Synthesis of a monophosphoryl derivative of Escherichia coli lipid A and Its efficient coupling to a tumor-associated carbohydrate antigen

Article abstract of DOI:10.1002/chem.200902153

Monophosphoryl lipid A is a safe and potent immunostimulant and vaccine adjuvant, which is potentially useful for the development of effective carbohydrate-based conjugate vaccines. This paper presents a convergent and efficient synthesis of a monophosphoryl derivative of E. coli lipid A that has an alkyne functionality at the reducing end, which is suitable for coupling with various molecules. The coupling of this derivative to an N-modified analogue of tumor-associated antigen GM3 through click chemistry is also presented.

Full text of DOI:10.1002/chem.200902153

FULL PAPER
DOI: 10.1002/chem.200902153
Synthesis of a Monophosphoryl Derivative of Escherichia coli Lipid A and
Its Efficient Coupling to a Tumor-Associated Carbohydrate Antigen
Shouchu Tang, Qianli Wang, and Zhongwu Guo*[a]
Abstract: Monophosphoryl lipid A is a
safe and potent immunostimulant and
vaccine adjuvant, which is potentially
useful for the development of effective
carbohydrate-based conjugate vaccines.
This paper presents a convergent and
efficient synthesis of a monophosphoryl
derivative of E. coli lipid A that has an
alkyne functionality at the reducing
end, which is suitable for coupling with
various molecules. The coupling of this
derivative to an N-modified analogue
of tumor-associated antigen GM3
through click chemistry is also present-
ed.
Keywords: cancer · carbohydrates ·
glycoconjugates · glycolipids · lipids
Introduction
useful immunostimulants, numerous lipid A derivatives have
been designed, synthesized, and biologically assayed.[7–10]
The structure–activity relationship studies of natural lipid A
and various synthetic analogues have shown that both the
number and lengths of acyl chains and the phosphorylation
state of lipid A have a significant impact on its endotoxicity.
It appears that the diphosphorylated hexaacyl form of
lipid A, such as Escherichia coli lipid A (Scheme 1), is opti-
mally recognized by TLR4 to exhibit the full spectrum of
endotoxicity.[1] Most importantly, it was observed that the
endotoxic activity of lipid A could be significantly reduced
after the removal of its anomeric phosphate group,[1] where-
as its immunostimulatory properties remained unaffected.
For example, although Salmonella minnesota lipid A
(Scheme 1) is a potent endotoxin, the 4’-O-monophosphory-
lated form is essentially nontoxic.[11,12] Monophosphoryl
lipid A (MPLA) is clinically safe as a vaccine adjuvant[13]
and has even been explored as a potential vaccine against
bacterial infections and cancer.[6]
The special carbohydrates expressed by bacterial and
cancer cells are important targets for the design and devel-
opment of bacterial and cancer vaccines.[14,15] However, a
major problem for carbohydrate antigens, especially the
tumor-associated carbohydrate antigens (TACAs), is that
they are typically poorly immunogenic.[14] To overcome this
problem, much recent effort has been focused on synthetic
multicomponent glycoconjugate vaccines, which consist of a
carbohydrate antigen, an immunostimulant, and/or other
functional epitopes.[16–22] It has been demonstrated that cova-
lently coupling an immunostimulant to a carbohydrate anti-
gen can remarkably improve the immunogenicity of the
latter and generate potent glycoconjugate vaccines. In this
Lipopolysaccharides (LPS), which constitute the major com-
ponents on the cell surface of Gram-negative bacteria,[1] are
particularly endotoxic and cause septicemia.[2] It has been
further demonstrated that the LPS anchor part, namely,
lipid A, is primarily responsible for the endotoxicity of LPS.
Lipid A binds to Toll-like receptor 4 (TLR4) to activate a
cascade of immunological responses, including the produc-
tion of a number of cytokines and chemokines, such as
tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), in-
terleukin-6 (IL-6), and interferon-b (IFN-b).[3] Thus, lipid A
has become a valuable molecular template in the discovery
of new immunostimulants,[4,5] for example, vaccine adju-
vants, and in the design and development of novel conjugate
vaccines.[6]
Lipid A is a highly hydrophobic glycolipid, consisting of a
b-1,6-linked diglucosamine with two phosphate or pyrophos-
phate groups and four to eight long lipid chains attached to
the 1,4’-O- and 2,2’-N-3,3’-O-positions, respectively. The
structures of lipid A derived from different bacteria can
vary significantly in terms of the number, structures, and lo-
cations of their lipids.[1] To understand and eventually miti-
gate the endotoxicity of lipid A for the development of
[a] Dr. S. Tang, Dr. Q. Wang, Prof. Dr. Z. Guo
Department of Chemistry
Wayne State University
5101 Cass Avenue, Detroit, Michigan 48202 (USA)
Fax : (+1)313-557-8822
E-mail: zwguo@chem.wayne.edu
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200902153.
Chem. Eur. J. 2010, 16, 1319 – 1325
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Scheme 2. Structure of the target MPLA derivative and the synthetic
plan. Bn=benzyl, Ph=phenyl, Tol=tolyl.
based on carboxylic acid–amine condensation reactions or
on reductive amination reactions. This report describes the
chemical synthesis of 1 and its coupling with a TACA by
means of click chemistry.
