Multivalent zanamivir-bovine serum albumin conjugate as a potent influenza neuraminidase inhibitor

Article abstract of DOI:10.1080/07328303.2017.1390577

A multivalent Zanamivir (ZA) conjugate as new anti-influenza agent was prepared. Specifically, multiple copies of ZA with a C7 azido-terminated linker were covalently attached onto alkynemodified bovine serum albumin (BSA) via copper-free click reaction. The loading number of ZA was determined by MALDI-TOF. Primary neuraminidase (NA) inhibition assay showed that this conjugate retained some NA inhibitory activity compared to its parent ZA monomer. Moreover, the ZA-BSA conjugate was not cytotoxic with concentrations up to 1 μM.

Full text of DOI:10.1080/07328303.2017.1390577

Journal of Carbohydrate Chemistry
ISSN: 0732-8303 (Print) 1532-2327 (Online) Journal homepage: http://www.tandfonline.com/loi/lcar20
Multivalent zanamivir-bovine serum albumin
conjugate as a potent influenza neuraminidase
inhibitor
Teng-Fei Zhao, Hai-Juan Qin, Yao Yu, Mei-Bing Yang, Hao Chang, Na Guo, Yun
He, Yang Yang & Peng Yu
To cite this article: Teng-Fei Zhao, Hai-Juan Qin, Yao Yu, Mei-Bing Yang, Hao Chang, Na
Guo, Yun He, Yang Yang & Peng Yu (2017): Multivalent zanamivir-bovine serum albumin
conjugate as a potent influenza neuraminidase inhibitor, Journal of Carbohydrate Chemistry, DOI:
10.1080/07328303.2017.1390577
To link to this article: http://dx.doi.org/10.1080/07328303.2017.1390577
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Date: 11 November 2017, At: 23:29

JOURNAL OF CARBOHYDRATE CHEMISTRY
, VOL. , NO. , –

https://doi.org/./..
Multivalent zanamivir-bovine serum albumin conjugate as a
potent influenza neuraminidase inhibitor
a
b
a
a
a
a
Teng-Fei Zhao , Hai-Juan Qin , Yao Yu , Mei-Bing Yang , Hao Chang , Na Guo ,
c
a
a
Yun He , Yang Yang , and Peng Yu
aChina International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal
Chemistry, Key Lab of Industrial Fermentation Microbiology of Ministry of Education, College of
b
Biotechnology, Tianjin University of Science and Technology, Tianjin, China; Research Centre of Modern
c
Analytical Technology, Tianjin University of Science and Technology, Tianjin, China; Research center for
molecular diagnostics and sequencing, Research Institute of Tsinghua University in Shenzhen, Nanshan
District, Shenzhen, China
ABSTRACT
ARTICLE HISTORY
Received  July 
Accepted  October 
A multivalent Zanamivir (ZA) conjugate as new anti-influenza
agent was prepared. Specifically, multiple copies of ZA with a C7
azido-terminated linker were covalently attached onto alkyne-
modified bovine serum albumin (BSA) via copper-free click reac-
tion. The loading number of ZA was determined by MALDI-TOF.
Primary neuraminidase (NA) inhibition assay showed that this
conjugate retained some NA inhibitory activity compared to its
parent ZA monomer. Moreover, the ZA-BSA conjugate was not
cytotoxic with concentrations up to 1 µM.
KEYWORDS
Multivalent effect; zanamivir;
bovine serum albumin
conjugate; nueraminidase
inhibitor; click chemistry
GRAPHICAL ABSTRACT
Introduction
Zanamivir (ZA)[1,2] and Oseltamivir phosphate (OS)
[3,4]
are two sialic acid (SA)
[5]
analogs that mimic the catalytic transitional state of viral surface neuraminidase
CONTACT Prof. Yang Yang
yyang@tust.edu.cn; Prof. Peng Yu
yupeng@tust.edu.cn
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lcar.
Teng-Fei Zhao and Hai-Juan Qin contributed equally to this work.
Supplemental data for this article can be accessed on the publisher’s website.
©
 Taylor & Francis Group, LLC

