Supporting information

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1 Supporting information Conformationally Induced Off-On Cell Membrane Chemosensor Targeting Receptor Protein-Tyrosine Kinases for in Vivo and in Vitro Fluorescence Imaging of Cancers Yang Jiao,, Jiqiu Yin, Haiyang He, Xiaojun Peng, Qianmiao Gao, Chunying Duan *, State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian , People s Republic of China College of Chemistry, Dalian University of Technology, Dalian , People s Republic of China Contents 1. Material and methods 2. Synthesis route of SP1 3. Synthesis, 1 H NMR, 13 C NMR and HRMS of 1 4. Synthesis, 1 H NMR, 13 C NMR and HRMS of 2 5. Synthesis, 1 H NMR, 13 C NMR and HRMS of 3 6. Synthesis, 1 H NMR, 13 C NMR and HRMS of 4 7. Synthesis, 1 H NMR, 13 C NMR and HRMS of 5 8. Synthesis, 1 H NMR, 13 C NMR and HRMS of SP1 9. NOESY and COSY spectra of SP1 10. Table of VEGFR2 enzyme activity influence 11. Fluorescence response of SP1 with VEGFR2 in addition of analytes 12. Relative growth rate of cells estimated by the CCK8 assay 13. Fluorescent imaging of SP1 in A549 cells 14. Fluorescence images of different cells co-stained with D4292 and SP1 15. VEGFR2 cellular immune experiments of the cells 16. Fluorescent imaging of SP1 in A549 and HUVECs adding with axitinib 17. 3D fluorescent imaging of chick embryo chorioallantoic membrane S1

2 Material and methods All chemicals used were of reagent grade or obtained from commercial sources and used without further purification except the solvents for physical measurements which were purified by classical methods. Tyrosine kinase was purchased from Sino Biological Inc.. ESI mass spectra were carried out on an Accurate-Mass TOF LC/MS spectrometer using methanol as mobile phase. Fluorescent spectra were performed on Edinburgh FS920 steady-state fluorescence spectrophotometer. 1 H NMR spectra were recorded on a Varian INOVA 400 MHz and 500 MHz NMR spectrometer. The enzyme activity assays were carried out using Kinase-Glo Plus luminescence kinase assay kit as described previously [1]. Cell lines were grown in RPMI-1640 and Dulbecco s modified Eagle s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS). Confocal fluorescence imaging was operated on an OLYMPUS FV1000 confocal microscopy. Synthesis of SP1 sensor Scheme S1 Synthesis route of SP1 S2

3 Synthesis of 5-Fluoro-1,3-dihydro-2H-indol-2-one (1) 5-Fluoroindoline-2, 3-dione (4.95 g, 30.0 mmol) and 3 ml hydrazine (100 %) were suspended in n-buoh (50 ml) and stirred at room temperature for 30 min [2]. After heated at 80 for 3 h, triethylamine (5 ml) was added and the suspension was stirred for 12 h at 100. Upon cooling to room temperature and solvent removal in vacuo, the crude product was dissolved in 100 ml ethyl acetate and washed with 10 % potassium hydrogen sulphate solution. The aqueous layer was extracted with ethyl acetate (1 100 ml), and the combined organic phases were washed with brine (50 ml) and evaporated in vacuo. The residue was dissolved in hot ethyl acetate (50 ml) and petroleum ether was added until the solution turned slightly cloudy. After filtration and cooling to room temperature, compound 1 was filtrated to afford light brown crystals. The yield was 57.4 % (2.84 g, mmol). 1 H NMR (500 MHz, DMSO-d 6 ) δ (s, 1H), 7.10 (d, J = 8.2 Hz, 1H), 6.99 (t, J = 9.0 Hz, 1H), 6.79 (dd, J = 8.4, 4.5 Hz, 1H), 3.50 (s, 2H). 13 C NMR (101 MHz, DMSO-d 6 ) δ , ( 2), ( 2), ( 2), ( 2), ( 2), ( 2), High resolution MS of [C 8 H 5 FNO] - : calcd ; measured Fig S1. 1 H NMR spectrum of 5-Fluoro-1,3-dihydro-2H-indol-2-one (1) in DMSO-d 6 solution S3

4 Fig S2. 13 C NMR spectrum of 5-Fluoro-1,3-dihydro-2H-indol-2-one (1) in DMSO-d 6 solution Fig S3. HRMS spectrum of 5-Fluoro-1,3-dihydro-2H-indol-2-one (1) Relative Abundance m/z S4

