Synthesis of S-linked NeuAc-α(2-6)-di-LacNAc bearing liposomes for H1N1 influenza virus inhibition assays

Article abstract of DOI:10.1016/j.bmc.2018.02.012

S-NeuAc-α(2-6)-di-LacNAc (5) was efficiently synthesized by a [2+2] followed by a [1+4] glycosylation, and later conjugated with 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) to form both single-layer and multi-layer homogeneous liposomes in the presence of dipalmitoyl phosphatidylcholine (DPPC) and cholesterol. These liposomes were found to be weak inhibitors in both the influenza virus entry assay and the hemagglutination inhibition assay. The single layer liposome was found to more efficiently interfere with the entry of the H1N1 influenza virus into MDCK cells than the multilayer liposome containing 5.

Full text of DOI:10.1016/j.bmc.2018.02.012

Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of S-linked NeuAc-a(2-6)-di-LacNAc bearing liposomes for
H1N1 influenza virus inhibition assays
Hou-Wen Cheng a, Hsiao-Wen Wang a, Tsung-Yun Wong a, Hsien-Wei Yeh a, Yi-Chun Chen a, Der-Zen Liu b,c
,
Pi-Hui Liang a,d,
⇑
a School of Pharmacy, College of Medicine, National Taiwan University, Taipei 100, Taiwan
b Graduate Institute of Biomedical Materials and Tissue Engineering, College of Oral Medicine, Taipei Medical University, Taipei 110, Taiwan
c Medical and Pharmaceutical Industry Technology and Development Center, Academia Sinica, Taipei 128, Taiwan
d The Genomics Research Center, Academia Sinica, Taipei 128, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
S-NeuAc-a(2-6)-di-LacNAc (5) was efficiently synthesized by a [2+2] followed by a [1+4] glycosylation,
Received 13 December 2017
Revised 7 February 2018
Accepted 10 February 2018
Available online xxxx
and later conjugated with 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) to form both sin-
gle-layer and multi-layer homogeneous liposomes in the presence of dipalmitoyl phosphatidylcholine
(DPPC) and cholesterol. These liposomes were found to be weak inhibitors in both the influenza virus
entry assay and the hemagglutination inhibition assay. The single layer liposome was found to more effi-
ciently interfere with the entry of the H1N1 influenza virus into MDCK cells than the multilayer liposome
containing 5.
Keywords:
Liposome
Influenza
Ó 2018 Elsevier Ltd. All rights reserved.
Sialic acid
Hemagglutinin
1. Introduction
decade.4 Oseltamivir resistance,5,6 however, is a growing problem;
influenza continues to threaten humans; and new drugs to combat
Influenza is an infectious disease caused by the influenza virus,
which infects humans and animals through contact with bronchial
epithelial cells. The mechanism by which the virus attacks the cell
entails recognition and cleavage of the terminal alpha-linked sialic
acid on the surface of the cell by the glycoprotein hemagglutinin
(HA), present on the surface of the virus. There are several different
types of alpha-sialic acid linkage present on the surfaces of human
mucosal cells, for which different types of influenza viruses have
different binding preferences. For example, avian influenza virus
it are urgently required. Meanwhile, anti-influenza inhibitors that
target HA are underdeveloped, compared to NA inhibitors.7,8
Although the targeting of HA is an option for the treatment of
influenza, the binding between multiple HA of an influenza virus
and the sialic acid surface receptors of an erythrocyte during viral
infection is estimated to occur with an affinity of 1013 Mꢀ1, whereas
the association constant of a single sialic acid-HA interaction
is 103 Mꢀ1 9
; and thus the high degree of multi-valency required
for a strong binding affinity between HA and HA-bound molecules
poses a challenge.10 For example, the sialic acid moieties presented
by human mucosal cells are densely packed together, and any
platform presenting an array of molecules intended to interact with
the HA trimer would also have to be very densely packed.
binds to sialic acid in a
enza virus is reported to bind sialic acid in either a
linkage with galactose; and human-adapted influenza viruses have
binding preferences for a
2-6 sialic acid linkage.1–3
a2-3-linkage with galactose; swine influ-
a
2-6 or 2-3
a
a
Following invasion of the cell in this way, a second influenza
surface glycoprotein, neuraminidase (NA), is implicated in the
release of the virus from the host cell. As both processes are inte-
gral to the proliferation of the virus, both HA and NA are promising
targets for the development of anti-influenza drugs. NA inhibitors,
such as oseltamivir and zanamivir, have seen clinical use for a
When covalently displayed on the surface of liposome, S-
Neu5Ac-
around 70–180
a
(2-6)-LacNAc (2, Fig. 1) was shown to have an EC50 of
l
M in a virus entry inhibitory assay.11 Since the
potency was not optimized, we sought to increase the inhibitory
effect of sialic conjugation. A model to describe the binding
between HA and the sialoside was recently disclosed.12 When HA
binds to
topology. However, when HA binds to
‘‘umbrella-like” topology with additional glycan conformational
flexibility. Longer 2-6 sialic acid bearing oligosaccharides would
a
2-3-sialoside, it is believed to assume a ‘‘cone-like”
a2-6-sialoside, it forms an
⇑
Corresponding author at: School of Pharmacy, College of Medicine, National
Taiwan University, Taipei 100, Taiwan.
a
E-mail address: phliang@ntu.edu.tw (P.-H. Liang).
https://doi.org/10.1016/j.bmc.2018.02.012
0968-0896/Ó 2018 Elsevier Ltd. All rights reserved.
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2
H.-W. Cheng et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
OH
HO
COONa
COOH
OH
AcHN
OH
HO
HO
HO
O
X
O
AcHN
OH
HO
O
HO
OH
O
HO
X
OH
HO
OH
O
OH
O
O
O
O
O
OH
O
OH
O
HO
AcHN
HO
HO
HO
HO
OH
NHAc
OH
NHAc
1
2
X=O
X=S
3
4
X=O
X=S
OH
COONa
OH
HO
HO
O
S
O
AcHN
OH
HO
HO
OH
OH
O
O
OH
O
O
O
O
O
O
O
O
HO
O
O
P
O
N
H
HO
HO
OH
NHAc
O
OH
NHAc
O
5
Fig. 1. Structure of sialic acid containing trisaccharides (1, 2), pentasaccharides (3, 4), and DLPE conjugation 5.
favor this conformation. In mammalian respiratory epithelial cells,
LacNAc is frequently displayed.12,13 Glycan microarray studies
revealed that the HAs of human influenzas bound best to N-glycans
with the linear di-LacNAc sequence, and also to N-glycans with the
be formulated with DPPC (dipalmitoyl phosphatidylcholine) to
form liposomes (Fig. 2) in aqueous solution. These compounds
and formulations were evaluated in the A/WSN/33 H1N1 influenza
entry inhibition and hemagglutination inhibition assays by red
blood cells (RBC)s.
tri-LacNAc repeat sequence.14 Neu5Ac-
a(2-6)-di-LacNAc (3) was
indeed found to have a stronger binding preference for influenza
than trisaccharide 1 (Fig. 1). Accordingly, we designed and synthe-
sized S-Neu5Ac-a(2-6)-di-LacNAc (4), wherein the aforementioned
2. Results and discussion
trisaccharide 2 has been extended by LacNAc and S-linked Neu5Ac
moieties.15,16 This compound is expected to serve as both a novel
synthetic recognition molecule of HA, and as a competitor of natu-
ral O-glycosides to NA.