2
T.-F. ZHAO ET AL.
Figure . Schematic representation of neuraminidase hydrolysis of terminal sialic acid residue on the
surface of infected cells to release influenza virus.
(
NA) to catalyze sialyl linkage hydrolysis (Figure 1) and are the first line drugs for
the treatment of influenza. Both drugs have significantly higher binding affinity
with NA active sites than the native substrate SA,[ leading to the interference
of freeing viral progeny from infected host cells. However, drug resistance induced
by the mutations of amino acid residues in and around the active sites of NA can
decrease these interactions. In recent years, more and more NA inhibitor-resistant
clinical isolates of viruses, such as the human seasonal H1N1/H3N2
H5N1 /H7N9 viruses, have been discovered.
6,7]
[8,9]
and avian
[10]
[11]
Two strategies for developing new generation of antiviral inhibitor with improved
NA affinity may deal with the resistance.[12] One strategy is to modify functional
groups of ZA, including carboxylic acid on C1, unsaturated bond on C2 and C3,
guanidine on C4, acetamide on C5 and glycerol side chain on C8/C9. The resulted
ZA derivatives can more or less enhance the antiviral activities on enzymatic or
[13,14]
animal models.
However, the complicated synthesis and purification steps
are always involved in these chemical derivatizations, making the process labor-
intensive and time-costing. Another approach is inspired by the natural principle
[15–17]
called “cluster effect”.
Structural studies of the influenza virus have shown that

JOURNAL OF CARBOHYDRATE CHEMISTRY
3
about 50–100 copies of tetrameric NA are tethered on a single virion,[18] which
provides adequate binding sites for multivalent interactions. Therefore, attaching
ZA to appropriate di- or tetra-valent scaffolds can enhance the potency against
broad range of influenza viruses, especially the drug-resistant strains.[
13,19]
Fur-
thermore, when biocompatible polymers are used as the backbones, higher anti-
influenza activity up to 2000-fold against ZA-resistant strains than monomeric
[20–25]
ZA could be achieved.
have prepared SA-bovine serum albumin (SA-BSA) and SA-human serum albumin
SA-HSA) conjugates. These conjugates were expected to have the following three
Inspired by this promising multivalent strategy, we
(
[26]
characters: (i) glycan-protein conjugate can closely mimic the 3D presentation of
oligosacchrides on the sialo-receptor on mammalian cell surface; (ii) the molecular
weight of the protein conjugates can be easily determined by Matrix-Assisted Laser
Desorption Ionization Time of Flight Mass (MALDI-TOF), which makes it feasible
to quantitatively analyze the interactions between the glycoconjugate and virus; (iii)
unlike conjugation with polymer backbone,[ the protein-small molecule conju-
gate will not cause uncontrollable complement activation. These synthetic sialyl pro-
tein conjugates showed enhanced NA inhibition activity compared with monomeric
SA.
27]
Herein, we directly attached ZA, which has stronger binding than SA to NA,
to BSA scaffold via copper free click chemistry[ to prepare potent anti-influenza
virus agent. We expected that the multivalent effect can further enhance the antiviral
activity of the synthetic ZA-BSA conjugate. Finally, the NA inhibition activity and
cytotoxicity of the conjugate were investigated. This paper presents the initial step
towards the development of multivalent ZA-protein conjugates as anti-influenza
inhibitors.
28]
Result and discussion
The design and synthesis of the ZA-BSA conjugate is shown in Scheme 1. A strain-
promoted alkyne–azide cycloaddition (SPAAC)[ strategy was adopted for the in
situ ZA attachment onto the BSA backbone, due to its high chemical selectivity
without compromising the protein function. We chose C7 azido-substituted ZA as
the functionalized molecule because X-ray structural analysis of NA-ZA complex
has illustrated the lack of hydrogen bond between the C7 hydroxyl group and the
29]
[6]
active site of NA. Thus, the linker attached to the C7 hydroxyl would orient away
from the catalytic center without substantial impact on the affinity of ZA analogs to
[30]
[31]
NA. Bicyclo[6.1.0]nonyne (BCN), which has been used extensively in imaging
and tracking of biomolecules, was adopted as the ring-strained alkyne for metal-free
cycloaddition reaction with azide for the alkynation of BSA.
The introduction of the linker on C7 was achieved by amidation of azido tri-
ethylene glycol linker with amine derived from active carbamate ester, following
the procedure reported in our previous publication.[ The protecting groups of t-
butyloxy carbonyl (Boc) and isopropylidene in 2 were removed with trifluoroacetic
acid (TFA), followed by guanidinylation of the C-4 amino group with 1, 3-bis
32]

