Supporting Information © Wiley-VCH 2005

69451 Weinheim, Germany

1

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 Lod,*

aInstitute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan bGenomics Research Center, Academia Sinica, Taipei 115, Taiwan cInstitute of Biochemical Sciences and dDepartment of Chemistry, National Taiwan University, Taipei 106, Taiwan §Both contributed equally to this work.

Experimental procedures and characterization, including copies of 1H and 13C 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. 1H, 19F and 13C NMR were recorded

using a Brucker AC-300 or Bruker Avance 400 spectrometer. The proton and carbon chemical

shifts are given in ppm using CDCl3 (δH 7.24 and 77.0) as internal standard. High resolution

mass spectra were recorded with a JEOL-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 (70-230 mesh) silica gel (Merck).

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Melting points are reported without correction.

O

AcO

AcNHAcO

O

OAc

AcO

NO2

O OMeCHO

O

AcO

AcNHAcO

O

OAc

AcO

NO2

O OMeCHF2

O

AcO

AcNHAcO

O

OAc

AcO

NH2

O OMeCHF2

O

AcO

AcNHAcO

O

OAc

AcO

NH

O OMeCHF2

OHO

O

O

AcO

AcNHAcO

Cl

OAc

AcOO OMe

O

HO

AcNHHO

OH

OH

HOO OMe

O

HO

AcNHHO

OH

OH

HOO OH

O

AcO

AcNHAcO

O

OAc

AcO

NH

O OMeCHF2

HN

O

ONH

O

SNH

HN O

TFAH2N N

H

O

SNH

HN O

i ii

6 7 8

iii iv

9 10

v vi

11 12

vii

13

14

viii O

HO

AcNHHO

O

HO

HO

NH

O OCHF2

HN

O

ONH

O

SNH

HN O

2

Na

Scheme 1. Synthesis of probe 2 for neuraminidase. Conditions: (i) MeOH, IR-120 (H+) resin, 92 percent; (ii) AcCl, AcOH; (iii) 2-hydroxy-5-nitrobenzaldehyde, Cs2CO3, Bu4NBr, H2O-CHCl3, 67 percent for two steps; (iv) DAST, CH2Cl2, 47 percent; (v) H2, 5% Pd/C, MeOH, 95 percent; (vi) succinic ahydride, TEA, CH2Cl2, 94 percent; (vii) EDCI, HOBt, 13, DIEA, DMF, 75 percent; (viii) Na2CO3, MeOH; aqueous Na2CO3, 52 percent.

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 MeOH. 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

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collected by filtration to obtain product 7 (0.96 g, 92%).

Methyl (2-O-(2-formyl-4-nitro)phenyl-5-acetamido-4,7,8,9-tetra-O-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 CHCl3, was added a solution of 2-hydroxy-5-nitrobenzaldehyde (1.50 g, 9.0 mmol) in 150

mL of Cs2CO3 (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 CHCl3. The combined CHCl3 extract was further washed

with saturated NaCl solution and dried over anhydrous Na2SO4. The desired product 9 (1.20 g,

67% from 7) was purified by silica gel column chromatography eluted with hexane/EtOAc (6/4).

mp 192-194oC; IR (KBr) 3257, 1759, 1739, 1646 cm-1; 1H-NMR (CDCl3, 300 MHz) δ 10.36 (s,

1 H, CHO), 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), 5.34-5.29 (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), 4.19-3.99 (m, 3 H, H-5 +

H-6 +H-9’), 3.63 (s, 3 H, OCH3), 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, OAc), 2.06 (s, 3 H, OAc), 2.02 (s, 3 H, OAc), 2.00 (s, 3 H, OAc),

1.88 (s, 3 H, NAc); 13C-NMR (CDCl3, 100 MHz) δ 187.0 (CH), 170.8 (C), 170.5 (C), 170.3 (C),

170.1 (C), 170.0 (C), 167.6 (C), 160.0 (C), 143.6 (C), 130.5 (CH), 126.3 (C), 124.2 (CH), 119.4

(CH), 100.0 (C), 73.9 (CH), 68.0 (CH), 67.8 (CH), 66.9 (CH), 62.2 (CH2), 53.6 (CH3), 49.4 (CH),

38.6 (CH2), 23.2 (CH3), 21.0 (CH3), 20.8 (CH3), 20.7 (CH3); MS m/z (%) 641 (7, M+ + H), 474

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(18), 414 (100); HRMS calcd for C27H33N2O16: 641.1830, found 641.1828.

Methyl (2-O-(2-difluoromethyl-4-nitro)phenyl-5-acetamido-4,7,8,9-tetra-O-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 CH2Cl2 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 MeOH. The mixture was

concentrated and the desired product was purified by silica gel column chromatography.