2.1. Synthesis of DLPE bearing 5
Our retrosynthesis of 5 is depicted in Scheme 1. The key steps
are the formation of the amide bond that connects the lipid with
the pentasaccharide; and two key glycosylation steps. Firstly, the
Several multivalent vectors (dendrimers,17 polymers,18 lipo-
somes,19 and nanoparticles20), have been functionalized with sialic
acid; and their abilities to inhibit the activities of their correspond-
ing lectins were measured. However, in all cases, either heteroge-
neous mixtures of Neu5Ac were submitted for conjugation, and/or
they contained only a Neu5Ac moiety for protein recognition,
which may be not sufficient for HA interaction, and cannot be used
to quantify the contribution of Neu5Ac in the virus interaction.14 In
order to procure a multivalent display of homogenous 4, it was
designed to conjugate with phospholipid – DLPE (1,2-dilauroyl-
sn-glycero-3-phosphoethanolamine) to give 5 (Fig. 1), which could
S-glycosidic a2-6 linkage, would be formed by nucleophilic substi-
tution of the triflate group at the galactose 6-position of compound
8 by the Neu5Ac thiolate of 7 ([1+4] glycosylation step); and sec-
ondly, the [2+2] glycosylation in step c, which would yield com-
pound 8. In order to control the selectivity of the glycosylations
of steps c, d and e, benzoyl (Bz) and trichloroethylcarbonyl (Troc)
groups would be introduced at the galactose 2-OH (compounds
11 and 14) and glucosamine 2-NH2 positions (compounds 12 and
13), respectively. These are expected to confer a degree of b-selec-
tivity on the glycosylation, by a way of neighboring-group partici-
Fig. 2. Approach to present NA S-linked NeuAc-a(2-6)-di-LacNAc bearing liposomes to HA of influenza virus.
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H.-W. Cheng et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
3
Scheme 1. Retrosynthetic analysis of S-Neu5Ac-a(2-6)Gal-b(1–4)GlcNAc-b(1–3)-Gal-b(1–4)GlcNAc containing 5. (a) Deprotection of benzyl, benzoyl, Troc groups; (b)
Formation of S-linkage, [1+4] glycosylation; (c) [2+2] glycosylation; (d), (e) Glycosylation; (f), (g) Glycosylation and selective ring opening reaction/or selective silyl group
protection.
pation. The anomeric position of the non-reducing end of GluNAc
(15) is modified by a methyl glycolate linker to give 12, which
can be hydrolyzed to the corresponding carboxylic acid, for conju-
gation with the phospholipid.
Our synthesis started with compound 15,11 which was coupled
with methyl glycolate under NIS/TfOH conditions to give com-
pound 16 in 85% yield (Scheme 2). The selective ring opening reac-
tion was conducted under NaBH3CN and HCl in diethyl ether to
Ph
O
O
O
O
Ph
O
BnO
a
O
O
O
STol
NHTroc
b
O
HO
BzO
O
O
BzO
OMe
BzO
OMe
NHTroc
NHTroc
15
16
12
OBz
O
O
OBz
OBn
O
d
HO
HO
O
c
OBn
O
O
O
O
O
O
OMe
BzO
O
O
OBz
NHTroc
OMe
BzO
OBz
h
NHTroc
17
9
OBz
OBz
LevO
LevO
OBz
O
LevO
g
OBn
e, f
O
O
O
O
O
O
BzO
STol
STol
O
O
LevO
OMe
OBz
OBz
OBz
NHTroc
19
18
11
Scheme 2. Preparation of 9. (a) NIS, TfOH, methyl glycolate, DCM, 4 Å MS, ꢀ40 °C, 1 h, 85%; (b) NaBH3CN, HCl ether/THF, 0 °C, 70%; (c) BSP, TTBP, Tf2O, DCM, 4 ÅMS, ꢀ60 °C,
1.5 h, 80%; (d) AcOH 60%, 60 °C; (e) Acetic acid 60%, 60 °C, 12 h; (f) levulinic acid, EDCI, DMAP/DCM, rt, 12 h, 80% two steps; (g) 12, BSP, TTBP, Tf2O/DCM, ꢀ60 °C, 1.5 h, 85%; (h)
1.0 N hydrazine acetate, toluene, rt, 10 min, 70%.
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H.-W. Cheng et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
give compound 12 in 70% yield. Compounds 11 and 12 underwent
glycosylation under BSP, TTBP and Tf2O conditions to afford the
disaccharide compound 17. In order to remove the dimethyl ace-
tonide group, compound 17 was treated with acetic acid (60%) at
60 °C. Unfortunately, these conditions resulted in hydrolysis of
the glycosidic bond, and none of the desired compound 9 was iso-
lated. Alternative reaction conditions (higher/lower temperatures)
and reagents (HClO4 at 0 °C or I2 dissolved in MeOH)21 were also
unsuccessful-hydrolysis of the glycosidic bond occurred in all
cases. Accordingly, we revised our protecting group strategy and
replaced the acetonide group of 11 with a levulinate (Lev) group.
Compound 11 was treated with 60% acetic acid at 60 °C to remove
the acetonide group, which was directly coupled with levulinic
acid using EDCI, and DMAP to give 18. Glycosylation of compounds
12 and 18 took place with BSP, TTBP and Tf2O at ꢀ60 °C to give the
glycosylated intermediate 19, whereupon the Lev groups were
removed by hydrazine acetate to afford compound 9 in a yield of
60% over two steps.
Glycolipid 5 could now be pursued (Scheme 3). Compound 15
underwent selective ring opening using NaBH3CN and HCl in
diethyl ether to give compound 20 in 70% yield. The thiol group
of 20 was removed smoothly using NBS to provide compound 21,
Scheme 3. Preparation of 5. (a) NaBH3CN, HCl ether/THF, 0 °C, 70%; (b)NBS/Acetone:H2O = 5:1, ꢀ25 °C, 0.5 h; (c) TBDMSCl, imidazole/DCM, rt, 12 h, 70%; (d) 14, NIS, TfOH/
DCM, 4 Å MS, ꢀ45 °C to ꢀ25 °C, 1.5 h, 70%;(e) TBAF, acetic acid/THF, rt, 1 h; (f) CCl3CN, DBU/DCM, rt, 4 h, two steps 70%; (g) 9, TMSOTf/DCM, ꢀ78 °C, 36 h; (h) Acetic acid 80%,
60 °C, 24 h, 60%; (i) Tf2O, pyridine/DCM, ꢀ25 °C, 4 h; (j) diethylamine/DMF, ꢀ20 °C, 2 h, 43% (3 steps); (k) i. Zn, Ac2O, Et3N, DCM, rt, 6 h; ii. NaOMe/MeOH, rt, 12 h; iii. 0.2 N
NaOH, rt, 2 h, 56% for 3 steps; (l) H2, Pd(OH)2, 4 bar, rt, 12 h, 70%; (m) i. AmberliteÒIR120 H+; ii. DLPE, EDC, HOBt, NMM/DMF, 35 °C, 9 h, 50%.
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H.-W. Cheng et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
5
the anomeric hydroxyl group of which reacted with TBDMSCl in
the presence of imidazole to give compound 13. Reaction of the
4-hydroxyl group of 21 with TBDMSCl was not observed, presum-
ably due to its lower reactivity and more congested steric environ-
ment, compared to the anomeric hydroxyl group. Further
glycosylation of 13 with thioglycoside donor 14 was performed
using NIS and TfOH activation to give 22 in 70% yield. Next, the silyl
protecting group of 22 was removed under TBAF and acetic acid
conditions, and then reacted with trichloroacetonitrile and DBU
to afford glycosyl imidate 10. Disaccharides 9 and 10 underwent
glycosylation by TMSOTf at ꢀ78 °C to give tetrasaccharide 23.
