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 116024, People s Republic of China College of Chemistry, Dalian University of Technology, Dalian 116024, 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
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
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, 18.80 mmol). 1 H NMR (500 MHz, DMSO-d 6 ) δ 10.40 (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 176.64, 158.17 ( 2), 140.35 ( 2), 128.08 ( 2), 113.95 ( 2), 112.59 ( 2), 110.03 ( 2), 36.63. High resolution MS of [C 8 H 5 FNO] - : calcd. 150.0361 ; measured 150.0360. Fig S1. 1 H NMR spectrum of 5-Fluoro-1,3-dihydro-2H-indol-2-one (1) in DMSO-d 6 solution S3
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) 100 150.0360 Relative Abundance 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 94.9809 209.9486 164.0151 119.9470 269.9845 146.5023 178.0418 199.6199 225.9262 252.2856 297.0474 100 120 140 160 180 200 220 240 260 280 300 m/z S4
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 12.12 (s, 2H), 9.61 (s, 1H), 2.46 (s, 3H), 2.42 (s, 3H). 13 C NMR (126 MHz, DMSO-d 6 ) δ 177.74, 165.94, 142.71, 134.07, 128.16, 113.28, 13.55, 10.37. High resolution MS of [C 8 H 8 NO 3 ] - : calcd 166.0510 ; measured 166.0509. Fig S4. 1 H NMR spectrum of 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2) in DMSO-d 6 solution S5
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) 100 166.0509 Relative Abundance 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 170.0455 160.9802 182.0457 122.0612 202.0273 89.9879 147.0563 196.0247 206.0817 231.9432 251.0279 278.1027 291.9216 311.1674 329.9884 100 120 140 160 180 200 220 240 260 280 300 320 340 m/z S6
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 13.83 (s, 1H), 12.12 (s, 1H), 10.89 (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 170.01, 166.38, 158.68 ( 2), 141.28, 135.14, 133.91, 127.43 ( 2), 126.50, 125.09, 116.09, 114.70, 113.07 ( 2), 110.47 ( 2), 106.54 ( 2), 14.94, 11.91. High resolution MS of [C 16 H 12 FN 2 O 3 ] - : calcd 299.0837 ; measured 299.0835. 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
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) 100 299.0835 Relative Abundance 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 313.0625 283.2639 149.0609 186.1135 255.2327 331.0730 393.0913 430.1998 491.3409 519.3685 562.5916 621.1558 655.3097 712.2586 150 200 250 300 350 400 450 500 550 600 650 700 m/z S8
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, 0.069 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 13.65 (s, 1H), 10.86 (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), 1.60-1.41 (m, 4H), 1.37-1.24 (m, 2H). 13 C NMR (126 MHz, DMSO-d 6 ) δ 173.33, 169.55, 164.54, 158.18 ( 2), 136.23, 134.44, 130.18, 127.14 ( 2), 125.71, 124.87, 121.15, 114.41 ( 2), 112.28 ( 2), 109.95 ( 2), 105.87 ( 2), 51.15, 38.42, 33.23, 28.95, 25.94, 24.16, 13.20, 10.44. High resolution MS of [C 23 H 25 FN 3 O 4 ] - : calcd 426.1835; measured 426.1826. 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
Fig S11. 13 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) 100 426.1826 Relative Abundance 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 462.1592 283.2636 325.1839 354.1614 412.1673 440.1622 494.1700 539.2664 575.2432 607.3257 641.3869 679.3285 706.6489 300 350 400 450 500 550 600 650 700 m/z S10
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, 0.937 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 13.67 (s, 1H), 10.88 (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), 1.59-1.47 (m, 4H), 1.35-1.25 (m, 2H). 13 C NMR (126 MHz, DMSO-d 6 ) δ 174.39, 169.52, 164.53, 158.17 ( 2), 136.23, 134.45, 130.21, 127.14 ( 2), 125.71, 124.87, 121.16, 114.40 ( 2), 112.27 ( 2), 109.96 ( 2), 105.86 ( 2), 38.47, 33.61, 29.02, 26.03, 24.21, 13.22, 10.45. High resolution MS of [C 22 H 23 FN 3 O 4 ] - : calcd 412.1678 ; measured 412.1675. 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
Fig S14. 13 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) 100 412.1675 95 90 85 80 75 70 65 Relative Abundance 60 55 50 45 40 448.1443 35 30 25 20 15 10 475.1629 5 255.2327 131.0719 205.5802 339.1995 381.8517 486.0999 563.2112 618.7536 675.6763 706.6726 760.6905 798.4267 0 150 200 250 300 350 400 450 500 550 600 650 700 750 800 m/z S12
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, 0.057 mmol). 1 H NMR (500 MHz, DMSO-d 6 ) δ 13.66 (s, 1H), 10.88 (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), 1.38-1.32 (m, 2H). 13 C NMR (126 MHz, DMSO-d 6 ) δ 171.93, 169.53, 164.54, 162.25, 136.25, 134.45, 133.07, 130.74, 130.28, 130.20, 130.03, 128.04, 127.45, 127.32, 127.14 ( 2), 126.93, 126.59, 126.17, 125.72, 125.16, 125.09, 124.84, 124.65, 124.01, 123.90, 123.19, 121.16, 114.40 ( 2), 112.26 ( 2), 109.94 ( 2), 105.84 ( 2), 40.32, 38.56, 35.74, 30.74, 26.23, 25.10, 13.22, 10.45. High resolution MS of [C 39 H 36 FN 4 O 3 +H] + : calcd 627.2766 ; measured 627.2768. 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
Fig S17. 13 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) 100 627.2768 95 90 85 80 75 70 649.2586 Relative Abundance 65 60 55 50 45 40 35 30 25 20 15 10 5 0 439.2329 619.3641 665.2338 728.2196 480.2266 533.1415 556.6789 592.3695 706.9188 759.2706 797.6846 850.5167 450 500 550 600 650 700 750 800 850 m/z S14
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 10 12 14 15 24 H25 N N 16 17 18 H 11 13 O N 9 H 7 O 6 8 5 22 21 20 19 S15
S16
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 11972 12338 12312 10 nm 11886 12094 12026 101 100 100 33 nm 12882 12001 12123 98 100 100 100 nm 14451 13510 14727 92 95 91 333 nm 14639 13791 15370 91 95 89 1 µm 20881 20430 21380 70 72 69 3.3 µm 29592 29164 30349 40 42 39 10 µm 38265 38006 38175 11 11 11 33µM 38856 38618 39363 9 9 9 100µM 39861 39898 40359 5 5 5 Background 41532 41182 41438 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
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
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
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, 70-83. [2] Nishino, S.; Hirotsu, K.; Takahashi, T. Chem. Abstr. 2007, 147, 95540. [3] Paul, W. E.; Michael, G. J. Label Compd. Radiopharm 2009, 52, 360-365. S20