Article pubs.acs.org/Biomac
Carbohydrate-Functionalized Chitosan Fiber for Influenza Virus Capture Xuebing Li,*,†,‡ Peixing Wu,†,§ George F. Gao,‡ and Shuihong Cheng‡ ‡
CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China § Lanzhou Institute of Animal Science and Veterinary Pharmaceutics, Chinese Academy of Agricultural Science, Lanzhou 730050, China S Supporting Information *
ABSTRACT: The high transmissibility and genetic variability of the influenza virus have made the design of effective approaches to control the infection particularly challenging. The virus surface hemagglutinin (HA) protein is responsible for the viral attachment to the host cell surface via the binding with its glycoligands, such as sialyllactose (SL), and thereby is an attractive target for antiviral designs. Herein we present the facile construction and development of two SL-incorporated chitosan-based materials, either as a water-soluble polymer or as a functional fiber, to demonstrate their abilities for viral adhesion inhibition and decontamination. The syntheses were accomplished by grafting a lactoside bearing an aldehyde-functionalized aglycone to the amino groups of chitosan or chitosan fiber followed by the enzymatic sialylation with sialyltransferase. The obtained water-soluble SL−chitosan conjugate bound HA with high affinity and inhibited effectively the viral attachment to host erythrocytes. Moreover, the SLfunctionalized chitosan fiber efficiently removed the virus from an aqueous medium. The results collectively demonstrate that these potential new materials may function as the virus adsorbents for prevention and control of influenza. Importantly, these materials represent an appealing approach for presenting a protein ligand on a chitosan backbone, which is a versatile molecular platform for biofunctionalization and, thereby, can be used for not only antiviral designs, but also extensive medical development such as diagnosis and drug delivery.
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virus, is responsible for the viral attachment to host cells via binding to its glycoligands, such as sialyllactose [SL, Neu5Acα(2,3)Galβ(1,4)Glc].11,12 The structural study on HA proteins complexed with SL or its analogues reveals that the specific binding is driven by multiple interactions consisting of hydrogen bonds, hydrophobic interactions, and van der Waals contacts.13 Similarly, E. coli O157 and its virulence determinant, Shiga toxin, recognize a cell surface trisaccharide, globotriose, to facilitate the bacterial colonization and toxin entry into the cells.14,15 The binding complex of Shiga toxin with globotriose is also well-defined by the crystal structure.16 These examples illustrate that carbohydrates can play an indispensable role in infectious diseases and suggest that pathogen−carbohydrate interactions can be an excellent target for medical development. Indeed, coupling of glycoligands with scaffolds (molecules or materials) may generate appealing multivalent systems that not only contribute to anti-infective drug designs, but also advance the field applications. The artificial multivalent glyco-materials have been shown to be powerful tools for biomedical research and development.17−20 Herein, we demonstrate two novel carbohydrate-functionalized chitosan-based materials (Figure 1). The first is a water-
