Design of Glycopolymers Carrying Sialyl Oligosaccharides for

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Design of glycopolymers carrying sialyl oligosaccharides for controlling the interaction with the influenza virus Masanori Nagao, Yurina Fujiwara, Teruhiko Matsubara, Yu Hoshino, Toshinori Sato, and Yoshiko Miura Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01426 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on November 8, 2017

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Design of glycopolymers carrying sialyl oligosaccharides for controlling the interaction with the influenza virus

Masanori Nagao,1 Yurina Fujiwara,2 Teruhiko Matsubara,2 Yu Hoshino,1 Toshinori Sato,2 and Yoshiko Miura1*

1

Department of Engineering, Graduate School of Chemical Engineering, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395, Japan Email: [email protected]

2

Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan

KEYWORDS: Reversible addition-fragmentation chain transfer (RAFT) polymerization, glycopolymers, click chemistry, siallylactose, influenza virus, acrylamide

ABSTRACT We designed glycopolymers carrying sialyl oligosaccharides by “post-click” chemistry and evaluated the interaction with the influenza virus. The glycopolymer structures were synthesized in a well-controlled manner by reversible addition-fragmentation chain transfer polymerization and the Huisgen reaction. Acrylamide-type monomers were copolymerized to give hydrophilicity to the polymer backbones, and the hydrophilicity enabled the successful introduction of the oligosaccharides into the polymer backbones. The glycopolymers with different sugar densities and polymer lengths were designed for the interaction with hemagglutinin on the virus surface. The 1

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synthesized glycopolymers showed the specific molecular recognition against different types of influenza viruses depending on the sugar units (6’- or 3’-sialyllactose). The sugar density and the polymer length of the glycopolymers affected the interaction with the influenza virus. Inhibitory activity of the glycopolymer against virus infection was demonstrated.

2


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Introduction Achieving precise control of the polymer structure is crucial in the synthesis of biopolymers. Biomacromolecules, such as proteins and DNA, are sequence-controlled biopolymers and have well-defined 3D structures.1,2 The precise 3D location of functional groups enables a specific interaction between biomacromolecules by multiple inetraction.1,2 Synthetic biopolymers are designed to carry similar functional groups, and to exhibit the biological interactions with biomolecules.3,

4

Development of living polymerization technique, has enabled control of the

structure of synthetic biopolymers, including the monomer composition and polymer backbone length.5–7 Carbohydrates are one of the crucial materials for biological interactions. Carbohydrates exist on the cell surface as oligosaccharides and are involved in biological phenomena, such as cell-cell communication and pathogen infection.8 The cluster structure of oligosaccharides on cell surfaces enables multivalent and strong interactions with lectins (the cluster glycoside effect).9, 10 Glycopolymers carrying side-chain carbohydrates make it possible to use the properties of carbohydrates for practical applications e.g., biosensing, pathogen removal, and nanomedicine.11–15 Glycopolymers have polyvalent structures and display several carbohydrates to exhibit the cluster glycoside effect.16 The ease of preparation is an advantage of glycopolymers compared with other glycoconjugates, such as dendrimers and glycopeptides.17,

18

The structures of glycopolymers

carrying monosaccharides can be controlled by direct polymerization of glyco-monomers, and the 3


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influence of the polymer structures on the interaction with lectins has been studied.13, 19–22 However, there have been few reports of well-controlled glycopolymers carrying oligosaccharides.23,24 Oligosaccharides have a bulky structure and high hydrophilicity, and these properties are problematic for the direct polymerization of the glyco-monomers. Achieving the design of glycopolymers carrying oligosaccharides would enable control of the interaction with practical targets such as pathogenic proteins, bacteria, and viruses. To achieve further precise control of glycopolymers, “post-click” chemistry is adopted as a method to prepare glycopolymers. The methodology of “post-click” chemistry was suggested by Hawker and coworkers, and Haddleton and coworkers applied the technique for glycopolymer synthesis.23, 25–27 In this method, polymer backbones are first prepared by living polymerization and sugar units are introduced by the “click” reaction. Our group has recently reported hydrophilic polymer backbones carrying alkyne groups that enabled the successful introduction of oligosaccharides as side chains.28,

