Supporting Information

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1 Supporting Information Wiley-VCH Weinheim, Germany

2 Supporting Information Design of a Mechanism-Based Probe for Neuraminidase to Capture Influenza Viruses Chun-Ping Lu, c, Chien-Tai Ren, a, Yi-Ning Lai, a Shih-Hsiung Wu, a,b,c,* Wei-Man Wang, d Jean-Yin Chen, d and Lee-Chiang Lo d,* a Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan b Genomics Research Center, Academia Sinica, Taipei 115, Taiwan c Institute of Biochemical Sciences and d Department of Chemistry, National Taiwan University, Taipei 106, Taiwan Both contributed equally to this work. Experimental procedures and characterization, including copies of 1 H and 13 C NMR, for compounds 2, and 9 14 are included. The synthesis of probe 2 begins with a commercially available N-acetylneuraminic acid 6 (Scheme 1). All the synthetic procedures were the combined efforts from previous publications. [1-5] General methods. All reagents and starting materials were obtained from commercial suppliers (Acros, Aldrich and Merck) and were used without further purification. IR spectra were recorded on a Nicolet 550 series II spectrometer. 1 H, 19 F and 13 C NMR were recorded using a Brucker AC-300 or Bruker Avance 400 spectrometer. The proton and carbon chemical shifts are given in ppm using CDCl 3 (δ H 7.24 and 77.0) as internal standard. High resolution mass spectra were recorded with a JEL-102A mass spectrometer. Analytical TLC (silica gel, 60F-54, Merck) and spots were visualized under UV light and/or phosphomolybdic acid-ethanol. Column chromatography was performed with Kiesegel 60 ( mesh) silica gel (Merck). 1

3 Melting points are reported without correction. H H H AcNH H H H H Me Ac Ac Me i ii H Ac H AcNH H AcNH Cl H Ac iii Ac Ac Ac AcNH Ac Me Ac Ac Me CH iv CHF2 Ac AcNH Ac 9 N 2 10 N 2 v vii viii Ac Ac Me Ac Ac Me CHF2 Ac vi CHF2 Ac AcNH AcNH Ac Ac 11 NH 2 12 N H Ac Ac Ac AcNH Ac H H H AcNH H 14 Me CHF2 N H Na CHF2 2 N H H N H N N H N H S S HN HN H NH NH H 2 N TFA 13 N H S HN Scheme 1. Synthesis of probe 2 for neuraminidase. Conditions: (i) MeH, IR-120 (H + ) resin, 92 percent; (ii) AcCl, AcH; (iii) 2-hydroxy-5-nitrobenzaldehyde, Cs 2 C 3, Bu 4 NBr, H 2 -CHCl 3, 67 percent for two steps; (iv) DAST, CH 2 Cl 2, 47 percent; (v) H 2, 5% Pd/C, MeH, 95 percent; (vi) succinic ahydride, TEA, CH 2 Cl 2, 94 percent; (vii) EDCI, HBt, 13, DIEA, DMF, 75 percent; (viii) Na 2 C 3, MeH; aqueous Na 2 C 3, 52 percent. NH Methyl 5-acetamido-3,5-dideoxy-D-galacto-2-nonulopyranosonate (7): N-Acetylneura- minic acid (6, 1.00 g, 3.2 mmol) was suspended in 25 ml of anhydrous MeH. Amberlite IR-120 (H + ) resin (0.67 g) was added. The reaction mixture was stirred until the suspension became a clear solution. After removal of the resin by filtration, the filtrate was concentrated under reduced pressure. White solid product was formed when ether was added. It was 2

