Molecular Design of Spacer-N-Linked Sialoglycopolypeptide as

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Biomacromolecules 2009, 10, 1894–1903

Molecular Design of Spacer-N-Linked Sialoglycopolypeptide as Polymeric Inhibitors Against Influenza Virus Infection Makoto Ogata,† Kazuya I. P. J. Hidari,‡ Wataru Kozaki,§ Takeomi Murata,† Jun Hiratake,# Enoch Y. Park,† Takashi Suzuki,‡ and Taichi Usui*,† Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, Ohya 836, Suruga ward, Shizuoka 422-8529, Japan, Department of Biochemistry, University of Shizuoka, School of Pharmaceutical Sciences and Global COE Program, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan, and Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan Received March 16, 2009; Revised Manuscript Received April 10, 2009

A series of spacer-N-linked glycopolymers carrying long/short R2,3/6 sialylated glycan were designed as polymeric inhibitors of influenza virus. Lactose (Lac) and N-acetyllactosamine (LN: Galβ1,4GlcNAc) were first converted to spacer-N-linked disaccharide glycosides, followed by consecutive enzymatic addition of GlcNAc and Gal residues to the glycosides. The resulting spacer-N-linked glycosides with di-, tetra-, and hexasaccharides carrying a Lac, LN, lacto-N-neotetraose (LNnT: Galβ1,4GlcNAcβ1,3Galβ1,4Glc), and LNβ1,3LNnT were coupled to the carboxy group of γ-polyglutamic acid (γ-PGA) and enzymatically converted to glycopolypeptides carrying R2,3/6 sialylated glycans. The interactions of a series of sialoglycopolypeptides with avian and human influenza virus strains were investigated using a hemagglutination inhibition assay. The avian virus A/Duck/HongKong/313/4/78 (H5N3) bound specifically, regardless of the structure of the asialo portion. In contrast, human virus A/Aichi/2/68 (H3N2) bound preferentially to long R2,6sialylated glycans with penta- or heptasaccharides in a glycan length-dependent manner. Furthermore, the Sambucus sieboldiana (SNA) lectin was also useful as a model of human virus hemagglutinin (HA) for understanding the carbohydrate binding properties, because the recognition motifs of the inner sugar in the receptor were very similar.

Introduction Influenza virus infection is initiated by the binding of hemagglutinin (HA), a viral carbohydrate-binding membrane protein, to sialoglycoproteins or sialoglycolipids on receptors on the host cell surfaces.1-3 The viruses recognize not only sialic acid (SA) on the receptors but also particular sugar chain structures, such as sialyllacto-series type I [SAR2-(3/6)Galβ13GlcNAcβ1] and type II [SAR2-(3/6)Galβ1-4GlcNAcβ1] structures.4 The host cell specificity of viruses is dependent on the linkages (R2-3 or R2-6) between SA and the penultimate galactose, as well as on the number of SA residues and the core structure.4-6 We recently designed and prepared spacer-O-linked sialoglycopolypeptides to block infection by avian and human influenza viruses.7,8 However, the method is not always practical, because of its low efficiency, especially from the Oglycosylation step through the total synthetic process. As a result of these studies, we were further interested in developing an efficient synthetic route to sialoglycopolymers as glycomimetics. We therefore planned a simple synthesis of a new type of spacerN-linked sialoglycopolymers starting with Lac or LN. In this paper, we describe our strategy, which provides a facile way to design strong polymeric inhibitors of infection by influenza virus by arranging multivalent short or long glycans attached to a polypeptide backbone. Using these glycopolymers, * To whom correspondence should be addressed. Fax: +81-54-238-4873. E-mail: [email protected]. † Shizuoka University. ‡ University of Shizuoka and Global COE Program. § Faculty of Agriculture, Shizuoka University. # Institute for Chemical Research, Kyoto University.

structure-activity relationships were investigated by measuring inhibition of hemagglutination mediated by influenza viruses.

Experimental Section Materials. γ-PGA-Na (MW 990000) from Bacillus subtilis was a kind gift from Meiji Food Materia Co. UDP-GlcNAc, UDP-Gal and CMP-Neu5Ac were gifts from Yamasa Corporation (Chiba, Japan). Bovine milk β1,4-galactosyltransferase (β4GalT), R2,3-(N)-sialyltransferase (R2,3SiaT, rat recombinant, Spodoptera frugiperda) and R2,6(N)-sialyltransferase (R2,6SiaT, rat recombinant, S. frugiperda) were purchased from Calbiochem-Novabiochem (San Diego, CA). β1,3-NAcetylglucosaminyltransferase (β3GnT) was prepared by our previously described methods.9 Biotin-labeled SNA was purchased from EY Laboratories, Inc. (San Mateo, CA). Spacer-O-linked sialoglycopolymers 23 poly[SAR2,3LNβ-O(CH2)5NH-/γ-PGA], 24 poly[SAR2,3LNnTβO(CH2)5NH-/γ-PGA], 25 poly[SAR2,3LNβ1,3LNnTβ-O(CH2)5NH-/ γ-PGA], 26 poly[SAR2,6LNβ-O(CH2)5NH-/γ-PGA], 27 poly[SAR2, 6LNnTβ-O(CH2)5NH-/γ-PGA], and 28 poly[SAR2,6LNβ1,3LNnTβO(CH2)5NH-/γ-PGA] were prepared by previously reported methods.8 The human and avian influenza virus strains used in this study were propagated and purified as described previously.2,10 Viral hemagglutination units (HAUs) of purified viruses were determined as described previously.11 All other reagents were of the highest quality commercially available and were used without further purification. Enzyme Assay. β3GnT and β4GalT activities were assayed by previously described methods.8 HPLC Determination of Neutral Sugar Units or Sialic Acids in the Artificial Glycopolypeptides. Neutral sugar units or sialic acids of the artificial glycopolypeptides were determined by previously described ABEE- or DMB-labeled HPLC methods.8 Analytical Methods. HPLC analysis was carried out using an Asahipak NH2-P50 4-E column (4.6 × 250 mm, Shodex) with a JASCO

10.1021/bm900300j CCC: $40.75  2009 American Chemical Society Published on Web 05/13/2009