INTRODUCTION Influenza is a severe viral disease of the respiratory tract that causes over 300000 human deaths annually.1 Recent outbreaks of avian H5N1 and 2009 A/H1N1 influenza have caused public panic and economic losses,2,3 emphasizing the urgent need for effective measures for virus prevention and treatment. Antivirals and vaccinations are critical in the fight against influenza; however, the effectiveness of the currently available drugs (rimantidine, amantadine, oseltamivir, and zanamivir) is increasingly diminished by the viral resistance,4−6 and the current process of constructing a new vaccine strain based on the newly circulating virus is quite time-consuming (over six months7), which can hardly meet the requirements of rapid mass vaccination for curtailing the pandemic. Developments of alternatives to the present drugs therefore must be a high priority to meet challenges of the emerging drug-resistant and novel pandemic viruses. Furthermore, the effective approaches for endowing fibrous materials with a virus-capture capability are strongly demanded as their potential applications, such as the virus filters for face mask, air conditioner, air cleaner, and so on, would significantly limit the spread of the virus infection.8 It is well established that many pathogens use host cell surface glycans as anchors for attachments, which subsequently lead to infection.9,10 Prominent examples of such pathogens include influenza virus, Escherichia coli O157, and Shiga toxin. Hemagglutinin (HA), the major surface antigen of the influenza © 2011 American Chemical Society
Received: July 14, 2011 Revised: October 4, 2011 Published: October 6, 2011 3962
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Measurements. 1H NMR spectra were recorded on a Bruker DRX 600 spectrometer at 600 MHz. Elemental analyses were performed using a CHN elemental analyzer (CHN corder MT-6, Yanaco Co.). IR spectra were recorded on an IR spectrometer (FT/ IR-4200, Jasco Co.) by the KBr pellet method. Surface morphology of the fibers was observed using a scanning electron microscope (SEM; S-2250, Hitachi, Ltd.) operated at 25 kV. Prior to analysis, the fibers were dried in vacuo and coated with gold−palladium alloy according to a standard procedure.37 The Mw and PDI of the starting chitosan and all soluble conjugates were measured by a GPC system (CTO20A, Shimadzu Co., Ltd.) equipped with a multiangle LLS detector (DAWN HELEOS-II, Wyatt Technology Corp.) in NaOAc buffer (pH 4.5). Dialysis was conducted using a cellulose ester (CE) membrane tube with the MWCO of 300 kDa (Thermo Fisher Scientific Inc.). Surface plasmon resonance (SPR) analysis was performed by a biosensor system (BIAcore 3000, Biacore, Inc.). Synthesis of Lactose−Chitosan Conjugate 5. O3 was successively bubbled through a stirred solution of alkene 3 (398 mg, 0.94 mmol) in MeOH (40 mL) at −78 °C until TLC (9:4:2 ethyl acetate−isopropanol−water) showed no starting material left. N 2 was subsequently bubbled through the solution for 10 min to remove the remaining O3. After adding Me2S (139 μL, 1.88 mmol), the reaction mixture was stirred at room temperature overnight followed by evaporation to quantitatively yield 4 (400 mg, white solid), which was directly subjected to the next coupling reaction with chitosan. Thus, to a stirred aqueous 5 wt % AcOH solution (10 mL) of chitosan (100 mg, 0.59 mmol, 0.47 mmol of GlcN) was added dropwise a solution of the above aldehyde 4 in methanol (6 mL). The mixture was stirred for 1 h at room temperature, and then NaCNBH3 (118 mg, 1.88 mmol) was added. After stirring the mixture overnight, the acidic solution was neutralized with 1 N aqueous solution of NaOH. The mixture was subjected to dialysis. After filtration to remove the precipitate, lyophilization gave 214 mg of conjugate 5 as a white amorphous solid. The DSLac is 0.80, as determined from the 1H NMR spectrum and elemental analysis. The yield is 73% on the basis of the 0.8 DS Lac. The Mw is 15000 kDa as measured by GPC. 1H NMR (D2O, 27 °C): δ 4.42 (1H, d, J = 7.8 Hz, H-1 of Gal), 4.38 (1H, d, J = 7.8 Hz, H-1 of Glc), 3.93−3.45 (19.5H, m, protons of sugar ring and linker OCH2), 3.25 (1H, br, H-2 of Gal), 2.88−2.66 (3H, brm, NHCH and NHCH2), 2.01 (0.75H, brs, COCH3), 1.59 (4H, brs, 2 × linker CH2), 1.36 (2H, brs, linker CH2). Anal. Calcd for (C8H13NO5)0.20(C23H41NO15)0.80(H2O)0.23: C, 47.81; H, 7.14; N, 2.79. Found: C, 47.59; H, 7.25; N, 2.78. Synthesis of SLCC 1. Conjugate 5, CMP-NANA, and α2,3-SiaT, calf intestine alkaline phosphatase (CIAP) in a 50 mM sodium cacodylate buffer (pH 7.3, 1−5 mL) containing 2.5 mM MnCl2 and 0.1% bovine serum albumin (BSA) was incubated at 37 °C for 1 day. The mixture was heated at 100 °C for a few minutes and then centrifuged. The supernatant was subjected to exhaustive dialysis against water using a cellulose ester membrane tube with MWCO of 300 kDa, which allowed differentiation of the conjugates (Mw > 5000 kDa) from the contamination of proteins and small molecule byproduct (the Mw of proteins used in the reaction including the enzyme were lower than 160 kDa). After filtration to remove the precipitate, lyophilization gave SLCC 1a−d as the white amorphous solids. The details of reactants, yield, and structural parameter of the products are listed in Table S1 (Supporting Information). 1H NMR (D2O, 27 °C) of SLCC 1a: δ 4.47, 4.42, 4.39 (2.76H, 3 × d, J = 7.8 Hz, respectively, H-1 of Gal and Glc), 4.05 (1H, d, J = 8.0 Hz, H-3 of Gal in SiaLac branch), 3.94−3.47 (32.9H, m, protons of sugar ring and linker OCH2), 3.25 (1.38H, br, H-2 of Gal in SiaLac branch), 3.20− 2.75 (4.14H, brm, NHCH and NHCH2), 2.70 (1H, brd, J = 7.8 Hz, H3eq of Neu5Ac), 2.06−1.97 (4.03H, brm, COCH3), 1.74−1.61 (6.52H, brm, H-3ax of Neu5Ac and 2 × linker CH2), 1.40 (2.76H, brs, linker CH2). SLCC 1b−d have the similar 1H NMR data. The elemental analysis data of SLCC 1a−d are summarized in Supporting Information. Preparation of Chitosan Fiber. Chitosan fiber was prepared according to a wet-spinning process as described previously. 38 Briefly, chitosan flake (10 g, same as that used for SLCC 1 preparation) was
Figure 1. Schematic illustrations of (a) inhibition of influenza virus adhesion by soluble SLCC 1 and (b) capture of influenza virus by fiber SLCF 2. Abbreviations: HA, hemagglutinin; SL, sialyllactose.
soluble SL−chitosan conjugate (SLCC 1) designed as a highly active influenza virus HA inhibitor. The second is a simple filtration device featured by a sheet of SL-modified chitosan fiber (SLCF 2) to effectively remove the virus from the medium. Sugar-incorporated systems reported in the past are diverse with various functions, which include conjugated polymers and dendrimers, nanoparticles, vesicles, magnetic beads, quantum dots, membrane, and microarrays.21−28 To the best of our knowledge, however, this work is the first to functionalize a fibrous material to confer the molecular recognition capability with the sugar. Importantly, the use of polysaccharide chitosan, which has been widely used in the pharmaceutical and medical areas due to its unique properties, such as biocompatibility, biodegradability, and antimicrobial activity,29−33 as the scaffold enables not only the improved safety of SLCC 1 as a potential anti-influenza agent, but also attractive applications of SLCF 2 as a filter material in the control of the virus transmission.
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EXPERIMENTAL SECTION
Materials. Chitosan flakes were purchased from Yaizu Suisankagaku Industry Co. Ltd. The molecular weight (Mw) and polydispersity index (PDI: Mw/Mn) were, respectively, 5000 kDa and 1.8, as measured by a gel permeation chromatography (GPC) system equipped with a laser light scattering (LLS) detector. The deacetylation degree was 80%, as determined by the 1H NMR spectrum. Alkene 3 was synthesized according to procedures described previously.34,35 α2,3-Sialyltransferase (α2,3-SiaT, rat recombinant, Spodoptera frugiperda, Mw 3.9 kDa) was purchased from Calbiochem Co. Sialyllactose and cytidine-5′-monophospho-N-acetylneuraminic acid sodium salt (CMP-Neu5Ac) was purchased from Sigma Co. Recombinant influenza virus hemagglutinin (HA, A/Equine/La Plata/ 93, H3N8) was purchased from Katakura Industries Co., Ltd. Human influenza A/PR/8/34 (H1N1) virus was propagated in the allantoic cavity of 10 day old embryonated chicken eggs. Infected allantoic fluid was pooled and clarified by sucrose-density gradient centrifugation. The hemagglutination unit (HAU) of the purified virus was determined by hemagglutination assay.36 3963
