The influence of the long chain fatty acid on the antagonistic activities of Rhizobium sin-1 lipid A

Article abstract of DOI:10.1016/j.bmc.2007.05.012

The lipid A from nitrogen-fixing bacterial species Rhizobium sin-1 is structurally unusual due to lack of phosphates and the presence of a 2-aminogluconolactone and a very long chain fatty acid, 27-hydroxyoctacosanoic acid (27OHC28:0), moiety. This struct

Full text of DOI:10.1016/j.bmc.2007.05.012

Bioorganic & Medicinal Chemistry 15 (2007) 4800–4812
The influence of the long chain fatty acid on the
antagonistic activities of Rhizobium sin-1 lipid A
Yanghui Zhang, Margreet A. Wolfert and Geert-Jan Boons*
Complex Carbohydrate Research Center, The University of Georgia, 315 Riverbend Road, Athens, GA 30602, USA
Received 13 March 2007; revised 23 April 2007; accepted 1 May 2007
Available online 6 May 2007
Abstract—The lipid A from nitrogen-fixing bacterial species Rhizobium sin-1 is structurally unusual due to lack of phosphates and
the presence of a 2-aminogluconolactone and a very long chain fatty acid, 27-hydroxyoctacosanoic acid (27OHC28:0), moiety. This
structurally unusual lipid A can antagonize TNF-a production by human monocytes induced by Escherichia coli LPS. To establish
the relevance of the unusual long chain 27-hydroxyoctacosanoic acid for antagonistic properties, a highly convergent strategy for the
synthesis of several derivatives of the lipid A of R. sin-1 has been developed. Compound 1 is a natural R. sin-1 lipid A having a 27-
hydroxyoctacosanoic acid at C-20, compound 2 contains an octacosanoic acid moiety at this position, and compound 3 is modified
by a short chain tetradecanoic acid. Cellular activation studies with a human monocytic cell line have shown that the octacosanoic
acid is important for optimal antagonistic properties. The hydroxyl of the natural 27-hydroxyoctacosanoic moiety does, however,
not account for inhibitory activity. The resulting structure–activity relationships are important for the design of compounds for the
treatment of septic shock.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
remain to be discovered,9,10 it is clear that cellular acti-
vation leads to the induction of cytokine gene expres-
sion, primarily through the activation of NF-jB, and
the MAP kinases, which results in the biosynthesis of
a diverse set of inflammatory mediators such as TNF-
a, IL-6, and IL-1b to eradicate the immediate infection.
Unfortunately, the presence of a large amount of LPS in
the blood can cause the overproduction of the media-
tors, which can lead to septic inflammatory response
syndrome (SIRS) and include life-threatening symptoms
such as vascular fluid leakage, tissue damage, hypoten-
sion, shock, and organ failure.
Septicemia is a serious worldwide health problem asso-
ciated with mortality rates of 40–60%. Currently, no
effective treatment exists for this life-threatening syn-
drome other than supportive therapy in an intensive
care setting.1,2 The development of septicemia is often
linked to a systemic inflammatory response to lipopoly-
saccharide (LPS) in the blood of affected patients.
LPS induces cellular responses after binding to the clus-
ter differentiation antigen CD14 on mononuclear phago-
cytes, or to soluble CD14 in plasma and then to cells
lacking CD14.3–5 As CD14 is a glycosylphosphatidyli-
nositol-anchored protein, it lacks transmembrane and
cytoplasmic domains, and therefore is unable to directly
transmit signals to the interior of the cell. The latter
function is performed by the Toll-like receptor 4
(TLR4),6,7 which contains extracellular transmembrane
and intracellular domains, and an accessory protein
MD2.8 While the precise mechanisms involved in the
interactions among LPS, CD14, TLR4, and MD2
LPS consists of an O-chain polysaccharide, a core oligo-
saccharide, and an amphiphilic moiety referred to as li-
pid A. The latter moiety has been shown to be the toxic
principle of LPS. Lipid A of most enteric bacteria con-
sists of a b(1-6)-linked glucosamine disaccharide back-
bone with phosphate monoesters at C-1 and C-40 and
b-hydroxyl fatty acyl groups and acyloxyacyl residues
at C-2, and C-3 and C-20 and C-30, respectively
(Fig. 1).11 Small modifications in the acylation pattern
of lipid A are thought to contribute to the virulence of
enteric pathogens. For example, fatty acyl components
can be present that have shorter chain length, sites of
unsaturation or keto functional groups.12,13 Other mod-
ifications include the addition of a palmitoyl residue, the
Keywords: Lipopolysaccharides; Lipid A; Antagonist; Tumor necrosis
factor; Septic shock.
*
Corresponding author. E-mail: gjboons@ccrc.uga.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.05.012

Y. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4800–4812
4801
Figure 1. Structures of E. coli lipid A and target R. sin-1 lipid A derivatives 1–3.
hydroxylation of a myristoyl substituent, and the addi-
tion of aminoarabinosyl and phosphoethanolamine
moieties.14
hydroxyoctacosanoic acid at C-20, compound 2 contains
an octacosanoic acid moiety at this position, and com-
pound 3 is modified by a short chain tetradecanoic acid.
A number of lipid A derivatives have been shown to lack
agonistic properties and instead can inhibit the produc-
tion of TNF-a induced by enteric LPS.15–19 Not surpris-
ingly, these compounds have attracted attention for the
treatment of septic shock. For example, the lipid As
from Rhodobacter sphaeroides, Rhodobacter capsulatus,
and Helicobacter pylori, deacylated LPS, and lipid IVa
lack toxic properties, but can antagonize cytokine pro-
duction induced by enteric LPS. Furthermore, chemical
synthesis of lipid A analogs patterned after R. sphaero-
ides/R. capsulatus lipid A has been reported. A number
of these compounds could prevent the pyrogenic effects
of enteric LPS in rabbits,20 protect against LPS-induced
lethality in mice,19 and block TLR4-mediated NF-jB
activation by LPS.21
2. Results and discussion
2.1. Chemical synthesis
In first instance, attention was focused on the prepara-
tion of compound 3 which is acylated with a simple tet-
radecanoic acid at the b-hydroxyl of the C-2 acyloxyacyl
residue of the distal sugar moiety. It was envisaged that
3 could be obtained by a regio-, chemo-, and stereoselec-
tive glycosylation of glycosyl donor 426 with glycosyl
acceptor 527,28 to give disaccharide 11, which after a
number of selective deprotections can be acylated at
the C-2, C-20, C-3, and C-30, oxidized to a lactone,
and deprotected. Thus, an N-iodosuccinimide (NIS)/tri-
fluoromethanesulfonic acid (TfOH)29 mediated coupling
of 4 with 5 in dichloromethane (DCM) at ꢀ75 ꢁC gave,
We have identified naturally occurring LPS from Rhizo-
bium sin-1, a nitrogen-fixing bacterial species, that does
not stimulate TNF-a production by human mono-
cytes,22–24 and that prevents the induction of TNF-a
by Escherichia coli LPS. The lipid A of R. sin-1 is per-
haps the most structurally unusual lipid A reported to
date, its structure (Fig. 1; ea compound 1) differing in al-
most every aspect from those known to contribute to the
toxicity of enteric lipid A.25 In particular, the disaccha-
ride moiety of Rhizobial lipid A is devoid of phosphate
and the glucosamine phosphate is replaced by 2-amino-
gluconolactone. Furthermore, it contains a very long
chain fatty acid, 27-hydroxyoctacosanoic acid
(27OHC28:0).
Previously reported phosphate-containing lipid A antag-
onists are metabolically labile. Hence, it is attractive to
study the structure–activity relationship of R. sin-1 lipid
A to identify specific structural features that render
R. sin-1 lipid A an antagonist. We have already investi-
gated the importance of the lactone moiety of R. sin-1
lipid A for antagonistic properties. Here, we report the
chemical synthesis of compounds 1–3 (Fig. 1) to estab-
lish the relevance of the unusual long chain 27-hydroxy-
octacosanoic acid for antagonistic properties. Thus,
compound 1 is a natural R. sin-1 lipid A having a 27-
Figure 2. Structures of building blocks 4–10.

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Y. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4800–4812
after purification by silica gel column chromatography,
disaccharide 11 in a yield of 84% (Fig. 2 and Scheme
1). The coupling exploited the higher reactivity of the
C-6 hydroxyl of glycosyl acceptor 5 compared to the
C-3 hydroxyl of glycosyl donor 4. Furthermore, the
reaction took advantage of the ability to selectively acti-
vate a selenoglycoside in the presence of a thioglyco-
side.30 Interestingly, it was found that NIS/TfOH gave
reagent to afford 12 in a yield of 78%. Reduction of the
azido moiety of 12 was easily accomplished by reaction
with propane-1,3-dithiol in a mixture of pyridine, trieth-
ylamine, and water, and the amine and hydroxyls of the
resulting compound were acylated with (R)-3-benzyl-
oxy-hexadecanoic acid (8) using DCC and 4-dimethyla-
minopyridine (DMAP) as activating reagents to afford
13 in an overall yield of 59%.
31
a higher yield of 11 than when AgOTf/K2CO3 was
used as the promoter system.
Hydrolysis of the thiophenyl moiety of 13 by treatment
with NIS and a catalytic amount of TfOH in wet DCM
proved problematic and two products were isolated
namely the required lactol 15 and the 1,2-oxazoline
derivative 14. The latter byproduct was formed by
neighboring participation of the C-2 amide after activa-
tion of the thioglycoside of 13 with NIS followed by
elimination of water. Fortunately, the 1,2-oxazoline
could be converted into compound 15 by treatment with
dibenzylphosphate in wet DCM34 in an 80% yield. The
Having advanced disaccharide 11 in hand, attention was
focused on removal of the protecting groups and selec-
tive introduction of the fatty acids. Thus, the phthalim-
ido group and acetyl ester of 11 were cleaved by
treatment with ethylenediamine in refluxing n-butanol.32
The resulting C-20 amino was selectively acylated with
(R)-3-tetradecanoyloxy-hexadecanoic acid 733 using
N,N0-dicyclohexyl carbodiimide (DCC) as the activating
˚
Scheme 1. Reagents and conditions: (a) NIS, TfOH, MS 3 A, DCM, ꢀ75 ꢁC; (b) 1—H2N(CH2)2NH2, n-BuOH, 90 ꢁC; 2—7, DCC, DCM; (c) 1—
˚
HS(CH2)3SH, pyridine, Et3N, H2O; 2—8, DCC, DMAP, DCM; (d) NIS, TfOH, DCM, H2O; (e) dibenzylphosphate, H2O, DCM; (f) PCC, MS 3 A,
DCM; (g) Pd/C, H2, t-BuOH, THF.

Y. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4800–4812
4803
anomeric hydroxyl of 15 was oxidized using pyridinium
chlorochromate (PCC) to give lactone 16 in high yield.
Finally, the benzyl ethers and benzylidene acetal of 16
were removed by catalytic hydrogenation over Pd/C to
afford target compound 3.
can more easily be trapped by water to give a lactol then
when the anomeric leaving group can be directly dis-
placed by the C-2 amide leading to an 1,2-oxazoline.
Furthermore, the acyloxyacyl moiety at C-20 was intro-
duced by first acylation with (R)-3-levulinoyloxy-hexa-
decanoic acid (9)35 followed by selective removal of
the Lev ester and acylation of the resulting alcohol with
an appropriate fatty acid. This approach is attractive be-
cause it provides an easy route to a variety of structural
analogs. Thus, NIS/TMSOTf mediated glycosylation of
glycosyl donor 4 with glycosyl acceptor 6 in DCM at
ꢀ75 ꢁC provided disaccharide 17 in 82% yield. Treat-
ment of 17 with ethylenediamine in refluxing n-butanol
followed by acylation of the resulting C-20 amino with
9 using DCC as the activating reagent afforded 18 in a
yield of 78%. The azido moiety of 18 was reduced with
To avoid the formation of the unwanted 1,2-oxazoline
observed during the preparation of 3, a different strategy
was employed for the preparation of compounds 1 and
2. In the new approach, glycosyl acceptor 627,28 was em-
ployed, which contains an h-instead of a b-anomeric thi-
ophenyl moiety (Scheme 2). It was hoped that the
hydrolysis of the a-anomeric thioglycoside would be less
problematic because the C-2 amide cannot directly dis-
place the anomeric leaving group. As a result, the hydro-
lysis will proceed through an oxa-carbenium ion, which
˚
Scheme 2. Reagents and conditions: (a) NIS, TfOH, MS 3 A, DCM, ꢀ75 ꢁC; (b) 1—H2N(CH2)2NH2, n-BuOH, 90 ꢁC; 2—9, DCC, DCM; (c)
1—HS(CH2)3SH, pyridine, Et3N, H2O; 2—8, DCC, DMAP, DCM; (d) 1—H2NNH2 Æ HOAc, DCM; 2—ROH, DCC, DMAP, DCM; (e) NIS,
˚
TfOTMS, DCM, H2O; (f) PCC, MS 3 A, DCM; (g) Pd/C, H2, t-BuOH, THF.