29

However, the influence of the

polymer structures on the interaction with the influenza virus has not been studied in detail. In the present study, we report the design of well-defined glycopolymers with sialyl oligosaccharides and the influence of the polymer structure on the interaction with influenza viruses. The “post-click” chemistry with the hydrophilic polymer backbones enabled to prepare the well-defined glycopolymers carrying sialyllactoses (SALacs) with controlled sugar densities and polymer lengths. The hydrophilic polymer backbones for “post-click” chemistry were prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization. Either 6’-SALac or 4


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3’-SALac was introduced into the designed polymer backbones by the Huisgen reaction. The interaction of the designed glycopolymers with the influenza virus was evaluated by a hemagglutination assay. This work revealed the relation between the structure design of the glycopolymers for hemagglutinin and the interaction with the influenza virus.

Experimental Section Materials. Methyl 2-bromopropionate (98.0%) and lithium bromide (LiBr, 99.0%) were purchased from Tokyo Chemical

Industry

(Tokyo,

Japan).

2,2’-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride

Acrylamide (AIPD,

98%),

(AAm, triethylamine

97%), (TEA,

99.0%), carbon disulfide (98.0%), 1-butanethiol (95.0%), crystal violet and acetic acid (99.7%) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Acrylamide was purified by recrystallization prior to use. N,N-Dimethylacetamide (DMAc, 99.0%), potassium hydroxide (KOH, 86%), copper sulfate (CuSO4, 97.5%) and sodium L-ascorbate (L･Asc･Na, 98%) were purchased from Kanto Chemical (Tokyo, Japan). Dimethyl sulfoxide (DMSO) was purchased from Kishida chemical (Osaka, Japan). Tetrabutylammonium fluroride (TBAF) solution (1.0 M in THF), acetonitrile (MeCN, 99.9%), bovine albumin, and acetyltrypsin were purchased from Sigma Aldrich (St. Louis, USA). Eagle's minimum essential medium (MEM), diethylaminoethyl-dextran, 1 M HEPES buffer, L-glutamin were purchased from nacalai tesque (Kyoto, Japan). Trypsin was 5


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purchased from Becton Dickinson. Fetal bovine serum was purchased from biosera. Blood cell suspension from a guinea pig was purchased from Nippon Bio-test Laboratory Inc (Saitama, Japan). 4-trimethylsilyl-3-butynyl acrylamide (TMS BtnAAm), 6’-sialyllactose aizde (6’-SALac azide), 3’-sialyllactose

aizde

(3’-SALac

azide),

tris(benzyltriazolylmethyl)

amine

(TBTA),

and

2-azidoethanol were prepared according to the previous paper.29, 30

Characterization. Proton nuclear resonance (1H NMR) spectra were recorded on a JEOL-ECP400 spectrometer (JEOL, Tokyo, Japan) using CDCl3, MeOD, d6-DMSO or D2O as a solvent. Size exclusion chromatography (SEC) and light scattering (LS) with water solvent was performed on a JASCO DG-980-50 degasser equipped with a JASCO PU-980 pump (JASCO Co., Tokyo, Japan), a Shodex OH pak SB-G guard column, a Shodex OH pak LB-806 HQ column (Showa Denko, Tokyo, Japan), a JASCO RI-2031 Plus RI detector, and a Viscotek TDA (Malvern Instruments Ltd, Worcestershire UK). SEC analyses and LS measurements were performed by injecting 20 µL of a polymer solution (2 g/L) in 100 mM sodium nitrate solution. The SEC system and the LS system were calibrated using a pullulan standard (Shodex) and polyethylene oxide standard (Mn = 24k, Malvern). SEC with organic solvent was performed on a HLC-8320 GPC Eco-SEC equipped with a TSKgel Super AW guard column and TSKgel Super AW (4000 and 2500) columns (TOSOH, Tokyo Japan). The SEC analyses were performed by injecting 20 µL of a polymer solution (5 g/L) in DMAc buffer with 10 mM LiBr. The 6


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SEC system was calibrated using a polystyrene standard (Shodex). All the samples for SEC and LS were previously been filtered through a 0.45 µm filter. The buffer solution was also used as the eluent at a flow rate of 0.5 mL/min.

