Highly Activatable and Rapidly Releasable Caged Fluorescein Derivatives

Transcription

1 Supporting information Highly Activatable and Rapidly Releasable Caged Fluorescein Derivatives Tomonori Kobayashi, Yasuteru Urano, Mako Kamiya, Tasuku Ueno, Hirotatsu Kojima and Tetsuo Nagano * Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo , Japan, and CREST, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan, and PREST, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan. tlong@mol.f.u-tokyo.ac.jp Abbreviations. AcEt: ethyl acetate, Bn: benzyl, Bu: butyl, DMF: N,N-dimethylformamide, DMS: dimethylsulfoxide, HPLC: high-performance liquid chromatography, MeH: methanol, MS: mass spectrometry, NMR: nuclear magnetic resonance, TBDMS: tert-butyldimethylsilyl, TFA: trifluoroacetic acid, THF: tetrahydrofuran, TLC: thin layer chromatography S1

2 Experimental Section Materials. General chemicals were of the best grade available, supplied by Tokyo Chemical Industries, Wako Pure Chemical, Kanto Chemical Co. Inc., or Aldrich Chemical Co., and were used without further purification. Special chemicals consisted of dimethyl sulfoxide (fluorometric grade, Dojindo), N,N-dimethylformamide (fluorometric grade, Dojindo) and CMNB-caged fluorescein (F7103, Invitrogen Corp., USA). All solvents were used after appropriate distillation or purification. Instruments. NMR spectra were recorded on a JEL JNM-LA300 instrument at 300 MHz for 1 H NMR and at 75 MHz for 13 C NMR or on a JEL JNM-AL400 instrument at 400 MHz for 1 H NMR and at 100 MHz for 13 C NMR. Mass spectra (MS) were measured with a JEL JMS-DX300 for EI and a JMS-T100LC AccuToF for ESI. UV/visible spectra were obtained on an Agilent 8453 UV/Vis spectrometer. Fluorescence spectroscopic studies were performed on a Perkin Elmer LS55. Photoirradiation experiments in cuvettes were carried out in a monochromator unit (Bunko-Keiki Co., Ltd., Japan) equipped with a 500 W xenon lamp (Usio Inc., Japan) as a light source. The light intensity was measured with a Nova Display (HPIR Japan Ltd.). HPLC analyses and purification were performed on a reverse-phase column, Inertsil DS-3 (GL Sciences, Tokyo, Japan, 5 µm, 4.6 x 250 mm for analyses and 5 µm, 10 x 250 mm for purification) using the eluent specified below with an S2

3 HPLC system (JASC, Japan) equipped with a pump (PU-2080 Plus) and a UV/Vis detector (MD-2010 Plus). Thin layer chromatography and preparative thin layer chromatography were carried out with 25TLC plates 20 x 20 cm, silica gel 60 F254 and 15PLC plates 20 x 20 cm, silica gel 60 F254 (Merck Ltd., Japan), respectively. Silica gel column chromatography was performed with Wakogel C-200 (Wako, Japan). Microinjection was carried out with Transjector5246 (Eppendorf, Germany) equipped with Injectman (Eppendorf, Germany). Measurement of Photochemical Properties. A 1 mm DMF stock solution of each compound was prepared. Absorption spectra were obtained with a 100 mm sodium phosphate buffer (ph 7.4) solution of each compound at the desired concentration, adjusted by appropriate dilution of the 1 mm DMF stock solution. For determination of the quantum efficiency of fluorescence (Φ fl ), fluorescein in 0.1 M NaH aqueous solution was used as a fluorescence standard. The quantum efficiency of fluorescence was obtained with the following equation (F denotes fluorescence intensity at each wavelength and [F] was calculated by summation of fluorescence intensity). Φ fl sample = Φ fl standard Abs standard [F sample ] /Abs sample [F sample ] Photoirradiation Experiments in Cuvette (Figure S1). A 3 µl aliquot of 1 mm DMF stock S3

4 solution of each compound was diluted by adding 2997 µl of 100 mm sodium phosphate buffer, ph 7.4 in a quartz cuvette equipped with a magnetic stirrer. The cuvette containing the prepared solution was held on a cell holder placed in the light path of a monochromator, and illuminated at around 350 nm (20 nm, 2.13 mw/cm 2 at 350 nm) with stirring for a specified period. After each irradiation, the fluorescence spectrum was recorded in the range from 480 nm to 550 nm, with 490 nm excitation. The set-up parameters were as follows: excitation slit: 2.5 nm, emission slit: 2.5 nm, scan speed: 60 nm/min, photomultiplier voltage: 700 V, response: 1.5 nm and spectral correction file: EMRED.cor. The maximum fluorescence intensity of each obtained spectrum (caged fluorescein: 513 nm, caged TokyoGreens: 516 nm) was plotted against irradiation time. Cytotoxicity Assay (Figure S3). An HBSS (2 ml) solution containing 1 µm TG-NPE AM (0.1% DMF as a cosolvent) was added, and the cells were incubated for 15 min at room temperature in the dark. The cells were then washed twice with HBSS (2 ml), and the extracellular solution was replaced with 1 µm Calcein/AM and 1 µm ethidium homodimer-1 (EthD-1) (LIVE/DEAD VIability/Cytotoxicity kit, Molecular Probes) SR1 in HBSS. Incubation was continued for 15 min at room temperature. Then, the cells were washed with HBSS and the two-color fluorescence cell viability test was conducted under a fluorescence microscope. The fluorescence image was acquired by using a BP excitation filter and a BA emission filter for S4