4
T.-F. ZHAO ET AL.
Scheme . Synthesis of Zanamivir-BSA conjugate. Reagents and conditions: (a)
HNCHCHOCHCHOCHCHN, DMAP/Py, .%; (b) i)TFA/CHCl; ii) MeSC = NBocNHBoc,
ꢀ
HgCl, EtN, % over two steps; (c) i) NaOH/HO; ii) TFA/CHCl (d) N,N -disuccinimidyl carbonate,
triethylamine /CHCN; (e) BSA, PBS pH .; (f) PBS pH ..
(
tert-butoxycarbonyl)-2-methylthiopseudourea in the presence of HgCl and Et N
2 3
[33]
to afford the fully protected guanidine 3. Finally, a two-step deprotection proce-
dure was performed. First, 0.1 M NaOH was added to remove the methyl ester and
+
the reaction mixture was neutralized with H resin. Next, the Boc protecting groups
were removed by treatment with TFA/CH Cl . The reaction mixture was evaporated
2
2
and the resulting syrup was purified by Sephadex LH-20, followed by lyophilization
[12]
to afford the deprotected ZA monomer 4. All of the key intermediates and final
products were characterized by NMR and HRMS (see Supplemental Materials).
We then prepared the BCN functionalized BSA BCN-BSA as the multivalent
scaffold. BCN alcohol 5 was synthesized according to the literature with rhodium
tetraacetate replaced with cheaper copper acetoacetonate as the catalyst in the initial
Simmons–Smith type reaction.[34] Treatment of 5 with N,N-disuccinimidyl carbon-
ate gave active BCN succinimidyl carbonate 6, which further reacted with primary
amines on lysine side chains of BSA in phosphate buffered saline (PBS) at pH 7.4
to afford BCN-BSA. After purification by Sephadex G-75, the molecular weight of
the resulting BCN-BSA was measured by MALDI-TOF MS[ (see Supplemental
Materials). Compared with BSA, mass analysis of BCN-BSA conjugate showed that
an average number of 14 BCN were attached to each BSA molecule (Table 1). Next,
BCN-BSA was stirred with azide functionalized ZA 4 for 12 h in PBS at room tem-
perature. After purification, the resultant ZA-BSA conjugate was also characterized
by MALDI-TOF, which showed that 4 ZA were loaded onto one molecule of BSA,
meaning that not all of the BCN on BSA surface were reacted with 4. This could be
due to the steric hindrance and the decomposition of the BCN carbonate,[ which
limited the number of available reactive site for the azide, resulting in the reduction
35]
36]

JOURNAL OF CARBOHYDRATE CHEMISTRY
5
Table . The conjugations of BCN and ZA with BSA.
a
Entry
Molecular weight
Number of loading ligand
BSA
BCN-BSA
ZA-BSA