Preparation of RAFT reagent (methyl 2-(butylthiocarbonothioylthio)propanoate, MCEBTTC). MCEBTTC was synthesized by following the literature.31 To a stirred solution of 1-butanethiol (10 mL, 93.4 mmol) and TEA (14.3 mL, 103 mmol) in dryCH2Cl2 (100 mL) under nitrogen, carbon disulfide (6.18 mL, 103 mmol) in dryCH2Cl2 (50 mL) was added drop wise over a period of 30 min at 0 °C. The solution gradually turned yellow during the addition. After complete addition, the solution was stirred at room temperature for 1 h. Methyl 2-bromopropionate (11.5 mL, 103 mmol) in dryCH2Cl2 (50 mL) was then added drop wise to the solution over 30 min at 0 °C. The reaction mixture was kept stirring for 1 h at 0 °C, and 20 h at room temperature under nitrogen atmosphere. CH2Cl2 (100 mL) was added, washed with 10 wt% HClaq (3 × 100 mL) and MilliQ (3 × 100 mL) and then dried over anhydrous MgSO4. The solution was filtered and concentrated under reduced pressure. The residual yellow oil was purified by column chromatography (1:19 = ethyl acetate:hexane) (23.6 g, 75%). 1

H NMR (CDCl3, δ in ppm): 4.84 (qu, J = 7.5 Hz, CH3–CH–S, 1H), 3.73 (s, –O–CH3, 3H), 3.36 (tr, J

= 7.5 Hz, S–CH2–, 2H), 1.65 (quin, J = 7.5 Hz, S–CH2-CH2–, 2H), 1.62 (d, J = 7.5 Hz, CH3–CH–S, 3H), 1.43 (mult, J = 7.5 Hz, –CH2–CH3, 2H), 0.92 (t, J = 7.5 Hz, –CH2–CH3, 3H). 7


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13

C NMR (CDCl3, δ in ppm): 222.1 (S–C–S), 171.8 (–O–C=O), 53.0 (CH3–O–), 47.8 (Ester-CH–S),

37.1 (–S–CH2–C3H6), 30.0 (–CH2–CH2–CH3), 22.1 (–CH2–CH3), 17.0 (CH3–CH–S–), 13.7 (–CH2– CH3). MS (ESI+) m/z = 275.0 [M+Na]+.

Preparation of polymer backbones. All polymer backbones were prepared by RAFT polymerization. Briefly, acrylamide (AAm), 4-trimethylsilyl-3-butynyl acrylamide (TMS BtnAAm) were dissolved in DMSO with MCEBTTC and AIPD. The monomer feed ratio and target degree of polymerization were varied. The solution was degassed by freeze-thaw cycles (three times) and put in an oil bath. The reaction progressed at 70 °C for 12 h. The reaction was stopped by exposing to air. The monomer conversions were judged by 1H NMR. The polymer backbones were purified by dialysis against DMSO (MWCO = 1 kDa). The solution was changed from DMSO to MeOH, and the product was obtained by evaporating the solvent. Polyacrylamide (PAAm) was prepared in the same method.

Deprotection of TMS groups from the polymer backbones. The polymer backbones with middle and high alkyne densities (P2–P7, 100 mg) was dissolved in dry THF (5 mL), and TBAF solution (1 mL, 1.0M in THF) was added into the solutions. The solution was kept stirring at room temperature for 9 h. Excess THF was removed under reduced 8


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pressure. The deprotected polymers were dialyzed against the mixture of MeOH and acetone (MeOH : acetone = 1 :1, MWCO = 1 kDa). The products were obtained by evaporating the solvent. The polymer backbone with low alkyne densities (P1, 100 mg) was dissolved in KOHaq (1 M, 3 mL). The solution was kept stirring at room temperature for 3 h. The deprotected polymer was dialyzed against MilliQ (MWCO = 3.5 kDa). The product was obtained by freeze-drying.

