Synthesis and toll-like receptor 4 (TLR4) activity of phosphatidylinositol dimannoside analogues

Article abstract of DOI:10.1021/jm2008419

A series of five PIM₂ analogues were synthesized and tested for their ability to activate primary macrophages and modulate LPS signaling. Structural changes included replacement of the fatty acid esters of the phosphatidyl moiety of PIM₂ with the corresponding ether or amide. An AcPIM₂ analogue possessing an ether linkage was also prepared. The synthetic methodology utilized an orthogonally protected chiral myo-inositol starting material that was conveniently prepared from myo-inositol in just two steps. Important steps in the synthetic protocols included the regio- and α-selective glycosylation of inositol O-6 and introduction of the phosphodiester utilizing phosphoramidite chemistry. Replacement of the inositol core with a glycerol moiety gave compounds described as phosphatidylglycerol dimannosides (PGM₂). Biological testing of these PIM compounds indicated that the agonist activity was TLR4 dependent. An ether linkage increased agonist activity. Removal of the inositol ring enhanced antagonist activity, and the presence of an additional lipid chain enhanced LPS-induced cytokine production in primary macrophages. Furthermore, the interruption of the LPS-induced 2:2 TLR4/MD-2 signaling complex formation by PIM₂ represents a previously unidentified mechanism involved in the bioactivity of PIM molecules.

Full text of DOI:10.1021/jm2008419

ARTICLE
pubs.acs.org/jmc
Synthesis and Toll-like Receptor 4 (TLR4) Activity of
Phosphatidylinositol Dimannoside Analogues
Gary D. Ainge,†,‡ William John Martin,§ Benjamin J. Compton,† Colin M. Hayman,† David S. Larsen,‡
Sung-il Yoon,|| Ian A. Wilson,||,^ Jacquie L. Harper,*,§ and Gavin F. Painter*,†
†Carbohydrate Chemistry Team, Industrial Research Limited, P.O. Box 31-310, Lower Hutt, New Zealand
‡Department of Chemistry, University of Otago, P.O. Box 56, Dunedin, New Zealand
§The Malaghan Institute of Medical Research, P.O. Box 7060, Wellington, New Zealand
Department of Molecular Biology and ^The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California
92037, United States
S Supporting Information
b
ABSTRACT: A series of five PIM2 analogues were synthesized
and tested for their ability to activate primary macrophages
and modulate LPS signaling. Structural changes included
replacement of the fatty acid esters of the phosphatidyl moiety
of PIM2 with the corresponding ether or amide. An AcPIM2
analogue possessing an ether linkage was also prepared. The
synthetic methodology utilized an orthogonally protected
chiral myo-inositol starting material that was conveniently
prepared from myo-inositol in just two steps. Important steps in the synthetic protocols included the regio- and α-selective
glycosylation of inositol O-6 and introduction of the phosphodiester utilizing phosphoramidite chemistry. Replacement of the
inositol core with a glycerol moiety gave compounds described as phosphatidylglycerol dimannosides (PGM2). Biological
testing of these PIM compounds indicated that the agonist activity was TLR4 dependent. An ether linkage increased agonist
activity. Removal of the inositol ring enhanced antagonist activity, and the presence of an additional lipid chain enhanced LPS-
induced cytokine production in primary macrophages. Furthermore, the interruption of the LPS-induced 2:2 TLR4/MD-2
signaling complex formation by PIM2 represents a previously unidentified mechanism involved in the bioactivity of PIM
molecules.
’ INTRODUCTION
novel PIM analogues, 3, 4, 5, 6, and 7 where the functionality on
the glyceryl moiety and the inositol ring has been varied to probe
SAR for this class of compound (Figure 1). The targets were
specifically designed to gain as much structural information as
possible: target 3 probes amide versus ether or ester functionality
at the sn-2 position; 4 is a positional isomer of 1 (sn-1 vs sn-2);
analogue 5 investigates the effect of introducing a third lipid
functionality; targets 6 and 7 probe the importance of the inositol
ring. The previously reported diether 816 was also tested to in-
vestigate the impact of two ether linkages. These compounds were
then assayed and compared for their ability to induce cytokine
production and suppress LPS-induced cytokine production in
primary macrophages. We also investigated the interactions of
PIMs with the TLR4 signaling pathway.
The cell wall of mycobacteria is a rich source of immunomo-
dulatory molecules that includes lipids, glycoplipids, phospho-
glycolipids, lipoproteins, and mycolyl-arabinogalactan-peptiglycan
motifs.1 Included among these is the family of mannosylated
phosphatidylinositols, commonly known as phosphatidylinositol
mannosides or PIMs,2 that are of great interest because of their
biological activity and synthetic accessibility. Biological activity
includes the ability to affect T-cell proliferation,3,4 recruit natural
killer T (NKT) cells,5À7 activate innate receptors,6,8 and function
as immune adjuvants.9À11 In contrast PIMs have also been shown to
suppress allergic airway disease,12,13 negatively regulate lipopo-
lysaccharide (LPS) signaling in macrophages,14 and suppress hu-
man T-cell proliferation.15
In a previous study16 we synthesized a PIM2 monoether analogue,
PIM2ME (1), where the glyceryl sn-2 acyl group (COC15H31)
had been replaced by an alkyl group (C16H33). Our motivation
for this change was to overcome the inherent lability of this acyl
group toward hydrolysis, giving rise to lyso-PIM species. This
analogue, 1, enhanced cytokine production by bone-marrow-
derived dendritic cells compared to the natural analogous PIM2
compound (2). Here, we report the syntheses and activity of five
’ RESULTS AND DISCUSSION
Previous syntheses of PIM compounds have utilized 1-O-allyl-
3,4,5-tri-O-benzyl-D-myo-inositol as a convenient starting ma-
terial.9,17À19 This compound is generally obtained from α-methyl
Received: June 28, 2011
Published: September 21, 2011
r
2011 American Chemical Society
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Figure 1. Structures of PIM analogues.
Scheme 1. Synthesis of Common Intermediate 16
D-glucopyranoside by a series of transformations that, although
often produce high yields, suffers from the use of stoichiometric
amount of a mercuric trifluoroacetate in a carbocyclization re-
action.20 Also, this reaction and the subsequent reduction steps
both suffer from the unwanted formation of isomers that are
difficult to separate resulting in a relatively unscalable process.
For these reasons, we decided to utilize a different approach starting
from the known diol 921 that can be prepared from myo-inositol via
the camphanilydene acetal in a scalable two-step process.22
Synthesis of 6-Mannosylated myo-Inositol 15. Glycosyla-
tion of diol 9 is known to be favored at the C-6 hydroxyl on
inositol. Moreover, Martin-Lomas has reported21 the selective
glycosylation of 9 with an 2-azido donor in the synthesis of
inositol phosphoglycans (IPGs), and Watanabe demonstrated
the synthesis of distearoyl PIM2 using the analogous racemic cyl-
cohexylidene acetal.23,24 In the current work, glycosylation of 9
with mannosyl donor 1025 gave the desired glycoside 11 as the
major product along with smaller amounts of the diglycosylated
product. Lowering the temperature of the glycosylation inhibited
the formation of the overglycosylated product, resulting in the
clean formation of 11. Routine functional group manipulation via
12 through 14 afforded diol 1526 that was regioselectively
allylated using a minor modification of the literature transforma-
tion to 16 (Scheme 1).26
The commercially available (R)-(+)-2-amino-3-benzyloxy-1-pro-
panol (22) was used as a starting material for the synthesis of the
2-amido containing phosphoramidite 18. Diacylation with palmitoyl
chloride and DMAP in chloroform29 afforded 23 (Scheme 2).
Hydrogenolysis of 23 over Pearlman’s catalyst proceeded smoothly
to give the desired alcohol 24. 1H-Tetrazole activated reaction of
alcohol with benzyloxy-bis(diisopropylamino)phosphine gave the
required phosphoramidite 18 in high yield over the three steps.
Synthesis of 3 and 4. Glycosylation of 16 with 10 afforded
an inseparable mixture of the anomeric glycosides. This result
again demonstrates the propensity of the O-2 inositol hydroxyl to
form unexpected β-anomers.30 Removal of the benzoate direct-
ing group allowed separation of the anomers and access to the
desired pseudo-trisaccharide 26. Subsequent benzylation fol-
lowed by deallylation provided the known inositol dimannoside
headgroup 28.31 The spectroscopic data collected for compound
28 prepared in this way were identical to those reported
previously. Inositol dimannoside 28 was then coupled with
phosphoramidite 18 to afford the benzyl protected target mole-
cule 30 in 62% yield (Scheme 3). Hydrogenolysis over Pearl-
man’s catalyst gave the target PIM2-monoamide analogue that
was redissolved in MeOH/H2O with the aid of triethylamine and
then passed through a short column of silica gel. Evaporation and
lyophilization of the residue gave 3 as the partial triethylammo-
nium salt in 75% yield. Coupling of 28 with phosphoramidite 17
followed by oxidation afforded the fully protected 29 that was
subsequently deprotected to afford the PIM sn-1 ether analogue
4 in good purity.
Synthesis of Phosphoramidites 17 and 18. The phosphor-
amidites 17 and 18 were prepared for the synthesis of targets 3
and 4, respectively. With this in mind, the known monoether
1927 was esterified and the p-methoxybenzyl group removed by
hydrogenoloysis to afford alcohol 2128 in good yield. The
phosphoramidite 17 was prepared by reaction with benzyloxy-
bis(diisopropylamino)phosphine and 1H-tetrazole.
For the synthesis of AcPIM2 monoether (5), the thioglycoside
donor 31 was employed (Scheme 4). This compound was prepared
from diol 3232 by regioselective acylation of O-6 with palmitoyl
chloride followed by acylation of O-2 with 2-azidomethylbenzoyl
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Scheme 2. Synthesis of Phosphoramidites 17 and 18
Scheme 3. Synthesis of 3 and 4
Scheme 4. Synthesis of Donor 31
of the phosphodiester linkage using the known phosphoramidite
3616 and deprotection afforded the target compound 5 in high
yield and purity.
To probe the effect of the inositol ring in combination with the
above changes to the phosphatidyl group, phosphatidylglycerol
dimannoside monoether analogue 6 (PGM2ME), where the
inositol ring is replaced by a glycerol unit, was prepared. Pre-
paration of the dimannosylated glycerol derivative was effected
by DDQ deprotection of the known PMB ether 3912 to give the
desired headgroup 40 in 72% yield. Reaction of the dimannosy-
lated glycerol alcohol 40 and phosphoramidite 36 provided the
protected lipid 41 (Scheme 6). Hydrogenolytic debenzylation to
41 and selective hydrolysis of the acetates gave the sn-2 ether
linked PGM2 analogue 6. In a similar manner the bis-acylated
congener 7 was prepared from dimannosylated glycerol 40 and
phosphoramidite 43.