 (BCN)
 (ZA with C- azido linker, 4)
aMolecular weight was determined by MALDI-TOF mass spectrometry.
of the protein coupling efficiency. A long and flexible linker instead of the carba-
mate in 6 and other more stable alkynyl ligand are now under investigation in our
laboratory for improving the conjugation efficiency.
The influenza A neuraminidase inhibition activity of the synthetic ZA-
BSA conjugate was analyzed and compared with ZA monomer and C-7
azide functionalized ZA 4 using three influenza strains: A/Hecheng, Hunan
Province /SWL1331/2014 (H1N1), A/Huairou, Beijing/11069/2014 (H3N2),
and A/Chicken/Beijing/AT609/2014 (H9N2). A fluorescent MUNANA [2´-(4-
Methylumbelliferyl)-α-D-N-acetylneuraminic acid]-based NA inhibition assay[
was used to evaluate the NA inhibition activity with BCN-BSA and ZA as controls
26]
(Table 2). Under our assay system, both of ZA-BSA and 4 were nanomolar NA
inhibitor and retained the antiviral activity. Our data proved that the modification
on C-7 of ZA (see 4) did not change the NA inhibition activity.[ Although
ZA-BSA was less potent than its corresponding monomeric ZA, this primary data
encouraged us to further study the antiviral activity of ZA-BSA on cell and animal
30]
[
37]
[19,38]
model. It has also been reported that some of the di- , tetra-
ZA conjugates
and polyvalent
[22,24,25]
showed enhanced anti-influenza activities in plaque yield
reduction assay and in vivo efficacy studies in mouse models, even though they
have less activity in NA inhibition assay. The antiviral activity of ZA-BSA conjugate
in a cell based cytopathic effect (CPE) reduction[ and plaque assay
39]
[40]
are now
under investigation in our lab.
Before performing the antiviral study on cell and mouse model, it is of impor-
tance to evaluate the cytotoxicity of the ZA-BSA. Human umbilical vein endothe-
lial cells (HUVEC) were incubated with ZA-BSA or BCN-BSA conjugate for 24 h.
Then, the cell activity was determined by MTT[ . Figure 2 showed that the ZA-
BSA and BCN-BSA were non-cytotoxic against normal mammalian cells at the
concentration of 1 µM, indicating these conjugate could be a candidate in medical
applications.
26]
Table . IC values in the neuramidinase inhibition assay.
Entry
HN
IC (nM) HN
HN
a
BCN-BSA
ZA-BSA
4
NA
NA



NA






Zanamivir
aNo activity.

6
T.-F. ZHAO ET AL.
Figure . Cytotoxicity study of BSA conjugates.
Conclusion
We have prepared a novel Zanamivir-BSA conjugate via copper-free cycloaddition
reaction. The primary neuraminidase inhibition assay showed that this conjugate
retained the antiviral activity of the corresponding monomeric ZA. The results of
our study also illustrated that further assay systems should be adopted in order to
demonstrate the efficacy of ZA-BSA in vivo. Moreover, other appropriate linker on
BCN should also be designed and attached which may increase the ZA coupling
efficiency onto the protein scaffold. We can also adopt our Zanamivir protein con-
jugate strategy in the antibody coupling antiviral drug design for the targeted drug
delivery. All of these are now under research in our laboratory.
Experimental
All the 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 com-
pounds were stained with 5% H SO in ethanol. Detection with UV light was
2
4
employed when possible. Flash column chromatography was performed on silica
gel 200–300 mesh. All of the final compounds and protein conjugates were purified
TM
TM
using Sephadex
LH-20 and Sephadex
G-75 (GE Healthcare), respectively.
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 key
intermediates and final target molecules were confirmed by electrospray ioniza-
tion mass spectra (ESI-MS) on a hybrid IT-TOF mass spectrometer (Shimadzu
LCMS-IT-TOF, Kyoto, Japan) and Bruker ultrafleXtreme MALDI-TOF/TOF mass
spectrometer (Bruker Daltonics, Bremen, Germany). Fluorescence intensity was
TM
measured using the Synergy H1/H1MF microplate reader (BioTek Instruments,
Inc. USA).
The influenza viruses were obtained from National Institute for Viral Disease
Control and Prevention, China CDC and propagated in 9 days old embryonated
−
1
chicken eggs. The allantoic fluid was collected, then centrifuged at 3000 rpm min

