Novel lipid A mimetics derived from pentaerythritol: Synthesis and their potent agonistic activity

Article abstract of DOI:10.1016/S0040-4020(02)01067-0

A pentaerythritol unit is substituted for glucosamine as its functional mimic at the reducing end of lipid A disaccharide. Two such pentaerythritol derived lipid A mimetics 3 and 4, carrying three identical fatty acyl moieties, have been designed and synt

Full text of DOI:10.1016/S0040-4020(02)01067-0

Tetrahedron 58 (2002) 8833–8842
Novel lipid A mimetics derived from pentaerythritol:
synthesis and their potent agonistic activity
*
Zi-Hua Jiang, Wladyslaw A. Budzynski, Lisa N. Skeels, Mark J. Krantz and R. Rao Koganty
Biomira Inc., 2011-94 Street, Edmonton, Alta., Canada T6N 1H1
Received 25 June 2002; revised 20 August 2002; accepted 21 August 2002
Abstract—A pentaerythritol unit is substituted for glucosamine as its functional mimic at the reducing end of lipid A disaccharide. Two such
pentaerythritol derived lipid A mimetics 3 and 4, carrying three identical fatty acyl moieties, have been designed and synthesized. An
efficient strategy, which involves simultaneous introduction of lipids onto two amino groups, is described for the synthesis of compounds 3
and 4. The biological activity profile of these two novel lipid A mimetics 3 and 4 is surprisingly similar to the natural lipid A product isolated
from Salmonella minnesota R595. An in vitro cell activation assay, using human adherent antigen presenting cells, has demonstrated that
both 3 and 4 induce the secretion of high levels of cytokines such as tumour necrosis factor-a (TNF-a), IL-6 and IL-8. Also, in a totally
synthetic liposomal vaccine system, 3 and 4 exhibit strong immunostimulatory adjuvant property in enhancing antigen specific T-cell
activation. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
in innate and adaptive immune responses has led to a better
understanding of LPS–lipid A functions: it has been
suggested that lipid A is a ligand for Toll-like receptor 4
(TLR4), a pattern-recognition receptor involved in the
mediation of immune responses to LPS/lipid A.8,9
Lipid A is the lipid anchor of lipopolysaccharide (LPS), the
outer cell membrane component of Gram-negative bacteria,
and widely considered responsible for strongly signalling
and activating the innate immunity in the host, following
bacterial infection. Structural investigations have shown
that lipid A is responsible for the biological activities of LPS
such as the typical endotoxic reactions, fever, lethal shock
as well as for its beneficial effects such as the adjuvant
characteristics and anti-tumour properties.1 However,
because of their toxicity, neither LPS nor lipid A has so
far been used for clinical applications. There have been
continuing efforts, through design, synthesis and testing of
structural analogues, to reduce the toxicity and enhance the
beneficial effects of lipid A.
Numerous analogues related to lipid A partial structures
have been synthesized with the aim of separating unwanted
endotoxic properties from potentially beneficial immuno-
stimulatory effects.10 Several reports11 have described the
design and synthesis of acyclic lipid A analogues to mimic
the functions of lipid A: in these molecules one mono-
saccharide unit or the whole disaccharide backbone of lipid
A has been replaced by an open chain spacer. Of particular
interest is the recently reported lipid A agonist ER-
112022,11e a symmetric phospholipid dimer connected by
a non-carbohydrate backbone. The authors suggested that
ER-112022 interacted directly with the LPS-signalling
receptor and mediated the stimulatory effects through the
TLR4 and MD-2 pathway in a CD14-independent manner.
Structure–activity relationship of lipid A has been a subject
of wide research interest over the last two decades.1,2 It
appears that an optimal number of lipid chains and chain
length are required on the disaccharide backbone in order to
exert strong endotoxic and related biological activities of
lipid A. Recently, Seydel et al. observed that the agonistic
and antagonistic activity of lipid A were governed by the
intrinsic conformation of lipid A, which in turn was defined
mainly by the number of charges and acyl chains of the
molecule.3–5 Furthermore, the recent discovery of mam-
malian Toll-like receptors (TLRs)6 and their essential role7
The endotoxic effect of the lipid A from Salmonella
minnesota R595 (2, Fig. 1) could be ameliorated by
selective hydrolysis of the 1-phosphate group,12 and the
lipid A modified in this way, called monophosphoryl lipid A
(1),13 is an effective adjuvant in both prophylactic and
therapeutic human vaccines.13b Lipid A preparations
purified from bacterial cultures suffer from lack of
consistency both in composition and performance. Its
heterogeneity is a major cause of large batch-to-batch
variations both in composition and activity. As a result, its
use in vaccine formulations adds to the compliance
requirements. We chose to pursue synthetic analogues14 of
Keywords: lipid A mimetic; pentaerythritol; immunostimulatory adjuvant;
cytokine; molecular vaccine.
*
Corresponding author. Fax: þ1-780-988-5936;
e-mail: rkoganty@biomira.com
0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)01067-0

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Z.-H. Jiang et al. / Tetrahedron 58 (2002) 8833–8842
Figure 1. The main component (2) of the natural lipid A product isolated from S. minnesota R595 and its chemically modified monophosphoryl lipid A (1), and
two lipid A mimetics 3 and 4.
similar activity, which have the advantage of reproducibility
and the consistency of pure single molecule. Such single
molecule adjuvants fulfil the needs of regulatory com-
pliance, which plays an important role in the approvability
of a vaccine. An ideal synthetic adjuvant must have
optimum activity balanced between benefits and toxicity,
trigger a specific cytokine profile, which in turn directs the
immune system to deliver an appropriate immune response
to an accompanying antigen, and its synthesis should be
adaptable for large-scale production. With this rationale, we
have designed two lipid A mimetics (3 and 4, Fig. 1) with
simplified structures as potential vaccine adjuvants. In this
paper, we report the design, synthesis, potent agonistic
activity, and adjuvant property of this two lipid A mimetics.
which is an important feature required for large-scale
synthesis of such molecules.
2.1. Synthesis
We have adapted a simplified strategy that requires the
introduction of both N-acyl groups simultaneously and
benzyl as a global protecting group. The preparation of the
aglycone 13 is described in Scheme 1. The readily available
mono-benzylated pentaerythritol 517 is first converted to
mono-tosylate 6 in 46% yield. Attempts to displace the tosyl
group in 6 with sodium azide in two-phase medium in the
presence of phase transfer catalyst have failed. Instead of
producing the expected product 7, the reaction results in the
formation of 8 through an intra-molecular substitution,18
together with some hydrolysed material 5. To overcome
this, compound 5 is first converted into its isopropylidene-
protected derivative 9, which is subsequently tosylated to
give 10. Under the same reaction conditions as above, azide
ion displaces the tosyl group affording 11 in 70%, together
with the recovery of 18% of the starting material. Treatment
of 11 with Zn–HOAc, followed by reacting with trichloro-
ethoxy chloroformate (Troc-Cl), provides 12 in 47% yield.
Inspite of the moderate yield of the azide reduction,
alternate conditions have not been tried since enough
material of 12 has been obtained to carry out further
synthesis. The removal of the isopropylidene group in 12
gives the desired glycosylation acceptor 13 in 90% yield.
2. Results and discussion
In both structure 3 and 4, the reducing end glucosamine of
lipid A disaccharide has been replaced by an amine-
substituted pentaerythritol unit. As a four directional core,
pentaerythritol has been successfully employed for the
synthesis of multi-functional dendrimers,15 and as a
molecular scaffold for combinatorial chemistry.16 We
perceive the amine-substituted pentaerythritol as a possible
structural mimic for glucosamine since the four functional
groups of the pentaerythritol core appear, from observing
the molecular models, to represent the 2-amine as well as
1,4 and 6-hydroxy functional groups of glucosamine.
