 3.51-3.47 (m, 1H,
H-2′), 2.61-2.18 (m, 12H, 2 × H-2L, 2 × H-2S, 2 × H-2L′), 1.74-
1.41 (m, 12H, 2 × H-4L, 2 × H-4S, 2 × H-3L′), 1.24 (broad, 104H, 52
9
5214 J. AM. CHEM. SOC. VOL. 129, NO. 16, 2007

Modulation of Innate Immune Responses
A R T I C L E S
H-2S, 3 × H-2L′), 1.62-1.53 (broad, 14H, 3 × H-4L, H-4S × 2, 3 ×
H-3L), 1.3 (broad, 120H, 60 × CH2, lipid), 0.87-0.85 (m, 21H, 7 ×
CH3, lipid). HR MS (m/z) calcd for C133H219N2O23PSi [M + Na]+,
2266.5664; found, 2266.8252. The anomeric hydroxyl of the
resulting lactol (12.0 mg, 0.0053 mmol) was phosphorylated in a manner
similar to the synthesis of 34 to afford 46 as a white solid (9.2 mg,
69%).
g/L), glucose (4.5 g/L), HEPES (10 mM), and sodium pyruvate (1.0
mM), and supplemented with penicillin (100 u/mL)/streptomycin (100
µg/mL; Mediatech) and fetal bovine serum (FBS, 10%; Hyclone).
Human embryonic kidney (HEK) 293T cells were grown in Dulbecco’s
modified Eagle’s medium (ATCC) with L-glutamine (4 mM), glucose
(4.5 g/L), and sodium bicarbonate (1.5 g/L), supplemented with
penicillin (100 u/mL)/streptomycin (100 µg/mL), Normocin (100 µg/
mL), and FBS (10%). Stably transfected HEK 293T cells with murine
TLR4, MD2, and CD14 (InvivoGen) were obtained from InvivoGen
and grown in the same growth medium as for HEK 293T cells
supplemented with the selective agents HygroGold (50 µg/mL; Invi-
voGen) and blasticidin (10 µg/mL; InvivoGen). All cells were
maintained in a humid 5% CO2 atmosphere at 37 °C.
6-O-{2-Deoxy-3-O-[(R)-3-dodecanoyloxy-dodecanoyl]-2-[(R)-3-
dodecanoyloxy-tetradecanoylamino]-â-D-glucopyranosyl}-2-deoxy-
3-O-[(R)-3-hydroxy-dodecanoyl]-2-[(R)-3-hydroxy-tetredecanoy-
lamino]-r-D-glucopyranose 1,4′-Bisphosphate (2). Compound 45 (8.0
mg, 0.0034 mmol) was deprotected in a manner similar to the synthesis
1
of 1 to provide 2 as a colorless film (4.7 mg, 81%). H NMR (600
MHz, CDCl3/CD3OD, 1/1, v/v): δ 5.08 (broad, 1H, H-1), 4.79-4.76
(m, 4H, H-3, H-3′, 2 × H-3L), 4.35 (d, 1H, J1′,2′ ) 7.8 Hz, H-1′), 3.82
(broad, 1H, H-4′), 3.77-3.75 (m, 1H, H-2), 3.67 (broad, 1H, H-5),
3.61 (d, J6a,6b or J6′a,6′b ) 11.4 Hz, H-6a or 6′a), 3.56 (m, 1H, H-3S),
3.49-3.40 (m, 5H, H-2′, H-6a or H-6′a, H-6b, H-6′b, H-3S), 3.12
(t, 1H, J3,4 ) J4,5 ) 9.0 Hz, H-4), 3.02 (broad, 1H, H-5′), 2.29-1.84
(m, 12H, 2 × H-2L, 2 × H-2S, 2 × H-2L′), 1.18 (broad, 12H,
2 × H-4L, 2 × H-4S, 2 × H-3L), 0.85 (broad, 80H, 40 × CH2, lipid),
0.47-0.45 (m, 18H, 6 × CH3, lipid). HR MS (m/z) (negative)
calcd for C88H166N2O25P2, 1713.1255; found, 1712.0845 [M - H],
1713.0880 [M].