5 Calcein imaging (living cells), and a BP excitation filter and a BA590 emission filter for EthD-1 imaging (dead cells). In the control experiment, 0.1% DMF in HBSS used as a live cell control, and 100% MeH as a dead cell control. Cell Assay (Figure S5-S8). HeLa cells were cultured in Dulbecco s modified Eagle s medium (DMEM, Invitrogen Corp.), supplemented with 10% (v/v) fetal bovine serum (Invitrogen Corp.), penicillin (100 units/ml) and streptomycin (100 µg/ml) in a humidified incubator containing 5% C 2 gas. For fluorescence microscopy, HeLa cells were plated in a 35-mm-diameter glass-bottomed dish (MatTek Corp., Ashland, MA) and cultured overnight in DMEM. The medium was removed after 24 hours, and the cells were washed twice with 1 ml of Hank s balanced salt solution (HBSS) buffer (Invitrogen Corp.). Then an HBSS (2 ml) solution containing 1 µm TG-NPE AM (0.1% DMF as a cosolvent) or the desired concentration (1 µm, 10 µm, and 20 µm) of BisCMNB-FL AM (0.1%, 1%, and 2% DMF as a cosolvent, respectively) was added and the cells were incubated for 15 min at room temperature. The cells were then washed twice with HBSS (1.5 ml), and selected cells were illuminated with a high-pressure mercury lamp (BH2-RFL-T3, lympus, Japan) via a nm band-pass filter (BP , lympus) through the objective lens (UPlanFL N 40x, lympus) of a fluorescence microscope (IX71, lympus). After illumination, the fluorescence image was acquired with a cooled CCD camera (Coolsnap HQ, lympus), using a xenon lamp (AH2-RX-T, lympus) S5

6 with a BP excitation filter and a BA emission filter. Preparation of TG-NPE-dextran Conjugate (Figure S9-S10). TG-NPE SE 10 mm DMS solution (320 µl) was added to dextran-amino (MW , Invitrogen) 1.0 mg/ml 200 mm sodium phosphate buffer (ph 8.4) solution (1000 µl) in a 1.5 ml plastic tube. The mixture was allowed to stand for 1 hour at room temperature in the dark. Then, it was applied to a PD-10 column (Amersham Biosciences, Sweden) with ph 7.4 PBS (Invitrogen Corp.) as the eluent. The desired fraction was collected, and applied to another column to afford TG-dextran conjugate stock solution. This stock solution was diluted to the desired concentration with 100 mm sodium phosphate buffer (ph 7.4), and used for cuvette experiments. For the microinjection experiment, the original stock solution was used. Determination of Uncaging Quantum Efficiency (Figure S11). TG-NPE (30 µm) or BisCMNB-FL (30 µm) in 100 mm sodium phosphate buffer (ph7.4) solution containing 3% DMF was illuminated at around 270 nm (±20 nm, 1.03 mw at 270 nm) with stirring for the specified period. After each irradiation, a 20 µl aliquot of the irradiated solution was subjected to HPLC analyses. Disappearance of caged substrates was monitored in terms of peak area at the appropriate detection wavelength (TG-NPE: 450 nm absorbance, BisCMNB-FL: 300 nm absorbance). Remaining caged substrate [%] was plotted versus irradiation time [sec]. Each plot was fitted to a linear equation, and S6

7 the slope (k TG-NPE, k BisCMNB-FL ) was determined. Uncaging quantum efficiency of TG-NPE ( Φ TG NPE uncage ) was determined according to the following equation Φ TG NPE uncage = ε ε BisCMNB FL 270nm TG NPE 270nm k k TG NPE BisCMNB FL Φ BisCMNB FL uncage BisCMNB FL Φuncage BisCMNB FL : uncaging quantum efficiency of BisCMNB-FL, ε 270 nm : molar extinction coefficient of BisCMNB-FL at 270 nm [M -1 cm -1 TG NPE ], ε 270 nm : molar extinction coefficient of TG-NPE at 270 nm [M -1 cm -1 ]. S7

8 (A) TG-NB TG-NPE Fluorescence Intensity (a.u.) sec 10sec 20sec 30sec 40sec 50sec 60sec Wavelength (nm) Fluorescence Intensity (a.u.) sec 10sec 20sec 30sec 40sec 50sec 60sec Wavelength (nm) Fluorescence Intensity (a.u.) TG-DMNB 0sec 10sec 20sec 30sec 40sec 50sec 60sec Fluorescence Intensity (a.u.) MonoNB-FL 0sec 10sec 20sec 30sec 40sec 50sec 60sec Wavelength (nm) Wavelength (nm) Fluorescence Intensity (a.u.) BisCMNB-FL 0sec 10sec 20sec 30sec 40sec 50sec 60sec Wavelength (nm) (B) Figure S1. (A) Change of fluorescence spectra of caged TokyoGreens and caged fluoresceins (sample concentration: 1 µm) upon UV irradiation ( nm, 1.88 mw/cm 2 at 350 nm) in 100 mm sodium phosphate buffer, ph 7.4, containing 0.1% DMF as a cosolvent. (B) Change of fluorescence intensity at the fluorescence maxima wavelength (caged TokyoGreen: 513 nm, caged fluorescein: 516 nm) upon irradiation. Fluorescence Intensity (a.u.) TG-NB TG-NPE TG-DMNB MonoNB-FL BisCMNB-FL Irradiation time (sec) S8

