Chemical synthesis and proinflammatory responses of monophosphoryl lipid a adjuvant candidates

Article abstract of DOI:10.1002/ejoc.200900973

Lipopolysaccharides (LPS), which are structural components of the outer-surface membrane of Gram-negative bacteria, trigger innate immune responses through activation of Tolllike receptor 4 (TLR4). Such responses may be exploited for the development of ad

Full text of DOI:10.1002/ejoc.200900973

FULL PAPER
DOI: 10.1002/ejoc.200900973
Chemical Synthesis and Proinflammatory Responses of Monophosphoryl Lipid
A Adjuvant Candidates
Kaustabh K. Maiti,[a][‡] Michael DeCastro,[a][‡] Abu-Baker M. Abdel-Aal El-Sayed,[a,b]
Matthew I. Foote,[a] Margreet A. Wolfert,[a] and Geert-Jan Boons*[a]
Keywords: Carbohydrates / Lipids / Lipopolysaccharides / Immunochemistry / Cytokines / Protecting groups / Adjuvant /
Tumor necrosis factor / Inflammation
Lipopolysaccharides (LPS), which are structural components
of the outer-surface membrane of Gram-negative bacteria,
trigger innate immune responses through activation of Toll-
like receptor 4 (TLR4). Such responses may be exploited for
the development of adjuvants and in particular monophos-
phoryl lipid A (MPLA) obtained by controlled hydrolysis of
LPS of Salmonella minnesota, exhibits low toxicity yet pos-
sesses beneficial immuno-stimulatory properties. We have
developed an efficient synthetic approach for the preparation
of a major component of MPLA (1), which has as a key fea-
ture the use of allyloxycarbonates (Alloc) as permanent pro-
tecting groups for the C-3 and C-4 hydroxy groups of the
proximal glucosamine unit. The latter protecting groups
N-2,2,2-trichloroethoxycarbamates (Troc), which performed
efficient neighboring-group participation to give selectively
1,2-trans-glycosides and could easily be removed under mild
conditions without affecting the permanent Alloc carbonates
and anomeric dimethylthexylsilyl (TDS) ether. The synthetic
methodology was also employed for the preparation of a
monophosphoryl lipid A (2) derivative that has the anomeric
center of the proximal sugar modified as a methyl glycoside.
Compound 1 was not able to induce cytokine production in
mouse macrophages whereas methyl glycoside 2 displayed
activity, however it has a lower potency and efficacy than
lipid A obtained by controlled hydrolysis S. minnesota. This
indicates compound 2 is an attractive candidate for adjuvant
greatly facilitated deprotection of the fully assembled com- development and that 1 is not the active substance of MPLA
pound. Furthermore, the amino functions were protected as
obtained by controlled hydrolysis of LPS.
Recent studies have indicated that adjuvant activity
arises in large part from activation of the innate immune
system.[2] The innate immune system is an evolutionarily
ancient system designed to detect the presence of microbial
invaders and activate protective reactions.[3] It responds
rapidly to compounds that are integral parts of pathogens
that are perceived as danger signals by the host. Recogni-
tion of these molecular patterns is mediated by sets of
highly conserved receptors,[4] whose activation results in
acute inflammatory responses. These responses include the
production of a diverse set of cytokines and chemokines,
direct local attack against the invading pathogen, and initia-
tion of responses that activate and regulate the adaptive
component of the immune system.[5]
Toll-like receptor (TLR) ligands have attracted consider-
able attention as lead compounds for adjuvant develop-
ment. However, a concern of the use of these compounds
is that they can over-activate innate immune responses lead-
ing to the clinical symptoms of septic shock.[6] Thus, an
important issue for the design of safe immune modulators
is a detailed knowledge of structure-activity relationships to
harness beneficial effects without causing toxicity.
Introduction
Adjuvants are compounds or macromolecular complexes
that boost the potency and longevity of immune response
to vaccine antigens without causing toxicity.[1] They can af-
fect the migration, maturation and antigen presentation of
dendritic cells (DC), which in turn improves immune re-
sponses to antigen. Adjuvants can also affect the nature
of CD4 and CD8 T-helper responses with some adjuvants
promoting Th1-related responses and others preferentially
inducing Th2-biased effects. Furthermore, some adjuvants
enhance cross-presentation by DCs of MHC I-restricted an-
tigens to CD8+ T cells. Adjuvants may also directly act on
T- or B-cells by enhancing their proliferation and/or conver-
sion into memory cells.
[a] Complex Carbohydrate Research Center,
The University of Georgia,
315 Riverbend Road, Athens, GA 30602, USA
Fax: +1-706-5424412
E-mail: gjboons@ccrc.uga.edu
[b] School of Molecular & Microbial Sciences,
The University of Queensland,
Queensland, Australia
[‡] These authors contributed equally to this research.
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200900973.
Lipopolysaccharides (LPS), which are structural compo-
nents of the outer surface membrane of Gram-negative bac-
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Synthetic Monophosphoryl Lipid A
Figure 1. Chemical structures of E. coli lipid A, a major component of MPLA (1) and a methyl glycoside analog (2).
teria, trigger innate immune responses through TLR4, a
As part of a program to develop a fully synthetic vaccine
member of the TLR family that participates in pathogen against cancer,[14] we required an efficient synthetic ap-
recognition. LPS consists of a hydrophobic domain known proach for the preparation of well-defined derivatives of
as lipid A, a non-repeating core oligosaccharide and a distal MPLA.[15] During previous efforts to prepare well-defined
(non-reducing end) polysaccharide (or O-antigen).[7] The li- lipid A derivatives, we observed that removal of a benzyl
pid A moiety of E. coli consists of a hexaacylated bis-1,4Ј- ether at C-4 of the proximal (reducing end) sugar by cata-
phosphorylated glucosamine disaccharide, which has (R)-3- lytic hydrogenation was very slow causing complica-
hydroxymyristyl residues at C-2, C-2Ј, C-3, and C-3Ј. tions.[16,17] Furthermore, sugar lactols are known to react
Furthermore, both of the (3)-hydroxyacyl chains in the dis- with a wide range of reagents compromising their stability.
tal glucosamine moiety are esterified by lauric and myristic Also, these compounds are difficult to analyze by NMR
acids, and the primary hydroxy at the C-6 position is linked due to mixtures of alpha- and beta-lactols. To address these
to the polysaccharide through a di-KDO carbohydrate moi- issues, we have developed an efficient approach for the
ety (Figure 1). It has been demonstrated unequivocally that chemical synthesis of monophosphoryl lipid A and deriva-
lipid A is the inflammation-inducing moiety of LPS.[8]
tives thereof by using common disaccharide intermediates,
Monophosphoryl lipid A, obtained by controlled hydrol- which are convenient starting material for the preparation
ysis of LPS of Salmonella minnesota (MPLA), has a much of a wide range of acylated derivatives. Importantly, an al-
reduced toxicity yet possesses useful immunostimulatory lyloxycarbonate (Alloc)[18] was employed as a permanent
properties.[9] It is among the first of a new generation of protecting group for the C-3 and C-4 hydroxy groups of the
TLR agonists likely to be approved as a vaccine adjuvant proximal glucosamine moiety to avoid difficulties during
for humans. In this respect, numerous preclinical and clin- deprotection. This protecting group can easily be removed
ical studies have demonstrated the favorable adjuvant prop- by treatment with Pd(PPh3)4, without effecting acyloxyacyl-
erties of MPLA and in general it has been found that it or phosphate esters. Furthermore, we have prepared a mon-
elicits a Th-1 or mixed Th1/Th2 response.
ophosphoryl lipid A derivative that has the anomeric center
It has been suggested that the low toxicity of MPLA is of the proximal sugar modified as a methyl glycoside. Pre-
associated with a bias toward Toll-interleukin-1 receptor vious studies have shown that replacement of an anomeric
(TIR)-domain-containing adapter inducing IFN-β (TRIF) phosphate by a glycine moiety did not compromise bio-
signaling, due to active suppression rather than passive loss logical activity of E. coli lipid A,[19] and thus it was hoped
of pro-inflammatory activity of the LPS derivative.[10] Al- that methyl glycoside 2 would have comparable immunolog-
ternatively, it has been proposed that MPLA’s lack of pro- ical properties to MPLA but be more stable and easier to
inflammatory activity is due to its inability to activate cas- characterize. The pro-inflammatory properties of com-
pase-1.[11] This protease is involved in the maturation of pounds 1 and 2 have been studied by exposing mouse ma-
several pro-inflammatory mediators such as IL-1β and IL- crophages to the compounds followed by the measurement
18 and the reduced production of these cytokines may ex- of the production of several cytokines, including tumor ne-
plain the low toxicity of MPLA. Yet another explanation crosis factor α (TNF-α).
for MPLA’s low toxicity adjuvant functions is that it stimu-
lates higher levels of the anti-inflammatory cytokine IL-
10.[12]
Results and Discussion
A major compound of MPLA is a lipid A derivative with
only one phosphate at C-4 of the distal GlcNAc moiety and
Compounds 1 and 2 have been prepared by an efficient
acyloxyacyl side chains at C-2, C-2’ and C-3Ј (e.g. com- approach using the monosaccharide building blocks 3, 4
pound 1, Figure 1).[13]
and 5 and fatty acid 6[20] (Figure 2). Our strategy for the
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preparation of compound 1 and 2 is based on glycosyl-
ations of trichloroacetimidate 3 with glycosyl acceptors 4
or 5, to give disaccharides that can easily be acylated and
phosphorylated to give the required lipid A derivatives. The
C-3 hydroxy of 3 is already modified by a (R)-3-dodeca-
noyltetradecanoic ester because previous studies had shown
that installation of this lipid at a late stage of the synthesis
leads to unwanted site reaction such as phosphate mi-
gration. The amino groups of 3 is protected as N-(2,2,2-
trichloroethoxy)carbamates (Troc), which can perform ef-
ficient neighboring group participation to provide selec-
tively 1,2-trans-glycosides. However, this protecting group
can easily be removed under mild conditions using activated
Zn in acetic acid to give a free amine, which can then be
acylated with 6. The benzylidene acetal of 3 can be regiose-
lectively opened to give a C-4 hydroxy, which can then be
converted into a phosphate ester. The C-3 and C-4 hydroxy
groups of 4 are permanently protected as Alloc carbonates
because previous studies had shown that protection by
common benzyl ethers gave difficulties during deprotection.
Similarly, the anomeric hydroxy of 5 was permanently pro-
tected with dimethylthexylsilyl group (TDS), which could
be removed prior to the final global reduction to yield the
requisite lactol.
Scheme 1. Chemical synthesis monosaccharide building blocks.
Reagent and conditions: a) TDSCl, imidazole, DMF; b) allylchlo-
roformate, N,N,NЈ,NЈ-tetramethylethylenediamine, CH2Cl2; c) HF/
pyridine, THF; d) guanidine·HCl, CH2Cl2, then dimethoxylethyl-
benzene, CSA, CH3CN; e) AcOH in toluene, 80 °C; f) (R)-3-do-
decanoyltetradecanoic acid, EDC, DMAP, CH2Cl2; g) HF-pyr-
idine, THF then CCl3CN, DBU, CH2Cl2.
(Scheme 1). Acylation of 11 with (R)-3-dodecanoyltetrade-
canoic acid (6)[20] using the activator EDC as the activating
reagent afforded 13 in a yield of 80%. Removal of the an-
omeric TDS moiety of 13 by treatment with HF-pyridine
followed by reaction of the resulting lactol with trichloro-
acetonitrile in the presence of catalytic amount of DBU
gave trichloroacetimidate 3 (α/β ≈ 1:1).
Glycosyl acceptor 5 was obtained by protection of the C-
3 hydroxy of 11 as an Alloc carbonate followed by selective
removal of the benzylidene acetal of the resulting com-
pound 12 using acetic acid.
Having glycosyl donor 3 and acceptors 4 and 5 at hand,
attention was focused on glycosylation, acylation and phos-
phorylation. A triflic acid (TfOH) mediated glycosyla-
tion[24] of glycosyl donor 3 with acceptor 4 in the presence
of molecular sieves in DCM at –40 °C gave the disaccharide
14 as exclusively the β-anomer in a yield of 82%
Figure 2. Building blocks for the synthesis of compounds 1 and 2.