Pentaerythritol not only simplifies the synthesis, but also
adds stability to the entire molecule, as it holds the anomeric
phosphate group well compared to that in glucosamine.
Thus, structure 4 retains two phosphoryl groups, mimicking
the natural diphosphoryl lipid A (2); and structure 3,
carrying only one phosphoryl group, is the structural
mimetic of the chemically modified monophosphoryl lipid
A (1). Both 3 and 4 carry three identical fatty acyl groups,
O-Glycosyl trichloroacetimidate method19 has been widely
used for complex oligosaccharide synthesis, and trichloro-
ethoxycarbonyl (Troc) group has been successfully
employed as the amine protecting group for glucosamine
to provide exclusively b-glycosyl linkages.20,21 We have
prepared trichloroacetimidate 20 as the glycosyl donor for
the synthesis of lipid A mimetic 3 and 4 (Scheme 2). The
coupling of the readily available glucosamine derivative
1422 and fatty acid 1523 is carried out in the presence of

Z.-H. Jiang et al. / Tetrahedron 58 (2002) 8833–8842
8835
Scheme 1. (a) Ts–Cl, Pyr., 08C!rt, 46% for 6 and 98% for 10; (b) NaN3, aliquat, NaHCO3, toluene–H2O, reflux, 50% for 8 and 70% for 11; (c) (i)
Me2C(OMe)2, TsOH, CH3CN, rt, (ii) TsOH·Et3N, MeOH, reflux, 82% (for two steps); (d) (i) Zn, HOAc, rt, (ii) Troc-Cl, NaHCO3, dioxane–H2O, rt, 47% (two
steps); (e) HOAc–H2O (4:1), 608C, 90%.
DCC and DMAP, which yields 16 in nearly quantitative
yield. Selective reductive ring opening of 4,6-di-O-benzyl-
idene in 16 by treatment with NaBH3CN and HCl(g)-
saturated diethyl ether solution24 results in the formation of
17 which is converted into 4-O-phosphorylated compound
18 through a two step procedure25 (in 86% yield). Phosphite
group is introduced by treating 17 with phosphoramidite and
tetrazole, followed by the oxidation of phosphite to
phosphate with m-chloroperbenzoic acid (m-CPBA).
Removal of the allyl group at the anomeric position follows
a two step procedure, first the isomerization of allyl double
bond using iridium complex26 followed by the hydrolysis of
the isomerized aglycone to provide 19 in 76% yield. The
conversion of 19 to the donor trichloroacetimidate 20 is
effected by treatment with trichloroacetonitrile and cesium
carbonate. The a-isomer (1H NMR d 6.47 ppm, d,
J¼3.5 Hz, 1H, H-1) is isolated in 76% yield. TLC showed
a lower spot in the reaction mixture in about 5–10% yield,
which is likely to be the b-isomer but has not been
characterized. When this reaction is carried out using DBU
Scheme 2. (a) 15, DCC, DMAP, CH2Cl2, rt, 98%; (b) NaBH3CN, HCl(g)–Et2O, THF, 08C, 85%; (c) (i) (BnO)2PN(iPr)2, tetrazole, CH2Cl2, rt, (ii) m-CPBA,
CH2Cl2, 08C, 86% for 19 and 80% for 24 (two steps); (d) (i) [bis(methyldiphenylphosphine)](1,5-cyclooctadiene) iridium(I) hexafluorophosphate, THF, rt,
(ii) NBS, THF–H2O, rt, 76% (two steps); (e) CNCCl3, Cs2CO3, CH2Cl2, rt, 76%; (f) TMSOTf, CH2Cl2, rt, 81%; (g) Zn, HOAc, rt; (h) 15, DCC, CH2Cl2, rt,
64% (two steps); (i) Pd–C, H2, THF–HOAc, rt, 100% for 3 and 96% for 4.

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Z.-H. Jiang et al. / Tetrahedron 58 (2002) 8833–8842
as the base, TLC indicates a more complex reaction profile
while the yield of the a-isomer is lowered to about 50–60%.
Table 1. Induction of cytokine TNF-a, IL-6 and IL-8 in matured human
adherent APCs by synthetic lipid A mimetic 3, 4, and reference R595 lipid
A (the natural detoxified lipid A product isolated from S. minnesota R595)
at the concentration of 1.0 mg/mL
The glycosylation reaction between donor 20 and acceptor
13 (at fourfold excess) in the presence of TMSOTf as
catalyst has provided the expected glycoside 21 in 81%
yield. No di-glycosylation product has been detected,
probably due to an enormous steric crowding that would
have ensued. Reductive cleavage of Troc-groups with Zn–
HOAc (!22) and N-acylation with fatty acid 15 in the
presence of DCC furnishes the crucial precursor 23 in 64%
yield. For the synthesis of lipid A analogues, fatty acyl
groups at both N-positions of lipid A disaccharide are
usually introduced separately; in one similar instance,
Johnson and coworkers13a tried the bis-N-acylation with
the same fatty acid (15) in a single step, but obtained the
desired product in very low yield (25–30%). The structure
of 23 (and the initial glycosylation product 21) has been
confirmed by 1H NMR spectral data: two sets of doublets at
d 4.35 and 4.65 (both J¼8.0 Hz, each 0.5H, H-1) indicate
the existence of two diastereoisomers in about 1:1 ratio,
both as b-glycoside. Due to the loss of its symmetry in the
pentaerythritol unit, compounds 21–24 and the final
product 4 exist as a mixture of two diastereoisomers in
about 1:1 ratio. Using the same reaction conditions for
phosphate group introduction, compound 23 was converted
to di-phosphate 24. The global de-benzylation of 23 and 24
by hydrogenolysis in the presence of palladium on charcoal
as catalyst provides, respectively, the designed lipid A
mimetics 3 and 4, and their structures have been confirmed
TNF-a (pg/mL)
IL-6 (pg/mL)
IL-8 (pg/mL)
3
4
3713
1513
2742
1
5812
1167
5049
0
32,197
12,296
34,288
359
R595 lipid A
Medium
liposomal vaccine formulation29 containing MUC1 derived
25 amino acid long lipopeptide BLP25,30 and a lipid A
analogue, either 3 or 4, or the reference natural lipid A
product, has been used to immunize mice. Preliminary
results show that both 3 and 4 have demonstrated
comparable adjuvant activity in promoting antigen specific
T-cell responses. Significantly the monophosphorylated 3
exhibits higher potency in the activation of human APCs
than the corresponding diphosphorylated analogue 4
(Table 1), while in the T-cell (C57B1/6) proliferation
assay compound 4 shows slightly higher activity. Such
change of potency level demonstrated by 3 and 4 might
suggest subtle differences between the murine and human
immune systems in response to lipid A molecules.
At the outset we were guided by the necessity to develop
well-defined synthetic structures as adjuvants with consist-
ent performance, rather than to achieve quantitative
improvement to adjuvant activity. The function of an
immunostimulatory adjuvant is to merely trigger innate
immunity and to alert the immune system to a perception of
‘pathogen invasion’, a path that is becoming widely
accepted as required for adaptive response to an accom-
panying antigen.31 Adjuvant activity must be balanced and
appropriate and not always measured in terms of ‘more or
less’. The present indications are that synthetic structure 3
and 4 exhibit adjuvant activity comparable to a natural
product under clinical evaluation. A cytokine profile that is
required to stimulate an immune system has been achieved
by these less complex single synthetic structures. With these
encouraging preliminary results, both compounds are
currently under more detailed evaluation as potential
1
by H NMR and MS spectroscopic data.