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Y. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4800–4812
propane-1,3-dithiol and the amine and hydroxyls of the
resulting compound were acylated with 8 using DCC
and DMAP as activating reagents to provide 19 in an
overall yield of 61%. Next, the Lev ester of 19 was selec-
tively removed by treatment with hydrazine acetate, and
the hydroxyl of the resulting compound was acylated
with 27-benzyloxyoctacosanoic- and octacosanoic acid
to give compounds 20 and 21, respectively. Gratifyingly,
no 1,2-oxazoline was formed when the anomeric thio-
glycosides of 20 and 21 were hydrolyzed with NIS/
TMSOTf and lactols 22 and 23 were isolated in high
yields. Finally, 22 and 23 were oxidized using PCC to
give lactones 24 and 25 which were globally deprotected
using standard conditions to afford the target com-
pounds 1 and 2, respectively.
Figure 4. Antagonism of E. coli LPS by R. sin-1 LPS, R. sin-1 lipid A,
and synthetic compounds 1–3 in human monocytic cells. TNF-a
concentrations were measured after preincubation of Mono Mac 6
cells with increasing concentrations of R. sin-1 LPS, R. sin-1 lipid A, 1,
2, or 3 as indicated for 1 h at 37 ꢁC, followed by 5.5 h of incubation
with E. coli LPS (10 ng/mL). Results are expressed as percentage of
cytokine concentration of control cells, which are incubated only with
E. coli LPS.
2.2. Biological evaluations
Compounds 1–3 were tested over a wide concentration
range for their ability to activate a human monocytic
cell line (Mono Mac 6) to produce TNF-a protein and
the resulting values were compared with similar data ob-
tained for E. coli LPS, R. sin-1 LPS, and R. sin-1 lipid A.
Thus, incubation of Mono Mac 6 cells with E. coli LPS
for 5.5 h yielded a clear dose response effect of TNF-a
production with maximal supernatant concentrations
of TNF-a being caused by 10 ng/mL (0.093 nM) of
E. coli LPS. Incubations of concentrations up to
10 lg/mL for R. sin-1 LPS (270 nM) and R. sin-1 lipid
A (6.6 lM) and up to 100 lg/mL (approximately
50 lM) for the synthetic compounds 1–3 did not induce
significant production of TNF-a (Fig. 3).
(concentration producing 50% inhibition) of 9.1 lM
(16.4 lg/mL) was established (Fig. 4). A similar inhibi-
tion experiment with compound 2 gave a similar IC50
value of 7.5 lM (13.2 lg/mL). However, compound 3
was only able to marginally inhibit the production of
TNF-a. Thus, these data show that the hydroxyl of
the 27-hydroxyoctacosanoic acid moiety of R. sin-1 lipid
A is not important for antagonistic properties. However
shortening the octacosanoic acid moiety as in compound
3 resulted in a significant reduction in inhibitory poten-
tial. Furthermore, the EC50 values for compounds 1 and
2 are similar to that of R. sin-1 lipid A, which was ob-
tained by mild acid hydrolysis of the corresponding
LPS. As expected, R. sin-1 LPS was a more potent inhib-
itor and an IC50 value of 6.5 nM (239 ng/mL) was deter-
mined when incubated with 10 ng/mL of E. coli LPS
(Fig. 4). Probably, the KDO moiety of R. sin-1 LPS ac-
counts for the greater biological activity.
Based on their lack of proinflammatory effects, com-
pounds 1–3 were tested over a wide concentration range
for their ability to antagonize TNF-a production by
monocytic cells incubated with E. coli LPS (10 ng/mL).
At the highest concentration tested, compound 1 antag-
onized the effect of E. coli LPS by 86%, and an IC50
3. Conclusions
Several studies have indicated that compounds that can
antagonize cytokine production induced by enteric LPS
may have the potential to be developed as therapeutics
for the treatment of Gram-negative septicemia.36 Suc-
cess in this area has been limited and most efforts have
been directed toward the synthesis of analogs of lipid
A of R. sphearoides18,19 and derivatives of lipid X.15–17
Unique features of R. sin-1 lipid A are lack of phos-
phates, an 2-aminogluconolactone moiety, and a very
long chain fatty acid 27-hydroxyoctacosanoic acid. Pre-
viously, we reported that the 2-aminogluconolactone is
in equilibrium with the corresponding 2-aminogluco-
nate.37 Furthermore, studies with a compound that
was locked in the latter form displayed antagonistic
activity. In this report, we have shown that acylation
of the C-20 acyloxyacyl moiety with octacosanoic acid
is important for optimal antagonistic properties. The
hydroxyl of the natural 27-hydroxyoctacosanoic moiety
does, however, not account for inhibitory activity.
Figure 3. Concentration–response curves of E. coli LPS, R. sin-1 LPS,
R. sin-1 lipid A, and synthetic compounds 1–3 in human monocytic
cells. Mono Mac 6 cells were incubated for 5.5 h at 37 ꢁC with
increasing concentrations of E. coli LPS, R. sin-1 LPS, R. sin-1 lipid A,
1, 2, or 3 as indicated. TNF-a protein in cell supernatants was
measured using ELISA (please note that R. sin-1 LPS, R. sin-1 lipid A,
and 1–3 show background values and therefore overlap in the figure).
Treatment with E. coli LPS, R. sin-1 LPS, R. sin-1 lipid A, and 1–3 did
not affect cell viability, as judged by cellular exclusion of Trypan blue.

Y. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4800–4812
4805
4. Experimental
4.1. Chemical synthesis
were filtered off through a pad of Celite, and the residue
was washed with DCM (3· 5 mL). The combined fil-
trates were concentrated in vacuo, and the residue was
purified by silica gel column chromatography (eluent:
DCM/methanol, 100:1, v/v) to afford 7 as a white solid
Column chromatography was performed 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 un-
der reduced pressure at <40 ꢁC. CH2Cl2 was distilled
from CaH2 and tetrahydrofuran (THF) was distilled
from sodium directly prior to the application. CH3OH
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
(1.04 g, 98%). Rf = 0.35 (toluene/ethyl acetate, 4:1,
24:9
D
v/v); ½hꢁ ¼ ꢀ1:2 ꢂ (c = 1.0, CHCl3). 1H NMR
(300 MHz, CDCl3): d 5.21–5.14 (m, 1H, H-3), 2.66–
0
0
2.51 (m, 2H, H-2a, 2b), 2.26 (t, 2H, J2 ,3 = 7.8 Hz, H-
20), 1.60–1.56 (m, 4H, H-3a0, H-3b0, H-4a, H-4b), 1.23
(bs, 42H, 21 · CH2), 0.88–0.83 (m, 6H, 2 · CH3). 13C
NMR (75 MHz, CDCl3): d 176.28 (C@O), 173.28
(C@O), 69.96 (C-3), 38.87 (C-2), 34.34 (C-20), 33.97-
22.69 [C-(4-15), (30-130)], 14.11 (C-140, 16). HR-MS
(m/z) calcd for C30H58O4[M+Na]+, 505.4233; found:
505.4313.
˚
and stored over molecular sieves (3 A). Molecular sieves
˚
(3 and 4 A), used for reactions, were crushed and acti-
4.1.2.
Phenyl
3-O-acetyl-6-O-(4,6-O-benzylidene-2-
vated in vacuo at 390 ꢁC during 8 h in the first instance
and then for 2–3 h at 390 ꢁC directly prior to applica-
tion. Optical rotations were measured with a Jasco mod-
deoxy-2-phthalimido-b-D-glucopyranosyl)-2-azido-4-O-
benzyl-2-deoxy-1-thio-b-D-glucopyranoside (11). A sus-
pension of glycosyl donor 4 (435 mg, 0.81 mmol), accep-
tor 5 (290 mg, 0.68 mmol), and activated molecular
1
el P-1020 polarimeter. H NMR and 13C NMR spectra
˚
were recorded with Varian spectrometers (model Ino-
va500) equipped with Sun workstations. 1H NMR spec-
tra were recorded in CDCl3 and referenced to residual
CHCl3 at 7.24 ppm, and 13C NMR spectra were refer-
enced to the center peak of CDCl3 at 77.0 ppm. Assign-
ments were made by standard gCOSY and gHSQC.
High resolution mass spectra were obtained on a Bruker
model Ultraflex MALDI-TOF mass spectrometer. Sig-
nals marked with a subscript L symbol belong to the
biantennary lipid at C-20, whereas signals marked with
subscript L0 symbol belong to the side chain. Signals
marked with a subscript S symbol belong to the mono-
antennary lipids at C-2, C-3, and C-30.
sieves (4 A, 500 mg) in DCM (15 mL) was stirred under
an atmosphere of argon at room temperature for 2 h.
The reaction mixture was cooled (ꢀ75 ꢁC), and NIS
(191 mg, 0.85 mmol) and TfOH (5.8 lL, 0.07 mmol)
were added. After being warmed up to ꢀ35 ꢁC in
10 min, the reaction mixture was quenched by addition
of pyridine (100 lL) and diluted with DCM (20 mL).
The reaction mixture was washed with aqueous
Na2S2O3 (15%, 2· 40 mL) and water (2 · 50 mL). The
organic phase was dried (MgSO4) and filtered, and the
filtrate was concentrated in vacuo. The residue was puri-
fied by silica gel column chromatography (eluent: hex-
ane/ethyl acetate, 4:1, v/v) to afford
amorphous solid (461 mg, 84%). Rf = 0.45 (hexane/ethyl
6 as an
24:9
D
1
4.1.1. (R)-3-Tetradecanoyloxy-hexadecanoic acid (7).
Myristoyl chloride (2.22 mL, 8.16 mmol) was added
dropwise to a solution of 2-(4-bromophenyl)-2-oxoethyl
(R)-3-hydroxyhexadecanoate (3.65 g, 7.77 mmol), pyri-
dine (1.26 mL, 15.54 mmol), and DMAP (47 mg,
0.39 mmol) in DCM (70 mL). After stirring the reaction
mixture at room temperature for 9 h, it was diluted with
DCM (50 mL) and then washed with saturated aqueous
NaHCO3 (2· 60 mL) and brine (2· 60 mL). The organic
phase was dried (MgSO4) and filtered, and the filtrate
was concentrated in vacuo. The residue was purified
by silica gel column chromatography (eluent: toluene)
to afford 2-(4-bromophenyl)-2-oxoethyl (R)-3-tetradeca-
noyloxy-hexadecanoate as a colorless syrup (4.95 g,
acetate, 5:2, v/v). ½aꢁ ¼ ꢀ32:7ꢂ (c = 1.0, CHCl3); H
NMR (300 MHz, CDCl3): d 6.94–7.76 (m, 19H, aro-
matic), 5.59 (s, 1H, CH, benzylidene), 5.41 (d, 1H,
J1 ,2 = 8.7 Hz, H-10), 5.02 (dd, 1H, J2,3 = J3,4 = 9.3 Hz,
H-3), 4.71–4.63 (m, 1H, H-30), 4.43–4.33 (m, 3H, H-1,
H-20, H-60a), 4.25 (d, 1H, J = 10.8 Hz, CHH, Bn), 4.19
(d, 1H, CHH, Bn), 4.09 (d, 1H, H-6a), 3.88–3.82 (m,
1H, H-60b), 3.74 (dd, J5,6a = 11.1 Hz, J6a,6b = 4.2 Hz,
H-6b), 3.68–3.64 (m, 2H, H-40, H-50), 3.46–3.42 (m,
1H, H-5), 3.45 (dd, 1H, J4,5 = 9.6 Hz, H-4), 3.18 (t,
1H, H-2), 1.91 (s, 3H, COCH3). 13C NMR (75 MHz,
CDCl3): d 169.57 (C@O), 168.09 (C@O), 137.13–
123.52 (aromatic), 102.01 (CH, benzylidene), 98.64 (C-
10), 85.46 (C-1), 82.25(C-40), 75.66(C-3 or 4), 75.59 (C-
3 or 4), 74.51 (CH2, Bn), 68.68 (C-30, 60), 67.64 (C-6),
66.20 (C-50), 62.84 (C-2), 56.37 (C-2), 23.72 (CH3).
HR-MS (m/z) calcd for C42H40N4O11S[M+Na]+,
831.2312; found: 831.2519.
0
0
1
94%). Rf = 0.68 (DCM). H NMR (300 MHz, CDCl3):
d 7.74 (d, 2H, J = 8.7 Hz, aromatic), 7.64 (d, 2H,
J = 8.7 Hz, aromatic), 5.29–5.25 (m, 1H, H-3), 5.25 (s,
2H, H-2), 2.78–2.65 (m, 2H, H-2a, 2b), 2.28 (t, 2H,
J2 ,3 = 7.5 Hz, H-2a0, H-2b0), 1.63–1.56 (m, 4H, H-3a0,
H-3b0, H-4a, H-4b), 1.23 [broad, 42H, 21 · CH2],
0.88–0.84 (m, 6H, 2 · CH3). HR-MS (m/z) calcd for
C38H63BrO5[M+Na]+, 701.3757; found: 701.3568. Zinc
dust (719 mg, 11.1 mmol) was added portionwise to 2-
0
0
4.1.3. Phenyl 2-azido-4-O-benzyl-6-O-{4,6-O-benzyli-
dene-2-deoxy-2[(R)-3-tetradecanoyloxy-hexadecanoyl-
amino]-b-D-glucopyranosyl}-1-thio-b-D-glucopyranoside (12).
Compound 11 (250 mg, 0.31 mmol) was dissolved in a
mixture of n-butanol (25 mL) and ethylenediamine
(5 mL, 75 mmol). The reaction mixture was stirred at
90 ꢁC for 20 h, after which it was concentrated in vacuo.
The residue was dissolved in DCM (30 mL), and the
(4-bromophenyl)-2-oxoethyl
(R)-3-tetradecanoyloxy-
hexadecanoate (1.50 g, 2.21 mmol) in acetic acid
(15 mL). The reaction mixture was stirred at 60 ꢁC for
2 h and then diluted with DCM (20 mL). The solids