4 collected by filtration to obtain product 7 (0.96 g, 92%). Methyl (2--(2-formyl-4-nitro)phenyl-5-acetamido-4,7,8,9-tetra--acetyl-3,5-dideoxy-D- glycero-α-d-galacto-2-nonulopyranosid)onate (9): To an ice-cooled solution of compound 7 (0.96 g, 3.0 mmol) in acetic acid (12 ml) was slowly added 22 ml of freshly distilled acetyl chloride. The reaction mixture was stirred for 48 h. It was then concentrated to dryness to offer chloride product 8, which was used for the next step without further purification. To a solution of chloride 8 (3.0 mmol) and tetrabutylammonium bromide (2.10 g, 6.6 mmol) in 100 ml of CHCl 3, was added a solution of 2-hydroxy-5-nitrobenzaldehyde (1.50 g, 9.0 mmol) in 150 ml of Cs 2 C 3 (0.1 M). The biphasic reaction mixture was stirred at rt overnight. When no more starting material was observed (~12 h), the organic layer was separated. The aqueous layer was extracted three times with CHCl 3. The combined CHCl 3 extract was further washed with saturated NaCl solution and dried over anhydrous Na 2 S 4. The desired product 9 (1.20 g, 67% from 7) was purified by silica gel column chromatography eluted with hexane/etac (6/4). mp o C; IR (KBr) 3257, 1759, 1739, 1646 cm -1 ; 1 H-NMR (CDCl 3, 300 MHz) δ (s, 1 H, CH), 8.62 (d, J = 2.9 Hz, 1 H, aromatic), 8.36 (dd, J = 9.1, 2.9 Hz, 1 H, aromatic), 7.39 (d, J = 9.1 Hz, 1 H, aromatic), 5.57 (d, J = 10.0 Hz, 1 H, NH), (m, 2 H, H-7 + H-8), 4.99 (ddd, J = 11.7, 10.4, 4.7 Hz, 1 H, H-4), 4.61 (d, J = 10.9 Hz, 1 H, H-9), (m, 3 H, H-5 + H-6 +H-9 ), 3.63 (s, 3 H, CH 3 ), 2.79 (dd, J = 12.2, 4.7 Hz, 1 H, H-3e), 2.33 (dd, J = 12.2, 11.7 Hz, 1 H, H-3a), 2.13 (s, 3 H, Ac), 2.06 (s, 3 H, Ac), 2.02 (s, 3 H, Ac), 2.00 (s, 3 H, Ac), 1.88 (s, 3 H, NAc); 13 C-NMR (CDCl 3, 100 MHz) δ (CH), (C), (C), (C), (C), (C), (C), (C), (C), (CH), (C), (CH), (CH), (C), 73.9 (CH), 68.0 (CH), 67.8 (CH), 66.9 (CH), 62.2 (CH 2 ), 53.6 (CH 3 ), 49.4 (CH), 38.6 (CH 2 ), 23.2 (CH 3 ), 21.0 (CH 3 ), 20.8 (CH 3 ), 20.7 (CH 3 ); MS m/z (%) 641 (7, M + + H), 474 3

5 (18), 414 (100); HRMS calcd for C 27 H 33 N 2 16 : , found Methyl (2--(2-difluoromethyl-4-nitro)phenyl-5-acetamido-4,7,8,9-tetra--acetyl-3,5-di- deoxy-d-glycero-α-d-galacto-2-nonulopyranosid)onate (10): To an ice-cooled solution of compound 9 (1.60 g, 2.5 mmol) in 6 ml of anhydrous CH 2 Cl 2 was slowly added DAST (1.6 ml, 10.0 mmol) through a syringe. The reaction was stirred overnight. When no more starting material was observed, it was cooled and quenched by adding MeH. The mixture was concentrated and the desired product was purified by silica gel column chromatography. Product 10 (778.4 mg, 47 %) was obtained. mp o C; IR (KBr) 3456, 1739, 1660 cm -1 ; 1 H-NMR (CDCl 3, 300 MHz) δ 8.42 (d, J = 2.7 Hz, 1 H, aromatic), 8.29 (dd, J =9.3, 2.7 Hz, 1 H, aromatic), 7.37 (d, J = 9.3 Hz, 1 H, aromatic), 6.86 (t, J = 54.9 Hz, 1 H, CHF 2 ), 5.44 (d, J = 9.9 Hz, 1 H, NH), (m, 2 H, H-7 + H-8), 4.97 (ddd, J = 11.5, 10.5, 4.6 Hz, 1 H, H-4), 4.60 (d, J = 11.2 Hz, 1 H, H-9), (m, 3 H, H-5 + H-6 + H-9 ), 3.62 (s, 3 H, CH 3 ), 2.75 (dd, J = 12.9, 4.6 Hz, 1 H, H-3e), 2.30 (dd, J = 12.9, 11.5 Hz, 1 H, H-3a), 2.15 (s, 3 H, Ac), 2.07 (s, 3 H, Ac), 2.02 (s, 3 H, Ac), 2.01 (s, 3 H, Ac), 1.90 (s, 3 H, NAc); 13 C-NMR (CDCl 3, 100 MHz) δ (C), (C), (C), (C), (C), (C), (C), (C), (CH), (C), (CH), (CH), (CH, t, J = Hz), (CH), 73.7 (CH), 68.0 (CH), 67.9 (CH), 66.8 (CH), 62.1 (CH2), 53.4 (CH 3 ), 50.6 (CH), 38.4 (CH 2 ), 23.0 (CH 3 ), 21.2 (CH 3 ), 20.8 (CH 3 ), 20.6 (CH 3 ), 20.5 (CH 3 ); MS m/z (%) 663 (67, M + + H), 603 (25), 414 (100); HRMS calcd for C 27 H 33 F 2 N 2 15 : , found Methyl (2--(2-difluoromethyl-4-amino)phenyl-5-acetamido-4,7,8,9-tetra--acetyl-3,5- dideoxy-d-glycero-α-d-galacto-2-nonulopyranosid)onate (11): To a solution of compound 10 (113.7 mg, 0.18 mmol) in 5 ml of MeH was added Pd/C (5%, 5 mg). The system was evacuated and filled with H 2 three times. It was kept under H 2 atmosphere with a balloon and 4