Sialoglycopolypeptide as Polymeric Inhibitors Intelligent system liquid chromatograph and detection at 210 nm. The bound material was eluted with 80% CH3CN at a flow rate of 1.0 mL/ min at 40 °C. FAB-mass analysis was carried out in positive ion mode using a JEOL JMS DX-303HF mass spectrometer coupled to a JEOL DA-800 mass data system. An accelerating voltage of 10 kV and a mass resolution of 1000 were employed. A sample in distilled water (1 µL) was loaded onto a probe tip and mixed with glycerol (1 µL) as a matrix. 1H and 13C NMR spectra were recorded on a JEOL JNM-LA 500 spectrometer at 25 °C. Chemical shifts are expressed in δ relative to sodium 3-(trimethylsilyl) propionate as an external standard. Synthesis of Lacβ-NH2 and LNβ-NH2. Lacβ-NH2 1 and LNβ-NH2 2 were prepared from Lac and LN according to a modified procedure of Kato et al.12 Lac (5.0 g, 0.014 mol) was added to 95% MeOH containing 0.5% CH3COOH (117 mL) saturated with ammonia gas at 0 °C in a pressure bottle. The bottle was tightly closed, and the mixture was stirred at 40 °C for 2 days. After the suspension became soluble, it formed a colorless precipitate within 12 h. EtOH (110 mL) was added to the reaction mixture, which was kept at 4 °C to precipitate the product. The crystalline precipitate was collected by filtration and washed with EtOH and ether, successively, to give pure Lacβ-NH2 (4.5 g, 95%). [R]D30 +53.7° (c 0.1, water); FAB-mass: m/z 342 [M + H]+ (matrix: glycerol); 1H NMR (D2O, 500 MHz) δ 4.46 (d, 1H, J1′,2′ ) 7.6 Hz, H-1′), 4.12 (d, 1H, J1,2 ) 8.9 Hz, H-1), 3.94 (dd, 1H, J5,6b ) 2.2, J6a,6b ) 12.2 Hz, H-6b), 3.92 (1H, H-4′), 3.78 (dd, 1H, J5,6a ) 4.9, J6a,6b ) 12.2 Hz, H-6a), 3.77-3.71 (2H, H-6′a, H-6′b), 3.73 (1H, H-5′), 3.66 (dd, 1H, J2′,3′ ) 10.1, J3′,4′ ) 3.4 Hz, H-3′), 3.65-3.60 (2H, H-3, H-4), 3.56 (1H, H-5), 3.54 (dd, 1H, J1′,2′ ) 7.6, J2′,3′ ) 10.1 Hz, H-2′), 3.20 (t, 1H, J1,2 ) 8.9, J2,3 ) 8.9 Hz, H-2); 13C NMR (D2O, 500 MHz) δ 105.7 (C-1′), 87.7 (C-1), 81.4 (C-4), 78.5 (C-5), 78.2 (C-5′), 77.9 (C-3), 76.7 (C-2), 75.3 (C-3′), 73.8 (C-2′), 71.4 (C-4′), 63.8 (C-6′), 63.0 (C-6). LN (1.0 g, 2.6 mmol) and ammonium bicarbonate (0.21 g, 2.6 mmol) were added to MeOH (8 mL) saturated with ammonia gas at 0 °C in a pressure bottle. The bottle was tightly closed, and the mixture was stirred at 40 °C for 5 days. After the suspension became soluble, it formed a colorless precipitate within 6 h. EtOH (8 mL) was added to the reaction mixture, which was kept at 4 °C to precipitate the product. The crystalline precipitate was collected by filtration and washed with EtOH and ether, successively, to give a mixture of LNβ-NH2 and its carbamate (9:1, 1H NMR in D2O). The crude product was dissolved in MeOH (20 mL) and was evaporated to dryness to decompose the carbamate. This evaporation process was repeated three times, yielding pure LNβ-NH2 as a colorless glassy hydroscopic form (0.71 g, 71%). [R]D30 +34.1° (c 0.1, water); FAB-mass: m/z 383 [M + H]+ (matrix: glycerol); 1H NMR (D2O, 500 MHz) δ 4.46 (d, 1H, J1′2′ ) 8.0 Hz, H-1′), 4.16 (d, 1H, J1,2 ) 8.6 Hz, H-1), 3.95 (dd, 1H, J5,6b ) 2.1, J6a,6b ) 12.2 Hz, H-6b), 3.92 (1H, H-4′), 3.79 (dd, 1H, J5,6a ) 5.2, J6a,6b ) 12.2 Hz, H-6a), 3.77 (dd, 1H, J5′,6′b ) 2.2, J6′a,6′b ) 10.4 Hz, H-6′b), 3.74 (dd, 1H, J5′,6′a ) 5.2, J6′a,6′b ) 10.4 Hz, H-6′a), 3.73 (1H, H-5′), 3.69-3.66 (3H, H-4, H-2, H-3), 3.66 (dd, 1H, J2′,3′ ) 10.1, J3′,4′ ) 3.4 Hz, H-3′), 3.55 (1H, H-5), 3.53 (dd, 1H, J1′,2′ ) 8.0, J2′,3′ ) 10.1 Hz, H-2′), 2.04 (s, 3H, CH3CONH-); 13C NMR (D2O, 500 MHz) δ 177.4 (CH3CONH-), 105.7 (C-1′), 87.0 (C-1), 81.5 (C-4), 78.5 (C-5), 78.2 (C-5′), 75.9 (C-3), 75.3 (C-3′), 73.8 (C-2′), 71.4 (C-4′), 63.8 (C-6′), 63.0 (C-6), 58.7 (C-2), 25.1 (CH3CONH-). Synthesis of Spacer-N-Linked Di-, Tetra-, and Hexasaccharide Glycosides. 1. Synthesis of Spacer-N-Linked Disaccharide Glycosides. 6-Trifluoroacetamidohexanoic acid (334 mg, 1.47 mmol) was dissolved in DMSO (2.63 mL). Next, N-ethyldiisopropylamine (1.28 mL, 7.35 mmol) and HBTU (557 mg, 1.47 mmol) were added to the solution, and the mixture was allowed to preactivate for approximately 5 min. In a separate vial, compound 1 (500 mg, 1.47 mmol) was dissolved in DMSO (5.25 mL). The solution was heated slightly to completely dissolve the disaccharide. After the solution was cooled to room temperature, it was added to the activated solution. The mixture was allowed to react at room temperature with constant stirring for 5 h. After the reaction mixture was concentrated to a syrup, it was

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dissolved in 5 mL of CHCl3/CH3OH/H2O ) 7/3/0.5 and then loaded onto a Silica Gel 60N column (3.5 × 60 cm). The column was developed with the same solvent at a flow rate of 10 mL/min and a fraction size of 30 mL/tube. Fractions 68-135 were pooled and concentrated. N-(ε-Trifluoroacetamidocaproyl)-β-lactosylamine 3 was obtained in a total yield of 82% (685 mg). [R]D30 +16.0° (c 0.1, water); FAB-mass: m/z 551 [M + H]+ (matrix: glycerol); 1H NMR (D2O, 500 MHz) δ 4.98 (d, 1H, J1,2 ) 9.5 Hz, H-1), 4.46 (d, 1H, J1′,2′ ) 7.6 Hz, H-1′), 3.94 (dd, 1H, J5,6b ) 1.6, J6a,6b ) 12.2 Hz, H-6b), 3.93 (1H, H-4′), 3.82 (dd, 1H, J5,6a ) 4.0, J6a,6b ) 12.2 Hz, H-6a), 3.79-3.69 (6H, H-3, H-4, H-5, H-5′, H-6′a, H-6′b), 3.67 (dd, 1H, J2′,3′ ) 10.1, J3′,4′ ) 3.4 Hz, H-3′), 3.56 (dd, 1H, J1′,2′ ) 7.6, J2′,3′ ) 10.1 Hz, H-2′), 3.44 (t, 1H, J1,2 ) 9.5, J2,3 ) 9.5 Hz, H-2), 3.33 (t, 2H, H-ε), 2.34 (t, 2H, H-R), 1.65 (q, 2H, H-β), 1.60 (q, 2H, H-δ), 1.36 (q, 2H, H-γ); 13C NMR (D2O, 500 MHz) δ 181.1 (-NHCO-), 158.9 (CF3CONH-), 117.6 (CF3CONH-), 105.7 (C-1′), 81.9 (C-1), 80.6 (C-4), 79.2 (C-5), 78.2 (C-5′), 77.9 (C-3), 75.3 (C-3′), 74.3 (C-2), 73.8 (C-2′), 71.4 (C-4′), 63.9 (C-6′), 62.7 (C-6), 42.4 (C-ε), 38.4 (C-R), 30.2 (C-δ), 28.2 (C-γ), 27.4 (C-β). N-(ε-Trifluoroacetamidocaproyl)-β-N-acetyllactosaminylamine 4 was obtained in a manner similar with a total yield of 75% (81 mg) from compound 2 and 6-trifluoroacetamidohexanoic acid. [R]D30 +27.3° (c 0.1, water); FAB-mass: m/z 592 [M + H]+ (matrix: glycerol); 1H NMR (D2O, 500 MHz) δ 5.08 (d, 1H, J1,2 ) 9.5 Hz, H-1), 4.49 (d, 1H, J1′,2′ ) 7.6 Hz, H-1′), 3.94 (dd, 1H, J5,6b ) 2.1, J6a,6b ) 12.2 Hz, H-6b), 3.93 (1H, H-4′), 3.87 (t, 1H, J1,2 ) 9.5, J2,3 ) 9.5 Hz, H-2), 3.85 (dd, 1H, J5,6a ) 4.6, J6a,6b ) 12.2 Hz, H-6a), 3.81-3.72 (5H, H-3, H-4, H-5′, H-6′a, H-6′b), 3.67 (dd, 1H, J2′,3′ ) 9.8, J3′,4′ ) 3.4 Hz, H-3′), 3.66 (1H, H-5), 3.55 (dd, 1H, J1′,2′ ) 7.6, J2′,3′ ) 9.8 Hz, H-2′), 3.32 (t, 2H, H-ε), 2.28 (t, 2H, H-R), 2.00 (s, 3H, CH3CONH-), 1.59 (4H, H-β, H-δ), 1.31 (q, 2H, H-γ); 13C NMR (D2O, 500 MHz) δ 180.5 (-NHCO-), 177.4 (CH3CONH-), 158.9 (CF3CONH-), 117.6 (CF3CONH-), 105.6 (C-1′), 81.1 (C-1), 80.6 (C-4), 79.3 (C-5), 78.2 (C-5′), 75.6 (C-3), 75.3 (C-3′), 73.8 (C-2′), 71.4 (C-4′), 63.9 (C-6′), 62.6 (C-6), 56.7 (C-2), 42.3 (C-ε), 38.4 (C-R), 30.2 (C-δ), 28.1 (C-γ), 27.5 (C-β), 24.8 (CH3CONH-). 2. Enzymatic Synthesis of Spacer-N-Linked Tetra- and Hexasaccharide Glycosides. N-(ε-Trifluoroacetamidocaproyl)-β-lacto-N-neotetraosylamine 5 was synthesized by alternating addition of β1-3 linked GlcNAc and β1-4 linked Gal to compound 3, using two kinds of glycosyltransferase. Compound 3 (50 mg, 0.09 mmol) and UDPGlcNAc (117 mg, 0.18 mmol) were dissolved in 150 mM Tris-HCl buffer, pH 6.8 (5.7 mL), containing MnCl2 (14.5 mg) and 1% (w/v) NaN3 (0.09 mL), followed by addition of 250 mU (3.3 mL) partially purified β3GnT preparation from the cell culture supernatant. The mixture was incubated for 144 h at 37 °C, and the reaction was terminated by boiling for 5 min. UDP-Gal (111 mg, 0.18 mmol) was dissolved in the mixture, followed by addition of 0.95 U (0.5 mL) β4GalT from bovine milk. After the mixture was incubated at 37 °C for 12 h, the reaction was terminated by boiling for 5 min. The precipitate was removed by centrifugation (8000 g, 20 min), and the supernatant was loaded onto an ODS column (2.0 × 50 cm) equilibrated with 5% MeOH at a flow rate of 2.0 mL/min. After washing the column with 760 mL of 5% MeOH, the adsorbed portion was eluted with a linear gradient of 5-15% MeOH in a total volume of 1.0 L and with a fraction size of 20 mL/tube. The chromatographic eluate was monitored by measuring the absorbance at 210 nm using a spectrometer. An aliquot from fractions 28-38 was then concentrated and lyophilized: compound 5 was obtained with a total yield of 80% (67 mg) based on the acceptor substrate 3. [R]D30 +14.8° (c 0.1, water); FAB-mass: m/z 916 [M + H]+ (matrix: glycerol); 1H NMR (D2O, 500 MHz) δ 4.97 (d, 1H, J1,2 ) 9.2 Hz, H-1), 4.70 (d, 1H, J1′′,2′′ ) 8.2 Hz, H-1′′), 4.47 (d, 1H, J1′′′,2′′′ ) 8.0 Hz, H-1′′′), 4.44 (d, 1H, J1′,2′ ) 7.9 Hz, H-1′), 4.15 (1H, H-4′), 3.96-3.83 (4H, H-6a, H-6b, H-6′a, H-6′b), 3.92 (1H, H-4′′′), 3.82-3.65 (13H of sugar protons), 3.66 (dd, 1H, J2′′′,3′′′ ) 9.8, J3′′′,4′′′ ) 4.0 Hz, H-3′′′), 3.59 (dd, 1H, J1′,2′ ) 7.9, J2′,3′ ) 10 Hz, H-2′), 3.58 (1H, H-5′′), 3.53 (dd, 1H, J1′′′,2′′′ ) 8.0, J2′′′,3′′′ ) 9.8 Hz, H-2′′′),