5 Synthesis of 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2) A solution of DMF (0.80 g, 11.0 mmol) and phosphorus (V) oxychloride (1.69 g, 11.0 mmol) in 10 ml CH 2 Cl 2 was prepared and cooled to 4, then ethyl 2,4-dimethyl-1Hpyrrole-3-carboxylate (1.67 g, 10.0 mmol) was added drop wise. The reaction mixture was stirred for 15 min and subsequently heated to reflux for 1 h. A solution of hydrochloric acid (5 ml, 10 M) was cooled to 10 and added with vigorous stirring. The layers were separated. The organic phase was extracted with hydrochloric acid (2 10 ml, 10 M). The aqueous extracts were washed with 20 ml CH 2 Cl 2, and then NaOH (25 ml, 10 M) was added to obtain yellow precipitate ethyl 5-formyl-2,4-dimethyl-1H-pyrrole-3 -carboxylate. Then the suspension (1.49 g, 8.0 mmol) with 90 % KOH (20 ml) was heated to reflux for 5 h. After cooling to room temperature, the solution was diluted with 30 ml water and washed with 40 ml CH 2 Cl 2. Then, ph was adjusted to 4 using hydrochloric acid to obtain the yellow precipitate 2. The yield was 76.5 % (1.14 g, 6.82 mmol). 1 H NMR (400 MHz, DMSO-d 6 ) δ (s, 2H), 9.61 (s, 1H), 2.46 (s, 3H), 2.42 (s, 3H). 13 C NMR (126 MHz, DMSO-d 6 ) δ , , , , , , 13.55, High resolution MS of [C 8 H 8 NO 3 ] - : calcd ; measured Fig S4. 1 H NMR spectrum of 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2) in DMSO-d 6 solution S5

6 Fig S5. 13 C NMR spectrum of 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2) in DMSO-d 6 solution Fig S6. HRMS spectrum of 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2) Relative Abundance m/z S6

7 Synthesis of 5-((5-Fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl)-2,4-dimethyl-1 H-pyrrole-3-carboxylic acid (3) [3] A solution of 1 (8.8 mmol, 1.33 g) and 2 (8.8 mmol, 1.47 g), and pyrrolidine (18.0 mmol, 1.5 ml) in ethanol (120 ml) was heated to reflux for 3 h. Upon cooling to room temperature hydrochloric acid (15 ml, 2 M) was added to the suspension, a crude precipitate was recovered by suction filtration and washed with ethanol (20 ml) followed by petroleum ether (20 ml) to afford compound 3 as a yellow powder. The yield was 88.5 % (2.34 g, 7.80 mmol). 1 H NMR (400 MHz, DMSO-d 6 ) δ (s, 1H), (s, 1H), (s, 1H), 7.74 (d, J = 9.4 Hz, 1H), 7.71 (s, 1H), 6.91 (t, J = 8.7 Hz, 1H), 6.81 (dd, J = 8.3, 4.5 Hz, 1H), 2.50 (s, 3H), 2.47 (s, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ , , ( 2), , , , ( 2), , , , , ( 2), ( 2), ( 2), 14.94, High resolution MS of [C 16 H 12 FN 2 O 3 ] - : calcd ; measured Fig S7. 1 H NMR spectrum of 5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimeth yl-1h-pyrrole-3-carboxylic acid (3) in DMSO-d 6 solution S7

8 Fig S8. 13 C NMR spectrum of 5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimeth yl-1h-pyrrole-3-carboxylic acid (3) in DMSO-d 6 solution Fig S9. HRMS spectrum of 5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl- 1H-pyrrole-3-carboxylic acid (3) Relative Abundance m/z S8

