Multivalent oleanolic acid human serum albumin conjugate as nonglycosylated neomucin for influenza virus capture and entry inhibition

Article abstract of DOI:10.1016/j.ejmech.2017.10.070

We report the synthesis of multivalent oleanolic acid (OA) protein conjugates as nonglycosylated neomucin mimic for the capture and entry inhibition of influenza viruses. Oleanolic acid derivatives bearing an amine-terminated linker were synthesized by esterification of carboxylic acid and further grafted onto the human serum albumin (HSA) via diethyl squarate method. The binding of hemagglutinin (HA) on the virion surface to the synthetic neomucin was evaluated by hemagglutination inhibition assay. The influenza virus capture ability of the PEGylated OA-HSA conjugate was further investigated by Dynamic Light Scattering (DLS), virus capture assay and Isothermal Titration Calorimeter (ITC) techniques. The pronounced agglutination of viral particles, the high capture efficiency and affinity constant indicate that this neoprotein is comparable to natural glycosylated mucin, suggesting that this material could potentially be used as anti-infective barriers to prevent virus from invading host cells. The study also rationalizes the feasibility of antiviral drug development based on OA or other antiviral small molecules conjugated protein strategies.

Full text of DOI:10.1016/j.ejmech.2017.10.070

Accepted Manuscript
Multivalent oleanolic acid human serum albumin conjugate as nonglycosylated
neomucin for influenza virus capture and entry inhibition
Yang Yang, Hao-Jie He, Hao Chang, Yao Yu, Mei-Bing Yang, Yun He, Zhen-Chuan
Fan, Suri S. Iyer, Peng Yu
PII:
S0223-5234(17)30872-3
10.1016/j.ejmech.2017.10.070
EJMECH 9862
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 August 2017
Revised Date: 22 October 2017
Accepted Date: 25 October 2017
Please cite this article as: Y. Yang, H.-J. He, H. Chang, Y. Yu, M.-B. Yang, Y. He, Z.-C. Fan, S.S.
Iyer, P. Yu, Multivalent oleanolic acid human serum albumin conjugate as nonglycosylated neomucin
for influenza virus capture and entry inhibition, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.10.070.
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Multivalent oleanolic acid human serum
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entry inhibition
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Yang Yang, Hao-Jie He, Hao Chang, Yao Yu, Mei-Bing Yang, Yun He, Zhen-Chuan Fan, Suri S. Iyer,
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European Journal of Medicinal Chemistry
journal homepage: www.elsevier.com
Multivalent oleanolic acid human serum albumin conjugate as nonglycosylated
neomucin for influenza virus capture and entry inhibition
Yang Yang a, Hao-Jie He a, Hao Chang a, Yao Yu a, Mei-Bing Yang a, Yun He b, Zhen-Chuan Fan c,
Suri S. Iyer d, , Peng Yu a,
,
a China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, College of Biotechnology, Tianjin University
of Science and Technology, Tianjin 300457, China
b Research Institute of Tsinghua University in Shenzhen, Nanshan District, Shenzhen 518057, China
c Key Laboratory of Food Nutrition and Safety of Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science and
Technology, Tianjin 300457, China
d Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30302, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
We report the synthesis of multivalent oleanolic acid (OA) protein conjugates as
nonglycosylated neomucin mimic for the capture and entry inhibition of influenza viruses.
Oleanolic acid derivatives bearing an amine-terminated linker were synthesized by esterification
of carboxylic acid and further grafted onto the human serum albumin (HSA) via diethyl squarate
method. The binding of hemagglutinin (HA) on the virion surface to the synthetic neomucin was
evaluated by hemagglutination inhibition assay. The influenza virus capture ability of the
PEGylated OA-HSA conjugate was further investigated by Dynamic Light Scattering (DLS),
virus capture assay and Isothermal Titration Calorimeter (ITC) techniques. The pronounced
agglutination of viral particles, the high capture efficiency and affinity constant indicate that this
neoprotein is comparable to natural glycosylated mucin, suggesting that this material could
potentially be used as anti-infective barriers to prevent virus from invading host cells. The study
also rationalizes the feasibility of antiviral drug development based on OA or other antiviral
small molecules conjugated protein strategies.
Available online
Keywords:
Oleanolic acid
Hemagglutinin inhibitor
Human serum albumin conjugate
PEGylation
Isothermal titration calorimetry
2009 Elsevier Ltd. All rights reserved
.
humans emphasizes the need for newer methods of prevention
and treatment. In fact, China CDC warrants continuous
surveillance of live poultry markets for the appearance of
pathogenic avian influenza since 2017 7. Also, novel methods and
strategies are required for the development of new antiviral
agents targeting different steps in the viral life cycle for resistant
1. Introduction
Influenza virus is a deadly respiratory virus capable of
causing significant harm to the population. For example, recent
outbreaks of avian H7N9 influenza in South China have caused
79 deaths and 192 hospitalizations in January 20171. This
outbreak is far from over as new cases of infection and mortality
are being reported2. Currently, Zanamivir (Renlenza®) and
Osetamivir (Tamiflue®) as neuraminidase inhibitors (NAIs),
which can competitively occupy the active site of the NA and
disturbing virus propagation3,4 have been approved in many
countries and used for the treatment of H7N9 virus infections.
However, resistance to these drugs have emerged with two
strains 8-10
.
In the past few years, a new strategy inspired by mucin11, a
key proteoglycan component of the mucosal layer covering
epithelial cells in many tissues of humans and animals as part of
defense systems, has been considered in the generation of new
antiviral conjugates12. Although the intricate mechanistic details
by which mucin prevents influenza from infection is still under
investigation, it is generally accepted that these highly
glycosylated protein with large molecular weight (>100 kD) can
strains containing the NAIs resistant mutations in this 2017
5
pandemic as reported by China CDC and WHO . Although the
resistant strains could be managed by oral administration of large
dose of Tamiflue®6, the side effects can be significant.