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Y. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4800–4812
solids were removed by filtration. The filtrate was con-
centrated in vacuo, and the residue was purified by silica
gel column chromatography (eluent: DCM/methanol,
30:1, v/v) to afford phenyl 6-O-(2-amino-4,6-O-benzyli-
dene-2-deoxy-b-D-glucopyranosyl)-2-azido-4-O-benzyl-
2-deoxy-1-thio-b-D-glucopyranoside as a white solid
(180 mg, 92%). Rf = 0.45 (DCM/methanol, 10:1, v/v).
1H NMR (300 MHz, CDCl3): d 7.59–7.25 (m, 15H, aro-
matic), 5.54 (s, 1H, CH, benzylidene), 4.78 (d, 1H, J=
10.4 Hz, CHH, Bn), 4.69 (d, 1H, CHH, Bn), 4.50 (d,
1H, J1,2 = 10.2 Hz, H-1), 4.33–4.28 (m, 2H, H-10, H-
60a), 4.14 (dd, 1H, J5,6a = 1.8 Hz, J6a,6b = 11.1 Hz, H-
6a), 3.80–3.50 (m, 6H, H-3, H-30, H-40, H-50, H-6b,
H-60b), 3.44–3.36 (m, 1H, H-5), 3.34–3.25 (m, 2H, H-
3, H-4), 2.78 (t, 1H, J2,3 = 9.6 Hz, H-2), 1.91 (s, 3H,
COCH3, acetyl). HR-MS (m/z) calcd for
C32H36N4O8S[M+Na]+, 659.2151; found: 659.2238. A
mixture of DCC (45 mg, 0.218 mmol) and 7 (70 mg,
0.145 mmol) in DCM (2 mL) was stirred at room tem-
perature for 10 min, and then the above-mentioned ami-
no derivative (58 mg, 91 lmol) in DCM (1 mL) was
added. The reaction mixture was stirred at room tem-
perature for 2 h, after which the solids were removed
by filtration, and the residue was washed with DCM
(2· 3 mL). The combined filtrates were concentrated in
vacuo, and the residue was purified by silica gel column
chromatography (eluent: DCM/methanol, 50:1, v/v) to
column chromatography (eluent: DCM/methanol,
60:1, v/v) to afford phenyl 2-amino-4-O-benzyl-6-O-
{4,6-O-benzylidene-2-deoxy-2-[(R)-3-tetradecanoyloxy-
hexadecanoylamino]-b-D-glucopyranosyl}-2-deoxy-1-thio-
b-D-glucopyranoside as a white solid (85.5 mg, 92.5%).
Rf = 0.40 (DCM/diethyl ether, 6:1, v/v). 1 H NMR
(500 MHz, CDCl3): d 7.26–7.53 (m, 15H, aromatic),
6.08 (d, 1H, JNH,2 = 5.0 Hz, NH), 5.57 (s, 1H, CH, ben-
zylidene), 5.06 (m, 1H, H-3L), 4.82 (d, 1H, J = 11.5 Hz,
CHH, Bn), 4.72 (d, 1H, J1 ,2 = 9.0 Hz, H-10), 4.66 (d,
1H, CHH, Bn), 4.53 (d, 1H, J1,2 = 9.5 Hz, H-1), 4.34
0
0
(dd, 1H, J5 ,6 a = 5.0 Hz, J6 a,6 b = 10.5 Hz, H-60a), 4.20
0
0
0
0
0
0
(d, 1H, H-6a), 4.05 (dd, 1H, J2 ,3 = 9.5 Hz,
0
J3 ,4 = 9.0 Hz, H-30), 3.79 (dd, 1H, J5 ,6 b = 10.0 Hz, H-
0
0
0
60b), 3.64–3.71 (m, 2H, H-5, H-6b), 3.58 (dd, 1H,
0
0
0
0
J4 5 = 9.0 Hz, H-4 ), 3.49–3.52 (m, 2H, H-2 , H-3),
3.45 (ddd, 1H, H-50), 3.26 (dd, 1H, J3,4 = J4,5 = 9.0 Hz,
H-4), 2.63 (dd, 1H, J2,3 = 10,0 Hz, H-2), 2.27–2.43 (m,
4H, H-2L, H-20L), 1.50–1.58 (m, 4H, H-4L, H-30L), 1.25
(m, 42H, 21 · CH2, lipid), 0.88–0.90 (m, 6H, 2 · CH3, li-
pid). 13C NMR (75 MHz, CDCl3): d 126.60–138.18 (aro-
matic), 102.18 (CH, benzylidene), 101.45 (C-10), 89.18
(C-1), 81.73 (C-40), 78.88 (C-5), 78.55 (C-3), 77.45 (C-
4), 74.71 (CH2, Bn), 72.04 (C-30), 71.59 (C-3L), 69.48
(C-6), 68.83 (C-60), 66.67 (C-50), 59.49 (C-20), 56.50 (C-
2). HR-MS (m/z) calcd for C62H94N2O11S[M+Na]+,
1097.6476; found: 1097.6069. A mixture of DCC
(57.5 mg, 0.279 mmol) and (R)-3-benzyloxy-hexadeca-
noic acid8 (85 mg, 0.232 mmol) in DCM (3 mL) was
stirred at room temperature for 10 min, after which
DMAP (6.2 mg, 0.051 mmol) and the above-mentioned
amino derivative (50 mg, 46.5 lmol) in DCM (1.5 mL)
were added. The reaction mixture was stirred at room
temperature for 5 h, and then the solids 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 (eluent: hexane/ethyl acetate, 5:1, v/v)
afford 12 as a white solid (85 mg, 85%). Rf = 0.60
24:8
D
(DCM/diethyl ether, 6:1, v/v); ½aꢁ ¼ ꢀ18:3ꢂ (c = 1.0,
1
CHCl3). H NMR (500 MHz, CDCl3): d 7.57–7.31 (m,
15H, aromatic), 5.50 (s, 1H, CH, benzylidene), 5.04
(m, 1H, H-3L), 4.76 (d, 1H, J = 11.5 Hz, CHH, Bn),
4.73 (d, 1H, J1 ,2 = 8.0 Hz, H-10), 4.67(d, 1H, CHH,
Bn), 4.56 (d, 1H, J1,2 = 10.0 Hz, H-1), 4.46 (br s, 1H,
0
0
OH0), 4.34 (dd, 1H, J5,6 a = 5.0 Hz, J6 a,6 b = 10.0 Hz,
0
0
0
H-60a), 4.16 (d, 1 H, H-6a), 4.06 (dd, 1H, J2 ,3 = 8.5 Hz,
0
0
J3 ,4 = 9.5 Hz, H-30), 3.79 (dd, 1H, J5 ,6 b = 10.5 Hz, H-
0
0
0
0
6b0), 3.60–3.70 (m, 3H, H-3, H-5, H-6b), 3.58 (dd, 1H,
J4 ,5 = 10.0 Hz, H-40), 3.52 (m, 1H, H-20), 3.45 (ddd,
1H, H-50), 3.27 (dd, 1H, J2,3 = 9.5 Hz, H-2), 3.27 (dd,
1H, J3,4 = J4,5 = 9.5 Hz, H-4), 2.76(b, 1H, OH), 2.25–
2.31 (m, 2H, H-2L), 1.53–1.58 (m, 4H, H-4L, H-30L),
1.25 (m, 42H, 21 · CH2, lipid), 0.87–0.90 (m, 6H,
2 · CH3, lipid). 13C NMR (75 MHz, CDCl3): d 174.31
(C@O), 171.72 (C@O), 137.65–126.36 (aromatic),
101.96 (CH, benzylidene), 101.13 (C-10), 85.57 (C-1),
81.49 (C-40), 78.59 (C-5), 77.86 (C-4), 77.21 (C-3),
74.84 (CH2, Bn), 71.72 (C-30), 71.48 (C-3L), 68.78 (C-
6), 68.58 (C-60), 66.44 (C-2), 59.14 (C-20). HR-MS
(m/z) calcd for C62H92N4O11S[M+Na]+, 1123.6381;
found: 1123.9268.
to afford 13 as a white solid (62 mg, 63%). Rf = 0.60
0
0
25:2
D
(hexane/ethyl acetate, 3:1, v/v). ½aꢁ ¼ þ9:1ꢂ (c = 1.0,
1
CHCl3). H NMR (300 MHz, CDCl3): d 7.14–7.43 (m,
30 H, aromatic), 6.46 (d, 1H, JNH,2 = 9.6 Hz, NH),
5.42 (s, 1H, CH, benzylidene), 5.36 (d, 1H,
JNH,2 = 9.0 Hz, NH0), 5.24 (dd, 1H, J2 .3 = 9.6 Hz,
0
0
0
J3 ,4 = 9.9 Hz, H-30), 5.10 (dd, 1H, J2,3 = 8.7 Hz,
J3,4 = 10.2 Hz, H-3), 4.96 (m, 1H, H-3L), 4.67 (d,
0
0
J1 ,2 = 8.4 Hz, H-10), 4.41–4.61 (m, 9H, H-1, 4 · CH2,
Bn), 4.32 (dd, 1H, J5,6a = 4.8 Hz, J6a,6b = 10.5 Hz, H-
6a), 4.04 (m, 1H, H-2), 3.96 (d, 1H, H-60a), 3.59–3.84
(m, 7H, H-20, H-40, H-6b, H-60b, 3 · H-3s), 3.36–3.49
(m, 3H, H-4, H-5, H-50), 2.09–2.69 (m, 10H, H-2L, H-
0
0
0
2L , 3 · H-2S), 1.57–0.98 (m, 118H, 59 · CH2, lipid),
4.1.4. Phenyl 4-O-benzyl-6-O-{4,6-O-benzylidene-3-O-
[(R)-3-benzyloxy-hexadecanoyl]-2-deoxy-2-[(R)-3-tetra-
decanoyloxy-hexadecanoylamino]-b-D-glucopyranosyl}-2-
[(R)-3-benzyloxy-exadecanoylamino]-3-O-[(R)-3-benzyl-
oxy-hexadecanoyl]-2-deoxy-1-thio-b-D-glucopyranoside (13).
1,3-Propanedithiol (0.18 mL, 1.75 mmol) was added to a
stirred solution of 12 (95 mg, 0.086 mmol) and triethyl-
amine (0.2 mL) in a mixture of pyridine and H2O
(7 mL, 6:1, v/v). The reaction mixture was stirred at
room temperature for 16 h and then concentrated in
vacuo to dryness. The residue was purified by silica gel
0.82 (m, 15H, 5 · CH3, lipid). 13C NMR (75 Hz,
CDCl3): d 126.34–138.73 (aromatic), 101.62 (C-10, CH,
benzylidene,), 86.95 (C-1), 79.68 (C-5 or 50), 79.13 (C-
40), 75.67–76.30 (C-3, 3 C-3s), 74.67 (C-4), 71.18-71.63
*
(C-30,3L, 4 · CH2, Bn), 68.86 (C-6 or 60), 68.33 (C-6
or 60), 66.43 (C-5 or 50), 55.24 (C-20), 53.10 (C-2); HR-
MS (m/z) calcd for
2130.4622; found: 2130.6140.
C
131H202N2O17S[M+Na]+,
4.1.5. 4-O-Benzyl-6-O-{4,6-O-benzylidene-3-O-[(R)-3-
benzyloxy-hexadecanoyl]-2-deoxy-a-2-[(R)-3-tetradeca-

