Supporting Information. Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2008

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1 Supporting Information Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2008

2 Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 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 P k antigen binding sites S2 3. Figure S3. The linker length of the P k antigen S3 4. Figure S4. Native polyacrylamide gel electrophoretic analysis of AuNP extracted B-Slt S3 5. Calculation of P k number on a particular size of AuNP S4 6. The IC 50 of P k AuNPs to B-Slt S5 7. NMR spectra of compounds 1 and 10 S6 S15

3 P k -s-aunp 13-P k -s-aunp 20-P k -s-aunp 0.6 Abs Wavelength (nm) Figure S1. The UV-visible spectra of AuNPs site1 14A site2 17A 17A 19A site3 Between two site 2 29A Between two site 1 27A Figure S2. The X-ray structure of B-Slt and distance between two P k antigen binding sites. S2

4 N H S 13.3A N H S 27.7A Figure S3. The linker length of the P k 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 P k 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)^ (1) S3

5 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, , ,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 o C 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, P k ligand on nanoparticle was calculated and these are given in Table 2. Combining the information of Tables 1 and 2, the number of P k antigen on each AuNP can be estimated. Table 2. Abbreviation Amount of sugar on 1 mg of P k -AuNP (µg/mg) No. of sugar per nanoparticle 4-P k -s-aunp P k -s-aunp P k -s-aunp 21 1,498 4-P k -l-aunp P k -l-aunp 67 1, P k -l-aunp ,970 S4

6 The IC 50 of P k AuNPs to B-Slt The IC 50 of P k AuNPs to B-Slt was estimated by using the Cheng-Prusoff equation 1 (eq. 2). IC 50 = K i * (1+ F/K d ) = Ki * (1+3.1E-8*1.16E8) = Ki *( ) (2) Where the parameters are defined as: K d is dissociation constant (1/Kd = Ka = 1.16E8 M -1 ), F is concentration of the B-Slt (F = 3.1E-8). Compound K i (M) IC 50 P k 1.14 x 10-4 P k -s-dimer 3.68 x 10-5 P k -l- dimer 6.38 x s-P k -AuNP 1.46 x s-P k -AuNP 8.64 x s-P k -AuNP 1.18 x l-P k -AuNP 1.95 x l-P k -AuNP 20-l-P k -AuNP 6.84 x x x x x x x x x x x Reference: 1. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, S5

7 N S ppm

8 N S ppm

9 H 2 N S ppm

10 H 2 N S ppm

11 Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac N H S ppm

12 Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac N H S ppm

13 Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac N H SAc S ppm

14 Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac N H SAc S ppm

15 N H SH S ppm

16 N H SH S ppm