9 Synthesis of Methyl 6-(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-py rrole-3-carboxamido)hexanoate (4) A mixture of 3 (1.008 g, 3.36 mmol), EDC (1.080 g, 7 mmol), HOBt (616 mg, 4.56 mmol), Triethylamine (2 ml, mmol) and Methyl 6-aminohexanoate hydrochlorid (726.9 mg, 4 mmol) were added into 50 ml DMF in stand-up flask. The reaction was allowed to stir for 48 h at 30. The mixture was poured into 5 % Na 2 CO 3 solution. Then it was filtered and dried. The mixture was separated and purified through column chromatography with CH 2 Cl 2 :CH 3 OH=500:6 to give a yellow solid, the yield was 81.1 % (818 mg, 1.91 mmol). 1 H NMR (400 MHz, DMSO-d 6 ) δ (s, 1H), (s, 1H), 7.72 (d, J = 9.3 Hz, 1H), 7.68 (s, 1H), 7.61 (t, J = 5.4 Hz, 1H), 6.89 (t, J = 9.0 Hz, 1H), 6.81 (dd, J = 8.3, 4.5 Hz, 1H), 3.56 (s, 3H), 3.18 (dd, J = 12.5, 6.3 Hz, 2H), 2.38 (d, J = 7.0 Hz, 6H), 2.29 (t, J = 7.3 Hz, 2H), (m, 4H), (m, 2H). 13 C NMR (126 MHz, DMSO-d 6 ) δ , , , ( 2), , , , ( 2), , , , ( 2), ( 2), ( 2), ( 2), 51.15, 38.42, 33.23, 28.95, 25.94, 24.16, 13.20, High resolution MS of [C 23 H 25 FN 3 O 4 ] - : calcd ; measured Fig S10. 1 H NMR spectrum of methyl 6-(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl) -2,4-dimethyl-1H-pyrrole-3-carboxamido)hexanoate (4) in DMSO-d 6 solution S9

10 Fig S C NMR spectrum of methyl 6-(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl) -2,4-dimethyl-1H-pyrrole-3-carboxamido)hexanoate (4) in DMSO-d 6 solution Fig S12. HRMS spectrum of 6-(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimet hyl-1h-pyrrole-3-carboxamido)hexanoate (4) Relative Abundance m/z S10

11 Synthesis of 6-(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3- carboxamido)hexanoic acid (5) Compound 4 (400 mg, mmol) was added into 300 ml methanol and heated at 70. Then 60 ml 8 g/l KOH was added. The solution was cooled to room temperature and stirred for 24 h. Then the mixture was treated with hydrochloric acid and adjusted ph to 3. Then the solution was filtered and dried to afford yellow solid, the yield was 56.4 % (232 mg, 0.56 mmol). 1 H NMR (500 MHz, DMSO-d 6 ) δ (s, 1H), (s, 1H), 7.76 (dd, J = 9.3, 2.2 Hz, 1H), 7.71 (s, 1H), 7.63 (t, J = 5.6 Hz, 1H), 6.92 (td, J = 9.3, 2.4 Hz, 1H), 6.84 (dd, J = 8.4, 4.5 Hz, 1H), 3.59 (s, 1H), 3.21 (dd, J = 12.7, 6.5 Hz, 2H), 2.41 (d, J = 9.4 Hz, 6H), 2.22 (t, J = 7.3 Hz, 2H), (m, 4H), (m, 2H). 13 C NMR (126 MHz, DMSO-d 6 ) δ , , , ( 2), , , , ( 2), , , , ( 2), ( 2), ( 2), ( 2), 38.47, 33.61, 29.02, 26.03, 24.21, 13.22, High resolution MS of [C 22 H 23 FN 3 O 4 ] - : calcd ; measured Fig S13. 1 H NMR spectrum of 6-(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dim ethyl-1h-pyrrole-3-carboxamido)hexanoic acid (5) in DMSO-d 6 solution S11

12 Fig S C NMR spectrum of 6-(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-di methyl-1h-pyrrole-3-carboxamido)hexanoic acid (5) in DMSO-d 6 solution Fig S15. HRMS spectrum of 6-(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimet hyl-1h-pyrrole-3-carboxamido)hexanoic acid (5) Relative Abundance m/z S12