13
block the virial hemagglutinin (HA)-based binding to the host
Additionally, the constant emergence of NAIs-resistant viruses in
———
Corresponding author. e-mail: fanzhen@tust.edu.cn ( Z. C. Fan)
Corresponding author. e-mail: siyer@gsu.edu (S. S. Iyer)
Corresponding author. e-mail: yupeng@tust.edu.cn (P. Yu)

2
European Journal of Medicinal Chemistry
cell and neuramidinase (NA)-based releasing14 from the infected
cell via multivalent sialic acid (SA)-HA/NA interactions.
Researchers have attempted to mimic Nature by attaching
multiple copies of SA or SA analogs to polymers, dendrimers,
were attached onto one molecule of HSA (Table 1). The relative
insolubility and steric hindrance of the functional ligands 5 and 6
may affect the coupling efficiency.
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To improve the hydrophilicity and chain flexibility, we chose
poly (ethylene glycol) (PEG), which has been widely used in
protein chemistry for crosslinking34,35 as the linker for the HSA
conjugation. An uncapped 2,000 average molecular weight PEG
diol HO-PEG-OH was selected and subjected to OA
functionalization. Mono tosylation to HO-PEG2000-OH can be
achieved by using Ag2O/KI 36. The resulting monotosylate was
converted to amine 8 via ammonolysis with ammonia. This
product was subsequently reacted with (Boc)2O to give amide 9.
Repeating the tosylation at the other end of the alcohol and
substitution of tosylate with LiBr gave the corresponding
bromide 10 with a yield of 85% 37. According to the established
liposome and even metal nanoparticles as effective antiviral
barrier for anti-influenza drug development
15
16. Some of the
studies have shown promise under various assay conditions.
However, there are two drawbacks to these multivalent
glycoconjugates. First, the natural O-linked sialosides could be
hydrolyzed by NA17 leading to the decrease of the binding
activity. To overcome this problem, studies using pseudo (S, N
and C) sialyl α-2,3 or α-2,6 galactosylsaccharide have been
synthesized 18 and conjugated onto different scaffolds 19,20, which
are stable towards NA cleavage. A second and more pressing
concern is that natural and modified SA conjugates are not
synthesis friendly, as numbers of protection/deprotection
strategies with multiple purification steps are required.
Chemoenzymatic strategies for the synthesis of complex
esterification method, PEG2000-OA,
4 was successfully
synthesized in 97% yield. After treatment of 4 with TFA, a
similar squaric acid diethyl ester procedure was applied for the
preparation of PEGylated OA decorated HSA. After dialysis,
purification on Sephadex G-75 and lyophilization, the HSA-OA
conjugates were characterized by MALDI-TOF mass
spectrometry again. Mass analysis of HSA-PEG2000-OA
conjugate showed that an average number of 5 PEG2000-OA
was attached per one molecule of HSA. Compared with
corresponding parent OA, all of the HSA-OA conjugates had
high solubility in PBS, indicating its further application in
bioassay system. Methoxypolyethylene glycols modified HSA
(HSA-PEG2000-OMe) was also prepared as negative control.
carbohydrates are gaining more recognition21-23
, but the
engineering enzymes and improving susbstrate specificity, etc.
are also labor and time intensive. While carbohydrate based drug
development will continue to be a major force in the near and
distant future, developing novel nonglycosylated influenza
inhibitors, which are relatively easy to synthesize is highly
desirable. Here, we report our initial studies with a naturally
occurring compound, oleanolic acid (OA) that functions as a
broad spectrum entry inhibitor of influenza viruses.
OA belongs to pentacyclic triterpene family, with only one
carboxyl and hydroxyl group has been recently reported to have
tighter binding to HA with disassociation constant KD around
micromolar range, which is much higher than monomeric SA
Table 1. Conjugation of OA derivatives with HSA
Molecular Weighta
Loading OA
residues
Entry
25,26
with millimolar KD 24. Conjugation of OA onto cyclodextrin
has been shown to significantly enhanced its influenza entry
inhibitory activity. Encouraged by the dramatic activity
enhancement induced by the cluster effect 27,28, we designed
design and prepare OA protein conjugates as multivalent
nonglycosylated neomucin for the development of influenza
capture and entry inhibitor. Specifically, natural protein presents
in plasma (Human Serum Albumin, HSA) was adopted as the
artificial mucin backbone because of its availability,
biodegradability, nontoxicity and nonimmunogenicity, which is
well established in our previous work29. The molecular weight of
this new neomucin was easily characterized by matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF-MS)30, which makes it feasible to quantitatively
measure the virus interactions. Finally, the virus capture and
entry inhibitory ability of the synthetic nonglycosylated
neomucin was demonstrated.
HSA
66740.67
79812.97
67820.62
69147.45
80251.87
0
0/6b
2
HSA-PEG2000-OMe
HSA-C6- OA
HSA-OEG-OA
HSA-PEG2000- OA
3
5
a Molecular weight was determined by MALDI-TOF
b
Number of molecules of the PEG2000OMe derivative, 13 attached to
HSA
2.2. Bioassay
2.2.1. HAI assay
An easy-to-perform hemagglutination inhibition (HAI) assay
38 was initially applied for evaluating the binding of the influenza
virus to the synthetic HSA-OA conjugates. We choose three
different virus strains that have different HA subtypes to
demonstrate broad specificity. Table 2 showed the results of Ki 39
which defined as the lowest concentration of the OA derivatives
that prevented hemagglutination, using human influenza virus
[A/Puerto Rico/8/1934 (H1N1), A/Huairou, Beijing/11069/2014
2. Results and discussion
2.1.Chemistry
,
The syntheses of the HSA-OA conjugates are summarized in
Schemes 1. To realize covalent coupling between OA and HSA,
azide as the precursor of amine was first introduced into the 28-
(H3N2)]
or
chicken
influenza
virus
position of OA.