PIM Analogues Induce Interleukin-6 (IL-6) Production and
Negatively Regulate LPS Signaling in Primary Peritoneal
Macrophages. TLRs play a key role in the innate immune re-
sponse of macrophages to exogenous pathogens via recognition
of pathogen-associated molecular patterns.34À36 Whether any spe-
cific interaction(s) occur between PIMs and TLRs is currently
chloride (AZMBCl).33 The AZMB protecting group was chosen
to provide α-selectivity in the glycosylation, together with an
orthogonal mode of deprotection.
Coupling of 16 and 31 using standard thioglycosylation con-
ditions afforded the α,α-product 34 in 34% yield (Scheme 5).
The two anomeric 13C NMR signals of 34 were coincident; how-
1
ever, JCH values for these signals (both 175 Hz) could be ex-
tracted from a HSQC experiment run without 13C decoupling
during acquisition. As observed previously (vide infra), some
β-coupled product was also formed which could be removed by
column chromatography.30 Deallylation followed by installation
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Scheme 5. Synthesis of 5
Scheme 6. Synthesis of Targets 6 and 7
not clear, but PIM molecules have been reported to act as agonists of
TLR2 and TLR46,8,37,38 and antagonists of LPS14 signaling in
macrophages. For these reasons, we assessed the ability of the
synthetic PIM analogues to both directly activate primary
macrophages and modulate LPS-induced macrophage activation.
Primary peritoneal macrophages were isolated from mice and
the cells treated with synthetic PIM analogues in vitro. Consis-
tent with previous reports of agonist activity of PIM molecules,8,38
all of the PIM analogues tested induced macrophage IL-6 pro-
duction (Figure 2B, Table 1). In all cases, peak IL-6 production
was observed between 0.6 and 3.1 μM PIM and decreased at higher
concentrations without the appearance of cytoxicity39 (Figure 2B
and Supporting Information). Self-regulation of TLR agonist
activity has been previously reported for both TLR4 and TLR2.40
Therefore, it is likely that the observed pattern of cytokine pro-
duction for the PIM analogues also results from the engagement
of negative feedback mechanisms designed to regulate PIM agonist
activity via TLRs.
contrast, inclusion of two ether groups, as with compound 8, or
exchanging the ether for an amide group, as with compound 3,
resulted in decreased cytokine production compared to synthetic
PIM2 (2). Interestingly, removal of the inositol ring but retention
of the natural glycerol diester (2 vs 7) resulted in a significant loss
of agonist activity that was recovered by introducing the sn-2
monoether (6 vs 7). Agonist activity was also lost with the
addition of a third lipid group, as exemplified by AcPIM2ME (5).
Taken together, these results indicate that the inositol ring
contributes to the agonist activity and that agonist activity is
enhanced with a combination of ester and ether linkages on the
phosphatidyl group.
Next, we investigated whether PIM treatment enhanced or
suppressed LPS induced IL-6 production by primary macro-
phages. Previous work on bone-marrow-derived macrophages
showed that PIM molecules are able to inhibit LPS-induced
cytokine production.14 Consistent with this finding, the majority
of the PIM analogues inhibited LPS-induced macrophage IL-6
production in a dose-dependent manner (Figure 2C and Table 2).
In general, suppressive activity fell in the same concentration range
as that observed for shutdown of agonist activity. Whether the same
regulatory mechanisms are involved in both the inhibition of PIM
agonist activity and PIM-dependent inhibition of LPS-induced IL-6
production has yet to be determined.
Consistent with our earlier study of dendritic cell responses,16
the inclusion of an ether rather than an ester group on the sn-2
position of glycerol in PIMs increased the induction of IL-6
(Table 1, 1 vs 2 or 6 vs 7). Moving the ether to the sn-1 position
resulted in a decrease in IL-6 production (Table 1, 1 vs 4); however,
compound 4 still enhanced IL-6 secretion compared to 2. In
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Figure 2. Agonist and antagonist activity of PIM compounds. Thioglycollate-induced peritoneal macrophages were treated with (A) LPS (10 ng/mL)
or (B) 1 alone for 18 h. Peritoneal macrophages were treated with LPS (10 ng/mL) in the presence of (C) 1 or (D) 5 for 18 h. Supernatants were
collected and the levels of IL-6 determined by ELISA. Data are representative of at least two replicate experiments. Statistical significance was calculated
by one-way ANOVA: (//) p < 0.01, (///) p < 0.001 compared to PBS control.
Table 1. PIM-Induced IL-6 Production by Thioglycollate-
Elicited Macrophages
compd
IL-6max (pg/mL)
SE
structural variation
1
2
3
4
5
6
7
8
1456
817
664
1130
275
923
143
494
161
210
332
33
PIM2, sn-2 ether, sn-1 ester
PIM2, sn-2 and sn-1 both esters
PIM2, sn-2 amide, sn-1 ester
PIM2, sn-2 ester, sn-1 ether
AcPIM2, sn-2 ether, sn-1 ester
PGM2, sn-2 ether, sn-1 ester
PGM2, sn-2 and sn-1 both esters
PIM2, sn-2 and sn-1 both ethers
62
197
31
Figure 3. Agonist activity of 2 in thioglycollate-induced macrophages is
TLR4 dependent. Data are representative of at least two replicate
experiments. Statistical significance between the curves was calculated
by two-way ANOVA: p < 0.0007.
161
Table 2. Inhibition of IL-6 Production by LPS-Stimulated
Thioglycollate-Elicited Macrophages
complex that leads to activation of the intracellular signaling
cascade.41À43 To reveal the molecular basis whereby 2 negatively
regulates LPS signaling, we examined the effect of 2 on LPS-
mediated formation of the active 2:2 TLR4/MD-2 homodimer.
As shown by native PAGE, LPS induces the homodimerization of
a purified protein complex between the TLR4 ectodomain
(sTLR4) and MD-2 (Figure 4A). This LPS-mediated sTLR4/
MD-2 homodimerization was partially inhibited by 2 (Figure 4A).
Given that for TLR4 activation LPS is deeply inserted into the
hydrophobic cavity of MD-2, we speculated that 2 would inhibit
2:2 TLR4/MD-2 complex formation by competing with LPS for
the MD-2 cavity. However, the direct interaction of MD-2 with
LPS or 2 could not be addressed because of technical difficulty in
recombinant expression of MD-2 alone. Instead, an MD-2 homo-
logue, MD-1, was used as an MD-2 model system. MD-1 has
been implicated in the regulation of LPS-induced immune re-
sponses and, like MD-2, houses a large hydrophobic cavity that is
able to accommodate LPS and presumably other lipo-gylcan
structures such as PIM.44,45 In native gels, 2 was shown to associate
with MD-1 in a dose-dependent manner and to compete with
LPS for MD-1 binding (Figure 4B), supporting that 2 directly
interacts with MD-2. Taken together, these data suggest that
PIM molecules, like MPL,43 act as partial agonists of TLR4 and
likely inhibit LPS signaling by interfering with LPS binding to the
TLR4/MD-2 complex.
CN
IC50 (μM)
SE
structural variation
1
2
3
4
6
7
8
30.3
40.2
22.7
28.8
42.8
12.9
23.3
4.9
6.1
1.8
3.8
5.1
3.0
5.0
PIM2, sn-2 ether, sn-1 ester
PIM2, sn-2 and sn-1 both esters
PIM2, sn-2 amide, sn-1 ester
PIM2, sn-2 ester, sn-1 ether
PGM2, sn-2 ether, sn-1 ester
PGM2, sn-2 and sn-1 both esters
PIM2, sn-2 and sn-1 both ethers
Removal of the inositol moiety in 7 (IC50 = 12.9 μM) resulted
in increased inhibitory activity and decreased agonist activity
compared to 2 (IC50 = 40.2 μM). Therefore, it appeared that the
presence of an inositol ring abrogated inhibitory activity.
Interestingly, only 5 enhanced LPS-induced IL-6 production
(Figure 2D). The difference between 1, which exhibited inhibi-
tory activity, and 5 is the addition of C-16 (palmitic acid) fatty
acyl chain to one of the mannosyl moieties. As such, the observed
switch from inhibition to enhancement of LPS-induced IL-6
production may be associated with the greater lipophilicity of 5.
PIM Analogue Activities Act via TLR4. To determine
whether PIMs and their analogues may induce cytokine produc-
tion via TLRs, macrophages from wild type C57 and TLR4-
deficient mice were treated with 2. IL-6 production was abro-
gated in TLR4-deficient macrophages, indicating that 2 activates
macrophages via the TLR4 signaling pathway (Figure 3).
A key event required for LPS signaling via the TLR4 pathway is
LPS binding to a heterodimeric 1:1 complex between TLR4 and
its co-receptor, myeloid differentiation factor 2 (MD-2), which
then induces homodimerization of the 1:1 complex to form a 2:2
In summary, we present concise syntheses of PIM analogues 3,
4, and 5, utilizing the readily available mannosylated chiral inositol
16, and 6 and 7 from a dimannosylated glycerol intermediate. In
general, the PIMs and their analogues exhibit partial agonist
activity, which is associated with the inositol ring. Furthermore,
this activity is enhanced by replacement of a fatty acid ester
moiety with that of the corresponding ether on the phosphatidyl
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Figure 4. Compound 2 interferes with LPS binding to TLR4/MD-2 (A) and MD-1 (B) as shown by native PAGE. 2 binding to MD-1 results in a band
shift (B, left), whereas 2-induced sTLR4/MD-2 band shift was not observed (A, left) potentially because of the large size of sTLR4/MD-2 (87 kDa)
compared to MD-1 (17 kDa).
group. SAR for the inhibition of LPS signaling resulted in two
notable observations. First, loss of the inositol ring enhanced the
antagonist activity, and somewhat surprisingly, addition of a third
lipid moiety may synergize with LPS to enhance proinflamma-
tory activation of macrophages. For the first time, we also show
that the activities of PIMs involve regulation of TLR4 signaling,
potentially via interactions with TLR4/MD-2. Together, these
findings identify key structural features that could be used to
direct the synthesis of lipoglycans with optimal agonist, antago-
nist, or adjuvant activities and identify an important role for TLR4 in
the bioactivity of PIMs.