Glycosyl acceptor 4 was prepared by a three-step pro- (Scheme 2). Regioselective reductive ring opening of the
cedure starting from compound 7[21] (Scheme 1). Thus, the benzylidene acetal of 14 using Et3SiH and of TfOH at
C-6 hydroxy group of 7 was selectively protected as TDS –78 °C gave 15.[24,25] The alcohol of 15 was phosphorylated
ether by reaction with chloro(dimethyl)thexylsilane to give 16 by reaction with N,N-diethyl-1,5-dihydro-2,3,4-
(TDSCl) in the presence of imidazole in DMF to give 8 in benzodioxaphosphepin-3-amine in the presence of 1H-
a yield of 90%. The free hydroxy groups of compound 8 tetrazole followed by in-situ oxidation with m-chloroper-
were protected as Alloc carbonates by reaction with allyl oxybenzoic acid (mCPBA).[26] The N-Troc groups of 16 was
chloroformate, in the presence of N,N,NЈ,NЈ-tetramethyl- removed using Zn-dust in acetic acid and the resulting
ethylenediamine in DCM to give fully protected 9. Removal amino groups were immediately acylated with (R)-3-do-
of the TDS ether of 9 by treatment with HF-pyridine gave decanoyltetradecanoic acid (6)[20] using EDC and DMAP
the target glycosyl acceptor 4.
as the coupling reagents to afford 17. It was observed that
Compounds 3 and 5 could conveniently be prepared the first acylation proceeded with a high reaction rate
from common building block 10.[22] Thus, the acetyl esters whereas the second amine reacted sluggishly. The relatively
of 10 were removed by treatment with guanidine-HCl in low overall yield of the two-step procedure is due to incom-
DCM[23] and the 4,6-diol of the resulting compound was plete acylations. Removal of the Alloc groups of 17 could
protected as a benzylidene acetal by treatment with dimeth- easily be accomplished by treatment with Pd(PPh3)4 in the
oxybenzaldehyde and a catalytic amount of camphorsul- presence of BuNH2 buffered with HCOOH[18] in THF to
fonic acid (CSA) in acetonitrile to give compound 11 give compound 18 after purification by size exclusion
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Synthetic Monophosphoryl Lipid A
chromatography using LH-20. Finally, hydrogenolysis over vinyl protons of the Alloc carbonate. Next, regioselective
Pd-black resulted in the deprotection of the phosphotriester reductive opening of the benzylidene acetal of 20 using Et3-
and removal of the benzyl ethers to provide monophospho- SiH and TfOH at –78 °C[25] gave 21, which after phosphor-
ryl lipid A derivative 2. The structure of 2 was confirmed ylation with N,N-diethyl-1,5-dihydro-2,3,4-benzodioxapho-
by NMR spectroscopy.
sphepin-3-amine in the presence of 1H-tetrazole followed
by in-situ oxidation with m-chloroperoxybenzoic acid
(mCPBA),[26] provided the phosphotriester 22. Removal of
the Troc protecting groups of 22 using activated Zn fol-
lowed by immediate acylation of the resulting amino group
with (R)-3-dodecanoyltetradecanoic acid (6) in the presence
of EDC gave compound 23. The latter derivative was de-
protected to give MPLA (1) by removal of the Alloc car-
bonates using Pd(PPh3)4, cleavage of the anomeric TDS
moiety employing HF in pyridine, and finally hydrogena-
tion over Pd-black to remove the phosphate protecting
group and benzyl ethers.
To examine the pro-inflammatory properties of the com-
pounds, RAW 264.7 γNO(–) mouse macrophages were ex-
posed over a wide range of concentrations to compounds 1
and 2, a commercial lipid A derivative obtained by con-
trolled hydrolysis of the LPS of S. minnesota (MPLA), a
synthetic lipid A derived from E. coli,[16] and E. coli 055:B5
LPS. After 5.5 h, the supernatants were harvested and ex-
amined for mouse TNF-α using a commercial capture EL-
ISA. Potencies (EC50, concentration producing 50% ac-
tivity) and efficacies (maximal level of production) were de-
Scheme 2. Reagents and conditions: a) TfOH, CH2Cl2, –40 °C, MS;
b) TfOH, Et3SiH, CH2Cl2, –78 °C; c) N,N-diethyl-1,5-dihydro-
2,4,3-benzodioxaphosphepin-3-amine, tetrazole, mCPBA, CH2Cl2;
d) Zn-dust, AcOH, CH2Cl2; e) (R)-3-dodecanoyltetradecanoic
acid, EDC, Et3N, CH2Cl2; f) Pd(PPh3)4, HCO2 H, nBuNH2,
THF;g) 10% Pd on charcoal (Degussa), H2 (1 atm), THF, tBuOH.
The synthesis of MPLA (1) commenced with a regiose- termined by fitting the dose-response curves to a logistic
lective glycosylation of trichloroacetimidate 3 with acceptor equation using PRISM software. As can be seen in Figure 3
5 to give formation of disaccharide 19 in high yield and Table 1, compound 2, the synthetic lipid A derived
(Scheme 3). The C-4 hydroxy group of 19 was permanently from E. coli and MPLA yielded clear dose response curves.
protected as an Alloc carbonate by employing standard re- Surprisingly, compound 1 gave only a marginal response at
action conditions to give fully protected 20, the structure of the highest concentration tested. As expected, LPS had a
which was supported by a down field shift of 4-H and the greater potency than the lipid As because previous studies
appearance of a multiplet at 4.53–4.45 corresponding to the had shown that the KDO moiety of the core region of LPS
Scheme 3. Reagents and conditions: a) TfOH, CH2Cl2, –40 °C; b) allyl chloroformate, N,N,NЈ,NЈ-tetramethylethylenediamine, CH2Cl2;
c) TfOH, Et3SiH, CH2Cl2, –78 °C; d) N,N-diethyl-1,5-dihydro-2,4,3-benzodioxaphosphepin-3-amine, tetrazole, mCPBA, CH2Cl2; e) Zn
dust, AcOH, CH2Cl2; f) (R)-3-dodecanoyltetradecanoic acid, EDC, Et3N, CH2Cl2; g) Pd(PPh3)4, HCO2H, nBuNH2, THF; h) HF/pyr-
idine, THF; i) 10% Pd on charcoal (Degussa), THF, tBuOH, H2 (1 atm).
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contributes significantly to potency.[17] Furthermore, TLR4/MD2 complex leading to cellular activation. The ef-
MPLA and lipid A from E. coli had similar potencies ficacy of cytokine induction by compound 2 was signifi-
whereas methyl glycoside 2 was significantly less active.
cantly lower than that of E. coli lipid A and LPS. The re-
duced potency and efficacy of 2 are expected to be benefi-
cial for adjuvant development because sufficient levels of
cytokines will be elicited for the induction of relevant re-
sponses, yet toxicity will be minimized.
The commercial lipid A from S. minnesota obtained by
controlled hydrolysis of isolated material showed activity at
a surprisingly low concentration indicating that it contains
compounds other than 1 that induces pro-inflammatory re-
sponses. Recently, we found that shortening the fatty acids
of monophosphoryl lipid A derivative derived from S. thy-
phimurium increased proinflammatory properties by as
much as a thousand fold.[17] It is possible that the sample
obtained by controlled hydrolysis may contain lipid A de-
rivatives that have shorter fatty acids or contaminants that
may account for the high activity. It is important to note
that therapeutic grade MPLA may not contain these com-
ponents and hence may exhibit a different proinflammatory
profile.
Figure 3. TNF-α production by murine macrophages after stimula-
tion with E. coli LPS, E. coli lipid A, MPLA and synthetic com-
pounds 1 and 2. Murine RAW 264.7 γNO(–) cells were incubated
for 5.5 h with increasing concentrations of E. coli LPS, E. coli lipid
A, MPLA and compounds 1 and 2 as indicated. TNF-α in cell
supernatants was measured using an ELISA. Treatment with E.
coli LPS, E. coli lipid A, MPLA, 1 and 2 did not affect cell viability,
as judged by cellular exclusion of trypan blue.
Methyl glycoside 2 will be an attractive compound for
incorporation into fully synthetic vaccines comprised of an
inbuilt adjuvant, a helper T-epitope and a cancer-associated
glycopeptide. Our previous studies have shown that such
compound can elicit robust antigenic responses against
tumor-associated carbohydrates.[14] It is to be expected that
the methyl glycoside of 2 will protect the anomeric center
making it possible to employ a variety of conjugation reac-
tions for attachment to a glycopeptide antigen using an ap-
propriate linker.
Table 1. EC50 values[a] [nM] of E. coli LPS and lipid A, MPLA and
compound 2.
E. coli LPS
TNF-α 0.0091
(0.0074–0.011)
IFN-β 0.011
E. coli lipid A MPLA
Lipid A (2)
35
11
184
(112–302)
299
(26–48)
229
(9–14)
30
(0.088–0.013)
0.010
(0.087–0.012)
0.039
(158–332)
161
(115–226)
184
(20–46)
33
(26–43)
66
(225–397)
139
(42–458)
3287
IP-10
IL-6
(0.029–0.053)
(115–292)
(56–77)
(2552–4237)
Conclusions
[a] Values of EC50 are reported as best-fit values and as minimum-
maximum range (best-fit valueϮstd. error).
In conclusion, we have developed an efficient chemical
synthesis of a monophosphoryl lipid A derivative from S.
minnesota. A key feature of the approach was the perma-
nent protection of the C-3 and C-4 hydroxy groups of the
proximal glucosamine moiety as Alloc carbonates, which
greatly facilitated the final deprotection steps. The methyl
glycoside 2 displayed lower pro-inflammatory activity than
a lipid A derivative obtained by controlled hydrolysis of
LPS of S. minnesota and LPS and lipid A derived from E.
coli. The adjuvant properties of the synthesis derivatives are
under investigations.
Importantly, the various compounds displayed differ-
ences in maximum response (efficacy) of TNF-α production
with compound 2 having a lower efficacy than MPLA,
which in turn had a lower maximum response than E. coli
lipid A and LPS (Table 2). The supernatants were also in-
vestigated for the presence of IFN-β, IP-10 and IL-6 and
similar trends for both potencies and efficacies were ob-
served as for the presence of TNF-α.
Table 2. Cytokine top values[a] (pg/mL) of dose-response curves of
E. coli LPS and lipid A, MPLA and compound 2.
E. coli LPS
E. coli lipid A MPLA
Lipid A (2)
Experimental Section
TNF-α 5782Ϯ169
IFN-β 654Ϯ19
6197Ϯ362
550Ϯ37
4783 Ϯ185
418Ϯ30
2216Ϯ177
134Ϯ6
2498Ϯ474
622Ϯ33
Chemical Synthesis
IP-10
IL-6
11847Ϯ367
2103Ϯ77
11879Ϯ692
1873Ϯ152
7898Ϯ353
1643Ϯ51
General Synthetic Methods: Column chromatography was per-
formed on silica gel 60 (EM Science, 70–230 mesh). Reactions were
monitored by thin-layer chromatography (TLC) on Kieselgel 60
F254 (EM Science), and the compounds were detected by examina-
tion under UV light and by charring with 10% sulfuric acid in
MeOH. Solvents were removed under reduced pressure at Ͻ40 °C.
DCM was distilled from NaH and stored over molecular sieves
[a] Top values are reported as best-fit values Ϯstd. error.
The observation that synthetic methyl glycoside 2 has a
significantly higher pro-inflammatory activity than its syn-
thetic hydroxy counter part 1 indicates that the anomeric
methyl moiety can make favorable interactions with the (3 Å). THF was distilled from sodium directly prior to the applica-
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Synthetic Monophosphoryl Lipid A
tion. MeOH was dried by refluxing with magnesium methoxide
and then was distilled and stored under argon. Pyridine was dried
by refluxing with CaH2 and then was distilled and stored over mo-
lecular sieves (3 Å). Molecular sieves (3 and 4 Å), used for reac-
tions, were crushed and activated in vacuo at 390 °C during 8 h in
the first instance and then for 2–3 h at 390 °C directly prior to
application. Optical rotations were measured with a Jasco model
P-1020 polarimeter. 1H NMR and 13C NMR spectra were recorded
with Varian spectrometers (models Inova300 and Inova500)
equipped with Sun workstations. NMR spectra were recorded in
CDCl3 and referenced to residual CHCl3 at δ = 7.24 ppm, and 13C
NMR spectra were referenced to the central peak of CDCl3 at δ =
77.0 ppm. Assignments were made by standard gCOSY and
gHSQC. High-resolution mass spectra were obtained with a Bruker
model Ultraflex MALDI-TOF mass spectrometer. Signals marked
with a subscript L symbol belong to the biantennary lipids, whereas
signals marked with a subscript LЈ symbol belongs to their side
chain.