Esterification of OA with 1-azido-6-
[A/Chicken/Beijing/AT609/2014 (H9N2)] as the sources of HA.
It is evident that all the HSA-OA conjugates bound influenza
virus more strongly than HSA and HSA-PEG2000-OMe, which
doesn’t possess OAs. Compared with OA monomer, the lower Ki
of the conjugates indicated the cluster effect
bromohexane or 1-azido-2-(2-(2-bromoethoxy)ethoxy)ethane
gave azide, C6-OA, 2 (42%) and OEG-OA, 3 (45%) ,
respectively31,32. After reducing the azide with Lindlar catalyst
under hydrogen atmosphere, the resultant amine was first reacted
with one of the esters of squaric acid diethyl esters in potassium
27,28
induced by the
covalent attachment of multiple OA residues onto the HSA
backbone. Moreover, HSA-PEG2000-OA was the best
agglutinating conjugate; its binding efficiency is comparable with
mucin, a naturally occurring protein from bovine submaxillary
glands (BSG, MW ~ 400 kDa), which has been used as a control
to define the potency of HA inhibitor40. The decreased Ki value
of HSA-PEG2000-OA also suggested that the PEGylation
phosphate buffer (pH 7.0) to give
5 and 6, respectively. After
purification with column chromatography, the remaining ester
group of 5 or 6 was coupled with the amines on the lysine residue
side chains of HSA in borate buffer (pH 9.0) to obtain the
conjugates HSA-C6-OA or HSA-OEG-OA 29,33. The number of
OA derivatives attached onto protein backbone was determined
by MALDI-TOF-MS (see Supplementary Material). Mass
analysis of 5 or 6 revealed that about only 2 or 3 OA molecules

3
ACCEPTED MANUSCRIPT
f
g
e
HO
OH
HO
NH2
HO
NHBoc
Br
NHBoc
O
O
O
O
43
43
43
43
HO-PEG2000-OH
8
9
10
MW~2000
HO-PEG2000-NH2
d
a
b
R'
R
C
C
COOH
O
O
HO
HO
2, R=
HO
O
O
OA
O
O
O
O
5, R'=
N3
NH
OEt
C6-OA
O
O
6, R'=
7, R'=
N3
O
O
O
O
3, R=
NH
OEt
OEG-OA
O
O
c
OEt
NH
4, R=
O
NHBoc
O
O
O
43
43
PEG2000-OA
HSA-OA conjugates
O
O
O
O
O
O
O
NH
N
O
C
O
H
C
O
NH
N
H
HO
HO
HSA-C6-OA
HSA-OEG-OA
O
O
O
O
N
NH
O
O
H
C
O
N
H
NH
H3CO
43
O
43
HO
HSA-PEG2000-OA
HSA-PEG2000-OMe
O
NH
13
O
d
b
h
N3
OH
H3CO
H3CO
OEt
O
H3CO
O
O
43
43
43
12
11
Scheme 1. Synthesis of HSA-OA conjugates. (a) K2CO3, bromide linker; (b) (i) H2, Lindlar catalyst (ii) squaric acid diethyl esters, phosphate
buffer saline (pH 7.0); (c) (i) TFA/CH2Cl2 (ii) squaric acid diethyl esters, phosphate buffer saline (pH 7.0); (d) HSA, borate buffer buffer (pH
9.0); (e) (i) Ag2O, KI, TosCl (ii) NH3 H2O, NH4Cl; (f) (Boc)2O, Et3N, CH2Cl2; (g) (i) TosCl, Et3N, CH2Cl2 (ii) LiBr/acetone, overnight; (h) (i)
TosCl, Et3N, CH2Cl2 (ii) NaN3/DMF, overnight.

4
is
European Journal of Medicinal Chemistry
for ACCEPTED MANUSCRIPT
a
useful modification strategy
improving the
the conjugation complex, which will be easily precipitated
biocompatibility and bioactive compound efficacy.
by centrifugation. This study strongly demonstrated HSA-
PEG2000-OA can capture the virus via specific adsorption
to the HA on the virial surface. These results clearly indicate
the OA modified neoprotein functions similarly to natural
mucin.
2.2.2. Dynamic Light Scattering, DLS
The successful synthesis and influenza binding
bioactivity verifications of biocompatible HSA-PEG2000-
OA encouraged us to further study the capturing ability of
Table 3. Capture of influenza virus by HSA-OA conjugate
HSA-PEG2000-OA
.
Table 2. Inhibition constant Ki of HAI assay
Compounds
Virus
Relative NA
activity
Entry Ki (µM)
H1N1
H3N2
H9N2
49%
56%
48%
H1N1
125
NA
60
H3N2
125
62.5
60
H9N2
125
62.5
30
HSA-PEG2000-OMe
HSA
HSA-PEG2000-OMe
OA
H1N1
H3N2
H9N2
15%
43%
19%
HSA-C6-OA
HSA-OEG-OA
HSA-PEG2000-OA
Mucin
108
54
54
54
HSA-PEG2000-OA
13.5
3
13.5
3
3
0.16
0.16
0.16
Virus capture behavior of the HSA-PEG2000-OA was
thoroughly investigated by Dynamic Light Scattering, a
technique that can provide a quantitative estimate of the
average diameter of the aggregates induced by the
adsorption of numerous virions with HSA-OA conjugate 41,42
From the DLS measurements, the size of the HSA
conjugates and viral particles were found to be around 15
nm and 120 nm, respectively. After incubation with virus for
1 h, the mean hydrodynamic diameters of HSA-PEG2000-
OA had increased to ~ 300 nm and the peak of the virus
almost disappeared, clearly indicating aggregate formation
(Figure 1). However, no aggregation was observed after the
addition of the virus to HSA-PEG2000-OMe solution, as no
larger standard deviation was measured. The DLS peaks
clearly showed our HSA-OA conjugate could cross-link
several virus particles and could be a promising capture
biomacromolecule for influenza inhibition and detection
development.