The benzylidene groups of tetrasaccharide 23 were removed by
treatment with acetic acid to give a major product 8, which was
immediately reacted with Tf2O in the presence of pyridine to yield
triflate 24. Nucleophilic substitution of the triflate and Neu5Ac thi-
olate, generated in situ by diethylamine from thioacetate 7,22 gen-
erated the S-linked pentasaccharide 6 in 70% yield. 6 was carried
out using Zn/Ac2O to remove Troc group and sequentially N-acety-
lated, and then global deprotected using cat. NaOMe in methanol
followed by basic hydrolysis with 0.1 N NaOH to provide com-
pound 25. Hydrogenolysis of compound 25 by H2 and Pd/C gave
the desired compound 26. Subsequently, 26 was conjugated via
amide bond formation with DLPE, a kind of phospholipid found
in biological membranes, which bears an amino group on the
hydrophilic side. In order to free the carboxylic acid from its
sodium salt, compound 26 was acidified with AmberliteÒ IR120
H+ and the free carboxylic group activated by EDC and HOBt, prior
to reaction with DLPE in the presence of NMM, to give the dipolar
compound 5. No DLPE was observed to conjugate with the 2-car-
boxylic group of sialyl 26, presumably because of steric hindrance.
tures were then concentrated for 30 min at 40 °C, then re-
suspended in PBS buffer (pH 7.4) to give multilayer liposomes
(namely 5-ML), which was used as a control. When the suspension
was pressed through extruders of membrane pore sizes 400 nm
and 100 nm, liposomes of 162.5 1.3 nm diameter (monitored by
Dynamic Light Scattering) were obtained, and henceforth referred
to as 5-SL1. The final concentrations were 20 mM for DPPC, and
165 lM for 5 (present as 5-SL1). By way of comparison, SLs both
without 5 and with a 0.4 mol% of 5 were also prepared by a similar
method, and resulted in liposomes bearing 5 at concentrations of 0
lM (lipo-control) and 83 lM (5-SL2), respectively. The zanamivir
encapsulated liposome 5-SL1-Za was also prepared by adding
zanamivir (2 mg) to a suspension of 5-SL1 (5 mL), and then press-
ing it through extruder of membrane pore size 400 nm and 100
nm. Compound 5 could also self-assemble to form micelles. The
physical parameters for each of these formulations are disclosed
in the Supporting Information.
2.3. Virus entry inhibition assay
To evaluate the ability of our compounds/formulations for
inhibiting influenza virus entry, we adapted an effective method
by co-incubating of our inhibitors with virus particles and
Madin-Darby canine kidney (MDCK) cells, and then quantifying
the amount of viral nucleoprotein (NP) present in the cells.23 The
presence of NP can be detected by ELISA with a primary mono-
clonal antibody to the influenza A NP, followed by incubation with
a secondary monoclonal antibody. The fluorescence value of
ex485/em535 is then detected. Here, compound 5, 5-SL1, 5-SL2,
and 5-ML were subjected to an influenza entry inhibition assay
with A/WSN/33 H1N1. Fluorescence values for each experiment
were obtained at different concentrations, and the data is pre-
sented in Table 1. The greater the fluorescence, the lower the inhi-
bitory ability of that compound. None of the multi-valent vectors
2.2. Preparation of liposome and zanamivir encapsulated liposomes
Single-layer (SL) and multi-layer liposomes (ML) are the most
common type of liposomes. A single-layer liposome was deemed
the most suitable for this study, because the surface ligands pre-
sent in ML are buried within layers, which reduces the surface den-
sity of the ligand.
Fig. 2 depicts the expected interaction between HA and the lipo-
some. In order for a trivalent interaction between the SL and the
HA to occur, the surface density of the ligand presented on the sur-
face of the liposome should be closed to the density of HA. For a
liposome composed of DPPC and cholesterol in a 4:1 ratio and of
an approximate diameter of 200 nm, the molar percentage of 5
on the liposome was estimated to be 0.8 mol% (see Supporting
Information). Accordingly, DPPC, cholesterol (CH), and 5 were dis-
solved in CHCl3 in a molar ratio of 4:1:0.008. The lipid sample mix-
tested displayed any inhibitory activity at 83
lM. Formulations
of both 5-SL1 and 5-SL2 at 165 M imparted weak inhibition
l
(17%). Liposome encapsulated zanamivir (5-SL1-Za, zanamivir is
1.4 mM concentration herein) showed 25% virus entry inhibitory.
These results indicate that zanamivir interfered with the entry of
the virus into the cells,24 and that our attempt to block influenza
entry by capturing the virus using a sialic acid containing liposome
failed, a result inconsistent with a recent study that demonstrated
that a liposome bearing sialylneolacto-N-tetraose could effectively
inhibit virus (Influenza A/Puerto Rico/8/34 virus) entry at 10 nM.25
The contrasting nature of our results with this study might be due
to the differences in virus strain and conditions for the influenza
entry inhibition assay.
Table 1
The data of entry inhibition and hemagglutination inhibition assay.
Compd/formulation
Surface density (%)a
Assay conc.b
Entry inhibition
Ki (l
M)c
Control/Single layer liposome
5/Self assemble micelle
0 mol%
100 mol%
0
83
165
83
165
42
83
83
165
83
0%
2%
6%
7%
17%
4%
17%
10%
25%
0%
0
NDd
15.6
15.6
ND
5-SL1/Single layer liposome
5-SL2/Single layer liposome
5-SL1-Za/Single layer liposome
5-ML/Multilayer liposome
0.8 mol%
0.4 mol%
0.8 mol%
0.8 mol%
31.2
165
0%
a
b
c
Indicated the molar concentration of 4 in percentage among the formulation.
Conc. of 5 in mM.
Hemagglutination inhibition assay.
Not determined.
d
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H.-W. Cheng et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
2.4. Hemagglutination inhibition assay
formed using a VG platform electrospray ESI/MS or a BioTOF II (Tai-
wan). The DLS experiments used a BIC 90 PLUS device (Brookhaven
Instrument Co. Holtsville, NY, USA).