In 1, the functionality that can be utilized to couple
MPLA with other epitopes is located at the disaccharide re-
ducing end. Structure–activity relationship studies of MPLA
have revealed that modifying the reducing end of MPLA
has little influence on its binding to the lipid A receptor and
that MPLA derivatives with spacers attached to the reduc-
ing end had activities similar to that of MPLA.[1,38] Thus, we
anticipated that glycoconjugates derived from 1, which will
have both the carbohydrate antigen and other molecular
epitopes linked to the MPLA reducing end, will retain the
immunostimulatory activities of MPLA and be highly immu-
nogenic.
Scheme 1. Selected lipid A structures derived from different Gram-nega-
tive bacteria. Dashed lines indicate partial structures, which result from
incomplete biosynthesis, and the numbers in parentheses indicate the po-
tential variation of chain lengths.
regard, MPLA can be particularly useful as an extremely
potent and nontoxic immunostimulant.
To exploit the immunostimulatory or adjuvant activity of
MPLA for the development of functional conjugate vac-
Results and Discussion
cines, we designed
a
monophosphoryl derivative
1
(Scheme 2) of E. coli lipid A,[1] which is suitable for coupling
with peptides/proteins and carbohydrates for the assembly
of multicomponent glycoconjugates. Although several syn-
theses of lipid A and MPLA have already been report-
ed,[23–37] compound 1 is unique in that it contains an alkyne
functionality, enabling its coupling with other molecules
through click chemistry. In addition, an alkyne functionality
can be readily transformed into a carboxyl or carbonyl
group to facilitate other coupling methods, such as those
Depending on the structures and the locations of the acyl
chains in the target molecule, there are two general strat-
egies for the synthesis of lipid A and MPLA. One strategy is
to assemble the disaccharide backbone first and then to in-
troduce individual lipid chains at the correct positions.[23–29]
In this case, various positions of the disaccharide unit have
to be differentially protected by orthogonal protecting
groups, especially when different lipid chains are present in
the synthetic target. The second strategy is to build the lipi-
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Chem. Eur. J. 2010, 16, 1319 – 1325

Synthesis of a Monophosphoryl Derivative of E. coli Lipid A
FULL PAPER
dated monosaccharides first and then to couple them togeth-
er through a glycosylation reaction.[30–37] In this case, the gly-
cosylation reaction can be challenging because of increased
steric hindrance. Our target molecule 1 has three different
types of lipids attached to 2,2’-N, 3,3’-O positions and we
chose to adopt the second synthetic strategy (Scheme 2).
For the monosaccharide 2, we planned to introduce the lipid
moiety at the 3’-O-position and to protect the 2’-N-position
with a trichloroethyloxycarbonyl (Troc) group to minimize
the steric hindrance of the glycosyl donor. The Troc group
can be readily removed later for the introduction of the
lipid moiety and the acylation of a free amino group should
be relatively easy. For the monosaccharide block 3, we plan-
ned to introduce both lipid chains at the initial stage be-
cause they were expected to have little influence on the rel-
atively reactive primary alcohol as a glycosyl acceptor. Com-
pound 3 was also designed to include an azidoethyl group
linked to the anomeric position, which can be used as a mo-
lecular handle to perform various chemical transformations.
The synthesis of 2 is outlined in Scheme 3. Compound 4
was prepared from glucosamine after a series of established
transformations.[29,39] The phthaloyl and acetyl groups pro-
Scheme 4. Synthesis of monosaccharide building block 3. TsOH=4-
methyl benzenesulfonic acid, TMSOTf=trimethylsilyl triflate, Pyr.=pyri-
dine.
For the assembly of the synthetic target 1 (Scheme 5), 3
was glycosylated by 2 using N-iodosuccinimide (NIS) and
silver triflate (AgOTf) as the promoters. This reaction was
stereoselective and afforded only the desired b anomer (J-
ACHUTNGREN(NUG 1,2)=3.2 Hz, JACHUTNGTREN(NGUN 1’,2’)=8.0 Hz), probably owing to neighbor-
ing group participation, and 13 was obtained in a 75% iso-
lated yield. For the regioselective opening of the benzyli-
dene acetal ring of 13, we were surprised to observe that 13
did not react with sodium cyanoborohydride (NaCNBH3)/
HCl (g) and the starting material was recovered completely.
After exploring a number of reactions and conditions, we
eventually found that the regioselective ring opening could
be accomplished smoothly by using Et3SiH and BF3·Et2O to
expose the C-4’ hydroxyl group. The regiochemistry of prod-
uct 14 was also confirmed by an acetylation experiment,
which resulted in a significant downfield shift of the signal
Scheme 3. Synthesis of monosaccharide building block 2. DCC=N,N’-di-
cyclohexylcarbodiimide,
phthalyl.
DMAP=4-dimethylaminopyridine,
Phth=
1
of H-4’ in the H NMR spectrum. Next, compound 14 was
phosphorylated by means of a one-pot protocol, using di-
benzyl phosphoramidite and tBuO2H as the phosphorylating
and oxidizing reagents, respectively, to furnish 15. We decid-
ed to introduce the alkyne functionality at this stage. Thus,
the azido group of 15 was reduced with propane-1,3-dithiol
and the free amine 16 was acylated with 4-pentynoic acid
(17) by using N’-(3-dimethylaminopropyl)-N-ethylcarbodi-
tecting the 2-N- and 3-O-positions, respectively, were re-
moved by treatment with ethylenediamine, then regioselec-
tive protection of the free amino group with a Troc group
was carried out to give 5. The lipidation of 5 by means of
coupling with (R)-3-tetradecanoyloxytetradecanoic acid (6)
was accomplished by using N,N’-dicyclohexylcarbodiimide
(DCC) as the coupling reagent to afford building block 2 in
an excellent yield.