Oligosaccharide addition by the Huisgen reaction. Each of the deprotected polymer backbones (10 mg, the molar numbers of alkyne units were calculated by each monomer ratio), 6’- or 3’-SALac azide (3 eq mol to alkyne unit) and CuSO4 (0.4 eq mol to alkyne unit) were dissolved in H2O (1 mL). TBTA (0.4 eq mol to alkyne unit) dissolved in MeCN (1 mL) was added. Finally, L･Asc･Na (2 eq mol to alkyne unit) in H2O (1 mL) was added and the mixture solution was kept at 60 °C for 6 h while purged with N2 bubbling. The mixture was freeze-dried, and 2-azido ethanol (500 µL), CuSO4 (2.6 mg), TBTA (8.7 mg), and L･Asc･Na (16 mg) were added with water (1.9 mL) and CH3CN (600 µL). The mixture was kept stirring for 9 h at room temperature. The products were purified by dialysis (Spectra/Por 7; MWCO 3,500) against water with hydrochloric acid (pH = 4) for 24 h. Water was changed to pure water (pH = 7) subsequently and kept for 12 h, and the product was obtained by freeze-drying.

Hemagglutination inhibition (HI) assay. 9


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Phosphate-buffered saline (PBS) was added into a 96-well plate (25 µL/well) except the first lane. Glycopolymer solution (2 mg/mL, 50 µL) was injected in the first lane. The solution in the first lane were diluted by two steps (25 µL). Influenza virus solution [A/Aichi/2/68 (H3N2) or A/Puerto Rico/8/34 (H1N1), 4 HAU] was injected in each well (25 µL/well). The 96-well plate was incubated for 1 h at 4°C. Red blood cells in the purchased blood cell suspension were washed by centrifugation with PBS three times. The concentrated red blood cells were resuspended in PBS (0.5 v/v%) and was injected in each well (50 µL). The 96-well plate was incubated for 2 h at 4 °C (n = 2). Precipitation of red blood cells was determined by visual inspection.

Plaque assay. The infection of Madin-Darby canine kidney (MDCK) cells by influenza virus was evaluated using a plaque assay. The glycopolymer solution at each concentration in PBS (calculated with sugar unit, 20, 66, 200, 666, 2000 µM, 315 µL) was mixed with 315 µL of influenza A/Aichi/2/68 (H3N2) or A/Puerto Rico/8/34 (H1N1) virus solution containing 50−200 plaque forming units (pfu). After incubation for 30 min at 4 °C, the mixture was incubated with a MDCK monolayer for 30 min at 4 °C (200 µL/well, n = 3). After washing, two milliliters of 2% agarose solution containing 0.01% O-(diethylaminoethyl)cellulose-dextran, 10 mM HEPES buffer, 0.01 mg/mL acetyltrypsin, and 0.2% bovine serum albumin in MEM was added and incubated for 2 days. After removing the gels, live cells were stained with crystal violet (1 mg/mL in 20% ethanol), and the number of plaques was 10


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counted. The inhibition rate was calculated using the equation 100 × (1 − (N/N0)), where N and N0 are the number of plaques in the presence and in the absence of glycopolymer, respectively. The IC50 value of the glycopolymer was obtained from the plot of log [percentage of inhibition] versus log [sugar unit]. Results and Discussion

Figure 1. Schematic illustration of the preparation of various glycopolymers by “post-click” chemistry.

Preparation of well-defined polymer backbones. AAm and TMS BtnAAm were adopted as spacer and alkyne monomers, respectively. AAm and TMS BtnAAm were copolymerized by RAFT polymerization (Figure 1). Previously, our group demonstrated that hydrophilic polymer backbones composed of acrylamide-type monomers were appropriate for the post-modification of oligosaccharides.29 However, RAFT polymerization of the polymer backbones were not controlled enough because the polydispersities were relatively broad. In this report, methyl 2-(butylthiocarbonothioylthio)propanoate (MCEBTTC) was used as the RAFT 11