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of LCF 6 afforded Ac-LCF 7a−c. The details of reactants, yield, and structural parameter of the products are listed in Table S3. IR (KBr) of Ac-LCF 7a: ν = 3415 (OH and NH), 2939, 2878 (CH), 1639, 1567, 1406, 1362, 1318 (NH2 and NHCOCH3), 1091 cm−1 (COC). Compounds 7b and 7c have the similar IR data. The elemental analysis data of Ac-LCF 7a−c are summarized in Supporting Information. Preparation of SLCF 2. Ac-LCF 7a−c were, respectively, immersed in a 50 mM sodium cacodylate buffer (pH 7.3, 5−10 mL mL) containing 2.5 mM MnCl2, 0.1% BSA, and CMP-NANA. To each mixture, α2,3-SiaT and CIAP were added with stirring. The mixtures were incubated at 37 °C for 2 days and the filaments were collected by filtration, washed with deionized water and methanol, and air-dried to afford SLCF 2a−c. The details of reactants, yield, and structural parameter of the products are listed in Table S4. IR (KBr) of SLCF 2a: ν = 3415 (OH and NH), 2941 (CH), 1679 (COOH), 1632, 1591, 1428, 1386 (NH2 and NHCOCH3), 1060 cm−1 (COC). Compounds 2b and 2c have similar IR data. The elemental analysis data of SLCF 2a−c are summarized in Supporting Information. Binding Assay. The binding of SLCC 1 with the HA protein was analyzed by a BIAcore biosensorsystem based on the SPR technique. The HA protein was covalently immobilized by a standard aminecoupling method onto a sensor chip (CM-5, research grade) that was precoated with carboxymethyl dextran. The serial dilutions of SLCC 1a−d (∼5 mg/mL) in running buffer [phosphate buffered saline (PBS), pH 7.2] were, respectively, injected over the immobilized HA at a flow rate of 20 μL/min for 5 min (association phase). During the dissociation phase, the sensor chip surface was exposed to running buffer at a flow rate of 20 μL/min. Conjugate 5 and monomeric SL were used as the negative controls. The Kd of the binding was calculated by a standard BIA evaluation software. Hemagglutination Inhibition (HAI) Assay. HAI assays were performed as described by Suzuki et al.11 Briefly, the 2-fold serial dilutions of SLCC 1a−d (in PBS, pH 7.2) were, respectively, added (25 μL) to 96-well microtiter plates followed by adding the influenza A/PR/8/34 (H1N1) virus suspensions (in PBS, 4 HAU, 25 μL) to each well. After 1 h of incubation (4 °C), 50 μL of 0.5% (v/v) guinea pig erythrocytes suspension in PBS was added to each well. The hemagglutinations and MIC values were recorded after 2 h of incubations. Conjugates 5, monomeric SL, and fetuin were used for controls. Virus Capture Assay. To examine the virus-capture capability of SLCF 2a−c, the simple filtration devices were built by packing the fiber (20 mg, cut into 0.5 cm length) onto the bottom of a 1 mL sterilized syringe (Terumo Co.) between 2 sheets of glass fiber. The filters were successively washed with 0.01% Tween 20 in PBS (10 mL) and 0.1% BSA in PBS (5 mL) for preventing the nonspecific adsorption of the virus. Influenza virus suspensions in PBS (512 HAU, 1 mL) containing 0.1% BSA were added to each syringe, and passed slowly through the filter. The virus-capture efficacy of the fibers was evaluated by hemagglutination assay36 of the filtrates. Chitosan fiber and Ac-LCF 7a were used as the controls.