(OCH2CH=CH2) 101.61 (C-1), 73.00 (C-3), 78.12 (C-4), 76.05 (C-
6), 73.80 (C-2), 72.42 (C-5), 32.96 (O-CH3), 24.85, 19.96, 18.47,
18.45 (CH3) ppm. HR-MALDI-ToF/MS: m/z calcd. for
C26H42Cl3NO11Si 677.1593; found 700.1597 [M + Na]+.
Methyl 3,4-O-Allyloxycarbonyl-2-[(2,2,2-trichloromethoxy)carbon-
ylamino]-2-deoxy-α-D-glucopyranoside (4): Compound 9 (1.5 g,
2.21 mmol) was dissolved in THF followed by the addition of HF/
pyridine (0.5 mL). The resulting reaction mixture was stirred for
8 h at room temperature. The reaction was quenched with
NaHCO3 and extracted with DCM (250 mL). The organic phase
was washed with water (2ϫ100 mL) and brine (2ϫ100 mL). The
organic phase was dried (MgSO4), filtered and the filtrate concen-
trated in vacuo. The crude product was purified by flash silica gel
column chromatography (hexane/ethyl acetate, 1:2, v/v) to yield
1
compound 4 as a white amorphous solid (1.02 g, 86%). H NMR
(300 MHz, [D6]acetone): δ = 6.79 (d, J = 9.9 Hz, 1 H, NH) 5.98–
5.84 (m, 2 H, OCH2CH=CH2) 5.60–5.23 (m, 4 H, OCH2CH=CH2)
5.22 (d, J1-2 = 4.8 Hz, 1 H, 1-H), 5.20 (dd, J2-3 = 4.8, J3-4 = 9.6 Hz,
1 H, 3-H), 4.96 (dd, J3-4 = 9.6, J4-5 = 9.7 Hz, 1 H, 4-H), 4.83 (br.
s, 2 H, OCH2CCl3), 4.62–4.55 (m, 4 H, OCH2CH=CH2) 4.10–4.02
(m, 2 H, 2-H, 6-H) 3.86–3.81 (m, 2 H, 5-H, 6-H), 3.74–3.62 (m, 1
H, 6-H) 3.47 (s, 3 H, OCH3) ppm. 13C NMR (75 MHz, [D6]ace-
tone): δ = 154.81 (C=O), 154.73, 154.26 (OCH2CH=CH2), 132.22
(OCH2CH=CH2), 117.94,117.84 (OCH2CH=CH2) 98.52 (C-1),
75.57 (CH2-O) 73.19 (C-3), 74.28 (C-4), 73.19 (C-6), 70.31 (C-2),
68.60 (C-5), 32.96 (O-CH3) ppm. HR-MALDI-ToF/MS: m/z calcd.
for C18H24Cl3NO11 536.0415; found 559.0419 [M + Na]+.
Methyl 6-O-Dimethylthexylsilyl-2-[(2,2,2-trichloromethoxy)carbon-
ylamino]-2-deoxy-α-D-glucopyranoside (8): Imidazole (6.05 g,
88.8 mmol) was added to a solution of compound 7 (31.2 g,
88.8 mmol) in DMF (25 mL). After stirring the mixture for 10 min,
chloro(dimethyl)thexylsilane (17.4 g, 97.7 mmol) was added. The
reaction mixture was stirred for 18 h and then the reaction was
quenched by the addition of a saturated solution of Na2CO3
(50 mL). The crude material was extracted with DCM (200 mL)
and washed with water (3ϫ100 mL) and brine (2ϫ100 mL). The
organic phase was dried (MgSO4), filtered and the filtrate concen-
trated in vacuo. The crude product was purified by flash silica gel
column chromatography (hexane/ethyl acetate, 5:1, v/v) to give
compound 8 as a clear oil (39.5 g, 90%). 1H NMR (300 MHz, [D6]
acetone): δ = 6.49 (d, J = 8.1 Hz, 1 H, NH), 4.78–4.67 (m, 2 H,
OCH2CCl3) 4.65 (d, J1-2 = 3.6 Hz, 1 H, 1-H), 4.13 (dd, J3-4 = 5.1,
J4-5 = 4.5 Hz, 1 H, 4-H), 3.89–3.71 (m, 2 H, 6-H) 3.65–3.54 (m, 1
H, 3-H), 3.45 (dd, J1-2 = 3.6, J2-3 = 2.7 Hz, 1 H, 2-H), 3.38–3.34
(m, 1 H, 5-H), 3.28 (s, 3 H, OCH3) 1.49–1.43 (m, 1 H,
CH3CHCH3), 0.733–0.633 (m, 12 H, CH3), 0.007–0.00 (m, 6 H,
CH3Si) ppm. 13C NMR (75 MHz, [D8]acetone): δ = 205.88 (C=O),
99.12 (C-1), 76.10 (C-4), 72.13 (C-3), 71.49 (C-6), 66.63 (C-2), 65.28
(C-5), 34.2, 24.85, 19.96, 18.47, 18.45 (CH3) ppm. HR-MALDI-
ToF/MS: m/z: calcd. for C18H34Cl3NO7Si 509.1170; found 532.1175
[M + Na]+.
Dimethylthexylsilyl
[(2,2,2-trichloromethoxy)carbonylamino]-β-
3-O-Allyloxycarbonyl-4,6-O-benzylidene-2-
-glucopyranoside (11):
D
Compound 1 (25 g, 50.3 mmol) was dissolved in acetonitrile
(100 mL) and dimethoxybenzaldehyde (15.3 g, 100.6 mmol) and
camphor sulfonic acid were added (1.17 g, 5.03 mmol) sub-
sequently. The reaction mixture was stirred at room temperature
for 8 h. The solvent was removed under vacuo and the crude prod-
uct was purified by flash silica gel column chromatography (hex-
ane/ethyl acetate, 3:1, v/v) to give 11 as a white amorphous solid
material (28.3 g, 96%). 1H NMR (300 MHz, [D8]acetone): δ =
7.34–7.15 (m, 5 H, aromatic), 6.72 (d, J = 9.6 Hz, 1 H, NH), 5.41
(s ϾCHPh) 4.72 (d, J1-2 = 5.7 Hz, 1 H, 1-H), 4.61–4.49 (m, 3 H,
2-H, COOCH2, Troc), 4.05 (dd, J3-4 = 10.5, J4-5 = 5.2 Hz, 1 H, 4-
H), 3.71 (m, 1 H, 3-H), 3.58 (dd, J1-2 = 5.2, J2-3 = 10.5 Hz, 1 H,
2-H), 3.34–3.33 (m, 2 H, 6-H), 3.29–3.21 (m, 1 H, 5-H), 1.49–1.43
(m, 1 H, CH3CHCH3), 0.733–0.633 (m, 12 H, CH3), 0.007–0.00
(m, 6 H, CH3Si) ppm. 13C NMR (300 MHz, [D8]acetone): δ =
205.88 (C=O), 154.89, 138.46, 128.96, 128.18, 126.72 (Ar), 101.52
(CHPh), 97.18 (C-1), 82.10 (C-3), 74.61 (C-4), 71.31 (C-6), 68.63
(C-2), 66.68 (C-5), 34.2, 24.85, 19.96, 18.47, 18.45 (CH3) ppm. HR
MS (m/z) calcd. for C24H36Cl3NO7Si 583.1301; found 603.1306 [M
+ Na]+.
Methyl 3,4-O-Allyloxycarbonyl-6-O-dimethylthexylsilyl-2-[(2,2,2-
trichloromethoxy)carbonylamino]-2-deoxy-α-D-glucopyranoside (9):
Compound 8 (36.0 g, 70.7 mmol) was dissolved in DCM (20 mL)
followed by the addition of N,N,NЈ,NЈ-tetramethylethylenediamine
(8.16 g, 70.4 mmol). The reaction temperature was dropped to 0 °C
followed by the addition of allyl chloroformate (8.52 g, 70.7 mmol).
Next, the reaction was warmed to room temperature over a period
of 24 h. The crude material was extracted with DCM (250 mL)
and the organic phase washed with water (2ϫ100 mL) and brine
(2ϫ100 mL). The organic phase was dried (MgSO4), filtered and
the filtrate concentrated in vacuo. The crude product was purified
by flash silica gel column chromatography (hexane/ethyl acetate,
Dimethylthexylsilyl 3,4-O-Allyloxycarbonyl-2-[(2,2,2-trichlorometh-
oxy)carbonylamino]-β-D-glucopyranoside (12): A solution of 11
(2.5 g, 4.27 mmol) in DCM (12 mL) and N,N,NЈ,NЈ-tetramethyl-
ethylenediamine (0.45 mL, 2.99 mmol) was cooled (0 °C) and allyl
chloroformate (0.55 mL, 5.12 mmol) was added dropwise. Stirring
1
3:1, v/v) to yield compound 9 as a light yellow oil (41 g, 87%). H
NMR (300 MHz, CDCl3): δ = 5.88–5.72 (m, 2 H, OCH2CH=CH2) was continued at room temperature for 18 h. The reaction mixture
5.28–5.17 (m, 4 H, OCH2CH=CH2) 5.15 (d, J1-2 = 6.6 Hz, 1 H, 1- was quenched with saturated solution of NaHCO3 (3.0 mL) and
H), 5.05 (dd, J2-3 = 9.6, J3-4 = 10.2 Hz, 1 H, 3-H), 4.84 (dd, J3-4
=
diluted with DCM (30 mL). The organic layer was washed with
water (2ϫ15 mL) and brine (1ϫ10 mL), dried (Na2SO4), filtered
and the filtrate was concentrated in vacuo. The residue was purified
by flash silica gel column chromatography (hexane/ethyl acetate,
9.6, J4-5 = 9.6 Hz, 1 H, 4-H), 4.55–4.49 (m, 2 H, OCH2CCl3), 4.00–
3.94 (m, 2 H, 2-H, 6-H) 3.75–3.71 (m, 2 H, 5-H, 6-H), 3.64–3.62
(m, 1 H, 6-H) 3.32 (s, 3 H, OCH3) ppm. 13C NMR (75 MHz,
CDCl3):
(OCH2CH=CH2),
δ
=
158.54, 157.69, 157.33 (C=O), 134.81 5:1, v/v Ǟ 4:1, v/v) to yield 12 as a white foam (2.1 g, 76%). Rf =
122.55 (OCH2CH=CH2),
122.34 3.9 (hexane/ethyl acetate, 1:1, v/v). 1H NMR (300 MHz, CDCl3): δ
Eur. J. Org. Chem. 2010, 80–91
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
85

G.-J. Boons et al.