δ 8.11À8.05 (m, 2H), 7.57À7.50 (m, 1H), 7.41À7.15 (m, 17H), 5.71
(dd, J = 1.8, 2.7 Hz, 1H), 5.40 (d, J = 1.8 Hz, 1H), 4.87À4.74 (m, 3H),
4.60À4.49 (m, 3H), 4.37À4.30 (m, 1H), 4.20 (dd, J = 3.9, 5.4 Hz, 1H),
4.16À4.05 (m, 2H), 4.01À3.85 (m, 4H), 3.84À3.70 (2H, m), 3.36 (ddd,
J = 1.8, 8.7, 10.1 Hz, 1H), 2.57 (d, J = 1.8 Hz, 1H), 2.00À1.80 (m, 2H),
1.74À1.57 (m, 2H), 1.46 (d, J = 12.9 Hz, 1H), 1.40À1.13 (m, 3H),
1.12À0.94 (m, 30H), 0.82 (s, 3H), 0.86 (s, 3H); 13C NMR (75 MHz,
CDCl3) δ 165.7, 138.9, 138.7, 138.3, 132.9, 130.0, 128.3, 128.24, 128.19,
128.1, 128.0, 127.9, 127.5, 127.4, 127.3, 117.7, 96.8, 79.9, 78.6, 77.4, 77.0,
76.9, 76.6, 76.3, 75.7, 75.1, 74.5, 73.3, 72.9, 72.6, 71.6, 71.5, 69.4, 69.2,
51.5, 48.0, 45.2, 45.1, 29.4, 27.1, 20.4, 20.2, 17.6, 17.4, 17.31, 17.26, 17.2,
17.1, 17.0, 13.0, 12.7, 12.5, 12.3, 12.1, 11.7, 9.7. HRMS-ESI [M + Na]+
calculated for C62H84O13Si2Na: 1115.5348. Found 1115.5365.
’ EXPERIMENTAL SECTION
6-(3,4,5-Tri-O-benzyl-α-D-mannopyranosyl)-3,4-O-(1,1,3,3-
tetraisopropyldisiloxanedi-1,3-yl)-1,2-O-((1S,4S)-1,7,7-trime-
thyl[2.2.1]bicyclohept-6-ylidene)-D-myo-inositol (12). Sodium
methoxide in MeOH (30% solution, 0.58 mL) was added dropwise to a
stirred solution of benzoate 11 (3.91 g, 3.58 mmol) in CH2Cl2/MeOH
(1:1, 50 mL). After 22 h, the mixture was partitioned between Et2O
(100 mL) and saturated NH4Cl (100 mL), dried (MgSO4), filtered
and the solvent removed. The crude residue was purified on silica gel
(EtOAc/petroleum ether = 1:9) to afford the title compound 12 (2.75 g,
78%) as a pale yellow oil. [α]20D +33 (c 0.5, CH2Cl2); 1H NMR (300
MHz, CDCl3) δ 7.38À7.15 (m, 15H), 5.38 (d, J = 1.2 Hz, 1H), 4.82
(d, J = 11.0 Hz, 1H), 4.75À4.61 (m, 3H), 4.59À4.45 (m, 2H), 4.31À4.21
(m, 1H), 4.21À4.15 (m, 1H), 4.15À4.08 (m, 1H), 3.97À3.83 (m, 5H),
3.83À3.71 (m, 2H), 3.71À3.61 (m, 1H), 3.31 (ddd, J = 1.5, 8.3, 9.8 Hz,
1H), 2.61À2.56 (m, 1H), 2.50À2.42 (m, 1H), 2.02À1.87 (m, 2H),
1.78À1.64 (m, 2H), 1.49À1.34 (m, 2H), 1.28À1.14 (m, 1H), 1.13À0.82
(m, 37H); 13C NMR (75 MHz, CDCl3) δ 138.7, 138.6, 138.2, 128.4,
128.2, 128.0, 127.9, 127.82, 127.76, 127.4, 127.3, 117.7, 98.2, 80.3, 79.7,
77.4, 77.2, 77.0, 76.9, 76.6, 76.4, 75.8, 74.9, 74.5, 73.2, 73.0, 72.4, 72.0,
70.9, 68.9, 68.8, 51.5, 48.0, 45.4, 45.1, 29.6, 27.1, 20.5, 20.2, 17.6, 17.4,
17.34, 17.28, 17.25, 17.22, 17.1, 17.0, 13.0, 12.7, 12.3, 12.1, 9.7. HRMS-
ESI [M + NH4]+ calculated for C55H80O12Si2NH4: 1006.5532. Found
1006.5491.
General Experimental Information. NMR spectra are refer-
enced to tetramethylsilane (TMS) (0.0 ppm) or the residual solvent
peak (1H CHCl3 δ 7.26; 13C CDCl3 δ 77.0) or to an external reference
(31P H3PO4 δ 0.0). Anhydrous solvents were sourced commercially and
used without further treatment unless otherwise stated. Powdered mole-
cular sieves were flame-dried under vacuum immediately prior to use.
Flash column chromatography was carried out using 40À63 μm silica
gel unless otherwise stated. All flash chromatography solvents were AR-
grade. Petroleum ether used was one with bp 60À80 ꢀC range. All
compounds were isolated after silica gel column chromatography, and
fractions collected were one spot by thin layer chromatography. Thin
layer chromatography (TLC) plates were visualized under an UV lamp
and/or with a spray consisting of 5% w/v dodecamolybdophosphoric
acid in ethanol with subsequent heating. See Supporting Information for
HPLC conditions.
HPLC purities of target compounds are as follows: 1 (97%), 2 (95%),
3 (94%), 4 (96%), 5 (95%), 6 (96%), 7 (95%), and 8 (97%).
6-(2-O-Benzoyl-3,4,5-tri-O-benzyl-α-D-mannopyranosyl)-
3,4-O-(1,1,3,3-tetraisopropyldisiloxanedi-1,3-yl)-1,2-O-((1S,4S)-
1,7,7-trimethyl-[2.2.1]bicyclohept-6-ylidene)-D-myo-inositol
(11). TMSOTf (0.20 mL, 1.1 mmol) was added to a mixture of diol 9
(6.00 g, 10.8 mmol), trichloroacetamide 10 (7.53 g, 10.77 mmol), and 4
Å molecular sieves in dry Et2O (100 mL) at À70 ꢀC. After the mixture
was stirred for 45 min, the reaction was quenched with saturated
NaHCO3 (100 mL) and extracted with Et2O (3 Â 60 mL) and washed
with water (100 mL), dried (MgSO4), filtered and the solvent removed.
The crude residue was purified on silica gel (EtOAc/petroleum ether = 1:9)
to afford compound 11 (8.95 g, 76%) as a white foam. [α]20D À1.5 (c 0.4,
CH2Cl2); [α]20365 À26 (c 0.4, CH2Cl2); 1H NMR (300 MHz, CDCl3)
3,4,5-Tri-O-benzyl-6-(2,3,4,5-tetra-O-benzyl-α-D-manno-
pyranosyl)-1,2-O-((1S,4S)-1,7,7-trimethyl[2.2.1]bicyclohept-
6-ylidene)-D-myo-inositol (14). TBAF (1 M, 7.8 mL) was added to
a solution of 12 (3.10 g, 3.13 mmol) in THF (50 mL). After the mixture
was stirred for 1 h, the solvent was removed. The residue was purified by
flash chromatography on silica gel (MeOH/CH2Cl2 = 3:97 to 1:24) to
afford partially purified 13 (2.8 g). 1H NMR (300 MHz, CDCl3) inter
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Journal of Medicinal Chemistry
ARTICLE
alia δ 7.38À7.16 (m, 15H), 5.18 (d, J = 1.5 Hz, 1H), 4.81 (d, J = 11.0 Hz,
1H), 4.72 (d, J = 11.4 Hz, 1H), 4.67 (d, J = 11.4 Hz, 1H), 4.61À4.45 (m,
3H), 4.28 (dd, J = 4.4, 5.4 Hz, 1H), 3.94À3.84 (m, 2H), 3.82À3.50 (m,
7H), 3.21 (dd, J = 3.0, 9.4 Hz, 1H), 3.06 (br s, 1H), 2.89 (br s, 1H), 2.67
(d, J = 2.3 Hz, 1H), 2.55 (d, J = 7.0 Hz, 1H), 2.06À1.85 (m, 2H),
1.80À1.64 (m, 1H), 1.53À1.33 (m, 2H), 1.28À1.14 (m, 1H), 0.98 (s,
3H), 0.86 (s, 3H), 0.82 (s, 3H). HRMS-ESI [M + NH4]+ calculated for
C43H54O11NH4: 764.4010. Found 764.4013.
the title compound 21 (207 mg, 66%) as an oil. [α]20 À2.8 (c 0.72,
consistent with those previously reported.28,46
Benzyl (2-O-Hexadecanoyl-1-O-hexadecyl-sn-glycero)di-
isopropylphosphoramidite (17). 1H-Tetrazole (35 mg, 0.50 mmol)
was added to a stirred solution of alcohol 21 (205 mg, 0.369 mmol) and
benzyloxy-bis(diisopropylamino)phosphine (262 mg, 0.775 mmol) in
dry CH2Cl2 (20 mL) at room temperature for 90 min. The solvent was
removed and the residue purified by column chromatography on silica
D
CHCl3); lit.46 = À2.6 (c 2.1, CHCl3); lit.28 = À1.2. Analytical data were
BnBr (3.7 mL, 31 mmol) was added dropwise to a stirred solution of
the crude tetraol 13 and NaH (0.94 g, 60% in mineral oil, 23.5 mmol) in
dry DMF (75 mL) at 0 ꢀC. The mixture was left to slowly warm to room
temperature over 80 min before being diluted with Et2O (300 mL) and
quenched by the slow addition of water (150 mL). The aqueous phase
was re-extracted with Et2O (2 Â 100 mL), dried (MgSO4), filtered and
the solvent removed. The crude residue was purified on silica gel
(EtOAc/petroleum ether = 3:7) to afford the title compound 14
gel (Et3N/EtOAc/petroleum ether = 3:10:90) to afford the title com-
1
pound 17 (267 mg, 91%) as an oil. [α]20 +4.9 (c 0.72, CHCl3). H
D
NMR (300 MHz, CDCl3) δ 7.37À 7.23 (m, 5H), 5.13 (quintet, J = 5.2
Hz, 1H), 4.74À 4.62 (m, 2H), 3.83À3.35 (m, 8H), 2.30 (t, J = 7.3 Hz,
2H), 1.65À1.48 (m, 4H), 1.32À1.16 (m, 62H), 0.90À0.83 (m, 6H);
13C NMR (75 MHz, CDCl3) δ 173.3, 139.6, 128.3, 127.3, 127.0, 72.2,
71.7, 69.2, 65.5, 65.3, 62.3, 62.1, 61.9, 43.2, 43.1, 34.6, 32.0, 29.8, 29.7,
29.6, 29.4, 29.2, 26.2, 25.1, 24.8, 24.7, 24.6, 22.8, 14.2. 31P NMR (121.5
MHz, CDCl3) δ 149.5, 149.2. HRMS-ESI [M + H]+ calculated for
C48H91NO5P: 729.6635. Found 792.6638.