According to the hemagglutination inhibition assay, the single
layer liposomes 5-SL1 and 5-SL2, which had 0.8 mol% and 0.4
mol% S-linked Neu5Ac-
faces, displayed the lowest Ki values of 15.6 mM (Table 1). Multi-
layer liposomes which had 0.8 mol% S-linked Neu5Ac- (2-6)-di-
LacNAc displayed the same Ki values of 31.2 M. In our previous
study, a liposome of mono-sialic acid conjugated with DLPE dis-
played a Ki of 500 M, and that of a trisaccharide 2 displayed a
Ki of 125 (2-6)-
M.11 The formulation bearing S-linked Neu5Ac-
di-LacNAc designed in this study demonstrated the ability to inhi-
bit the virus from binding with red blood cells (RBC) bearing sialic
acid. This phenomenon suggests that our design of liposomal S-
a(2-6)-di-LacNAc on their respective sur-
4.2. Synthesis and characterization of compounds
4.2.1. 3-O-Benzoyl-6-O-benzyl-2-deoxy-2-(2,2,2-
trichloroethoxycarbonylamino)-1-O-methylglycolate-b-
glucopyranoside (12)
a
l
D-
l
Sodium cyanoboronhydride (2.2 g, 35.2 mmol) and 4 Å MS were
added to a stirred solution of compound 1611 (2.4 g, 3.91 mmol), in
dry THF (50 mL) at 0 °C over 15 min. After 8 h, 1.0 N HCl (in diethyl
ether) was added slowly, until effervescence had ceased. The reac-
tion was quenched by sat. NaHCO3 solution, filtered to remove 4 Å
MS, and concentrated in vacuo to give a residue that was extracted
with dichloromethane. The organic layers were separated, dried
over MgSO4, filtered and concentrated in vacuo. The residue was
purified by column chromatography (silica gel, hexanes/ethyl acet-
ate, 2/1) to afford 12 (1.7 g, 70% yield) as yellow oil; 1H NMR
(CDCl3, 600 MHz) d 8.02–7.27 (m, 10H, Ar-H), 5.66 (d, J = 9.6 Hz,
1H, N-H), 5.29 (t, J = 9.6 Hz, 1H, H-3), 4.75 (d, J = 12 Hz, 1H, Troc-
CH), 4.71 (d, J = 8.4 Hz, 1H, H-1), 4.54 (q, J = 12 Hz, 2H), 4.46 (d, J
= 12 Hz, 1H, Troc-CH), 4.33 (q, J = 16.8 Hz, 2H), 3.93 (q, J = 9.6 Hz,
1H, H-2), 3.87 (t, J = 9.6 Hz, 1H, H-4), 3.78 (d, J = 4.2 Hz, 2H, H-6),
3.72 (s, 3H, OCH3), 3.60 (t, J = 4.2 Hz, 1H, H-5) ppm; 13C NMR
(CDCl3, 150 MHz) d 170.3, 167.3, 154.7, 137.6, 133.5, 130.1,
129.1, 128.5, 128.4, 127.9, 127.7, 100.2 (C-1), 95.4 (Troc-CCl3),
l
a
linked Neu5Ac-a(2-6)-di-LacNAc did have a stronger binding affin-
ity with the virus than liposomal S-Neu5Ac-a(2-6)LacNAc (2) and
liposomal mono-sialic acid. Since the sialic conjugation was
homogenously displayed on the surface of liposome and yet still
led to negative results in our virus entry inhibition assay, it is pos-
tulated that the binding to HA is unable to alleviate virus infectiv-
ity, a finding which merits further investigation. Perhaps the
surface of the virus bears too high density of HA for its complete
inhibition to be possible; and/or the binding of weaponized lipo-
some to HA is simply insufficient for virus infectivity to be
disabled.
3. Conclusion
77.0, 76.2, 74.8, 74.3, 73.8, 70.6, 69.8, 64.7, 55.6, 52.0 (OCH3)
ppm; HRMS (ESI) calcd. forC26H28Cl3NO10Na [M+Na]+ 642.0671,
found m/z 642.0698.
Lipid conjugated S-linked NeuAc-a(2-6)-di-LacNAc (5) was suc-
cessfully synthesized by [2+2] and [1+4] glycosylation methods.
Liposomes bearing 5 at concentrations of 0.4 mol% and 0.8 mol%
were prepared by combining 5 with DPPC and cholesterol. It was
hoped that these liposomes would function as multivalent ligand
inhibitors of HA, and all formulations/compounds were subjected
to the HA inhibition assay by RBCs. 5-SL1 was determined to inhi-
4.2.2. 4-Methylphenyl-2,6-di-O-benzoyl-3,4-di-O-levulinoyl-1-thio-b-
D-galactopyranoside (18)
Compound 11 (3.0 g, 5.6 mmol) was stirred in 60% acetic acid
aqueous solution (30 mL) at 60 °C for 12 h. The reaction was
quenched with sat. NaHCO3 solution and brine and the organic lay-
ers were collected, dried over MgSO4, filtered and concentrated in
vacuo to give residue (2.8 g). The residue (2.8 g), EDCI (2.4 g, 12.3
mmol) and DMAP (12 mg, 0.112 mmol) in dichloromethane were
added levulinic acid (1.3 mL, 12.3 mmol). After stirring for 2 h, the
reaction was quenched with sat. NaHCO3 solution and brine and
the organic layers were collected, dried over MgSO4, filtered and
concentrated in vacuo to give a residue that was purified by column
chromatography (silica gel, toluene/ethyl acetate, 6/1) to afforded
18 (3.1 g, 80% yield in two steps) as a yellow solid; mp 121 °C; 1H
NMR (CDCl3, 400 MHz) d 8.00–6.92 (m, 14H, Ar-H), 5.57 (d, J = 3.2
Hz, 1H), 5.48 (t, J = 10 Hz, 1H), 5.20 (dd, J = 3.2, 10 Hz, 1H), 4.81
(d, J = 10 Hz, 1H, H-1), 4.52 (dd, J = 7.6, 11.2 Hz, 1H), 4.35 (dd, J =
5.2, 11.2 Hz, 1H), 4.11 (t, J = 6.4 Hz, 1H), 2.76–2.38 (m, 8H, –COCH2-
CH2CO–), 2.25, (s, 3H, CH3), 2.17 (s, 3H, CH3), 1.98 (s, 3H, CH3) ppm;
13C NMR (CDCl3, 100 MHz) d 206.0 (2C), 171.9, 171.8, 166.0, 165.3,
138.3, 133.4, 133.2, 133.1, 129.9, 129.8, 129.6, 129.5, 129.3, 128.8,
128.5, 128.4, 87.3 (C-1), 74.7, 72.2, 67.8, 67.7, 62.3, 37.8, 37.7,
29.7, 29.4, 27.8, 27.8, 21.1 ppm; HRMS (ESI) calcd. for C37H38O11SNa
[M+Na]+ 713.2027, found m/z 713.2061.
bit influenza virus binding with RBCs with a Ki of 15.6 lM. Disap-
pointingly, however, none of the formulations tested could
efficiently inhibit A/WSN/33/H1N1 influenza virus entry. The sin-
gle layer liposome 5-SL1 (0.8 mol%) was found to impart superior
inhibitory activity than the multilayer liposome 5-ML, but both
were inferior to the liposome encapsulated with Za (5-SL1-Za).
Although S-linked Neu5Ac-a(2-6)-di-LacNAc (4) decoy liposome
and micelles did not prove capable of inhibiting influenza entry,
the synthesis of the sialyloligosaccharides used in this study is
expected to useful in the future due to the homogenous nature
of the products.