9 -1 min 0 min 5 min 10 min 15 min 20 min 25 min 30 min Figure S2. Differential interference contrast images of TG-NPE AM (1 µm)-loaded HeLa cells to evaluate the cytotoxicity of TG-NPE AM. No apparent toxicity was observed under these conditions. S9

10 (A) TG-NPE AM DIC Calcein/AM EthD-1 (B) Live cells (DMF treated) DIC Calcein/AM EthD-1 (C) Dead cells (MeH treated) DIC Calcein/AM EthD Figure S3. Differential interference contrast images and fluorescence images of HeLa cells loaded with Calcein/AM (living cell marker) and EthD-1 (dead cell marker) after (A) incubation for 15 min with TG-NPE AM (1 µm), (B) addition of DMF (0.1%) (control for living cells), and (C) addition of MeH (2 ml) (control for dead cells). S10

11 0 sec 1 sec 5 sec Irradiation 10 sec 20 sec 30 sec Figure S4. Differential interference contrast images of TG-NPE AM (1 µm)-loaded HeLa cells after UV light irradiation to evaluate the cytotoxicity due to UV irradiation. No apparent toxicity was observed under these conditions. S11

12 500 DIC 0 sec 1 sec Irradiation sec 3 sec 4 sec 5 sec 6 sec 7 sec 8 sec 9 sec 10 sec Figure S5. Fluorescence images of TG-NPE AM (1 µm)-loaded HeLa cells after UV light illumination for the period specified in each image. S12

13 500 DIC 0 sec 1 sec Irradiation sec 3 sec 4 sec 5 sec 6 sec 7 sec 8 sec 9 sec 10 sec Figure S6. Fluorescence images of BisCMNB-FL AM (1 µm)-loaded HeLa cells after UV light illumination for the period specified in each image. S13

14 165 DIC 0 sec 1 sec Irradiation sec 3 sec 4 sec 5 sec 6 sec 7 sec 8 sec 9 sec 10 sec Figure S7. Fluorescence images of BisCMNB-FL AM (10 µm)-loaded HeLa cells after UV light illumination for the period specified in each image. S14

15 250 DIC 0 sec 60 sec Irradiation sec 180 sec 240 sec 300 sec 360 sec 420 sec 480 sec 540 sec 600 sec Figure S8. Fluorescence images of BisCMNB-FL AM (20 µm)-loaded HeLa cells after UV light illumination for the period specified in each image. S15

16 (A) Absorbance Wavelength (nm) x1/10 x1/20 x1/40 (B) Fluorescence Intensity (a.u.) Wavelength (nm) 60 [sec] Figure S9. (A) Absorption spectra of TG-NPE-dextran conjugate in 100 mm sodium phosphate buffer, ph 7.4. riginal stock solution eluted from a PD-10 column was diluted to 1/10, 1/20, and 1/40 respectively. (B) Change of fluorescence spectra of TG-NPE-dextran conjugate (1/40 diluted solution) upon UV irradiation ( nm, 2.37 mw/cm 2 at 350 nm). S16

17 DIC (Before) Injection + Irr Irradiation sec sec 20 sec 30 sec 40 sec 50 sec 60 sec Figure S10. Fluorescence images of TG-NPE-dextran conjugate (original stock solution)-injected HeLa cells after UV light illumination for the period specified in each image. S17

18 (A) Remaining caged substrate (%) (C) TG-NPE BisCMNB-FL 85 y = x R 2 = y = x R 2 = Irradiation time (sec) (B) Absorbance TG-NPE BisCMNB-FL Wavelength (sec) Φ TG NPE uncage = ε ε uncaging quantum efficiency TG-NPE 0.03 BisCMNB-FL 0.13 a: Determined by following equation BisCMNB FL 270nm TG NPE 270nm k k TG NPE BisCMNB FL Φ a b BisCMNB FL uncage k TG-NPE = k BisCMNB-FL = TG NPE ε 270 nm = BisCMNB FL ε 270 nm = b: See SR2. Figure S11. (A) Remaining caged substrate (TG-NPE, BisCMNB-FL) versus irradiation time plot. (B) Absorption spectra ( nm) of TG-NPE and BisCMNB-FL (1 µm) in 100 mm sodium phosphate buffer, ph7.4. (C) Uncaging quantum efficiency of TG-NPE. S18