6 stirred overnight. The Pd/C catalyst was filtered off through Celite 535, and the filtrate concentrated to offer product 11 (108.2 mg, 95%). mp o C; 1 H-NMR (CDCl 3, 300 MHz) δ 7.08 (d, J = 8.8 Hz, 1 H, aromatic), (m, 3 H, aromatic + CHF 2 ), 5.34 (s, 2 H, H-7 + H-8), 5.19 (d, J = 10.0 Hz, 1 H, NH), 4.92 (ddd, J = 12.5, 10.4, 4.6 Hz, 1 H, H-4), (m, 4 H, H-5 + H-6 + H-9), 3.62 (s, 3 H, Ac), 2.68 (dd, J = 12.5, 4.6 Hz, 1 H, H-3e), (m, 4 H, H-3a + Ac), 2.11 (s, 3 H, Ac), 2.03 (s, 3 H, Ac), 2.01 (s, 3 H, Ac), 1.89 (s, 3 H, NAc); 13 C-NMR (CDCl 3, 100 MHz) δ (C), (C), (C), (C), (C), (C), (CH), (CH), (CH), (CH, t, J = Hz), (C), 73.2 (CH), 68.9 (CH), 68.7 (CH), 67.2 (CH), 62.0 (CH 2 ), 53.0 (CH 3 ), 49.5 (CH), 37.7 (CH 2 ), 23.3 (CH 3 ), 21.0 (CH 3 ), 20.8 (CH 3 ), 20.8 (CH 3 ), 20.7 (CH 3 ); MS m/z (%) 633 (24, M + + H), 414 (100); HRMS calcd for C 27 H 35 F 2 N 2 13 : , found Acetoxy-5-acetylamino-2-[4-(3-carboxy-propionylamino)-2-difluoromethyl-phenoxy]-6-(1,2,3-triacetoxy-propyl)-tetrahydro-pyran-2-carboxylic acid methyl ester (12): To a solution of 11 (237 mg, 0.37 mmol) and succinic anhydride (50 mg, 0.50 mmol) in CH 2 Cl 2 (5.0 ml) was added TEA (0.10 ml, 0.71 mmol). The mixture was stirred at room temperature for 3 h, diluted with EtAc (150 ml), and washed successively with 5% aqueous citric acid (10 ml X3) and water (10 ml X2). The combined aqueous layer was extracted once with EtAc (150 ml), and the organic layer was washed with water (10 ml X2). The organic layers were combined, dried over anhydrous Na 2 S 4, filtered, and concentrated under reduced pressure to afford 12 (258 mg, 94%) as light brown foam. 1 H-NMR (CD 3 D, 400 MHz) δ 7.84 (d, J = 2.2 Hz, 1 H, aromatic), 7.56 (dd, J = 9.0, 2.2 Hz, 1 H, aromatic), 7.26 (d, J = 9.0 Hz, 1 H, aromatic), 6.96 (t, J = 55.3 Hz, 1 H, CHF 2 ), (m, 2 H, H-7 + H-8), (m, 1 H), 4.49 (d, J = 10.9 Hz, 1 H), 4.29 (d, J = 11.3 Hz, 1 H), 4.10 (dd, J = 12.3, 2.0 Hz, 1 H), 4.03 (dd, J = 10.5, 5