AHCTUNGTREGiNNNU mide (EDC) hydrochloride as the coupling reagent. It is
worth noting that functionalities other than a carbon–carbon
triple bond can also be introduced at this stage to facilitate
alternative methods for the coupling of MPLA to various
structures later on. Moreover, we found that other tradition-
al methods employed to reduce azides, such as those using
phosphines or the Lindlar catalyst/H2, did not work for 15.
The former reaction gave a good yield of an iminophosphor-
ane derivative that was difficult to hydrolyze and the latter
reaction was slow and inefficient. Finally, the Troc group of
18 was removed by treatment with Zn in AcOH, and the re-
sulting amine was coupled with (R)-3-dodecanoyloxy-tetra-
decanoic acid (20) by using (7-azabenzotriazol-1-yl)tetrame-
thyluronium hexafluorophosphate (HATU)/N,N-diisopropyl-
ethylamine (DIPEA) to afford the synthetic target 1 in a
The synthesis of 3 (Scheme 4) started with the preparation
of 7 by following a reported procedure.[40] The 4,6-O-posi-
tions in 7 were then selectively protected with a benzylidene
group, followed by removal of the acetyl group under basic
conditions. The amino and hydroxyl groups of 9 were simul-
taneously acylated by using (R)-3-benzyloxytetradecanoic
acid (10), with DCC as a coupling reagent, to yield 11. The
benzylidene acetal ring was then opened regioselectively by
using BH3·THF as the reducing reagent and trimethylsilyl
triflate (TMSOTf) as the catalyst to afford building block 3.
The regiochemistry of 3 was confirmed by the acetylation of
1
3, which resulted in the signals in the H NMR spectrum for
protons 6a and 6b shifting downfield significantly.
Chem. Eur. J. 2010, 16, 1319 – 1325
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Z. Guo et al.
mixture to give a very good
yield
GM3NPhAc
of
the
conjugate
MPLA–
22
(70% isolated yield after silica
gel column chromatography).
Conclusion
A highly efficient synthesis of a
monophosphoryl derivative 1 of
E. coli lipid A has been devel-
oped. This synthesis is based
upon the convergent assembly
of the MPLA framework
through the coupling of two
lipidated monosaccharide build-
ing blocks 2 and 3. The synthet-
ic method should be applicable
to similar MPLA structures.
The synthetic derivatives of
MPLA are useful for the explo-
ration of novel MPLA-derived
immunostimulants or immu-
noadjuvants.
Furthermore,
these MPLA derivatives can be
readily coupled with various
structures, as illustrated by the
coupling of 1 and TACA 21 to
form
a
MPLA–carbohydrate
conjugate 22, for the explora-
tion of effective conjugate vac-
cines. Finally, glycoconjugate 22
was
readily
deprotected
through Pd-catalyzed hydroge-
nolysis and both 22 and its de-
protected product are being
evaluated as cancer vaccines in
our laboratory.
Scheme 5. Synthesis of the target MPLA derivative 1. NIS=N-iodosuccinimide, EDC=N’-(3-dimethylamino-
propyl)-N-ethylcarbodiimide hydrochloride, HATU=(7-azabenzotriazol-1-yl)tetramethyluronium hexafluoro-
phosphate, DIPEA=N,N-diisopropylethylamine.
70% yield. We observed that using DCC or EDC for the
final condensation reaction only provided a low yield of the
desired product. Compound
1 was characterized by
1H NMR spectroscopy and high-resolution mass spectrome-
try.
To illustrate that 1 can be conveniently and efficiently
coupled to suitably modified carbohydrates for investigating
multicomponent conjugate vaccines, we then examined the
reaction between 1 and an azido derivative 21 of N-phenyl-
ACHTUNGTRENNUNG
acetyl GM3 (GM3NPhAc) by means of a click reaction[41]
(Scheme 6). GM3NPhAc, an unnatural analogue of GM3,[42]
can form promising vaccines that are presently being investi-
gated in our laboratory for cancer immunotherapy.[43,44] To
our satisfaction, the reaction between 1 and 21 in the pres-
ence of CuI/DIPEA proceeded smoothly in a MeOH/THF
Scheme 6. Coupling of 1 and GM3NPhAc through click chemistry.
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Chem. Eur. J. 2010, 16, 1319 – 1325

Synthesis of a Monophosphoryl Derivative of E. coli Lipid A
FULL PAPER
2H; OCH2CH2N3), 3.12–3.07 (m, 1H; CH2N3), 2.56 (dd, J=7.2, 16.0 Hz,
1H; CHH), 2.39 (dd, J=5.6, 16.0 Hz, 1H; CHH), 2.29–2.27 (m, 2H;
CH2), 1.66–1.43 (m, 4H; CH2), 1.37–1.16 (m, 36H; lipid), 0.89–0.86 ppm
(t, J=6.4 Hz, 6H; lipid-CH3); 13C NMR (CDCl3, 100 MHz): d=172.3,
171.6, 139.0, 138.8, 137.9, 128.7, 128.6, 128.5, 128.2, 128.1, 128.0, 127.9,
127.9, 127.8, 127.7, 127.6, 97.9, 76.7, 75.7, 75.3, 74.9, 73.3, 71.7, 71.6, 71.5,
70.8, 67.2, 61.7, 52.4, 50.4, 42.1, 40.0, 34.5, 34.2, 34.1, 32.2, 30.0, 29.93,
29.89, 29.8, 29.6, 26.7, 26.6, 25.5, 25.4, 25.2, 22.9, 14.4 ppm; HRMS (ESI):
m/z: calcd for C57H86N4O9Na [M+Na]+: 993.6293; found: 993.6284.