(3.12 g, 90%). [α]20 +19 (c 1.7, CH2Cl2); 1H NMR (300 MHz,
D
CDCl3) δ 7.45À7.00 (m, 35H), 5.53 (d, J = 1.4 Hz, 1H), 4.89 (d, J = 11.0
Hz, 1H), 4.82À4.50 (m, 12H), 4.35 (d, J = 12.1 Hz, 1H), 4.25 (dd,
J = 3.4, 6.4 Hz, 1H), 4.09 (dd, J = 8.8, 18.4 Hz, 1H), 4.03À3.95 (m, 2H),
3.90 (dd, J = 3.1, 9.4 Hz, 1H), 3.86À3.71 (m, 4H), 3.63 (dd, J = 3.7, 11.3
Hz, 1H), 3.50 (dd, J = 1.3, 11.3 Hz, 1H), 3.27 (dd, J = 7.1, 9.7 Hz, 1H),
2.00À1.87 (m, 2H), 1.80À1.65 (m, 2H), 1.49À1.33 (m, 2H), 1.29À1.12
(m, 1H), 1.06 (s, 3H), 0.88 (s, 3H), 0.86 (s, 3H); 13C NMR (75 MHz,
CDCl3) δ 139.1, 138.8, 138.6, 138.5, 138.1, 128.4, 128.3, 128.23, 128.16,
128.1, 127.9, 127.81, 127.77, 127.6, 127.5, 127.4, 127.2, 117.9, 96.1, 81.1,
80.7, 79.6, 77.9, 76.8, 76.4, 75.0, 74.91, 74.85, 74.7, 74.5, 73.8, 73.1, 72.5,
72.1, 71.8, 68.9, 51.6, 47.9, 45.1, 44.9, 29.9, 27.2, 20.6, 20.4, 9.8. HRMS-ESI
[M + NH4]+ calculated for C71H78O11NH4: 1124.5888. Found 1124.5896.
3,4,5-Tri-O-benzyl-6-(2,3,4,5-tetra-O-benzyl-α-D-manno-
pyranosyl)-D-myo-inositol (15). HCl (37%, 2 mL) was added to a
stirred solution of 9 (322 mg, 0.291 mmol) in dioxane/methanol (2:5,
35 mL) at room temperature. After 48 h, the mixture was diluted with
Et2O (100 mL) and washed with water (100 mL), the aqueous layer was
re-extracted with Et2O (50 mL), the combined organic fractions were
washed with saturated NaHCO3 (100 mL), brine (100 mL), dried
(MgSO4), and filtered, and the solvent was removed. The crude residue
was purified on silica gel (EtOAc/petroleum ether = 1:3 to 2:3) to afford
the title compound 15 (245 mg, 86%). Analytical data were consistent
with those previously reported.26
3-O-Benzyl-2-deoxy-1-O-hexadeconyl-2-hexadeconyla-
mino-sn-glycerol (23). Palmitoyl chloride (1.54 mL, 5.08 mmol)
was added dropwise to a stirred solution of (R)-(+)-2-amino-3-benzyl-
oxy-1-propanol (22) (230 mg, 1.27 mmol) and DMAP (621 mg, 5.08
mmol) in dry CHCl3 (25 mL) at 0 ꢀC. After warming to room tem-
perature over 6 h, the mixture was diluted with CHCl3 (30 mL) and
washed with H2O (30 mL), then 0.5 M HCl (30 mL), dried (MgSO4),
filtered and the solvent removed. The crude residue was purified by
column chromatography (EtOAc/petroleum ether = 1:4) to afford the
1
title compound 23 (705 mg, 84%) as a gummy solid. H NMR (300
MHz, CDCl3) δ 7.38À7.26 (m, 5H), 4.79 (d, J = 8,6 Hz, 1H), 4.51 (br s,
2H), 4.42À4.31 (m, 1H), 4.25 (dd, J = 6.1, 10.9 Hz, 1H), 4.13 (dd, J =
6.0, 10.9 Hz, 1H), 3.59 (dd, J = 3.4, 9.6 Hz, 1H), 3.48 (dd, J = 4.7, 9.6 Hz,
1H), 2.26 (dd, J = 7.4, 7.6 Hz, 2H), 2.14 (dd, J = 7.4, 7.8 Hz, 2H),
1.65À1.53 (m, 4H), 1.30À1.24 (m, 48H), 0.91À0.85 (m, 6H); 13C
NMR (75 MHz, CDCl3) δ173.8, 172.9, 137.8, 128.5, 128.0, 127.8, 73.4,
68.8, 63.1, 48.0, 36.9, 34.3, 32.0, 29.9, 29.2, 25.8, 25.0, 22.8, 14.2. HRMS-
ESI [M + Na]+ calculated for C42H75NO4Na: 680.5594. Found 680.5588.
2-Deoxy-1-O-hexadeconyl-2-N-hexadeconylamino-sn-gly-
cerol (24)29. Pd(OH)2/C (20%, 200 mg) was added to a mixture of 23
(663 mg, 1.01 mmol) in AcOH/EtOH (1:10, 33 mL). The mixture was
stirred under an H2 atmosphere for 16 h at room temperature, then filtered
through Celite and the solvent removed. The residue was purified by
column chromatography on silica gel (EtOAc/CH2Cl2 = 3:7) to afford
the title compound 24 (555 mg, 0.98 mmol, 97%) as a white solid. 1H
NMR (300 MHz, CDCl3) δ 6.02 (d, J = 6.6 Hz, 1H), 4.27À4.12 (m,
3H), 3.72À3.56 (m, 2H), 2.96 (br s, 1H), 2.33 (dd, J = 7.6, 7.6 Hz, 2H),
2.19 (dd, J = 7.6, 7.6 Hz, 2H), 1.67À1.55 (m, 4H), 1.35À1.19 (m, 48H),
0.91À0.84 (m, 6H); 13C NMR (75 MHz, CDCl3) δ 177.6, 173.8, 62.5,
62.0, 50.5, 36.8, 34.3, 32.0, 30.0, 29.2, 25.8, 25.0, 22.8. HRMS-ESI
[M + Na]+ calculated for C35H69NO4Na: 590.5124. Found 590.5118
2-Deoxy-1-O-hexadeconyl-2-N-hexadeconylamino-sn-gly-
cero-3-O-benzyl-(N,N-diisopropyl)phosphoramidite (18). 1H-
Tetrazole (28 mg, 0.397 mmol) was added to a stirred solution of
alcohol 24 (205 mg, 0.361 mmol) and benzyloxy-bis(diisopropylamino)-
phosphine (244 mg, 0.722 mmol) in dry CH2Cl2 (10 mL) at room
temperature for 60 min. The solvent was removed and the residue
purified by column chromatography on silica gel (Et3N/EtOAc/petroleum
ether = 3:10:90) to afford the title compound 18 (280 mg, 96%) as a clear
oil that solidified on standing. 1H NMR (300 MHz, CDCl3) δ 7.37À7.25
(m, 5H), 4.89À4.58 (m, 2H), 4.38À4.25 (m, 1H), 4.22À4.04 (m, 2H),
3.87À3.56 (m, 2H), 2.28 (dd, J = 7.6, 7.6 Hz, 2H), 2.12À2.04 (m, 1H),
1.94À1.87 (m, 1H), 1.64À1.45 (m, 4H), 1.33À1.17 (m, 60H), 0.91À0.85
(m, 6H); 31P NMR (121.5 MHz, CDCl3) δ 150.3, 149.9. HRMS-ESI
[M + Na] calculated for C48H89N2O5Na: 827.6407. Found 827.6407.
2-O-Hexadecanoyl-1-O-hexadecyl-3-O-(4-methoxybenzyl)-
sn-glycerol (20). Palmitoyl chloride (0.300 mL, 0.982 mmol) was added
dropwise to a stirred solution of alcohol 19 (270 mg, 0.618 mmol) and dry
pyridine (0.300 mL, 3.71 mmol) in dry CH2Cl2 (15 mL) at 0 ꢀC. The
reaction mixture was allowed to warm to room temperature over 17 h
before being quenched with water (100 mL). The mixture was extracted
with Et2O (2 Â 150 mL) and the ethereal extract washed with 0.5 M HCl
(100 mL), saturated NaHCO3 (100 mL), dried(MgSO4), filtered, and the
solvent removed. The crude residue was purified by column chromatog-
raphy on silica gel (EtOAc/petroleum ether = 0:1 to 1:9) to afford the title
compound 20 (384 mg, 92%) as a colorless oil. [α]20 +1.0 (c 7.7,
D
CHCl3). 1H NMR (300 MHz, CDCl3) δ 7.27À7.21 (m, 2H), 6.90À6.83
(m, 2H), 5.16 (quintet, J = 5.1 Hz, 1H), 4.49À4.43 (m, 2H), 3.79 (s, 3H),
3.60À3.55 (m, 4H), 3.44À3.35 (m, 2H), 2.32 (t, J = 7.5 Hz, 2H),
1.64À1.49 (m, 4H), 1.40À1.20 (m, 50H), 0.92À0.83 (m, 6H); 13C NMR
(75 MHz, CDCl3) δ173.4, 159.3, 130.2, 129.3, 113.8, 73.0, 71.7, 71.4, 69.3,
68.6, 55.3, 34.5, 32.0, 29.8, 29.6, 29.4, 29.2, 26.1, 25.1, 22.8, 14.2. HRMS-
ESI [M + Na]+ calculated for C43H78O5Na: 697.5747. Found 697.5734.
2-O-Hexadecanoyl-1-O-hexadecyl-sn-glycerol (21). Pd-
(OH)2/C (20%, 88 mg) was added to a mixture of 20 (380 mg, 0.563
mmol) in AcOH/EtOH (1:10, 16.5 mL). The mixture was stirred under
an H2 atmosphere for 4 h at room temperature, then filtered through
Celite and the solvent removed. The residue was purified by column
chromatography on silica gel (EtOAc/CH2Cl2 = 1:49 to 1:19) to afford
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Journal of Medicinal Chemistry
ARTICLE
1-O-Allyl-3,4,5-tri-O-benzyl-6-(2,3,4,5-tetra-O-benzyl-α-D-
mannopyranosyl)-2-O-(3,4,5-tri-O-benzyl-α-D-mannopyra-
nosyl)-D-myo-inositol (26). TMSOTf (12 μL, 0.066 mmol) was
added to a mixture of alcohol 1626 (0.339 g, 0.39 mmol) and trichlor-
oacetamide 10 (0.46 g, 0.66 mmol) and 4 Å molecular sieves in dry Et2O
(50 mL) at À40 ꢀC. After the mixture was stirred for 1 h, the reaction
was quenched with Et3N, filtered through Celite, and evaporated to
dryness. The crude residue was purified on silica gel (EtOAc/petroleum
ether = 1:9 to 1:4) to afford the intermediate compound 25 (0.473 g) as
an inseparable mixture of anomers. 1H NMR (300 MHz, CDCl3) inter
alia δ 5.92 (d, J = 2.9 Hz, 0.2H), 5.79À5.60 (m, 2H), 5.58À5.53 (m,
0.8H); 13C NMR (75 MHz, CDCl3) inter alia δ 99.2 (1JCH = 175 Hz),
0.89À0.82 (m, 6H). 13C (75 MHz, CDCl3) selected signals δ 173.3,
99.9, 98.9. 31P NMR (121.5 MHz, CDCl3) δ 0.26, 0.00. HRMS-ESI [M
+ Na]+ calculated for C137H173O22PNa: 2224.2054. Found 2224.2051.