.
2.2.3. Virus capture assay
To further examine the virus-capture capability of HSA-
PEG2000-OA, a simple assay was applied using the
solution mixture after DLS measurement. The mixture in the
sample cell for the DLS analysis was centrifuged at 3000
rpm for 5 min. The supernatant was added into black 384-
well
plate
and
25
µL
MUNANA
[2´-(4-
Methylumbelliferyl)-α-
D
-N-acetylneuraminic acid] in 0.1 M
sodium acetate buffer (pH 5.5) containing 10 mM CaCl2 was
added. The hydrolysis of MUNANA by neuraminidase (NA)
on the virial surface producing the fluorescent signal was
monitored for the NA activity43, 44. The NA activity in the
absence of HSA conjugates was defined as 100% and in the
presence of HSA-PEG2000-OMe was used as negative
control, respectively.
As shown in Table 3, the virus activity for H1N1 and H9N2
was dramatically decreased when incubated with HSA-
PEG2000-OA conjugate, indicating its capture efficiency,
For H3N2, the activity was less pronounced; we are not
clear why this is the case at this time. It was also found that
HSA-PEG2000-OMe has a moderate adsorption activity,
which may be attributed to the nonspecific electrostatic
interactions between the positive charges of cations on the
viral surface and negative charges on the macromolecule
surface45. Thus the virus would be captured and further form
Fig 1. Hydrodynamic size distribution curves of the influenza virus a)
Influenza A/Puerto Rico/8/1934 (H1N1), b) Influenza A/Huairou,
Beijing/11069/2014 (H3N2), c) Influenza A/Chicken/Beijing/AT609/2014 (H9N2)
before and after mixed with HSA-PEG2000-OA or HSA-PEG2000-OMe
.

5
2.2.4. Isothermal Titration Calorimeter, ITC
carrying luciferase reporter gene to simulate the actual
ACCEPTED MANUSCRIPT
infection process was used to evaluate the inhibitory activity
To quantitatively analyze the interactions between virus and
the HSA-OA conjugate, ITC was then performed to characterize
the thermodynamic binding affinity46,47. The number of the virus
was determined by haemagglutination titration which converted
to virus particles by multiplying by 109. The concentration of
virus particles was calculated on the basis of 105 particles/L
equivalent 10-13 mol/L48,49. In each ITC experiment, the HSA-
PEG2000-OA solution in PBS was loaded into the syringe and
titrated into the influenza virus solution in the calorimeter cell.
The heat released at each injection was used to calculate the
thermodynamic parameters as described in the experimental
section. HSA-PEG2000-OMe was used as the negative control
(Table 4).
50
of HSA-PEG2000-OA
. This assay system is an ideal
platform for the virus entry inhibition study (Figure 3). The
quantity of virus represented by luciferase activity, was
determined by the luciferase reporter gene assay using
fluorescence microplate reader for chemiluminescence
following the protocols provided by the manufacture
(Promega).
As shown in the insert column in Figure 3, unimolecular OA
or HSA-PEG2000-OMe exhibited low inhibitory activity against
H7N9 pseudovirus at 20 µM, probably due to the nonspecific
electrostatic interactions with viral particles. In contrast, the
HSA-PEG2000-OA inhibitory activity is much higher. These
data suggested that our non sialyl protein conjugate could
potentially be considered as a simpler alternative strategy for the
construction of mucin mimic as antiviral barriers and entry
inhibitor.
Table 4. Disassociation constant KD of HSA conjugate−influenza virus
interactions measured by ITC
Virus
H1N1
H3N2
HSA-PEG2000-OMe
HSA-PEG2000-OA (µM)
NBa
NB
NB
32.6
20.9
21.4
H9N2
a No binding
Significant binding of HSA-PEG2000-OA to all strains was
observed with KD in the µM range. A representative figure of the
ITC graphs with influenza virus H9N2 (Figure 2a) depicts large
favorable enthalpic and entropic contributions. In contract, no
binding was detected for HSA-PEG2000-OMe (Figure 2b),
indicating the specific binding was derived from the OA motif.
To the best of our knowledge, this is the first time measuring the
KD value with intact virus from ITC experiments, which clearly
highlighted the feasibility for the quantitative investigation of the
interactions between biomacromolecules and intact viruses.
Fig 3. Schematic illustration of pseudovirus infection assay. Insert: inhibitory
activity of HSA-PEG2000-OA (data derived from the mean of three independent
experiments)
.
3. Conclusions
OA functionalized HSA was prepared via squaric acid diethyl
esters strategy as nonglycan protein conjugates for mucin mimic.
The multivalent binding to influenza virus was probed by HAI,
DLS, ITC and virus capture assay. The antiviral activity was also
investigated by in vitro infection assays. The results showed that
this PEGylated OA-HSA conjugate exhibit strong virial capture
capability and entry inhibitory activity. Our results compare well
with other reports that use cyclodextrin-OA conjugates25,26 for
virus inhibition.
b)
a)
4. Experimental
4.1. Material
All materials were obtained from commercial suppliers, and
were used without further purification. All solvents were
commercially available grade. Thin-layer chromatography (TLC)
was purchased from EMD Co. Ltd. (German). All compounds
were stained with 5% phosphomolybdic acid in ethanol or iodine
vapors. Spots of compounds were also visualized with UV light
when possible. Flash column chromatography was performed on
silica gel 200-300 mesh. The final OA analogs and protein
conjugates were purified using SephadexTM LH-20 and
SephadexTM G-75 (GE Healthcare), respectively. Human Serum
Albumin was purchased from Sigma-Aldrich. Influenza viruses
were obtained from National Institute for Viral Disease Control
and Prevention, China CDC and propagated in 9 days old
embryonated chicken eggs. The allantoic fluid was collected,
then centrifuged at 3000 rpm min-1 for 10 min and the
supernatant was stored at -80 ℃. Virus was inactivated by the
addition of β -propiolactone ( β -PL) when assays were
conducted. H7N9 pseudoviruses was kindly provided by Dr. Hai-
Fig 2. ITC titration curves obtained at 298 K for the titration of HSA-
PEG2000-OA (60 µM) with a) 16 nM Influenza A/Chicken/Beijing/AT609/2014
(H9N2); b) 16 nM Influenza A/Chicken/Beijing/AT609/2014 (H9N2). The top
panel showed the raw calorimetric data. Binding parameters were auto-generated
after curve fitting using Microcal Origin 7.