FULL PAPER
=
7.41–7.22 (m,
5
H, aromatic), 5.82–5.69 (m,
1
H,
3-O-[(R)-3-(Dodecanoyloxy)tetradecanoyl]-4,6-O-benzylidene-2-
OCH2CH=CH2), 5.38 (s, 1 H, ϾCHPh), 5.22–5.06 (m, 3-H, NH, [(2,2,2-trichloromethoxy)carbonylamino]-α-
OCH2CH=CH2), 4.74 (d, J = 8.8 Hz, 1 H, 1-H), 4.64 (d, J = chloroacetimidate (3): HF/pyridine (0.5 mL) was added to a solu-
9.5 Hz, H, NH), 4.51–4.46 (m, H, OCH2CCl3, tion of compound 13 (1.5 g, 2.21 mmol) in THF. After stirring the
D-glucopyranosyl Tri-
1
4
OCH2CH=CH2), 4.16 (dd, J4,5 = 8.8, J5,6a = 6.8 Hz, 1 H, 5-H),
3.70–3.27 (m, 4 H, 4-H, 6ab-H, 2-H), 1.54–1.46 [m, 1 H, OSi(CH3)
reaction mixture for 8 h at room temperature, it was quenched with
NaHCO3 and extracted with DCM (250 mL). The organic phase
3C(CH3)3CH(CH3)3], 0.75–0.71 (m, 12 H, 4CH3), 0.02 (s, 3 H, was washed with water (2ϫ100 mL) and brine (2ϫ100 mL), dried
SiCH3), –0.03 (s, 3 H, SiCH3) ppm. 13C NMR (75 MHz, CDCl3): (MgSO4), filtered and the filtrate was concentrated in vacuo. The
δ = 158.35 (C=O), 157.39 (C=O), 140.34, 134.53, 132.52, 131.61,
crude product was purified by flash silica gel column chromatog-
129.64, 122.48, 104.88, 99.96, 82.25, 78.53, 78.10, 72.36, 72.00, raphy (hexane/ethyl acetate, 1:2, v/v) to yield a lactol as a white
1
69.66, 62.36, 37.33, 28.19, 23.34, 23.31, 21.91, 1.50 (SiCH3), –0.00
(SiCH3) ppm. HR-MALDI-ToF/MS: m/z: for [M + Na]+ calcd.
C28H40Cl3NO9SiNa 692.0630; found 692.7123.
amorphous solid (1.02 g, 86%). H NMR (300 MHz, CDCl3): δ =
7.48–7.31 (m, 5 H, aromatic), 5.88 (d, J = 8.4 Hz, 1 H, NH), 5.48–
5.46 (m, 2 H, ϾCHPh, 1-H), 5.42 (t, J = 10.6 Hz, 1 H, 3-H), 5.29
[br. s, 1 H, 3(R) CH of dodecanoyl moiety], 4.74 (d, J = 17.3 Hz,
1 H, OCHHCCl3), 4.62 (d, J = 18.4 Hz, 1 H, OCHHCCl3), 4.24–
3.88 (m, 3 H, 4-H, 2-H, 5-H), 3.73–3.68 (m, 2 H, 6ab-H), 2.54 (dd,
Dimethylhexylsilyl 3-O-[(R)-3-(Dodecanoyloxy)tetradecanoyl]-4,6-
O-benzylidene-2-[(2,2,2-trichloromethoxy)carbonylamino]-β-D-gluco-
pyranoside (13): (R)-3-dodecanoyltetradecanoic acid (0.64 g,
0.150 mmol) was added to a solution of compound 11 (0.80 g,
0.137 mmol) in DCM (5 mL). EDC (0.29 g, 0.150 mmol) and
DMAP (8 mg) were added sequentially and the resulting reaction
mixture was stirred for 8 h at room temperature. The mixture was
diluted with DCM (20 mL) and washed with 1  HCl (2ϫ100 mL),
water (2ϫ100 mL) and brine (2ϫ100 mL). The organic phase was
dried (MgSO4), filtered and the filtrate concentrated in vacuo. The
crude product was purified by flash silica gel column chromatog-
raphy (hexane/ethyl acetate, 15:1, v/v Ǟ 6:1, v/v) to yield com-
pound 13 as a clear oil (1.08 g, 80%). 1H NMR (300 MHz, CDCl3):
δ = 7.37–7.14 (m, 5 H, aromatic), 6.77 (d, J = 9.6 Hz, 1 H, NH),
5.46 (s, 1 H, ϾCHPh), 5.22 (t, J = 9.2 Hz, 1 H, 3-H), 5.14–5.11
[m, 1 H, 3(R) CH of dodecanoyl moiety], 4.78 (d, J = 9.7 Hz, 1 H,
1-H), 4.64 (d, J = 13.4 Hz, 1 H, OCHHCCl3), 4.48 (d, J = 14.1 Hz,
1 H, OCHHCCl3), 4.19 –4.16 (m, 1 H, 2-H), 3.68 (t, J = 10.4 Hz,
1 H, 4-H), 3.58–3.36 (m, 3 H, 6ab-H, 5-H), 2.50 (dd, J2Sa,2Sb = 16.2,
J2Sa,2Sb = 16.2, J2Sa,3S = 8.1 Hz, 1 H, 2Sa-H), 2.47 (dd, J2Sa,2Sb
=
17.4, J2Sa,3S = 7.8 Hz, 1 H, 2Sb-H), 2.12 (t, J = 9.5 Hz, 2 H), 1.49
(br. s, 4H CH2 lipids), 1.27–1.16 (br. s, 34 H, CH2 lipids) 0.83 (t, J
= 7.3 Hz, 6 H, 2ϫ CH3, lipid) ppm. 13C NMR (75 MHz, CDCl3):
δ = 173.94 (C=O), 170.65 (C=O), 154.73, 137.86, 129.31, 128.45,
128.43, 126.36, 79.63, 74.83, 70.32, 62.98, 57.63, 34.58, 32.14,
29.74, 25.71, 22.91 ppm. HR-MALDI-ToF/MS: m/z: for [M + Na]+
calcd. C42H66Cl3NO10Na 874.3331; found 874.2731. The resulting
lactol (0.70 g, 0.824 mmol) was diluted in dry DCM (1.0 mL) under
a blanket of argon. Trichloroacetonitrile (0.82 mL) and DBU
(0.02 mL, 0.164 mmol) were added to the reaction mixture and the
mixture was stirred for 3 h at room temperature. The crude was
purified directly using flash silica gel column chromatography (hex-
ane/ethyl acetate/Et3N, 7:1:0.01, v/v/v) to give 3 as a light yellow
oil material (0.792 g, 97%). 1H NMR (300 MHz, CDCl3): δ = 8.89
[s, 1 H, OC(CCl3)NH], 7.58–7.38 (m, 5 H, aromatic), 6.48 (d, J =
6.4 Hz, 1 H, NH), 5.55 (s, 1 H, ϾCHPh), 5.53 (d, J1-2 = 4.2 Hz, 1
H, 1-H), 5.46 (t, J = 9.8 Hz, 1 H, 3-H), 5.18 [t, J = 7.2 Hz, 1
H, 3(R) CH of dodecanoyl moiety], 4.81 (d, J = 16.3 Hz, 1 H,
OCHHCCl3), 4.65 (d, J = 15.6 Hz, 1 H, OCHHCCl3), 4.45–4.22
(m, 2 H, 4-H, 2-H), 4.10–4.01 (m, 1 H, 5-H), 3.84–3.79 (m, 2 H,
J2Sa,3S = 6.1 Hz, 1 H, 2Sa-H), 2.43 (dd, J2Sa,2Sb = 13.4, J2Sa,3S
=
6.7 Hz, 1 H, 2Sb-H), 2.07 (t, J = 6.9 Hz, 2 H, CH2 of lipids), 1.40–
1.45 (m, 4 H), 1.20–1.10 (m, 34 H, CH2 lipids), 0.82–0.76 (m, 12
H, 4CH3), 0.06 (s, 3 H, SiCH3), 0.02 (s, 3 H, SiCH3) ppm. 13C
NMR (75 MHz, CDCl3): δ = 172.49, 169.53, 154.60 (C=O), 138.46,
128.93, 128.14, 126.49, 101.52 (CHPh), 96.88 (C-1), 79.19 (C-3),
74.38 (C-4), 72.00 (C-6), 68.48 (C-2), 66.59 (C-5), 38.79, 34.17,
34.05, 33.69, 32.00, 29.96, (CH2-lipids), 34.2, 24.85, 19.96, 18.47,
18.45 (CH3) ppm. HR MS (m/z) calcd. for C50H84Cl3NO10Si
991.4921; found 1014.4929 [M + Na]+.
6ab-H), 2.62 (dd, J2Sa,2Sb = 14.2, J2Sa,3S = 7.1 Hz, 1 H, 2Sa-H), 2.59
(dd, J2Sa,2Sb = 14.4, J2Sa,3S = 6.8 Hz, 1 H, 2Sb-H), 2.22 (t, J =
8.5 Hz, 2 H), 1.63–1.45 (m, 4 H, CH2 lipids), 1.27–1.16 (br. s, 34
H, CH2 lipids) 0.85 (t, J = 7.3 Hz, 6 H, 2ϫ CH3, lipid) ppm. 13C
NMR (75 MHz, CDCl3): δ = 172.12 (C=O), 170.43 (C=O), 161.21,
156.21, 138.86, 131.32, 128.85, 126.19, 101.64, 78.36, 76.22, 67.73,
66.87, 64.76, 58.63, 38.08, 34.86, 32.54, 30.37, 29.62, 23.71, 22.27
ppm. HR-MALDI-ToF/MS: m/z: for [M + Na]+ calcd.
C45H68Cl6N2O9Na 1016.7474; found 1016.9106.
Dimethylhexylsilyl 3-O-Allyloxycarbonyl-2-[(2,2,2-trichlorometh-
oxy)carbonylamino]-β-D-glucopyranoside (5): Glacial acetic acid in
toluene (90%, 10 mL) was added to a solution of compound 12
(1.5 g, 2.24 mmol) in toluene (5.5 mL), and the reaction mixture
was heated to 80 °C for 15 h. The reaction mixture was concen-
trated in vacuo and the residue co-evaporated three times from tol-
uene to remove residues of acetic acid. The residue was recrys-
tallized from ethyl acetate/hexane (2:1, v/v) to give 5 as a white
solid (1.1 g, 82%). Rf = 2.1 (hexane/ethyl acetate, 1:1, v/v); m.p.
Methyl 4Ј,6Ј-O-Benzylidene-2Ј-[(2,2,2-trichloromethoxy)carbon-
ylamino]-3Ј-O-[{(R)-3-(dodecanoyloxy)tetradecanoyl-2Ј-deoxy-β-D-
glucopyranosyl}]-2-[(2,2,2-trichloromethoxy)carbonylamino]-3,4-O-
Allyloxycarbonyl-2-deoxy-α-D-glucopyranoside (14): A mixture of
115 °C. 1H NMR (300 MHz, CDCl3): δ = 5.92–5.81 (m, 1 H, glycosyl acceptor 4 (228 mg, 0.424 mmol), trichloroacetamidate do-
OCH2CH=CH2), 5.33–5.21 (m, 3 H, NH, OCH2CH=CH2), 4.91
(d, J = 9.2 Hz, 1 H, 3-H), 4.80 (d, J = 9.5 Hz, 1 H, 1-H), 4.73 (d,
nor 3 (470 mg, 0.472 mmol) and molecular sieves (4 Å, 700 mg) in
DCM (2.5 mL) was placed under an atmosphere of Ar and cooled
J = 16.2 Hz, 1 H, OCH HCCl3), 4.59–4.56 (m, 3 H, OCH HCCl3, to –40 °C. TfOH (8.35 µL, 0.094 mmol, in 0.1 mL DCM) was
OCH2CH=CH2), 3.83–3.71 (m, 3 H, 6ab-H, 4-H), 3.55–3.39 (m, 2 added and stirring was continued for 20 min until the temperature
H, 2-H, 5-H), 1.60–1.51 [m, 1 H, OSi(CH3)3C(CH3)3CH(CH3)3], had reached 0 °C. The reaction was quenched by the addition of
0.82–0.76 (m, 12 H, 4CH3), 0.10 (s, 3 H, SiCH3), 0.09 (s, 3 H,
triethylamine (35 µL). Next, the mixture was diluted with DCM
(30 mL) and washed with satd. NaHCO3 solution (10 mL), water
SiCH3) ppm. 13C NMR (75 MHz, CDCl3): δ = 159.00 (C=O),
157.43 (C=O), 134.40, 122.73, 99.21, 98.69, 82.24, 78.35, 78.00, (2 ϫ 10 mL) and brine (10 mL). The organic layer was dried
72.97, 72.51, 65.73, 61.51, 37.28, 28.15, 23.31, 23.29, 21.86, 1.53
(SiCH3), –0.09 (SiCH3) ppm. HR-MALDI-ToF/MS: m/z: for [M +
Na]+ calcd. C21H36Cl3NO9 SiNa 603.9563; found 603.0630.