7 10.5 Hz, 1 H), 3.64 (s, 3 H, CH 3 ), 2.80 (dd, J = 13.0, 4.7 Hz, 1 H, H-3e), 2.66 (s, 4 H), (m, 4 H, H-3a + Ac), 2.10 (s, 3 H, Ac), 2.01 (s, 3 H, Ac), 1.99 (s, 3 H, Ac), 1.86 (s, 3 H, NAc); 13 C-NMR (CD 3 D, 100 MHz) δ (C), (C), (C), (C), (C), (C), (C), (C), (C), (C), (C), (CH), (CH), (CH), (CHF 2, t, J = Hz), (C), 74.6 (CH), 70.5 (CH), 70.1 (CH), 68.7 (CH), 63.4 (CH 2 ), 53.9 (CH 3 ), 50.2 (CH), 39.5 (CH 2 ), 32.6 (CH 2 ), 30.2 (CH 2 ), 23.0 (CH 3 ), 21.3 (CH 3 ), 21.0 (CH 3 ), 21.0 (CH 3 ), 21.0 (CH 3 ); 19 F NMR (CD 3 D) δ (dd, J = 320, 60 Hz), (dd, J = 320, 60 Hz); MS m/z (%) 733 (35, M + + H), 673 (24), 474 (23), 414 (100); HRMS calcd for C 31 H 39 F 2 N 2 16 : , found Acetoxy-5-acetylamino-2-[2-difluoromethyl-4-(3-{6-[5-(2-oxo-hexahydro-thieno[3,4-d]imi dazol-6-yl)-pentanoylamino]-hexylcarbamoyl}-propionylamino)-phenoxy]-6-(1,2,3-triaceto xy-propyl)-tetrahydro-pyran-2-carboxylic acid methyl ester (14): To a solution of TFA salt 13 (124 mg, 0.27 mmol) [5] and 12 (150 mg, 0.22 mmol) in anhydrous DMF (6.0 ml) was added sequentially HBt (12 mg, 0.09 mmol), EDCI (104 mg, 0.54 mmol), and diisopropylethylamine (0.15 ml, 0.85 mmol). The reaction mixture was stirred at room temperature for 16 h, and the solvent was removed under reduced pressure. The resulting residue was purified by flash silica gel chromatography (10-30% gradient MeH in CH 2 Cl 2 ) to afford 14 (177 mg, 75%) as colorless foam. 1 H-NMR (CD 3 D, 400 MHz) δ 7.87 (d, J = 2.3 Hz, 1 H, aromatic), 7.55 (dd, J = 9.0, 2.3 Hz, 1 H, aromatic), 7.26 (d, J = 9.0 Hz, 1 H, aromatic), 6.96 (t, J = 55.3 Hz, 1 H, CHF 2 ), (m, 2 H, H-7 + H-8), (m, 1 H), (m, 2 H), (m, 2 H), 4.09 (dd, J = 14.0, 4.0 Hz, 1 H), 4.03 (dd, J = 10.5, 10.5 Hz, 1 H), 3.64 (s, 3 H, CH 3 ), (m, 6 H), 2.91 (dd, J = 12.8, 5.0 Hz, 1 H), 2.81 (dd, J = 13.0, 4.6 Hz, 1 H), (m, 3 H), (m, 2 H), (m, 2 H), 2.12 (s, 3 H, Ac), 2.10 (s, 3 H, Ac), 2.01 (s, 6