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Table 1. Synthesis of Spacer-N-Linked Asialoglycopolypeptides with Different Degrees of Polymerization of Glutamic Acid Residues products poly[Lacβ-NHCO(CH2)5NH-/γ-PGA] poly[LNβ-NHCO(CH2)5NH-/γ-PGA] poly[LNnTβ-NHCO(CH2)5NH-/γ-PGA] poly[LNβ1,3LNnTβ-NHCO(CH2)5NH-/γ-PGA]

compound γ-PGAa (mg) BOPb (mg) HOBtc (mg) amino-sugard (mg) yield (mg) DSe (%) 11 12 13 14

30 30 12 12

237 237 95 95

30 30 12 12

45 49 33 47

34 37 20 27

31 32 32 34

a γ-PGA: M.W. ) 990000. b Benzotriazol-1-yloxytris-(dimethylamino)phosphonium hexafluorophosphate. c 1-Hydroxybenzotriazole hydrate. N-(ε-Aminocaproyl)-β-lactosylamine for 11, N-(ε-aminocaproyl)-β-N-acetyllactosaminylamine for 12, N-(ε-aminocaproyl)-β-lacto-N-neotetraosylamine for 13, and N-(ε-aminocaproyl)-β-LacNAcβ1,3lacto-N-neotetraosylamine for 14. e Degree of substitution of sugar derivatives based on DP of γ-PGA as 100%. Calculated from 1H NMR data at 25 °C.

d

3.42 (t, 1H, J1,2 ) 9.2, J2,3 ) 9.2 Hz, H-2), 3.32 (t, 2H, H-ε), 2.33 (t, 2H, H-R), 2.03 (s, 3H, CH3CONH′′-), 1.63 (q, 2H, H-β), 1.59 (q, 2H, H-δ), 1.35 (q, 2H, H-γ); 13C NMR (D2O, 500 MHz) δ 181.1 (-NHCO-), 177.7 (CH3CONH′′-), 158.9 (CF3CONH-), 117.6 (CF3CONH-), 105.7 (C-1′′′, C-1′), 105.6 (C-1′′), 84.8 (C-3′), 81.9 (C-1), 81.0 (C-4′′), 80.5 (C-4), 79.2 (C-5), 78.2 (C-5′′′), 77.9 (C-3), 77.7 (C-5′), 77.4 (C-5′′), 75.3 (C-3′′′), 75.0 (C-3′′), 74.2 (C-2), 73.8 (C-2′′′), 73.0 (C-2′), 71.4 (C-4′′′), 71.2 (C-4′), 63.8 (C-6′′′, C-6′), 62.7 (C-6′′, C-6), 58.0 (C-2′′), 42.4 (C-ε), 38.4 (C-R), 30.2 (C-δ), 28.2 (C-γ), 27.4 (C-β), 25.0 (CH3CONH′′-). N-(ε-Trifluoroacetamidocaproyl)-β-LNβ1,3lacto-N-neotetraosylamine 6 was synthesized by alternating addition of GlcNAc and Gal residues to compound 5 in a similar manner. Compound 6 was obtained with a total yield of 75% (151 mg) based on the acceptor substrate 5. [R]D30 +11.9° (c 0.1, water); FAB-mass: m/z 1281 [M + H]+ (matrix: glycerol); 1H NMR (D2O, 500 MHz) δ 4.97 (d, 1H, J1,2 ) 9.2 Hz, H-1), 4.70 (d, 2H, J1′′′′,2′′′′ ) 8.2, J1′′,2′′ ) 8.2 Hz, H-1′′′′, H-1′′), 4.48 (d, 1H, J1′′′′′,2′′′′′ ) 7.0 Hz, H-1′′′′′), 4.46 (d, 1H, J1′′′,2′′′ ) 7.7 Hz, H-1′′′), 4.44 (d, 1H, J1′,2′ ) 7.9 Hz, H-1′), 4.15 (2H, H-4′′′, H-4′), 3.96-3.83 (6H, H-6ab, H-6′′ab, H-6′′′′ab), 3.92 (1H, H-4′′′′′), 3.82-3.64 (21H of sugar protons), 3.62-3.52 (5H, H-2′, H-5′′′′, H-5′′, H-2′′′′′, H-2′′′), 3.42 (t, 1H, J1,2 ) 9.2, J2,3 ) 9.2 Hz, H-2), 3.32 (t, 2H, H-ε), 2.34 (t, 2H, H-R), 2.03 (s, 6H, CH3CONH′′′′-, CH3CONH′′-), 1.63 (q, 2H, H-β), 1.60 (q, 2H, H-δ), 1.36 (q, 2H, H-γ); 13C NMR (D2O, 500 MHz) δ 181.1(-NHCO-),177.7(CH3CONH′′′′-,CH3CONH′′-),158.9(CF3CONH-), 117.6 (CF3CONH-), 105.7 (C-1′′′′′, C-1′′′, C-1′), 105.6 (C-1′′′′, C-1′′), 84.9 (C-3′′′), 84.8 (C-3′), 81.9 (C-1), 81.0 (C-4′′′′, C-4′′), 80.5 (C-4), 79.2 (C-5), 78.2 (C-5′′′′′), 77.9 (C-3), 77.7 (C-5′′′, C-5′), 77.4 (C-5′′′′, C-5′′), 75.3 (C-3′′′′′), 75.0 (C-3′′′′, C-3′′), 74.2 (C-2), 73.8 (C-2′′′′′), 72.8 (C-2′′′, C-2′), 71.4 (C-4′′′′′), 71.2 (C-4′′′, C-4′), 63.8 (C-6′′′′′, C-6′′′, C-6′), 62.7 (C-6′′′′, C-6′′, C-6), 58.0 (C-2′′′′, C-2′′), 42.4 (C-ε), 38.4 (C-R), 30.2 (C-δ), 28.2 (C-γ), 27.4 (C-β), 25.0 (CH3CONH′′′′-, CH3CONH′′-). Synthesis of ε-Aminocaproyl Di-, Tetra-, and Hexasaccharide Glycosides. N-(ε-Aminocaproyl)-β-lactosylamine 7, N-(ε-aminocaproyl)-β-N-acetyllactosaminylamine 8, N-(ε-aminocaproyl)-β-lacto-Nneotetraosylamine 9, and N-(ε-aminocaproyl)-β-LNβ1,3lacto-N-neotetraosylamine 10 were synthesized from 3∼6 by alkaline hydrolysis according to a previously reported method.7 Synthesis of Asialoglycopolypeptides with γ-PGA Backbones. The amino functions of oligosaccharide glycosides (7∼10) were coupled with the carboxy group of γ-PGA by a condensation reaction. The degrees of substitution (DSs) of the substituted residues in the glycopolypeptides were adjusted to 31∼34%. Poly[LNβ1,3LNnTβNHCO-(CH2)5NH-/γ-PGA] 14 was synthesized as follows. The compositions of aminocaproyl oligosaccharide glycosides, γ-PGAs and reagents used for the coupling reaction are summarized in Table 1. The DS in the mole fraction of substituted residues in the glycopolypeptides was calculated as a percentage from the relative intensities of the 1H NMR signal areas of peptide γ-methylene protons and aglycon ε-methylene protons. γ-PGA (M.W. 990000, 12.0 mg) was dissolved in 1.44 mL of 100 mM Na2CO3/Na2HCO3 buffer (pH 10.0). BOP (94.7 mg) and HOBt (11.8 mg) in Me2SO (4.36 mL) were then added and the resulting mixture was stirred magnetically at room temperature for 15 min. Compound 10 (47.1 mg) in 100 mM Na2CO3/Na2HCO3 buffer (pH 10.0, 0.72 mL) was added with continuous stirring for 24 h under