Product 10 (778.4 mg, 47 %) was obtained. mp 72-76oC; IR (KBr) 3456, 1739, 1660 cm-1;

1H-NMR (CDCl3, 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, CHF2), 5.44 (d, J = 9.9

Hz, 1 H, NH), 5.34-5.29 (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), 4.21-4.02 (m, 3 H, H-5 + H-6 + H-9’), 3.62 (s, 3 H, OCH3), 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, OAc), 2.07 (s,

3 H, OAc), 2.02 (s, 3 H, OAc), 2.01 (s, 3 H, OAc), 1.90 (s, 3 H, NAc); 13C-NMR (CDCl3, 100

MHz) δ 170.6 (C), 170.5 (C), 170.4 (C), 170.0 (C), 169.8 (C), 167.4 (C), 156.4 (C), 143.1 (C),

127.7 (CH), 125.2 (C), 122.4 (CH), 118.3 (CH), 109.9 (CH, t, J = 237.2 Hz), 100.0 (CH), 73.7

(CH), 68.0 (CH), 67.9 (CH), 66.8 (CH), 62.1 (CH2), 53.4 (CH3), 50.6 (CH), 38.4 (CH2), 23.0

(CH3), 21.2 (CH3), 20.8 (CH3), 20.6 (CH3), 20.5 (CH3); MS m/z (%) 663 (67, M+ + H), 603 (25),

414 (100); HRMS calcd for C27H33F2N2O15: 663.1849, found 663.1808

Methyl (2-O-(2-difluoromethyl-4-amino)phenyl-5-acetamido-4,7,8,9-tetra-O-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 MeOH was added Pd/C (5%, 5 mg). The system was

evacuated and filled with H2 three times. It was kept under H2 atmosphere with a balloon and

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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 82-86oC; 1H-NMR (CDCl3, 300 MHz) δ

7.08 (d, J = 8.8 Hz, 1 H, aromatic), 7.01-6.64 (m, 3 H, aromatic + CHF2), 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), 4.33-4.02 (m,

4 H, H-5 + H-6 + H-9), 3.62 (s, 3 H, OAc), 2.68 (dd, J = 12.5, 4.6 Hz, 1 H, H-3e), 2.19-2.12 (m,

4 H, H-3a + OAc), 2.11 (s, 3 H, OAc), 2.03 (s, 3 H, OAc), 2.01 (s, 3 H, OAc), 1.89 (s, 3 H, NAc);

13C-NMR (CDCl3, 100 MHz) δ 170.9 (C), 170.6 (C), 170.2 (C), 170.0 (C), 167.4 (C), 143.4 (C),

122.1 (CH), 118.1 (CH), 111.9 (CH), 111.3 (CH, t, J = 234.1 Hz), 100.9 (C), 73.2 (CH), 68.9

(CH), 68.7 (CH), 67.2 (CH), 62.0 (CH2), 53.0 (CH3), 49.5 (CH), 37.7 (CH2), 23.3 (CH3), 21.0

(CH3), 20.8 (CH3), 20.8 (CH3), 20.7 (CH3); MS m/z (%) 633 (24, M+ + H), 414 (100); HRMS

calcd for C27H35F2N2O13: 633.2107, found 633.2134.

3-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 CH2Cl2 (5.0

mL) was added TEA (0.10 mL, 0.71 mmol). The mixture was stirred at room temperature for 3

h, diluted with EtOAc (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 EtOAc (150

mL), and the organic layer was washed with water (10 mL X2). The organic layers were

combined, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to

afford 12 (258 mg, 94%) as light brown foam. 1H-NMR (CD3OD, 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, CHF2), 5.38-5.36 (m, 2 H, H-7 + H-8), 4.91-4.89 (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,

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10.5 Hz, 1 H), 3.64 (s, 3 H, OCH3), 2.80 (dd, J = 13.0, 4.7 Hz, 1 H, H-3e), 2.66 (s, 4 H),

2.16-2.15 (m, 4 H, H-3a + OAc), 2.10 (s, 3 H, OAc), 2.01 (s, 3 H, OAc), 1.99 (s, 3 H, OAc), 1.86

(s, 3 H, NAc); 13C-NMR (CD3OD, 100 MHz) δ 176.6 (C), 173.9 (C), 173.1 (C), 172.7 (C), 172.0

(C), 171.9 (C), 171.7 (C), 169.2 (C), 149.2 (C), 136.9 (C), 127.6 (C), 124.5 (CH), 121.9 (CH),

118.8 (CH), 112.9 (CHF2, t, J = 233.3 Hz), 102.3 (C), 74.6 (CH), 70.5 (CH), 70.1 (CH), 68.7

(CH), 63.4 (CH2), 53.9 (CH3), 50.2 (CH), 39.5 (CH2), 32.6 (CH2), 30.2 (CH2), 23.0 (CH3), 21.3

(CH3), 21.0 (CH3), 21.0 (CH3), 21.0 (CH3); 19F NMR (CD3OD) δ -115.4 (dd, J = 320, 60 Hz),

-117.6 (dd, J = 320, 60 Hz); MS m/z (%) 733 (35, M+ + H), 673 (24), 474 (23), 414 (100);

HRMS calcd for C31H39F2N2O16: 733.2268, found 733.2272.