19 Syntheses of caged TokyoGreen and caged fluorescein derivatives H CH C C a b c H CH N 2 N MonoNB-FL Scheme S1. Synthesis of MonoNB-FL (a) tert-buh, conc. H 2 S 4, reflux. (b) 2-Nitrobenzyl bromide, Cs 2 C 3, DMF, r.t. (c) TFA, CH 2 Cl 2, r.t. H BnC H TBDMS Br Me a Me Br Me b c H H Me BnC HC d R Me e R Me R = Me N 2 N 2 Me Me N 2 NB NPE DMNB 6 (R = NB) 7 (R = NPE) 8 (R = DMNB) TG-NB (R = NB) TG-NPE (R = NPE) TG-DMNB (R = DMNB) SchemeS2. Syntheses of caged TokyoGreens (a) TBDMS-Cl, imidazole, DMF, r.t. (b) (i) tert-buli, -78 r.t. (ii) 3,6-Bis-(tert-butyldimethylsilanyloxy)xanthen-9-one, THF, -78 r.t. (iii) 4N HCl aq., r.t. (c) Benzyl bromoacetate, Cs 2 C 3, DMF, r.t. (d) corresponding nitrobenzyl bromide, Cs 2 C 3, DMF, r.t. (e) THF/ NaH aq., 70 HC MAC Me Me AM-Br, DIEA DMF, rt Me Me N 2 TG-NPE N 2 TG-NPE AM KC CK AM-Br, DIEA DMF, rt MAC CAM N 2 2 N N 2 2 N BisCMNB-FL SchemeS3. Syntheses of AM esters AM = BisCMNB-FL AM S19

20 Synthesis of 2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid tert-butyl ester (1) Fluorescein 1.13 g (3.4 mmol) was suspended in tert-buh liquid (50 ml) in a round-bottomed flask, and conc. H 2 S 4 (5 ml) was added dropwise. The flask containing the mixture was equipped with a dropping funnel filled with activated Molecular Sieves 4A to trap generated H 2 and a reflux condenser. The suspended mixture was heated to reflux at 120 and stirred for 14 hours at this temperature, then cooled to room temperature and evaporated to a small volume. The residue was poured into ice water (50 ml), and a yellow solid precipitated. The precipitate was corrected by filtration and washed with H 2, then dissolved in AcEt (50 ml). The organic solution was washed with brine, dried over anhydrous Na 2 S 4, and filtered. The filtrate was added to 5 g of silica gel and evaporated to dryness. The residue was purified on a silica gel column (AcEt/MeH=100/0 95/5) to afford compound 1 76 mg (0.20 mmol, 5.8%, orange solid). 1 H-NMR (CDCl 3 /CD 3 D=80/20, 400 MHz) δ: 1.05 (s, 9H), 6.71 (dd, 2H, J = 9.0 Hz, 2.0 Hz), 6.76 (d, 2H, J = 2.0 Hz), 7.00 (d, 2H, J = 9.3 Hz), 7.29 (dd, 1H, J = 6.8 Hz, 1.5 Hz), (m, 2H), 8.19 (dd, 1H, J = 7.3 Hz, 1.5 Hz). 13 C-NMR (CDCl 3 /CD 3 D=80/20, 100 MHz) δ: 27.0, 82.2, 103.4, 115.1, 121.5, 129.6, 129.8, 130.4, 130.8, 131.9, 132.1, 133.0, 154.1, 157.2, 164.9, HRMS (ESI + ): m/z calcd for M+H; found; (-2.37 mmu). S20

21 Synthesis of 2-[6-(2-nitrobenzyloxy)-3-oxo-3H-xanthen-9-yl]benzoic acid tert-butyl ester (2) Compound mg (0.16 mmol), 2-nitrobenzyl bromide 34.7 mg (0.16 mmol) and Cs 2 C mg (0.19 mmol) were suspended in DMF (1 ml). The mixture was stirred for 26 hours under an Ar atmosphere at room temperature, then poured into CH 2 Cl 2 (30 ml). The organic solution was washed with H 2 (100 ml x 3), dried over anhydrous Na 2 S 4, and filtered. The filtrate was added to a small amount of silica gel and evaporated to dryness. The residue was purified on a silica gel column (AcEt/n-hexane=70/30 100/0) to afford compound mg (0.090 mmol, 57%, orange solid). 1 H-NMR (CDCl 3, 300 MHz) δ: 1.08 (s, 9H), 5.61 (s, 2H), 6.46 (d, 1H, J = 1.8 Hz), 6.57 (dd, 1H, J = 9.6 Hz, 1.9 Hz), 6.87 (dd, 1H, J = 8.8 Hz, 2.4 Hz), (m, 2H), 7.06 (d, 1H, J = 2.4 Hz), (m, 1H), 7.55 (t, 1H, J = 7.8 Hz), (m, 3H), 7.83 (d, 1H, J = 7.7 Hz), (m, 2H). 13 C-NMR (CDCl 3, 75 MHz) δ: 27.4, 67.4, 82.1, 101.6, 105.9, 113.3, 115.7, 118.0, 125.3, 128.4, 128.9, 129.5, 129.7, 130.1, 130.1, 130.4, 131.1, 132.0, 132.2, 132.6, 133.2, 134.1, 147.0, 150.1, 154.1, 158.7, 162.3, 164.8, HRMS (ESI + ): m/z calcd for M+H; found; (-3.07 mmu). S21