the same conditions. The reaction mixture was loaded onto a Sephadex G-25 M PD-10 column (Amersham Biosciences Corp., NJ) equilibrated with 100 mM PBS (pH 7.4). The high-molecular-weight fraction collected was dialyzed against distilled water for 3 days and lyophilized to afford compound 14 (27.2 mg). 1H NMR (D2O, 500 MHz) δ 4.97 (d, 1H, J1,2 ) 9.2 Hz, H-1), 4.70 (d, 2H, J1′′′′,2′′′′ ) 8.2, J1′′,2′′ ) 8.2 Hz, H-1′′′′, H-1′′), 4.48 (d, 1H, J1′′′′′,2′′′′′ ) 7.4 Hz, H-1′′′′′), 4.46 (d, 1H, J1′′′,2′′′ ) 6.7 Hz, H-1′′′), 4.45 (d, 1H, J1′,2′ ) 8.3 Hz, H-1′), 4.35-4.18 (1H, R-methine, γ-PGA), 4.15 (2H, H-4′′′, H-4′), 3.97-3.80 (6H, H-6ab, H-6′′ab, H-6′′′′ab), 3.92 (1H, H-4′′′′′), 3.80-3.65 (21H of sugar protons), 3.61-3.52 (5H, H-2′, H-5′′′′, H-5′′, H-2′′′′′, H-2′′′), 3.44 (t, 1H, J1,2 ) 9.2, J2,3 ) 9.2 Hz, H-2), 3.19 (2H, H-ε), 2.41 (2H, γ-methylene, γ-PGA), 2.31 (2H, H-R), 2.25-1.90 (2H, β-methylene, γ-PGA), 2.03 (s, 6H, CH3CONH′′′′-, CH3CONH′′-), 1.61 (2H, H-β), 1.51 (2H, H-δ), 1.31 (2H, H-γ); 13C NMR (D2O, 500 MHz) δ 181.0 (-NHCO-), 177.7 (CH3CONH′′′′-, CH3CONH′′-), 105.7 (C-1′′′′′, C-1′′′, C-1′), 105.6 (C-1′′′′, C-1′′), 84.8 (C-3′′′), 84.7 (C-3′), 81.9 (C-1), 80.9 (C-4′′′′, C-4′′), 80.5 (C-4), 79.2 (C-5), 78.2 (C-5′′′′′), 77.9 (C-3), 77.7 (C-5′′′, C-5′), 77.4 (C-5′′′′, C-5′′), 75.3 (C-3′′′′′), 75.0 (C-3′′′′, C-3′′), 74.3 (C-2), 73.8 (C-2′′′′′), 72.8 (C-2′′′, C-2′), 71.4 (C-4′′′′′), 71.2 (C4′′′, C-4′), 63.8 (C-6′′′′′, C-6′′′, C-6′), 62.7 (C-6′′′′, C-6′′, C-6), 58.0 (C-2′′′′, C-2′′), 56.4 (R-methine, γ-PGA), 42.0 (C-ε), 38.5 (C-R), 33.4 (γ-methylene, γ-PGA), 30.8 (C-δ), 29.7 (β-methylene, γ-PGA), 28.4 (C-γ), 27.5 (C-β), 25.0 (CH3CONH′′′′-, CH3CONH′′-). Poly[LacβNHCO-(CH2)5NH-/γ-PGA] 11, poly[LNβ-NHCO-(CH2)5NH-/γ-PGA] 12, and poly[LNnTβ-NHCO-(CH2)5NH-/γ-PGA] 13 were prepared from 7∼9 in a manner similar, respectively (see Table 1). Synthesis of Sialoglycopolypeptides with γ-PGA Backbones. Asialoglycopolypeptides 11∼14 were sialylated to the R2,3sialo-γ-PGAs 15∼18, respectively. Poly[SAR2,3LNβ1,3LNnTβ-NHCO(CH2)5NH-/γ-PGA] 18 was synthesized enzymatically from compound 14 as follows. A mixture containing 7.0 mg of compound 14, 16.0 mM CMP-β-Neu5Ac, 40 mU/mL of rat recombinant R2,3-(N)sialyltransferase, 2.5 mM MnCl2, 0.1% BSA, and 10 U/mL of calf intestine alkaline phosphatase (Boehringer-Mannheim, Mannheim, Germany) in 50 mM MOPS buffer (pH 7.4) was incubated at 37 °C for 48 h in a total volume of 550 µL. After heating to 100 °C and centrifugation, the supernatant from the reaction mixture was loaded onto a Sephadex G-25 M PD-10 column equilibrated with 100 mM PBS (pH 7.4). The high-molecular-weight fraction collected was dialyzed against distilled water for 3 days and lyophilized to afford compound 18 (7.4 mg). 1H NMR (D2O, 500 MHz) δ 4.97 (d, 1H, J1,2 ) 9.2 Hz, H-1), 4.70 (d, 2H, J1′′′′,2′′′′ ) 8.2, J1′′,2′′ ) 8.2 Hz, H-1′′′′, H-1′′), 4.55 (d, 1H, J1′′′′′,2′′′′′ ) 7.9 Hz, H-1′′′′′), 4.46 (d, 1H, J1′′′,2′′′ ) 7.7 Hz, H-1′′′), 4.45 (d, 1H, J1′,2′ ) 8.5 Hz, H-1′), 4.35-4.18 (1H, R-methine, γ-PGA), 4.13 (2H, H-4′′′, H-4′), 4.12 (1H, H-3′′′′′), 3.97-3.52 (39H of sugar protons), 3.43 (t, 1H, J1,2 ) 9.2, J2,3 ) 9.2 Hz, H-2), 3.19 (2H, H-ε), 2.75 (dd, 1H, J3′′′′′′ax, 3′′′′′′eq ) 12.2, J3′′′′′′eq, 4′′′′′′ ) 3.3 Hz, H-3′′′′′′eq), 2.42 (2H, γ-methylene, γ-PGA), 2.31 (2H, H-R), 2.25-1.90 (2H, β-methylene, γ-PGA), 2.03 (s, 9H, CH3CONH′′′′′′-, CH3CONH′′′′-, CH3CONH′′-), 1.80 (t, 1H, J3′′′′′′ax, 3′′′′′′eq ) 12.2, J3′′′′′′ax, 4′′′′′′ ) 12.2 Hz, H-3′′′′′′ax), 1.61 (2H, H-β), 1.50 (2H, H-δ), 1.31 (2H, H-γ); 13 C NMR (D2O, 500 MHz) δ 181.0 (-NHCO-), 177.7 (CH3CONH′′′′′′-, CH3CONH′′′′-, CH3CONH′′-), 175.9 (HOOC′′′′′′-), 105.7 (C-1′′′, C-1′), 105.6 (C-1′′′′, C-1′′), 105.3 (C-1′′′′′), 102.6 (C-2′′′′′′), 84.8 (C-3′′′, C-3′), 81.9 (C-1), 81.0 (C-4′′′′), 80.8 (C-4′′), 80.5 (C-4), 79.2 (C-5), 78.3 (C-

Sialoglycopolypeptide as Polymeric Inhibitors


Table 2. Spacer-N-Linked Glycopolypeptides for Inhibition of Binding by Influenza Viruses DS (%) sugar moiety LacβLNβLNnTβLNβ1,3LNnTβSAR2,3LacβSAR2,3LNβSAR2,3LNnTβSAR2,3LNβ1,3LNnTβSAR2,6LacβSAR2,6LNβSAR2,6LNnTβSAR2,6LNβ1,3LNnTβSAR2,3LNβSAR2,3LNnTβSAR2,3LNβ1,3LNnTβSAR2,6LNβSAR2,6LNnTβSAR2,6LNβ1,3LNnTβ-

linkage compound N-linked N-linked N-linked N-linked N-linked N-linked N-linked N-linked N-linked N-linked N-linked N-linked O-linked O-linked O-linked O-linked O-linked O-linked

11 12 13 14 15 16 17 18 19 20 21 22 23g 24g 25g 26g 27g 28g

NSa 31 (ND)f 32d (31)e 32 (33) 34 (29) 5 0 0 0 15 0 0 0 0 0 0 0 0 0

SAb

26 32 32 34 16 32 32 34 40 35 31 40 35 31

(26) (30) (33) (33) (17) (29) (35) (36) (37) (32) (33) (39) (39) (33)

kDac 1800 1900 2600 3500 2400 2600 3200 4200 2300 2600 3200 4200 2800 3400 3900 2800 3400 3900

a Neutral sugar derivatives substituted. b Sialyl sugar derivatives substituted ()Sia contents). c Calculated kDa. d Degree of substitution of asialo- or sialo-sugar derivatives based on DP of γ-PGA as 100%. Calculated from 1H NMR data at 25 °C. e Degree of substitution of asialoor sialo-sugar derivatives based on DP of γ-PGA as 100%. Calculated from two type HPLC analyses (ABEE and DMB methods). All data normalized to those of 1H NMR. f Not determined. g Ref. 8.