dissolved in an aqueous solution of AcOH (3 v/v %) to afford the dope (5 wt %), which was subsequently filtered through flannel and injected through a stainless-steel spinneret into a coagulation bath containing a mixture of 5% aqueous NaOH solution and ethanol (7:3 v/v ). The resulting filaments were stretched in water (15 °C) at the ratio of 1.2−1.4 and exhaustively washed with hot water (60 °C, in washing bath), deionized water, and methanol. The fibers were airdried and stored in a dry cabinet before use. Chitosan fiber was characterized by 1H NMR spectroscopy, IR spectroscopy, and CHN elemental analysis. The obtained data is same as that of starting chitosan flakes. Preparation of LCF 6. Modification of chitosan fiber with aldehyde 4 was carried out according to the N-alkylation procedure reported by Hirano et al.38 Chitosan fiber was immersed in a solution of aldehyde 4 in methanol (5−10 mL). After removing of air bubbles on the filaments surface by stirring under reduced pressure for a few minutes, the mixture was incubated with shaking at room temperature overnight. A solution of NaCNBH3 in methanol (0.5−3 mL) was added dropwise to the mixture with stirring. The reaction mixture was incubated with shaking at room temperature for an additional 1 day. The resultant filaments were collected by filtration, sufficiently washed with methanol, and air-dried to afford LCF 6a−c. The details of reactants, yield, and structural parameter of the products are listed in Table S2. 1H NMR (DCl/D2O) data of LCF 6a−c are similar to that of conjugate 5. The 1H NMR spectrum of 6a is typically illustrated in Figure 2. IR (KBr) of LCF 6a: ν = 3415 (OH and NH), 2930, 2879
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RESULTS AND DISCUSSION Synthesis and Characterizations of SLCC 1. The synthesis of SLCC 1 is shown in Scheme 1. With our glycoconjugation approach, 34,35 a lactose (Lac)-branched chitosan conjugate 5 was prepared by reductive N-alkylation of chitosan with an aldehyde 4, which was quantitatively obtained from an alkene 3 by ozonolysis. Complete Nsubstitution has been achieved in this reaction by the excess use of 4 (2 equiv), yielding the water-soluble conjugate 5 (degree of substitution of Lac branch, DSLac = 80%) that allowed the next enzymatic sialylation to proceed smoothly under the homogeneous aqueous condition. The transfer of sialic acid (Sia) residue from donor CMP-Neu5Ac to the lactose branch of 5 by an α2,3-sialyltransferase (α2,3-SiaT) afforded SLCC 1a−d in excellent yields. The degree of
Figure 2. 1H NMR spectra (600 MHz, 27 °C) of chitosan (DCl/ D2O), Lac-chitosan conjugate 5 (D2O), SLCC 1a (D2O), and LCF 6a (DCl/D2O). (CH), 1639, 1406, 1362 (NH2 and NHCOCH3), 1091 cm−1 (COC). Compounds 6b and 6c have the similar IR data. The elemental analysis data of LCF 6a−c are summarized in Supporting Information. Preparation of Ac-LCF 7. The N-acylation of LCF 6 was carried out according to the known procedures.39−41 Briefly, LCF 6a−c were respectively immersed in methanol (5−10 mL). To each mixture, acetic anhydride was added with stirring. The mixtures were incubated with shaking at 40 °C overnight. Workups as described for preparation 3964
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Scheme 1. Synthesis of SLCC 1a
sugar branch, alkyl linker, and acetyl group of the chitosan backbone, as typically illustrated in Figure 2. The DSLac and DSSL determined on the basis of an integral ratio of the branchderived peaks to chitosan acetyl peak were in good agreement with the CHN elemental analysis data. Moreover, the successive increase in the Mw of chitosan, conjugate 5, and SLCC 1d−a measured by GPC equipped with a LLS detector provided additional evidence of the incorporated sugar units (Supporting Information). However, an attempt for further characterization by 13C NMR spectroscopy has failed because of the rigid chitosan backbone42 and increased macromolecule size of the conjugates. Bindings of SLCC 1 with Influenza Virus HA Protein. To establish the binding capability of SL residues bound to chitosan scaffold, interactions between the conjugates and a viral HA protein (A/Equine/La Plata/93, H3N8) were analyzed by a BIAcore biosensor system based on the surface plasmon resonance (SPR) technique. The HA protein was covalently immobilized on a carboxymethylated dextran-coated sensor chip by the general amine-coupling method.43 The solutions containing various concentrations of SLCC 1a−d were respectively injected over the sensor chip surface, and the binding affinity was determined. As illustrated in a typical SPR sensorgram (Figure 3, in resonance units, RU), significant
a
Reagents and conditions: (i) O3, Me2S, −78 °C, quantitatively; (ii) chitosan (80% GlcN, 20% GlcNAc, Mw = 5000 kDa), NaCNBH3, AcOH (aq. 5 wt %), rt, 24 h, 73%; (iii) α-2,3-SiaT, CMP-Neu5Ac, sodium cacodylate buffer (pH 7.3), BSA, 37 °C, 24 h, 91−98%. b Degree of substitution (DS) of Lac branch. cDS of sialyllactose (SL) branch. Abbreviations: SiaT, sialyltransferase; CMP, cytidine 5′monophosphate; BSA, bovine serum albumin.