Experimental Section
General methods: NMR spectra were recorded on a 400 or 500 MHz
Varian spectrometer with the chemical shifts reported in ppm (d) in ref-
erence to tetramethylsilane (TMS), unless otherwise specified. Coupling
constants (J) are reported in hertz (Hz). Optical rotations were obtained
with an Autopol III polarimeter. High-resolution electrospray-ionization
mass spectra (HR-ESIMS) were recorded by using a Waters Micromass-
LCTPremier-XE mass spectrometer. MALDI-TOF mass spectra were re-
corded on a Bruker Ultraflex mass spectrometer. TLC was performed on
silica gel GF254 plates and visualized by staining with phosphomolybdic
acid or 1% H2SO4 in EtOH. Molecular sieves were dried under high
vacuum at 170–1808C for 6–10 h before use. Commercial anhydrous sol-
vents and other reagents were used without further purification.
Compound 13:
A mixture of 2 (162 mg, 0.165 mmol), 3 (150 mg,
0.155 mmol), and 4 ꢁ molecular sieves (0.5 g) was stirred in anhydrous
CH2Cl2 (2 mL) at RT for 0.5 h. The reaction mixture was then cooled to
À508C, and NIS (85 mg, 10 mmol) was added. The reaction mixture was
allowed to warm to À308C and stirred for 1 h, then a catalytic amount of
AgOTf was added. When TLC analysis showed that the glycosyl donor 2
was completely consumed, the reaction mixture was quenched with the
addition of triethylamine (0.5 mL). The mixture was filtered through a
pad of Celite and the filtrate was washed with water (1.0 mL), dried, and
evaporated under vacuum. The residue was purified by silica gel column
chromatography to give 13 as a white solid (212 mg, 75%). [a]D =+11.2
(c=1.0 in CHCl3); 1H NMR (CDCl3, 400 MHz): d=7.42–7.20 (m, 20H;
Ar-H), 6.12 (d, J=9.6 Hz, 1H; NH), 5.49 (s, 1H; PhCH), 5.36 (t, J=
9.2 Hz, 1H; H-3), 5.29–5.25 (m, 1H; H-3’), 5.20–5.17 (m, 1H; lipid), 4.72
(d, J=3.2 Hz, 1H; H-1), 4.68–4.46 (m, 8H; Troc, 3PhCH2), 4.42 (d, J=
8.0 Hz, 1H; H-1’), 4.33 (dd, J=4.8, 10.4 Hz, 1H; H-6a’), 4.29–4.23 (m,
1H; H-2), 4.01 (brd, J=10.4 Hz, 1H; H-6b), 3.87–3.75 (m, 4H;
2BnOCH, H-5, H-6b’), 3.69–3.63 (m, 5H; H-2’, H-4, H-4’, H-6a,
OCH2CH2N3), 3.47–3.40 (m, 2H; H-5’, lipid), 3.25–3.19 (m, 2H;
OCH2CH2N3), 3.12–3.07 (m, 1H; CH2N3), 2.62–2.53 (m, 3H; CH2), 2.41
(dd, J=4.8, 16.0 Hz, 1H; CHH), 2.28 (d, J=6.0 Hz, 2H; CH2), 2.19 (t,
J=7.2 Hz, 2H; CH2), 1.62–1.44 (m, 8H; CH2), 1.38–1.25 (m, 86H; lipid),
0.94–0.88 ppm (m, 12H; lipid-CH3); 13C NMR (CDCl3, 100 MHz): d=
173.7, 172.2, 171,4, 170.3, 154.3, 138.9, 138.8, 138.2, 137.1, 129.4, 128.8,
128.6, 128.5, 128.2, 128.1, 128.0, 127.7, 126.4, 101.7, 97.8, 95,6, 78.9, 76.7,
75.7, 74.8, 74.5, 73.7, 71.6, 71.3, 70.8, 70.4, 70.2, 68.7, 67.2, 66.6, 57.0, 52.1,
50.4, 42.1, 40.0, 39.5, 34.6, 34.4, 34.3, 34.1, 32.2, 29.9, 29.8, 29.6, 29.4, 26.7,
25.4, 25.2, 22.9, 14.4 ppm; HRMS (ESI): m/z: calcd for
Compound 2: DCC (184 mg, 0.90 mmol) and DMAP (28 mg, 0.23 mmol)
were added to a stirred solution of 6 (200 mg, 0.44 mmol) in CH2Cl2
(6.0 mL) at RT. After stirring at RT for 10 min, a solution of 5 (200 mg,
0.36 mmol) in CH2Cl2 (1.5 mL) was added. The reaction mixture was
stirred for 16 h at RT, then the solid materials were removed by filtration,
and the residue was washed with CH2Cl2 (10 mL). The filtrate was con-
centrated under vacuum and the residue was purified by silica gel
column chromatography to afford 2 as a white solid (330 mg, 93%).