2.2. Biological evaluation
LPS and its smaller structural component lipid A are well-
known activators of innate immunity and thus display strong
adjuvant activity and often acute toxicity to the host. As a
strong immune stimulant, LPS or lipid A induces an acute
inflammatory response in humans resulting in the release of
cytokines and cellular mediators, including tumour necrosis
factor-a (TNF-a), IL-1, IL-6 and IL-8.2 Endogenous
production of TNF-a seems to be crucial for induction of
normal immune responses against invading pathogens,
although it also, when released in excess, plays a central
role in LPS and lipid A associated endotoxicity. IL-6 and
IL-8 play important roles in activating and attracting various
types of cells at the site of invading organism. LPS and lipid
A have also been shown to induce the production of IL-12
by dendritic cells and macrophages27 and trigger the
maturation of dendritic cells,28 which as antigen presenting
cells (APCs), are important in the development of adaptive
immune responses. The synthetic lipid A mimetics 3 and 4
have been investigated for their potency to induce TNF-a,
IL-6, and IL-8 in human GM-CSF and IL-4 activated APCs
(Table 1). Even at concentration of 1 mg/mL, both 3 and 4
induce high level of cytokines TNF-a, IL-6 and IL-8, at
magnitudes similar to that of detoxified lipid A product
(R595 lipid A) isolated from S. minnesota R595.
Figure 2. In vitro antigen specific proliferation of T-cells from C57Bl/6
mice immunized with a single dose of BLP25 liposomal vaccine
formulation. The vaccine dose contains 0.2 mg of MUC1 derived 25-mer
lipopeptide as an antigen and 0.1 mg of synthetic lipid A mimetic 3, 4, or
reference R595 lipid A (the natural detoxified lipid A product isolated from
S. minnesota R595) as an adjuvant.
The immune stimulatory (adjuvant) properties of 3 and 4
have been evaluated for their ability to induce antigen
specific T-cell proliferation responses in comparison with
the natural lipid A product, R595 lipid A (Fig. 2). A

Z.-H. Jiang et al. / Tetrahedron 58 (2002) 8833–8842
8837
adjuvants for vaccine formulations. Lien et al.11e have
recently reported that an acyclic lipid A-like agonist ER-
112022 activates the TLR4 signalling pathway. We believe
that lipid A mimetic 3 and 4 also function by activating
TLR4 in exhibiting their agonistic activities. Further studies
are necessary in order to provide evidence of the underlying
mechanism of their action.
to 08C. p-Toluenesulfonyl chloride (0.51 g, 2.65 mmol) is
added and the mixture stirred for 3 h while the temperature
is allowed to warm up to room temperature. Pyridine is
removed by co-distillation with toluene. The resultant crude
solid is dissolved in dichloromethane (100 mL) and the
solution is washed with saturated sodium chloride solution
(30 mL). The organic layer is dried with sodium sulfate and
concentrated. The residue, upon purification by flash
chromatography (hexane–ethyl acetate, 1:1 and then 2:3)
yields 6 (401 mg, 46%) as white powder. Rf 0.55 (hexane–
3. Conclusions
1
ethyl acetate, 1:4). H NMR (300 MHz, CDCl3): d 2.45 (s,
5H, CH3, 2OH), 3.50 (s, 2H, CH2), 3.62 (d, J¼11.0 Hz, 2H),
3.67 (d, J¼11.0 Hz, 2H), 4.15 (s, 2H, CH2), 4.40 (s, 2H,
CH2Ph), 7.30 (m, 7H, Ar-H), 7.80 (m, 2H, Ar-H).
In summary, we have demonstrated that, (a) a penta-
erythritol unit can be a functional substitute for glucosamine
in lipid A disaccharide, (b) the synthetic lipid A mimetics 3
and 4 exhibit biological activities similar to those of more
complex mixtures of natural lipid A from S. minnesota
R595, and, (c) both 3 and 4 can be easily synthesized in pure
form.
4.2.2. 2-(Benzyloxymethyl)-1,3-epoxy-2-(hydroxy-
methyl)-propane (8). Compound 6 (200 mg, 0.526 mmol)
is dissolved in toluene (5 mL) and saturated NaHCO3
solution (5 mL), sodium azide (262 mg, 4.04 mmol) and an
aliquat of phase transfer catalyst (0.1 g, 0.11 mL,
0.247 mmol) are added. The mixture is maintained at reflux
temperature for 16 h and let cool to room temperature. The
organic layer is separated and the aqueous layer is extracted
with ethyl acetate (10 mL£3). The combined organic phase
is washed with cold water (10 mL), dried with sodium
sulfate and the solvent removed under vacuum. The residue,
upon purification by flash chromatography (hexane–ethyl
acetate–methanol, 1:1:1% and then 1:1:5%), gives 8
(55 mg, 50%) and 5 (48 mg, 40%) as colorless syrup. Rf
There is a tremendous potential for molecular adjuvants
since their activities can be reproduced impeccably. Unlike
those derived from bacterial cultures, which are mixtures of
several undefined structures, the synthetic structures
described in this report are simpler to produce and constitute
an excellent counterpart to molecular vaccines. Though a
formal toxicity analysis of 3 and 4 has not yet been carried
out, based on the tolerance by mice, they are expected to be
at least comparable to the detoxified natural lipid A
preparation from S. minnesota R595.
1
0.50 (hexane–ethyl acetate–methanol, 1:1:2%). H NMR
(300 MHz, CDCl3): d 2.37 (br s, 1H, OH), 3.80 (s, 2H,
CH2), 3.95 (s, 2H, CH2), 4.43 (d, J¼6.0 Hz, 2H), 4.49 (d,
J¼6.0 Hz, 2H), 4.57 (s, 2H), CH2Ph). ESIMS calcd for
C12H16O3: 208.1; found: 231.1 (MþNa).
4. Experimental
4.1. General
4.2.3. 5-(Benzyloxymethyl)-5-(hydroxymethyl)-2,2-
dimethyl-1,3-dioxane (9). Compound
All air and moisture sensitive reactions have been
performed under nitrogen atmosphere. Anhydrous THF,
DMF, acetonitrile, and dichloromethane are purchased from
Aldrich and other dry solvents are prepared in accordance
with standard procedures. ACS grade solvents are pur-
chased from Fisher and used for chromatography without
distillation. TLC plates (silica gel 60 F254, thickness
0.25 mm, Merck) and flash silica gel 60 (35–75 mm) for
column chromatography are purchased from Rose
Scientific, Canada. 1H NMR spectra are recorded on
Brucker AM 300 MHz, Varian Unity 500 MHz, or Brucker
DRX 600 MHz spectrometer with tetramethylsilane as
internal standard. Chemical shifts are reported in ppm (d),
and signals are expressed as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), or br (broad). Optical
rotations are measured on a Perkin–Elmer 241 Polarimeter
at room temperature (20–228C). Elemental analysis data are
obtained from the Micro-analytical laboratory in the
University of Alberta, Canada. Electron-spray mass spectro-
metric analyses (ESIMS) are performed, in positive mode,
either on MS50B or MSD1 SPC mass spectrometer, and the
data are reported in m/z.
5
(672 mg,
2.97 mmol) is dissolved in dry acetonitrile (10 mL) and
2,2-dimethoxypropane (560 mg, 0.66 mL, 5.35 mmol) and
p-toluenesulfonic acid (56 mg, 0.279 mmol) are added. The
mixture is stirred at room temperature for 1 h and then
triethylamine (0.5 mL) is added to quench the reaction. The
mixture is evaporated under vacuum and the residue purified
by flash chromatography (hexane–ethyl acetate, 2:1) to
obtain 9 (614 mg, 82%). Rf0.67 (hexane–ethyl acetate, 1:2).
1H NMR (300 MHz, CDCl3): d 1.41 (s, 3H, CH3), 1.42 (s,
3H, CH3), 2.40 (br s, 1H, OH), 3.59 (s, 2H, CH2), 3.69 (s,
2H, CH2), 3.74 (s, 4H, 2CH2), 4.55 (s, 2H, CH2Ph), 7.30 (m,
5H, Ar-H).