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reagent because RAFT reagents with a secondary “R” group (see Figure 1) are suitable for the polymerization of acrylamide monomers.32 To compare the influence of ligand densities on target recognition, the monomer feed ratio of [AAm]: [TMS BtnAAm] was varied from 9:1 to 0:10, while the target degree of polymerization was fixed at 100. The alkyne densities in the obtained polymer backbones were confirmed by 1H NMR, and the values corresponded to the monomer feed ratios (Table 1, P1–P5). Size exclusion chromatography (SEC) analysis revealed that these polymer backbones have narrow polydispersities and the same relative molecular weight (Table 1 and Figure 2). Polymer backbones with different degrees of polymerization were also prepared to compare the influence of polymer length on target recognition. The target degree of polymerization was set as 25 and 50 for P6 and P7, respectively, while the monomer feed ratio was fixed ([TMS BtnAAm]: [AAm] = 7:3). SEC analysis revealed that P6 has the smallest molecular weight and P7 has a molecular weight half that of P4. (Table 1 and Figure 2). RAFT polymerization with MCEBTTC allowed the preparation of well-defined polymer backbones with acrylamide-type monomers. The slight decrease in monomer conversion observed with higher TMS BtnAAm concentrations was caused by the lack of solubility of the obtained polymers in DMSO (P3−P5). The relatively short polymers (P6 and P7) were capable of dissolving in DMSO and the monomer conversions were high (> 80%). Degree of polymerization (D.P.) of the polymer backbones was calculated by 1H NMR. To activate the alkyne groups in the polymer backbones, TMS groups in P1–P7 were deprotected in THF solution with TBAF or in aqueous solution with KOH. In the 1H NMR spectra of the 12


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polymers, the peak of TMS group at 0.13 ppm disappeared, while the peak of alkyne group appeared after deprotection step (Figure S3). This indicates TMS groups in the polymer backbones were removed, and the alkyne groups were activated.

Table 1. Properties of RAFT polymerization of polymer backbones.

[M] (10-2 mol/L)

[M]/ [RAFT]

Conv.a (%)

Alkyne unitsa (%)

Calculated D.P.b (mer)

Mnc Mw/Mnc (×10 g/mol)

No.

AAm

TMS BtnAAm

P1

90

10

100

85

12

106

24d

1.34

P2

70

30

100

81

31

108

12

1.24

P3

50

50

100

67

49

114

17

1.20

P4

30

70

100

68

68

108

18

1.20

P5

0

100

100

68

100

100

21

1.17

P6 P7

30 30

70 70

25 50

82 81

67 67

25 48

4.8 9.0

1.15 1.18

-3

PAAm 100 0 100 92 0 134 9.0d 1.18 a The ratio of initiator ([RAFT]/[Initiator]) was fixed at 5. Monomr conversion and ratio of alkyne units were determined by 1H NMR. bDegree of polymerization was calculated by 1H NMR. c Molecular weight and polydispersity index were determined by SEC analysis (DMAc as eluent) calibrated to polystyrene standard. dMolecular weight and polydispersity index of P1 and PAAm were determined by SEC analysis (PBS as eluent) calibrated to pullulan standard after deprotection.

Figure 2. SEC chromatograph of polymer backbones with various alkyne densities (a) and with different 13


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degrees of polymerization (b). (Solvent: DMAc with 10 mM LiBr, calibrated by polystyrene standard).

Preparation of designed glycopolymers. Glycopolymers were obtained by post-modification of previously azidated SALac (either 6’- or 3’-SALac) into the polymer backbones (GP1-6’−GP7-6’ and GP1-3’−GP7-3’). The progress of the Huisgen reaction was confirmed by the additional Huisgen reaction with 2-azidoethanol as described previously.29 Introduction of 2-azidoethanol into the polymer backbones would provide a peak of triazole at 7.6–7.7 ppm in 1H NMR spectrum, which can be distinguished from the peak of triazole of sialyllactose units (8.0 ppm). However, there is only one peak at 8.0 ppm in the 1H NMR spectra of the glycopolymers (Figure S4). This demonstrates all alkyne groups in the polymer backbones were converted to triazole groups with sialyllactoses (Table 2). Relative molecular weights and absolute molecular weights of the glycopolymers were obtained by SEC analysis and light scattering (LS) measurement, respectively (Table 2 and FigureS5). For GP1s–GP5s (both -6’ and -3’), the absolute molecular weights were dependent on the sugar densities of the glycopolymers, while the relative molecular weights were almost the same value independent of the sugar densities. These results suggest that GP1–GP5 polymers have the same excluded volume in aqueous solution even though they have different sugar densities, because the polymer backbones were defined with the same target degree of polymerization. GP6s and GP7s have smaller molecular weights compared with GP4s, which corresponds to the results for the polymer backbones (P6, P7, and P4). 14


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Table 2. Properties of the synthesized glycopolymers carrying SALacs. No.