8 3 H, Ac), 1.99 (s, 3 H, Ac), 1.86 (s, 3 H, NAc), (m, 14 H); 13 C-NMR (CD 3 D, 100 MHz) δ (C), (C), (C), (C), (C), (C), (C), (C), (C), (C), (C), (C), (C), (CH), (CH), (CH), (CHF 2, t, J = Hz), (C), 74.6 (CH), 70.5 (CH), 70.1 (CH), 68.7 (CH), 63.4 (CH 2 ), 61.9 (CH), 57.3 (CH), 53.9 (CH 3 ), 50.3 (CH), 41.4 (CH 2 ), 40.6 (CH 2 ), 40.5 (CH 2 ), 39.5 (CH 2 ), 37.1 (CH 2 ), 33.3 (CH 2 ), 32.2 (CH 2 ), 30.6 (CH 2 ), 30.5 (CH 2 ), 30.1 (CH 2 ), 29.8 (CH 2 ), 27.9 (CH 2 ), 27.8 (CH 2 ), 27.2 (CH 2 ), 23.0 (CH 3 ), 21.4 (CH 3 ), 21.3 (CH 3 ), 21.1 (CH 3 ), 21.0 (CH 3 ); 19 F NMR (CD 3 D) δ (dd, J = 324, 60 Hz), (dd, J = 324, 60 Hz); MS m/z (%) 1057 (20, M + + H), 663 (100), 647 (56); HRMS calcd for C 47 H 67 F 2 N 6 17 S: , found Sodium Salt of Probe 2: To a solution of compound 14 (81 mg, mmol) in dried MeH (5.0 ml) was added anhydrous Na 2 C 3 (26 mg, 0.24 mmol). The mixture was stirred at room temperature for 2 h, and concentrated under reduced pressure to remove solvent and volatile material. The residual mixture was dissolved in water (5.0 ml), and stirred for 16 h. After the water was removed under reduced pressure, the resulting residue was purified by chromatography over Sephadex LH-20 with MeH, and the fractions containing the product were concentrated to afford probe 2 (36 mg, 52%) as a white foam. 1 H-NMR (CD 3 D, 400 MHz) δ 7.79 (s, 1 H, aromatic), (m, 2 H, aromatic), 7.10 (t, J = 55.6 Hz, 1 H, CHF 2 ), 4.47 (dd, J = 7.8, 4.6 Hz, 1 H), 4.28 (dd, J = 7.8, 4.6 Hz, 1 H), (m, 5 H), 3.63 (dd, J =11.5, 5.4 Hz, 1 H), 3.54 (d, J = 9.2 Hz, 1 H), (m, 5 H), 2.98 (dd, J = 10.7, 3.2 Hz, 1 H), 2.90 (dd, J = 11.8, 4.9 Hz, 1 H), (m, 3 H), 2.52 (dd, J = 7.2, 6.7 Hz, 2 H), 2.18 (t, J = 7.3 Hz, 2 H), 2.00 (s, 3 H, NAc), (m, 15 H); 13 C-NMR (CD 3 D, 100 MHz) δ (C), (C), (C), (C), (C), (C), (C), (C, t, J = 22 Hz), (CH), (CH), (CH), (CHF 2, t, J = 233 Hz), 75.6 (CH), 73.4 (CH),

