Supporting Information for: Indocyanine-Based Activatable Fluorescence Turn-On Probe for -Glutamyltranspeptidase and Its Application to the Mouse Model of Colon Cancer Seokan Park, a Soo-Yeon Lim, a Sang Mun Bae, b,c Sang-Yeob Kim, b,c Seung-Jae Myung, *b,d Hae-Jo Kim *a a Department of Chemistry, Hankuk University of Foreign Studies, Yongin 449-791, Republic of Korea. Tel.: +82 31 33 473; Fax: +82 31 33 4566; E-mail: haejkim@hufs.ac.kr (H.-J. Kim) b Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul 138-736, Republic of Korea c Department of Medicine, University of Ulsan College of Medicine, Seoul 138-736, Republic of Korea d Department of Gastroenterology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 138-736, Republic of Korea. Tel.: +82 2 31 3917; Fax: +82 2 476 824; E-mail: sjmyung@amc.seoul.kr (S.-J. Myung) Contents 1. NMR spectra S2 2. Mass spectra S5 3. LOD and Titration S8 4. ph profile S9 5. Inhibitor assay S1 6. HPLC profile S11 7. Cell viability assay S12 S1
5 1. NMR spectra 4 3 25 2 2 1 15 ppm (t1) 1. 5. 1 5 1 2 15 Fig. S1. ppm 4 (t1) Hz and 1 Hz spectra of 1 in CD 3 OD. 1 5 35 5 3 25 2 ppm (f1) 1. 5. 15 1 5 2 15 Fig. S2. ppm 4 (t1) Hz and 1 Hz spectra of 2 in CD 3 OD. 1 5 S2
3 3 2 25 1 2 ppm (f1) 1. 5. 15 3 1 25 5 2 2 15 Fig. S3. ppm 4 (t1) Hz and 1 Hz spectra of 3 in CD 3 OD. 1 5 15 7 1 6 5 5 4 ppm (f1) 1. 5. 3 2 1 2 15 Fig. ppm (t1) S4. 4 Hz and 1 Hz spectra of AP-Glu in CD 3 OD. 1 5 S3
Synthesis of AP To a solution of 1. mmol of 4-hydroxybenzaldehyde, 1. mmol of 1-(5-carboxypentyl)-2,3,3-trimethyl- 3H-indolium bromide in 2 ml of EtOH was stirred at 8 o C overnight. After the reaction was complete, the solvent was removed under reduced pressure. The crude product was purified by chromatography on silica gel (DCM-MeOH as eluent, 1:1 v/v) to afford 238 mg of AP in 52 % yield as a red solid. 1 H NMR (4 MHz, CD 3 OD): 8.4 (d, J = 16., 1H), 8.9 (d, J = 8.8, 2H), 7.76-7.73 (m, 2H), 7.64-7.56 (m, 2H), 7.39 (d, J = 16., 1H), 6.95 (d, J = 8.8, 2H), 4.59 (t, J = 7.2), 2.27 (t, J = 7.2, 2H), 2.2-1.93 (m, 2H), 1.85 (s, 6H), 1.76-1.68 (m, 2H), 1.6-1.52 (m, 2H). 13 C NMR (1 MHz, CD 3 OD): 182.76, 18.5, 166.89, 156.68, 144.63, 142.29, 135.16, 13.45, 13.12, 127.6, 124.2, 118.31, 115.5, 18.78, 53.31, 47.43, 36.69, 29.3, 27.37, 27.16, 26.36. 1 18.59 182.761 166.89 156.684 135.165 142.294 144.636 13.12 13.455 127.68 118.318 124.21 115.54 18.784 53.315 47.436 36.692 26.363 27.164 27.377 29.3 5 15 1 ppm (t1) 1. 5. 5 15 Fig. S5. ppm 4 (f1) Hz and 1 Hz spectra of AP in CD 3 OD. 1 5 S4
2. Mass spectra 431.2133. Fig. S6. Low resolution of 1 (MALDI +, DHB): m/z found 431.2133 calcd. 431.2154 for C 21 H 32 N 2 O 6 Na ([M+Na] + ). 535.266. Fig. S7. MALDI-TOF mass spectra of 2 (MALDI +, DHB): m/z found 535.266 calcd. 535.2416 for C 28 H 36 N 2 O 7 Na ([M+Na] + ). S5