[a]D =+14.1 (c=1.0 in CHCl3); 1H NMR (CDCl3, 400 MHz): d=7.42–
7.26 (m, 7H; Ar-H), 7.12 (d, J=7.2 Hz, 2H; Ar-H), 5.51 (d, J=9.6 Hz,
1H; NH), 5.48 (s, 1H; PhCH), 5.38 (t, J=9.6 Hz, 1H; H-3), 5.20–5.14
(m, 1H; lipid), 4.90 (d, J=10.4 Hz, 1H; H-1), 4.76 (d, J=4 Hz, 2H;
CH2CCl3), 4.34 (dd, J=4.8, 10.4 Hz, 1H; H-6a), 3.78 (t, J=10.0 Hz, 1H;
H-6b), 3.68–3.60 (m, 2H; H-4, H-2), 3.56–3.50 (m, 1H; H-5), 2.62–2.48
(m, 2H; CH2), 2.35 (s, 3H; CH3), 2.17 (t, J=7.2 Hz, 2H; CH2), 1.55–1.52
(m, 4H; CH2), 1.32–1.20 (m, 40H; lipid), 0.88 ppm (t, J=7.6 Hz, 6H;
lipid-CH3); 13C NMR (CDCl3, 100 MHz): d=173.7, 170.2, 154.3, 138.8,
137.0, 133.7, 130.1, 129.4, 128.5, 126.4, 101.7, 88.1, 78.8, 74.8, 72.5, 70.7,
70.2, 68.7, 56.0, 39.5, 34.6, 34.1, 32.1, 29.9, 29.8, 29.59, 29.56, 29.4, 25.3,
25.2, 22.9, 21.4, 14.4 ppm; HRMS (ESI): m/z calcd for C51H76Cl3NO9SNa
[M+Na]+: 1006.4204; found: 1006.4201.
Compound 11: DCC (0.64 g, 3.2 mmol) and DMAP (0.10 g, 0.08 mmol)
were added to a stirred solution of 10 (0.70 g, 2.1 mmol) in CH2Cl2
(10 mL) at RT. After stirring for 10 min at RT, a solution of 9 (0.27 g,
0.81 mmol) in CH2Cl2 (1.5 mL) was added and the reaction mixture was
stirred for 16 h at RT. The solid materials were removed by filtration and
the residue was washed with CH2Cl2 (10 mL). The filtrate was concen-
trated under vacuum and the residue was purified by silica gel column
chromatography to afford 11 as a white solid (0.56 g, 71%). [a]D =+26.8
(c=1.0 in CHCl3); 1H NMR (CDCl3, 400 MHz): d=7.40–7.21 (m, 15H;
Ar-H), 6.20 (d, J=9.6 Hz, 1H; NH), 5.46 (s, 1H; PhCH), 5.38 (t, J=
9.6 Hz, 1H; H-3), 4.73 (d, J=3.2 Hz, 1H; H-1), 4.56–4.35 (m, 5H;
2PhCH2, H-2), 4.25 (dd, J=9.6, 4.4 Hz, 1H; H-5), 3.92–3.68 (m, 6H; H-
6a, H-6b, H-4, 2 BnOCH, OCH2CH2N3), 3.25 (dd, J=7.6, 16.4 Hz, 2H;
OCH2CH2N3), 3.15–3.09 (m, 1H; CH2N3), 2.66 (dd, J=6.8, 10.8 Hz, 1H;
CHH), 2.44–2.36 (m, 1H; CHH), 2.33–2.26 (m, 2H; CH2), 1.47–1.43 (m,
4H; CH2), 1.30–1.19 (m, 36H; lipid), 0.89–0.86 ppm (t, J=6.4 Hz, 6H;
lipid); 13C NMR (CDCl3, 100 MHz): d=172.0 171.6, 138.9, 138.8, 137.1,
129.3, 128.6, 128.5, 128.4, 128.0, 127.9, 127.8, 127.6, 126.3, 101.8, 98.5,
79.4, 76.7, 75.7, 71.6, 71.3, 70.0, 69.0, 67.3, 63.4, 52.3, 50.4, 42.0, 40.0, 34.8,
34.1, 32.2, 29.92, 29.89, 29.8, 29.6, 25.5, 25.4, 22.9, 14.4 ppm; HRMS
(ESI): m/z: calcd for C57H84N4O9Na [M+Na]+: 991.6136; found:
991.6152.
C
101H154Cl3N5O18Na [M+Na]+: 1853.0252; found: 1853.0221.