22 Synthesis of 2-[6-(2-nitrobenzyloxy)-3-oxo-3H-xanthen-9-yl]benzoic acid (MonoNB-FL) Compound mg (29.2 µmol) was dissolved in CH 2 Cl 2 (1 ml) and TFA (1 ml), and the solution was stirred for 3.5 hours at room temperature. After confirmation of disappearance of the starting materials, the mixture was evaporated to dryness. The residue was purified by means of preparative reverse-phase HPLC to give MonoNB-FL 4.37 mg (9.4 µmol, 32%, light yellow solid). 1 H-NMR (DMS-d 6, 300 MHz) δ: 5.53 (s, 2H), 6.56 (m, 2H), (m, 2H), 6.78 (dd, 1H, J = 8.9 Hz, 2.5 Hz), 7.03 (d, 1H, J = 2.4 Hz), 7.28 (d, 1H, J = 7.5 Hz), (m, 1H), (m, 4H), 8.00 (d, 1H, J = 7.0 Hz), 8.13 (d, 1H, J = 7.9 Hz), 10.2 (br, 1H). 13 C-NMR (DMS-d 6, 100 MHz) δ: 66.7, 82.6, 101.8, 102.2, 109.4, 111.7, 112.4, 112.8, 124.0, 124.7, 124.9, 126.0, 129.1, 129.1, 129.2, 129.3, 130.2, 131.9, 134.0, 135.7, 147.5, 151.7, 151.8, 152.4, 159.5, 159.5, HRMS (ESI + ): m/z calcd for M+H; found; (-0.71mmu). Synthesis of (4-bromo-3-methylphenoxy)-tert-butyldimethylsilane (3) 4-Bromo-3-methylphenol 998 mg (5.3 mmol) and imidazole 1.85 g (27 mmol) were dissolved in DMF (2 ml), and then a DMF (3 ml) solution of TBDMS-Cl 1.24 g (8.2 mmol) was added dropwise with stirring at room temperature. The mixture was stirred for 1.5 hours under an Ar atmosphere at room S22

23 temperature. After confirmation of the disappearance of the starting materials, CH 2 Cl 2 (50 ml) was poured into the reaction mixture. The organic solution was washed with H 2 (100 ml), 0.1 N NaH aqueous solution (50 ml) and brine (30 ml). The organic layer was dried over anhydrous Na 2 S 4, filtered and evaporated to afford a colorless oil. This crude product was purified on a silica gel column (n-hexane 100%) to afford compound g (5.4 mmol, quant., colorless oil). 1 H-NMR (CDCl 3, 300 MHz) δ: 0.18 (s, 6H), 0.97 (s, 9H), 2.32 (s, 3H), 6.54 (dd, 1H, J = 8.6 Hz, 2.8 Hz), 6.72 (d, 1H, J = 2.8 Hz), 7.33 (d, 1H, J = 8.6 Hz). 13 C-NMR (CDCl 3, 75 MHz) δ: - 4.5, 18.2, 23.0, 25.6, 116.2, 119.1, 122.6, 132.8, 138.8, LRMS (EI + ): m/z 300 : 302 = 1 : 1 [M + ]. Synthesis of 6-hydroxy-9-(4-hydroxy-2-methylphenyl)xanthen-3-one (4) Compound g (5.3 mmol) in THF (5 ml) was cooled to 78 on a dry ice/acetone bath, and 1.5 M tert-buli in n-pentane solution 4.2 ml (6.4 mmol) was added dropwise under an Ar atmosphere. Stirring was continued for 5 min at 78, then the reaction mixture was gradually warmed to room temperature over 30 min. The prepared lithium reagent was added dropwise to a THF (6 ml) solution of xanthone ditbdms ether 1.22 g (2.7 mmol) at 78 under an Ar atmosphere and the mixture was stirred for 5 min at 78, then warmed gradually to room temperature over 20 min. Next, 4 N HCl aqueous solution (5 ml) was poured into the solution and the whole was stirred for 30 min at S23

24 room temperature in order to deprotect TBDMS groups. Then, resultant yellow suspension was alkalized with 2 N NaH aqueous solution (20 ml), affording a red solution. Saturated NaH 2 P 4 aqueous solution (5 ml) was added to acidify the solution (about ph 4). The solution was then evaporated to remove THF and n-pentane, yielding an orange precipitate. This precipitate was extracted with AcEt (300 ml) and the organic layer was evaporated to afford a crude product, which was recrystallized from AcEt/MeH (200 ml/50 ml) to afford compound mg. The filtrate after recrystallization was evaporated to dryness and purified on a silica gel column (AcEt/MeH=90/10) to afford compound mg. The two lots of 4 were confirmed to be as the same compound by NMR and MS spectral examination. Thus, compound mg (2.4 mmol, 88%, orange solid) was obtained. 1 H-NMR (CD 3 D and a drop of 40% KD in D 2 solution, 300 MHz) δ: 1.89 (s, 3H), 6.49 (d, 2H, J = 2.2 Hz), 6.55 (dd, 2H, J = 9.4 Hz, 2.2 Hz), 6.61 (dd, 1H, J = 8.3 Hz, 2.4 Hz), 6.66 (d, 1H, J = 2.4 Hz), 6.80 (d, 1H, J = 8.3 Hz), 7.15 (d, 2H, J = 9.4 Hz). 13 C-NMR (CD 3 D and a drop of 40% KD in D 2 solution, 75 MHz) δ: 20.2, 104.4, 113.7, 117.6, 119.2, 121.8, 124.0, 131.3, 132.8, 137.6, 160.3, 160.4, 170.0, HRMS (ESI + ): m/z calcd for M+H; found; (-3.46 mmu). Synthesis of [4-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-3-methylphenoxy]acetic acid benzyl ester (5) S24