4. Experimental section
4.1. General methods
All reagents and solvents were of reagent grade, and were used
without further purification unless otherwise stated. Reaction pro-
gress was monitored by analytical TLC on 0.25 mm E. Merck silica
gel 60 F254 using p-anisaldehyde, ninhydrine, and cerium as visual-
izing agents. NMR spectra were recorded on Bruker-AV-400 (400
MHz) and Bruker-AV-600 (600 MHz) instruments, with a TCI cryo
probe and a BACS 60 sample changer. Chemical shifts (d) are given
in parts per million relative to 1H: 7.24 ppm, 13C: 77.0 ppm for
CDCl3, 1H: 4.80 ppm for D2O, and 1H: 4.78 ppm, 13C: 49.2 ppm for
methanol-D4. The splitting patterns are reported as s (singlet), bs
(broad singlet), d (doublet), bd (broad doublet), t (triplet), q (quar-
tet), dd (double doublet), m (multiplet). Coupling constants (J) are
given in Hz. All peak assignments were confirmed using 2DNMR
(COSY, HSQC) techniques. Exact mass measurements were per-
4.2.3. 4-O-[2,6-Di-O-benzoyl-b-D-galactopyranosyl]-(1 ? 4)-3-O-
benzoyl-6-O-benzyl-2-deoxy-2-(2,2,2-
trichloroethoxycarbonylamino)-1-O-methylglycolate-b-
glucopyranoside (9)
D-
Thioglycoside 18 (1.7 g, 2.7 mmol) in dry dichloromethane (40
mL) was added 4 Å MS, BSP (848 mg, 4.05 mmol) and TTBP (1.3 g,
5.4 mmol). The solution was cooled to ꢀ60 °C, after which Tf2O
(0.8 mL, 4.59 mmol) was added slowly. After 10 min, 12 was then
added into the solution. After stirring for 1.5 h, the cooling bath
Please cite this article in press as: Cheng H.-W., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.02.012

H.-W. Cheng et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
7
was removed and the reaction diluted with dichloromethane.
4.2.6. 4-O-[2,3-Di-O-Benzoyl-4,6-di-O-benzylidene-b-D-
Then, the mixture was filtered to remove the 4 Å MS and washed
with sat. NaHCO3 and brine. The organic layers were collected,
dried over MgSO4, filtered and concentrated in vacuo to give a resi-
due which was purified by column chromatography (silica gel,
hexanes/ethyl acetate, 1/1) to afford a white foam (2.7 g, 85%).
Hydrazine acetate (1.0 N solution in MeOH) was added to a solu-
tion of this residue (2.0 g, 1.7 mmol) in toluene (40 mL). After 10
min, the reaction was concentrated in vacuo to give a residue
was purified by column chromatography (silica gel, hexanes/ethyl
acetate, 1/1) to afford 9 (1.2 g, 70% yield) as a white solid; mp 97
°C; 1H NMR (CDCl3, 400 MHz) d 8.05–7.26 (m, 20H, Ar-H), 5.58 (d, J
= 8.8 Hz, 1H), 5.31 (t, J = 9.2 Hz, 1H), 5.16 (t, J = 8.8 Hz, 1H), 4.75 (d,
J = 9.2 Hz, H-1), 4.61–4.44 (m, 4H), 4.3 (m, 3H), 4.10 (t, J = 9.2 Hz,
1H), 4.00 (m, 2H), 3.69 (s, 3H, OCH3), 3.62–3.73 (m, 3H), 3.50 (q,
J = 8.8 Hz, 2H), 3.40 (d, J = 10.8 Hz, 2H) ppm; 13C NMR (CDCl3,
100 MHz) d 170.3, 166.4, 166.2, 154.7, 1379, 133.5, 129.9, 129.8,
129.7, 129.5, 129.4, 128.6, 128.5, 128.4, 127.9, 127.8, 100.5 (C-1),
10.3 (C0-1), 95.4 (CCl3), 75.1, 74.7, 74.3, 73.8, 73.5, 73.4, 72.5,
72.2, 68.5, 67.3, 64.7, 61.8, 55.8, 52.0 ppm; HRMS (ESI) calcd.
forC46H46Cl3NO17Na [M+Na]+ 1012.1724, found m/z 1012.1766.
galactopyranosyl]-(1 ? 4)-3-O-benzoyl-6-O-benzyl-1-O-tert-
butyldimethylsily-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-
b-
D-glucopyranoside (22)
NIS (0.95 g, 4.2 mmol) were added to a stirred solution of com-
pound 13 (1.4 g, 2.1 mmol), 14 (2.46 g, 4.2 mmol) and 4 Å MS in
dry dichloromethane (40 mL) at rt. After the solution was cooled
to ꢀ60 °C, it was added TfOH (37
lL, 0.42 mmol) and warming to
ꢀ45 °C. After 2 h later, the reaction was quenched by 20% aqueous
sodium thiosulfate, and then the mixture was diluted with dichlor-
omethane, washed with sat. NaHCO3 solution and brine. The
organic layers were collected, dried over MgSO4, filtered and con-
centrated in vacuo to give a residue which was purified by column
chromatography (silica gel, toluene/ethyl acetate, 9/1) to afforded
22 (1.6 g, 70%) as white oil; 1H NMR (CDCl3, 400 MHz) d 8.06 (d, J
= 7.2 Hz, 2H, Ar-H), 7.42–7.92 (m, 21H, Ar-H), 7.17 (d, J = 6.8 Hz,
2H), 5.69 (t, J = 10 Hz, 1H), 5.42 (t, J = 10.4 Hz,1H), 5.30 (s, 1H,
CHPh), 5.25 (d, J = 9.6 Hz, 1H), 5.10 (d, J = 10.4 Hz, 1H), 4.84 (d, J =
8 Hz, 1H), 4.65 (m, 2H), 4.42 (d, J = 12 Hz, 1H), 4.33 (m, 2H), 4.15
(m, 2H), 3.83 (m, 2H), 3.57 (m, 4H), 2.98 (s, 1H), 0.84 (s, 9H, t-butyl),
0.09 (s, 3H, CH3), 0.04 (s 3H, CH3) ppm; 13C NMR (CDCl3, 100 MHz) d
166.1, 166.1, 164.1 (COO), 154.3 (NCO), 138.2, 137.4, 133.3, 133.2,
129.8, 129.8, 129.7, 129.5, 129.3, 128.9, 128.7, 128.5, 128.3, 128.3,
127.9, 127.6, 127.5, 126.3, 101.0 (C-1), 100.6 (C0-1), 96.5 (CHPh),
95.3 (CCl3), 76.0, 74.5, 74.3, 73.8, 73.1, 72.7, 69.5, 68.1, 68.0, 66.4,
58.5, 25.5, 17.8, ꢀ4.18, ꢀ5.32 ppm; HRMS (ESI) calcd. for C56H60Cl3-
NO15SiNa [M+Na]+ 1142.2690, found m/z 1142.2743.
4.2.4. 4-Methylphenyl 3-O-benzoyl-6-O-benzyl-2-deoxy-2-(2,2,2-
trichloroethoxycarbonylamino)-1-thio-b- -glucopyranoside (20)
D
1.0 N HCl (in diethylether) was slowly added to a stirred solution
of compound 1511 (4.0 g, 6.1 mmol), sodium cyanoboronhydride
(3.5 g, 54.9 mmol) and 4 Å MS in dry THF (100 mL) at 0 °C for 15
min until the bubble disappeared. After 10 min, the reaction was
quenched by sat. NaHCO3 solution and the mixture was filtered to
remove 4 Å MS, concentrated in vacuo, and extracted with dichloro-
methane. The combined organic layers were dried over MgSO4, fil-
tered, and concentrated in vacuo to give a residue which was
purified by column chromatography (silica gel, hexanes/ethyl acet-
ate, 2/1) to afford 20 (2.8 g, 70% yield) as white foam; 1H NMR
(CDCl3, 600 MHz) d 7.99–8.00 (d, J = 7.2 Hz, 2H, Ar-H), 7.18–7.57
(m, 10H, Ar-H), 7.06–7.07 (d, J = 7.98 Hz, 2H, Ar-H), 5.25–5.33 (m,
2H), 4.73 (d, J = 2.6 Hz, 1H), 4.71 (d, J = 4.3 Hz, 1H), 4.45–4.59 (m,
3H), 3.80–3.92 (m, 4H), 3.60–3.66 (m, 1H), 2.31 (s, 3H, STol-CH3)
ppm; 13C NMR (CDCl3, 100 MHz)d 167.2 (COO), 154.2 (NCO),
133.6, 133.2, 130.0, 129.7, 128.5, 128.4, 127.9, 127.7, 95.3 (C-1),
87.4 (CCl3), 78.4, 74.5, 73.8, 70.7, 60.6, 54.9, 29.8, 21.2; HRMS (ESI)
calcd. forC30H30Cl3NO7SNa [M+Na]+ 676.0701, found m/z 676.0727.