5′′′′′), 78.0 (C-3), 77.9 (C-3′′′′′), 77.7 (C-5′′′, C-5′), 77.4 (C-5′′′′, C-5′′), 75.7 (C-6′′′′′′), 75.0 (C-3′′′′, C-3′′), 74.5 (C-8′′′′′′), 74.3 (C-2), 72.8 (C-2′′′, C-2′), 72.2 (C-2′′′′′), 71.1 (C-4′′′′′′, C-4′′′, C-4′), 70.9 (C-7′′′′′′), 70.3 (C-4′′′′′), 65.4 (C-9′′′′′′), 63.8 (C-6′′′′′, C-6′′′, C-6′), 62.7 (C-6′′′′, C-6′′, C-6), 58.0 (C-2′′′′, C-2′′), 56.4 (R-methine, γ-PGA), 54.5 (C5′′′′′′), 42.4 (C-3′′′′′′), 42.0 (C-ε), 38.5 (C-R), 34.4 (γ-methylene, γ-PGA), 30.8 (C-δ), 29.5 (β-methylene, γ-PGA), 28.4 (C-γ), 27.5 (Cβ),25.0(CH3CONH′′′′-,CH3CONH′′-),24.9(CH3CONH′′′′′′-).Poly[SAR2,3LacβNHCO-(CH2)5NH-/γ-PGA] 15, poly[SAR2,3LNβ-NHCO-(CH2)5NH-/ γ-PGA] 16, and poly[SAR2,3LNnTβ-NHCO-(CH2)5NH-/γ-PGA] 17 were prepared from 11∼13 in a similar manner, respectively (see Table 2). Asialoglycopolypeptides 11∼14 were also sialylated to the R2,6sialo-γ-PGAs 19∼22, respectively. Poly[SAR2,6LNβ1,3LNnTβ-NHCO(CH2)5NH-/γ-PGA] 22 was synthesized enzymatically from compound 14 as follows. A mixture containing 7.0 mg of compound 14, 16.0 mM CMP-β-Neu5Ac, 40 mU/mL of rat recombinant R2,6-(N)sialyltransferase, 2.5 mM MnCl2, 0.1% BSA, and 10 U/mL of calf intestine alkaline phosphatase (Boehringer-Mannheim, Mannheim, Germany) in 50 mM MOPS buffer (pH 7.4) was incubated at 37 °C for 48 h in a total volume of 550 µL. After heating to 100 °C and centrifugation, the supernatant from the reaction mixture was loaded onto a Sephadex G-25 M PD-10 column equilibrated with 100 mM PBS (pH 7.4). The high-molecular-weight fraction collected was dialyzed against distilled water for 3 days and lyophilized to afford compound 22 (7.6 mg). 1H NMR (D2O, 500 MHz) δ 4.97 (d, 1H, J1,2 ) 8.9 Hz, H-1), 4.70 (d, 1H, J1′′′′,2′′′′ ) 8.0 Hz, H-1′′′′), 4.70 (d, 1H, J1′′,2′′ ) 8.0 Hz, H-1′′), 4.47 (d, 1H, J1′′′,2′′′ ) 7.7 Hz, H-1′′′), 4.45 (d, 2H, J1′′′′′,2′′′′′ ) 7.6, J1′,2′ ) 7.6 Hz, H-1′′′′′, H-1′), 4.37-4.15 (1H, R-methine, γ-PGA), 4.15 (2H, H-4′′′, H-4′), 4.03-3.50 (40H of sugar protons), 3.43 (t, 1H, J1,2 ) 8.9, J2,3 ) 8.9 Hz, H-2), 3.19 (2H, H-ε), 2.67 (dd, 1H, J3′′′′′′ax,3′′′′′′eq ) 12.2, J3′′′′′′eq,4′′′′′′ ) 4.3 Hz, H-3′′′′′′eq), 2.41 (2H, γ-methylene, γ-PGA), 2.31 (2H, H-R), 2.25-1.90 (2H, β-methylene, γ-PGA), 2.05 (s, 3H, CH3CONH′′′′′′-), 2.03 (s, 6H, CH3CONH′′′′-, CH3CONH′′-), 1.73 (t, 1H, J3′′′′,ax, 3′′′′′,eq ) 12.2, J3′′′′′,ax, 4′′′′′′ ) 12.2 Hz, H-3′′′′′,ax), 1.61 (2H, H-β), 1.50 (2H, H-δ), 1.31 (2H, H-γ); 13 C NMR (D2O, 500 MHz) δ 181.0 (-NHCO-), 177.7 (CH3CONH′′′′′′-, CH3CONH′′′′-, CH3CONH′′-), 176.0 (HOOC′′′′′′-), 106.3 (C-1′′′′′), 105.7 (C-1′′′, C-1′), 105.6 (C-1′′), 105.4 (C-1′′′′), 102.9 (C-2′′′′′′), 84.8 (C-3′′′, C-3′), 83.3 (C-4′′′′), 81.9 (C-1), 81.0 (C-4′′), 80.5 (C-4), 79.2 (C-5), 77.9 (C-3), 77.7 (C-5′′′, C-5′), 77.3 (C-5′′), 77.1 (C-5′′′′′), 76.5

1897

(C-5′′′′), 75.4 (C-6′′′′′′), 75.2 (C-3′′′′′), 75.1 (C-3′′′′), 75.0 (C-3′′), 74.5 (C-8′′′′′′), 74.3 (C-2), 73.5 (C-2′′′′′), 72.8 (C-2′′′, C-2′), 71.2 (C-4′′′′′′, C-4′′′′′, C-4′′′, C-4′), 71.0 (C-7′′′′′′), 66.1 (C-6′′′′′), 65.5 (C-9′′′′′′), 63.8 (C-6′′′, C-6′), 62.9 (C-6′′′′), 62.7 (C-6′′, C-6), 57.8 (C-2′′′′), 58.0 (C2′′), 56.4 (R-methine, γ-PGA), 54.7 (C-5′′′′′′), 42.8 (C-3′′′′′′), 42.0 (Cε), 38.5 (C-R), 34.4 (γ-methylene, γ-PGA), 30.8 (C-δ), 29.6 (βmethylene, γ-PGA), 28.5 (C-γ), 27.5 (C-β), 25.1 (CH3CONH′′′′-), 25.0 (CH3CONH′′-), 24.9 (CH3CONH′′′′′′-). Poly[SAR2,6Lacβ-NHCO(CH2)5NH-/γ-PGA] 19, poly[SAR2,6LNβ-NHCO-(CH2)5NH-/γ-PGA] 20 and poly[SAR2,6LNnTβ-NHCO-(CH2)5NH-/γ-PGA] 21 were prepared from 11∼13 in a similar manner (see Table 2). Hemagglutination Inhibition Assay. The hemagglutination inhibition (HI) assay was carried out using 96-well microtiter plates as described previously.13 Phosphate-buffered saline (PBS, pH 6.5) was used as a dilution buffer. Virus and an SNA lectin suspension (22 HA units in 0.025 mL of PBS) were added to each well containing the synthetic glycopolypeptides (200-0.024 nM) in a 2-fold serial dilution in dilution buffer, respectively. After incubation at 4 °C for 1 h, 0.05 mL of 0.6% (v/v) guinea-pig suspension erythrocytes was added to the plates, and the solutions were allowed to settle for 2 h at 4 °C. The maximum dilution of the samples showing complete inhibition of hemagglutination was defined as the HI titer. Direct Binding Assay. The assay measuring direct binding activity of SNA lectin to glycopolypeptides was performed by a previously described method.6,14 Glycopolymers (0.5 nM, 50 µL/well) in 50 mM sodium acetate buffer (pH 4.0) were briefly immobilized on polystyrene Universal-Bind microplates (Corning-Costar, universal binding) using an ultraviolet irradiation method. After blocked with PBS containing 2.5% bovine serum albumin, the plates were incubated in solutions containing biotin-labeled SNA lectin in PBS containing 0.05% Tween 20 (PBS-Tween) corresponding to 12.5∼1000 ng/mL at 25 °C for 1 h. After four washes with PBS-Tween, the plates were incubated in a substrate solution containing 2.0 µg/mL of streptavidin-HRP in PBSTween at 25 °C for 1 h. After washing the plate in the same manner, bound biotin-labeled SNA were detected with 100 µL of peroxidasesubstrate solution containing 3,3′,5,5′-tetramethylbenzidine and H2O2 from the POD substrate TMB kit HYPER (Nacalai tesque, Kyoto Japan). The reaction was stopped after 3 min with 100 µL 1 M phosphate solution, and the absorbance was measured at 450 nm.