768.4311. Fig. S8. Low (MALDI +, DHB) and high resolution mass spectra of 3 (FAB +, m-nba): m/z found 769.432, calcd. 769.431 for C 45 H 59 N 3 O 8 ([M+H] + ). S6
612.3253 Fig. S9. Low and high resolution mass spectra of AP-Glu (FAB +, m-nba): m/z found 612.373, calcd. 612.374 for C 36 H 42 N 3 O 6 ([M] + ). S7
3. Limit of detection (LOD) and Titration F 557 nm 2 1 y =.2416x +.335 R 2 =.995 1 2 3 4 5 U/L Fig. S1. Fluorescence intensity of GGT was measured between and 5 U/L of GGT. [AP-Glu] = 2 M in PBS buffer (1 X, ph 7.4). Excitation wavelength: 461 nm. Determination of the detection limit: First the calibration curve was obtained from the plot of fluorescence intensity as a function of the analyte concentration (GGT). The regression curve equation was then obtained for the lower concentration part. The detection limit = 3 S.D. / m where m is the slope of the linear equation, and S.D. represents the standard deviation for the fluorescence intensity ratio of the assay system in the absence of GGT. 557nm =.335 +.2416 [GGT] (R =.995) LOD = 3.12 /.3979 =.15 U/L 4 3 F 557 nm 2 1 1 2 3 4 5 U/L Fig. S11. Titration of GGT was measured between and 5 U/L of GGT. [AP-Glu] = 2 M in PBS buffer (1 X, ph 7.4). Excitation wavelength: 461 nm. S8
Flu at 557nm 4. ph profile 6 4 2 AP-Glu AP 3 4 5 6 7 8 9 1 ph Fig. S12. ph profile of AP-Glu and AP (2 M). Excitation wavelength: 461 nm S9
5. Inhibitor assay 1.8 1.2 A 525 /A 424.6 AP-Glu AP-Glu+gGT AP-Glu+gGT+GGsTop 4 8 12 time/h Fig. S13. Inhibitor assay of AP Glu (2 M in PBS, ph 7.4) in presence of GT (5 unit/l) and an enzyme inhibitor (GGsTop TM, 2 mm). S1
6. HPLC profile 59 49 59 39 49 29 59 39 19 49 29 9 6 39 19-1 D 29 9 4 19-1 C 5 A424nm 2 9-1 B 1 Time / min 7 1 13 16 19 22 T R Fig. S14. HPLC profile (OPTIMAPAK, MeOH:H 2 O = 43:57, 2 ml/min) of AP-Glu (1 mm, t R = 15 min) in presence of 5 U/mL of GT in PBS buffer (1X, ph 7.4). (A) AP-Glu + GT after min, (B) AP-Glu + GT after 1 min, (C) AP-Glu+ GT after 5 min, (D) AP (1 mm, t R = 9 min). A S11
Cell viability (%) 7. Cell viability assay Cell viability assay. HCT116 cells were plated in 1 l of media at 6, cells per well onto white clear-bottom 96-well plates (Corning Costar, NY, USA). Cells were incubated for 24h and 48h with or without compound before carrying out the viability assay. Using the Cell Titer-Glo Luminescent Cell Viability assay kit (Promega, WI, USA) and instructions, luminescent measurements were taken on an EnSpire 23 multilabel reader (Perkin Elmer, MA, USA). The graphically represented values are means s.d. for three independent samples. 12 24h treatment 48h treatment 1 8 6 4 2 DMSO.5 1 1 1 Fig. S15. Viability of HCT116 cells treated with AP-Glu, where HCT116 cells were treated with AP-Glu or control (DMSO), and cell viability was determined by Cell Titer-Glo assay after incubation for 24h or 48 h. S12