4.2.7. 4-O-[2,3-Di-O-benzoyl-4,6-di-O-benzylidene-b-
galactopyranosyl]-(1 ? 4)-3-O-[3-O-benzoyl-6-O-benzyl-2-deoxy-2-
(2,2,2-trichloroethoxycarbonylamino)-b- -glucopyranosyl]-(1 ? 3)-
4-O-[2,6-di-O-benzoyl-b- -galactopyranosyl]-(1 ? 4)-3-O-benzoyl-
6-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-1-O-
methylglycolate-b- -glucopyranoside (23)
TBAF (0.4 mL, 1.32 mmol) and acetic acid (75
D-
D
D
D
l
L, 1.32 mmol)
were added to a stirred solution of compound 22 (1.2 g, 1.1 mmol)
in THF (20 mL). After the reaction completed, it was diluted with
water and evaporated. After, the residue was extracted with
dichloromethane, concentrated in vacuo to give a residue which
was purified by column chromatography (silica gel, hexanes/ethyl
acetate, 1/1) to afforded 1-OH disaccharide as a white solid. This
disaccharide dissolved in dichloromethane (20 mL) was further
reacted with trichloroacetonitrile (0.55 mL, 5.5 mmol) in the pres-
4.2.5. 3-O-Benzoyl-6-O-benzyl-1-O-tert-butyldimethylsily-2-deoxy-2-
ence DBU (82 lL, 0.55 mmol). After stirring for 2 h, the reaction
(2,2,2-trichloroethoxycarbonylamino)-b- -glucopyranoside (13)
D
was concentrated in vacuo to give a residue which was purified
by column chromatography (silica gel, hexanes: ethyl acetate =
1:1) to afforded 10 (0.9 g, 70% yield over 2 steps) as yellow oil.
Compound 10 (0.9 g, 0.77 mmol) and 9 (509 mg, 0.51 mmol) were
dissolved in dry dichloromethane (20 mL) and stirred with 4 Å MS
NBS (2.2 g, 12.4 mmol) was added to a stirred solution of com-
pound 20 (2.0 g, 3.1 mmol) in acetone/H2O (5/1, 20 mL) was added
slowly at ꢀ25 °C. After 30 min, the reaction was quenched by 20%
aqueous sodium thiosulfate, and organic solvent was evaporated.
The mixture was diluted with dichloromethane, washed with sat.
NaHCO3 solution and brine. The organic layers were collected,
dried over MgSO4, filtered and concentrated in vacuo. The purified
residue was added with TBDMSCl (561 mg, 3.72 mmol) and imida-
zole (498 mg, 7.44 mmol) in dichloromethane (40 mL). After stir-
red 12 h, the reaction was concentrated in vacuo to give a residue
which was purified by column chromatography (silica gel, hex-
anes/ethyl acetate, 2/1) to afford 13 (1.4 g, 70% yield in two steps)
as yellow oil; 1H NMR (CDCl3, 600 MHz) d 8.07 (d, J = 7.8 Hz, 2H,
Ar-H), 7.30–7.60 (m, Ar-H), 5.80 (t, J = 10.2 Hz, 1H), 5.44 (t, J =
9.6 Hz, 1H), 4.83 (d, J = 7.8 Hz, 1H, H-1), 4.56–4.70 (m, 4H), 3.89–
3.96 (m, 2H), 3.83 (s, 2H), 3.71 (t, J = 4.8 Hz, 1H), 3.39 (s, –OH),
0.92 (s, -t-butyl, 9H), 0.17 (s ꢁ 2, 6H, CH3) ppm; 13C NMR (CDCl3,
150 MHz) d 167.4 (COO), 154.4 (NCO), 137.7, 133.5, 130, 129.1,
128.4, 127.7, 127.6, 96.4 (C-1), 95.3 (-CCl3), 76.0, 74.3, 74.2, 73.7,
71.1, 70.3, 57.8, 25.5, 17.8 ppm; HRMS (ESI) calcd. forC29H38Cl3-
NO8SiNa [M+Na]+ 684.1324, found m/z 684.1353.
at ꢀ78 °C. After TMSOTf (7
lL, 0.077 mmol) was added, the reac-
tion was stirred for 36 h and quenched by TEA (1 drop). The reac-
tion was concentrated in vacuo to give a residue which was
purified by column chromatography (silica gel, hexanes/ethyl acet-
ate, 1/1) to afford 22 (1.2 g, 80% yield) as white foam; 1H NMR
(CDCl3, 600 MHz) d 8.04–7.82 (m, 13H, Ar-H), 7.54–7.25 (m, 30H,
Ar-H), 7.06–7.03 (m, 2H, Ar-H), 5.66 (t, J = 9.6 Hz, 1H), 5.26 (s,
1H, –CHPh), 5.33–5.25 (m, 2H), 5.08 (d, J = 9.6 Hz, 1H), 4.86 (s,
1H), 4.74 (d, J = 7.8 Hz, 1H, H-1), 4.69 (d, J = 12 Hz, 1H, H0-1), 4.57
(d, J = 12.6 Hz, 1H, H00-1), 4.52 (d, J = 7.8 Hz, 1H, H000-1), 4.47–4.40
(m, 4H), 4.28–4.23 (m, 4H), 4.07–4.13 (m, 3H), 3.92–3.99 (m,
3H), 3.89 (s, 1H), 3.82–3.70 (m, 5H), 3.67 (s, 3H, OMe), 3.55–3.41
(m, 9H), 3.31–3.25 (m, 1H), 2.87–2.85 (m, 1H) ppm; 13C NMR
(CDCl3, 150 MHz)
d 170.2, 166.3, 166.1, 165.8, 165.0, 164.4
(COO), 154.1 (NCO), 137.6, 137.4, 133.5, 133.2, 130.1, 129.9,
129.8, 129.6, 129.3, 129.0, 128.9, 128. 6, 128.5, 128.2, 128.0,
Please cite this article in press as: Cheng H.-W., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.02.012

8
H.-W. Cheng et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
127.7, 126.3, 101.8 (C-1), 101.2 (C0-1), 100.6 (C00-1), 100.5 (C000-1),
ing bath at rt for 6 h. Then the mixture was filtered through Celite
pad, and the filtrate was washed with NaHCO3 solution, followed
by brine. The organic layers were collected and evaporated in vacuo
to give a residue which was dissolved in dry MeOH (2 mL) and then
sodium methoxide (1 mg) was added. After stirring at rt for12 h,
the solution was evaporated to give a residue which was treated
with 0.1 N NaOH (0.5 mL) for 1 h. The solution was neutralized with
AmberliteÒ IR 120H+ resin, filtered and concentrated in vacuo to
give a residue which was purified by Sephadex G-10 column chro-
matography to afford 25 (18 mg, 56% yield for 3 steps) as a yellow
solid; 1H NMR (D2O, 600 MHz) d 7.46–7.41 (m, 9H, Ar-H), 7.33 (m,
1H, Ar-H), 4.62 (d, J = 7.8 Hz, 1H, H-1), 4.57 (d, J = 8.4 Hz, 1H, H0-1),
4.52 (d, J = 12 Hz, 1H, H00-1), 4.33 (d, J = 8.4 Hz, 1H, H000-1), 4.17–4.06
(m, 3H), 4.00 (d, J = 11.4 Hz, 1H), 3.95 (d, J = 7.8 Hz, 2H), 3.90–3.80
(m, 13H), 3.71–3.66 (m, 9H), 3.59–3.57 (m, 3H), 3.54–3.47 (m, 4H),
3.36–3.31 (m, 3H), 3.01–2.92 (m, 2H), 2.80 (dd, J = 5.4, 13.2 Hz, 1H,
100.2 (CHPh), 95.5 (CCl3), 95.2 (CCl3), 76.2, 74.9, 74.6, 74.4, 74.2,
73.8, 73.5, 73.2, 73.1, 72.6, 72.2, 70.9, 69.6, 68.2, 68.0, 66.5, 56.5,
53.5, 52.1, 29.8, 25.7, 14.2 ppm; HRMS (ESI) calcd. forC96H90Cl6N2-
O
31Na2 [M+2Na]2+ 1012.1731, found m/z 1012.1742.