2,6-(Di-O-α-D-mannopyranosyl)-1-O-(2-O-hexadecanoyl-
1-O-hexadecyl-sn-glycero-3-phosphoryl)-D-myo-inositol (4).
Pd(OH)2/C (20%, 25 mg) was added to a stirred solution of 29 (68
mg, 0.031 mmol) in THF/MeOH (2:3, 5 mL). The mixture was stirred
under an H2 atmosphere for 4 h at room temperature, then filtered
through Celite and the solvent removed. The residue was purified by
column chromatography on silica gel (CHCl3/MeOH/H2O = 70:40:0
to 70:60:6) to afford the title compound 4 (26 mg, 74%) as a white
powder. [α]20D +39 (c 0.40, CHCl3/MeOH/H2O = 70:60:6); 1H NMR
(500 MHz, CDCl3/CD3OD/D2O = 70:40:6) δ 5.28À5.20 (m, 1H),
5.15 (br s, 1H), 5.12 (br s, 1H), 4.33 (m, 1H), 4.12À3.96 (m, 7H),
3.87À3.60 (m, 12H), 3.57À3.42 m, 3H), 3.30 (t, J = 7.0 Hz, 1H), 2.41
(t, J = 7.2 Hz, 2H), 1.65À1.55 (m, 4H), 1.33À1.22 (m, 50H), 0.92À0.85
(m, 6H); 13C (125 MHz, CDCl3/CD3OD/D2O = 70:40:6) δ 176.0,
103.0, 102.9, 79.9, 79.8, 78.2, 78.1, 74.8, 74.4, 74.4, 74.2, 73.5, 73.5, 73.0,
72.1, 72.1, 71.8, 71.6, 70.7, 68.4, 68.4, 65.4, 65.4, 62.8, 62.6, 35.7, 33.2,
31.0, 30.9, 30.7, 30.6, 30.4, 27.4, 26.4, 23.9, 15.0, 10.0; 31P NMR (121.5
MHz, CDCl3/CD3OD/D2O = 70:40:6) δ 0.63. HRMS-ESI [MÀH]À
calculated for C53H100O22P: 1119.6444. Found 1119.6456.
3,4,5-Tri-O-benzyl-2,6-di-O-(2,3,4,6-tetra-O-benzyl-α-D-man-
nopyranosyl)-1-O-(2-deoxy-3-benzylphosphoryl-1-O-hexa-
deconyl-2-O-hexadeconylamino-sn-glycero)-D-myo-inositol
(30). 1H-Tetrazole (10 mg, 0.135 mmol) was added to a stirred solution
of alcohol 28 (68 mg, 0.045 mmol) and phosphoramidite 18 (109 mg,
0.135 mmol) in dry CH2Cl2 (10 mL) at 0 ꢀC under argon. After 1 h, the
reaction mixture was cooled to À40 ꢀC and a dried (MgSO4) solution of
m-CPBA (∼50%, 47 mg, 0.135 mmol) in CH2Cl2 (5 mL) was added to
the reaction. After the mixture was warmed to room temperature over
2 h the reaction was quenched by addition of Na2SO3 (10%, 50 mL) and
extracted with Et2O (50 mL). The organic phase was washed with
saturated NaHCO3 (50 mL), dried (MgSO4), filtered and the solvent
removed. The crude residue was purified by column chromatography on
silica gel (EtOAc/petroleum ether = 1:9 to 1:4) to afford the title com-
pound 30 (61 mg, 62%) as an oil. 1H NMR (300 MHz, CDCl3) mixture
of isomers δ 7.48À7.03 (m, 60H), 6.54 (d, J = 7.5 Hz, 3H), 6.40 (d,
J = 8.6 Hz, 7H), 5.48 (br s, 3H), 5.44 (br s, 7H), 5.10À5.42 (m, 21H),
4.51À4.27 (m, 5H), 4.26À3.66 (m, 15H), 3.52À3.12 (m, 6H), 2.37À2.27
(m, 1H), 2.17À2.09 (m, 1H), 2.03À1.95 (m, 2H), 1.71À1.37 (m, 4H),
1.35À1.12 (m, 48H), 0.91À0.84 (m, 6H); 13C NMR (125 MHz,
CDCl3) selected signals δ 173.8, 173.5, 173.2, 173.1, 98.8, 98.7, 98.4,
98.1; 31P NMR (121.5 MHz, CDCl3) δ 0.94, À0.45. HRMS-ESI
[M+Na]+ calculated for C137H172NO5PNa: 2237.2006. Found 2237.2007.
2,6-(Di-O-α-D-mannopyranosyl)-1-O-(2-deoxy-1-O-hexa-
decanoyl-2-N-hexadecanoylamino-sn-glycero-3-phosphoryl)-
D-myo-inositol (3). Pd(OH)2/C (20%, 40 mg) was added to a stirred
solution of 28 (28 mg, 24.8 mmol) in THF/MeOH (2:3, 5 mL). The
mixture was stirred under an H2 atmosphere for 16 h at room tem-
perature, then filtered through Celite and the solvent removed. The
residue was purified by column chromatography on silica gel (CHCl3/
MeOH/H2O = 70:40:4) to afford the title compound 3 (21 mg, 75%) as
a white powder. [α]20D +35 (c 0.15, CHCl3/MeOH/H2O = 40:40:10).
1H NMR (500 MHz, CDCl3/CD3OD/D2O = 40:40:10) δ 5.15 (d, J =
1.6 Hz, 1H), 5.10 (d, J = 1.5 Hz, 1H), 4.32À4.28 (m, 2H), 4.15 (dd, J =
8.0, 11.4 Hz, 1H), 4.08À4.03 (m, 3H), 4.01À3.93 (m, 4H), 3.85À3.78
(m, 5H), 3.77À3.59 (m, 5H), 3.48 (dd, J = 7.6, 10.1 Hz, 1H), 3.30 (dd,
J = 9.2, 9.3 Hz, 1H), 3.16 (q, J = 7.3 Hz, 6H), 2.33 (dd, J = 8.3, 6.9 Hz,
2H), 2.24 (dd, J = 7.5, 7.5 Hz, 2H), 1.65À1.55 (m, 4H), 1.31 (t, J = 7.3
Hz, 9H), 1.29À1.26 (m, 48H), 0.91À0.87 (m, 6H); 13C NMR (125
MHz, CDCl3/CD3OD/D2O = 40:40:10) δ176.3, 175.5, 102.4, 102.2,
79.3, 77.55, 77.50, 74.1, 73.8, 73.7, 73.6, 71.5, 71.4, 71.11, 71.07, 70.9,
67.8, 67.7, 65.2, 65.1, 64.2, 62.2, 62.0, 47.4, 37.0, 34.8, 32.6, 30.5, 30.42,
98.9 (1JCH = 173 Hz). HRMS-ESI [M + Na]+ calculated for C98H100
-
O17Na: 1571.6858. Found 1571.6818.
Sodium methoxide in MeOH (30% solution, 0.1 mL) was added
dropwise to a stirred solution of 25 (323 mg) in CH2Cl2/MeOH (1:1,
20 mL). After 24 h, the mixture was quenched by the careful addition of
saturated NH4Cl (50 mL) and extracted with Et2O (2 Â 50 mL), dried
(MgSO4), filtered and the solvent removed. The crude residue was
purified on silica gel (EtOAc/petroleum ether = 1:9 to 3:7) to afford the
title compound 26 (208 mg, 63%) as a colorless oil. 1H NMR (300 MHz,
CDCl3) δ 5.77 (ddd, J = 5.5, 5.5, 10.4, 17.2 Hz, 1H), 4.50 (d, J = 1.4 Hz,
1H), 5.22 (dd, J = 1.4, 17.2 Hz, 1H), 5.21 (d, J = 1.1 Hz, 1H), 5.14 (dd,
J = 1.2, 10.4 Hz, 1H), 4.95À4.50 (m, 16H), 4.49À4.41 (m, 2H),
4.38À4.28 (m, 2H), 4.24À3.75 (m, 13H), 3.52À3.20 (m, 6H), 3.16 (dd,
J = 1.6, 9.7 Hz, 1H), 2.36 (d,, J = 1.9 Hz, 1H); 13C NMR (75 MHz,
CDCl3) δ 139.1, 138.8, 138.71, 138.68, 138.6, 138.5, 138.2, 138.1, 138.0,
137.9, 133.9, 128.6, 128.3, 128.23, 128.17, 128.13, 128.05, 128.02,
127.96, 127.91, 127.87, 127.8, 127.6, 127.5, 127.4, 127.32, 127.27,
127.2, 117.7, 100.1, 98.6, 81.6, 81.5, 81.4, 80.1, 79.5, 78.9, 77.4, 77.2,
77.0, 76.6, 76.1, 75.9, 75.6, 75.3, 75.0, 74.9, 74.8, 74.1, 73.4, 73.1, 72.3,
72.2, 72.1, 71.8, 71.0, 70.84, 70.77, 68.7, 68.2. HRMS-ESI [M + Na]+
calculated for C91H96O16Na: 1467.6596. Found 1467.6563.
