Supporting Information

© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2008

© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2008

Supporting Information

for

Globotriose-Functionalized Gold Nanoparticles as Multivalent Probes

for Shiga-like Toxin

Yuh-Yih Chien, Mi-Dan Jan, Avijit Kumar Adak, Hsiao-Chien Tzeng,

Yen-Ping Lin, Yu-Ju Chen, Ken-Tseng Wang, Chien-Tien Chen,

Chia-Chun Chen,* and Chun-Cheng Lin*

Contents

1. Figure S1. The UV-visible spectra of AuNPs S2

2. Figure S2. The X-ray structure of B-Slt and distance between two Pk

antigen binding sites S2

3. Figure S3. The linker length of the Pk antigen S3

4. Figure S4. Native polyacrylamide gel electrophoretic analysis of AuNP

extracted B-Slt S3

5. Calculation of Pk number on a particular size of AuNP S4

6. The IC50 of Pk –AuNPs to B-Slt S5

7. NMR spectra of compounds 1 and 10 S6 – S15

400 450 500 550 600 650 700

0.0

0.2

0.4

0.6

0.8

1.0

Abs

Wavelength (nm)

4-Pk-s-AuNP 13-Pk-s-AuNP 20-Pk-s-AuNP

Figure S1. The UV-visible spectra of AuNPs

14Asite1

site2

site3

17A17A

Between two site 127A

Between two site 229A

19A

14Asite1

site2

site3

17A17A

Between two site 127A

Between two site 229A

19A

Figure S2. The X-ray structure of B-Slt and distance between two Pk antigen binding sites.

S2

Figure S3. The linker length of the Pk antigen

Figure S4. Native polyacrylamide gel electrophoretic analysis of AuNP extracted B-Slt. Lane 1:

Alcohol dehydrogenase; Lane 2: Enolase; Lane 3: B-Slt; Lane 4: Marker

Calculation of Pk number on a particular size of AuNP

The number of gold atom composing a particular size of AuNP is directly related to its diameter

(eq. 1).

Gold atom number = 31.05* (diameter of AuNP)^3.0007 …………………………. (1)

S3

O

O O

HO

HOHOO

HOHO

OHO

OHO

NH

O

OH

OHOH

O

O O

HO

HOHOO

HOHO

OHO

OHO

NH

O

OH

OHOH

S

OO

O S

27.7A

13.3A

1 2 3 4

From eq 1, number of gold atom composing a particular size of AuNP can be calculated. For

example, total numbers of gold atoms for 2 and 10 nm size AuNPs are ~ 249 and ~ 31,097,

respectively. The number of gold atom of different size AuNP is given in Table 1.

Table 1.

Diameter (nm) Total atoms

4 1,989

13 68,339

20 248,776

Because the gold atom number composing a particular size of AuNP is known, the molecular weight

of this particular size AuNP can be calculated.

The amount of sugar on AuNP can be easily determined by anthrone-sulfuric acid assay. Briefly,

a standard curve was plotted for galactose using different concentrations ranging between 20 to 1 µg

in deionized water. A freshly prepared 0.5% anthrone (w/w) in 95% sulfuric acid was added to 1 mL

of sugar solutions. The mixture was carefully heated to 100 oC for 10 min and then allowed to cool.

The absorbance was recorded at 620 nm. Similarly, absorbances of glyconanoparticles (100 to 500

μg) were also recorded. From the standard curve, Pk ligand on nanoparticle was calculated and these

are given in Table 2. Combining the information of Tables 1 and 2, the number of Pk antigen on each

AuNP can be estimated.

Table 2.

Abbreviation Amount of sugar on 1 mg of Pk-AuNP

(µg/mg)

No. of sugar per nanoparticle

4-Pk-s-AuNP 90 60

13-Pk-s-AuNP 37.8 826

20-Pk-s-AuNP 21 1,498

4-Pk-l-AuNP 187 113

13-Pk-l-AuNP 67 1,298

20-Pk-l-AuNP 31.2 1,970

S4

The IC50 of Pk –AuNPs to B-Slt

The IC50 of Pk –AuNPs to B-Slt was estimated by using the Cheng-Prusoff equation1 (eq. 2).

IC50 = Ki * (1+ F/Kd) = Ki * (1+3.1E-8*1.16E8) = Ki *(1+3.596)………………(2)

Where the parameters are defined as: Kd is dissociation constant (1/Kd = Ka = 1.16E8 M-1), F is

concentration of the B-Slt (F = 3.1E-8).

Compound Ki (M)

Pk

4-s-Pk-AuNP 1.46 x 10-9

13-s-Pk-AuNP 8.64 x 10-11

20-s-Pk-AuNP 1.18 x 10-12

4-l-Pk-AuNP 1.95 x 10-11

13-l-Pk-AuNP

20-l-Pk-AuNP

6.84 x 10-13

2.54 x 10-13

Pk-s-dimer 3.68 x 10-5

Pk-l- dimer 6.38 x 10-6

1.14 x 10-4

IC50

5.11 x 10-4

1.65 x 10-4

2.86 x 10-5

6.54 x 10-9

3.87 x 10-10

5.29 x 10-12

8.74 x 10-11

3.06 x 10-12

1.14 x 10-12

Reference:

1. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099-3108.

S5

10 9 8 7 6 5 4 3 2 1 ppm

N3O

OO

S6

2030405060708090100110120130140150160170180190 ppm

N3O

OO

S7

10 9 8 7 6 5 4 3 2 1 0 ppm

H2NO

OO

S8

2030405060708090100110120130140150160170180190 ppm

H2NO

OO

S9

10 9 8 7 6 5 4 3 2 1 ppm

OAcO

OAc

OAcO

OAc

OAc

O O

O OAcO

OAcO

AcO OAc

AcO

NH

OO

O

S10

2030405060708090100110120130140150160170180190 ppm

OAcO

OAc

OAcO

OAc

OAc

O O

O OAcO

OAcO

AcO OAc

AcO

NH

OO

O

S11

10 9 8 7 6 5 4 3 2 1 ppm

OAcO

OAc

OAcO

OAc

OAc

O O

O OAcO

OAcO

AcO OAc

AcO

NH

OO

O SAc

S12

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

OAcO

OAc

OAcO

OAc

OAc

O O

O OAcO

OAcO

AcO OAc

AcO

NH

OO

O SAc

S13

10 9 8 7 6 5 4 3 2 1 ppm

OHO

OH

OHO

OH

OH

O O

O OHO

OHO

HO OH

HO

NH

OO

O SH

S14

2030405060708090100110120130140150160170180190 ppm

OHO

OH

OHO

OH

OH

O O

O OHO

OHO

HO OH

HO

NH

OO

O SH

S15