9 (CH), 69.5 (CH), 64.7 (CH 2 ), 63.6 (CH), 61.9 (CH), 57.3 (CH 3 ), 54.3 (CH), 42.9 (CH 2 ), 41.4 (CH 2 ), 40.6 (CH 2 ), 40.5 (CH 2 ), 37.1 (CH 2 ), 33.4 (CH 2 ), 32.4 (CH 2 ), 30.6 (CH 2 ), 30.5 (CH 2 ), 30.1 (CH 2 ), 29.8 (CH 2 ), 27.9 (CH 2 ), 27.8 (CH 2 ), 27.2 (CH 2 ), 22.9 (CH 3 ); 19 F NMR (CD 3 D) δ (dd, J = 320, 60 Hz), (dd, J = 320, 60 Hz); MS m/z (%) 919 (23, M + + Na), 897 (42, M + + H), 606 (100); HRMS calcd for C 38 H 56 F 2 N 6 Na 13 S: , found NA Inhibition Assay: Probe 2 or zanamivir (3.3 mm) was preincubated for 45 min with influenza A virus (9x10 3 PFU) or other bacterial NA (AU: 5 mu, CP: 10 U, VC: 3.7 mu) in MES buffer (32.5 mm MES, ph 6.5, 4 mm CaCl 2 ). The reactions were initiated by the addition of a small aliquot of 4-methylumbelliferyl-N-acetylneuraminic acid substrate (3.3 µm MUNANA, Sigma Chemical Co.) in a final volume of 150 µl in black 96-well plates. After 2 h of incubation at 37 o C, the reaction was stopped by the addition of 100 µl of freshly prepared 0.14 M NaH in 83% ethanol. Fluorometric determination was quantified immediately with a fluorometer (Fluoroskan Ascent from ThermoLabsystems, Sweden). The excitation wavelength was 355 nm and the emission wavelength was 460 nm. Experiments for the determination of the fifty percent inhibitory concentration (IC 50 ) values of probe 2 against four different NA activities were similarly carried out as describe above in the inhibition assay, except different buffers were used. Probe 2 (0 3.3 mm) was incubated with individual NA activity in the following buffers; influenza virus (32.5 mm MES buffer, ph 6.5), Arthrobacter ureafaciens (AU), Clostridium perfringens (CP), and Vibro cholerae (VC) (80 mm sodium acetate buffer, ph 5.0). The residual activities were measured as above and the IC 50 values were calculated from concentration-response curves using Microcal rigin Software. The IC 50 values together with the concentration-response curves of probe 2 against the four NA 8

10 activities are shown in Figure 1. Figure 1. Determination of the IC 50 values of probe 2 against four NA activities; (a) influenza A virus, (b) Arthrobacter ureafaciens, (c) Clostridium perfringens, and (d) Vibro cholerae. Virus Particles ELISA Assay: Probe 2 was added to the streptavidin coated 96-well ELISA plate (NUNC Immobilizer ). BSA-biotin conjugate was used as a negative control. After 1 h incubation, plate was blocked with 0.1% BSA/PBS for another 1 h and washed with PBS. Serial fourfold dilutions of influenza A virus were added and incubated for 1 h at room temperature. After another wash, captured viruses were detected by treatment with a polyclonal anti- FluA antibody, followed by a goat antirabbit-horseradish peroxidase conjugate and 9

11 treatment with a TMB substrate. For the selective capturing experiment, plates were assayed as above, except serial fivefold dilutions of a mixture of influenza virus and JEV were used. The captured viruses were detected individually with either monoclonal anti-flu A HA (abcam) or monoclonal anti-jev antibody. The result is shown in Figure 2. Figure 2. Selective capturing of influenza virus particles by the probe 2. A mixture of influenza A (A/WSN/33) and JEV was added to the probe 2-coated plate. It was washed and then detected with anti-flu A or anti-jev antibody. Acknowledgement: We thank Mr. Chi-Yuan Chu for the assistance in collecting some of the NMR spectra and Mr. Fong-Ku Shi at MicroMass Taiwan for the high resolution mass spectra of compounds 2, 12, and 14. Influenza A virus (A/WSN/33) was a gift from Dr. Shin-Ru Shih, Chang Gung University, Taiwan), and polyclonal anti-flua antibody was a gift from Dr. Hour-Young Chen (Center for Disease Control Taiwan). Japanese encephalitis virus (JEV, Taiwanese strain, RP-9) and mouse monoclonal antibody specific for this virus were kindly provided by Dr. Yi-Ling Lin (IBMS Academia Sinica). Zanamivir was a gift from Glaxo Wellcome Research and Development Ltd. 10

12 (Stevenage, United Kingdom) and from Professor Ching-Shih Chen (The hio State University). References: [1] R. Kuhn, P. Lutz, P. L. MacDonald, Chem. Ber. 1966, 99, 611. [2] P. Meindl, H. Tuppy, Monatsch. Chem. 1965, 96, 802. [3] F. Baumberger, A. Vasella, Helv. Chim. Acta 1986, 69, [4] S. Halazy, V. Berges, A. Ehrhard, C. Danzin, Bioorg. Chem. 1990, 18, 330. [5] G. Sabatino, M. Chinol, G. Paganelli, S. Papi, M. Chelli, G. Leone, A. M. Papini, A. De Luca, M. Ginanneschi, J. Med. Chem. 2003, 46,

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