1-O-Allyl-3,4,5-tri-O-benzyl-2,6-di-O-(2,3,4,6-tetra-O-ben-
zyl-α-D-mannopyranosyl)-D-myo-inositol (27). NaH (0.054 g,
60% dispersion in mineral oil, 1.4 mmol) was added to a stirred solution
of 26 (0.98 g, 0.68 mmol) and benzyl bromide (0.20 mL, 1.7 mmol) in
dry DMF (30 mL) at 0 ꢀC. The mixture was allowed to warm to room
temperature over 18 h before being diluted with Et2O (50 mL) and
quenched by the slow addition of water (50 mL). The aqueous phase
was re-extracted with Et2O (2 Â 25 mL), dried (MgSO4), filtered and
the solvent removed The crude residue was purified on silica gel
(EtOAc/petroleum ether = 1:9 to 3:7) to afford the title compound
27 (1.01 g, 97%). Analytical data were consistent with those previously
reported.17,31
3,4,5-Tri-O-benzyl-2,6-di-O-(2,3,4,6-tetra-O-benzyl-α-D-man-
nopyranosyl)-1-O-(2-O-hexadecanoyl-1-O-hexadecyl-sn-
glycero-3-benzylphosphoryl)-D-myo-inositol (29). 1H-Tetra-
zole (21 mg, 0.30 mmol) was added to a stirred solution of alcohol 28
(90 mg, 0.060 mmol) and phosphoramidite 17 (135 mg, 0.170 mmol) in
dry CH2Cl2 (7 mL) at 0 ꢀC under argon. After 2 h the reaction mixture
was cooled to À40 ꢀC and a dried (MgSO4) solution of m-CPBA
(∼55%, 90 mg, 0.29 mmol) in CH2Cl2 (10 mL) was added to the
reaction. After the mixture was warmed to room temperature over 1 h,
the reaction was quenched by addition of Na2SO3 (10%, 50 mL) and
extracted with Et2O (50 mL). The organic phase was washed with
saturated NaHCO3 (50 mL), dried (MgSO4), filtered and the solvent
removed. The crude residue was purified by column chromatography on
silica gel (EtOAc/petroleum ether = 1:9 to 3:7) followed by further
purification on silica gel (EtOAc/CH2Cl2 = 1:49 to 1:19) to afford the
title compound 29 (68 mg, 52%) as an oil. 1H NMR (300 MHz, CDCl3)
mixture of isomers δ 7.38À7.00 (m, 60H), 5.53À5.51 (m, 1H), 5.33À5.32
(m, 1H), 5.05À4.40 (m, 23H), 4.30À3.78 (m, 17H), 3.56À3.20 (m,
9H), 2.23À2.10 (m, 2H), 1.58À1.41 (m, 4H), 1.31À1.15 (m, 50H),
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Journal of Medicinal Chemistry
ARTICLE
30.37, 30.35, 30.31, 30.30, 30.26, 30.1, 30.01, 30.00, 29.9, 26.7, 25.5, 23.3,
14.5. 31P NMR (121.5 MHz, CDCl3) δ 4.63. HRMS-ESI [M À H]À
calculated for C53H99NO22P: 1132.6396. Found 1132.6382.
removed. The residue was purified by column chromatography on silica
gel (EtOAc/petroleum ether = 15:85) followed by a second column
(CH2Cl2/petroleum ether/diethyl ether = 30:20:1) to afford the title
compound 34 (88 mg, 34%). [α]20D +19 (c 0.96, CH2Cl2); 1H NMR
(300 MHz, CDCl3) δ 8.03 (dd, J = 1.1, 7.8 Hz, 1H), 7.59À7.50 (m, 2H),
7.41À7.08 (m, 46H), 5.82À5.68 (m, 2H), 5.57 (d, J = 1.6 Hz, 1H),
5.26À5.19 (m, 2H), 5.08À5.03 (m, 1H), 4.94À4.45 (m, 19H),
4.31À3.82 (m, 15H), 3.40 (dd, J = 3.1, 11.3 Hz, 1H), 3.33À3.25 (m,
3H), 3.17 (dd, J = 1.7, 9.7 Hz, 1H), 2.24À2.19 (m, 2H), 1.59À1.51 (m,
2H), 1.33À1.21 (m, 24H), 0.90À0.86 (m, 3H); 13C NMR (75 MHz,
CDCl3) δ 173.3, 165.1, 139.1, 138.85, 138.77, 138.6, 138.4, 138.2, 138.1,
137.7, 137.6, 133.8, 132.7, 131.3, 129.4, 128.7, 128.5, 128.4, 128.32,
128.29, 128.2, 128.1, 128.04, 127.95, 127.82, 127.78, 127.7, 127.5,
127.40, 127.35, 127.21, 127.15, 117.4, 98.9, 81.5, 81.4, 80.9, 80.0, 78.5,
75.9, 75.7, 75.3, 75.1, 74.9, 74.8, 73.6, 73.2, 72.55, 72.50, 72.1, 72.0, 71.5,
70.8, 69.8, 69.1, 68.6, 62.8, 52.9, 34.1, 31.9, 29.7, 29.63, 29.59, 29.5, 29.3,
29.23, 29.20, 24.8, 22.7, 14.1. HRMS-ESI [M + Na]+ calculated for
C108H125N3O18Na: 1774.8856. Found 1774.8870.
2-O-(2-O-[2-Azidomethylbenzoyl]-3,4-di-O-benzyl-6-O-hexa-
decanoyl-α-D-mannopyranosyl)-6-O-(2,3,4,6-tetra-O-ben-
zyl-α-D-mannopyranosyl)-3,4,5-tri-O-benzyl-D-myo-inositol (35).
(1,5-Cyclooctadiene)bis(methyldiphenylphosphine)iridium(I) hexa-
fluorophosphate (29 mg, 34 mmol) was stirred with a solution of 34
(301 mg, 172 mmol) in THF (10 mL) under Ar at room temperature
for 10 min. The mixture was exposed to an atmosphere of H2 for ∼30 s,
during which time the color changed from red to pale yellow, then
stirred under Ar for 45 min, at which point TLC (20% EtOAc/
petroleum ether) indicated complete consumption of the starting
material. The mixture was concentrated under reduced pressure and
dissolved in CH2Cl2/MeOH (2:1, 12 mL) containing 0.45 mL of AcCl.
After 1 h at room temperature, solid NaHCO3 was added and the
mixture stirred for a further 5 min. The reaction mixture was diluted
with water, extracted with CH2Cl2, dried (MgSO4), filtered and the
solvent removed. The crude residue was purified by column chroma-
tography on silica gel (EtOAc/petroleum ether = 1:4) to afford the title
compound 35 (240 mg, 82%). [α]20D +32 (c 1.1, CH2Cl2); 1H NMR
(500 MHz, CDCl3) δ 8.07, (d, J = 7.9 Hz, 1H), 7.59À7.50 (m, 2H),
7.39À7.14 (m, 46H), 5.70À5.69 (m, 1H), 5.40 (d, J = 1.6 Hz, 1H), 5.30
(d, J = 2.0 Hz, 1H), 4.91À4.26 (m, 20H), 4.19À3.76 (m, 11H),
3.64À3.56 (m, 2H), 3.47 (dd, J = 6.6, 10.3 Hz, 1H), 3.36À3.27 (m,
3H), 2.26À2.21 (m, 2H), 1.61À1.52 (m, 2H), 1.33À1.21 (m, 24H),
0.90À0.86 (m, 3H); 13C NMR (75 MHz, CDCl3) δ 173.4, 165.3,
138.53, 138.49, 138.4, 138.2, 138.1, 137.80, 137.76, 137.7, 132.8, 131.4,
129.3, 128.6, 128.44, 128.41, 128.38, 128.30, 128.26, 128.2, 128.0,
127.9, 127.8, 127.7, 127.63, 127.59, 127.54, 127.46, 127.4, 99.0, 96.0,
81.2, 80.4, 80.1, 79.4, 78.2, 76.1, 75.6, 75.3, 75.1, 75.02, 74.97, 74.5,
73.7, 73.3, 72.5, 72.2, 72.14, 72.09, 71.6, 71.1, 69.7, 69.3, 69.2, 62.8,
52.9, 34.1, 31.9, 29.7, 29.62, 29.58, 29.5, 29.3, 29.23, 29.20, 24.8, 22.7,
14.1. HRMS-ESI [M + Na]+ calculated for C105H121N3O18Na: 1734.8543.
Found 1734.8586.
Phenyl 3,4-Di-O-benzyl-6-O-hexadecanoyl-1-thio-α-D-man-
nopyranoside (33). Hexadecanoyl chloride (2.6 mL, 8.5 mmol)
was added dropwise to a stirred solution of diol 3232 (3.50 g, 7.73
mmol) and pyridine (6.25 mL, 77 mmol) in dry CH2Cl2 (30 mL) at
0 ꢀC, and the mixture was allowed to warm to room temperature
overnight. The reaction mixture was diluted with CH2Cl2 (70 mL),
washed with 1 M aqueous HCl (100 mL), saturated aqueous NaHCO3,
dried (MgSO4), filtered and the solvent removed. The residue was
purified by column chromatography on silica gel (EtOAc/petroleum
ether = 1:9 to 3:7) to afford the title compound 33 (4.82 g, 90%) as a
yellow oil. [α]20D +148 (c 1.3, CH2Cl2); 1H NMR (300 MHz, CDCl3)
δ 7.47À7.43 (m, 2H), 7.39À7.21 (m, 13H), 5.57 (d, J = 1.5 Hz, 1H),
4.87 (d, J = 10.8 Hz, 1H), 4.71 (s, 2H), 4.58 (d, J = 10.8 Hz, 1H),
4.38À4.30 (m, 3H), 4.26À4.23 (m, 1H), 3.90, (dd, J = 3.1, 9.0 Hz, 1H),
3.81 (app t, J = 9.2 Hz, 1H), 2.65 (d, J = 2.5 Hz, 1H), 2.27À2.22 (m, 2H),
1.62À1.52 (m, 2H), 1.33À1.24 (m, 24H), 0.90À0.86 (m, 3H); 13C
NMR (75 MHz, CDCl3) δ 173.5, 137.8, 137.4, 133.6, 131.6, 129.0,
128.6, 128.5, 128.2, 128.01, 127.99, 127.9, 127.5, 87.1, 80.3, 75.2, 74.4,
72.2, 70.5, 69.7, 63.1, 34.1, 31.9, 29.65, 29.62, 29.57, 29.4, 29.3, 29.2,
29.1, 24.8, 22.7, 14.1. HRMS-ESI [M + Na]+ calculated for C42H58O6S-
Na: 713.3852. Found 713.3881.