JOURNAL OF CARBOHYDRATE CHEMISTRY
7
for 10 min and the supernatant was stored at −80°C. Virus was inactivated by the
addition of β-propiolactone (β-PL) when assays were conducted.
Methyl {5-acetamido-2, 6-anhydro-7-O-[2-(2-(2-azidoethoxy) ethoxy)
ethan-1-amide]-8, 9-O-isopropylidene-4-N-tert-butyloxycarbonyl-3, 4,
5
-trideoxy-D-glycero-D-galacto} non-2-enonate (2)
To a solution of 2-[2-(2-azidoethoxy) ethoxy] ethan-1-amine (572 mg, 3.3 mmol)
in pyridine (10 mL), 4-dimethylaminopyridine (401 mg, 3.3 mmol) was added.
The solution was stirred at rt for 30 min then 1 (1 g, 1.64 mmol) was added and
the reaction mixture was kept stirring overnight. The progress of the reaction was
monitored by TLC. Upon completion, the reaction mixture was washed by HCl
(
1 M, 300 ml), extracted by CH Cl (3× 50 mL), the organic phases were combined
2
2
and dried over Na SO . Solvent was removed under vacuum and the product
2
4
was purified by column chromatography (petrol ether/acetone, 2:1 v/v) to give 2
1
(
(
1
3
0.92 g, 86.8%). H NMR (400 MHz, CDCl ) δ 5.93 (d, J = 8.8 Hz, 1H, NHAc), 5.86
3
d, J = 1.5 Hz, 1H), 5.29 (s, 1H), 5.18 (d, J = 4.1 Hz, 1H), 4.88 (d, J = 8.8 Hz,
H), 4.50 (t, J = 8.2 Hz, 1H), 4.32–4.27 (m, 2H), 4.08–3.91 (m, 3H), 3.71 (s, 3H),
.62–3.48 (m, 8H), 3.34–3.29 (m, 4H), 1.89 (s, 3H), 1.35 (s, 9H), 1.31 (s, 3H), 1.29
13
(
s, 3H). C NMR (100 MHz, CDCl ) δ 171.15, 162.11, 156.18, 155.57, 144.30,
3
1
43.33, 126.40, 111.32, 108.97, 80.06, 74.81, 70.49, 70.20, 70.00, 69.83, 65.86, 52.37,
50.59, 49.90, 47.60, 40.99, 28.27, 26.41, 25.44, 23.13. HRMS (ESI): m/z calcd for
+
C H N O Na [M+Na] : 667.2909, found: 667.2899.
27
44
6
12
Methyl {5-acetamido-2, 6-anhydro-7-O-[2-(2-(2-azidoethoxy) ethoxy)
ethan-1-amide]- 4-(bis-N, N’-tert-butyloxycarbonyl)-guanidino −3, 4,
5
-trideoxy-D-glycero-D-galacto} non-2-enonate (3)
To a solution of 2 (1.2 g, 1.98 mmol) in CH Cl (5 ml), TFA (5 ml) was added, and
2
2
the reaction mixture was stirred at rt for 1 h, then the solvent was removed in vacuo.
The residue was dissolved in CH Cl , and then Et N (1 mL) was added. The mixture
2
2
3
was stirred at rt for 30 min, after that HgCl (253 mg, 0.94 mmol) and 1, 3-Bis(tert-
2
butoxycarbonyl)-2-methyl-2-thiopseudourea (648 mg, 2.23mmol) was added. The
reaction mixture was further stirred at rt for another 12 h, then washed with HCl
(
1 M, 25 ml), extracted with CH Cl (3× 10 ml) and after concentration the residue
2
2
was purified by column chromatography (acetone/petrol ether, 1:1 v/v) to give the
1
product 3 (500 mg, 36%). H NMR (400 MHz, CDCl ) δ 11.32 (s, 1H), 8.43 (d, J =
3
9
.0 Hz, 1H), 6.69 (d, J = 9.2 Hz, 1H), 5.81 (s, 1H), 5.43 (t, J = 5.3 Hz, 1H), 5.11 (t,
J = 9.4 Hz, 1H), 4.76 (d, J = 9.5 Hz, 1H), 4.40 (d, J = 10.5 Hz, 1H), 4.31–4.24 (m,
1
1
1
5
H), 4.00 (s, 1H), 3.72 (s, 3H), 3.62–3.50 (m, 10H), 3.34–3.31 (m, 4H), 2.16 (s, 1H),
13
.88 (s, 3H), 1.42 (s, 18H). C NMR (100 MHz, CDCl ) δ 195.70, 170.82, 162.09,
3
56.17, 155.57, 144.34, 111.36, 108.99, 80.02, 74.77, 70.19, 70.14, 69.83, 69.73, 65.92,
2.34, 49.84, 47.68, 41.03, 30.56, 28.76, 28.28, 26.45, 25.46, 23.22. HRMS (ESI): m/z
+
calcd for C H N O [M+H] : 747.3519, found: 747.3513.
30
51
8
14