4.2.4. 5-(Benzyloxymethyl)-2,2-dimethyl-5-(toluene-4-
sulfonyloxymethyl)-1,3-dioxane (10). To a cold (08C)
solution of 9 (572 mg, 2.26 mmol) in dry pyridine (3 mL),
p-toluenesulfonyl chloride (517 mg, 2.71 mmol) is added
and the mixture is stirred for 3 h. More p-toluenefulfonyl
chloride (430 mg, 2.26 mmol) is added and the stirring is
continued at room temperature overnight. Methanol (1 mL)
is added to quench the reaction and the solvent is removed
by co-distillation with toluene under vacuum. The residue is
dissolved in dichloromethane (100 mL) and washed with
saturated NaHCO3 (30 mL). The organic layer is separated
and the aqueous layer is extracted with dichloromethane
(30 mL) and the combined organic layer is dried with
4.2. Synthetic procedures
4.2.1. 2-(Benzyloxymethyl)-2-(toluene-4-sulfonyloxy-
methyl)-1,3-propanediol (6). Compound 517 (500 mg,
3.21 mmol) is dissolved in dry pyridine (5 mL) and cooled

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Z.-H. Jiang et al. / Tetrahedron 58 (2002) 8833–8842
sodium sulfate and concentrated. The residue, upon
purification by flash chromatography (hexane–ethyl
acetate, 5:1), gives 10 (930 mg, 98%). Rf 0.65 (hexane–
acetate, 1:1). 1H NMR (300 MHz, CDCl3): d 3.03 (t,
J¼6.5 Hz, 2H, 2OH), 3.43 (s, 2H, CH2), 3.44 (d, J¼6.5 Hz,
2H, CH2N), 3.51 (d, J¼6.5 Hz, 4H, 2CH2OH), 4.55 (s, 2H),
4.73 (s, 2H), 5.35 (t, J¼6.5 Hz, 1H, NH), 7.35 (m, 5H, Ar-
H). ESIMS calcd for C15H20Cl3NO5: 399.0; found: 422.0
(MþNa), 424.0 (MþNa, 37Cl).
1
ethyl acetate, 2:1). H NMR (300 MHz, CDCl3): d 1.30 (s,
3H, CH3), 1.40 (s, 3H, CH3), 2.42 (s, 3H, CH3), 3.35 (s, 2H,
CH2), 3.63 (d, J¼12.0 Hz, 2H), 3.72 (d, J¼12.0 Hz, 2H),
4.20 (s, 4H, 2CH2), 4.50 (s, 2H, CH2Ph), 7.30 (m, 7H,
Ar-H), 7.78 (m, 2H, Ar-H). ESIMS calcd for C22H28O6S:
420.2; found: 443.2 (MþNa).
4.2.8. Allyl (R)-4,6-O-benzylidene-2-deoxy-3-O-[(R)-3-
(tetradecanoyloxy)tetradecanoyl]-2-(2,2,2-trichloro-
ethoxycarbonylamino)-a-D-glucopyranoside (16). To a
mixture of 1422 (312 mg, 0.65 mmol) and 1523 (200 mg,
0.44 mmol) in dry dichloromethane (5 mL), DCC (136 mg,
0.66 mmol) and 4-N,N-dimethylaminopyridine (DMAP,
27 mg, 0.22 mmol) are added. The reaction is stirred at
room temperature for 4 h. The solid is filtered off and
washed with ethyl acetate (5 mL). The filtrate is concen-
trated and the residue, upon purification by flash chroma-
tography (hexane–ethyl acetate, 8:1), gives 16 (398 mg,
98%) as a colorless syrup. Rf 0.69 (hexane–ethyl acetate,
3:1). [a]2D2¼þ32.0 (c 0.5, chloroform). 1H NMR (300 MHz,
CDCl3): d 0.90 (t, J¼6.5 Hz, 6H, 2CH3), 1.25 (m, 38H,
19CH2), 1.52 (m, 4H, 2CH2), 2.16 (t, J¼7.5 Hz, 2H, CH2),
2.50 (dd, J¼16.0, 6.0 Hz, 1H, CHH), 2.63 (dd, J¼16.0,
6.0 Hz, 1H, CHH), 3.71 (dd, J¼9.5, 9.5 Hz, 1H, H-4), 3.78
(dd, J¼10.0, 10.0 Hz, 1H, H-6a), 3.94 (m, 1H, H-5), 3.98–
4.08 (m, 2H, H-2, CHHCHvCH2), 4.21 (m, 1H,
CHHCHvCH2), 4.29 (dd, J¼10.0, 5.0 Hz, 1H, H-6b),
4.69, 4.76 (2d, J¼12.0 Hz, each 1H, Troc-CH2), 4.94 (d,
J¼3.6 Hz, 1H, H-1), 5.16 (m, 1H, lipid-3-H), 5.30 (m, 2H,
CHvCH2), 5.39 (dd, J¼9.5, 9.5 Hz, 1H, H-3), 5.42 (d,
J¼10.0 Hz, 1H, NH), 5.53 (s, 1H, CHPh), 5.90 (m, 1H,
CHvCH2), 7.30–7.35 (m, 15H, Ar-H). Anal. calcd for
C47H74Cl3NO10 (919.46): C, 61.40; H, 8.11; N, 1.52; found:
C, 61.40; H, 8.19; N, 1.58.
4.2.5. 5-(Azidomethyl)-5-(benzyloxymethyl)-2,2-
dimethyl-1,3-dioxane (11). Compound 10 (907 mg,
2.16 mmol) is dissolved in toluene (30 mL) and saturated
NaHCO3 (30 mL), sodium azide (561 mg, 8.63 mmol) and
the phase transfer catalyst aliquat (433 mg, 0.49 mL,
1.08 mmol) are added. The mixture is refluxed for 16 h
and more sodium azide (1.40 g, 21.60 mmol) is added. The
reflux is continued for 24 h and let cool to room
temperature. The organic layer is separated and the aqueous
layer is extracted with ethyl acetate (30 mL£3). The
combined organic layer is washed with water (30 mL),
dried with sodium sulfate and the solvent is removed under
vacuum. The residue, upon purification by flash chroma-
tography (hexane–ethyl acetate, 8:1), gives 11 (440 mg,
70%) as colorless syrup and the starting material 10
1
(163 mg, 18%). Rf 0.34 (hexane–ethyl acetate, 6:1). H
NMR (500 MHz, CDCl3): d 1.42 (s, 6H, 2CH3), 3.40 (s, 2H,
CH2), 3.52 (s, 2H, CH2), 3.64 (d, J¼12.0 Hz, 2H), 3.73 (d,
J¼12.0 Hz, 2H), 4.50 (s, 2H, CH2Ph), 7.30 (m, 5H, Ar-H).
ESIMS calcd for C15H21N3O3: 291.2; found: 314.1
(MþNa).
4.2.6. 5-(Benzyloxymethyl)-2,2-dimethyl-5-(trichloro-
ethoxycarbonylaminomethyl)-1,3-dioxane (12). Com-
pound 11 (40 mg, 0.137 mmol) and zinc powder (1.0 g) in
acetic acid (10 mL) is stirred at room temperature for 1 h.
The solid is filtered and washed with acetic acid (10 mL),
and the filtrate is concentrated under vacuum. The residue is
dissolved in dioxane–sat. NaHCO3 mixture (2:1, 6 mL, pH
8–9) and the mixture is stirred at room temperature for 6 h
in the presence of 2,2,2-trichloroethoxylchloroformate
(123 mg, 0.08 mL, 0.568 mmol). Dioxane is removed
under vacuum followed by the addition of water (10 mL).