Compound 14: Et3SiH (15 equiv, 0.10 mL) and BF3·Et2O (2 equiv, 10 mL)
were added to a stirred solution of 13 (70 mg, 0.038 mmol) in CH2Cl2
(2.0 mL) at 08C. The mixture was warmed to 258C over a period of 2 h,
then diluted with CH2Cl2 (30 mL), washed with an aqueous solution of
NaHCO3 (5 mL), dried, and concentrated. The residue was purified by
silica gel column chromatography to provide 14 (53 mg, 76%). [a]D =+
17.4 (c=1.0 in CHCl3); 1H NMR (CDCl3, 400 MHz): d=7.36–7.17 (m,
20H; Ar-H), 6.10 (d, J=9.6 Hz, 1H; NH), 5.31 (dd, J=8.8, 10.4 Hz, 1H;
H-3), 5.12–5.10 (m, 1H; lipid), 5.09 (d, J=8.4 Hz, 1H; NH’), 4.91 (t, J=
9.6 Hz, 1H; H-3’), 4.70 (d, J=4.0 Hz, 1H; H-1), 4.63–4.42 (m, 10H; Troc,
4PhCH2), 4.28–4.20 (m, 2H; H-2, H-1’), 4.06–4.02 (m, 1H; H-6b), 3.84–
3.72 (m, 5H; 2BnOCH, H-5, H-6a’), 3.67–3.60 (m, 5H; H-2’, H-4, H-4’,
H-6a, H-6b’), 3.48–3.38 (m, 2H; H-5’, OCH2CH2N3), 3.24–3.17 (m, 2H;
OCH2CH2N3), 3.11–3.05 (m, 1H; CH2N3), 2.60–2.51 (m, 3H; CH2), 2.41
(dd, J=4.8, 15.2 Hz, 1H; CH2), 2.30–2.26 (m, 4H; CH2), 1.62–1.44 (m,
8H; CH2), 1.38–1.25 (m, 86H; lipid), 0.94–0.88 ppm (m, 12H; lipid-CH3);
13C NMR (CDCl3, 100 MHz): d=174.6, 172.3, 171,7, 171.5, 154.3, 138.9,
138.7, 138.3, 138.0, 128.7, 128.5, 128.2, 128.0, 127.9, 127.5, 101.3, 97.7,
95,7, 76.7, 75.7, 75.5, 75.4, 74.7, 73.9, 71.6, 71.2, 70.8, 70.5, 70.2, 67.7, 67.2,
55.8, 52.1, 50.4, 42.1, 40.3, 40.1, 36.9, 34.9, 34.7, 34.4, 34.3, 32.4, 32.2, 30.1,
29.9, 29.7, 29.6, 26.7, 25.4, 25.2, 24.9, 23.6, 22.9, 14.4 ppm; HRMS (ESI):
Compound 3: TMSOTf (0.08 mL, 0.46 mmol) was added dropwise to a
stirred solution of compound 11 (365 mg, 0.38 mmol) in BH3·THF/
THF(2 mL, 2.0 mmol) at 08C. After stirring for 1 h, TLC analysis showed
that the reaction was complete. The reaction was then quenched with
triethylamine (0.5 mL) and MeOH (0.5 mL), the mixture was concentrat-
ed, and the residue was purified by silica gel column chromatography to
m/z: calcd for
C
101H159Cl3N5O18Na [M+Na]+: 1855.0409; found:
1855.0339.
Compound 15: 1H-tetrazole (0.45m, 0.5 mL) and dibenzyl diisopropyl-
phosphoramidite (0.05 mL, 0.15 mmol) were added to a stirred solution
of 14 (50 mg, 0.027 mmol) in dry CH2Cl2 (5 mL). The mixture was stirred
for 2 h at RT, then cooled to À308C, and tBuOOH (0.4 mmol) was
added. The mixture was stirred for another 0.5 h at 08C. The resulting so-
lution was washed sequentially with a saturated aqueous solution of
NaHCO3, then the organic phase was dried and concentrated. The resi-
1
afford 3 as a white solid (213 mg, 58%). H NMR (CDCl3, 400 MHz): d=
7.33–7.20 (m, 15H; Ar-H), 6.13 (d, J=8.8 Hz, 1H; NH), 5.39–5.34 (m,
1H; H-3), 4.71 (d, J=4.0 Hz, 1H; H-1), 4.65–4.43 (m, 6H; 3PhCH2),
4.28–4.22 (m, 1H; H-2), 3.85–3.62 (m, 7H; H-6b, H-4, H-5, H-6a,
2BnOCH, OCH2CH2N3), 3.47–3.38 (m, 2H; OCH2CH2N3), 3.25–3.19 (m,
Chem. Eur. J. 2010, 16, 1319 – 1325
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1323

Z. Guo et al.
due was purified by silica gel column chromatography to give 15 as an oil
(43 mg, 75%). [a]D =+5.2 (c=0.5 in CHCl3); 1H NMR (CDCl3,
400 MHz): d=7.36–7.22 (m, 30H; Ar-H), 6.10 (d, J=9.6 Hz, 1H; NH),
5.45 (d, J=8.4 Hz, 1H; NH’), 5.38–5.32 (m, 2H; H-3, H-3’), 5.18 (m, 1H;
lipid), 4.90–4.80 (m, 4H; 2 PhCH2), 4.71(d, J=3.2 Hz, 1H; H-1), 4.65–
4.58 (m, 3H; H-1’, PhCH2), 4.54–4.38 (m, 8H; H-4’, Troc, PhCH2), 4.29–
4.24 (m, 1H; H-2), 4.06–4.03 (m, 1H; H-6b), 3.85–3.77 (m, 4H; 2
BnOCH, H-5, H-6a’), 3.69–3.56 (m, 5H; H-4, H-5’, H-6a), 3.54–3.45 (m,
1H; H-2’), 3.44–3.38 (m, 1H; OCH2CH2N3), 3.26–3.17 (m, 2H;
OCH2CH2N3), 3.10–3.04 (m, 1H; CH2N3), 2.55 (dd, J=7.2, 16.0 Hz, 1H;
CHH), 2.47–2.33 (m, 1H; CHH), 2.29–2.21 (m, 5H; CH2), 1.94–1.87 (m,
1H; CH2), 1.62–1.44 (m, 8H; CH2), 1.38–1.25 (m, 86H; lipid), 0.94–
0.88 ppm (m, 12H; lipid-CH3); 13C NMR (CDCl3, 100 MHz): d=173.9,
172.2, 171.4, 170.5, 154.2, 138.9, 138.8, 138.3, 138.2, 135.7, 129.7, 128.8,
128.6, 128.3, 128.1, 127.8, 127.6, 100.6, 97.8, 95.5, 76.6, 75.7, 74.7, 74.6,
74.4, 73.6, 72.5, 71.6, 70.8, 70.5, 70.3, 69.8, 68.8, 68.1, 67.6, 67.1, 56.7, 52.1,
50.4, 42.1, 40.0, 39.8, 36.9, 34.7, 34.4, 34.3, 32.2, 29.9, 29.6, 29.4, 26.7, 25.4,
24.9, 23.7, 23.2, 22.9, 14.3 ppm; 31P NMR (CDCl3, 162 MHz): d=
À0.92 ppm; HRMS (ESI): m/z: calcd for C115H169Cl3N5O21PNa [M+Na]+:
2115.1011; found: 2115.0942.