Sugar densities (%)

Calculated D.P. (mer)

Mn,th (×10-3 g/mol)

Mn,SEC (×10-3 g/mol)

Mn,LS (×10-3g/mol)

Mw/Mn,SEC

GP1–6’

12

106

17

22

21

1.42

GP2–6’

31

108

31

18

31

1.40

GP3–6’

49

114

48

25

39

1.37

GP4–6’

68

108

60

24

50

1.48

GP5–6’

86

100

78

28

80

1.52

GP6–6’

67

25

14

10

13

1.26

GP7–6’

67

48

27

15

13

1.31

GP1–3’

12

106

17

21

18

1.36

GP2–3’

31

108

35

20

32

1.36

GP3–3’

49

114

52

25

45

1.35

GP4–3’

68

108

60

26

56

1.38

GP5–3’

86

100

78

25

68

1.49

GP6–3’

67

25

14

11

13

1.25

GP7–3’

67

48

27

15

23

1.31

Mn,th = [(MWSugar × Sugar density/100 + MWAAm × (100−Sugar density)/100] × Calculated D.P.

Evaluation of the interaction with the influenza virus. The influenza virus was adopted as a model target to demonstrate the properties of the glycopolymers. The influenza virus is an important target in biological and medical fields, and studies about glycopolymers targeting the influenza virus have been reported.23,24, 34–38 The virus

15


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possess sialic acid binding lectin called hemagglutinin on the surface.39–40 There are 16 subtypes of hemagglutinin based on its antigenic difference.41 Human influenza virus and avian influenza virus have hemagglutinin recognizing either Neu5Ac-α(2,6)-Gal or Neu5Ac-α(2,3)-Gal structures, which are contained in 6’-SALac and 3’-SALac, respectively.38–41 In the infectious mechanism, the influenza viruses recognize their host based on the specific interaction of hemagglutinin with oligosaccharides because a human has Neu5Ac-α(2,6)-Gal and a bird has Neu5Ac-α(2,3)-Gal structures in bodies.38–41 In terms of biological application, it is important to recognize the type of influenza virus depending on the type of oligosaccharide. Here it was expected that glycopolymers carrying these SALac would recognize the corresponding type of influenza virus. Both of the influenza viruses used in this work were human influenza virus, however, it was reported the virus strain of [A/Puerto Rico/8/34 (H1N1)] showed the specific interaction with Neu5Ac-α(2,3)-Gal

structure because of variation through a long term propagation with chicken eggs.42 The interaction between the glycopolymers and the influenza viruses was evaluated by a hemagglutination inhibition (HI) assay. The presence of the influenza virus causes the aggregation of red blood cells (RBCs) because of the existence of hemagglutinin on the virus surface. The glycopolymers were expected to inhibit the aggregation of RBCs by interacting with the hemagglutinin on the viruses. The concentrations of the glycopolymers were sequentially diluted to determine the minimum concentration required for inhibiting the aggregation of RBCs. Whitesides and coworkers expressed the minimum sugar concentration for HI as Ki, and evaluated the efficacy of glycopolymers.34–37 We 16


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also adopted the Ki value to compare the efficacy of the glycopolymers. PolyAAm (PAAm) was used as a negative control.

Figure 3. The plots of the Ki from HI assay of the glycopolymers against the influenza viruses [A/Aichi/2/68 (H3N2) (A) and A/Puerto Rico/8/34 (H1N1) (B)] (n =2). The unit is sugar concentration.

The specific molecular recognition of the sugar units and the influence of three factors in the glycopolymer structures; sugar density, polymer length, and localization of sugars, on the interaction with the influenza virus were evaluated by the HI assay. The glycopolymers carrying 6’-SALac (GP1-6’−GP7-6’) inhibited RBC aggregation caused by the H3N2 type of the virus, but not H1N1 [Figure S6 (A)]. Conversely, glycopolymers carrying 3’-SALac (GP1-3’−GP7-3’) inhibited the aggregation caused by the H1N1 type, but not H3N2 [Figure S6 (B)]. PAAm did not show any inhibition. As expected, the glycopolymers recognized the types of influenza viruses depending on 17