Y. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4800–4812
4807
noyloxy-hexadecanoylamino]-b-D-glucopyranosyl}-2-[(R)-
3-benzyloxy-hexadecanoylamino]-3-O-[(R)-3-benzyloxy-
hexadecanoyl]-2-deoxy-a-D-glucopyranoside (15). TfOH
(0.5 lL, 4.4 lmol) was added to a stirred solution of
13 (31 mg, 14.7 lmol) and NIS (9.9 mg, 44 lmol) in a
mixture of DCM and H2O (4 mL, 100:1, v/v) at 0 ꢁC.
The reaction mixture was vigorously stirred for 30 min
until TLC analysis indicated that the reaction had gone
to completion, and then it was diluted with DCM
(10 mL) and washed with aqueous Na2S2O3 (15%,
15 mL) and water (2· 10 mL). The organic phase was
dried (MgSO4) and filtered, and the filtrate was concen-
trated in vacuo. The residue was purified by silica gel
column chromatography (eluent: hexane/ethyl acetate,
6/1:4/1, v/v) to afford 15 as a white solid (10.5 mg,
35%) and its 1,2-oxazoline derivative 14 (15 mg, 51%).
A mixture of the resulting 1,2-oxazoline and dibenzyl-
phosphate (4 mg, 15 lmol) in wet 1,2-dichloroethane
(1 mL) was stirred at room temperature for 1 h. The
reaction mixture was diluted with DCM (10 mL) and
washed with saturated aqueous NaHCO3 (2· 8 mL)
and brine (2· 8 mL). The organic phase was dried
(MgSO4) and filtered, and the filtrate was concentrated
in vacuo. The residue was purified by silica gel column
chromatography (eluent: hexane/ethyl acetate, 4:1, v/v)
to afford 15 as a white solid (13.5 mg, 90%). The overall
yield of 15 for the two steps was 80%. Rf = 0.2 (hexane/
ethyl acetate, 2.5:1, v/v). 1H NMR (500 MHz, CDCl3): d
7.18–7.40 (m, 25H, aromatic), 6.28 (d, 1H,
(500 MHz, CDCl3): d 7.20–7.38 (m, 25H, aromatic),
6.82 (d, 1H, JNH,2 = 8.5 Hz, NH), 6.61 (d, 1H,
JNH ,2 = 7.5 Hz, NH0), 5.60 (t, 1H, J2 ,3 = J3 ,4
=
0
0
0
0
0
0
10.0 Hz, H-30), 5.39 (s, 1H, CH, benzylidene), 5.34
(dd, 1H, J2,3 = J3,4 = 10.0 Hz, H-3), 5.07 (m, 1H, H-
3L), 4.95 (d, 1H, J1 ,2 = 8.0 Hz, H-10), 4.79 (t, 1H, H-
2), 4.37–4.60 (m, 9H, H-5, 4 · CH2, Bn), 4.28 (dd, 1H,
0
0
J5 ,6 a = 3.5 Hz, J6 a,6 b = 9.5 Hz, H-60a), 4.02–4.06 (m,
2H, H-4, H-6a), 3.80–3.85 (3H, 3 · 3s), 3.75 (t, 1H,
0
0
0
0
J5 ,6 b = 10.0 Hz, H-60b), 3.52–3.60 (m, 4H, H-20, H-40,
0
0
0
0
H-5 , H-6a), 2.25–2.68 (m, 10H, H-2L, H-2L , 3 · H-
2S), 1.60–1.03 (m, 118H, 59 · CH2, lipid), 0.86
(m, 15H, 5 · CH3, lipid). HR-MS(m/z) calcd for
C125H196N2O18[M+Na]+, 2036.4381; found: 2036.8009.
4.1.7. 2-Deoxy-6-O-{2-deoxy-3-O-[(R)-3-hydroxy-hexa-
decanoyl]-2-[(R)-3-tetradecanoyloxy-hexadecanoylamino-
b-D-glucopyranosyl}-2-[(R)-3-hydroxy-hexadecanoylami-
no]-3-O-[(R)-3-hydroxy-hexadecanoyl]-D-glucono-1,5-lac-
tone (3). Pd/C (10 mg) was added to the solution of
lactone 16 (7 mg, 3.5 lmol) in a mixture of THF and
t-BuOH (3 mL, 1:1, v/v). The reaction mixture was sha-
ken under an atmosphere of H2 (15 psi) at room tem-
perature for 24 h, after which the catalyst was
removed by filtration and washed with THF (3·
0.5 mL). The combined filtrates were concentrated in
vacuo to afford 3 as a white solid (5 mg, 81%). 1H
NMR (500 MHz, CDCl3):
J2,3 = J3,4 = 10.0 Hz, H-3), 5.10 (m, 1H, H-3L), 5.00
d
5.34 (t, 1H,
JNH,2 = 9.0 Hz, NH), 5.95 (d, 1H, JNH ,2 = 8.5 Hz,
(t, 1H, J2 ,3 = J3 ,4 = 10.0 Hz, H-30), 4.56 (d, 1H,
0 0 0 0
0
0
NH0), 5.44 (s, 1H, CH, benzylidene), 5.39–5.43 (m,
J1 ,2 = 8.5 Hz, H-10), 4.26 (m, 1H, H-5), 4.21 (d, 1H,
H-2), 4.19 (d, 1H, J6a,6b= 11.0 Hz, H-6a), 3.74–3.92
(m, 7H, H-4, H-20, H-60a, H-6b, 3 · H-3s), 3.63 (m,
0
0
0
2H, H-3, H-30), 5.17 (d, 1H, J1 ,2 = 8.5 Hz, H-1 ), 5.09
(m, 1H, H-1), 4.95 (m, 1H, H-3L), 4.40–4.61 (m, 8H,
0
0
4 · CH2, Bn), 4.35 (dd, 1H, J5 ,6 a = 5.0 Hz,
1H, H-60b), 3.47 (dd, 1H, J4 ,5 = 9.0 Hz, H-40), 3.27
0
0
0
0
J6 a,6 b = 10.0 Hz, H-60a), 4.20 (m, 1H, H-2), 4.07 (m,
1H, H-5), 3.96 (d, 1H, H-6a), 3.74–3.85 (m, 4H, H-
60b, 3 · H-3S), 3.61–3.68 (m, 3H, H-20, H-40, H-6b),
3.52 (ddd, 1H, H-50), 3.37 (t, 1H, J3,4 = J4,5 = 10.0 Hz,
(m, 1H, H-5 ), 2.27–2.18 (m, 10H, H-2L, 2L , 3 · H-
0
0
0
0
2S), 1.63–0.96 (m, 118 H, 59 · CH2, lipid), 0.84 (m,
15H, 5 · CH3, lipid). HR-MS (m/z) calcd for
C90H168N2O18[M+Na]+, 1588.2190; found: 1588.4548.
0
H-4), 2.20–2.66 (m, 10H, H-2L, H-2L , 3 · H-2S), 1.59–
1.10 (m, 118H, 59 · CH2, lipid), 0.84 (m, 15H,
5 · CH3, lipid). 13C NMR (75 Hz, CDCl3): d 126.0–
138.6 (aromatic), 101.5 (CH, benzylidene), 100.4 (C-
10), 91.5 (C-1), 79.0 (C-40), 76.4 (C-4), 75.5–76.4 (C-
3 · 3S), 74.4 (CH2, Bn), 73.5 (C-3 or 30), 71.6 (C-5),
71.0–71.4 (C-3L, 3 or 30, 3 · CH2, Bn), 68.7 (C-60),
67.5 (C-6), 66.5 (C-50), HR-MS (m/z) calcd for
4.1.8.
Phenyl
3-O-acetyl-6-O-(4,6-O-benzylidene-2-
deoxy-2-phthalimido-b-D-glucopyranosyl)-2-azido-4-O-
benzyl-2-deoxy-1-thio-b-D-glucopyranoside (17). The gly-
cosylation of 4 (87 mg, 0.20 mmol) and 6 (58 mg,
0.136 mmol) was performed similar to the synthesis of
11 using NIS (47 mg, 0.21 mmol) and TfOH (1.7 lL,
0.02 mmol) in DCM (2 mL) to afford 13 as an amor-
C
125H198N2O18[M+Na]+, 2038.4537; found: 2038.3850.
phous solid (90 mg, 82%). Rf = 0.41 (hexane/ethyl ace-
24:9
D
tate, 5:2, v/v). ½aꢁ ¼ ꢀ40:5ꢂ (c = 1.0, CHCl3). 1H
4.1.6. 4-O-Benzyl-6-O-{4,6-O-benzylidene-3-O-[(R)-3-ben-
zyloxy-hexadecanoyl]-2-deoxy-2-[(R)-3-tetradecanoyloxy-
hexadecanoylamino]-b-D-glucopyranosyl}-2-[(R)-3-ben-
zyloxy-hexadecanoylamino]-3-O-[(R)-3-benzyloxy-hexadeca-
noyl]-2-deoxy-D-glucono-1,5-lactone (16). A suspension
of 15 (8.0 mg, 4.0 lmol) and activated molecular sieves
NMR (300 MHz, CDCl3): d 7.70–6.88 (m, 19H, aro-
matic), 5.56–5.55 (m, 2H, H-1, CH, benzylidene),
5.32–5.23 (m , 2H, H-10, H-3), 4.71–4.63 (m, 1H, H-
30), 4.40–4.27 (m, 3H, H-20, H-5, H-60a), 4.25 (d, 1H,
J= 10.5 Hz, CHH, Bn), 4.03–3.96 (m, 2H, H-6a,
CHH, Bn), 3.85–3.77 (m, 3H, H-2, H-6b, H-60b),
3.66–3.61 (m, 2H, H-40, H-50), 3.47 (dd, 1H,
J3,4 = J4,5 = 9.6 Hz, H-4), 1.90 (s, 3H, COCH3, acetyl).
13C NMR (75 MHz, CDCl3): d 169.30 (C@O), 136.89–
123.38 (aromatic), 101.79 (CH, benzylidene), 98.59 (C-
10), 86.92 (C-1), 81.75 (C-40), 75.98 (C-4), 74.26 (CH2,
Bn), 73.01 (C-3), 70.51 (C-5), 68.36 (C-30, C-60), 67.26
(C-6), 66.17 (C-50), 61.93 (C-2), 56.28 (C-2), 20.59
˚
(3 A, 15 mg) in DCM (2.0 mL) was stirred at room tem-
perature under an atmosphere of argon for 1 h. PCC
(4.3 mg, 20 lmol) was then added and the reaction mix-
ture was stirred for 1 h until TLC analysis indicated the
reaction had gone to completion. The reaction mixture
was purified by Iatro beads column chromatography
(eluent: hexane/ethyl acetate, 4:1, v/v) to afford 16 as a
23
D
white solid (7 mg, 87%). ½aꢁ ¼ ꢀ12:5ꢂ (c = 1, CHCl3).
(CH3,
acetyl).
HR-MS
(m/z)
calcd
for
Rf = 0.65 (hexane/ethyl acetate, 2.5:1, v/v). 1H NMR
C42H40N4O11S[M+Na]+, 831.2312; found: 831.1761.