25 Compound mg (1.0 mmol) and Cs 2 C g (4.6 mmol) were suspended in DMF (10 ml) and benzyl bromoacetacetate 231 mg (1.0 mmol) in DMF (1 ml) was added with stirring. The reaction mixture was stirred for 11 hours at room temperature under an Ar atmosphere, then saturated NaH 2 P 4 aqueous solution (40 ml) and H 2 (30 ml) were added to acidify the solution. Next, AcEt (150 ml) was added. The organic layer was washed with brine, dried over anhydrous Na 2 S 4, and evaporated to dryness. The residue was purified on a silica gel column (CH 2 Cl 2 /MeH=97/3) to afford compound mg (0.62 mmol, 62%, orange solid). 1 H-NMR (CDCl 3 /CD 3 D=90/10, 300 MHz) δ: 2.01 (s, 3H), 4.78 (s, 2H), 5.30 (s, 2H), 6.70 (dd, 2H, J = 9.2 Hz, 2.2 Hz), 6.73 (d, 2H, J = 2.2 Hz), 6.89 (dd, 1H, J = 8.4 Hz, 2.4 Hz), 6.93 (d, 1H, J = 2.4 Hz), 7.02 (d, 2H, J = 9.2 Hz), 7.07 (d, 1H, J = 8.4 Hz), 7.39 (m, 5H). 13 C-NMR (CDCl 3 /CD 3 D=80/20, 75 MHz) δ: 19.6, 65.1, 67.0, 103.5, 111.9, 115.4, 116.6, 121.6, 125.5, 128.2, 128.4, 130.1, 130.7, 134.8, 137.8, 152.9, 157.4, 158.4, 168.7, HRMS (ESI + ): m/z calcd for M+H; found; (+1.92 mmu). Synthesis of {3-methyl-4-[6-(2-nitrobenzyloxy)-3-oxo-3H-xanthen-9-yl]phenoxy}acetic acid benzyl ester (6) S25

26 Compound 5 70 mg (0.15 mmol), 2-nitrobenzyl bromide 36 mg (0.16 mmol) and Cs 2 C 3 51 mg (0.16 mmol) were suspended in DMF (1 ml). The suspension was stirred for 2 hours under an Ar atmosphere at room temperature, then poured into CH 2 Cl 2 (30 ml). The organic solution was washed with H 2 (50 ml x 3) and brine. The organic layer was dried over anhydrous Na 2 S 4, and filtered. The filtrate was added to 1 g of silica gel and evaporated to dryness, and the residue was purified on a silica gel column (CH 2 Cl 2 /MeH=100/0 99/1) to afford compound mg (0.16 mmol, quant., orange oil). 1 H-NMR (400 MHz, CDCl 3 ) δ: 2.03 (s, 3H), 4.78 (s, 2H), 5.29 (s, 2H), 5.60 (s, 2H), 6.40 (d, 1H, J = 2.0 Hz), 6.55 (dd, 1H, J = 9.8 Hz, 2.0 Hz), (m, 2H), (m, 2H), (m, 2H), 7.08 (d, 1H, J = 8.3 Hz), (m, 5H), (m, 1H), 7.73 (td, 1H, J = 7.7 Hz, 1.1 Hz), 7.85 (d, 1H, J = 7.3 Hz), 8.20 (dd, 1H, J = 8.3 Hz, 1.0 Hz). 13 C-NMR (100 MHz, CDCl 3 ) δ: 19.8, 65.1, 66.9, 67.3, 101.5, 105.6, 112.0, 113.3, 115.0, 116.6, 118.8, 125.1, 125.4, 128.3, 128.4, 128.5, 128.5, 128.8, 129.6, 130.0, 130.3, 130.4, 131.2, 134.1, 134.9, 138.0, 146.7, 148.7, 154.2, 158.4, 158.6, 162.3, 168.4, HRMS (ESI + ): m/z calcd for M+H; found; (+0.44 mmu). Synthesis of {3-methyl-4-[6-(2-nitrobenzyloxy)-3-oxo-3H-xanthen-9-yl]phenoxy}acetic acid (TG-NB) S26