Results Practical Synthetic Route to a New Type of Artificial Sialoglycopolypeptides. 1. Synthesis of Spacer-N-Linked Oligosaccharide Glycosides. Spacer-N-linked di-, tetra-, and hexasaccharide glycosides containing LN repeats were prepared as shown in Scheme 1. Lac and LN were first converted into the corresponding Lacβ-NH2 and LNβ-NH2 with high yields of 95 and 71%, respectively, by treatment with ammonia solution according to the modified method of Kato et al.12 The resulting β-diglycosylamines were then condensed with the carboxy group of 6-trifluoroacetamidohexanoic acid with HBTU and DIEA in DMSO. The target products 3 and 4 were purified by chromatography on a single silica gel column to give high yields of 82 and 75%, respectively, based on the amount of β-diglycosylamines added. The structures of the spacer-N-linked disaccharide glycosides 3 and 4 were elucidated by 1H and 13C NMR analyses. The N-linked β-anomeric bond between the glycosyl and the aglycon moieties was confirmed by the following 1H and 13C NMR assignments. The protons of the N-linked β-anomeric bond between the glycosyl and aglycon moieties showed lower chemical shifts with larger coupling constants (3, δ 4.98 ppm (J1,2 ) 9.5 Hz); 4, δ 5.08 ppm (J1,2 ) 9.5 Hz)). In the 13C NMR spectra, the N-β-linked C-1β signal was characterized by higher chemical shifts (3, δ 81.9 ppm; 4, δ 81.1 ppm). The N-glycosylation proceeded stereospecifically

1898


Ogata et al.

Scheme 1. Chemoenzymatic Synthesis of LN-Repeating Spacer-N-Linked Di-, Tetra-, and Hexasaccharide Glycosides

within a few hours to give only the β-glycoside without the need for any protection and deprotection steps. N-(ε-Trifluoroacetamidocaproyl)-β-lacto-N-neotetraosylamine 5 was synthesized by alternating addition of β-(1-3) linked GlcNAc and β-(1-4) Gal to compound 3, using two kinds of glycosyltransferases. To the resulting glycoside 5 was further added GlcNAc and Gal residues in a similar manner to form N-(ε-trifluoroacetamidocaproyl)-β-LNβ1,3-lacto-N-neotetraosylamine 6. 2. Synthesis of Asialoglycopolypeptides Carrying Spacer-NLinked Oligosaccharide Glycosides. The resulting spacer-Nlinked oligosaccharide glycosides with different glycans in the array were similarly deacylated to the corresponding amino group by alkali treatment as described above (Scheme 2). The amino functions of the resulting oligosaccharide glycosides 7∼10 were reacted with the carboxy groups of γ-PGA in the presence of the condensation reagents BOP and HOBt, as described earlier.7,8 In the synthesis, γ-PGA of 990 kDa was used as the polypeptide base of the glycopolymers. The reaction solution was applied to a column of Sephadex G-25 M PD-10 to separate the glycosylated γ-PGA from the low molecular weight reactants. The DSs in the mole fraction of the substituted residues in the asialoglycopolypeptides 11∼14 were calculated by two methods: 1H NMR and chemical analyses. The 1H NMR results were based on the relative intensities of signals due to peptide methylene protons and those due to spacer-linked methylene protons in the aglycon. The chemical analysis procedure used the HPLC method based on p-aminobenzoic

ethyl ester-derivatized monosaccharides of Yasuno et al.15 The DS in those asialoglycopolypeptides was limited to 31∼34% by controlling the coupling conditions of the aminated spacerlinked glycosides with γ-PGAs (Tables 1 and 2). 3. Synthesis of Sialoglycopolypeptides Carrying Spacer-NLinked Sialyloligosaccharide Glycosides. A series of asialoglycopolypeptides with different glycans in the array were sialylated to 15∼18 carrying SAR2,3Gal and 19∼22 carrying SAR2,6Gal by R2,3- and R2,6-sialyltransferases, respectively (Scheme 2). The target glycopolypeptides were obtained by separation on a Sephadex G-25 M PD-10 column. The structures of the synthesized glycopolypeptides were elucidated by 1H and 13C NMR analyses based on spectral analysis of spacer-O-linked glycopolymers as described previously.7,8 The DS of sialyl sugar derivatives in the sialoglycopolypeptides was determined by 1H NMR and chemical analyses together with the HPLC method based on 1,2-diamino-4,5-methylenedioxybenzene-derivatized sialic acids, for example, of the asialoglycopolypeptides mentioned above. The structures of spacer-N-linked sialoglycopolypeptides 18 and 22 as representative examples were similarly analyzed by 1 H NMR spectrometry (Figure 1). In the 1H NMR spectrum of 18, characteristic signals at δ 2.75 (dd, 1H, J3′′′′′,ax,3′′′′′,eq ) 12.2, J3′′′′′′eq, 4′′′′′′ ) 3.3 Hz, H-3′′′′′′eq) and δ 1.80 (t, 1H, J3′′′′′,ax,3′′′′′′eq ) 12.2, J3′′′′′′ax,4′′′′′′ ) 12.2 Hz, H-3′′′′′′ax) were assigned to the H-3′′′′′′ proton (Figure 1a). In 22, δ 2.67 (dd, 1H, J3′′′′′′ax,3′′′′′′eq ) 12.2, J3′′′′′′eq, 4′′′′′′ ) 4.3 Hz, H-3′′′′′′eq) and δ 1.73 (t, 1H,



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Scheme 2. (1) Chemoenzymatic Synthesis of LN-Repeating Oligosaccharide Glycosides, (2) Coupling of the Resulting Glycosides with γ-PGA, and (3) Sialylation to Highly Water-Soluble Glycopolypeptides Carrying Clustered Identical Sialooligosaccharide Segments

J3′′′′′′ax,3′′′′′′eq ) 12.2, J3′′′′′′ax,4′′′′′′ ) 12.2 Hz, H-3′′′′′′ax) were assigned to the H-3′′′′′′ proton (Figure 1b). It was possible to evaluate the extent of sialylation by integrating the resulting proton signals (Table 2). The properties of the synthesized glycopolypeptides are summarized in Table 2. The chemical analysis was performed using the method of Sato et al.16 The degree of substitution of neutral sugar derivatives (NS) and sialyl sugar derivatives (SA) was based on the DP of γ-PGA, which was arbitrarily given a value of 100% (Table 2). In the 13C NMR spectra of 18 and 22, the respective C-3′′′′′ and C-6′′′′′ signals were distinguished by their downfield positions with chemical shifts at δ 77.9 and 66.1, respectively. These data indicated that only the terminal Gal residues of the sugar chain moiety were regiospecifically and quantitatively sialylated in the R2,3 and R2,6 linkages.17 R2,3/6-Sialylations of the asialoglycopolypeptides 12, 13, and 14 were almost quantitative, but R2,3/6sialylation of the asialoglycopolypeptide 11 carrying a lactose unit occurred at low efficiency, around 52 and 84%, respectively (Table 2). Interaction of Synthetic Glycopolypeptides with Influenza Virus. Various substances carrying sialooligosaccharides are known to inhibit hemagglutination by binding to viral HA.7,8,14 We therefore tested the inhibitory effect of the spacer-N-linked glycopolypeptides with different glycans in the array on hemagglutination using avian and human influenza virus (Table 3). The avian virus A/Duck/HongKong/313/4/78 reacted with spacer-N-linked glycopolypeptides 15∼18 carrying R2,3 sialylated glycans regardless of the glycan length, whereas the human virus did not. In contrast, the human virus A/Aichi/2/68 bound

preferentially to sialoglycopolypeptides carrying SAR2,6Gal and bound to those carrying SAR2,3Gal to a lesser extent. The virus recognizes not only specific linkages of SAs, but also elongated core carbohydrates in the receptors. Binding of the human virus was also inhibited strongly by R2,6-sialopolymers 21 and 22 carrying LNnT and LN-LNnT units, which had 4∼16-fold higher inhibitory activities than 19 and 20, which carried a single Lac/LN (Table 3). Among the glycopolypeptides tested, 21 and 22 inhibited binding most potently at a concentration of 0.29 nM, and their relative binding affinities were 7.9 × 103-fold higher than that of fetuin itself, which binds both avian and human types of influenza virus due to the presence of both SA linkages, R2,3 and R2,6.18,19 The effects of different glycosidic linkages were further examined by comparing the N- and O-linked glycopolypeptides in Figure 2. The spacer-N-linked glycopolypeptides inhibited binding to a similar extent as the spacer-O-linked glycopolypeptides. The avian virus reacted specifically with the sialoglycopolymers carrying an R2,3 glycan (Figure 2A). The inhibition activities of spacer-N-linked R2,6-sialoglycopolymers 20-22 differ by 2∼4-fold of the corresponding spacer-O-linked glycopolymers 26-28 (Figure 2B). The spacer-N-linked glycopolymers were, as a whole, observed to bind the virus HA more efficiently in the receptor than spacer-O-linked glycopolymer, regardless of the linkage mode of the SA residue. Influenza virus-mediated hemagglutination was not affected by the N- and O-linked asialoglycopolypeptides or by the polymer backbone (γ-PGA) without oligosaccharides.

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Figure 1. 1H NMR spectra of (a) compound 18 and (b) compound 22. Solvent, D2O; temperature, 25 °C; concentration, 5 mg/mL; 500 MHz.