3-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 HOBt (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 MeOH in CH2Cl2) to afford 14 (177 mg, 75%) as

colorless foam. 1H-NMR (CD3OD, 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,

CHF2), 5.38-5.36 (m, 2 H, H-7 + H-8), 4.89-4.85 (m, 1 H), 4.51-4.48 (m, 2 H), 4.30-4.28 (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, OCH3),

3.21-3.13 (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), 2.71-2.65

(m, 3 H), 2.56-2.52 (m, 2 H), 2.20-2.16 (m, 2 H), 2.12 (s, 3 H, OAc), 2.10 (s, 3 H, OAc), 2.01 (s,

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3 H, OAc), 1.99 (s, 3 H, OAc), 1.86 (s, 3 H, NAc), 1.76-1.32 (m, 14 H); 13C-NMR (CD3OD, 100

MHz) δ 176.2 (C), 174.8 (C), 173.8 (C), 173.3 (C), 172.7 (C), 172.0 (C), 171.9 (C), 171.7 (C),

169.2 (C), 166.4 (C), 149.2 (C), 136.9 (C), 127.6 (C), 124.5 (CH), 121.9 (CH), 118.8 (CH),

112.9 (CHF2, t, J = 234.0 Hz), 102.3 (C), 74.6 (CH), 70.5 (CH), 70.1 (CH), 68.7 (CH), 63.4

(CH2), 61.9 (CH), 57.3 (CH), 53.9 (CH3), 50.3 (CH), 41.4 (CH2), 40.6 (CH2), 40.5 (CH2), 39.5

(CH2), 37.1 (CH2), 33.3 (CH2), 32.2 (CH2), 30.6 (CH2), 30.5 (CH2), 30.1 (CH2), 29.8 (CH2), 27.9

(CH2), 27.8 (CH2), 27.2 (CH2), 23.0 (CH3), 21.4 (CH3), 21.3 (CH3), 21.1 (CH3), 21.0 (CH3); 19F

NMR (CD3OD) δ -115.3 (dd, J = 324, 60 Hz), -117.5 (dd, J = 324, 60 Hz); MS m/z (%) 1057 (20,

M+ + H), 663 (100), 647 (56); HRMS calcd for C47H67F2N6O17S: 1057.4251, found 1057.4301.

Sodium Salt of Probe 2: To a solution of compound 14 (81 mg, 0.077 mmol) in dried MeOH

(5.0 mL) was added anhydrous Na2CO3 (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 MeOH, and the fractions containing the product

were concentrated to afford probe 2 (36 mg, 52%) as a white foam. 1H-NMR (CD3OD, 400

MHz) δ 7.79 (s, 1 H, aromatic), 7.51-7.45 (m, 2 H, aromatic), 7.10 (t, J = 55.6 Hz, 1 H, CHF2),

4.47 (dd, J = 7.8, 4.6 Hz, 1 H), 4.28 (dd, J = 7.8, 4.6 Hz, 1 H), 3.86-3.74 (m, 5 H), 3.63 (dd, J

=11.5, 5.4 Hz, 1 H), 3.54 (d, J = 9.2 Hz, 1 H), 3.20-3.12 (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), 2.70-2.63 (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), 1.83-1.28 (m, 15 H); 13C-NMR (CD3OD, 100 MHz) δ 176.3

(C), 175.9 (C), 174.8 (C), 173.1 (C), 166.4 (C), 150.5 (C), 136.2 (C), 128.7 (C, t, J = 22 Hz),

124.1 (CH), 123.9 (CH), 118.2 (CH), 113.4 (CHF2, t, J = 233 Hz), 75.6 (CH), 73.4 (CH), 70.4

8

(CH), 69.5 (CH), 64.7 (CH2), 63.6 (CH), 61.9 (CH), 57.3 (CH3), 54.3 (CH), 42.9 (CH2), 41.4

(CH2), 40.6 (CH2), 40.5 (CH2), 37.1 (CH2), 33.4 (CH2), 32.4 (CH2), 30.6 (CH2), 30.5 (CH2), 30.1

(CH2), 29.8 (CH2), 27.9 (CH2), 27.8 (CH2), 27.2 (CH2), 22.9 (CH3); 19F NMR (CD3OD) δ -112.8

(dd, J = 320, 60 Hz), -120.4 (dd, J = 320, 60 Hz); MS m/z (%) 919 (23, M+ + Na), 897 (42, M+ +

H), 606 (100); HRMS calcd for C38H56F2N6NaO13S: 897.3492, found 897.3475.

NA Inhibition Assay: Probe 2 or zanamivir (3.3 mM) was preincubated for 45 min with

influenza A virus (9x103 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 CaCl2). 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 37oC, the reaction was stopped by the addition of 100 µL of freshly prepared 0.14

M NaOH 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 (IC50) 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 IC50

values were calculated from concentration-response curves using Microcal Origin Software.

The IC50 values together with the concentration-response curves of probe 2 against the four NA

9

activities are shown in Figure 1.

Figure 1. Determination of the IC50 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

10

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.

11

(Stevenage, United Kingdom) and from Professor Ching-Shih Chen (The Ohio 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, 1927.

[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, 3170.

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