The mixture is extracted with ethyl acetate (10 mL£3) and
the organic layer is dried with sodium sulfate and
concentrated under vacuum. The residue is purified by
flash chromatography (hexane–ethyl acetate, 4:1) to
obtain 12 (28 mg, 47%) as colorless syrup. Rf 0.17
4.2.9. Allyl 6-O-benzyl-2-deoxy-3-O-[(R)-3-(tetradeca-
noyloxy)tetradecanoyl]-2-(2,2,2-trichloroethoxycar-
bonylamino)-a-D-glucopyranoside (17). A solution of 16
(1.45 g, 1.60 mmol) in dry THF (20 mL) and molecular
˚
sieves (4 A, 3.0 g) is stirred at room temperature under
nitrogen for 20 min. Sodium cyanoborohydride (1.0 g,
15.96 mmol) is added and the mixture is cooled to 08C
followed by drop wise addition of dry ethereal-HCl(g) till
no gas is evolved. The mixture is poured into saturated
sodium bicarbonate solution (50 mL) and extracted with
dichloromethane (100 mL£3). The combined organic
phase is washed with saturated sodium chloride solution
(20 mL), dried with sodium sulphate and concentrated. The
residue is purified by flash silica gel chromatography
(hexane–ethyl acetate, 5:1 and 4:1) to obtain 17 (1.23 g,
85%) as colorless syrup. Rf 0.20 (hexane–ethyl acetate,
1
(hexane–ethyl acetate, 6:1). H NMR (300 MHz, CDCl3):
d 1.39 (s, 3H, CH3), 1.41 (s, 3H, CH3), 3.32 (d, J¼6.0 Hz,
2H, CH2), 3.54 (s, 2H, CH2), 3.67 (d, J¼12.0 Hz, 2H), 3.75
(d, J¼12.0 Hz, 2H), 4.57 (s, 2H), 4.72 (s, 2H), 5.50 (t,
J¼6.0 Hz, 1H, NH), 7.35 (m, 5H, Ar-H). ESIMS calcd for
C18H24Cl3NO5: 439.1; found: 462.1 (MþNa), 464.1
(MþNa, 37Cl).
4:1). [a]2D0¼þ47.5 (c 1.0, CHCl3). H NMR (300 MHz,
1
CDCl3): d 0.88 (t, J¼6.5 Hz, 6H, 2CH3), 1.25 (br s,
38H, 19CH2), 1.50 (m, 4H, 2CH2), 2.28 (t, J¼7.5 Hz, 2H,
CH2), 2.48 (dd, J¼14.0, 4.0 Hz, 1H), 2.58 (dd, J¼14.0,
7.5 Hz, 1H), 3.27 (d, J¼3.5 Hz, 1H, OH), 3.70–3.86
(m, 4H), 3.92–4.03 (m, 2H), 4.58 (d, J¼12.0 Hz, 1H),
4.64 (d, J¼12.0 Hz, 1H), 4.66 (d, J¼12.0 Hz, 1H), 4.76
(d, J¼12.0 Hz, 1H), 4.92 (d, J¼3.5 Hz, 1H, H-1), 5.13
(m, 2H), 5.19–5.31 (m, 2H, CH2vCH), 5.40 (d, J¼9.5 Hz,
1H, NH), 5.88 (m, 1H, CH2vCH), 7.30 (m, 5H, Ar-H).
ESIMS calcd for C47H76Cl3NO10: 919.5; found: 920.8
(MþH).
4.2.7. 2-(Benzyloxymethyl)-2-(trichloroethoxycarbonyl-
aminomethyl)-1,3-propanediol (13). Compound 12
(18.3 mg, 0.0417 mmol), in acetic acid and water (4:1,
10 mL), is stirred at 608C for 45 min. The solvent is
removed under vacuum and the residue is purified by flash
chromatography (hexane–ethyl acetate, 1:1) to obtain 13
(15 mg, 90%) as white powder. Rf 0.27 (hexane–ethyl

Z.-H. Jiang et al. / Tetrahedron 58 (2002) 8833–8842
8839
4.2.10. Allyl 6-O-benzyl-2-deoxy-4-O-(di-O-benzyl-phos-
phono)-3-O-[(R)-3-(tetradecanoyloxy)tetradecanoyl]-2-
(2,2,2-trichloroethoxycarbonylamino)-a-D-glucopyra-
noside (18). To compound 17 (1.20 g, 1.30 mmol) in dry
nosyl trichloroacetimidate (20). To a solution of 19
(800 mg, 0.70 mmol) in dry dichloromethane (8 mL),
trichloroacetonitrile (504 mg, 0.35 mL, 3.51 mmol) and
cesium carbonate (343 mg, 1.05 mmol) are added. The
mixture is stirred at room temperature for 1 h and the solid is
filtered off through celite and washed well with dichloro-
methane. The filtrate is concentrated in vacuum and the
residue, upon purification by flash chromatography
(hexane–ethyl acetate, 4:1, with 0.5% of triethyl amine),
gives 20 (685 mg, 76%) as colorless syrup. Rf 0.36
(hexane–ethyl acetate, 3:1). [a]2D0¼þ12.5 (c 0.4, CHCl3).
1H NMR (300 MHz, CDCl3): d 0.88 (t, J¼6.5 Hz, 6H,
2CH3), 1.24 (br s, 38H, 19CH2), 1.50 (m, 4H, 2CH2), 2.19 (t,
J¼7.0 Hz, 2H, CH2), 2.46 (m, 2H, CH2), 3.71 (m, 2H), 4.04
(m, 1H). 4.15 (ddd, J¼1.0, 8.5, 3.5 Hz, 1H, H-2), 4.43 (d,
J¼12.0 Hz, 1H), 4.52 (d, J¼12.0 Hz, 1H), 4.61 (d,
J¼12.0 Hz, 1H), 4.71 (ddd, J¼9.5, 9.5, 9.5 Hz, 1H, H-4),
4.77 (d, J¼12.0 Hz, 1H). 4.94 (m, 4H), 5.12 (m, 1H), 4.39
(dd, J¼10.0, 9.5 Hz, 1H, H-3), 5.65 (d, J¼8.5 Hz, 1H, NH),
6.47 (d, J¼3.5 Hz, 1H, H-1), 7.32 (m, 15H, Ar-H), 8.72 (s,
1H, NH). ESIMS calcd for C60H85Cl6N2O13P: 1282.4;
found: 1284.0 (MþH).
dichloromethane
(20 mL),
1H-tetrazole
(273 mg,
3.90 mmol) and dibenzyl N,N-diisopropylphosphoramidite
(900 mg, 0.875 mL, 2.61 mmol) are added and stirred at
room temperature for 30 min. m-Chloroperbenzoic acid
(m-CPBA, 1.63 g, 55%, 5.22 mmol) is added at 08C and the
mixture is stirred for 30 min. The mixture is poured into a
solution of 10% sodium hydrogen sulfite (40 mL) and
extracted with dichloromethane (40 mL£3). The organic
layer is washed with saturated sodium bicarbonate solution
(20 mL), dried with sodium sulfate and concentrated. The
residue is purified by repeated flash chromatography
(hexane–ethyl acetate, 4:1 and then 3:1) to obtain 18
(1.33 g, 86%) as colorless syrup. Rf 0.31 (hexane–ethyl
acetate, 3:1). [a]D20¼þ35.0 (c 1.0, CHCl3). 1H NMR
(300 MHz, CDCl3): d 0.88 (t, J¼6.5 Hz, 6H, 2CH3), 1.24
(br s, 38H, 19CH2), 1.50 (m, 4H, 2CH2), 2.17 (t, J¼7.0 Hz,
2H, CH2), 2.41 (dd, J¼16.5, 5.5 Hz, 1H), 2.51 (dd, J¼16.5,
7.5 Hz, 1H), 3.66 (dd, J¼11.0, 4.5 Hz, 1H), 3.74 (dd,
J¼11.0, 2.0 Hz, 1H), 3.91 (m, 1H), 4.00 (m, 2H), 4.20 (m,
1H), 4.44 (d, J¼12.0 Hz, 1H), 4.53 (m, 1H, H-4), 4.54 (d,
J¼12.0 Hz, 1H), 4.63 (d, J¼12.0 Hz, 1H), 4.88–4.95 (m,
5H), 5.11 (m, 1H), 5.20–5.32 (m, 2H, CH2vCH), 5.35 (dd,
J¼10.5, 9.0 Hz, 1H, H-3), 5.41 (d, J¼9.5 Hz, 1H, NH), 5.88
(m, 1H, CH2vCH), 7.30 (m, 15H, Ar-H). ESIMS calcd for
C61H89Cl3NO13P: 1179.6; found: 1181.0 (MþH).