4.2.8. S-[Methyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
glycero- -galacto-non-2-ulopyranosylonate]-(2 ? 6)-4-O-[2,3-di-
O-benzoyl-b- -galactopyranosyl]-(1 ? 4)-3-O-[3-O-benzoyl-6-O-
benzyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-b-
glucopyranosyl]-(1 ? 3)-4-O-[2,6-di-O-benzoyl-b-
galactopyranosyl]-(1 ? 4)-3-O-benzoyl-6-O-benzyl-2-deoxy-2-
D-
a-D
D
D-
D-
(2,2,2-trichloroethoxycarbonylamino)-1-O-methylglycolate-b-
glucopyranoside (6)
D-
Compound 23 (110 mg, 0.058 mmol) was suspended in 80%
acetic acid and heated at 60 °C. After 12 h, the reaction was
quenched by sat. NaHCO3 solution and brine. The organic layers
were collected, dried over MgSO4, filtered and concentrated in
vacuo to give a residue, which was purified by column chromatog-
raphy (silica gel; hexanes/ethyl acetate, 1/1) to afford 4,6-dihy-
droxyl intermediate (68 mg, 0.036 mmol) which was dissolved in
H00000-3eq), 2.05–2.03 (s ꢁ 3, 9H, CH3), 1.77 (t, J = 12 Hz, H00000-3ax
)
ppm; 13C NMR (D2O, 150 MHz) d 175.0, 175.0, 174.9, 174.9,
174.9, 137.3, 128.8, 128.6, 128.4, 128.3, 102.8 (C-1), 102.6 (C0-1),
102.3 (C00-1), 100.2 (C000-1), 85.3, 81.5, 79.1, 76.9, 74.8, 74.0, 73.7,
73.4, 73.1, 72.9, 72.6, 72.4, 72.2, 71.8, 70.6, 69.8, 69.6, 69.3, 68.6,
68.3, 68.1, 67.8, 67.5, 62.6, 61.1, 55.2, 55.0, 51.7, 40.8, 29.6, 22.3,
22.2, 22.0 ppm; HRMS (ESI) calcd. for C55H79N3O30SNa [M+Na]+
1316.4367, found m/z 1316.4374.
dry dichloromethane (1.2 mL) and pyridine (14.6
5 equiv.) and stirred under N2. The solution was cooled to ꢀ25 °C,
then trifluoromethanesulfonic anhydride (7.3 L, 0.043 mmol,
lL, 0.18 mmol,
l
1.2 equiv.) was added dropwise over 1 min. After stirring at ꢀ25 °C
for 1.5 h, the reaction mixture was diluted with dichloromethane
(10 mL), quickly washed with 1.0 N HCl, saturated NaHCO3
solution, ice water, and brine. The organic layers were dried over
MgSO4, filtered, and concentrated. The resting crude residue was
added with 7 (20 mg, 0.036 mmol, 1 equiv.) in DMF (2 mL) and
4.2.10. S-[5-Acetamido-3,5-dideoxy-
ulopyranosylonate]-(2 ? 6)-4-O-[b-
O-[2-deoxy-2-acetamido-b- -glucopyranosyl]-(1 ? 3)-4-O-[b-
galactopyranosyl]-(1 ? 4)-2-deoxy-2-acetamido-1-O-glycolic-2-
deoxy-b- -glucopyranosidedisodum (26)
D
-glycero-
a-D-galacto-non-2-
D-galactopyranosyl]-(1 ? 4)-3-
D
D-
D
Compound 25 (18 mg, 0.014 mmol) in H2O (1 mL) and metha-
nol (1 mL) was added Pd(OH)2 (20 mg) and stirred under H2 (4
bar) for 12 h. The mixture was filtered through a pad of Celite
and washed with water. The filtrates were combined and concen-
trated in vacuo. The residue was purified by Sephadex G-10 column
chromatography to afford 26 (11 mg, 70% yield) as white foam; 1H
NMR (D2O, 400 MHz) d 4.40 (d, J = 6.9 Hz, 2H), 4.26 (t, J = 7.6 Hz,
3H), 3.95–3.97 (m, 4H), 3.75–3.91 (m, 5H), 3.31–3.66 (m, 31H),
3.17 (s, 1H), 2.36 (dd, J = 6.1, 12.2 Hz, 1H, H0000’-eq), 2.17 (d, J =
12.2 Hz, 1H), 1.86 (s, 3H, CH3), 1.85 (s, 3H, CH3), 1.81(s, 3H, CH3)
ppm; 13C NMR (D2O, 100 MHz) d 179.9, 179.9, 175.0, 175.9,
174.8, 103.0, 102.9, 102.7, 100.3, 82.1, 81.7, 78.9, 78.2, 75.2, 74.9,
74.8, 74.4, 73.9, 73.5, 72.7, 72.2, 72.1, 71.6, 71.2, 71.1, 70.8, 70.7,
70.6, 70.2, 70.0, 68.4, 68.4, 68.3, 68.3, 68.1, 68.0, 67.9, 63.0, 62.8,
62.5, 61.0, 59.95, 59.91, 55.1, 55.0, 52.57, 52.26, 52.23, 48.9, 48.3,
stirred under N2. After cooled to ꢀ20 °C, diethylamine (37
lL,
0.36 mmol, 10 equiv.) was added and the solution was stirred for
3 h, the mixture was concentrated in vacuo to give a residue which
was purified by column chromatography (silica gel, hexanes/
ethylacetate, 1/2) to afford 6 (60 mg, 43% yield for 3 steps) as white
foam; 1H NMR (CDCl3, 600 MHz) d 8.10 (d, J = 7.2 Hz, 2H), 8.05–
8.01 (m, 4H), 7.95–7.91 (m, 6H), 7.58–7.50 (m, 8H), 7.46–7.37
(m, 18H), 7.19–7.17 (d, J = 7.2 Hz, 2H), 5.53 (m, 2H), 5.37–5.34
(m, 3H), 5.28–5.24 (m, 3H), 5.18–5.17 (m, 1H), 4.94.93 (m, 2H),
4.82 (d, J = 10.2 Hz, 1H, H-1), 4.75 (d, J = 11.4 Hz, 1H, H0-1), 4.60
(d, J = 13.8 Hz, 1H, H00-1), 4.55 (d, J = 9 Hz, 1H, H000-1), 4.53–4.47
(m, 4H), 4.37–4.31 (m, 3H), 4.27–4.22 (m, 3H), 4.17–4.11
(m, 3H), 4.08–4.05 (m, 1H), 4.00–3.96 (m, 3H), 3.86 (s, OMe),
3.83-0.753 (m, 4H), 3.73 (s, OMe), 3.67–3.59 (m, 4H), 3.56–3.50
(m, 6H), 3.42–3.38 (m, 1H), 2.75 (dd, J = 5.4, 13.2 Hz, 1H),
2.42–2.38 (m, 2H), 2.14 (s, 3H), 2.11 (s, 3H), 2.03 (s, 3H), 1.96
(s, 3H), 1.88 (s, 3H) ppm;13C NMR (CDCl3, 100 MHz) d 171.0,
170.5, 170.3, 170.2, 167.4, 166.4, 166.3, 165.7, 165.6, 165.1,
164.6, 164.4, 133.4, 130.1, 129.8, 129.7, 129.6, 128.6, 128.3,
128.2, 128.0, 127.9, 127.6, 101.9(C-1), 100.5(C0-1), 100.4(C00-1),
36.7, 34.3, 22.1, 22.0, 20.0, 15.4 ppm; HRMS (ESI) calcd. for C41H66
-
N3Na2O30S [M+2Na]2+ 1158.3242, found m/z 1158.3298.