Interaction of Synthetic Glycopolypeptides with SNA. 1. Hemagglutination Inhibition Assay. Bark lectin from elderberry (SNA) specifically binds to the SAR2,6Gal/GalNAc sequence and has long been used for the analysis of sialoglycoconjugates that play important roles in many biological phenomena.20 The carbohydrate binding specificity of SNA with guinea pig hemagglutinin was studied comparatively with regard to hemagglutination inhibition using spacer-N-linked sialoglycopolypeptides with spacer-O-linked sialoglycopolypeptides as control samples (Table 3, Figure 2C). Inhibition by spacer-Nlinked R2,6-sialoglycopolypeptides 20-22 for SNA increased in a glycan length-dependent manner as well as that by spacerO-linked sialoglycopolypeptides 26-28 (Figure 2C). Compound 22, which carries a long heptasaccharide glycan, was shown to be the most effective inhibitor (IC50 0.03 nM) among the

glycopolymers and its relative binding affinity was 2.4 × 104fold higher than that of fetuin itself. The polymer showed 16fold higher inhibitory activity than 20 (IC50 0.47 nM), which carries a short trisaccharide glycan. Replacement of the LN unit by a Lac unit in polymer 19 (IC50 7.5 nM) markedly decreased the inhibition, making it 16-fold lower affinity than that of polymer 20. The length of glycans beyond the trisaccharide level was shown to critically influence the lectin binding constants. These results showed almost identical tendencies as those observed in the hemagglutination inhibition assay using human viral HA. 2. Direct Binding Assay. We have recently established a direct binding assay for human and avian influenza viruses utilizing artificial glycopolypeptides which are O-linked polymers carrying long/short R2,3/6 sialylated glycans.6,8,14 To



Table 3. Inhibition of Avian and Human Influenza Virus and SNA Lectin Hemagglutination by Artificial Sialoglycopolypeptides IC50a (nM) [IC50b (Sia µM)] avian virus compound γ-PGAc 15 16 17 18 19 20 21 22 Fetuin

human virus

A/Duck/HongKong/313/4/78 A/Aichi/2/68 (H5N3) (H3N2) NDd 38 [64] 38 [79] 38 [79] 38 [84] ND ND ND ND 23000 [315]

ND 38 [64] 38 [79] 9.4 [20] 19 [42] 4.7 [4.9] 1.2 [2.5] 0.29 [0.61] 0.29 [0.65] 2300 [31.5]

lectin SNA ND ND ND ND ND 7.5 [7.9] 0.47 [0.98] 0.06 [0.12] 0.03 [0.07] 730 [9.8]

a Minimumconcentrationsrequiredforcompleteinhibitionofhemagglutination. Concentration of Sia units. c γ-PGA: M.W. ) 990000. d ND: not detected (no activity). b

extend this approach, the assay to determine direct binding dynamics of artificial glycopolypeptides carrying long glycan units to SNA lectin was applied. Binding signals were detected when R2,6-sialoglycopolypeptides, but not asialo- and R2,3sialoglycopolypeptides, were used as ligands. As the optical activity from SNA bound to glycopolymers increased, it obeyed good first-order kinetic with respect to the concentration of SNA (Figure 3). Thus, the binding by R2,6-sialoglycopolypeptides 20-22 carrying an LN, LNnT, and LN-LNnT increased in a glycan length-dependent manner. These carbohydrate binding properties were comparable with those obtained by hemagglutination inhibition experiments. Taken together, the results indicate that SNA lectin bind exclusively to R2,6-sialopolypeptides carrying elongated core carbohydrate with higher affinity.

Discussion Numerous types of synthetic multivalent glycoconjugates have been synthesized in accordance with the multivalency principle that governs viral HA interactions with glycans.21-28 However, the contribution of the asialo portion of the glycoconjugate to this inhibitory activity has not been examined extensively, because the length of glycans has been limited to mono-, di-, and trisaccharides.21-27 We designed spacer-Nlinked asialo-type polymers with multiple LN repeats starting with Lac or LN, which consist of three parts: glycan, spacedlinker, and polypeptide backbone. Schemes 1 and 2 represent the pathway from Lac or LN via the following three steps: (1) chemical glycosylation of the respective spacer-N-linked disaccharide (Lac/LN) glycoside; (2) enzymatic sugar elongation of the Lac unit to the resulting disaccharide glycosides by consecutive use of β3GnT and β4GalT to produce tetra- and hexasaccharide glycosides; (3) coupling of the resulting glycoside to γ-PGA. The efficiency of N-glycosylation to Lac/LN was remarkably high, making this method more practical than O-glycosylation. Enzymatic O-glycosylation, which was carried out by cellulase-mediated condensation reaction between Lac/ LN and alkanol, has been shown to be a useful method for obtaining spacer-O-linked glycopolypeptides.7 However, the efficiency of these reactions were extremely low (1% yield based on Lac/LN). The efficient glycosylation protocol described here was therefore a key step in the present study. Thus, Nglycosylation was carried out by a two-step procedure by conversion to an amino function of the anomer hydroxy group of Lac and LN followed by coupling with the carboxy group.

1901

The reaction proceeded stereoselectively within a few hours to give only the β-glycoside in high yield, without requiring any protection and deprotection steps. The resulting N-β-diglycosylamines were condensed with 6-trifluoroacetamidohexanoic acid as a spacer. This method affords an easy and efficient synthesis of spacer-N-linked glycosides bearing Lac and LN from the corresponding glycosylamine. High molecular-weight γ-PGA was used as a backbone for each glycopolymer, based on the effects of molecular weights on inhibition of viral infection. The DS (%) of asialoglycan in the resulting glycopolypeptides was adjusted to a proportion of around 30% by controlling the coupling reaction (Table 1). A series of asialoglycopolypeptides was finally sialylated to obtain spacer-N-linked glycopolypeptides carrying R2,3/6 sialylated glycans in the array by the use of R2,3- and R2,6-SiaTs. Spacer-O-linked asialoglycopolypeptide with γ-PGA backbone can be used as an acceptor of a CMP-Neu5Ac donor by utilizing rat recombinant R2,3/6-SiaTs.7,8 This method was applicable to the sialylation of the present asialoglycopolypeptides. Sialylation of the terminal LN unit was quantitative, regardless of the length of glycan, but sialylation of the Lac unit occurred at lower efficiency, around 50∼80% (Table 2). The fact that sialylation proceeded more efficiently with an LN unit compared to a Lac unit in the asialo portion was consistent with results obtained for spacer-O-linked glycopolypeptides. To date, infection by influenza viruses has been successively inhibited by sialoglycopolymers carrying short glycan such as sialyllactose and sialyl-LN, even when the glycans have a short chain length.21,25 However, recent studies have demonstrated that the length of the glycan beyond a trisaccharide critically affects influenza virus HA binding contacts.8,14,29,30 The contribution of the asialo portion of sugar chains has been evaluated in hemagglutination by using spacer-O-linked glycopolypeptides carrying different length of glycans in the array.8 In the present study, the contribution of the asialo portion of the synthetic spacer-N-linked sialoglycopolypeptides was examined and compared to that of the corresponding spacer-O-linked glycopolypeptides. In the hemagglutination inhibition assay, the inhibitory activity of the spacer-N-linked sialoglycopolypeptides showed a distinct difference when applied to the avian and human viral strains (Table 3). The avian virus A/Duck/HongKong/313/4/78 specifically recognized a motif consisting of an R2,3 sialylated glycan to a Lac/LN in a trisaccharide, regardless of the glycan length (Figure 2A). In addition, the virus did not bind to the terminal SA-linked R2,6 of the Lac/LN motif. In contrast to this result, the human virus A/Aichi/2/68 had a stronger preference for the R2,6 glycan compared to the R2,3 glycan, and it bound to a long R2,3/6 sialylated glycan with LN repeats that were at least pentasaccharides in length (Figure 2B). This observation is comparable to recent results of inhibition experiments showing that the human virus was consistently inhibited by spacer-O-linked glycopolypeptides carrying multiple repeating LN units.8 As a result, whereas the avian HA interactions with the glycans is characteristic of short R2,3 glycans such as a single Lac/LN, the human interactions are specific for R2,6 glycans, which are typically adopted by long glycans with multiple LN repeats that are at least the length of pentasaccharides. The SNA lectin was further investigated as a model of human virus HA by carrying out the hemagglutination inhibition assay, in order to better understand the roles of inner sugars. Because the recognition of specific glycan motifs is known to be similar to that of the human virus HA, high affinity binding to the SAR2,6LN sequence was observed (Figure 2C). However,

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Ogata et al.