(Na2SO4), filtered and the filtrate concentrated in vacuo. The resi-
due was purified by silica gel column chromatography (hexane/
ethyl acetate, 9:1, v/v Ǟ 5:1, v/v) to yield disaccharide 14 (480 mg,
86
www.eurjoc.org
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 80–91

Synthetic Monophosphoryl Lipid A
82%) as a white foamy solid. Rf = 0.46 (hexane/ethyl acetate, 4:1,
MALDI-ToF/MS: m/z: for [M + Na]+ calcd. C60H90Cl6N2O20Na
1395.0760; found 1395.6084.
1
v/v). [α]2D4 = –8.6 (c = 1.0, CHCl3). H NMR (500 MHz, CDCl3):
δ = 7.43–7.42 (m, 2 H, aromatic), 7.35–7.32 (m, 3 H, aromatic),
6.76 (br. s, 1 H, NH), 6.42 (br. s, 1 H, NHЈ), 5.94–5.87 (m, 2 H,
OCH2CH=CH2), 5.50 (s, 1 H, PhCH), 5.38–5.33 (m, 4 H,
OCH2CH=CH2), 5.19 [br. s, 1 H, 3(R) CH of dodecanoyl moiety],
5.14 (t, J = 8.4 Hz, 1 H, 3Ј-H), 5.10 (t, J = 8.9 Hz, 1 H, 3-H), 4.49
(t, J = 9.1 Hz, 1 H, 4-H), 4.84 (t, J = 8.9 Hz, partially merged with
1Ј-H, 4Ј-H), 4.82 (d, J = 9.8 Hz, 1 H, 1Ј-H), 4.74 (d, J = 3.8 Hz, 1
H, 1-H), 4.64–4.56 (m, 8 H, 2 ϫ OCH2CH=CH2, OCH2CCl3),
4.37–4.34 (m, 1 H), 4.11–4.07 (m, 2 H, 2-H, 2Ј-H), 3.94–3.92 (m,
Methyl 6Ј-O-Benzyl-4-O-(1,5-dihydro-3-oxo-3λ5-3H-2,4,3-benzo-
dioxaphosphepin-3-yl)-2Ј-[(2,2,2-trichloromethoxy)carbonylamino]-
3Ј-O-[{(R)-3-(dodecanoyloxy)tetradecanoyl-2Ј-deoxy-β-D-
glucopyranosyl}]-2-[(2,2,2-trichloromethoxy)carbonylamino]-3,4-O-al-
lyloxycarbonyl-2-deoxy-α-D-glucopyranoside (16): Disaccharide 15
(120 mg, 0.087 mmol) in DCM (2.5 mL) was placed under Ar,
charged with 3 % tetrazole solution in acetonitrile (0.8 mL,
0.145 mmol), and N,N-diethyl-1,5-dihydro-2,4,3-benzodioxaphos-
phepin-3-amine (55 µL, 0.11 mmol) was added. After 45 min, the
reaction mixture was cooled to –20 °C and mCPBA (103 mg,
0.255 mmol) in DCM (2 mL) was added. The reaction mixture was
stirred at 0 °C for another 1 h and then diluted with DCM (25 mL),
washed with a satd. aq. NaHCO3 solution (10 mL), water
(2 ϫ 10 mL) and brine (10 mL). The organic layer was dried
(Na2SO4), filtered and the filtrate was concentrated in vacuo. The
residue was purified by silica gel column chromatography (hexa-
ne:ethyl acetate, 4:1, v/v Ǟ 1:1, v/v) to give 16 (110 mg, 83%) as
white foamy solid. Rf = 0.39 (hexane/ethyl acetate, 4:1, v/v). [α]2D4
2 H, 5-H, 5Ј-H), 3.77 (dd, J5Ј,6Јa = 4.9, J6Јa,6Јb = 16 Hz, 2 H, 6Јab
H), 3.67–3.58 (m, 2 H, 6ab-H), 3.53 (s, 3 H, OCH3), 2.62 (dd,
J2Sa,2Sb = 15.2, J2Sa,3S = 7.5 Hz, 1 H, 2Sa-H), 2.51 (dd, J2Sa,2Sb
-
=
15.0, J2Sa,3S = 6.4 Hz, 1 H, 2Sb-H), 2.15 (t, J = 7.5 Hz, 2 H), 1.53–
1.52 (m, 5 H), 1.31–1.29 [m, 1 H, CH(CH3)2], 1.28–1.20 (m, 33 H),
0.88 (t, J = 7.0 Hz, 6 H, 2CH3) ppm. 13C NMR (75 MHz, CDCl3):
δ = 174.34 (C=O), 170.11 (C=O), 164.21 (C=O), 156.31 (C=O),
155.87 (C=O), 138.92 (OCH2CH=CH2), 132.86 (OCH2CH=CH2),
128.86, 126.12 (OCH2CCl3), 103.32 (C-1Ј), 98.65 (C-1), 78.12 (H-
3), 76.19 (C-3Ј), 72.34 (C-4), 71.12 (PhCH), 68.30 (OCH2CCl3),
67.26 (C-5), 66.71 (C-5Ј), 64.23 (C-6ab), 63.19 (C-6Јab), 59.23 (C-
2Ј), 58.21 (C-2), 56.72 (OCH3), 38.23, 34.12, 32.10, 28.43, 23.19
(CH2 of lipid chains), 15.13 (terminal CH3 of lipid chain), 14.89
(terminal CH3 of lipid chain) ppm. HR-MALDI-ToF/MS: m/z: for
[M + Na]+ calcd. C60H88Cl6N2O20Na 1393.9021; found 1393.9182.
1
= +9.0 (c = 1.0, CHCl3). H NMR (500 MHz, CDCl3): δ = 7.38–
7.17 (m, 9 H, aromatic), 5.95–5.82 (m, 2 H, OCH2CH=CH2), 5.65
(d, J = 7.5 Hz, 1 H, NH), 5.44 (t, J = 9.5 Hz, 1 H, 4Ј-H), 5.32–5.23
(m, 4 H, OCH2CH=CH2), 5.12 [br. s, 1 H, 3(R) CH of dodecanoyl
moiety], 5.10 (s, 2 H, PhCH2), 5.08–5.05 (m, 2 H), 4.92 (t, J =
9.6 Hz, 1 H, 3Ј-H), 4.78 (d, J = 10.1 Hz, 1 H, 1Ј-H), 4.76 (d, J =
3.0 Hz, 1 H, 1-H), 4.71 (t, J = 9.8 Hz, 1 H, 4-H), 4.63–4.58 (m, 8
H, 2ϫ OCH2CH=CH2, OCH2CCl3), 4.10–4.03 (m, 2 H, 2-H, 2Ј-
H), 3.93–3.89 (m, 2 H, 5-H, 5Ј-H), 3.86–3.83 (m, 2 H, 6Јab-H),
3.73–3.70 (m, 2 H, 6ab-H), 3.39 (s, 3 H, OCH3), 2.68 (dd, J2Sa,2Sb
= 15.2, J2Sa,3S = 7.5 Hz, 1 H, 2Sa-H), 2.48 (dd, J2Sa,2Sb = 15.0,
J2Sa,3S = 6.4 Hz, 1 H, 2Sb-H), 2.26 (t, J = 7.5 Hz, 2 H), 1.58–1.42
(m, 5 H), 1.28–1.25 (m, 33 H), 0.87 (t, J = 5.9 Hz, 6 H, 2CH3)
ppm. 13C NMR (gHSQC, CDCl3): δ = 133.16 (OCH2CH=CH2),
120.41, 119.36 (OCH2CCl3), 98.93 (C-1Ј), 97.05 (C-1), 76.33 (C-3),
74.03 (OCH2CH=CH2), 71.92 (C-4), 69.01 (OCH2CCl3), 67.40 (C-
Methyl 6Ј-O-Benzyl-2Ј-[(2,2,2-trichloromethoxy)carbonylamino]-3Ј-
O-[{(R)-3-(dodecanoyloxy)tetradecanoyl-2Ј-deoxy-β-D-gluco-
pyranosyl}]-2-(2,2,2-trichloromethoxy carbonylamino)-3,4-O-al-
lyloxycarbonyl-2-deoxy-α-D-glucopyranoside (15): A mixture of di-
saccharide 14 (212 mg, 0.154 mmol) and molecular sieves (4 Å,
200 mg) in DCM (3.0 mL) was placed under Ar and then cooled
to –78 °C. TfOH (27.5 µL, 0.471 mmol) and triethylsilane (62 µL,
0.554 mmol) were added and the reaction mixture was stirred at
–78 °C for 1 h. The reaction was quenched by the addition of tri-
ethylamine (50 µL) and methanol (0.2 mL). Next, the mixture was
diluted with DCM (35 mL), and washed with satd. aq. NaHCO3
solution (10 mL), water (2ϫ10 mL) and brine (10 mL). The or-
ganic layer was dried (Na2SO4), filtered and the filtrate was concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (hexane/ethyl acetate, 9:1, v/vǞ 4:1, v/v) to yield
the disaccharide 15 (148 mg, 69%) as colorless syrup. Rf = 0.38
6ab), 65.31 (C-5Ј), 64.29 (C-6Јab), 58.83 (C-2Ј), 56.24 (C-2), 39.33,
38.04, 36.75, 27.65, 24.30 (CH2 of lipid chains), 15.22 (terminal
CH3 of lipid chain), 14.98 (terminal CH3 of lipid chain) ppm. HR-
MALDI-ToF/MS: m/z: for [M + Na]+ calcd. C68H97Cl6N2O23P Na
1574.4321; found 1574.6469.
Methyl [6Ј-O-Benzyl-3Ј-O-(R)-3-(dodecanoyloxy)tetradecanoyl-4-O-
(1,5-dihydro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-3-yl)-2Ј-
[(R)-3-(dodecanoyloxy)tetradecanoylamino-{2Ј-deoxy-β-D-
glucopyranosyl}]-2-(R)-3-(dodecanoyloxy)tetradecanoylamino-3,4-
1
(hexane/ethyl acetate, 4:1, v/v). [α]2D4 = +6.1 (c = 1.0, CHCl3). H
NMR (500 MHz, CDCl3): δ = 7.43–7.31 (m, 5 H, aromatic), 5.88–
5.83 (m, 2 H, OCH2CH=CH2), 5.58 (d, J = 10.2 Hz, 1 H, NH),
5.32 (s, 2 H, PhCH2), 5.30–5.22 (m, 4 H, OCH2CH=CH2), 5.13 O-allyloxycarbonyl-2-deoxy-α-D-glucopyranoside (17): A solution of
[br. s, 1 H, 3(R) CH of dodecanoyl moiety], 5.12 (t, J = 9.1 Hz, 1
H, 3Ј-H), 5.11 (t, 1 H, 3-H merged with 3Ј-H), 5.02 (t, J = 10.1 Hz,
disaccharide 16 (105 mg, 0.029 mmol) in DCM (2 mL) was placed
under Ar, and Zn dust (134 mg, 0.868 mmol) and acetic acid
1 H, 4-H), 4.98 (t, J = 9.1 Hz, 1 H, 4Ј-H), 4.84 (d, J = 9.9 Hz, 1 (0.05 mL) were added. After stirring at room temperature for 1.5 h,
H, 1Ј-H), 4.75 (d, J = 3.5 Hz, 1 H, 1-H), 4.66–4.58 (m, 8 H, 2ϫ
OCH2CH=CH2, OCH2CCl3), 4.42 (d, J = 8.0 Hz, 1 H, NH), 4.10–
4.02 (m, 2 H, 2-H, 2Ј-H), 3.81–3.75 (m, 2 H, 5-H, 5Ј-H), 3.67 (dd,
the reaction mixture was diluted with DCM (20 mL), and filtered
through a bed of Celite. The filtrate was concentrated and the resi-
due was co-evaporated three times with toluene, and then dried in
vacuo. The removal of the Troc protecting group was confirmed by
MALDI-TOF MS analysis. The crude free amine (80 mg) was di-
luted in dry DCM (1.5 mL) followed by the addition of EDC
(50.9 mg, 0.027 mmol) and (R)-3-dodecanoyltetradecanoic acid
(85.2 mg, 0.02 mmol) in DCM (2 mL) at room temperature under
J5Ј,6Јa = 4.9, J6Јa,6Јb = 16 Hz, 2 H, 6Јab-H), 3.57–3.52 (m, 2 H, 6ab
H), 3.39 (s, 3 H, OCH3), 2.59 (dd, J2Sa,2Sb = 15.2, J2Sa,3S = 7.5 Hz,
1 H, 2Sa-H), 2.48 (dd, J2Sa,2Sb = 15.0, J2Sa,3S = 6.4 Hz, 1 H, 2Sb
-
-
H), 2.27 (t, J = 7.5 Hz, 2 H), 1.59–1.55 (m, 5 H), 1.31–1.25 (m, 33
H), 0.88 (t, J = 5.9 Hz, 6 H, 2CH3) ppm. 13C NMR (gHSQC,
CDCl3): δ = 129.56 (OCH2CH=CH2), 119.81, 118.54 (OCH2CCl3), Ar. After 24 h of stirring at room temperature, the reaction mixture
98.08 (C-1Ј), 96.75 (C-1), 75.32 (C-3), 74.19 (C-3Ј), 73.81 was diluted with DCM (30 mL) and washed with satd. aq.