Phenyl 2-O-(2-Azidomethylbenzoyl)-3,4-di-O-benzyl-6-
O-hexadecanoyl-1-thio-α-D-mannopyranoside (31). 2-Azido-
methylbenzoyl chloride (AZMBCl) was prepared as described47 and
used without purification. A solution of 32 (1.35 g, 1.95 mmol) in
pyridine (10 mL) was added to ice-cooled AZMBCl (0.49 g, 2.5 mmol),
and the stirred mixture was allowed to warm to room temperature
overnight. Excess pyridine was concentrated under reduced pressure,
and the residue was taken up in EtOAc (150 mL), washed with 1 M
aqueous HCl (2 Â 50 mL), saturated aqueous NaHCO3 (2 Â 50 mL),
dried (MgSO4), filtered and the solvent removed. The crude residue was
purified by column chromatography on silica gel (EtOAc/petroleum
ether = 7.5:92.5) to afford the title compound 31 (1.016 g, 61%) as an
oil. [α]20D +55 (c 0.9, CH2Cl2); 1H NMR (300 MHz, CDCl3) δ 8.04,
(dd, J = 1.2, 7.8 Hz, 1H), 7.58 (dt, J = 1.2, 7.4 Hz, 1H), 7.52À7.48 (m,
3H), 7.42À7.24 (m, 14H), 5.83 (dd, J = 1.6, 3.0 Hz, 1H), 5.60 (d, J = 1.6
Hz, 1H), 4.92 (d, J = 10.9 Hz, 1H), 4.80 (d, J = 11.3 Hz, 1H), 4.75 (s,
2H), 4.64 (d, J = 11.3 Hz, 1H), 4.61 (d, J = 10.9 Hz, 1H), 4.48À4.32 (m,
3H), 4.07, (dd, J = 3.0, 9.2 Hz, 1H), 3.92 (app t, J = 9.3 Hz, 1H),
2.29À2.24 (m, 2H), 1.63À1.53 (m, 2H), 1.32À1.23 (m, 24H),
0.90À0.86 (m, 3H); 13C NMR (75 MHz, CDCl3) δ 173.4, 165.6,
137.8, 137.6, 137.4, 133.2, 133.0, 132.1, 131.4, 129.7, 129.1, 128.4, 128.2,
128.1, 127.95, 127.1, 86.1, 78.5, 75.3, 74.3, 71.9, 70.9, 70.8, 63.1, 52.9,
34.1, 31.9, 29.7, 29.63, 29.58, 29.5, 29.3, 29.23, 29.20, 24.8, 22.7, 14.1.
HRMS-ESI [M + Na]+ calculated for C50H63N3O7SNa: 872.4284.
Found 872.4283.
1-O-Allyl-2-O-(2-O-[2-azidomethylbenzoyl]-3,4-di-O-ben-
zyl-6-O-hexadecanoyl-α-D-mannopyranosyl)-6-O-(2,3,4,6-
tetra-O-benzyl-α-D-mannopyranosyl)-3,4,5-tri-O-benzyl-D-myo-
inositol (34). Acceptor 1626 (160 mg, 0.16 mmol) was dissolved in dry
CH2Cl2 (5 mL) and cooled to À60 ꢀC. In a separate flask, a mixture of
donor 31 (125 mg, 0.147 mmol), diphenyl sulfoxide (83 mg, 0.412
mmol), and 2,6-di-tert-butyl-4-methylpyridine (DTBMP, 91 mg, 0.441
mmol) was coevaporated from dry CH2Cl2 (10 mL). The reagents were
redissolved in CH2Cl2 (5 mL) and cooled to À60 ꢀC with stirring before
the addition of Tf2O (35 μL, 0.21 mmol). After 5 min, the solution of
acceptor was transferred to the reaction vessel (cannula), rinsing with
CH2Cl2 (2 mL), and the reaction mixture was allowed to warm to 0 ꢀC
over 3 h. The reaction was quenched with saturated aqueous NaHCO3
and extracted with CH2Cl2. The organic extracts were washed with
saturated aqueous NaHCO3, dried (MgSO4), filtered and the solvent
2-O-(2-O-[2-Azidomethylbenzoyl]-3,4-di-O-benzyl-6-O-
hexadecanoyl-α-D-mannopyranosyl)-1-O-(1-O-hexadeca-
noyl-2-O-hexadecyl-sn-glycero-3-benzylphosphoryl)-6-O-
(2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl)-3,4,5-tri-O-
benzyl-D-myo-inositol (37). 1H-Tetrazole (13 mg, 0.19 mmol)
was added to a stirred solution of alcohol 35 (106 mg, 0.062 mmol) and
phosphoramidite 36 (147 mg, 0.186 mmol) in dry CH2Cl2 (8 mL)
under Ar at 0 ꢀC. After the mixture was stirred at room temperature for
3 h, the reaction mixture was cooled to À40 ꢀC and a solution of m-
CPBA (50%, 86 mg, 0.25 mmol) in CH2Cl2 (10 mL) was transferred by
cannula into the reaction mixture. After the mixture was stirred at room
temperature for 1 h, the reaction was quenched by addition of 10%
aqueous Na2SO3 (50 mL) and the combined mixture extracted with
Et2O (100 mL). The ethereal extract was washed with saturated aqueous
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Journal of Medicinal Chemistry
ARTICLE
NaHCO3 (3 Â 50 mL), dried (MgSO4), filtered and the solvent
removed. The residue was purified by column chromatography on silica
gel (EtOAc/petroleum ether = 1:9 to 1:4) followed by a second column
(acetone/toluene = 1:50 to 3:97) to afford the title compound 37 (59
mg, 0.024 mmol, 39%) as an oil. 1H NMR (300 MHz, CDCl3) δ
8.04À7.98 (m, 1H), 7.58À7.43 (m, 2H), 7.36À7.03 (m, 51H), 5.66À5.62
(m, 1H), 5.56À5.62 (m, 1H), 5.33À5.29 (m, 1H), 5.11À5.04 (m, 2H),
4.95À4.39 (m, 20H), 4.36À3.82 (m, 17H), 3.60À3.22 (m, 7H),
2.35À2.13 (m, 4H), 1.61À1.48 (m, 4H), 1.42À1.17 (m, 76H),
0.90À0.86 (m, 9H); 13C NMR (75 MHz, CDCl3) selected signals δ
173.2, 99.6, 98.6; 31P NMR (121 MHz, CDCl3) δ 0.1, 0.0. HRMS-ESI
[M + Na]+ calculated for C147H196N3O24NaP: 2441.3845. Found
2441.3855.
(m, 3H), 3.89À3.78 (m, 4H), 3.78À3.72 (m, 2H), 3.72À3.65 (m, 3H),
3.62 (dd, J = 6.2, 10.7 Hz, 1H), 3.56 (dd, J = 4.5, 10.7 Hz, 1H), 3.51 (dd,
J = 6.6, 10.6 Hz, 1H), 2.13 (s, 6H); 13C NMR (125 MHz, CDCl3)
δ 170.3,138.09, 138.07, 137.9, 128.34, 128.28, 128.26, 128.0, 127.8,
127.7, 127.6, 127.5, 98.5 (J1CH = 170 Hz), 98.3 (J1CH = 170 Hz), 78.1,
78.0, 75.11, 75.09, 74.2, 73.4, 71.89, 71.85, 71.7, 69.8, 69.6, 69.2, 68.79,
68.77, 68.7, 68.6, 21.0. HRMS-ESI [M + Na]+ calculated for C61H68O15Na:
1063.4456. Found 1063.4445.
2-O-(Benzyloxy-1-O-hexadecanoyl-2-O-hexadecyl-sn-gly-
cero-3-phosphoryl)-1,3-bis(2-O-acetyl-3,4,5-tri-O-benzyl-α-
D-mannopyranosyl)glycerol (41). A mixture of alcohol 40 (86 mg,
0.083 mmol) and phosphoramidite 36 (98 mg, 0.124 mmol) was
coevaporated from dry CH2Cl2 (10 mL) and then placed under high
vacuum for 1 h. The reagents were dissolved in dry CH2Cl2 (2 mL) and
stirred for 1 h at room temperature over 4 Å molecular sieves. The
solution was cooled to 0 ꢀC before the addition of 4,5-dicyanoimidazole
(16 mg, 0.14 mmol) and then warmed to room temperature over 18 h.
The solution was then cooled to À15 ꢀC before the addition of a dried
(MgSO4) solution of mCPBA (∼60%, 50 mg) in CH2Cl2. After warming
to room temperature over 1 h, the reaction mixture was diluted with
Et2O (30 mL) and washed with Na2S2O3 (10%, 30 mL), saturated
NaHCO3 (3 Â 20 mL), brine (30 mL), dried (MgSO4), filtered and the
solvent removed. The crude residue was purified by column chroma-
tography on silica gel (EtOAc/petroleum ether = 1:4 to 2:3) to afford
2-O-(3,4-Di-O-benzyl-6-O-hexadecanoyl-α-D-mannopyra-
nosyl)-1-O-(1-O-hexadecanoyl-2-O-hexadecyl-sn-glycero-3-
benzylphosphoryl)-6-O-(2,3,4,6-tetra-O-benzyl-α-D-manno-
pyranosyl)-3,4,5-tri-O-benzyl-D-myo-inositol (38). Tri-n-butyl-
phosphine (34 μL, 0.14 mmol) was added to a degassed solution of
AZMB ester 37 (58 mg, 0.024 mmol) in THF/H2O (9:1, 10 mL) under
Ar. After the mixture was stirred at room temperature for 3 h, toluene
(20 mL) was added and the solvent removed in vacuo. The residue was
purified by column chromatography on silica gel (EtOAc/petroleum
ether = 1:4 to 3:7) to afford the title compound 38 (42 mg, 78%) as an
oil. 1H NMR (300 MHz, CDCl3) δ 7.40À6.99 (m, 50H), 5.46À5.42 (m,
1H), 5.19 (m, 1H), 5.11À4.39 (m, 20H), 4.19À3.78 (m, 18H),
3.62À3.20 (m, 7H), 2.54À2.49 (m, 1H), 2.28À2.18 (m, 4H),
1.60À1.40 (m, 6H), 1.33À1.22 (m, 74H), 0.90À0.86 (m, 9H); 13C
NMR (75 MHz, CDCl3) selected signals δ 173.5, 101.5, 98.4; 31P NMR
(121 MHz, CDCl3) δ 0.0, À0.2; HRMS-ESI [M + Na]+ calculated for
1
the title compound 41 (93 mg, 64%). H NMR (500 MHz, CDCl3)
mixture of isomers δ 7.37À7.19 (m, 31H), 7.17À7.10 (m, 4H), 5.11À5.01
(m, 2H), 4.90À4.79 (m, 4H), 4.70À4.57 (m, 5H), 4.52À4.39 (m, 6H),
4.17À4.10 (m, 1H), 4.09À3.73 (m, 10H), 3.71À3.58 (m, 4H),
3.58À3.52 (m, 1H), 3.50À3.37 (m, 2H), 2.26À2.21 (m, 2H), 2.12 (s,
3H), 2.11 (s, 3H), 1.62À1.51 (m, 2H), 1.50À1.41 (m, 2H), 1.35À1.15
(m, 50H), 0.93À0.80 (m, 6H); 13C NMR (125 MHz, CDCl3) δ 173.3,
170.2, 138.4, 138.2, 137.9, 135.8, 135.75, 135.71, 135.69, 128.6, 128.5,
128.4, 128.32, 128.30, 128.25, 128.2, 128.1, 128.0, 127.81, 127.80,
127.78, 127.7, 127.5, 98.32, 98.31, 98.15, 98.11, 78.20, 78.19, 75.79,
75.77, 75.73, 75.71, 75.4, 75.31, 75.26, 75.10, 75.07, 74.04, 74.00, 73.40,
73.38, 71.92, 71.88, 71.85, 71.83, 70.65, 70.61, 69.42, 69.38, 68.71, 68.66,
68.6, 68.4, 68.1, 66.72, 66.68, 66.42, 66.38, 66.3, 62.5, 34.1, 31.9, 29.9,
29.7, 29.64, 29.62, 29.5, 29.32, 29.28, 29.1, 26.0, 24.8, 22.6, 21.0; 31P
NMR (202 MHz, CDCl3) δ À1.44, À1.48. HRMS-ESI [M + Na]+
calculated for C103H143O21PNa: 1769.9757. Found 1769.9751 .