2.2.5. Inhibition of H7N9 pseudovirus
Finally, a cell based assay was used to demonstrate the
anti-infective properties of our lead compound. Briefly,
a
single-cycle H7N9 pseudovirus enveloped with influenza
surface protein HA and NA, and packed with HIV backbone

6
European Journal of Medicinal Chemistry
ACCEPTED MANUSCRIPT
Xia Xiao, Tianjin Institute of Industrial Biotechnology, Chinese
Academy of Sciences.
with CH2Cl2. The solvent was evaporated and the residue was
directly dissolved in 25% ammonia and NH4Cl (0.69 g, 12.94
mmol) was added. The reaction mixture was stirred at rt for 12 h,
and extracted with CH2Cl2. The organic extracts were dried over
Na2SO4. The solvent was removed under reduced pressure and
the residue purified by gradient flash silica gel column
chromatography to give H2N-PEG-OH (1.4 g, 87.5%). The
product was further purified by recrystallization from diethyl
4.2. Measurements
NMR spectra were recorded on Bruker AVANCE III (400
MHz) instruments. Chemical shifts (δ) were reported in parts per
million downfield from TMS, the internal standard; J values were
given in Hertz. The molecular weights of the OA conjugates
were confirmed by Bruker ultrafleXtreme MALDI-TOF/TOF
mass spectrometer (Bruker Daltonics, Bremen, Germany).
Fluorescence and chemiluminescence intensity was measured
using the SynergyTM H1/H1MF microplate reader (BioTek
Instruments, Inc. USA). Dynamic light scattering was recorded
on a Zata Sizer Nano apparatus (ZEN3690, Malvern Instruments
Ltd, U.K.). Microcalorimetric experiments were performed with
an Isothermal Titration Calorimeter (ITC200) (Microcal Inc.,
Northampton, MA). Solutions of the analytes in buffer were
filtered through a 0.22 µm micro membrane and degassed before
being injected.
1
ether. H NMR (400 MHz, CDCl3) δ 3.84 – 3.82 (t, J = 4.8 Hz,
2H), 3.58 (br, 173H), 3.14 – 3.11 (t, J = 4.8 Hz, 2H).
4.3.5. Br-PEG2000-NHBoc, 10
TEA (211 mg, 2.07 mmol) was added to a stirred solution of
HO-PEG2000-NH2 in CH2Cl2. After stirring for 0.5 h, (Boc)2O
was slowly added to the reaction mixture, and further stirred at rt
overnight. The reaction mixture was washed using 1 M HCl and
extracted with CH2Cl2 three times, and the organic extracts were
dried over Na2SO4. The solvent was removed under reduced
pressure and the residue (OH-PEG2000-NHBoc, 9) was
dissolved in CH2Cl2/Et3N and TsCl (3 eq.) was added at 0 ℃.
The resulting solution was stirred for 8 h at rt. Upon completion,
the reaction mixture was washed by HCl (1 M) and extracted
with CH2Cl2 three times. The organic phase was combined,
washed with brine, dried over Na2SO4 and evaporated to dryness.
The residue was dissolved in dry acetone and lithium bromide (4
eq.) was added. The reaction mixture was stirred and refluxed for
12 h. The suspension was filtered and the filtrate was
concentrated. The residue was washed with distilled water and
extracted with CH2Cl2 three times. The organic phase was
combined, washed with brine, dried over Na2SO4 and evaporated
to dryness. The residue was purified by gradient flash silica gel
4.3. Chemistry
4.3.1. General procedure for the esterification of oleanolic acid
To a solution of oleanolic acid (1.1 eq) and bromide (1 eq) in
DMF K2CO3, (2 eq) was added. The reaction mixture was stirred
at rt for 6–18 h and concentrated in vacuo. The residue was
diluted with CH2Cl2, and the extract was washed successively
with 1 M HCl, satd. NaHCO3, and brine, dried with Na2SO4,
filtered, and concentrated in vacuo. The residue was purified by
column chromatography.
4.3.2. 6-Azidohexyl 3β-hydroxyolean-12-en-28-oate (C6-OA, 2)
Prepared from OA (2.44 g, 5.34 mmol) and 1-azido-6-
bromohexane (1 g, 4.85 mmol) according to the general
procedure 4.3.1. The residue was purified by column
chromatography [petroleum ether (PE)/EtOAc, 20/1 v/v] to give
1
column chromatography to give Br-PEG2000-NHBoc, 10. H
NMR (400 MHz, CDCl3) δ 5.06 (s, 1H), 3.74 – 3.23 (m, 213 H),
1.37 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 155.93, 71.13, 70.50,
70.13, 40.29, 30.33, 28.39.