4808
Y. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4800–4812
4.1.9. Phenyl 2-azido-4-O-benzyl-6-O-{4,6-O-benzyli-
dene-2-deoxy-2-[(R)-3-levulinoyloxy-hexadecanoylamino]-
b-D-glucopyranosyl}-2-deoxy-1-thio-a-D-glucopyranoside
(18). The phthalimido and acetyl group of 17 (210 mg,
24.7 mmol) were removed similar to the deprotection
of 11 in a mixture of n-butanol (15 mL) and ethylenedi-
amine (3 mL, 45 mmol) to afford phenyl 6-O-(2-amino-
4,6-O-benzylidene-2-deoxy-b-D-glucopyranosyl)-2-azido-
1-thio-a-D-glucopyranoside as
a
colorless syrup
(397 mg, 91%). Rf = 0.45 (DCM/methanol, 15:1, v/v).
A mixture of (R)-3-benzyloxy-hexadecanoic acid 8
(226 mg, 624 lmol) and DCC (154 mg, 749 lmol) was
stirred for 10 min, and then the above-described amine
(150 mg, 156 lmol) in DCM (2 mL) and DMAP
(3.0 mg, 24.7 lmol) were added. After stirring the reac-
tion mixture for 16 h, the solids were filtered off, and
the residue was washed with DCM (2· 1 mL). The com-
bined filtrates were concentrated in vacuo, and the resi-
due was purified by silica gel column chromatography
(eluent: toluene/ethyl acetate, 10:1, v/v) to afford 19 as
4-O-benzyl-2-deoxy-1-thio-a-D-glucopyranoside as
a
colorless syrup (142 mg, 91%). Rf = 0.52 (DCM/metha-
nol, 1:9, v/v). DCC (67 mg, 32.4 mmol) was added to a
solution of (R)-3-levulinoyloxyhexadecanoic acid 9
(100 mg, 27.0 mmol) in DCM (5 mL) and the resulting
solution was stirred for 10 min. Next, the above-de-
scribed amino derivative (142 mg, 22.5 mmol) in DCM
(2 mL) was added and the reaction mixture was stirred
for 12 h at room temperature. The solids were filtered-
off and the residue was washed with DCM (2· 3 mL).
The combined filtrates were concentrated in vacuo and
the residue was purified by silica gel column chromatog-
raphy (eluent: DCM/methanol, 50:1, v/v) to afford 18 as
a white solid (209 mg, 67%). Rf = 0.56 (DCM/methanol,
23
D
60:1, v/v). ½aꢁ ¼ þ11:5ꢂ (c = 1.0, CHCl3). 1H NMR
(600 MHz, CDCl3): d 7.38–7.20 (m, 30H, aromatic),
6.55 (d, 1H, JNH ,2 = 9.6 Hz, NH0), 6.45 (d, 1H,
JNH,2 = 8.4 Hz, NH), 5.71 (d, 1H, J1,2 = 4.8 Hz, H-1),
5.33–5.31 (m, 2H, H-30, CH, benzylidene), 5.24 (t, 1H,
J2,3 = J3,4 = 9.6 Hz, H-3), 4.88 (m, 1H, H-3L), 4.83 (d,
0
0
1H, J1 ,2 = 8.4 Hz, H-10), 4.62–4.41 (m, 9H, H-2,
4 · CH2, Bn), 4.41–4-27 (m, 2H, H-5, H-60a), 4.17 (m,
1H, H-20), 4.01 (d, 1H, J6a,6b = 10.8 Hz, H-6a), 3.92 (d,
1H, H-6b), 3.80–3.76 (m, 4H, H-4, 3 · H-3S), 3.69 (t,
0
0
a white solid (189 mg, 86%). Rf = 0.55 (DCM/diethyl
23
ether, 6:1, v/v). ½aꢁ ¼ þ4:9ꢂ (c = 1.0, CHCl3). 1H
D
NMR (500 MHz, CDCl3): d 7.53–7.18 (m, 15H, aro-
1H, J5 ,6 b = J6 a,6 b = 10.6 Hz, H-60b), 3.62 (t, 1H,
0 0 0 0
matic), 6.34 (d, 1H, JNH ,2 = 7.0 Hz, NH0), 5.61 (d,
1H, J1,2 = 5.0 Hz, H-1), 5.56 (s, 1H, CH, benzylidene),
J3 ,4 = J4 ,5 = 9.6 Hz, H-40), 3.47 (m, 1H, H-50), 2.87–
2.21 (m, 12H, H-2L, 3 · H-2S, 2 · CH2, Lev), 2.07(s,
3H, CH3, Lev), 1.59–1.51 (m, 8H, H-4L, 3 · H-4S),
1.23 (broad, 88H, 44 · CH2, lipid), 0.88–0.86 (m, 12H,
0
0
0
0
0
0
0
0
5.07–5.02 (m, 1H, H-3L), 4.91 (d, 1H, J1 ,2 = 8.5 Hz,
H-10), 4.84 (d, 1H, J = 11.5 Hz, CHH, Bn), 4.74 (d,
1H, CHH, Bn), 4.37 (d, 1H, J = 10.0 Hz, H-5), 4.32
4 · CH3,
lipid).
HR-MS
(m/z)
calcd
for
(dd, 1H, J5 ,6 a = 5.0 Hz, J6 a,6 b = 10.5 Hz, H-60a), 4.23
C122H182N2O18S[M+Na]+, 2018.3006; found: 2018.2588.
0
0
0
0
(t, 1H, J2 ,3 = J3 ,4 = 9.5 Hz, H-30), 4.13 (d, 1H,
J6a,6b = 10.0 Hz, H-6a), 3.98 (dd, 1H, J = 10.0 Hz,
J = 9.0 Hz, H-3), 3.89–3.86 (m, 2H, H-2, H-60b), 3.80
(t, 1H, J5,6b = 10.0 Hz, H-6b), 3.63 (t, 1H,
0
0
0
0
4.1.11. Phenyl 4-O-benzyl-6-O-{4,6-O-benzylidene-3-O-
[(R)-3-benzyloxy-hexadecanoyl-2-deoxy-2-[(R)-3-(27-ben-
zyloxy-octacosanoyloxy)-hexadecanoylamino]-b-D-gluco-
pyranosyl}-2-[(R)-3-benzyloxy-hexadecanoylamino]-3-O-
[(R)-3-benzyloxy-hexadecanoyl]-2-deoxy-1-thio-a-D-gluco-
pyranoside (20). To a solution of 19 (50.0 mg, 25 lmol)
in DCM (2 mL) was added dropwise hydrazine acetate
(2.4 mg, 26 lmol) in methanol (0.2 mL). The resulting
mixture was stirred at room temperature for 3 h after
which TLC indicated completion of the reaction. The
reaction mixture was concentrated in vacuo, and the res-
idue was purified by preparative silica gel TLC chroma-
tography (eluent: DCM/methanol, 60:1, v/v) to afford
the alcohol as a white solid (43 mg, 90%). Rf = 0.48
(DCM/methanol, 60:1, v/v). 1H NMR (600 MHz,
CDCl3): d 7.37–7.21 (m, 30H, aromatic), 6.48 (d, 1H,
JNH,2 = 7.8 Hz, NH), 5.68–5.65 (m, 2H, H-1, NH0),
0
0
J3,4 = J4,5 = 9.5 Hz, H-4), 3.58 (dd, 1H, J4 ,5 = 9.0 Hz,
H-40), 3.54–3.48 (m, 2H, H-20, H-50), 2.97-2.24 (m, 6H,
H-2L, 2 · CH2, Lev), 2.17 (s, 3H, CH3, Lev), 1.59–1.51
(m, 2H, H-4L), 1.24 (broad, 22H, 11 · CH2, lipid),
0.92–0.90 (m, 3H, CH3, lipid). 13C NMR (75 MHz,
CDCl3): d 137.76–123.74 (aromatic), 102.02 (CH, ben-
zylidene), 100.86 (C-10), 87.64 (C-1), 81.62(C-40), 78.43
(C-4), 75.26 (CH2, Bn), 74.31 (C-3), 72.6 4(C-3L),
71.62 (C-3, 5), 71.45 (C-30), 69.02 (C-60), 68.33 (C-6),
66.71 (C-50), 64.19 (C-2), 59.41 (C-20). HR-MS (m/z)
calcd for C53H72N4O11S[M+Na]+: 1012.2143; found:
1012.3649.
4.1.10. Phenyl 4-O-benzyl-6-O-{4,6-O-benzylidene-3-O-
[(R)-3-benzyloxy-hexadecanoyl-2-deoxy-2-[(R)-3-levulinoyl-
oxy-hexadecanoylamino]-b-D-glucopyranosyl}-2-[(R)-3-
benzyloxy-hexadecanoylamino]-3-O-[(R)-3-benzyloxy-hexa-
decanoyl]-2-deoxy-1-thio-a-D-glucopyranoside (19). Tri-
ethylamine (1.0 mL) was added to a stirred solution of
18 (450 mg, 0.455 mmol) and propanedithiol (491 mg,
45.5 mmol) in pyridine (15 mL) and H2O (1.5 mL).
The reaction mixture was stirred at room temperature
for 14 h, after which it was concentrated in vacuo to
dryness. The residue was co-evaporated with toluene
(2· 30 mL) and ethanol (2· 20 mL) and then purified
by silica gel column chromatography (eluent: DCM/
methanol, 30:1, v/v) to afford phenyl 2-amino-4-O-ben-
zyl-6-O-{4,6-O-benzylidene-2-deoxy-2-[(R)-3-levulinoyl-
oxy-hexadecanoylamino]-b-D-glucopyranosyl}-2-deoxy-
5.46 (t, 1H, J2 ,3 = J3 ,4 = 9.6 Hz, H-30), 5.41 (s, 1H,
0
0
0
0
CH, benzylidene), 5.25 (t, 1H, J2,3 = J3,4 = 10.2 Hz, H-
0
0
0
3), 4.79 (d, 1H, J1 ,2 = 7.8 Hz, H-1 ), 4.62–4.41 (m, 9H,
H-2, 4 · CH2, Bn), 4.32 (m, 1H, H-5), 4.29 (dd, 1H,
J5 ,6 a = 4.8 Hz, J6 a,6 b = 10.2 Hz, H-60a), 3.98 (d, 1H,
J6a,6b = 9.6 Hz, H-6a), 3.84–3.69 (m, 8H, H-H-20, H-4,
0
0
0
0
H-6b,
0
H-60b,
0
4 · H-3S),
3.63
(t,0
1H,
J3 ,4 = J4 ,5 = 9.6 Hz, H-40), 3.48 (m, 1H, H-5 ), 2.62–
2.56 (m, 2H, H-2S), 2.49 (dd, 1H, J = 5.4 Hz,
J = 15.0 Hz, H-2Sa), 2.43(dd, 1H, J = 5.4 Hz,
J = 15.6 Hz, H-2Sb), 2.32–2.27 (m, 2H, H-2S), 1.96–
1.88 (m, 2H, H-2S), 1.57–1.55 (m, 8H, 4 · H-4S), 1.24
(broad, 88H, 44 · CH2, lipid), 0.87–0.85 (m, 12H,
4 · CH3, lipid). 13C NMR (75 MHz, CDCl3): d 172.63
(C@O), 172.10 (C@O), 171.44 (C@O), 171.26 (C@O),
0
0