(OCH2CH=CH2), 71.84 (C-4), 69.81 (OCH2CCl3), 66.36 (C-6ab),
65.71 (C-5Ј), 64.09 (C-6Јab), 59.13 (C-2Ј), 56.06 (C-2), 39.13, 38.82,
36.10, 27.83, 21.19 (CH2 of lipid chains), 15.13 (terminal CH3 of
lipid chain), 14.89 (terminal CH3 of lipid chain) ppm. HR-
NaHCO3 solution (3 ϫ 10 mL), water (2 ϫ 10 mL) and brine
(10 mL). The organic layer was dried (Na2SO4), filtered and the
filtrate was concentrated in vacuo. The residue was purified by sil-
ica gel column chromatography (hexane/ethyl acetate, 5:1, v/v Ǟ
Eur. J. Org. Chem. 2010, 80–91
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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87

G.-J. Boons et al.
FULL PAPER
2:1, v/v) to give 17 (61 mg, 45%) as a white foamy solid. Rf = 0.41
7.4 Hz, 1 H, 2Sa-H), 2.70 (dd, J2Sa,2Sb = 16.8, J2Sa,3S = 7.0 Hz, 1 H,
2Sb-H), 2.65 (dd, J1 = 15.8, J2 = 8.4 Hz, 2 H), 1.77–1.63 (m, 12 H),
(hexane/ethyl acetate, 4:1, v/v). [α]2D4 = –7.0 (c = 1.0, CHCl3). 1H
NMR (500 MHz, CDCl3): δ = 7.39–7.19 (m, 9 H, aromatic), 6.02 1.36–1.21 (m, 114 H), 1.16 (t, J = 7.1 Hz, 18 H, 6CH3) ppm. HR-
(d, J = 8.0 Hz, 1 H, NH), 5.92 (d, J = 9.9 Hz, 1 H, NH), 5.88–5.79 MALDI-ToF/MS: m/z: for [M + Na]+ calcd. C91H171N2O21P Na
(m, 2 H, OCH2CH=CH2), 5.32 (t, J = 10.0 Hz, 1 H, 4Ј-H), 5.25–
5.13 (m, 4 H, OCH2CH=CH2), 5.22 [br. s, 1 H, 3(R) CH of dodeca-
noyl moiety partially merged with OCH2CH=CH2], 5.03 (s, 2 H,
PhCH2), 5.00–4.89 (m, 2 H, 3-H, 3Ј-H), 4.89 (t, J = 10.6 Hz, 1 H,
4-H), 4.68 (d, J = 10.6 Hz, 1 H, 1Ј-H), 4.64 (d, J = 3.1 Hz, 1 H, 1-
H), 4.55–4.51 (m, 4 H, OCH2CH=CH2), 4.31–4.24 (m, 2 H, 2-H,
2Ј-H), 3.82–3.76 (m, 2 H, 6Јab-H), 3.69–3.58 (m, 2 H, 6ab-H), 3.47–
1682.3060; found 1682.1757.
Dimethylthexylsilyl 4Ј,6Ј-O-Benzylidene-2Ј-[(2,2,2-trichlorometh-
oxy)carbonylamino]-3Ј-O-[{(R)-3-(dodecanoyloxy)tetradecanoyl-2Ј-
deoxy-β-
D
-glucopyranosyl}]-2-[(2,2,2-trichloromethoxy)carbonyl-
-glucopyranoside (19): A
amino]-3-O-allyloxycarbonyl-2-deoxy-α-
D
mixture of glycosyl acceptor 3 (210 mg, 0.361 mmol), trichloroacet-
amidate donor 5 (300 mg, 0.301 mmol) and molecular sieves (4 Å,
650 mg) in DCM (2.5 mL) was placed under Ar and cooled to
–40 °C. TfOH (1.34 µL, 0.015 mmol, in 0.1 mL DCM) was added
and the resulting reaction mixture was stirred for 15 min until the
temperature had reached 0 °C. The reaction was quenched by the
addition of triethylamine (30 µL). Next, the mixture was diluted
with DCM (35 mL), and washed with satd. aq. NaHCO3 solution
(10 mL), water (2ϫ10 mL) and brine (10 mL). The organic layer
was dried (Na2SO4), filtered and the filtrate was concentrated in
vacuo. The residue was purified by silica gel column chromatog-
raphy (hexane/ethyl acetate, 9:1, v/v Ǟ, 4:1, v/v) to yield the disac-
charide 19 (349 mg, 82%) as a white foamy solid. Rf = 0.44 (hex-
ane/ethyl acetate, 4:1, v/v). [α]2D4 = –11.6 (c = 1.0, CHCl3). 1H NMR
(500 MHz, CDCl3): δ = 7.41–7.33 (m, 2 H, aromatic), 5.95–5.86
(m, 2 H, OCH2CH=CH2), 5.46 (d, J = 8.2 Hz, 1 H, NH), 5.42 (s,
1 H, PhCH), 5.38–5.22 (m, 4 H, OCH2CH=CH2), 5.20 [br. s, 1 H,
3(R) CH of dodecanoyl moiety], 5.13–5.05 (m, 2 H, 3-H, 3Ј-H),
4.79 (dist. t, J = 10.1 Hz, 1 H, 4-H), 4.71–4.60 (m, 3 H, 1-H, 1Ј-H,
4Ј-H, all partially merged), 4.58–4.51 (m, 8 H, 2ϫ OCH2CH=CH2,
OCH2CCl3), 4.28–4.24 (m, 1 H), 3.90 (d, J6b,6a = 15.7 Hz, 1 H, 6b-
3.38 (m, 2 H, 5-H, 5Ј-H), 3.28 (s, 3 H, OCH3), 2.37 (dd, J2Sa,2Sb
=
14.2, J2Sa,3S = 8.5 Hz, 1 H, 2Sa-H), 2.30 (dd, J2Sa,2Sb = 15.0, J2Sa,3S
= 6.4 Hz, 1 H, 2Sb-H), 1.73–1.52 (m, 8 H), 1.33–1.18 (m, 122 H),
0.91 (t, J = 7.0 Hz, 18 H, 6CH3) ppm. HR-MALDI-ToF/MS: m/z:
for [M + Na]+ calcd. C114H191N2O25P Na 2042.3507; found
2042.1571.
Methyl [6Ј-O-Benzyl-3Ј-O-(R)-3-(dodecanoyloxy)tetradecanoyl-4-O-
(1,5-dihydro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-3-yl)-2Ј-
[(R)-3-(dodecanoyloxy)tetradecanoylamino-{2Ј-deoxy-β-D-gluco-
pyranosyl}]-2-(R)-3-(dodecanoyloxy)tetradecanoylamino-2-deoxy-α-
D
-glucopyranoside (18): A mixture of butylamine (7 µL,
0.065 mmol) and formic acid (4 µL, 0.030 mmol) was added to a
solution of compound 17 (40 mg, 0.0198 mmol) in THF (1.5 mL)
under Ar. Tetrakis(triphenylphosphane)palladium(0) (8 mg) was
added to the stirring solution. After 1 h, the reaction mixture was
concentrated and the residue was purified by flash silica gel column
chromatography (hexane/ethyl acetate, 3:1, v/v Ǟ 1:1, v/v) to afford
18 as an amorphous solid (24 mg, 66%). Rf = 0.24 (hexane/ethyl
acetate, 1:1, v/v). [α]2D4 = –18.0 (c = 1.0, CHCl3). 1H NMR
(500 MHz, CDCl3): δ = 7.38–7.17 (m, 9 H, aromatic), 6.26 (d, J = H), 3.71 (t, J6a,6b = 16.4 Hz, 1 H, 6a-H), 3.64–3.42 (m, 6 H, 6Јab
-
6.5 Hz, 1 H, NH), 5.95 (d, J = 8.5 Hz, 1 H, NH), 5.35 (t, J =
H, 2-H, 2Ј-H, 5-H, 5Ј-H), 3.94–3.92 (m, 2 H, 5-H, 5Ј-H), 2.52 (dd,
9.5 Hz, 1 H, 4Ј-H), 5.15–5.11 (m, 1 H), 5.09–4.92 [m, 4 H, 3-H, 3Ј- J2Sa,2Sb = 14.2, J2Sa,3S = 7.8 Hz, 1 H, 2Sa-H), 2.46 (dd, J2Sa,2Sb
=
H, 4-H, 3(R) CH of dodecanoyl moiety], 4.57 (d, J = 10.8 Hz, 1
H, 1Ј-H), 4.51 (d, J = 3.4 Hz, 1 H, 1-H), 4.05–4.3.94 (m, 2 H, 2- (br. s, 5 H), 1.37–1.29 (m, 33 H), 0.94–0.85 (m, 12 H, 4 CH3), 0.06
H, 2Ј-H), 3.79–3.49 (m, 6 H, 6Јab-H, 6ab-H, 5-H, 5Ј-H), 3.26 (s, 3
(s, 3 H, SiCH3), 0.0 (s, 3 H, SiCH3) ppm. 13C NMR (gHSQC,
14.4, J2Sa,3S = 7.4 Hz, 1 H, 2Sb-H), 2.23 (t, J = 7.4 Hz, 2 H), 1.54
H, OCH3), 2.67 (dd, J2Sa,2Sb = 15.2, J2Sa,3S = 8.0 Hz, 1 H, 2Sa-H), CDCl3): δ = 131.72 (OCH2CH=CH2), 122.86, 119.02 (OCH2CCl3),
2.61 (dd, J2Sa,2Sb = 15.8, J2Sa,3S = 7.4 Hz, 1 H, 2Sb-H), 2.26 (dd, J 98.42 (C-1Ј), 96.65 (C-1), 77.02 (C-3), 76.19 (C-3Ј), 74.91
= 16.8, J = 7.0 Hz, 4 H), 1.74–1.52 (m, 8 H), 1.23–1.18 (m, 122
(OCH2CH=CH2), 72.01 (PhCH), 71.34 (C-4), 68.81 (OCH2CCl3),
H), 0.80 (t, J = 7.8 Hz, 18 H, 6CH3) ppm. HR-MALDI-ToF/MS: 68.26 (C-6ab), 66.70 (C-5Ј), 63.19 (C-6Јab), 58.13 (C-2Ј), 58.06 (C-
m/z: for [M + Na]+ calcd. C106H183N2O21P Na 1874.5618; found
1874.1497.
2), 38.23, 38.12, 35.10, 28.83, 22.19 (CH2 of lipid chains), 14.13
(terminal CH3 of lipid chain), 13.89 (terminal CH3 of lipid chain)
ppm. HR-MALDI-ToF/MS: m/z: for [M + Na]+ calcd.
C58H98Cl6N2O18 SiNa 1375.2048; found 1375.3142.