400 MHz): d=7.31–7.19 (m, 30H; Ar-H), 6.31 (d, J=9.2 Hz, 1H; NH),
6.25 (d, J=7.2 Hz, 1H; NH’), 5.41 (t, J=9.6 Hz, 1H; H-3’), 5.27 (t, J=
9.6 Hz, 1H; H-3), 5.16 (m, 1H; lipid), 5.07 (m, 1H; lipid), 4.90–4.88 (m,
4H; PhCH2), 4.64 (d, J=4.0 Hz, 1H; H-1), 4.56–4.41 (m, 10H; H-1’, H-
4’, 4PhCH2), 4.28–4.22 (m, 1H; H-2), 4.06–4.03 (m, 1H; H-6b), 3.88–3.75
(m, 4H; 2ROCH, H-5, H-6a’), 3.66–3.48 (m, 6H; H-4, H-5’, H-6a, H-2’,
OCH2CH2NHCO), 3.22–3.10 (m, 3H; OCH2CH2NHCO), 2.56–2.19 (m,
15H), 2.13 (d, J=2.0 Hz, 1H; CH2), 1.55–1.42 (m, 12H; CH2), 1.24–1.03
(m, 116H; lipid), 0.98–0.88 ppm (m, 18H; lipid-CH3); 13C NMR (CDCl3,
100 MHz): d=173.8, 172.1, 171.5, 171.3, 170.5, 138.8, 138.5, 138.3, 137.7,
135.8, 128.8, 128.7, 128.5, 128.3, 128.2, 128.0, 127.9, 127.8, 127.7, 100.5,
97.3, 95.5, 83.7, 76.9, 76.1, 75.6, 74.7, 74.4, 73.8, 73.5, 72.7, 71.4, 71.2, 70.7,
70.4, 70.0, 69.8, 68.8, 62.0, 57.9, 52.2, 50.7, 41.9, 39.9, 35.2, 34.7, 34.5, 34.2,
32.2, 29.9, 29.6, 29.5, 29.5, 25.5, 25.4, 25.3, 22.9, 15.1, 14.4 ppm; 31P NMR
(CDCl3, 162 MHz): d=À0.91 ppm; HRMS (ESI): m/z: calcd for
C143H222N3O23PNa [M+Na]+: 2403.5930; found: 2403.5903.
Compound 22: DIPEA (10 equiv) was added to a stirred solution of 1
(10 mg, 0.004 mmol), 21 (4 mg, 0.005 mmol), and CuI (10 equiv) in
MeOH/THF (2:1, 0.6 mL). The resulting mixture was stirred for 24 h at
RT, then filtered through a Celite pad, and the solvent was evaporated
under high vacuum. The crude product was purified by flash silica gel
column chromatography (eluent: from AcOEt/hexane (4:1) to AcOEt
and then to MeOH/CH2Cl2 1:5) to give 22 as a white solid (9 mg, 70%).