Y. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4800–4812
4809
138.45–126.26 (aromatic), 101.49 (CH, benzylidene),
100.03 (C-1), 87.53 (C-1), 78.70 (C-40), 77.20–75.96 (C-
4, 3 · C-3S), 75.96 (C-4), 75.63 (CH2, Bn), 74.56 (C-3),
74.56, 73.46, 71.41, 71.19, 70.95 (C-30, 5, 3 · CH2, Bn),
68.54 (C-60), 68.19 (C-3S), 66.94 (C-6), 66.43 (C-50),
55.40 (C-20), 52.72 (C-2). A mixture of 27-hydroxyocta-
cosanoic acid 10 (22 mg, 42 lmol) and DCC (13 mg,
65 lmol) in DCM (1.5 mL) was stirred at room temper-
ature for 10 min, and then the above alcohol (50 mg,
26 lmol) in DCM (1 mL) and DMAP (2.5 mg, 21 lmol)
were added. The reaction mixture was stirred at room
temperature for 15 h, the solids were filtered off, and
the residue was washed with DCM (2· 1 mL). The com-
bined filtrates were concentrated in vacuo, and the resi-
due was purified by preparative silica gel TLC
chromatography (eluent: DCM/methanol, 60:1, v/v) to
afford 20 as a white fluffy solid (35.7 mg, 63%).
Rf = 0.65 (DCM/methanol, 60:1, v/v).1H NMR
(500 MHz, CDCl3): d = 7.40–7.18 (m, 30H, aromatic),
6.48 (d, 1H, JNH,2 = 8.5 Hz, NH), 5.72 (d, 1H,
J3 ,4 = 9.3 Hz, H-30), 5.24 (t, 1H, J2,3 = J3,4 = 9.0 Hz,
0 0
0
0
H-3), 4.95 (m, 1H, H-3L), 4.80 (d, 1H, J1 ,2 = 8.4 Hz,
H-10), 4.37–4.61 (m, 9H, H-2, 4 · CH2, Bn), 4.33 (m,
0 0
J5 ,6 a = 5.4 Hz,
1H,
0
H-5),
4.29
(dd,
1H,
J6 a,6 b = 11.1 Hz, H-60a), 3.64–4.00 (m, 8H, H-20, H-4,
H-6a, H-6b, H-60b, 3 · H-3S), 3.60 (dd, 1H,
0
J4 ,5 = 9.3 Hz, H-40), 3.48 (m, 1H, H-50), 1.90–2.64 (m,
0
0
0
10H, H-2L, H-2L , 3 · H-2S), 1.63–1.05 ((m, 146H,
73 · CH2, lipid), 0.85 (m, 15H, 5 · CH3, lipid). 13C
NMR (75 MHz, CDCl3): d 173.91 (C@O), 172.35
(C@O), 171.45 (C@O), 171.28 (C@O), 169.91 (C@O),
135.59–125.00 (aromatic), 101.23 (CH, benzylidene),
100.92 (C-10), 87.03 (C-1), 78.08 (C-40), 75.93 (C-4,
2 · C-3S), 75.21 (C-3S), 74.52 (CH2, Bn), 73.00 (C-3),
71.2 (C-5), 70.91 (C-30), 70.42–70.64 (3 · CH2, Bn),
70.33 (C-3L), 68.26 (C-60), 67.41 (C-6), 66.03 (C-50),
55.52 (C-20), 52.66 (C-2). HR-MS (m/z) calcd for
C
145H230N2O17S[M+Na]+, 2326.6847; found: 2326.7550.
4.1.13. 4-O-Benzyl-6-O-{4,6-O-benzylidene-3-O-[(R)-3-
benzyloxy-hexadecanoyl]-2-deoxy-2-[(R)-3-(27-benzyloxy-
octacosanoyloxy)-hexadecanoylamino]-b-D-glucopyrano-
syl}-2-[(R)-3-benzyloxy-hexadecanoylamino]-3-O-[(R)-3-
benzyloxy-hexadecanoyl]-2-deoxy-D-glucopyranose (22).
NIS (10.0 mg, 44.4 lmol) and TMSOTf (0.3 lL,
1.5 lmol) were added to a stirred solution of 20
(25 mg, 10.4 lmol) in DCM/H2O (3 mL, 100:1, v/v) at
0 ꢁC. The reaction mixture was vigorously stirred at
room temperature for 20 min until TLC analysis indi-
cated that reaction had gone to completion. The reac-
tion mixture was diluted with DCM (5 mL), and then
washed with aqueous Na2S2O3 (10%, 5 mL) and water
(2· 5 mL). The organic phase was dried (MgSO4) and
filtered. The filtrate was concentrated in vacuo, and
the residue was purified by silica gel column chromato-
graphy (eluent: toluene/ethyl acetate, 20:1, v/v) to afford
lactol 22 as a white solid (18 mg, 78%). Rf = 0.45 (DCM/
J1,2 = 4.5 Hz, H-1), 5.61 (d, 1H, JNH ,2 = 8.5 Hz, NH0),
0
0
5.39 (s, 1H, CH, benzylidene), 5.39 (t, 1H,
0
J2 ,3 = J3 ,4 = 9.5 Hz, H-30), 5.27 (dd, 1H, J = 9.5 Hz,
J = 10.0 Hz, H-3),0 4.97 (m, 1H, H-3L), 4.89 (d, 1H,
0
0
0
0
0
J1 ,2 = 8.5 Hz, H-1 ), 4.62–4.42 (m, 11H, H-2, 5 · CH2,
Bn), 4.38 (m, 1H, H-5), 4.31 (dd, 1H, J5 ,6 a = 5.0 Hz,
0
0
J6 a,6 b = 11.0 Hz, H-60a), 3.98 (d, 1H, J6a,6b = 10.5 Hz,
H-6a), 3.89-3.83 (m, 2H, H-6b, H-3S), 3.80–3.79 (m,
0
0
2H, 2 · H-3S), 3.73–3.66 (m, 3H, H-20, H-4, H-60b),
0
3.63 (t, 1H, J4 ,5 = 9.5 Hz, H-4 ), 3.52–3.47 (m, 2H, H-
0
0
0
5 , H-27L ), 2.65–2.57 (m, 2H, H-2S), 2.51 (dd, 1H,
0
J = 5.5 Hz, J = 15.0 Hz, H-2Sa), 2.45 (dd, 1H,
J = 5.5 Hz, J = 15.5 Hz, H-2Sb), 2.33–2.19 (m, 4H,
0
2 · H-2S, H-2La, H-2La ), 2.09–1.98 (m, 2H, H-2Lb, H-
0
0
0
2
Lb ), 1.59–1.40 (m, 12H, H-4L, H-3L , H-26L , 3 · H-
4S), 1.25 (broad, 176H, 88 · CH2, lipid), 1.18 (d, 3H,
0
0
0
J27L ,28L = 6.0 Hz, H-28L ), 0.89–0.85 (m, 12H,
4 · CH3, lipid). 13C NMR (75 MHz, CDCl3): d 173.98
(C@O), 172.45 (C@O), 171.40 (C@O), 171.26 (C@O),
169.96 (C@O), 139.65–126.31 (aromatic), 101.96 (CH,
benzylidene), 101.01 (C-10), 87.5 8 (C-1), 79.05 (C-40),
1
methanol, 50:1, v/v). H NMR (600 MHz, CDCl3): d
7.39–7.16 (m, 25H, aromatic), 6.28 (d, 1H,
0
0
JNH,2 = 9.0 Hz, NH), 5.92 (d, 1H, JNH ,2 = 7.8 Hz,
NH0), 5.43 (s, 1H, CH, benzylidene), 5.42–5.39 (m,
77.26 (C-3S), 76.68 (C-3S), 75.96 (C-3S), 75.26 (C-27L ),
2H, H-3, H-30), 5.20 (d, 1H, J1 ,2 = 8.5 Hz, H-10), 5.09
(m, 1H, H-1), 4.96 (m, 1H, H-3L), 4.61–4.40 (m, 8H,
0
0
0
74.51 (CH2, Bn), 73.88 (C-3), 71.86 (C-5), 70.96 (C-30),
70.68–70.42 (C-4, C-3L, 3 · CH2, Bn), 68.85 (C-60),
67.78 (C-6), 66.56 (C-50), 55.86 (C-20), 53.12 (C-2).
HR-MS (m/z) calcd for C152H236N2O18S[M+Na]+,
2432.7232; found: 2432.9846.
0
0
4 · CH2, Bn), 4.35 (dd, 1H, J5 ,6 a = 5.4 Hz,
J6 a,6 b = 10.8 Hz, H-60a), 4.20 (m, 1H, H-2), 4.06 (m,
1H, H-5), 3.95 (d, 1H, J6a,6b = 12.0 Hz, H-6a), 3.84–
0
0
3.740 (m, 4H, H-60b, 3 · H-3S), 3.69–3.58 (m, 3H, H-20,
0
0
H-4 , H-6b), 3.54–3.48 (m, 2H, H-5 , H-27L ), 3.37 (t,
4.1.12. Phenyl 4-O-benzyl-6-O-{4,6-O-benzylidene-3-O-
[(R)-3-benzyloxy-hexadecanoyl]-2-deoxy-2-[(R)-3-octaco-
sanoyloxy-hexadecanoylamino]-b-D-glucopyranosyl}-2-[(R)-
3-benzyloxy-hexadecanoylamino]-3-O-[(R)-3-benzyloxy-
hexadecanoyl]-2-deoxy-1-thio-a-D-glucopyranoside (21).
The above alcohol intermediate (40 mg, 20 lmol) was
acylated similar to the synthesis of 20 with octacosanoic
acid (12 mg, 30 lmol), using DCC (9.3 mg, 45 lmol)
and DMAP (2 mg, 16.5 lmol) as activating agents, to
1H, J4,3 = J4,5 = 9.6 Hz, H-4), 2.65–2.56 (m, 2H, H-2S),
2.50 (dd, 1H, J = 5.4 Hz, J = 15.0 Hz, H-2Sa), 2.41 (dd,
1H, J = 4.8 Hz, J = 15.6 Hz, H-2Sb), 2.35–2.20 (m, 4H,
0
2 · H-2S, H-2La, H-2La ), 2.12–2.02 (m, 2H, H-2Lb
,
0
0
0
2Lb ), 1.59–1.40 (m, 12H, H-4L, H-3L,26L , 3 · H-4S),
1.25 (broad, 176H, 88 · CH2, lipid), 1.18 (d, 3H,
J27L ,28L = 6.0 Hz, H-28L ), 0.89–0.87 (m, 12H,
0
0
0
4 · CH3,
lipid).
HR-MS
(m/z)
calcd
for
C
146H232N2O19[M+Na]+, 2340.7147; found: 2340.7925.
afford 21 as a white fluffy solid (30 mg, 65%). Rf = 0.65
26
D
(DCM/methanol, 60:1, v/v). ½aꢁ ¼ þ14:8ꢂ (c = 1.0,
4.1.14. 4-O-Benzyl-6-O-{4,6-O-benzylidene-3-O-[(R)-3-
benzyloxy-tetradecanoyl-2-deoxy-2-[(R)-3-octacosanoyl-
oxy-hexadecanoylamino]-b-D-glucopyranosyl}-2-[(R)-3-ben-
zyloxy-hexadecanoylamino]-3-O-[(R)-3-benzyloxy-tetra-
decanoyl]-2-deoxy-D-glucopyranose (23). Compound 21
1
CHCl3). H NMR (300 MHz, CDCl3): d 7.41–7.11 (m,
30H, aromatic), 6.46 (d, 1H, JNH,2 = 8.4 Hz, NH), 5.70
(d, 1H, J1,2 = 5.3 Hz, H-1), 5.59 (d, 1H, JNH ,2 = 8.1 Hz,
NH0), 5.37 (s, 1H, CH, benzylidene), 5.37 (dd, 1H,
0
0