Methyl 3Ј-O-{(R)-3-(Dodecanoyloxy)tetradecanoyl-4-O-phosphono-
2Ј-[(R)-3-(dodecanoyloxy)tetradecanoyl]-2Ј-deoxy-β-D-gluco-
pyranosyl}-2-[(R)-3-(dodecanoyloxy)tetradecanoylamino]-2-deoxy-α-
Dimethylthexylsilyl 4,6Ј-O-Benzyl-2Ј-[(2,2,2-trichloromethoxy)-
D-glucopyranoside (2): A mixture of 18 (12 mg, 0.0064 mmol) and carbonylamino]-3Ј-O-[{(R)-3-(dodecanoyloxy)tetradecanoyl-2Ј-de-
Pd-black (10% wt. Pd on charcoal, Degussa, 15.0 mg) was sus-
pended in anhydrous THF (1.5 mL) and tBuOH (1.5 mL). The
mixture was placed under H2 (1 atm) and the resulting reaction
mixture was stirred at room temperature for 10 h, after which it
was neutralized with triethylamine (0.1 mL). The catalyst was re-
moved by filtration through a bed of Celite, which was washed
with THF (2ϫ2 mL). The combined filtrates were concentrated in
vacuo to afford 2 as a colorless film (6.8 mg, 68%). [α]2D4 = –31.0
oxy-β-
2Ј-[(2,2,2-trichloromethoxy)carbonylamino]-3Ј-O-{(R)-3-(dodeca-
noyloxy)tetradecanoyl-2Ј-deoxy-β- -glucopyranosyl}]-2-[(2,2,2-tri-
chloromethoxy)carbonylamino]-3,4-O-allyloxycarbonyl-2-deoxy-α-
-glucopyranoside (21): Disaccharide 19 (350 mg, 0.247 mmol) in
DCM (2.5 mL) was placed under an atmosphere of Ar and charged
with N,N,NЈ,NЈ-tetramethylethylenediamine (0.18 mL, 1.23 mmol).
The reaction mixture was cooled to 0 °C and allyloxycarbonyl chlo-
ride (0.13 mL, 1.23 mmol) was added slowly. The progress of reac-
D-glucopyranosyl}]-2-(2,2,2-dimethylthexylsilyl)-[6Ј-O-benzyl-
D
D
1
(c = 1.0, CHCl3). H NMR (500 MHz, CDCl3/CD3OD, 1:1, v/v):
δ = 5.29–5.27 (m, 1 H, 3-H), 4.78 (1 H, 1-H, merged with CD3OD tion was monitored by TLC and MALDI-TOF MS. After 12 h of
peak), 4.74 (d, J = 9.0 Hz, 1 H, 1Ј-H), 4.41 (dd, J1 = 17.8, J2
=
stirring at room temperature, the reaction mixture was diluted with
DCM (30 mL), washed with satd. aq. NaHCO3 solution (10 mL),
water (2ϫ10 mL) and brine (10 mL). The organic layer was dried
(Na2SO4), filtered and the filtrate was concentrated in vacuo. The
10.1 Hz, 1 H, 4-H), 4.29 (dist. t, J = 9.0 Hz, 1 H, 4Ј-H), 4.14 (dd,
J5,6a = 2.9, J6a,6b = 14.1 Hz, 1 H, 6a-H), 4.07–4.03 (m, 2 H, 2-H,
2Ј-H), 3.92 (d, J6b,6a = 15.7 Hz, 1 H, 6b-H), 3.80 (dist. quartet,
J6Јa,6Јb = 16.4 Hz, 1 H, 6Јa-H), 3.76 (t, J = 10.2 Hz, 1 H, 3Ј-H), structure of the compound was confirmed by MS and the com-
3.49 (d, J6Јb,6Јa = 16.7 Hz, 1 H, 6Јb-H), 3.44–3.41 (m, 2 H, 5-H, 5Ј-
H), 3.32 (br. s, 3 H, OCH3), 2.82 (dd, J2Sa,2Sb = 15.3, J2Sa,3S
pound was used in the next step without further purification. A
mixture of the crude 20 (270 mg) and molecular sieves (4 Å,
=
88
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© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 80–91

Synthetic Monophosphoryl Lipid A
270 mg) in DCM (3.0 mL) was placed under an atmosphere of Ar
and cooled to –78 °C. TfOH (32 µL) and triethylsilane (72 µL,
0.091 mmol) were added to the reaction mixture. The reaction mix-
ture was stirred at –78 °C for 1 h after which the reaction was
quenched by the addition of triethylamine (50 µL) and methanol
(0.2 mL). Next, the mixture was diluted with DCM (35 mL), and
washed with satd. aq. NaHCO3 solution (10 mL), water
(2 ϫ 10 mL) and brine (10 mL). The organic layer was dried
(Na2SO4), filtered and the filtrate was concentrated in vacuo. The
residue was purified by silica gel column chromatography (hexane/
ethyl acetate, 9:1, v/v Ǟ 3:1, v/v) to yield the disaccharide 21
(168 mg, 62%) as a colorless thick syrup. Rf = 0.32 (hexane/ethyl
acetate, 4:1, v/v). [α]2D4 = –10.6 (c = 1.0, CHCl3). 1H NMR
(500 MHz, CDCl3): δ = 7.32–7.18 (m, 5 H, aromatic), 5.78–5.71
(m, 2 H, OCH2CH=CH2), 5.31 (d, J = 9.2 Hz, 1 H, NH), 5.19–5.10
(m, 4 H, OCH2CH=CH2), 5.05 [br. s, 1 H, 3(R) CH of dodecanoyl
moiety], 4.99–4.94 (m, 2 H, 3-H, 3Ј-H), 4.91 (d, J = 10.0 Hz, 1 H,
1Ј-H), 4.88 (br. s, 1 H, 1-H), 4.73 (t, J = 9.1 Hz, 1 H, 4-H), 4.71–
4.69 (m, 1 H, 4Ј-H, partially merged with OCH2CH=CH2), 4.53–
2Sb-H), 2.41 (t, J = 7.3 Hz, 2 H), 1.79–1.62 (m, 6 H), 1.10–0.88 (m,
33 H), 0.66–0.53 (m, 18 H, 6 CH3), 0.05 (s, 3 H, SiCH3), 0.0 (s, 3
H, SiCH3) ppm. 13C NMR (gHSQC, CDCl3): δ = 131.20
(OCH2CH=CH2), 120.09, 118.87 (OCH2CCl3), 98.71 (C-1Ј), 97.64
(C-1), 88.71 (C-3), 76.75 (OCH2CH=CH2), 74.64 (C-4Ј), 68.06 (C-
6
abЈ), 66.52 (C-6Јab), 63.31.13 (C-2Ј), 63.01 (C-2), 34.03, 32.32,
30.91, 28.32, 20.11 (CH2 of lipid chains), 15.13 (terminal CH3 of
lipid chain), 12.61 (terminal CH3 of lipid chain) ppm. HR-
MALDI-ToF/MS: m/z: for [M + Na]+ calcd. C75H113Cl6N2O23
PSiNa 1705. 4766; found 1705.3152.
Dimethylthexylsilyl [6Ј-O-Benzyl-3Ј-O-(R)-3-(dodecanoyloxy)tetra-
decanoyl-4-O-(1,5-dihydro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphe-
pin-3-yl)-2Ј-(R)-3-(dodecanoyloxy)tetradecanoylamino-{2Ј-deoxy-β-
D-glucopyranosyl}]-2-(R)-3-(dodecanoyloxy)tetradecanoylamino-3,4-
O-allyloxycarbonyl-2-deoxy-α- -glucopyranoside (23): Disaccharide
D
22 (80 mg, 0.0475 mmol) in DCM (2.5 mL) was placed under an
atmosphere of Ar, and Zn dust (90 mg, 1.426 mmol) and acetic acid
(0.05 mL) were added. After stirring at room temperature for 1.5 h,
the reaction mixture was diluted with DCM (20 mL), and filtered
through a bed of Celite. The filtrate was concentrated and the resi-
due co-evaporated three times with toluene, and then dried in
vacuo. The deprotection of the Troc group was confirmed by
MALDI-TOF MS analysis. The crude free amine (63 mg,
0.0473 mmol), dissolved in DCM (1.5 mL) and triethylamine
(25 µL), was added to a stirring solution of EDC (37 mg,
0.189 mmol) and (R)-3-dodecanoyltetradecanoic acid (60.5 mg,
0.142 mmol) in DCM (2 mL) at room temperature under an atmo-
sphere of Ar. After stirring for 24 h at room temperature, the reac-
tion mixture was diluted with DCM (30 mL) and washed with satd.
aq. NaHCO3 solution (3ϫ10 mL), water (2ϫ10 mL) and brine
(10 mL). The organic layer was dried (Na2SO4), filtered and the
filtrate was concentrated in vacuo. The residue was purified by flash
silica gel column chromatography (hexane/ethyl acetate, 4:1, v/vǞ
2:1, v/v) to give 23 (49 mg, 48%) as a white foam. Rf = 0.38 (hex-
ane/ethyl acetate, 3:1, v/v). [α]2D4 = 21.6 (c = 1.0, CHCl3). 1H NMR
(500 MHz, CDCl3): δ = 7.39–7.16 (m, 9 H, aromatic), 5.89–5.80
(m, 2 H, OCH2CH=CH2), 5.66 (d, J = 6.7 Hz, 1 H, NH), 5.48 (t,
J = 8.2 Hz, 1 H, 4Ј-H), 5.27–5.21 (m, 4 H, OCH2CH=CH2), 5.17–
4.89 [m, 4 H, 3(R) CH of dodecanoyl moiety, 4-H, PhCH2], 4.80–
4.76 (m, 2 H, 3-H, 3Ј-H), 4.68 (d, J = 8.8 Hz, 1 H, 1Ј-H), 4.66 (br.
s, 1 H, 1-H), 4.55–4.43 (m, 2 H, 3-H, 3Ј-H), 3.85 (d, J6b,6a = 9.7 Hz,
1 H, 6b-H), 3.80 (d, J6Јb,6Јa = 8.6 Hz, 1 H, 6Јb-H), 3.64 –3.57 (m, 4
H, 6a-H, 6Јa-H, 2-H, 2Ј-H), 3.41–3.27 (m, 2 H, 5-H, 5Ј-H), 2.46–
2.51 (m, 2 H, 2Sa-H, 2Sb-H), 1.63–1.10 (m, 130 H), 1.79–1.62 (m,
6 H), 1.10–0.88 (m, 33 H), 0.71–0.63 (m, 18 H, 6 CH3), 0.05 (s, 3
H, SiCH3), 0.0 (s, 3 H, SiCH3) ppm. HR-MALDI-ToF/MS: m/z:
for [M + Na]+ calcd. C121H207N2O25 PSiNa 2171.9959; found
2171.3125.
4.45 (m, 8 H, 2ϫ OCH2CH=CH2, OCH2CCl3), 3.98 (d, J6b,6a
=
15.7 Hz, 1 H, 6b-H), 3.68–3.52 (m, 2 H, 2-H, 2Ј-H), 3.55–3.49 (m,
3 H, 6a-H, 6Јab-H), 3.43–3.37 (m, 2 H, 5-H, 5Ј-H), 2.43 (dd, J2Sa,2Sb
= 16.2, J2Sa,3S = 6.8 Hz, 1 H, 2Sa-H), 2.39 (dd, J2Sa,2Sb = 14.7,
J2Sa,3S = 7.9 Hz, 1 H, 2Sb-H), 2.14 (t, J = 7.3 Hz, 2 H), 1.53–1.55
(m, 6 H), 1.12–0.88 (m, 33 H), 0.69–0.58 (m, 12 H, 4 CH3), 0.04
(s, 3 H, SiCH3), 0.0 (s, 3 H, SiCH3) ppm. 13C NMR (gHSQC,
CDCl3): δ = 133.72 (OCH2CH=CH2), 119.06, 115.87 (OCH2CCl3),
96.22 (C-1Ј), 95.01 (C-1), 75.02 (C-3), 74.71 (C-3Ј), 74.01
(OCH2CH=CH2), 69.30 (C-4), 63.70 (C-5Ј), 63.19 (C-6Јab), 58.13
(C-2Ј), 58.16 (C-2), 36.03, 35.12, 30.10, 26.83, 20.85 (CH2 of lipid
chains), 15.23 (terminal CH3 of lipid chain), 14.21 (terminal CH3
of lipid chain) ppm. HR-MALDI-ToF/MS: m/z: for [M + Na]+
calcd. C67H106Cl6N2O20 SiNa 1521.3476; found 1521.4486.