4.2.13. 2-(Benzyloxymethyl)-3-hydroxy-2-(trichloro-
ethoxycarbonylaminomethyl)-propan-1-yl 6-O-benzyl-
2-deoxy-4-O-(di-O-benzyl-phosphono)-3-O-[(R)-3-(tetra-
decanoyloxy)tetradecanoyl]-2-(2,2,2-trichloroethoxycar-
bonylamino)-b-D-glucopyranoside (21). A solution of 20
(620 mg, 0.484 mmol) and 13 (750 mg, 1.936 mmol) in dry
˚
dichloromethane (15 mL) and molecular sieves (4 A, 2.0 g)
4.2.11. 6-O-Benzyl-2-deoxy-4-O-(di-O-benzyl-phos-
phono)-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-
(2,2,2-trichloroethoxycarbonylamino)-a/b-D-glucopyra-
nose (19). Into the suspension of [bis(methyldiphenyl-
phosphine)](1,5-cyclooctadiene) iridium(I) hexafluoro-
phosphate (14 mg, 0.0165 mmol) in dry THF (5 mL)
hydrogen gas is bubbled until a yellowish solution is
formed (about 5 min), which is added to a solution of 18
(1.30 g, 1.10 mmol) in dry THF (10 mL). The mixture is
stirred at room temperature for 2 h. Water (0.5 mL) and
N-bromosuccinimide (NBS, 294 mg, 1.62 mmol) are added
and the reaction is stirred further for 1 h. The residue
obtained after solvent removal is dissolved in ethyl acetate
(200 mL) and washed with saturated sodium bicarbonate
solution (20 mL£2). The combined organic phase is dried
with sodium sulfate and concentrated. The residue, upon
purification by flash chromatography (hexane–ethyl
acetate, 2:1), yields 19 (950 mg, 76%) as colorless syrup.
Rf 0.31 (ethyl acetate–hexane, 1:2). [a]2D0¼þ17.5 (c 1.0,
CHCl3). 1H NMR (300 MHz, CDCl3): d 0.88 (t, J¼6.5 Hz,
6H, 2CH3), 1.24 (br s, 38H, 19CH2), 1.50 (m, 4H, 2CH2),
2.18 (t, J¼7.0 Hz, 2H, CH2), 2.39 (m, 2H, CH2), 3.59 (dd,
J¼11.0, 6.0 Hz, 1H), 3.71 (dd, J¼11.0, 1.5 Hz, 1H), 3.94
(m, 1H), 4.16 (m, 1H), 4.40 (m, 3H), 4.49 (d, J¼12.0 Hz,
1H), 4.65 (d, J¼12.0 Hz, 1H), 4.72 (d, J¼12.0 Hz, 1H), 4.90
(m, 4H), 5.09 (m, 1H), 5.39 (t, J¼3.5 Hz, 1H, H-1), 5.37
(dd, J¼10.0, 9.5 Hz, 1H, H-3), 5.70 (d, J¼9.5 Hz, 1H, NH),
7.30 (m, 15H, Ar-H). ESIMS calcd for C58H85Cl3NO13P:
1139.5; found: 1141.0 (MþH).
is stirred under nitrogen for 10 min at room temperature. A
solution of TMSOTf (0.01 M in dichloromethane, 3.0 mL)
is added drop wise in about 5 min. The mixture is stirred at
room temperature for 1 h and saturated sodium bicarbonate
solution (10 mL) is added to quench the reaction. Usual
aqueous work-up and flash chromatography (hexane–
acetone, 2.8:1 and 2:1) yields 21 (590 mg, 81%), colorless
syrup of diastereomeric mixture in the ratio of about 1:1. Rf
0.27 (hexane–acetone, 2.5:1). [a]2D0¼27.6 (c 0.8, chloro-
1
form). H NMR (300 MHz, CDCl3): d 0.88 (t, J¼6.5 Hz,
6H, 2CH3), 1.25 (br s, 36H, 18CH2), 1.45 (m, 2H, CH2),
1.58 (m, 2H, CH2), 1.68 (m, 2H, CH2), 2.23 (t, J¼7.5 Hz,
2H, CH2), 2.41 (m, 2H, CH2), 3.10 (m, 0.5H), 3.30–3.62
(m, 7.5H), 3.68–3.83 (m, 2H), 4.40–4.56 (m, 7H), 4.65–
4.80 (m, 5H), 4.90 (m, 5H), 5.19 (m, 2H), 5.53 (d, J¼9.0 Hz,
0.5H, NH), 5.72 (m, 1.5H, NH), 7.30 (m, 20H, Ar-H).
ESIMS calcd for C73H103Cl6N2O17P: 1520.5; found: 1543.5
(MþNa, 42), 1544.4 (MþNa, 13C-isotope, 34), 1545.5
(MþNa, 37Cl-isotope, 100).
4.2.14. 2-(Benzyloxymethyl)-3-hydroxy-2-(amino-
methyl)-propan-1-yl 6-O-benzyl-2-deoxy-4-O-(di-O-ben-
zyl-phosphono)-3-O-[(R)-3-(tetradecanoyloxy)tetradeca-
noyl]-2-amino-b-D-glucopyranoside (22). To a solution of
compound 21 (450 mg, 0.30 mmol) in acetic acid (50 mL)
zinc powder (4.0 mg) is added. The mixture is stirred at
room temperature for 1 h and filtered. The solid is washed
with acetic acid (50 mL) and the filtrate was concentrated in
vacuum. The residue is dissolved in dichloromethane
(150 mL) and the solution is washed with saturated aqueous
sodium bicarbonate solution (20 mL). After separating the
organic layer, the aqueous phase is extracted with
dichloromethane (20 mL£2). The combined organic phase
4.2.12. 6-O-Benzyl-2-deoxy-4-O-(di-O-benzyl-phos-
phono)-3-O-[(R)-3-(tetradecanoyloxy)tetradecanoyl]-2-
(2,2,2-trichloroethoxycarbonylamino)-a-D-glucopyra-

8840
Z.-H. Jiang et al. / Tetrahedron 58 (2002) 8833–8842
is dried with sodium sulphate and concentrated under
vacuum to obtain the di-amine 22 (346 mg) which is used
directly in the next step.
(300 MHz, CDCl3): d 0.87 (t, J¼6.5 Hz, 18H, 6CH3), 1.30
(m, 108H, 54CH2), 1.48–1.70 (m, 18H), 2.10–2.53 (m,
11H), 2.90–3.35 (m, 5H), 3.55 (m, 2H), 3.75–3.90 (m, 4H),
4.00 (m, 1H), 4.36–4.52 (m, 6H), 4.85–5.01 (m, 8H), 5.08–
5.22 (m, 4H), 6.30 (m, 1H, NH), 6.88 (d, J¼8.5 Hz, 0.5H,
NH), 7.00 (d, J¼8.0 Hz, 0.5H, NH), 7.30 (m, 30H, Ar-H).
