Highly Specific near-infrared Fluorescent probe for the Real-Time Detection of β-glucuronidase in Various Living Cells and Animals

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1 Supplementary information Highly Specific near-infrared Fluorescent probe for the Real-Time Detection of β-glucuronidase in Various Living Cells and Animals Yinzhu Jin,, Xiangge Tian,, Lingling Jin, Yonglei Cui, Tao Liu, Zhenlong Yu, Xiaokui Huo, Jingnan Cui, Chengpeng Sun, Chao Wang, Jing Ning, Baojing Zhang, Lei Feng,*,, Xiaochi Ma,*, College of Pharmacy, Academy of Integrative Medicine, Dalian Medical University, Lvshun South Road No 9, Dalian , China State Key Laboratory of Fine Chemicals, Dalian University of Technology, Ganjingzi District, Linggong Road No.2, Dalian , China. Correspondence Author: X.C. Ma and Lei Feng ( and Tel.: , Fax: These authors contributed equally to this work. Table of Contents Synthesis and structural characterization of HC-glu...S2-S4 Figure S7. HRMS spectrum of HC-glu.....S5 Figure S8. HPLC chromatograms of HC-glu upon addition of GLU...S5 Figure S9. The mass spectrum of HC product of HC-glu by GLU..S6 Figure S10. The effects of ph values on the fluorescence intensity of HC and the activity of biotrsansformation.. S6 Figure S11. The linear relationship between fluorescence intensity and time...s7 Figure S12. Fluorescence responses of HC-glu to various analytes...s7 Figure S13. The inhibition IC 50 curve of Baicalin toward GLU.....S8 Figure S14. The Hydrolysis kinetics of HC-glu in different source of GLU...S8 Figure S15. The Eadie-Hofstee plot of HC-glu kinetic......s9 Figure S16. The potential molecular interaction model of HC-glu and GLU..S10 Figure S17. Cytotoxicity assay of HC-glu....S10 Figure S18. Confocal fluorescence images of Hep3B cells by HC-glu...S11 Figure S19. Evaluation of HC-glu photostability in living LoVo cells S12 Figure S20. Fluorescence images of the control group of zebrafish grown...s13 Table S1. Kinetic parameters for HC-glu in different enzyme sources....s14 S1

2 Figure S1. 1 H NMR spectra of HC(CDCl 3 ). Figure S2. 13 C NMR spectra of HC (CDCl 3 ). S2

3 Relative Abundance FL-HC #6-9 RT: AV: 4 NL: 3.31E7 T: FTMS + p ESI Full ms [ ] m/z Figure S3. HRMS spectra of HC Figure S4. The synthetic route of HC-glu. S3

4 Figure S5. The 1 H-NMR spectrum of HC-glu (500 MHz, DMSO-d6). Figure S6. The 13 C-NMR spectrum of HC-glu (125 MHz, DMSO-d6). S4

5 Relative Abundance Glu-1 #12-14 RT: AV: 3 SB: NL: 4.82E5 T: FTMS + p ESI Full ms [ ] m/z Figure S7. HRMS spectrum of HC-glu. Figure S8. Representative HPLC chromatograms of HC-glu (50 μm) incubation samples at 37, UV detector was set at 650 nm. S5

6 Figure S9. The mass spectrum of HC as a reaction product of HC-glu by GLU. Figure S10. The effects of ph values on the fluorescence intensity of HC (10 μm, blue line), and the effects of ph values on the activity of biotrsansformation (peak area of metabolism). HC (red line) from the reaction mixtures of HC-glu (10 μm) with GLU (50 μg/ml). S6

7 Figure S11. The linear relationship between fluorescence intensity and time (0-60 min) in Buffer acetonitrile (v:v = 2: 1, ph = 7.0). λ ex = 670 nm. Figure S12. Fluorescence responses of HC-glu (10 μm) to various analytes in aqueous solution (Buffer/acetonitrile = 2: 1). S7

8 Figure S13. The inhibition IC 50 curve of Baicalin toward GLU. Figure S14. The Hydrolysis kinetics of HC-glu in (a) GLU from bovine liver (10 μg/ml), (b) GLU from E. coli (0.02 μg/ml), (c) GLU from E. coli Type IX-A (2 μg/ml), (d) GLU from E. coli Type VII-A (0.01 μg/ml), (e) GLU from E. coli K 12 S8

9 ( μg/ml). The K m values were 15.0, 6.23, 5.83, 19.7, 43.4 μm for GLU from bovine liver, E. coli, E. coli Type IX-A, E. coli Type VII-A and E. coli K 12, respectively. Figure S15. The Eadie-Hofstee plot of HC-glu kinetic in (a) GLU from bovine liver, (b) GLU from E. coli, (c) GLU from E. coli Type IX-A, (d) GLU from E. coli Type VII-A, (e) GLU from E. coli K 12. S9

10 Figure S16. The potential molecular interaction model of HC-glu and GLU established by docking. Figure S17. Cytotoxicity assay of HC-glu in (a) LoVo cells, (b) HepG2 cells, and (c) Hep3B cells. S10

11 Figure S18. Confocal fluorescence images of Hep3B cells co-stained with HC-glu and Hoechst33342 (nuclear staining); (A-F) Cells treated with HC-glu (50 μm) and Hoechst33342 (2 μm) at 37 for 60 min. A, D. Bright and fluorescence fields of cells stained with Hoechst33342 and untreated with HC-glu. B, E. Bright and fluorescence fields of cells treated with Hoechst33342 and HC-glu. C, F. Bright and Fluorescence images from cells pretreated with Baicalin (100 μm) and stained with HC-glu and Hoechst S11

12 Figure S19. Evaluation of HC-glu photostability in living LoVo cells. The LoVo cells were incubated with HC-glu (50 μm) for 60 min at 37. Whereafter, images were taken by fluorescent microscopy for different time points (every 5 minutes, 5 min (a), 10 min (b), 15 min (c), 20 min (d), 25 min (e), 30 min (f), and 35 min (g)), with excitation at 633 nm. Scale bar = 50 μm. Each time of exciting is 30 sec. S12

13 Figure S20. Fluorescence images of the control group of zebrafish grown for different days (1 7 days). (a, d, g, j) Fluorescent fields of zebrafish; (b, e, h, k) The bright fields of the above corresponding samples; (c, f, i, l) The merge images of bright and fluorescence fields for the different growth periods of zebrafish. Scale bar, 500 μm. S13

14 Table S1. Kinetic parameters for HC-glu in different enzyme sources. Each data point represents the mean ± SD of three replicates. Enzyme sources V max (nmol/min/μg) K m (μm) CL int (μl/min/μg) Bovine liver 0.005± E. coli Type IX-A ± E. coli 3.53± E. coli Type VII-A 2.45± E. coli K ± S14