4810
Y. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4800–4812
(30 mg, 13 lmol) was hydrolyzed similar to the synthesis
of 22 with NIS (12 mg, 52 lmol) and TMSOTf (1.0 lL,
2.7 lmol) in DCM/H2O (4 mL, 100:1) to afford 23 as a
white solid (23.4 mg, 80%). Rf = 0.45 (DCM/methanol,
50:1, v/v). 1H NMR (300 MHz, CDCl3): d 7.38–7.12
(m, 26H, aromatic), 6.26 (d, 1H, JNH,2 = 9.6 Hz, NH),
dene), 5.31 (dd, 1H, J2,3 = J3,4 = 9.5 Hz, H-3), 5.04 (m,
1H, H-3L), 4.93 (d, 1H, J1 ,2 = 8.5 Hz, H-10), 4.78 (t,
1H, J2,3 = 10.5 Hz, H-2), 4.59–4.36 (m, 9H, H-5,
0
0
0
0
4 · CH2, Bn), 4.27 (dd, 1H, J5 ,6 a = 5.5 Hz,
J6 a,6 b = 10.5 Hz, H-60a), 4.04–4.01 (m, 2H, H-4, H-
6a), 3.87–3.75 (m, 3H, 3 · H-3S), 3.69 (t, 1H,
0
0
5.90 (d, 1H, JNH ,2 = 8.1 Hz, NH0), 5.41 (s, 1H, CH,
J5,6 b = 9.5 Hz, H-60b), 3.57–3.50 (m, 4H, H-20, H-40,
0
0
0
benzylidene), 5.40–5.36 (m, 2H, H-3, H-30), 5.18 (d,
H-5 , H-6b), 2.30–1.93 (m, 10H, H-2L, H-2L , 3 · H-
0
0
1H, J1 ,2 = 8.4 Hz, H-10), 5.10 (d, 1H, J1,2 = 3.3 Hz, H-
1), 4.95 (m, 1H, H-3L), 4.59–4.37 (m, 9H, H-2,
4 · CH2, Bn), 4.36 (m, 1H, H-60a), 4.17 (m, 1H, H-2),
4.03 (m, 1H, H-5), 3.92 (m, 1H, H-6a), 3.83–3.47 (m,
8H, H-20, H-40, H-50, H-6b, H-60b, 3 · H-3S), 3.35 (t,
2S), 1.00–1.75 (m, 146H, 73 · CH2, lipid), 0.82 (m,
15H, 5 · CH3, lipid). HR-MS (m/z) calcd for
0
0
C
139H224N2O18[M+Na]+, 2232.6572; found: 2232.6703.
4.1.17. 2-Deoxy-6-O-{2-deoxy-3-O-[(R)-3-hydroxy-tetra-
decanoyl]-2-[(R)-3-(27-hydroxy-octacosanoyloxy)-hexa-
decanoylamino]-b-D-lucopyranosyl}-2-[(R)-3-hydroxy-hexa-
decanoylamino]-3-O-[(R)-3-hydroxy-hexadecanoyl]-D-gluc-
ono-1,5-lactone (1). Compound 24 (7.0 mg, 3.0 lmol)
was dissolved in THF/t-BuOH (2 mL, 1:3, v/v) and Pd/
C (10 mg) was added. The reaction mixture was shaken
under an atmosphere of H2 (15 psi) at room tempera-
ture for 36 h, the catalyst was filtered off, and the res-
idue was washed with THF (2· 1 mL). The combined
filtrates were concentrated to afford 1 as a colorless
film (4.2 mg, 75%). 1H NMR (600 MHz, CDCl3/
CD3OD, 1:1, v/v): d = 5.40 (t, 1H, J2,3 = J3,4 = 9.6 Hz,
0
1H, J4,5 = 9.6 Hz, H-4), 1.64–1.99 (m, 10H, H-2L, H-2L ,
3 · H-2S), 1.63–1.05 (m, 146H, 73 · CH2, lipid), 0.88–
0.84 (m, 15H, 5 · CH3, lipid). HR-MS (m/z) calcd for
C139H226N2O18[M+Na]+, 2234.6728; found: 2234.6657.
4.1.15. 4-O-Benzyl-6-O-{4,6-O-benzylidene-3-O-[(R)-3-
benzyloxy-tetradecanoyl-2-deoxy-2-[(R)-3-(27-benzyloxy-
octacosanoyloxy)-hexadecanoylamino]-b-D-glucopyrano-
syl}-2-[(R)-3-benzyloxy-hexadecanoylamino]-3-O-[(R)-3-
benzyloxy-tetradecanoyl]-2-deoxy-D-glucono-1,5-lactone
(24). A suspension of 22 (10 mg, 4.3 lmol) and activated
˚
molecular sieves (3 A, 25 mg) in DCM (2 mL) was stir-
0
0
0
0
red at room temperature under an atmosphere of argon
for 1 h. PCC (9.3 mg, 43 lmol) was then added and the
reaction mixture was stirred for another 1.5 h until TLC
analysis indicated completion of the reaction. The reac-
tion mixture was purified by Iatro beads column chro-
matography (eluent: hexane/ethyl acetate, 5/1:3/1, v/v)
to afford lactone 24 as a colorless film (7.0 mg, 70%).
Rf = 0.35 (hexane/ethyl acetate, 3:1, v/v). 1H NMR
(600 MHz, CDCl3): d = 7.39–7.18 (m, 25H, aromatic),
H-3), 5.12 (br s, 1H, H-3L), 4.98 (t, 1H, J2 ,3 = J3 ,4 =
9.6 Hz, H-30), 4.62 (br s, 1H, H-10), 4.30–3.95 (m, H-2,
H-4, H-5, H-60a, H-60b), 3.81–3.33 (m, H-20, H-40,
0
H-5 , H-6a, H-6b, H-27L ), 2.44–2.24 (m, 10H, H-2L,
0
0
H-2L ,
C
3 · H-2S).
HR-MS
(m/z)
calcd
for
104H196N2O19[M+Na]+, 1800.4330; found: 1800.6962.
4.1.18. 2-Deoxy-6-O-{2-deoxy-3-O-[(R)-3-hydroxy-tetra-
decanoyl]-2-[(R)-3-octacosanoyloxy-hexadecanoylamino]-
b-D-glucopyranosyl}-2-[(R)-3-hydroxy-hexadecanoylami-
no]-3-O-[(R)-3-hydroxy-hexadecanoyl]-D-glucono-1,5-lac-
tone (2). Compound 25 (7.5 mg, 3.4 lmol) was
deprotected similar to the synthesis of 1 by hydrogena-
tion (H2, 15 psi) over Pd/C (10 mg) in THF/t-BuOH
(2 mL, 1:3, v/v) to afford 2 as a colorless film (4.8 mg,
5.60 (t, 1H, J2 ,3 = J3 ,4 = 9.6 Hz, H-30), 5.34 (s, 1H,
CH, benzylidene), 5.32 (t, 1H, J2,3 = J3,4 = 9.6 Hz, H-
0
0
0
0
0
0
3), 5.06 (m, 1H, H-3L), 4.94 (d, 1H, J1 ,2 = 7.8 Hz, H-
10), 4.78 (t, 1H, J1,2 = J2,3 = 10.2 Hz, H-2), 4.59–4.36
(m, 8H, 4 · CH2, Bn), 4.40 (m, 1H, H-5), 4.26 (dd,
1H, J5 ,6 a = 4.8 Hz, J6 a,6 b = 9.6 Hz, H-60a), 4.04–4.01
0
0
0
0
1
(m, 2H, H-4, H-6a), 3.83 (m, 1H, H-3S), 3.80–3.76 (m,
0
80%). H NMR (600 MHz, CDCl3/CD3OD, 1:1, v/v):
2H, 2 · H-3S), 3.69 (t, 0 1H, J5 ,6 b = 10.2 Hz, H-60b),
d 5.39 (t, 1H, J3,4 = J3,4 = 9.6 Hz, H-3), 5.09 (br s, 1H,
0
0
0
3.56–3.47 (m, 5H, H-2 , H-4 , H-5 , H-6b, H-27L ),
2.64 (dd, 1H, J = 6.0 Hz, J = 14.4 Hz, H-2Sa), 2.52 (dd,
1H, J = 7.2 Hz, 15.6 Hz, H-2Sb), 2.46 (dd, 1H,
J = 6.0 Hz, J = 18.0 Hz, H-2Sa), 2.39–2.23 (m, 7H, H-
H-3L), 4.97 (t, 1H, J2 ,3 = J3 ,4 = 9.6 Hz, H-30), 4.55
(br s, 1H, H-10), 4.32 (m, 1H, H-5), 4.17 (m, 1H, H-2),
4.13 (m, 1H, H-60a), 3.95 (m, 1H, H-4), 3.85–3.81 (m,
2H, H-20, H-60b), 3.76–3.70 (m, 2H, H-6a, H-6b), 3.54
0
0
0
0
0
2S · 3, H-2L, 2L ), 1.59–1.40 (m, 12H, H-4L, H-3L ,26L ,
3 · H-4S), 1.24 (broad, 176 H, 88 · CH2, lipid), 1.17
(d, 3H, J27L ,28L = 6.0 Hz, H-28L ), 0.88–0.86 (m, 12H,
(t, 1H, J4 ,5 = 9.3 Hz, H-40), 3.33 (m, 1H, H-50), 2.55–
0
0
0
0
0
0
2.13 (m, 10H, H-2L, H-2L , 3 · H-2S), HR-MS (m/z)
calcd for C104H196N2O18[M+Na]+, 1784.4381; found:
1784.4586.
0
0
0
4 · CH3,
lipid).
HR-MS
(m/z)
calcd
for
C
146H230N2O19[M+Na]+, 2338.6990; found: 2338.8489.
4.2. Biological experiments
4.1.16. 4-O-Benzyl-6-O-{4,6-O-benzylidene-3-O-[(R)-3-
benzyloxy-tetradecanoyl-2-deoxy-2-[(R)-3-octacosanoyl-
oxy-hexadecanoylamino]-b-D-glucopyranosyl}-2-[(R)-3-ben-
zyloxy-hexadecanoylamino]-3-O-[(R)-3-benzyloxy-tetradec-
anoyl]-2-deoxy-D-glucono-1,5-lactone (25). Compound
23 (12 mg, 5.4 lmol) was oxidized similar to the synthe-
sis of 24 with PCC (9.4 mg, 43.4 lmol) to afford lactone
25 as a colorless film (8.5 mg, 71%). Rf = 0.35 (hexane/
4.2.1. Reagents. Escherichia coli 055:B5 LPS was ob-
tained from List Biologicals, and R. sin-1 LPS and lipid
A were kindly provided by Dr. R. Carlson (CCRC, Ath-
ens, GA). All data presented in this study were gener-
ated using the same batches of E. coli 055:B5 LPS and
R. sin-1 LPS. Synthetic compounds 1–3 were stored
lyophilized at ꢀ80 ꢁC and reconstituted in dry THF
on the day of the experiment; final concentrations of
THF in the biological experiments never exceeded
0.5% to avoid toxic effects.
1
ethyl acetate, 3:1, v/v). H NMR (500 MHz, CDCl3): d
7.38–7.17 (m, 25H, aromatic), 5.60 (t, 1H,
J2 ,3 = J3 ,4 = 9.5 Hz, H-30), 5.39 (s, 1H, CH, benzyli-
0
0
0
0