ESIMS calcd for C137H218N2O22P: 2305.5; found: 2328.5
(MþNa, 78), 2329.5 (MþNa, 13C-isotope, 100).
4.2.15. 2-(Benzyloxymethyl)-3-hydroxy-2-[(R)-3-tetra-
decanoyloxytetradecanamidomethyl]-propan-1-yl 6-O-
benzyl-2-deoxy-4-O-(di-O-benzyl-phosphono)-3-O-[(R)-
3-(tetradecanoyloxy)tetradecanoyl]-2-[(R)-3-(tetradeca-
noyloxy)tetradecanamido]-b-D-glucopyranoside (23). A
mixture of the di-amine 22 (346 mg), lipid acid 1523
(545 mg, 1.20 mmol) and DCC (371 mg, 1.80 mmol) in dry
dichloromethane (10 mL) is stirred at room temperature for
20 h. Water (0.05 mL) is added and the reaction mixture is
stirred for 10 min. The solid is filtered through a sintered
glass funnel with a bed of sodium sulphate. The filtrate is
concentrated and the residue purified by flash chromato-
graphy (hexane–acetone, 5:1 and 4.5:1) to obtain 23
(390 mg, 64% over two steps) as a colorless syrup. Rf
0.20 (hexane–acetone, 4:1). [a]2D0¼29.4 (c 0.5, chloro-
4.2.17. 2-(Hydroxymethyl)-3-hydroxy-2-[(R)-3-(tetra-
decanoyloxy)tetradecanamidomethyl]-propan-1-yl 2-
deoxy-4-O-(phosphono)-3-O-[(R)-3-(tetradecanoyloxy)-
tetradecanoyl]-2-[(R)-3-(tetradecanoyloxy)tetradecan-
amido]-b-D-glucopyranoside (3). A solution of compound
23 (96 mg, 0.047 mmol) in THF–HOAc (10:1, 77 mL) and
palladium on charcoal (100 mg) is stirred under hydrogen
atmosphere for 24 h. The catalyst is filtered and washed
with chloroform–methanol (1:1, 30 mL). The filtrate is
concentrated under vacuum and the residue is purified by
flash chromatography (chloroform–methanol–water, 9:1:0
and then 4:1:0.1) to obtain 3 which is freeze-dried from tert-
butanol to afford the product as white powder (80 mg,
100%). Rf 0.16 (chloroform–methanol–water–acetic acid,
6:1:0.1:0.1). [a]2D0¼26.5 (c 0.2, chloroform). 1H NMR
(600 MHz, CDCl3–CD3OD, 1:1): d 0.89 (t, J¼6.5 Hz, 18H,
6CH3), 1.26 (m, 114H, 57H), 1.60 (m, 12H, 6CH2), 2.30 (m,
6H, 3CH2), 2.37 (dd, J¼15.0, 6.0 Hz, 1H), 2.45 (dd, J¼15.0,
7.0 Hz, 1H), 2.50 (dd, J¼15.0, 5.0 Hz, 1H), 2.54 (dd,
J¼15.0, 8.0 Hz, 1H), 2.57 (dd, J¼15.0, 5.0 Hz, 1H), 2.67
(dd, J¼15.0, 7.0 Hz, 1H), 3.04 (dd, J¼14.0, 6.0 Hz, 1H),
3.18 (dd, J¼14.0, 6.0 Hz, 1H), 3.25 (d, J¼10.0 Hz, 1H),
3.29 (d, J¼10.0 Hz, 1H), 3.36 (m, 3H), 3.37 (d, J¼10.0 Hz,
1H), 3.64 (d, J¼10.0 Hz, 1H), 3.77 (br d, J¼12.0 Hz, 1H),
3.89 (dd, J¼10.0, 9.0 Hz, 1H), 3.96 (br d, J¼12.0 Hz, 1H),
4.25 (m, 1H, H-4), 4.43 (d, J¼8.5 Hz, 1H, H-1), 5.07 (dd,
J¼10.0, 10.0 Hz, 1H, H-3), 5.17 (m, 2H), 5.23 (m, 1H).
ESIMS calcd for C95H181N2O19P: 1685.3; found: 1686.3
(MþH), 1708.3 (MþNa), 1730.3 (Mþ2Na–H).
1
form). H NMR (500 MHz, CDCl3): d 0.88 (t, J¼7.0 Hz,
18H, 6CH3), 1.25–1.50 (m, 112H, 56CH2), 1.58 (m, 11H),
1.71–1.81 (m, 3H), 1.97 (m, 1H, OH), 2.21 (t, J¼7.5 Hz,
2H, CH2), 2.24–2.63 (m, 10H, 5CH2), 3.06 (dd, J¼14.0,
5.0 Hz, 0.5H), 3.17 (dd, J¼14.0, 6.0 Hz, 0.5H), 3.30–3.40
(m, 3H), 3.43–3.53 (m, 2H), 3.57–3.63 (m, 2.5H), 3.67–
3.80 (m, 2H), 3.87 (m, 1H), 3.97 (m, 0.5H), 4.35 (d,
J¼8.0 Hz, 0.5H, H-1), 4.38–4.53 (m, 5H), 4.65 (d,
J¼8.0 Hz, 0.5H, H-1), 4.90 (m, 4H), 5.07–5.24 (m, 4H),
6.04 (d, J¼8.5 Hz, 0.5H, NH), 6.38 (d, J¼7.5 Hz, 0.5H,
NH), 6.65 (dd, J¼6.5, 6.5 Hz, 0.5H, NH), 6.79 (dd, J¼7.0,
6.0 Hz, 0.5H, NH), 7.30 (m, 20H, Ar-H). ESIMS calcd for
C123H205N2O19P: 2045.5; found: 2068.5 (MþNa, 63),
2069.5 (MþNa, 13C-isotope, 100).
4.2.16. 2-(Benzyloxymethyl)-3-O-(di-O-benzyl-phos-
phono)-2-[(R)-3-(tetradecanoyloxy)tetradecanamido-
methyl]-propan-3-ol-1-yl 6-O-benzyl-2-deoxy-4-O-(di-O-
benzyl-phosphono)-3-O-[(R)-3-(tetradecanoyloxy)tetra-
decanoyl]-2-[(R)-3-tetradecanoyloxytetradecanamido]-
b-D-glucopyranoside (24). To a solution of compound 23
(220 mg, 0.108 mmol) in dry dichloromethane (5 mL),
dibenzyl N,N-diisopropyl phosphoramidite (74.3 mg,
74.3 mL, 0.215 mmol) and 1H-tetrazole (22.7 mg,
0.324 mmol) are added and the mixture is stirred at
room temperature for 30 min and then cooled to 08C.
m-Chloroperbenzoic acid (m-CPBA, 55%, 118 mg,
0.379 mmol) is added with stirring at 08C for 30 min. The
mixture is diluted with dichloromethane (100 mL) and
washed with aqueous sodium bisulphite solution (10%,
20 mL). Following the separation of two layers, the aqueous
layer is further extracted with dichloromethane (20 mL).
The combined organic layer is washed with saturated
sodium bicarbonate solution (20 mL) and again the aqueous
layer is back extracted with dichloromethane (20 mL). The
combined organic layer is dried with sodium sulphate and is
concentrated under vacuum. The residue is purified by
repeated flash chromatography (hexane–acetone, 5:1
followed by 4.5:1; dichloromethane–methanol, 100:1
followed by 100:1.5; hexane–ethyl acetate, 2:1 followed
by 1.5:1) to obtain 24 (200 mg, 80%), colorless syrup, as a
diastereomeric mixture in a ratio of about 1:1. Rf (upper
spot) 0.29 and Rf (lower spot) 0.25 (hexane–acetone, 3:1).