1
C6-OA, 2 as colorless oil (1.18 g, 42%). H NMR (400 MHz,
4.3.6. PEG2000-OA, 4
DMSO-d6) δ 5.18 (t-like, 1H), 4.28 (d, J = 5.1 Hz, 1H), 3.94 (dd,
J = 6.0, 4.4 Hz, 2H), 3.31 (d, J = 5.3 Hz, 2H), 3.02 – 2.97 (m,
1H), 2.79 (dd, J = 13.6, 3.9 Hz, 1H), 1.99 – 1.92 (m, 1H), 1.81
(d, J = 6.9 Hz, 2H), 1.63 – 1.40 (m, 16H), 1.34 – 1.30 (m, 5H),
1.24 – 1.20 (m, 2H), 1.10 (s, 4H), 0.89, 0.88, 0.85, 0.67, 0.66 (5s,
21H). 13C NMR (100 MHz, DMSO-d6) δ 176.97, 143.93, 122.33,
79.67, 77.25, 64.00, 55.26, 51.03, 47.51, 46.48, 45.87, 41.69,
41.37, 38.83, 38.53, 37.02, 33.67, 33.21, 32.86, 32.58, 30.82,
28.74, 28.67, 28.46, 27.54, 27.42, 26.22, 26.05, 25.64, 23.76,
23.40, 23.02, 18.45, 17.16, 16.46, 15.48.
Prepared from OA (68 mg, 0.15 mmol) and Br-PEG2000-
NHBoc (0.3 g, 0.14 mmol) according to the general procedure
4.3.1. The residue was purified by column chromatography
(CH2Cl2/MeOH, 20/1 v/v) to yield 4 as colorless oil(340 mg,
1
97%). H NMR (400 MHz, CDCl3) δ 5.21 (s, 1H), 5.06 (s, 1H),
4.15 – 4.06 (m, 2H), 3.75 (s, 2H), 3.58 (s, 188H), 3.48 – 3.46 (m,
3H), 3.40 (d, J = 4.4 Hz, 2H), 3.24 (d, J = 4.5 Hz, 2H), 3.14 –
3.12 (d, J = 6.7 Hz, 1H), 2.84 – 2.78 (m, 1H), 2.50 (s, 2H), 1.93 –
1.80 (m, 2H), 1.62 – 1.46 (m, 9H), 1.37 (s, 9H), 1.30 – 1.19 (m,
4H), 1.06, 0.91, 0.85, 0.83, 0.71, 0.66 (6s, 21H).13C NMR (100
MHz, CDCl3) δ 177.54, 155.97, 143.67, 122.36, 78.79, 70.53,
70.18, 69.12, 63.31, 55.19, 47.57, 46.62, 45.84, 41.64, 41.24,
40.32, 39.28, 38.71, 38.42, 36.99, 33.83, 33.09, 32.69, 32.33,
30.66, 28.41, 28.11, 27.61, 27.16, 25.85, 23.60, 23.38, 22.94,
18.30, 16.96, 15.61, 15.31.
4.3.3. 2-(2-(2-azidoethoxy) ethoxy) ethyl 3β-hydroxyolean-12-en-
28-oate (OEG-OA, 3)
prepared from OA (2.1 g, 4.62 mmol) and 1-azido-2-(2-(2-
bromoethoxy)ethoxy)ethane (1 g, 4.2 mmol) according to the
general procedure 4.3.1. The residue was purified by column
chromatography (PE/EtOAc, 15/1 v/v) to give OEG-OA, 3 as
1
colorless oil (1.15 g, 45%). H NMR (400 MHz, DMSO-d6) δ
4.3.7. General procedure for the squaric acid monoamination
A solution of azide in methanol and CH2Cl2 (1/1 v/v) was
hydrogenated with Lindlar catalyst under an atmosphere of
hydrogen for 12 h. Then the suspension was filtered through a
pad of Celite, and washed with methanol. The filtrate was
concentrated under reduced pressure to give colorless foam,
which was dissolved in carbonate buffer (7.0 mL, pH 7.0).
Diethyl squarate (2 eq, based on azide) was added, and the
solution was slowly stirred at rt for 14 h. The solvent was
removed in vacuo and the residue was purified by flash column
chromatography.
5.24 (t-like, J = 3.7 Hz, J = 3.3 Hz, 1H), 4.34 (d, J = 4.9 Hz, 1H),
4.19 – 4.06 (m, 2H), 3.67 – 3.64 (t, J = 4.8 Hz, 4H), 3.61 (s, 3H),
3.45 – 3.42 (m, 2H), 3.06 – 3.04 (m, 1H), 2.87 – 2.83 (m, 1H),
2.05 – 1.98 (m, 1H), 1.87 – 1.86 (m, 1H), 1.69 – 1.61 (m, 4H),
1.57 – 1.48 (m, 8H), 1.39 – 1.35 (m, 2H), 1.32 – 1.08 (m,6H),
0.95, 0.94, 0.91, 0.73 (4s, 21H). 13C NMR (100 MHz, DMSO-d6)
δ 177.02, 143.83, 122.37, 77.28, 70.33, 70.24, 69.82, 68.86,
63.71, 55.27, 50.49, 47.53, 46.49, 45.89, 41.70, 41.35, 38.85,
38.56, 37.04, 33.67, 33.22, 32.82, 32.47, 30.83, 29.52, 28.68,
27.57, 27.43, 26.07, 23.79, 23.41, 23.01, 18.45, 17.11, 16.48,
15.54.