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the sugar ligands in the polymer structures. The influence of the sugar density on the interaction was evaluated by comparing GP1−GP5 polymers. GP4-6’and GP5-3’ showed the lowest Ki values (48 and 81 µM) indicating the strongest interaction in the two groups, GP1-6’−GP5-6’ and GP1-3’−GP5-3’, respectively (Figure 3). The interaction of the glycopolymers was enhanced by the cluster glycoside effect and was expected to be maximized at maximum sugar density (100%). However, GP4-6’ showed the strongest interaction even though the sugar density was 68% while the sugar density of GP5-3’ was 100%. These results indicated the contribution of the cluster glycoside effect was different for the type of sugar units in the glycopolymers. The flexibility of 6’-SALac in the side chain was considered to be lower than that of 3’-SALac because of steric hindrance, and the poor flexibility of 6’-SALac caused the decrease of the interaction between GP5-6’ (sugar density: 100%) and hemagglutinin.43–45 These results suggested that a glyco-homopolymer is not always the best structure, and that appropriate sugar density is required in terms of exhibiting a cluster glycoside effect because of a potential lack of flexibility. The influence of the polymer lengths on the interaction was evaluated by comparing GP6s and GP7s with GP4s. The polymer lengths of GP6s (25 mer) and GP7s (48 mer) were shorter than that of GP4s (108 mer) while the sugar densities were the same (68%). For both sugar types, GP6s and GP7s showed the highest Ki values indicating the low interaction. The Ki value for GP6-6’ and GP7-6’ was 1.2 mM, and the value for GP6-3’ and GP7-3’ was 0.92 mM (Figure 3). GP4-6’ and 18


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GP4-3’, which have the longest polymer structures, exhibited the strongest interaction with the influenza viruses at a µM order. (KiGP4-6’ = 48 µM and KiGP4-3’ = 120 µM). The decrease in the Ki values from a mM to a µM order indicates the interaction with hemagglutinin was greatly enhanced by elongating the polymer structures. To compare the influence of the polymer length, the average distances between the terminals of the glycopolymers in solution were calculated theoretically according to the literature (the detail of the calculation is in Supporting Information).46,

47

The

distances for GP6, GP7, and GP4 polymers were ca. 2.8, 4.1, and 6.7 nm, respectively. Hemagglutinin has three binding sites on the surface, and the distance between two of the binding sites is ca. 4.5 nm.48 The theoretical distance between the terminals of the GP4 molecules are longer than the distance between the binding sites of hemagglutinin. This suggests that the polymer lengths of the GP4 molecules were long enough to bind to two binding sites of hemagglutinin, and thus the interaction was enhanced. The results of the HI assay demonstrated that specific design of the polymer length using living radical polymerization can enhance the interaction of glycopolymers with the influenza virus. Comparison of the Ki values for GP2 and GP7 polymers revealed the influence of localizing the sugar units on the interaction with the influenza virus. The sugar densities of GP2 and GP7 polymers are 31 and 68%, respectively, and the polymer lengths of GP2 and GP7 polymers are 108 and 48 mer, respectively. The numbers of sugar units in GP2s and GP7s were calculated as 33 and 32 units, respectively. The polymer lengths of GP2s are longer than that of GP7s, and the sugar densities of 19


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GP7s are higher than that of GP2s. Interestingly, GP2-6’ and GP2-3’ (KiGP2-6’ = 0.13 mM and KiGP2-3’ = 0.40 mM) showed lower Ki values than GP7-6’ and GP7-3’ (KiGP7-6’ = 1.2 mM and KiGP2-3’ = 0.92 mM) despite having the same number of sugar groups in the polymer structures (Figure 3). The polymer lengths of GP2s are long enough to interact with two binding sites of hemagglutinin while GP7s are expected to interact with only one binding site. This result demonstrated that multivalent binding to two sites contributes to the polymer–virus interaction better than the multiple display of sugar units toward one binding site.