substitution of the SL branch (DSSL) in SLCC 1 was adjusted by the molar ratio of CMP-Neu5Ac to the Lac branch of conjugate 5. The maximum DSSL was obtained at 58% by using 1.6 equiv of CMP-Neu5Ac. More excessive use of the donor and enzyme had been tested without effect on further increasing the DSSL, probably due to the increased steric hindrance of SLCC 1. Like the conjugate 5, SLCC 1a−d showed good solubility in the neutral water (solubility ≥ 1.0 mg/mL, PBS, pH 7.2) in contrast to the water-insoluble chitosan, owing to the incorporated hydrophilic sugar branches. All the conjugate 1a−d and 5 were characterized by 1H NMR spectroscopy, CHN elemental analysis, and GPC analysis. The structural compositions were verified by 1H NMR assignments of several baseline-separated signals of the
Figure 3. Representative sensorgrams for the bindings of SLCC 1 with influenza virus HA protein analyzed by a BIAcore biosensor system. The hemagglutinin (HA) was immobilized on a sensor chip (CM-5, research grade). The solution containing SLCC 1, Lac-chitosan conjugate 5, or sialyllactose (SL) was, respectively, injected over the sensor chip surface. Data was outputted in resonance unit (RU). Abbreviations: SPR, surface plasmon resonance; PBS, phosphate buffered saline; Kd, dissociation constant.
bindings of SLCC 1a−d to the HA were observed, with the decreased intensity (RU) in accordance with the DSSL value due to the cluster glycoside effect (multivalent glyco-systems 3965
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with the higher density of sugar moiety often achieve increased binding to the target proteins44−47). In contrast, no activity was detected for the conjugate 5, proving that the specific binding was derived from SL branches of SLCC 1. The interaction between monomeric SL and influenza HA has been reported to be typically low affinity with the dissociation constant (Kd) in the millimolar range (approximately 2−3 mM48,49). Here the SPR study also revealed a commercially available SL had no visible binding with the HA at the concentration of 1 mg/mL (1.57 mM) or less. The result of high avidity of SLCC 1a−d to HA (K d = 0.59−1.93 μM) demonstrates multivalent presentation of SL ligands on chitosan backbone is highly effective to achieve strong binding. Inhibitions of Influenza Virus Attachment to Host Cells by SLCC 1. The above binding study provides a baseline for the follow-up hemagglutination inhibition (HAI) assay, in which the inhibitory efficacy of SLCC 1 on the viral adhesion to host erythrocytes was verified (Table 1). SLCC 1a−d
modifications of chitosan fiber using aldehydes (N-alkylation) or carboxylic anhydrides (N-acylation) have been well established by several groups. 38−41 According to these published methods and our own experience in the synthesis of SLCC 1, we first attached Lac residues onto the fiber via the N-alkylation with aldehyde 4 under a heterogeneous condition (Scheme 2). The adjusted DSLac of resultant fiber 6a−c (LacScheme 2. Heterogeneous Modification of Chitosan Fiber
Table 1. Inhibition of Influenza Virus-Induced Hemagglutination of Erythrocytes by SLCC 1a sample
DSSL (%)
MIC (μg/mL)b
SLCC 1a SLCC 1b SLCC 1c SLCC 1d SLc conjugate 5 fetuine
58 39 18 5
63 (54 μM) 109 (70 μM) 219 (72 μM) 950 (93 μM) NAd NAd 156 (31 μM)
a
Influenza A/PR/8/34 (H1N1) virus and guinea pig erythrocytes were used. A/PR/8/34 is a typical human influenza virus that has been widely used in laboratory investigations. This virus binds specifically to structures of Siaα(2,3)Gal and Siaα(2,6)Gal. See ref 61 for more details. bMinimum inhibitory concentration: μg/mL of sample (μM of SL residue). The MIC value was calculated as the mean of a set of data obtained from at least 3 individual experiments, and the standard deviations were generally very low. cCommercially available sialyllactose. dNo activity at the concentration of 1 mg/mL (for SL, equal to 1.6 mM). eOn average, 1 molecule of fetuin (Mw ≈ 50 kDa) contains approximately 10 Sia-terminated carbohydrate branches. See refs 59 and 60.