8
T.-F. ZHAO ET AL.
5
-acetamido-2, 6-anhydro-7-O-[2-(2-(2-azidoethoxy) ethoxy) ethan-1-amide]−4-
guanidino −3, 4, 5-trideoxy-D-glycero-D-galacto-non-2-enonic acid (4)
3
(100 mg) in methanol (4 ml) and water (4 mL) was treated with 0.1 M sodium
+
hydroxide solution. After 1 h, the reaction mixture was neutralized by H resin to
pH 7.0, filtered and the solvent was evaporated. The residue was stirred in 50% TFA
(
1
10 mL) solution in CH Cl at 0°C for 10 mins and at room temperature for another
h then the solvent was evaporated. The resulting syrup was dissolved in distilled
2 2
water and purified by Sephadex LH-20 then lyophilisation to afford 4 (61 mg,
®
1
8
(
2
(
5.5%) as a foam and stored at 4°C until use. H NMR (400 MHz, D O) δ 5.61
2
d, J = 2.0 Hz, 1H), 4.86 (d, J = 9.2 Hz, 1H), 4.44 (d, J = 10.4 Hz, 1H), 4.34 (dd, J =
.1 Hz, 1H), 4.06–4.00 (m, 2H), 3.66–3.60 (m, 7H), 3.54 (t, J = 5 Hz, 2H), 3.45–3.41
13
m, 3H), 3.29–3.23 (m, 1H), 3.19–3.13 (m, 1H), 1.90 (s, 3H). C NMR (100 MHz,
D O) δ 173.75, 168.47, 156.96, 156.67, 149.13, 104.75, 75.19, 69.83, 69.50, 69.38,
2
6
9.21, 69.11, 68.51, 62.47, 51.71, 50.09, 47.35, 40.13, 21.82. HRMS (ESI): m/z calcd
+
for C H N O [M+H] : 533.2314, found: 533.2304.
19
33
8
10
(
1R, 8S, 9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl (2, 5-dioxopyrrolidin-1-yl)
carbonate and (1R, 8S, 9r)-bicyclo[6.1.0]non-4-yn-9-ylmethyl
2,5-dioxopyrrolidin-1-yl) carbonate (6)
(
Triethylamine (4 mL, 2.9 g, 29.0 mmol) was added under argon to a solution of 5
ꢀ
(
1.45 g, 9.65 mmol) and N, N -Disuccinimidyl carbonate (4.95 g, 19.3 mmol) in
acetonitrile. The mixture was stirred overnight under an inert atmosphere, con-
centrated, and the residue was purified by flash column chromatography (hex-
1
anes/EtOAc, 3:1 v/v) on silica gel to give the product 6 (2.3 g, 81%). H NMR
(
2
400 MHz, CDCl ) δ 4.38 (d, J = 8.4 Hz, 2H), 4.19 (d, J = 6.9 Hz, 4H), 2.77 (s, 12H),
3
13
.41–2.04 (m, 18H), 1.60–1.24 (m, 8H), 1.06–0.92 (m, 2H), 0.83–0.67 (m, 6H). C
NMR (100 MHz, CDCl ) δ 167.72, 167.70, 150.65, 150.62, 97.63, 74.97, 69.33, 32.00,
3
27.96, 24.47, 22.40, 21.83, 20.30, 20.21, 19.70, 16.17.
Alkynylation of BSA (BCN-BSA)
To a solution of 24 mg BSA in l mL 0.1 M sodium phosphate buffer (pH 7.4), 6
(
10 mg, 34 µmol) in 5 mL 0.1 M sodium phosphate buffer (pH 7.4) was added drop-
wise. After gentle shaking overnight at room temperature, the reaction mixture was
purified by Sephdex G-75 then freeze dried to give BCN-BSA and stored at 4°C
until use.
Copper-Free click reactions
To a solution containing 25 mg BCN-BSA in l mL 0.1 M sodium phosphate buffer
(
pH 7.4), 4 (10 mg, 18 µmol) in 5 mL 0.1 M sodium phosphate buffer (pH 7.4) was
added. After gentle shaking for 24 h at room temperature, the reaction mixture was