Compound 18: Triethylamine (0.2 mL) was added to a stirred solution of
15 (50 mg, 0.024 mmol) and 1,3-propanedithiol (0.15 mL) in pyridine
(4 mL) and H2O (0.2 mL). The mixture was stirred at 08C until the start-
ing material disappeared (monitored by TLC). The solvent was then
evaporated under vacuum and the residue was coevaporated with toluene
(2ꢂ10 mL) and ethanol (2ꢂ10 mL). The residue was purified by silica
gel column chromatography to give 16, which was then dissolved in
CH2Cl2 (1.5 mL). EDC (9 mg, 0.048 mmol) and a solution of 17 (7 mg,
0.072 mmol) in CH2Cl2 (3 mL) was added to this solution. The reaction
mixture was stirred for 2 h at RT. The mixture was diluted with CH2Cl2
and washed with H2O and brine. The organic phase was dried, concen-
trated, and the residue was purified by silica gel column chromatography
to afford 18 (30 mg, 58%). [a]D =+9.2 (c=0.65 in CHCl3); 1H NMR
(CDCl3, 400 MHz): d=7.32–7.20 (m, 30H; Ar-H), 6.28 (d, J=9.6 Hz,
1H; NH), 5.94 (s, 1H; NH), 5.68 (d, J=7.6 Hz, 1H; NH’), 5.42 (dd, J=
9.6 Hz, 8.4 Hz, 1H; H-3’), 5.28 (dd, J=11.2, 8.4 Hz, 1H; H-3), 5.17 (m,
1H; lipid), 4.91–4.88 (m, 4H; PhCH2), 4.67–4.39 (m, 13H; H-1, H-1’, H-
4’, Troc, PhCH2), 4.27–4.21 (m, 1H; H-2), 4.06–4.04 (m, 1H; H-6b), 3.91–
3.76 (m, 4H; 2BnOCH, H-5, H-6a’), 3.63–3.53 (m, 5H; H-4, H-5’, H-6a,
OCH2CH2NHCO), 3.45–3.41 (m, 1H; H-2’), 3.14–3.08 (m, 2H;
OCH2CH2NHCO), 3.00 (m, 1H, OCH2CH2NHCO), 2.58–2.20 (m, 12H;
CH2), 1.65–1.38 (m, 10H; CH2), 1.38–1.25 (m, 90H; lipid), 0.87 ppm (t,
J=6.4 Hz, 12H; lipid-CH3); 13C NMR (CDCl3, 100 MHz): d=173.8,
172.3, 171.5, 170.4, 154.3, 138.8, 138.4, 138.3, 137.9, 135.7, 128.8, 128.6,
128.3, 127.9, 127.8, 127.7, 127.6, 127.0, 100.4, 97.2, 95.5, 83.5, 77.1, 73.6,
72.3, 71.4, 71.2, 70.5, 70.3, 69.8, 68.8, 68.3, 67.6, 66.8, 56.7, 52.1,41.9, 39.9,
39.6, 39.0, 35.2, 34.7, 34.4, 34.1, 32.2, 29.9, 29.8, 29.6, 29.4 , 25.4, 25.2, 22.9,
15.0, 14.3 ppm; 31P NMR (CDCl3, 162 MHz): d=À0.91; HRMS (ESI): m/
z: calcd for C120H175Cl3N5O22PNa [M+Na]+: 2169.1368; found: 2169.1301.
1
[a]D =4.4 (c=0.45 in CHCl3); H NMR (CDCl3/CD3OD (3:1), 500 MHz):
d=7.45 (s, 1H; triazole-H), 7.19–6.90 (m, 35H; Ar-H), 5.24–5.21 (m, 1H;
H-3’), 5.15–5.08 (m, 1H; H-3), 4.98 (m, 1H; lipid), 4.88 (m, 1H; lipid),
4.72–4.65 (m, 4H; PhCH2), 4.43 (d, J=3.5 Hz, 1H; H-1), 4.39–4.42 (m,
12H; H-1’, H-4’, PhCH2), 4.09 (d, J=7.0 Hz, 1H; H-sugar), 4.28–4.22 (m,
1H; H-2), 3.96 (m, 1H; H-6b), 3.78 (m, 1H; H-sugar), 3.68–3.35 (m, 4H;
2ROCH, H-5, H-6a’), 2.98–2.92 (m, 2H; CH2), 2.75 (m, 2H; CH2), 2.35–
1.99 (m, 12H; CH2), 1.86–1.80 (m, 3H; CH2), 1.55–1.42 (m, 12H; CH2),
1.24–1.03 (m, 116H; lipid), 0.98–0.88 ppm (m, 18H; lipid); 13C NMR
(CDCl3/CD3OD (3:1), 125 MHz): d=129.0, 128.7, 128.6, 128.4, 128.3,
128.2, 128.1, 128.0, 127.8, 127.7, 127.6, 127.5, 127.0, 123.2, 103.0, 100.2,
97.4, 77.6, 76.7, 75.9, 75.4, 75.0, 74.6, 74.5, 74.0, 73.4, 73.3, 73.1, 72.6, 71.5,
71.2, 70.6, 70.0, 69.8, 68.5, 68.0, 62.0, 57.9, 52.2, 51.7, 50.2, 49.3, 49.1, 49.0,
48.8, 48.6, 41.4, 39.6, 38.8, 35.1, 34.5, 34.2, 31.9, 29.6, 29.3, 29.2, 25.4, 25.1,
25.0, 22.7, 21.2, 13.9 ppm; 31P NMR (CDCl3/CD3OD 3:1, 162 MHz): d=
À1.50 ppm; MALDI-TOF MS: m/z: calcd for C174H268N7O42P: 3158.9
[M]+; found: 3181.8 [M+Na]+, 3203.8 [MÀH+2Na]+.
Acknowledgements
This work was supported by NIH (R01A95142). The authors thank Dr.
B. Shay and Dr. L. Hryhorczuk for HRMS measurements and Dr. B.
Ksebati for help with some NMR spectroscopy measurements.
Compound 1: Zn powder (100 mg) was added to a stirred solution of 18
(52 mg, 0.024 mmol) in acetic acid (5 mL) and the resulting mixture was
stirred at RT for 24 h. The solid materials were removed by filtration
through a pad of Celite. The filtrate was concentrated and coevaporated
with toluene three times. The residue was dissolved in CH2Cl2 (40 mL)
and washed sequentially with a saturated aqueous solution of NaHCO3
(20 mL), H2O (10 mL), and brine (10 mL). The organic phase was dried
and concentrated. The residue was purified by silica gel column chroma-
tography to afford 19 as a foamy solid.
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Published online: November 26, 2009
Chem. Eur. J. 2010, 16, 1319 – 1325
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www.chemeurj.org
1325