modified chitosan fiber, LCF) was 16, 7, and 2%, respectively, as estimated by the 1H NMR spectra and elemental analyses (attempts to increase the DSLac over 16% had failed, probably due to the heterogeneous reaction condition). However, a preliminary test of sialylation (α2,3-SiaT, CMP-Neu5Ac) of LCF 6a had failed to yield product displaying satisfactory Sialoading (lower than 1%). We reasoned that electrostatic attractions between CMP-Neu5Ac and the alkalic primary amino groups abundantly present in LCF 6a had interfered with the enzymatic reaction and resulted in the low SL-loading. The heterogeneous N-acetylation of LCF 6a−c was therefore performed to convert the amines to amides to reduce the alkalinity. As a consequence, fiber products (Ac-LCF 7a−c) with the degree of acetylation over 72% were afforded by respective treatment of LCF 6a−c with acetic anhydride in menthol, and the desired SLCF 2a−c with the full SL-loadings (DSSL = 16, 7, and 2%, respectively) were obtained successfully via the subsequent enzymatic sialylation of Ac-LCF 7a−c. The SLCF 2a−c and Ac-LCF 7a−c were tested to be stable in neutral, alkalic, and acidic waters, but chitosan fiber and LCF 6a−c were soluble under acidic conditions due to the abundant free protonable amino groups.
significantly inhibited the agglutination of erythrocytes induced by the viral attachment, in contrast to monomeric SL and conjugate 5 that had no activity at the concentration tested, suggesting that the high affinity bindings of SLCC 1a−d with virus surfaces HAs resulted in the effective inhibitions. The MIC value of SLCC 1 strongly relies on the DSSL, and 1a, with the highest density of SL branch and affinity to the HA protein, was the most active inhibitor among the conjugates. Previous studies have demonstrated that a verity of multivalent Siacontaining molecules could act as active influenza virus HA inhibitors.50−58 Here the fact that inhibitory efficiency of SLCC 1a (MIC = 54 μM) is comparable with fetuin, a naturally occurring sialoglycoprotein which has been well used as an experimental control to define the potency of HA inhibitors,50−54,59,60 highlights its potential as a new HA blocker in explorations of safe and effective anti-influenza agents. Preparation and Characterizations of SLCF 2. The successful synthesis and bioactivity verifications of soluble SLCC 1 led us to embark on modification of chitosan fiber with the SL functionality. Chitosan fiber was prepared using a wetspinning procedure as described previously.38 The chemical 3966
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suggesting that the modification processes had little affect on the surface morphologies. Capture of Influenza Virus by SLCF 2. To examine the virus-capture capability of SLCF 2, a simple filtration device, as illustrated in Figure 1b, was built by packing the fiber onto the bottom of a 1 mL sterilized syringe between two sheets of glass fiber. After successive treatment of the fiber with Tween 20 and BSA to prevent nonspecific adsorption,62,63 influenza virus suspended in PBS buffer (512 HAU) was passed through the filter. The capture efficacy was evaluated by hemagglutination assay of the filtrate. As shown in Table 2, the virus
All the fibers described here were characterized by CHN elemental analysis and IR spectroscopy. Chitosan fiber and LCF 6a−c were additionally characterized by 1H NMR spectroscopy (the spectrum of 6a is shown in Figure 2). As typically illustrated by the IR spectra of 6a, 7a, and 2a in Figure
Table 2. Capture of Influenza Virus by SLCF 2a HAUc fiber none chitosan fiber Ac-LCF 7a SLCF 2a SLCF 2b SLCF 2c
DSSLb (%)
before filtration
after filtration
16 7 2
512 512 512 512 512 512
512 256 256