6-O-{2-Deoxy-3-O-[(R)-3-dodecanoyloxy-dodecanoyl]-2-[(R)-3-
dodecanoyloxy-tetradecanoylamino]-â-D-glucopyranosyl}-2-deoxy-
2-[(R)-3-hexadecanoyloxy-tetredecanoylamino]-3-O-[(R)-3-hydroxy-
dodecanoyl]-r-D-glucopyranose 1,4′-Bisphosphate (4). Compound 46
(9.2 mg, 0.0041 mmol) was deprotected in a manner similar to the
synthesis of 1 to provide 4 as a colorless film (5.5 mg, 69%). 1H NMR
(500 MHz, CDCl3/CD3OD, 1/1, v/v): δ 5.33 (broad, 1H, H-1), 5.11-
5.03 (m, 5H, H-3, H-3′, 3 × H-3L), 4.61 (d, 1H, J1′,2′ ) 8.5 Hz, H-1′),
4.16-3.10 (m, 1H, H-4′), 4.09-4.07 (m, 1H, H-2), 4.04 (broad, 1H,
H-5), 3.94-3.89 (m, H-6a or H-6′a, H-3S), 3.75-3.67 (m, H-2′), 3.39
(dd, J ) 8.5 Hz, J ) 9.5 Hz, H-4), 3.31-3.29 (m, 1H, H-5′), 2.63-
2.19 (m, 14H, 3 × H-2L, H-2S, 3 × H-2L′), 1.52 (broad, 14H,
3 × H-4L, H-4S, 3 × H-3L), 1.18 (broad, 120H, 60 × CH2, lipid),
0.81-0.78 (m, 21H, 7 × CH3, lipid). HR MS (m/z) (negative)
calcd for C104H196N2O26P2, 1951.3552; found, 1950.4846 [M - H],
1951.4910 [M].
Cytokine Induction and ELISAs. RAW 264.7 γNO(-) cells were
plated on the day of the exposure assay as 2 × 105 cells/well in 96-
well tissue culture plates (Nunc). Cells were incubated with different
stimuli for 5.5 and 24 h in replicates of five. Culture supernatants were
then collected, pooled, and stored frozen (-80 °C) until assayed for
cytokine production. After removal of the supernatant, cells were lysed
by adding PBS containing Tween 20 (0.01%) and BSA (1%) in the
same volume as that of the supernatant and sonicating for 5 min. The
cell lysates were pooled and stored frozen (-80 °C) until assayed for
cytokine production.
All cytokine ELISAs were performed in 96-well MaxiSorp plates
(Nunc). Cytokine DuoSet ELISA Development Kits (R&D Systems)
were used for the cytokine quantification of mouse TNF-R, IL-6, IP-
10, RANTES, and IL-1â according to the manufacturer’s instructions.
The absorbance was measured at 450 nm with wavelength correction
set to 540 nm using a microplate reader (BMG Labtech). Concentrations
of IFN-â in culture supernatants were determined as follows. ELISA
MaxiSorp plates were coated with rabbit polyclonal antibody
against mouse IFN-â (PBL Biomedical Laboratories). IFN-â in
standards and samples was allowed to bind to the immobilized antibody.
Rat anti-mouse IFN-â antibody (USBiological) was then added,
producing an antibody-antigen-antibody “sandwich”. Next, horserad-
ish peroxidase (HRP) conjugated goat anti-rat IgG (H+L) antibody
(Pierce) and a chromogenic substrate for HRP 3,3′,5,5′-tetramethyl-
benzidine (TMB; Pierce) were added. After the reaction was stopped,
the absorbance was measured at 450 nm with wavelength correction
set to 540 nm. All cytokine values are presented as the mean ( SD of
triplicate measurements, with each experiment being repeated three
times.