Dimethylthexylsilyl [6Ј-O-Benzyl-2Ј-deoxy-4-O-(1,5-dihydro-3-oxo-
3λ5-3H-2,4,3-benzodioxaphosphepin-3-yl)-2Ј-[(2,2,2-trichlorometh-
oxy)carbonylamino]-3Ј-O-{(R)-3-(dodecanoyloxy)tetradecanoyl]-2Ј-
deoxy-β-
D
-glucopyranosyl}]-2-[(2,2,2-trichloromethoxy)carbonyl-
-glucopyranoside (22):
amino]-3,4-O-allyloxycarbonyl-2-deoxy-α-
D
Disaccharide 21 (130 mg, 0.0866 mmol) in DCM (2.5 mL) was
placed under an atmosphere of Ar and 3% tetrazole solution in
acetonitrile (0.32 µL, 0.346 mmol) and N,N-diethyl-1,5-dihydro-
2,4,3-benzodioxaphosphepin-3-amine (74 µL, 0. 346 mmol) were
added. After 45 min, the reaction mixture was cooled to –20 °C
and mCPBA (104 mg, 0.606 mmol) in DCM (2 mL) was added.
The reaction mixture was stirred at 0 °C for another 1 h and then
diluted with DCM (30 mL), washed with satd. aq. NaHCO3 solu-
tion (10 mL), water (2ϫ10 mL) and brine (10 mL). The organic
layer was dried (Na2SO4), filtered and the filtrate was concentrated
in vacuo. The residue was purified by flash silica gel column
chromatography (hexane/ethyl acetate, 4:1, v/v Ǟ 1:1, v/v) to give
22 (122 mg, 84%) as a white foam. Rf = 0.35 (hexane/ethyl acetate,
3:1, v/v). [α]2D4 = –23.6 (c = 1.0, CHCl3). 1H NMR (500 MHz,
CDCl3): δ = 7.67 (d, J = 9.8 Hz, 2 H, aromatic), 7.47 (t, J =
10.2 Hz, 2 H, aromatic), 7.36–7.16 (m, 5 H, aromatic), 5.88–5.77
(m, 2 H, OCH2CH=CH2), 5.67 (d, J = 6.7 Hz, 1 H, NH), 5.58 (t,
Dimethylthexylsilyl [6Ј-O-Benzyl-3Ј-O-(R)-3-(dodecanoyloxy)tetra-
decanoyl-4-O-(1,5-dihydro-3-oxo-3λ5-3H-2,4,3-benzodioxaphosphe-
pin-3-yl)-2Ј-(R)-3-(dodecanoyloxy)tetradecanoyl-{2Ј-deoxy-β-
glucopyranosyl}]-2-(R)-3-(dodecanoyloxy)tetradecanoylamino-2-de-
oxy-α- -glucopyranoside (24): A mixture of butylamine (7.4 µL,
D-
D
0.0744 mmol) and formic acid (2.8 µL, 0.0744 mmol) was added to
a solution of compound 23 (40 mg, 0.0186 mmol) in THF (1.5 mL)
J = 8.8 Hz, 1 H, 4Ј-H), 5.35–5.21 (m, 4 H, OCH2CH=CH2), 5.21– under Ar. Tetrakis(triphenylphosphane) palladium(0) (8 mg) was
4.88 [m, 4 H, 3(R) CH of dodecanoyl moiety, 4-H, PhCH2], 4.99– added to the resulting solution and stirring was continued for 1 h.
4.94 (m, 2 H, 3-H, 3Ј-H), 4.91 (d, J = 10.0 Hz, 1 H, 1Ј-H), 4.88 The reaction mixture was concentrated and the residue was purified
(br. s, 1 H, 1-H), 4.61–4.55 (m, 2 H, 3-H, 3Ј-H), 4.52–4.48 (m, 4 H,
OCH2CCl3), 3.97 (d, J6b,6a = 10.7 Hz, 1 H, 6b-H), 3.88 (d, J6Јb,6Јa
by flash silica gel column chromatography (hexane/ethyl acetate,
=
3:1, v/v Ǟ 1:1, v/v) to afford 24 as a sticky solid (25.7 mg, 70%).
9.8 Hz, 1 H, 6Јb-H), 3.73 –3.62 (m, 4 H, 6a-H, 6Јa-H, 2-H, 2Ј-H), Rf = 0.28 (hexane/ethyl acetate, 2:1, v/v). [α]2D4 = 19.0 (c = 1.0,
3.46–3.37 (m, 2 H, 5-H, 5Ј-H), 2.86 (dd, J2Sa,2Sb = 16.2, J2Sa,3S
=
CHCl3). 1H NMR (500 MHz, CDCl3): δ = 7.43–7.18 (m, 9 H, aro-
matic), 5.87 (br. s, 1 H, NH), 5.53 (t, J = 8.6 Hz, 1 H, 4Ј-H), 5.33–
6.8 Hz, 1 H, 2Sa-H), 2.85 (dd, J2Sa,2Sb = 14.7, J2Sa,3S = 7.9 Hz, 1 H,
Eur. J. Org. Chem. 2010, 80–91
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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89

G.-J. Boons et al.
FULL PAPER
5.30 (m, 1 H, 4-H), 5.27–5.00 [m, 3 H, 3(R) CH of dodecanoyl
formed in 96-well MaxiSorp plates (Nunc). Cytokine DuoSet EL-
moiety, PhCH2], 4.80–4.66 (m, 4 H, 1-H, 1Ј-H, 3-H, 3Ј-H), 4.15 (d, ISA Development Kits (R&D Systems) were used for the cytokine
J6b,6a = 9.1 Hz, 1 H, 6b-H), 3.88 (d, J6Јb,6Јa = 8.6 Hz, 1 H, 6Јb-H), quantification of TNF-α, IP-10 and IL-6 according to the manu-
3.74–3.67 (m, 4 H, 6a-H, 6Јa-H, 2-H, 2Ј-H), 3.51–3.29 (m, 2 H, 5- facturer’s instructions. The absorbance was measured at 450 nm
H, 5Ј-H), 2.76–2.61 (m, 2 H, 2Sa-H, 2Sb-H), 1.63–1.10 (m, 130 H),
with wavelength correction set to 540 nm using a microplate reader
2.24 (t, J = 9.0 Hz, 2 H), 1.78–1.70 (m, 6 H), 1.42–1.21 (m, 130 (BMG Labtech). Concentrations of IFN-β in culture supernatants
H), 0.71–0.63 (m, 18 H, 6 CH3), 0.06 (s, 3 H, SiCH3), 0.0 (s, 3 were determined as follows. ELISA MaxiSorp plates were coated
H, SiCH3) ppm. HR-MALDI-ToF/MS: m/z: for [M + Na]+ calcd.
C113H199N2O21 PSiNa 2003.8492; found 2003.5231.
with rabbit polyclonal antibody against mouse IFN-β (PBL Bio-
medical Laboratories). IFN-β in standards and samples was al-
lowed to bind to the immobilized antibody. Rat anti-mouse IFN-β
antibody (USBiological) was then added, producing an antibody-
antigen-antibody “sandwich”. Next, horseradish peroxidase (HRP)
conjugated goat anti-rat IgG (H+L) antibody (Pierce) and a chro-
mogenic substrate for HRP 3,3Ј,5,5Ј-tetramethylbenzidine (TMB;
Pierce) were added. After the reaction was stopped, the absorbance
was measured at 450 nm with wavelength correction set to 540 nm.
3Ј-O-(R)-3-(Dodecanoyloxy)tetradecanoyl-4-O-phosphono-2Ј-[(R)-3-
(dodecanoyloxy)tetradecanoyl]-2Ј-deoxy-β-
D-glucopyranosyl-2-[(R)-
3-(dodecanoyloxy)tetradecanoylamino]-2-deoxy-α-
D
-glucopyranose
(1): A mixture of 24 (24 mg, 0.0121 mmol) and HF/pyridine (18 µL,
0.726 mmol, 65–70%) in anhydrous THF (1.5 mL) was stirred at
room temperature for 10 h. The reaction mixture was quenched by
the addition of solid NaHCO3 (25 mg) and then diluted with DCM
(35 mL), filtered and the filtrate was concentrated in vacuo. The
residue was purified by flash silica gel column chromatography
(DCM/MeOH, 15:1, v/v Ǟ 10:1, v/v) to yield a lactol (14 mg, 68%)
as a colorless thick syrup. The lactol (14 mg, 0.007 mmol) was
added to a suspension of Pd-black (10% wt. Pd on charcoal, De-
gussa, 18.0 mg) in a mixture of anhydrous THF (1.5 mL) and
tBuOH (1.5 mL). The mixture was placed under H2 (1 atm) and
the resulting reaction mixture was stirred at room temperature for
10 h, after which it was neutralized with triethylamine (0.12 mL),
and the catalyst removed by filtration through a bed of Celite,
which was washed with THF (2ϫ2 mL). The combined filtrates
were concentrated in vacuo to afford 1 as a colorless flim (7.8 mg,
Data Analysis: Concentration-response data were analyzed using
nonlinear least-squares curve fitting in Prism (GraphPad Software,
Inc.). These data were fit with the following four parameter logistic
equation: Y = Emax/(1 + (EC50/X)Hill slope), where Y is the cytokine
response, X is the concentration of the stimulus, Emax is the maxi-
mum response and EC50 is the concentration of the stimulus pro-
ducing 50% stimulation. The Hill slope was set at 1 to be able to
compare the EC50 values of the different inducers. All values are
presented as the meansϮSD of triplicate measurements, with each
experiment being repeated three times.
Supporting Information (see also the footnote on the first page of
this article): NMR spectra synthetic compounds.
1
68%). [α]2D4 = 19.0 (c = 1.0, CHCl3). H NMR (500 MHz, CDCl3/
CD3OD, 1:1, v/v): δ = 5.34–5.08 (m, 5 H), 4.78–4.44 (m, 4 H, 4Ј-
H, 3-H, 1Ј-H, 1-H peaks are partially merged with CD3OD), 4.33
[br. s, 1 H, 3(R) CH of dodecanoyl moiety], 4.23–4.02 (m, 2 H, 4-
H, 3Ј-H), 3.82–3.31 (m, 6 H, 6ab-H, 6Јab-H, 2-H, 2Ј-H, 5-H, 5Ј-H),
2.78–2.51 (m, 2 H, 2Sa-H, 2Sb-H), 2.21 (br. s, 4 H), 1.73–1.09 (m,
130 H), 0.81–0.73 (m, 18 H, 6 CH3) ppm. HR-MALDI-ToF/MS:
m/z: for [M + Na]+ calcd. C90H169N2O21 PNa 1669.2794; found
1669.0132.
Acknowledgments
We thank Dr Yanghui Zhang for synthesizing the E. coli lipid A
and Dr Jidnyasa Gaekwad for performing the cell activation stud-
ies. This research was supported by the National Institute of Gene-
ral Medical Sciences (NIGMS) of the National Institutes of Health
(NIH) (R01GM061761) and the National Cancer Institute (NCI)
(R01CA088986). M. DeCastro was supported by a Carl Storm
Postdoctoral Fellowship - National Institutes of Health and
A.-B. M. Abdel-Aal El-Sayed’s stay in Dr Boons’ laboratory was
supported by Dr I. Toth (University of Queensland) and Francine
Kroesen, Arlo D. Harris and UQ-GSRTG scholarships.
Biological Experiments
Reagents for Biological Experiments: E. coli 055:B5 LPS was ob-
tained from List Biologicals and monophosphorylated (detoxified)
lipid A obtained by controlled hydrolysis of the LPS of S. minne-
sota (MPLA) was obtained from Avanti Polar Lipids Inc. The syn-
thesis of E. coli lipid A has been reported elsewhere[16] and was
reconstituted in PBS with DMSO (10%). The synthetic compounds
1 and 2 were reconstituted in PBS containing THF (10%). Final
concentrations of DMSO and THF in the biological experiments
never exceeded 0.5% to avoid toxic effects.
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Received: August 25, 2009
Published Online: November 18, 2009
Eur. J. Org. Chem. 2010, 80–91
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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91