C
139H191O23PNa: 2282.3412. Found 2282.3401.
1-O-(1-O-Hexadecanoyl-2-O-hexadecyl-sn-glycero-3-phos-
phoryl)-2-O-(6-O-hexadecanoyl-α-D-mannopyranosyl)-6-O-
(α-D-mannopyranosyl)-D-myo-inositol (5). Pd(OH)2/C (20%,
36 mg) was added to a stirred solution of compound 38 (42 mg, 0.019
mmol) in THF/MeOH (2:3, 5 mL). The mixture was stirred under a H2
atmosphere for 3.5 h at room temperature, then filtered through Celite
and the solvent removed. The residue was purified by column chroma-
tography on silica gel (CHCl3/MeOH/H2O = 70:40:0 to 70:40:8),
affording the title compound 5 (19 mg, 75%) as a white powder. It was
95% pure by HPLC system 1. [α]20 +30 (c 0.10, CHCl3/CH3OH/
D
H2O = 70:40:6); 1H NMR (500 MHz, CDCl3/CD3OD/D2O =
70:40:6) δ 5.15 (br s, 1H), 5.10 (br s, 1H), 4.38À4.08 (m, 8H), 4.05
(dd, J = 1.7, 3.3 Hz, 1H), 4.01À3.96 (m, 2H), 3.94À3.89 (m, 1H),
3.84À3.73 (m, 6H), 3.70À3.65 (m, 3H), 3.60 (app t, J = 9.5 Hz, 1H),
3.55À3.51 (m, 1H), 3.46 (dd, J = 2.5, 10 Hz, 1H), 3.29 (t, J = 9.3 Hz,
1H), 2.39À2.33 (m, 4H), 1.65À1.54 (m, 6H), 1.37À1.27 (m, 74H),
0.90À0.87 (m, 9H); 13C NMR (126 MHz, CDCl3/CD3OD/D2O =
70:40:6) δ 175.7, 175.3, 102.3, 102.2, 79.4, (br d), 79.2, 77.5, 77.4, 74.0,
73.8, 73.5, 71.44, 71.35, 71.3, 71.2, 71.0, 70.9, 70.7, 67.8, 67.6, 64.9, 64.7,
(br d), 64.3, 61.9, 34.9, 34.5, 32.51, 30.46, 30.40, 30.37, 30.3, 30.23,
30.18, 30.03, 29.97, 29.9, 29.8, 26.7, 25.6, 25.4, 23.2, 14.5; 31P NMR (202
MHz, CDCl3/CD3OD/D2O = 70:40:6) δ À0.3; HRMS-ESI [M À H]À
calculated for C69H130O23P: 1357.8741. Found 1357.8729.
1,3-Bis(2-O-acetyl-3,4,5-tri-O-benzyl-α-D-mannopyranosyl)-
2-hydroxypropane (40). DDQ (35 mg, 0.154 mmol) was added to a
stirred solution of 39 (149 mg, 0.128 mmol) in CH2Cl2/H2O (9:1,
4 mL). After 50 min the mixture was diluted with Et2O (80 mL), washed
with water (3 Â 20 mL), saturated NaHCO3 (3 Â 20 mL), dried
(MgSO4), filtered and the solvent removed. The crude residue was
purified by column chromatography on silica gel (EtOAc/petroleum
ether = 2:3) to afford the title compound 40 (96 mg, 72%) as waxy solid.
[α]20D +30 (c 0.82, CH2Cl2); 1H NMR (500 MHz, CDCl3) δ 7.35À7.22
(m, 26H), 7.18À7.13 (m, 4H), 5.38À5.35 (m, 2H), 4.87À4.82 (m, 4H),
4.71À4.62 (m, 4H), 4.55À4.50 (m, 2H), 4.50À4.45 (m, 4H), 3.97À3.90
2-O-(1-O-Hexadecanoyl-2-O-hexadecyl-sn-glycero-3-phos-
phoryl)-1,3-bis(2-O-acetyl-α-D-mannopyranosyl)glycerol (42).
A suspension of 41 (90 mg, 51 mmol) and Pd(OH)2 in 2:3 THF/
MeOH (5 mL) was stirred under an atmosphere of H2 at ambient
temperature for 4 h. The mixture was filtered through Celite, concen-
trated, and purified by purified by column chromatography (neat CHCl3
to 2:1 CHCl3/MeOH) to give 42 (40 mg, 70%) as a white powder.
1
[α]20 +27 (c 0.74, 2:1 CHCl3/MeOH); H NMR (500 MHz, 2:1
D
CDCl3/CD3OD) δ 5.08À5.03 (m, 2H), 4.83 (d, J = 0.9 Hz, 1H), 4.81
(d, J = 1.1 Hz), 4.54À4.46 (m, 1H), 4.29 (dd, J = 2.8, 11.6 Hz),
4.17À4.11 (m, 1H), 4.06À3.86 (m, 8H), 3.80À3.51 (m, 11H), 2.35 (t,
J = 7.5 Hz, 1H), 2.12 (2 Â s, 6H), 1.67À1.52 (m, 4H), 1.38À1.22 (m,
50H), 0.92À0.85 (m, 6H); 13C NMR (125 MHz, 2:1 CDCl3/CD3OD)
δ 174.8, 171.61, 171.58, 98.1, 98.0, 76.8, 76.7, 74.4, 74.3, 73.7, 73.6,
72.72, 72.69, 71.2, 69.8, 68.7, 68.5, 67.0, 65.43, 65.41, 64.0, 62.5, 62.4,
34.6, 32.3, 30.3, 30.05, 30.00, 29.90, 29.87, 29.7, 29.5, 26.4, 25.3, 23.0,
20.9, 14.2; 31P NMR (202 MHz, 2:1 CDCl3/CD3OD) δ 2.7. HRMS-ESI
[M À H]À calculated for C54H100O21P: 1115.6495. Found 1115.6488.
2-O-(1-O-Hexadecanoyl-2-O-hexadecyl-sn-glycero-3-phos-
phoryl)-1,3-bis-O-(α-D-mannopyranosyl)glycerol (6). Sodium
methoxide in MeOH (0.5 M solution, 50 μL) was added dropwise to
a stirred solution of 42 (26 mg, 0.023 mmol) in MeOH (5 mL). After 2 h
the mixture was quenched with Dowex 50W8-100 (H+) resin, filtered,
and subsequently made neutral by treatment with Dowex 50W8-100
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(Na+) resin. After filtration and concentration, the crude residue was
purified on silica gel (CHCl3/MeOH/H2O = 70:35:1.75 to 70:35:6) to
afford the title compound 6 (17 mg, 71%). [α]20D +39 (c 0.14, CHCl3/
MeOH/H2O = 70:40:6); 1H NMR (500 MHz, CDCl3/CD3OD/D2O =
70:35:6) δ 4.87 (br, 1H), 4.84 (br, 1H), 4.39 (br, 1H), 4.30 (dd, J = 2.7,
11.8 Hz, 1H), 4.11 (dd, J = 7.3, 11.7 Hz, 1H), 3.95À3.83 (m, 8H),
3.77À3.59 (m, 12H), 3.53 (dt, J = 7.0, 9.4 Hz, 1H), 2.35 (t, J = 7.6 Hz,
2H), 1.65À1.60 (m, 2H), 1.59À1.53 (m, 2H), 1.34À1.27 (m, 50H),
0.90À0.87 (m, 6H); 13C NMR (125 MHz, CDCl3/CD3OD/D2O =
70:35:6) δ 175.2, 100.8, 100.7, 77.3, 77.2, 73.5, 73.4, 71.5, 71.3, 70.94,
70.90, 67.8, 67.7, 67.3, 67.1, 64.9, 64.7, 64.6, 61.9, 61.8, 34.8, 32.4, 30.4,
30.23, 30.19, 30.14, 30.08, 30.05, 29.8, 29.7, 26.5, 25.5, 23.1, 14.4; 31P
NMR (202 MHz, CDCl3/CD3OD/D2O = 70:35:6) δ À0.8. HRMS-ESI
[M-H]À calculated for C50H96O19P: 1031.6283. Found 1031.6288.
Cytokine Release Assay. Thioglycollate-elicited peritoneal mur-
ine macrophages were generated as previously described.48 Macrophages
were cultured in 96-well plates at 2 Â 105 cells/well in supplemented
RPMI-1640 (100 U/mL penicillinÀstreptomyocin, 10% fetal calf
serum). Compounds and 10 ng/mL LPS were added to macrophage
cultures to a final culture volume of 200 μL/well and incubated at 37 ꢀC,
5% CO2 for 18 h. Supernatants were collected and IL-6 levels measured
by ELISA. Cell viability was measured by trypan blue exclusion.
Expression and Purification of sTLR4/MD-2. A complex
between TLR4 ectodomain (sTLR4) and MD-2 was expressed by a
baculovirus expression system using a modified dual expression vector,
pAcUW51 (BD Biosciences). sTLR4 and MD-2 are appended to a
C-terminal His6-tag and a Strep-tag II, respectively. sTLR4/MD-2
expressing baculovirus was generated by co-transfecting SF9 insect cells
with the sTLR4/MD-2 expression vector and linearized baculovirus
DNA, BaculoGold (BD Biosciences). For sTLR4/MD-2 expression,
Hi5 insect cells were infected with the amplified recombinant virus. The
secreted sTLR4/MD-2 protein was initially purified by Ni-NTA and
Strep-Tactin affinity chromatography, and its C-terminal tags were
removed by thrombin. The resultant protein was further purified by
size exclusion chromatography.
Dr. Regan Anderson for his very helpful contributions in the
preparation of this manuscript.
’ ABBREVIATIONS USED
TLR, Toll-like receptor; PIM, phosphatidylinositol mannoside;
NKT, natural killer T; LPS, lipopolysaccharide; MD-2, myeloid
differentiation factor 2
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’ ASSOCIATED CONTENT
S
Supporting Information. NMR spectra, HPLC data,
b
and viability data. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
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irl.cri.nz.
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The work was supported by the New Zealand Foundation for
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AI042266 (I.A.W.), and the Skaggs Institute for Chemical
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