4.2.11. S-[5-Acetamido-3,5-dideoxy-
ulopyranosylonate]-(2 ? 6)-4-O-[b-
O-[2-deoxy-2-acetamido-b- -glucopyranosyl]-(1 ? 3)-4-O-[b-
galactopyranosyl]-(1 ? 4)-2-deoxy-2-acetamido-1-O-(glycolic-1,2-
D
-glycero-a-D-galacto-non-2-
D-galactopyranosyl]-(1 ? 4)-3-
D
D-
99.0(C000-1), 95.6(CCl3), 95.2(CCl3), 75.1, 74.9, 74.8, 74.6, 74.4,
74.1, 74.0, 73.5, 73.4, 72.5, 72.3, 72.3, 71.8, 70.0, 69.4, 68.4, 68.2,
67.9, 67.5, 67.4, 67.3, 63.2, 62.5, 62.0, 56.2, 53.5, 52.8, 38.2, 37.9,
37.4, 37.2, 32.0, 29.8, 27.1, 23.3, 21.3 ppm; HRMS (ESI) calcd. for
didodecanoyl-sn-glycero-3-phosphoethanolamido)-2-deoxy-b-
glucopyranoside (5)
D-
Compound 26 (10 mg, 0.009 mmol) in methanol was added
AmberliteÒIR 120H+ in one portion. After stirred for 30 min, the
solution was filtered and evaporated under reduced pressure. The
dry residue was dissolved in DMF (2 mL), followed by addition of
EDC-HCl (3 mg, 0.018 mmol, 2 equiv.), HOBt (6 mg, 0.045 mmol,
C
109H113Cl6N3O42SNa [M+Na]+ 2400.4512, found m/z 2400.4566.
4.2.9. S-[5-acetamido-3,5-dideoxy-
ulopyranosylonate]-(2 ? 6)-4-O-[b-
O-[6-O-benzyl-2-deoxy-2-acetamido-b-
O-[b- -galactopyranosyl]-(1 ? 4)-6-O-benzyl-2-deoxy-2-acetamido-
1-O-glycolic-2-deoxy-b- -glucopyranoside (25)
Freshly acid washed activated Zn dust (60 mg) was added to a
stirred solution of compound 6 (60 mg, 0.025 mmol), Ac2O (6 L,
0.063 mmol) and Et3N (18 L, 0.126 mmol) in dichloromethane
(1.2 mL). The reaction vessel was sonicated in an ultrasonic clean-
D
-glycero-
-galactopyranosyl]-(1 ? 4)-3-
-glucopyranosyl]-(1 ? 3)-4-
a-D-galacto-non-2-
D
D
5 equiv.), DLPE (5 mg, 0.009 mmol, 1 equiv.), and NMM (5 lL,
D
0.045 mmol, 5 equiv.) gradually. After the desired product was
detected, the solution was evaporated and purified by column
chromatography three times (Sephadex LH-20, chloroform/metha-
nol, 1/9) to afford compound 5 (7.6 mg, 50% yield) as brown oil; 1H
NMR (CD3OD and CDCl3, ratio = 4:1, 400 MHz) d 5.32–5.30 (m, 1H),
5.20–5.19 (m, 1H), 4.88–4.85 (d, J = 18 Hz, 1H), 4.55–4.51 (m, 2H),
D
l
l
Please cite this article in press as: Cheng H.-W., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.02.012

H.-W. Cheng et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
9
4.15–4.11 (m, 2H), 4.01–3.95 (m, 10H), 3.56–3.50 (m, 6H), 3.36–
3.34 (m, 2H), 3.34 (d, J = 12 Hz, 1H), 3.15–3.05 (m, 6H), 2.71 (s,
1H), 2.67–2.65 (m, 1H), 2.52 (t, J = 12 Hz, 1H), 2.45 (t, J = 10 Hz,
1H), 2.39–2.38 (m, 2H), 2.31–2.31 (s, 2H) 2.28 (s, 3H, CH3), 2.06–
1.95 (m, 7H), 1.79–1.64 (m, 8H), 1.56–1.52 (m, 9H), 1.07 (t, J =
10.8 Hz, 3H), 085–0.82 (m, 15H) ppm; 13C NMR (CD3OD and CDCl3,
ratio = 4:1, 100 MHz) d 181.6, 181.5, 174.9, 175.0, 174.8, 173.9,
173.5, 103.0, 102.9, 102.7, 100.3, 78.9, 75.9, 75.2, 75.0, 74.8, 74.5,
73.9, 73.5, 72.7, 72.2, 72.1, 71.6, 71.1, 70.7, 70.6, 70.5, 70.4, 70.3,
70.0, 69.95, 69.91, 69.8, 69.7, 69.3, 68.5, 68.5, 68.4, 68.1, 68.0,
68.0, 63.66, 63.62, 62.4, 61.66, 61.63, 61.0, 55.0, 52.5, 40.5, 40.4,
33.9, 33.8, 31.8, 29.5, 29.4, 29.2, 29.0, 24.7, 23.3, 22.5, 20.1, 13.5
2627-M-002-009; 106-2627-M-002-001) and Academia Sinica in
Taiwan for financial support. We gratefully thank Professor Y. S.
Edmond Cheng at the Genomics Research Center of Academia
Sinica for the influenza virus assay and valuable discussion.
A. Supplementary data
Supplementary data (synthesis of compound 11, as well as
physical parameter of liposomes and the calculation mol% of 5
on the liposome surface.) associated with this article can be found,
in the online version, at https://doi.org/10.1016/j.bmc.2018.02.
012.
ppm; HRMS (ESI) calcd. for
C
70H123N4NaO37PS [M+Na]+
1697.7217, found m/z.1697.7259.
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Acknowledgements
(LSTc)-bearing liposomal decoys capture influenza
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A virus. J Biol Chem.
We acknowledge the Ministry of Science and Technology
(MOST 104-2320-B-002-008-MY3; 104-2627-M-002-011; 105-
Please cite this article in press as: Cheng H.-W., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.02.012