4.3.8. 6-(2-ethoxy-3, 4-dioxocyclobut-1-en-1-amino) hexyl 3β-
hydroxyolean-12-en-28-oate (5)
4.3.4. OH-PEG2000-NH2, 8
To a stirred solution of diol HO-PEG2000-OH (2g, 1 mmol)
in CH2Cl2, fresh Ag2O (350 mg, 1.5 mmol), TsCl (210 mg, 1.12
mmol) and KI (33 mg, 0.2 mmol) was added. The reaction
mixture was stirred at rt for 4 h, filtered with celite and washed
Prepared from 2 (1.18 g, 2.03 mmol) according to the general
procedure 4.3.7. The residue was purified by column
chromatography (PE/EtOAc, 2/1 v/v) to give 5 as colorless syrup
1
(560 mg, 50%). H NMR (400 MHz, DMSO-d6) δ 5.18 (t-like,

7
1H), 4.69 – 4.64 (m, 2H), 4.32 (d, J = 5.0 Hz, 1H), 3.95 (t, J =
5.6 Hz, 2H), 3.48 – 3.45 (m, 1H), 3.30 – 3.25 (m, 2H), 3.01 –
2.97 (m, 1H), 2.80 (d, J = 10.0 Hz, 1H), 2.00 – 1.92 (m, 1H),
1.81 (s, 2H), 1.67 – 1.43 (m, 16H), 1.39 – 1.36 (m, 5H), 1.34 –
1.31 (m, 6H), 1.10 (s, 4H), 0.90, 0.89, 0.83, 0.67, 0.66 (5s, 21H).
13C NMR (100 MHz, DMSO-d6) δ 189.73, 182.47, 176.94,
143.91, 122.34, 79.66, 77.28, 69.13, 64.01, 55.29, 47.52, 46.47,
45.89, 44.20, 43.87, 41.69, 41.38, 39.35, 38.82, 38.55, 37.01,
33.69, 33.21, 32.88, 32.58, 30.80, 30.40, 28.66, 28.44, 27.55,
27.41, 26.04, 25.89, 25.62, 23.76, 23.40, 23.04, 18.45, 17.16,
16.44, 16.10, 15.47.
as eluent then lyophilisation. The characterization of the
ACCEPTED MANUSCRIPT
conjugate was performed using MALDI-TOF-MS in positive
linear ion mode using 2, 5-dihydroxybenzoic acid (DHB) as
matrix. FlexAnalysis was used for analysis of the data.
4.4. Bioevaluation
4.4.1. Hemagglutination inhibition (HAI) assay
HAI assays were performed according to the method described
previously29.
4.4.2. Binding Assay
4.4.2.1. Dynamic Light Scattering (DLS)
4.3.9. 2-[2-(2-2-ethoxy-3, 4-dioxocyclobut-1-en-1-amino) ethoxy]
ethoxy ethyl 3β-hydroxyolean-12-en-28-oate (6)
The hydrodynamic diameters of the HSA-PEG2000-OA with
or without the influenza virus were measured by dynamic light
scattering. HSA-PEG2000-OA or virus in phosphate buffered
saline (pH 7.4) was individually filtered through 0.22 µm water
membrane filters. Glycoconjugates and different strains of
influenza virus were mixed. The resulted solution without further
filtration was transferred into a sample cell for measurement.
DLS measurement was done on a Zata Sizer Nano apparatus
(ZEN3690, Malvern Instruments Ltd, U.K.). Data analysis was
performed using the software provided with the instrument.
Prepared from 3 (1.15 g, 1.87 mmol) according to the general
procedure 4.3.7. The residue was purified by column
chromatography (PE/EtOAc, 2/1 v/v) to give 6 as white syrup
1
(350 mg, 36%). H NMR (400 MHz, DMSO-d6) δ 5.23 (t-like, J
= 3.7 Hz, J = 3.3 Hz, 1H), 4.71 (q, J = 6.7 Hz, 2H), 4.37 (d, J =
5.0 Hz, 1H), 4.18 – 4.04 (m, 2H), 3.68 – 3.66 (m, 1H), 3.63 –
3.62 (t, J = 4.1 Hz, 2H), 3.58 (s, 6H), 3.49 – 3.48 (m, 1H), 3.06 –
3.02 (m, 1H), 2.85 – 2.81 (m, 1H), 2.03 – 1.97 (m, 1H), 1.86 –
1.85 (m, 1H), 1.68 – 1.60 (m, 4H), 1.56 – 1.50 (m, 8H), 1.46 –
1.43 (m, 5H), 1.14 (s, 4H), 0.95, 0.93, 0.90, 0.72, 0.71 (5s, 21H).
13C NMR (100 MHz, DMSO-d6) δ 189.71, 189.56, 182.67,
182.53, 176.93, 143.77, 122.37, 79.64, 77.30, 70.31, 70.20,
69.77, 69.18, 68.86, 63.62, 55.30, 47.55, 46.47, 45.89, 44.18,
41.68, 41.34, 40.65, 40.44, 40.23, 40.02, 39.82, 39.61, 39.40,
38.84, 38.58, 37.03, 33.71, 33.23, 32.84, 32.46, 30.80, 28.66,
27.57, 27.42, 26.06, 23.77, 23.41, 23.01, 18.46, 17.09, 16.44,
16.11, 15.54.
4.4.2.2. Virus Capture Assay
The resulted mixtures of HSA-PEG2000-OA/ HSA-
PEG2000-OMe and different strains of influenza virus in 4.2.2.1
were centrifuged (3000 rpm,
supernatant was transferred, and the quantity of virus was
represented by the NA activity with 4-methylumbelliferyl-a- -N-
5 min), respectively. The
D
acetylneuraminic acid sodium salt (4-MUNANA) as the
fluorescent substrate. The solution in the absence of conjugates
with virus was used as control.