4.2.18. 2-(Hydroxymethyl)-3-phosphoryl-2-[(R)-3-(tetra-
decanoyloxy)tetradecanamidomethyl]-propan-1-yl 2-
deoxy-4-O-(phosphono)-3-O-[(R)-3-(tetradecanoyloxy)-
tetradecanoyl]-2-[(R)-3-(tetradecanoyloxy)tetradecan-
amido]-b-D-glucopyranoside (4). Adapting the procedure
described for 3, compound 24 (104 mg, 0.045 mmol) is,
following flash chromatographic purification (chloroform–
methanol–water, 9:1:0 and then 6:4:0.5), converted to 4
(77 mg, 96%). Rf 0.50 (chloroform–methanol–water,
6:4:0.5). [a]2D0¼23.0 (c 0.2, chloroform). 1H NMR
(600 MHz, CDCl3–CD3OD, 1:1): d 0.89 (t, J¼6.5 Hz,
18H, 6CH3), 1.25 (m, 114H, 57CH2), 1.60 (m, 12H, 6CH2),
2.30 (m, 6H, 3CH2), 2.37–2.70 (m, 6H, 3CH2), 3.03 (d,
J¼14.0 Hz, 0.5H), 3.13 (d, J¼14.0 Hz, 0.5H), 3.24 (d,
J¼14.0 Hz, 0.5H), 3.27 (d, J¼14.0 Hz, 0.5H), 3.29–3.36
(m, 3H), 3.45 (br s, 1H), 3.55–3.95 (m, 6H), 4.06–4.32 (m,
2H), 5.14–5.27 (m, 4H). ESIMS calcd for C95H181N2O19P:
1685.3; found: 1686.3 (MþH), 1708.3 (MþNa), 1730.3
(Mþ2Na–H).
4.3. Bioassay
4.3.1. Measurement of cytokines secreted by human
adherent antigen presenting cells. APCs isolated from
1
[a]2D0(,1:1 mixture)¼27.6 (c 0.5, chloroform). H NMR

Z.-H. Jiang et al. / Tetrahedron 58 (2002) 8833–8842
8841
8. Hoshino, K.; Takeuchi, O.; Kawai, T.; Sanjo, H.; Ogawa, T.;
Takeda, Y.; Takeda, K.; Akira, S. J. Immunol. 1999, 162,
3749–3752.
human peripheral blood by adherence to plastic are
incubated for four days in complete RPMI-1640 medium
with 5% human AB serum in the presence of GM-CSF
(50 ng/mL) and IL-4 (10 ng/mL). The lipid A analogues are
added at 1 mg/mL to the matured cells containing 106 cells.
After 24 h incubation, the supernatants are harvested and the
presence of the cytokines is determined using ELISA kits
(BD Pharmingen). TNF-a, IL-6 and IL-8 levels are
measured and the data are listed in Table 1.
9. Kutuzova, G. D.; Albrecht, R. M.; Erickson, C. M.; Qureshi,
N. J. Immunol. 2001, 167, 482–489.
10. (a) Matsuura, M.; Yamamoto, A.; Kojima, Y.; Homma, J. Y.;
Kiso, M.; Hasegawa, A. J. Biochem. 1985, 98, 1229–1237.
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1999, 67, 6286–6292. (d) Lynn, W. A.; Golenbock, D. T.
Immunol. Today 1992, 13, 271–276.
4.3.2. Mice immunized with liposomal vaccines. Groups
of C57Bl/6 mice are immunized intradermally with
BLP2530 liposomal vaccine containing 0.2 mg of MUC1-
based 25-mer lipopeptide as an antigen and 0.1 mg of
compound 3, 4, or the reference R595 lipid A as an adjuvant.
Nine days after vaccine injection, mice are sacrificed and
lymphocytes are taken from the draining lymph nodes (local
response) to determine the immune response by measure-
ment of antigen specific T-cell proliferation in vitro.
¨
11. (a) Bulusu, M. A. R. C.; Waldstatten, P.; Hildebrandt, J.;
Schu¨tze, E.; Schulz, G. J. Med. Chem. 1992, 35, 3463–3469.
(b) Eustache, J.; Grob, A.; Retscher, H. Carbohydr. Res. 1994,
251, 251–267. (c) Johnson, D. A.; Sowell, C. G.; Johnson,
C. L.; Livesay, M. T.; Keegan, D. S.; Rhodes, M. J.; Ulrich,
J. T.; Ward, J. R.; Cantrell, J. L.; Brookshire, V. G. Bioorg.
Med. Chem. Lett. 1999, 9, 2273–2278. (d) Ikeda, K.; Asahara,
T.; Achiwa, K. Chem. Pharm. Bull. 1993, 41, 1879–1881.
(e) Lien, E.; Chow, J. C.; Hawkins, L. D.; McGuinness, P. D.;
Miyake, K.; Espeviks, T.; Gusovsky, F.; Golenbock, D. T.
J. Biol. Chem. 2001, 276, 1873–1880.
4.3.3. Measurement of T-cell proliferation. T-cell pro-
liferation is evaluated using a standard 3H thymidine
incorporation assay. Briefly, nylon wool passed inguinal
lymph node lymphocytes, at 0.25£106/well, pooled from
each mouse group, are added to a culture containing naive
12. Qureshi, N.; Mascagni, P.; Ribi, E.; Takayama, K. J. Biol.
Chem. 1985, 260, 5271–5278.
13. The monophosphoryl lipid A (MPLw) provided by Ribi
ImmunoChem, Inc., is obtained after selective hydrolysis of
the (R)-3-hydroxytetradecanoyl group at 3-O-position. The
removal of this 3-O-acyl group is believed to reduce its
toxicity and the major component of MPLw has been claimed
to be hexaacyl substituted. On the other hand, the lipid A
product provided by Avanti Polar Lipids, Inc., does not
undergo selcetive hydrolysis of this 3-O-acyl group and its
major component has been claimed to be heptaacyl sub-
stituted. Our experimental results (data not shown) indicate
that this 3-O-acyl group does not have much effect on the
toxicity profile of lipid A product from S. minnesota R595. For
more informations, please see (a) Johnson, D. A.; Keegan,
D. S.; Sowell, C. G.; Livesay, M. T.; Johnson, C. L.; Taubner,
L. M.; Harris, A.; Myers, K. R.; Thompson, J. D.; Gustafson,
G. L.; Rhodes, M. J.; Ulrich, J. T.; Ward, J. R.; Yorgensen,
Y. M.; Cantrell, J. L.; Brookshire, V. G. J. Med. Chem. 1999,
42, 4640–4649. (b) Ulrich, J. T.; Myers, K. R. In Vaccine
Design: The Subunit and Adjuvant Approach. Powell, M. F.,
Newman, M. J., Eds.; Plenum: New York, 1995; pp 495–524.
14. Jiang, Z.-H.; Bach, M. V.; Budzynski, W. A.; Krantz, M. J.;
Koganty, R. R.; Longenecker, B. M. Bioorg. Med. Chem. Lett.
2002, 12, 2193–2196.
mitomycin
C-treated
syngeneic
splenocytes
at
0.25£106/well, which serve as APCs. To each well 20 mg
of MUC1-based 25-mer peptide30 is added as boosting
antigen. The culture is incubated for 72 h in a total volume
of 300 mL/well, followed by the addition of 1 mCi of
3H-thymidine in a volume of 50 mL. The plates are
incubated for an additional 18–20 h. Cells are harvested
and [3H]dTh incorporation is measured by liquid scintil-
lation counter. T-cell proliferation results corresponding to
various liposomal vaccines adjuvanted with lipid A mimic
3, 4, or reference natural R595 lipid A are shown in Fig. 2.
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