27 Compound mg (0.17 mmol) was dissolved in THF (9 ml)/ 2 N NaH aqueous solution (1 ml) and the solution was heated to 70 for 30 min, with stirring. Then, saturated NaH 2 P 4 aqueous solution (5 ml) was added to the mixture, and the THF was evaporated. The remaining solution was extracted with AcEt after acidification with 2 N HCl aqueous solution. The organic layer was washed with brine, dried over anhydrous Na 2 S 4, filtered and evaporated to dryness. The residue was purified by means of reverse-phase preparative HPLC (MeH/0.2 M triethylamine-acetic acid buffer=10/90 100/0 over 30 min, flow: 5.0 ml/min) to afford TG-NB 26.4 mg (0.052 mmol, 31%, orange solid). 1 H-NMR (DMS-d 6, 300 MHz) δ: 1.99 (s, 3H), 4.77 (s, 2H), 5.67 (s, 2H), (m, 1H), (m, 1H), (m, 2H), (m, 3H), 7.21 (d, 1H, J = 8.4 Hz), 7.41 (s, 1H) (m, 1H), (m, 2H), 8.16 (d, 1H, J = 7.9 Hz). 13 C-NMR (DMS-d 6, 100 MHz) δ: 19.4, 64.5, 67.6, 100.1, 101.6, 104.2, 112.2, 115.1, 116.5, 118.0, 124.4, 125.1, 128.2, 129.4, 129.6, 130.0, 130.5, 131.2, 134.2, 137.5, 147.5, 154.8, 157.9, 158.3, 158.7, 163.6, 170.0, 181.9, HRMS (ESI + ): m/z calcd for M+H; found; (+3.85 mmu). Synthesis of (3-methyl-4-{6-[1-(2-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl}phenoxy)acetic acid benzyl ester (7) S27

28 Compound mg (0.30 mmol), 1-(1-bromoethyl)-2-nitrobenzene 77 mg (0.33 mmol) and Cs 2 C mg (0.33 mol) were suspended in DMF (2 ml). The suspension was stirred at room temperature under an Ar atmosphere for 15 hours, then CH 2 Cl 2 (50 ml) and H 2 (150 ml) were added. The organic layer was separated, washed with H 2 (150 ml x 3), dried over anhydrous Na 2 S 4, and filtered. The filtrate was added to 2 g of silica gel and evaporated to dryness. The residue was purified on a silica gel column (CH 2 Cl 2 100%) to afford compound mg (0.27 mmol, 90%, orange solid). 1 Η NMR (CDCl 3, 300 MHz) δ: 1.76 (d, 3H, J = 6.2 Hz), 1.98 (s, 3H), 4.75 (s, 2H), 5.28 (s, 2H), 6.19 (q, 1H, J = 6.4 Hz), (m, 1H), 6.54 (dd, 1H, J = 9.7 Hz, 2.0 Hz), (m, 1H), (m, 5H), 7.02 (d, 1H, J = 8.3 Hz), (m, 5H), (m, 1H), 7.63 (t, 1H, J = 7.6 Hz), 7.73 (d, 1H, J = 7.9 Hz), 8.06 (d, 1H, J = 8.3 Hz). 13 C-NMR (CDCl 3, 75 MHz) δ: 19.8, 23.3, 23.3, 53.4, 65.2, 67.0, 72.2, 72.2, 102.3, 102.5, 105.7, 112.0, 112.1, 113.4, 113.7, 114.9, 116.7, 116.7, 118.8, 118.8, 124.9, 125.5, 127.0, 128.3, 128.5, 128.8, 129.6, 130.0, 130.3, 130.3, 130.5, 134.2, 135.0, 137.5, 137.6, 138.0, 147.2, 148.7, 154.2, 154.2, 158.4, 158.6, 161.6, 161.6, 168.4, HRMS (ESI + ): m/z calcd for M+H; found; (-2.28 mmu). Synthesis of (3-methyl-4-{6-[1-(2-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl}phenoxy)acetic acid (TG-NPE) A mixture of compound mg (0.16 mmol) in THF (5 ml) and 0.2 N NaH aqueous solution (5 S28

29 ml) was heated to 70 for 5 min, with stirring. The reaction mixture was acidified with saturated NaH 2 P 4 aqueous solution and evaporated to remove THF. The residue was taken up in 2 N HCl aqueous solution (5 ml) and extracted with CH 2 Cl 2 (100 ml). The organic layer was washed with brine, dried over anhydrous Na 2 S 4, and filtered. The filtrate was added to 1 g of silica gel and evaporated to dryness. The residue was purified on a silica gel column (CH 2 Cl 2 /MeH=100/0 93/7) to afford a brown oil, which was further purified by means of reverse -phase HPLC (CH 3 CN/H 2 (0.1%TFA)=30/70 100/0 over 60 min, flow: 5.0 ml/min) to afford compound TG-NPE 40 mg (0.076 mmol, 49%, orange solid). 1 H-NMR (300 MHz, DMS-d 6 ) δ: 1.71 (d, 3H, J = 6.2 Hz), 1.95 (s, 3H), 4.76 (s, 2H), 6.18 (q, 1H, J = 6.4 Hz), 6.32 (d, 1H, J = 1.7 Hz), 6.52 (d, 1H, J = 9.7 Hz), (m, 5H), (m, 2H), (m, 1H), (m, 2H), 8.06 (d, 1H). 13 C-NMR (100 MHz, DMS-d 6 ) δ: 19.4, 22.4, 22.4, 64.5, 72.4, 102.0, 102.2, 104.2, 112.1, 112.2, 115.2, 115.4, 115.8, 116.5, 118.0, 124.3, 124.8, 127.3, 127.9, 129.5, 130.2, 130.5, 131.3, 134.3, 136.0, 136.1, 137.4, 137.4, 147,4, 147,5, 154.7, 158.7, 158.7, 162.6, HRMS (ESI + ): m/z calcd for M+H; found; (-0.26 mmu). Synthesis of {4-[6-(4, 5-dimethoxy-2-nitrobenzyloxy)-3-oxo-3H-xanthen-9-yl]-3-methylphenoxy}acetic acid benzyl ester (8) S29