Y. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4800–4812
4811
4.2.2. Cell maintenance. Mono Mac 6 (MM6) cells, pro-
References and notes
vided by Dr. H.W.L. Ziegler-Heitbrock (Institute for
Inhalationbiology, Munich, Germany), were cultured
in RPMI 1640 medium with L-glutamine (BioWhittaker)
supplemented with penicillin (100 lg/mL) / streptomycin
(100 lg/mL; Mediatech, OPI supplement (1%; Sigma;
containing oxaloacetate, pyruvate, and bovine insulin),
and fetal calf serum (FCS; 10%; HyClone). New batches
of frozen cell stock were grown up every 2 months and
growth morphology evaluated. Before each experiment,
MM6 cells were incubated with calcitriol (10 ng/mL;
Sigma) for 2 days to differentiate into macrophage like
cells. The cells were maintained in a humid 5% CO2
atmosphere at 37 ꢁC.
1. Pittet, D.; Tarara, D.; Wenzel, R. P. JAMA J. Am. Med.
Assoc. 1994, 271, 1598.
2. Roberts, F. J.; Geere, I. W.; Coldman, A. Rev. Infect. Dis.
1991, 13, 34.
3. Rietschel, E. T.; Schletter, J.; Weidemann, B.; El-
Samalouti, V.; Mattern, T.; Zahringer, U.; Seydel, U.;
Brade, H.; Flad, H. D.; Kusumoto, S.; Gupta, D.;
Dziarski, R.; Ulmer, A. J. Microb. Drug Resist. 1998,
4, 37.
4. Rietschel, E. T.; Kirikae, T.; Schade, F. U.; Mamat, U.;
Schmidt, G.; Loppnow, H.; Ulmer, A. J.; Zahringer, U.;
Seydel, U.; Di Padova, F.; Schreier, M.; Brade, H. FASEB
J. 1994, 8, 217.
5. Gupta, D.; Kirkland, T. N.; Viriyakosol, S.; Dziarski, R.
J. Biol. Chem. 1996, 271, 23310.
4.2.3. Cytokine induction and TNF-a ELISA. On the day
of the exposure assay differentiated MM6 cells were har-
vested by centrifugation and gently suspended (106 cells/
mL) in prewarmed (37 ꢁC) medium. MM6 cells were then
incubated with different combinations of stimuli for
5.5 h. Culture supernatants were then collected and
stored frozen (ꢀ80 ꢁC) until assayed for TNF-a produc-
tion. Concentrations of human TNF-a protein in culture
supernatants were determined by a solid phase sandwich
ELISA. Plates (96-well MaxiSorp plates; Nalge Nunc
International) were coated with purified mouse anti-hu-
man TNF-a antibody (Pharmingen). TNF-a in standards
and samples was allowed to bind to the immobilized anti-
body. Biotinylated mouse anti-human TNF-a antibody
(Pharmingen) was then added, producing an antibody–
antigen–antibody ‘sandwich’. After addition of avidin-
horseradish peroxidase conjugate (Pharmingen) and
ABTS peroxidase substrate (Kirkegaard and Perry Lab-
oratories), a green color was produced in direct propor-
tion to the amount of TNF-a present in the sample.
The reaction was stopped by adding peroxidase stop solu-
tion (Kirkegaard and Perry Laboratories) and the absor-
bance was measured at 405 nm using a microplate reader
(BMG Labtech). TNF-a values are presented as
means SD of triplicate measurements, with each exper-
iment being repeated three times.
6. Poltorak, A.; He, X.; Smirnova, I.; Liu, M. Y.; Huffel, C.
V.; Du, X.; Birdwell, D.; Alejos, E.; Silva, M.; Galanos,
C.; Freudenberg, M.; Ricciardi-Castagnoli, P.; Layton, B.;
Beutler, B. Science 1998, 282, 2085.
7. Lien, E.; Means, T. K.; Heine, H.; Yoshimura, A.;
Kusumoto, S.; Fukase, K.; Fenton, M. J.; Oikawa, M.;
Qureshi, N.; Monks, B.; Finberg, R. W.; Ingalls, R. R.;
Golenbock, D. T. J. Clin. Invest. 2000, 105, 497.
8. Akashi, S.; Shimazu, R.; Ogata, H.; Nagai, Y.; Takeda,
K.; Kimoto, M.; Miyake, K. J. Immunol. 2000, 164,
3471.
9. Takeda, K.; Kaisho, T.; Akira, S. Annu. Rev. Immunol.
2003, 21, 335.
10. van Amersfoort, E. S.; van Berkel, T. J. C.; Kuiper, J.
Clin. Microbiol. Rev. 2003, 16, 379.
11. Raetz, C. R. Annu. Rev. Biochem. 1990, 59, 129.
12. Raetz, C. R.; Whitfield, C. Annu. Rev. Biochem. 2002, 71,
635.
13. Caroff, M.; Karibian, D.; Cavaillon, J. M.; Haeffner-
Cavaillon, N. Microbes Infect. 2002, 4, 915.
14. Suda, Y.; Ogawa, T.; Kashihara, W.; Oikawa, M.;
Shimoyama, T.; Hayashi, T.; Tamura, T.; Kusumoto, S.
J. Biochem.(Tokyo) 1997, 121, 1129.
15. Lam, C.; Hildebrandt, J.; Schutze, E.; Rosenwirth, B.;
Proctor, R. A.; Liehl, E.; Stutz, P. Infect. Immun. 1991, 59,
2351.
16. Kawata, T.; Bristol, J. R.; Rossignol, D. P.; Christ,
W. J.; Asano, O.; Dubuc, G. R.; Gavin, W. E.;
Hawkins, L. D.; Lewis, M. D.; McGuinness, P. D.;
Mullarkey, M. A.; Perez, M.; Robidoux, A. L.; Wang,
Y.; Kishi, Y.; Kobayashi, S.; Kimura, A.; Hishinima,
I.; Katayama, K.; Yamatsu, I. In Novel Therapeutic
Strategies in the Treatment of Sepsis; Morrison, D. C.,
Ryan, J. L., Eds.; Marcel Dekker: New York, 1995; p
171.
17. Golenbock, D. T.; Hampton, R. Y.; Qureshi, N.; Takay-
ama, K.; Raetz, C. R. J. Biol. Chem. 1991, 266, 19490.
18. Christ, W.; McGuinness, P.; Asano, O.; Wang, Y.;
Mullarkey, M.; Perez, M.; Hawkins, L.; Blythe, T.;
Dubuc, G.; Robidoux, A. J. Am. Chem. Soc. 1994, 116,
3637.
19. Christ, W. J.; Asano, O.; Robidoux, A. L.; Perez, M.;
Wang, Y.; Dubuc, G. R.; Gavin, W. E.; Hawkins, L. D.;
McGuinness, P. D.; Mullarkey, M. A.; Lewis, M. D.;
Kishi, Y.; Kawata, T.; Bristol, J. R.; Rose, J. R.;
Rossignol, D. P.; Kobayashi, S.; Hishinuma, I.; Kimura,
A.; Asakawa, N.; Katayama, K.; Yamatsu, I. Science
1995, 268, 80.
4.2.4. Data analysis. Concentration–response and inhibi-
tion data were analyzed using nonlinear least-squares
curve fitting in Prism (GraphPad Software, Inc.). Con-
centration–response data were fit with the following four
parameter logistic equation: 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 maxi-
mum response, and EC50 is the concentration of the stim-
ulus producing 50% stimulation. Inhibition data were fit
with the following logistic equation: Y = Bot-
)
tom + (Top ꢀ Bottom)/(1 + 10(Xꢀlog IC ), where Y is
50
the TNF-a response, X is the logarithm of the concentra-
tion of the inhibitor, and IC50 is the concentration of the
inhibitor that reduces the TNF-a response by half.
Acknowledgment
20. Asai, Y.; Nozu, Y.; Ikeuchi, T.; Narazaki, R.; Iwamoto,
K.; Watanabe, S. Biol. Pharm. Bull. 1999, 22, 432.
21. Chow, J. C.; Young, D. W.; Golenbock, D. T.; Christ, W.
J.; Gusovsky, F. J. Biol. Chem. 1999, 274, 10689.
This research was supported by the Institute of General
Medicine of the National Institutes of Health
(GM061761).

4812
Y. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 4800–4812
22. Demchenko, A. V.; Wolfert, M. A.; Santhanam, B.;
Moore, J. N.; Boons, G. J. J. Am. Chem. Soc. 2003, 125,
6103.
23. Santhanam, B.; Wolfert, M. A.; Moore, J. N.; Boons, G.
J. Chem. -Eur. J. 2004, 10, 4798.
30. Duffels, A.; Ley, S. V. J. Chem. Soc. Perkin Trans. 1 1999,
375.
31. Mehta, S.; Pinto, B. M. Tetrahedron Lett. 1991, 32, 4435.
32. Kanie, O.; Crawley, S. C.; Palcic, M. M.; Hindsgaul, O.
Carbohydr. Res. 1993, 243, 139.
24. Vandenplas, M. L.; Carlson, R. W.; Jeyaretnam, B.;
McNeill, B.; Barton, M. H.; Norton, N.; Murray, T. F.;
Moore, J. N. J. Biol. Chem. 2002, 277, 41811.
25. Jeyaretnam, B.; Glushka, J.; Kolli, V. S. K.; Carlson, R.
W. J. Biol. Chem. 2002, 277, 41802.
26. Mehta, S.; Pinto, B. M. J. Org. Chem. 1993, 58, 3269.
27. Xue, J.; Guo, Z. W. Tetrahedron Lett. 2001, 42, 6487.
28. Sakairi, N.; Hayashida, M.; Amano, A.; Kuzuhara, H.
J. Chem. Soc. Perkin Trans. 1 1990, 1301.
33. Fukase, K.; Fukase, Y.; Oikawa, M.; Liu, W. C.; Suda,
Y.; Kusumoto, S. Tetrahedron 1998, 54, 4033.
34. Nakabayashi, S.; Warren, C. D.; Jeanloz, R. W. Carbo-
hydr. Res. 1988, 174, 279.
35. Santhanam, B.; Boons, G. J. Org. Lett. 2004, 6, 3333.
36. Rossignol, D. P.; Hawkins, L. D.; Christ, W. J.; Kobay-
ashi, S.; Kawata, T.; Lynn, M.; Yamatsu, I.; Kishi, Y. In
Endotoxin in Health and Disease; Helmut, B., Opal, S. M.,
Vogel, S. N., Morrison, D. C., Eds.; Marcel Dekker: New
York, 1999; vol. 1, p 699.
29. Zuurmond, H. M.; van der Klein, P. A. M.; de Wildt, J.;
van der Marel, G. A.; van Boom, J. H. J. Carbohydr.
Chem. 1994, 13, 323.
37. Lee, H. S.; Wolfert, M. A.; Zhang, Y. H.; Boons, G. J.
ChemBioChem 2006, 7, 140.