6-O-{2-Deoxy-3-O-[(R)-3-dodecanoyloxy-dodecanoyl]-2-[(R)-3-
dodecanoyloxy-tetradecanoylamino]-â-D-glucopyranosyl}-2-deoxy-
2-[(R)-3-hexadecanoyloxy-tetredecanoylamino]-3-O-[(R)-3-hydroxy-
dodecanoyl]-r-D-glucopyranose (5). The resulting lactol in the
synthesis of 46 (8.5 mg, 0.0038 mmol) was deprotected in a manner
similar to the synthesis of 1 to provide 5 as a colorless film (5.1 mg,
Transfection and NF-KB Activation Assay. The day before
transfection, HEK 293T wild-type cells and HEK 293T cells stably
transfected with murine TLR4/MD2/CD14 were plated in 96-well tissue
culture plates (16 000 cells/well). The next day, cells were transiently
transfected using PolyFect Transfection Reagent (Qiagen) with expres-
sion plasmids pELAM-Luc (NF-κB-dependent firefly luciferase reporter
plasmid, 50 ng/well)43 and pRL-TK (Renilla luciferase control reporter
vector, 1 ng/well; Promega) as an internal control to normalize
experimental variations. The empty vector pcDNA3 (Invitrogen) was
used as a control and to normalize the DNA concentration for all of
the transfection reactions (total DNA 70 ng/well). Forty-four hours
post-transfection, cells were exposed to the stimuli at the indicated
concentrations for 4 h, after which cell extracts were prepared. The
luciferase activity was measured using the Dual-Luciferase Reporter
Assay System (Promega) according to the manufacturer’s instructions
and the Fluoroskan Accent FL combination luminometer/fluorometer
(Thermo Electron Corp.). Expression of the firefly luciferase reporter
gene was normalized for transfection efficiency with expression of
Renilla luciferase. The data are reported as the means ( SD of
triplicate treatments. The transfection experiments were repeated at
least twice.
1
71%). H NMR (600 MHz, CDCl3/CD3OD, 1/1, v/v): δ 5.01-4.91
(m, 5H, H-3, H-3′, 3 × H-3L), 4.89 (broad, 1H, H-1), 4.48 (d, 1H, J1′,2′
) 8.4 Hz, H-1′), 4.06 (broad, 1H, H-4′), 3.90-3.85 (m, 3H, H-2, H-5,
H-6a or H-6′a), 3.75 (broad, H-3S), 3.70 (broad, 1H, H-6a or H-6′a),
3.67-3.62 (m, 2H, H-2′, H-6b or H-6′b), 3.58 (broad, 1H, H-6b or
6′b), 3.28-3.20 (m, 2H, H-4, H-5′), 2.61 (m, 1H, H-2Sa), 2.53 (m, 1H,
H-2Sb), 2.40-2.12 (m, 6H, 3 × H-2L), 2.11-2.08 (m, 6H, 3 × H-2L′),
1.45 (broad, 14H, 3 × H-4L, H-4S, 3 × H-3L), 1.12 (broad, 120H, 60
× CH2, lipid), 0.76-0.83 (m, 21H, 7 × CH3, lipid). HR MS (m/z)
(negative) calcd for C104H195N2O23P, 1871.3888; found, 1870.4127 [M
- H], 1871.4128 [M].
Reagents for Biological Experiments. E. coli 055:B5 LPS was
obtained from List Biologicals. All data presented in this study were
generated using the same batch of E. coli 055:B5 LPS. Synthetic lipid
A’s were reconstituted in PBS with DMSO (10%) and stored at
-80 °C.
Cell Maintenance. RAW 264.7 γNO(-) cells, derived from the
RAW 264.7 mouse monocyte/macrophage cell line, were obtained from
ATCC. The cells were maintained in RPMI 1640 medium (ATCC)
with L-glutamine (2 mM), adjusted to contain sodium bicarbonate (1.5
(43) Chow, J. C.; Young, D. W.; Golenbock, D. T.; Christ, W. J.; Gusovsky, F.
J. Biol. Chem. 1999, 274, 10689-10692.
9
J. AM. CHEM. SOC. VOL. 129, NO. 16, 2007 5215

A R T I C L E S
Zhang et al.
Data Analysis. Concentration-response data were analyzed using
nonlinear least-squares curve fitting in Prism (GraphPad Software, Inc.).
These data were fit with the following four-parameter logistic equa-
tion: Y ) Emax/(1 + (EC50/X)Hill slope), where Y is the cytokine response,
X is logarithm of the concentration of the stimulus, Emax is the maximum
response, and EC50 is the concentration of the stimulus producing 50%
stimulation. The Hill slope was set at 1 to be able to compare the EC50
values of the different inducers.
Acknowledgment. This research was supported by the
Institute of General Medicine of the National Institutes of Health
(GM061761).
Supporting Information Available: Copies of NMR spectra
of synthetic compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
JA068922A
9
5216 J. AM. CHEM. SOC. VOL. 129, NO. 16, 2007