Figure 2. Inhibitory effect of glycopolypeptides with different glycans and spacer linkage types (spacer-N-linked: a, c, and e; spacer-O-linked: b, d, and f) on hemagglutination using two strains of influenza viruses and SNA: (A) Avian strain A/Duck/HongKong/313/4/78 (H5N3), (B) human strain A/Aichi/2/68 (H3N2), and (C) SNA.

the role of inner sugars has been not well elucidated. SAR2,6glycopolymers inhibited the binding of the lectin in a glycan length-dependent manner: spacer-N-linked 20 < 21 < 22 and spacer-O-linked 26 < 27 < 28. These results were further supported by direct binding assays demonstrating that elongation of the core carbohydrate portion markedly enhances binding affinity of R2,6-sialoglycopolymers with the SNA lectin (Figure 3). Thus, in structure-activity relationships, the SNA lectin may serve as a satisfactory model of human virus HA for investigating carbohydrate-binding properties. In our study, the spacer-N-linked glycopolymers were, as a whole, observed to bind the avian and human virus HA more efficiently than the spacer-O-linked glycopolymers. This type of structure is suitable to build up glycopolypeptides carrying

spacer-N-linked glycans, because the N-glycosylation step is much shorter than the enzymatic O-glycosylation step. Thus, replacement of glycosidic oxygen by nitrogen in the asialo portion should be useful and practical improvement applicable to large-scale preparations of these types of glycopolypeptides. Furthermore, γ-PGA is an obvious starting point to prepare polymeric inhibitors. Indeed, the γ-PGA backbone from B. subtilis is commercially available in large quantities. We have demonstrated that sialoglycopolymers with an R-PGA backbone have relatively low immunogenicity.25 Generally, conventional glycopolymers could present several problems for use in vivo, such as low solubility, significant cytotoxicity, and immunogenicity.13,31-33 For example, a previous study showed that the acrylamide monomer, which is known to be a potent neurotoxin,


Figure 3. Direct binding activity of SNA lectin to glycopolypeptides was determined.6,14

and PAA-Na were cytotoxic to MDCK cells under the conditions used for the test.25

Conclusions We have developed a practical method to synthesize artificial glycopolypeptides carrying long/short sialylated glycans, which is suitable for large-scale preparations. Our strategy provides a facile way to design strong polymeric inhibitors of avian and human influenza virus infection. The SNA lectin was also found to be a useful model of human virus HA for understanding its carbohydrate binding properties. Furthermore, precise analysis of the asialo moiety in the array of glycopolypeptides provides valuable information with which to explore the roles of glycan sequences in the binding of glycopolymers to the viruses, as well as increasing our understanding of the molecular basis of influenza infection. Acknowledgment. We are grateful to the Yamasa Corporation for the generous gift of the CMP-Neu5Ac, UDP-GlcNAc, and UDP-Gal. This work was supported by a grant-in-aid for Scientific Research (No. 19310141) and Scientific Research on Priority Areas (No. 18570135 to K.I.H.) from the Ministry of Education, Science, Sports, and Culture of Japan, and a research grant from Cooperation of Innovative Technology and Advanced Research in Evolutional Area (CITY AREA) in the Central Shizuoka Area-, Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. We thank Mr. Takeshi Hattori of Shizuoka University for NMR measurements.

References and Notes (1) Paulson, J. C.; Sadler, J.; Hill, R. L. J. Biol. Chem. 1979, 254, 2120– 2124. (2) Suzuki, Y.; Nagano, Y.; Kato, H.; Matsumoto, M.; Nerome, K.; Nakajima, K.; Nobusawa, E. J. Biol. Chem. 1986, 261, 17057–17061.


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(3) Rogers, G. N.; D’Souza, B. L. Virology 1989, 173, 317–322. (4) Suzuki, Y. Prog. Lipid Res. 1994, 33, 429–457. (5) Hidari, K. I.-P. J.; Shimada, S.; Suzuki, Y.; Suzuki, T. Glycoconjugate. J. 2007, 24, 583–590. (6) Yamada, S.; Suzuki, Y.; Suzuki, T.; Li, M. Q.; Nidom, C. A.; SakaiTagawa, Y.; Muramoto, Y.; Ito, M.; Kiso, M.; Hiromoto, T.; Shinya, K.; Sawada, T.; Kiso, M.; Usui, T.; Murata, T.; Lin, Y.; Hay, A.; Haire, L. F.; Stevens, D. J.; Russell, R. J.; Gamblin, S. J.; Skehel, J. J.; Kawaoka, Y. Nature 2006, 444, 378–382. (7) Ogata, M.; Murata, T.; Murakami, K.; Suzuki, T.; Hidari, K. I.-P. J.; Suzuki, Y.; Usui, T. Bioorg. Med. Chem. 2007, 15, 1383–1393. (8) Ogata, M.; Hidari, K. I.-P. J.; Murata, T.; Shimada, S.; Kozaki, W.; Park, E. Y.; Suzuki, T.; Usui, T. Bioconjugate Chem. 2009, 20, 538– 549. (9) Kato, T.; Murata, T.; Usui, T.; Park, E. Y. Biosci. Biotechnol. Biochem. 2003, 67, 2388–2395. (10) Suzuki, Y.; Matsumoto, M. J. Biol. Chem. 1985, 260, 1362–1365. (11) Suzuki, Y.; Suzuki, T.; Matsumoto, M. J. Biochem. (Tokyo, Jpn.) 1983, 93, 1621–1633. (12) Kato, M.; Uno, T.; Hiratake, J.; Sakata, K. Bioorg. Med. Chem. 2005, 13, 1563–1571. (13) Furuike, T.; Aiba, S.; Suzuki, T.; Takahashi, T.; Suzuki, Y.; Yamada, K. J. Chem. Soc., Perkin Trans. 1 2000, 1, 3000–3005. (14) Hidari, K. I.-P. J.; Murata, T.; Yoshida, K.; Minamijima, Y.; Adachi, S.; Ogata, M.; Usui, T.; Suzuki, Y.; Suzuki, T. Glycobiology 2008, 18, 779–788. (15) Yasuno, S.; Murata, T.; Kokubo, K.; Yamaguchi, T.; Kamei, M. Biosci. Biotech. Biochem. 1997, 61, 1944–1946. (16) Sato, C.; Inoue, S.; Matsuda, T.; Kitajima, K. Anal. Biochem. 1998, 261, 191–197. (17) Blixt, O.; Allin, K.; Bohorov, O.; Liu, X.; Andersson-sand, H.; Hoffmann, J.; Razi, N. Glycoconjugate J. 2008, 25, 59–68. (18) Baenziger, J. U.; Fiete, D. J. Biol. Chem. 1979, 254, 789–795. (19) Edge, A. S.; Spiro, R. G. J. Biol. Chem. 1987, 262, 16135–16141. (20) Shibuya, N.; Goldstein, I. J.; Broekaert, W. F.; Nsimba-Lubaki, M.; Peeters, B.; Peumans, W. J. J. Biol. Chem. 1987, 262, 1596–1601. (21) Tsuchida, A.; Kobayashi, K.; Matsubara, N.; Muramatsu, T.; Suzuki, T.; Suzuki, Y. Glycoconjugate J. 1998, 15, 1047–1054. (22) Gamian, A.; Chomik, M.; Laferriere, C.; Roy, R. Can. J. Microbiol. 1991, 37, 233–237. (23) Oka, H.; Onaga, T.; Koyama, T.; Guo, C.-T.; Suzuki, Y.; Esumi, Y.; Hatano, K.; Terunuma, D.; Matsuoka, K. Bioorg. Med. Chem. Lett. 2008, 18, 4405–4408. (24) Ohta, T.; Miura, N.; Fujitani, N.; Nakajima, F.; Niikura, K.; Sadamoto, R.; Guo, C.-T.; Suzuki, T.; Suzuki, Y.; Monde, K.; Nishimura, S.-I. Angew. Chem., Int. Ed. 2003, 42, 5186–5189. (25) Totani, K.; Kubota, T.; Kuroda, T.; Murata, T.; Hidari, K. I.-P. J.; Suzuki, T.; Suzuki, Y.; Kobayashi, K.; Ashida, H.; Yamamoto, K.; Usui, T. Glycobiology 2003, 13, 315–326. (26) Spaltenstein, A.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 686–687. (27) Bovin, N. V. Glycoconjugate J. 1998, 15, 431–446. (28) Umemura, M.; Itoh, M.; Makimura, Y.; Yamazaki, K.; Umekawa, M.; Masui, A.; Matahira, Y.; Shibata, M.; Ashida, H.; Yamamoto, K. J. Med. Chem. 2008, 51, 4496–4503. (29) Chandrasekaran, A.; Srinivasan, A.; Raman, R.; Viswanathan, K.; Raguram, S.; Tumpey, T. M.; Sasisekharan, V.; Sasisekharan, R. Nat. Biotechnol. 2008, 26, 107–113. (30) Srinivasan, A.; Viswanathan, K.; Raman, R.; Chandrasekaran, A.; Raguram, S.; Tumpey, T. M.; Sasisekharan, V.; Sasisekharan, R. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2800–2805. (31) Mammen, M.; Dahmann, G.; Whitesides, G. M. J. Med. Chem. 1995, 38, 4179–4190. (32) Sigal, G. B.; Mammen, M.; Dahmann, G.; Whitesides, G. M. J. Am. Chem. Soc. 1996, 118, 3789–3800. (33) Reuter, J. D.; Myc, A.; Hayes, M. M.; Gan, Z.; Roy, R.; Qin, D.; Yin, R.; Piehler, L. T.; Esfand, R.; Tomalia, D. A.; Baker, J., Jr. Bioconjugate Chem. 1999, 10, 271–278.

BM900300J

Molecular Design of Spacer-N-Linked Sialoglycopolypeptide as

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