JOURNAL OF CARBOHYDRATE CHEMISTRY
9
purified by Sephdex G-75 then freeze dried. The conjugate was characterized by
MALDI-TOF mass spectrometry and stored at 4°C until use.
Characterization of BSA conjugates with MALDI-TOF
The characterization of the BCN-BSA and ZA-BSA were carried out by matrix
assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrome-
try in positive linear ion mode using 2, 5-dihydroxybenzoic acid (DHB) as matrix.
BSA conjugate solutions were diluted with the matrix (5 mg/mL matrix in 50%
acetonitrile containing 0.1% trifluoroacetic acid) at a ratio of 1:5 by volume and
deposited onto a MALDI target plate and dried in air. FlexAnalysis was used for
analysis of the data.
Neuraminidase inhibition assay
Assays were conducted in 0.1 M sodium acetate buffer (pH 5.5) containing
1
0 mM CaCl . 4-methylumbelliferyl-α-D-N-acetylneuraminic acid sodium salt (4-
2
MUNANA) was used as the fluorescent substrate. Serial dilutions of inhibitors were
premixed with the inactive virus for 30 min. The enzymatic reactions were carried
out for 2 h at 37°C. The fluorescent signal was monitored using the kinetics func-
TM
tion of Synergy H1/H1MF microplate reader with excitation and emission wave-
lengths of 360 and 440 nm, respectively. The IC was calculated as the concentration
50
of the inhibitor resulting in a 50% decrease of the rate compared to the control.
In vitro analysis of cytotoxicity of BSA conjugates on HUVEC cells. HUVEC cells
4
were seeded at 5× 10 cells/well in a 96-well plate and grown overnight in dul-
becco minimum essential medium (DMEM) containing 10% fetal bovine serum.
Cells were incubated with serially diluting BCN-BSA or ZA-BSA for 24 h at 37°C
under 5% CO . Then 20 µL of MTT was added to each well and the plates were incu-
2
bated for 4 h. After discarding the supernatants, 200 µL DMSO was added to each
well. The absorbance of each well was measured using a microplate reader at 492 nm,
and the percent viability was calculated by (Asample /Acontrol) × 100%, where A
sample
and Acontrol are the absorbance of a well containing the BCN-BSA or ZA-BSA and
the DMEM as control, respectively. All experiments were performed in triplicate.
Acknowledgments
This work was financially supported by the Natural Science Foundation of China (21402140,
8
1773583), Shenzhen Peacock Plan (KQTD2016053114253158), Natural Science Foundation of
Tianjin City (16JCTPJC46000), Key Laboratory of Industrial Fermentation Microbiology of Min-
istry of Education and Tianjin Key Lab of Industrial Microbiology (2015IM107) and Youth Inno-
vation Research Foundation of Tianjin University of Science and Technology (2016LG08). The
authors are thankful to the Research Centre of Modern Analytical Technology, Tianjin University
of Science and Technology for NMR, HRMS and MALDI-TOF mass measurements. The authors
also appreciate National Institute for Viral Disease Control and Prevention, China CDC for the
viral strains.

1
0
T.-F. ZHAO ET AL.
ORCID
Yang Yang http://orcid.org/0000-0003-1792-4130
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