4.3.10. PEG2000-OA-amino squaric acid monoethyl ester, (7)
To a solution of PEG2000-OA, 4 in CH2Cl2, trifluoroacetic
acid /CH2Cl2 (1/1 v/v) was added under 0 ℃, and the reaction
mixture was stirred for 20 min at room temperature. The solution
was evaporated to dryness. The crude material was directly used
in next step. PEG2000-OA-monoethyl ester was prepared
according to the general procedure 4.3.7. The residue was
purified by column chromatography (CH2Cl2/MeOH, 15/1 v/v) to
give 7 as white syrup (180 mg, 63%). 1H NMR (400 MHz,
CDCl3) δ 5.21 (t-like, 1H), 4.69 (q-like, J = 6.7 Hz, 2H), 4.16 (t,
J =4.5 Hz, 2H), 3.58 (s, 187H), 3.40 (t, J =5.2 Hz, 2H), 3.17 –
3.13 (m, 1H), 2.80 (d, J = 9.6 Hz, 1H), 2.67 (d, J = 11.7 Hz, 1H),
2.42 (s, 4H), 2.12 (d, J = 6.8 Hz, 1H), 1.90 – 1.80 (m, 3H), 1.70 –
1.53 (m, 8H), 1.48 – 1.44 (m, 3H), 1.42 – 1.37 (m, 4H), 1.18 (s,
3H), 1.07 (d, J = 8.6 Hz, 4H), 0.91, 0.85, 0.83, 0.80, 0.71, 0.69,
0.66 (7s, 21H). 13C NMR (100 MHz, CDCl3) δ 176.60, 143.78,
137.88, 128.10, 122.09, 86.28, 70.54, 69.43, 69.12, 63.32, 63.19,
55.25, 50.59, 48.44, 47.50, 46.62, 45.83, 44.29, 41.66, 41.31,
39.30, 38.32, 38.00, 37.93, 37.19, 36.87, 36.80, 35.79, 34.78,
33.84, 33.08, 32.93, 32.65, 32.16, 30.67, 29.65, 27.86, 26.98,
25.82, 25.07, 24.13, 23.60, 23.19, 21.63, 20.96, 18.13, 17.50,
16.94, 16.35, 15.34.
4.4.2.3. Isothermal titration calorimetry (ITC)
ITC experiments were performed using an ITC200
Microcalorimeter in PBS buffer. The number of the viral
particles was determined by haemagglutination titration which
converted to virus particles by multiplying 109 as described
previously48,49
. The concentration of virus particles was
calculated on the basis of 105 particles/L equivalent to 10-13
mol/L. The concentration of HSA conjugates was 60 µM, and
that of influenza virus H1N1, H3N2, H9N2 was 4, 8 and 16 nM,
respectively to get a good fit. In each individual experiment, ∼38
µL of 40 µM conjugate solution was injected through the
computer-controlled 40 µL microsyringe at an interval of 2 min
into the viral solution in the same buffer (cell volume = 200 µL)
while stirring at 750 rpm. The experimental data were fitted to a
theoretical titration curve using the software supplied by
MicroCal. A standard one-site model was used with ∆H
(enthalpy change, in kcal/mol), K (association constant, in M−1),
and N (number of binding sites) as the variables.
4.4.3. Entry inhibitory activity against H7N9 pseudovirus
MDCK cells (104/well) were seeded in 96-well plates and
grown overnight. H7N9 pseudotyped particles [HA plasmid from
the H7 subtype strain A, NA plasmid from the N1 subtype strain,
HIV backbone plasmid (pNL4-3.luc.R_E)] was incubated with
appropriate concentrations of tested OA derivates for 30 min at
37 ℃ . Subsequently, the virus-OA derivate mixture was
transferred to the cells and incubated for an additional 48 h. Cells
were washed with phosphate buffer saline (PBS) and lysed with
luciferase cell culture lysis reagent (Promega, Madison, WI).
Aliquots of cell lysates were transferred to 96-well flat bottom
luminometer plates (Costar), followed by the addition of
luciferase assay substrate (Promega). The luciferase activity was
measured in a microplate reader (BioTek Instruments, Inc. USA).
As a negative control, H7N9 pseudotyped particles were
incubated with HSA-PEG2000-OMe. The yield of
pseudoviruses in MDCK cells were reflected by luminescence
intensity.
4.3.11. CH3O-PEG- amino squaric acid monoethyl ester, (13)
Prepared from 12 (2.3 g, 1.13 mmol) according to the general
procedure 4.3.7. The residue was recrystallized in ethyl ether to
give (1.40 g, 82%). 1H NMR (400 MHz, CDCl3) δ 4.76 (q-like, J
= 6.9 Hz, 2H), 3.64 (s, 185H), 3.38 (s, 3H), 1.46 (t, J = 7.1 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 71.89, 70.53, 69.40, 58.98,
15.84.
4.3.12. General procedure for the modification of HSA with OA
monoethyl ester
HSA (1 eq) was dissolved in carbonate buffer (pH 9.0). When
all protein had dissolved, OA monoethyl ester (20 eq) was added,
and the soln was slowly stirred at rt for 18 h. The reaction
mixture was transferred to an Amicon ultrafiltration device
(MWCO 10 kDa, Millipore) and desalted by repeatedly adding
deionized water to the upper chamber. The water was evaporated
to 2 mL and purified by Sephadex® G-75 using deionized water

8
European Journal of Medicinal Chemistry
ACCEPTED MANUSCRIPT
(16) Sun, X. L. Recent anti-influenza strategies in multivalent
Acknowledgments
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This work was financially supported by the Natural Science
Foundation of China (21402140, 81773583), Shenzhen Peacock
Plan (KQTD2016053114253158) and Youth Innovation
Research Foundation of Tianjin University of Science and
Technology (2016LG08). We thank personnel in charge of NMR
and MALDI-TOF mass spectroscopy facilities in the Research
Centre of Modern Analytical Technology at Tianjin University of
Science and Technology. The authors also appreciate National
Institute for Viral Disease Control and Prevention, China CDC
for the viral strains.
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Supplementary Material
1H and 13C NMR spectra of key intermediates and final
glycopolymers and ITC raw data associated with this article can
be found in the online version, at
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ACCEPTED MANUSCRIPT
¶Oleanolic acid−human serum albumin conjugates as nonglycosylated
neomucin mimic were prepared.
¶ITC technology was used in quantificational analysis of the conjugate −
intact influenza affinity.
¶The conjugate can be used as influenza virus adsorbent and entry
inhibitor.