13 Synthesis of 5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-N-(6-oxo-6-((pyr en-1-ylmethyl)amino)hexyl)-1h-pyrrole-3-carboxamide (SP1) A mixture of 5 (104.5 mg, 0.25 mmol), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (101 mg, 0.53 mmol), 1-hydroxybenzotriazole (58 mg, 0.43 mmol), Triethylamine (0.2 ml) and 1-Pyrenemethylamine hydrochloride (96.37 mg, 0.36 mmol) were added into 10 ml DMF into stand-up flask. The reaction was allowed to stir for 48 hours at 30. The mixture was poured into 5 % sodium chloride solution. Then the solution was filtered and dried. The mixture was separated and purified through column chromatography with CH 2 Cl 2 :CH 3 OH=500:6 to give a yellow solid. The yield was 34.2 % (35.8 mg, mmol). 1 H NMR (500 MHz, DMSO-d 6 ) δ (s, 1H), (s, 1H), 8.51 (t, J = 5.4 Hz, 1H), 8.37 (d, J = 9.2 Hz, 1H), 8.29 (t, J = 7.4 Hz, 2H), 8.25 (t, J = 8.0 Hz, 2H), 8.15 (s, 2H), 8.07 (t, J = 7.6 Hz, 1H), 8.02 (d, J = 7.8 Hz, 1H), 7.74 (dd, J = 9.4, 2.2 Hz, 1H), 7.69 (s, 1H), 7.61 (t, J = 5.5 Hz, 1H), 6.92 (td, J = 9.5, 2.3 Hz, 1H), 6.84 (dd, J = 8.4, 4.5 Hz, 1H), 5.01 (d, J = 5.5 Hz, 2H), 3.21 (d, J = 6.1 Hz, 2H), 2.41 (d, J = 8.3 Hz, 6H), 2.21 (t, J = 7.4 Hz, 2H), 1.62 (dt, J = 14.7, 7.4 Hz, 2H), 1.52 (dt, J = 14.0, 6.9 Hz, 2H), (m, 2H). 13 C NMR (126 MHz, DMSO-d 6 ) δ , , , , , , , , , , , , , , ( 2), , , , , , , , , , , , , ( 2), ( 2), ( 2), ( 2), 40.32, 38.56, 35.74, 30.74, 26.23, 25.10, 13.22, High resolution MS of [C 39 H 36 FN 4 O 3 +H] + : calcd ; measured Fig S16. 1 H NMR spectrum of 5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimet hyl-n-(6-oxo-6-((pyren-1-ylmethyl)amino)hexyl)-1h-pyrrole-3-carboxamide (SP1) in DMSO-d 6 solution S13

14 Fig S C NMR spectrum of 5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimet hyl-n-(6-oxo-6-((pyren-1-ylmethyl)amino)hexyl)-1h-pyrrole-3-carboxamide (SP1) in DMSO-d 6 solution Fig S18. HRMS spectrum of 5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl -N-(6-oxo-6-((pyren-1-ylmethyl)amino)hexyl)-1H-pyrrole-3-carboxamide (SP1) Relative Abundance m/z S14

15 Fig S19. NOESY spectrum of sensor SP1 in DMSO-d 6 (up), NOESY spectra of se nsor SP1 in DMSO-d 6 solution after titrating D 2 O (middle), and COSY spectrum of SP1 in DMSO-d 6 (bottom) 1 F 2 N 3 H 4 23 O H25 N N H O N 9 H 7 O S15

16 S16

17 Table S1. Compound SP1 result of VEGFR2 enzyme activity influence SP1 VEGFR2 Luminescence % Activity Repeat 1 Repeat 2 Repeat 3 Repeat 1 Repeat 2 Repeat 3 No compound nm nm nm nm µm µm µm µM µM Background Fig S20. Left bars: Fluorescence response of SP1 in addition of analytes (BSA, G lutathione, Nitroreductase, Cytochrome C). Right bars: Fluorescence response of S P1 with VEGFR2 in addition of analytes. Data were acquired in Tris-HCl (ph = 7.4, 40mM, containing 10% DMSO). λex = 460 nm S17

18 Fig S21. Relative growth rate (%) of cells estimated by the CCK8 assay that wer e cultured in the presence of 0-20 μm SP1 for 24 h. (a) A549 cells; (b) HUVECs; (c) Hela cells Fig S22. Fluorescent imaging of SP1 (a-c, 0.5 μm; d-f, 1 μm) in A549 cells S18

19 Fig S23. Fluorescence images of HT-29 cells (a-c), A549 cells (d-f), HUVECs (g-i) and HeLa cells (j-l) co-stained with D4292 (green channel) and SP1 (0.07 μm, re d channel) Fig S24. VEGFR2 cellular immune experiments of the cells S19

20 Fig S25. Fluorescent imaging of SP1 (0.5 μm) in A549 and HUVECs with additio n of axitinib (0.5 μm) Fig S26. 3D fluorescent imaging of Chick embryo chorioallantoic membrane References [1] Kashem, M. A.; Nelson, R. M.; Yingling, J. D.; Pullen, S. S.; Prokopowicz, A. S.; Jones, J. W.; Wolak, J. P.; Rogers, G. R.; Morelock, M. M.; Snow, R. J.; Homon, C. A.; Jakes, S. J. Biomol. Screen 2006, 12, [2] Nishino, S.; Hirotsu, K.; Takahashi, T. Chem. Abstr. 2007, 147, [3] Paul, W. E.; Michael, G. J. Label Compd. Radiopharm 2009, 52, S20