Inhibitory activity of the glycopolymers against the virus infection. The ability of the glycopolymers carrying 6’-SALac to inhibit virus infection was evaluated by plaque assay.49 MDCK cells were prepared as a monolayer on a well and the influenza virus solution with the glycopolymers was added into the well. The MDCK cells were incubated at 4 °C to prevent the virus from entering the cells by endocytosis. The glycopolymers were expected to inhibit the virus adsorption onto the cell surface by binding to hemagglutinin on the virus surface. The number of plaques formed by infected cells was counted, and the infection percentage was calculated. GP1-6’−GP7-6’ polymers were evaluated, and the polymer concentration was calculated based on the sugar units in the polymers (Figure 4 and S7). GP4-6’ showed inhibition of the virus infection with an IC50 value of 320 µM. The other glycopolymers did not show obvious inhibition of virus infection even at 1000 µM. Interestingly, only one glycopolymer (GP4-6’) showed a clear inhibitory 20


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effect on virus infection even though other glycopolymers also showed an interaction with hemagglutinin in the HI assay. These results suggest that the hemagglutinin−oligosaccharide interaction is not the only factor affecting inhibition against the virus infection. It has been reported that hydrophobic chains in polymer structures can penetrate lipid bilayer membranes.50 The synthesized glycopolymers in this report are all hydrophilic, and will not be introduced into a lipid bilayer membrane. Thus, these glycopolymers will not be incorporated in the virus envelope and do not use hydrophobic interactions for the inhibition activity. In future work, the introduction of hydrophobic parts into the glycopolymer structure will be considered as a design criteria, with the aim to incorporate the polymer into the virus envelope and enhance the inhibition activity against the virus infection.

Figure 4. Inhibition of influenza virus infection of MDCK cells by glycopolymers (GP3-6’, GP4-6’, and GP5-6’). H3N2 (A/Aichi/2/68) virus was used. The number of plaques was counted, and the percentage inhibition was plotted against the sugar concentration. The data are average values ± the standard deviation (n = 3).

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Conclusions In summary, we have demonstrated that the design of polymer structures have great contribution for the interaction of synthetic glycopolymers with the influenza virus. Various glycopolymers carrying sialyl oligosaccharides were prepared by “post-click” chemistry. The polymer structures were controlled by RAFT polymerization and sialyllactoses were introduced by the Huisgen reaction. An HI assay demonstrated that the glycopolymers exhibited molecular recognition against different types of influenza viruses depending on the sugar units in the polymer structures. The HI assay of the glycopolymers carrying different sugar densities indicated that a glyco-homopolymer is not always the best structure for exhibiting a cluster glycoside effect because of a lack of flexibility of side chains. It is also important to render the glycopolymers long enough to bind to multiple sites of hemagglutinin to enhance the interaction. Interestingly, the comparison of the Ki values for the glycopolymers carrying the same number of sugar units revealed that a multivalent interaction with multiple binding sites was more efficient than the multiple display of sugar units toward one binding site. Finally, the ability of the glycopolymers to inhibit the virus infection was demonstrated. Lipidation of the polymer structures is considered as an essential factor for improving the inhibition in future work. The interactions of synthetic glycopolymers can be enhanced by the considered design of the structures, and such synthetic polyvalent glycoconjugates would be a good candidate for nano-medicine and as inhibitors against pathogens.

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Supporting Information 1

H NMR spectra, 13C NMR spectrum, and ESI-MS spectrum of the obtained compounds (FigureS1–

S4), detailed properties of RAFT polymerization (Table S1), properties of the Huisgen reaction (Table S2), SEC chromatographs of the synthesized glycopolymers (Figure S5), result of the HI assay (Figure S6), and result of plaque assay (Figure S7) are shown in the Supporting Information.

Acknowledgment We acknowledge financial support from a Grant-in- Aid for Scientific Research (B) (15H03818) and a Grant-in- Aid for Challenging Exploratory Research (16K140007). We appreciate assistance from Ms. Akane Kubo for helping us with a HI assay.

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For Table of Contents Use Only

Title: Design of glycopolymers carrying sialyl oligosaccharides for controlling the interaction with the influenza virus.

Authors: Masanori Nagao,1 Yurina Fujiwara,2 Teruhiko Matsubara,2 Yu Hoshino,1 Toshinori Sato,2 and Yoshiko Miura1* 1

Department of Engineering, Graduate School of Chemical Engineering, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395, Japan Email: [email protected]

2

Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan

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Design of Glycopolymers Carrying Sialyl Oligosaccharides for

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