30 Compound mg (0.30 mmol), 4,5-dimethoxy-2-nitrobenzyl bromide 82 mg (0.30 mmol) and Cs 2 C 3 98 mg (0.30 mmol) were suspended in DMF (2 ml). The mixture was stirred for 15 hours under an Ar atmosphere at room temperature. Compound 8 was confirmed to be present by TLC, so more 4,5-dimethoxy-2-nitrobenzyl bromide 81 mg (0.30 mmol) was added and the reaction mixture was heated to 50 for 30 min, with stirring, then cooled to room temperature. CH 2 Cl 2 (50 ml) was added and the organic solution was washed with H 2 (100 ml x 3). The organic layer was dried over anhydrous Na 2 S 4 and filtered. The filtrate was added to 2 g of silica gel and evaporated to dryness. The residue was purified on a silica gel column (CH 2 Cl 2 /MeH=100/0 98/2) to afford compound mg (0.29 mmol, 98%, orange solid). 1 H-NMR (300 MHz, CDCl 3 ) δ: 2.04 (s, 3H), 3.98 (s, 3H), 3.98 (s, 3H), 4.76 (s, 2H), 5.29 (s, 2H), 5.59 (s, 2H), 6.42 (d, 1H, J = 2.0 Hz), 6.55 (dd, 1H, J = 9.7 Hz, 1.8 Hz), (m, 4H), (m, 3H), 7.26 (s, 1H), (m, 5H), 7.79 (s, 1H). 13 C-NMR (75 MHz, CDCl 3 ) δ: 19.8, 56.3, 56.4, 65.2, 67.0, 67.6, 101.8, 105.7, 108.2, 109.4, 112.1, 113.2, 115.2, 116.7, 118.9, 125.5, 127.2, 128.3, 128.5, 128.5, 129.6, 130.1, 130.3, 130.5, 135.0, 138.0, 139.2, 148.2, 148.7, 153.9, 154.3, 158.5, 158.7, 162.4, 168.4, HRMS (ESI + ): m/z calcd for M+H; found; (-1.43 mmu). Synthesis of {4-[6-(4, S30

31 5-dimethoxy-2-nitrobenzyloxy)-3-oxo-3H-xanthen-9-yl]-3-methylphenoxy}acetic acid (TG-DMNB) Compound mg (0.20 mmol) in THF (9 ml) was added 2 M K 2 C 3 aqueous solution (1 ml). The mixture was heated to 70 for 30 min, with stirring, then, more 2 M K 2 C 3 aqueous solution (8 ml) was added, and stirring was continued for 60 min at 70. Next, the reaction mixture was heated to reflux at 100 for 60 min, but hydrolysis was not completed. Therefore, the mixture was cooled to room temperature, 2 N NaH aqueous solution (2 ml) was added, and the whole was stirred for 30 min. It was next poured into saturated NaH 2 P 4 aqueous solution and evaporated to remove THF. The residue was taken up in 2 N HCl aqueous solution (15 ml) and extracted with AcEt (200 ml). The organic layer was dried over anhydrous Na 2 S 4, filtered and evaporated to afford a brown oil. This was dissolved in DMS (500 µl), and upon addition of CH 3 CN (2 ml) and H 2 (2 ml) containing 0.1% TFA, an orange solid was deposited. The deposit was collected by filtration and identified as TG-DMNB 61 mg (0.11 mol, 53%, orange solid). It was purified by means of reverse-phase HPLC (CH 3 CN/H 2 (0.1%TFA)=30/70 100/0 over 30 min, flow: 5.0 ml/min) for use in photoirradiation experiments. 1 H-NMR (300 MHz, DMS-d 6 ) δ: 2.06 (s, 3H), 3.88 (s, 3H), 3.88 (s, 3H), 4.77 (s, 2H), 5.58 (s, 2H), 6.31 (d, 1H, J = 1.7 Hz), 6.51 (dd, 1H, J = 9.7 Hz, 1.9 Hz), (m, 5H), 7.21 (d, 1H, J = 8.4 Hz), S31

32 7.34 (s, 1H), 7.40 (d, 1H, J = 2.2 Hz), 7.74 (s, 1H). 13 C-NMR (75 MHz, DMS-d 6 ) δ: 19.4, 56.1, 56.3, 64.5, 67.5, 101.7, 104.7, 108.4, 111.7, 112.1, 113.9, 114.5, 116.4, 117.9, 124.5, 125.9, 129.4, 129.6, 130.4, 130.6, 137.4, 139.9, 148.1, 148.8, 153.1, 153.8, 158.3, 158.5, 162.6, 170.0, HRMS (ESI + ): m/z calcd for M+H; found; (-3.14 mmu). Synthesis of (3-methyl-4-{6-[1-(2-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl}phenoxy)acetic acid acetoxymethyl ester (TG-NPE AM) TG-NPE 17.4 mg (33 µmol) was dissolved in DMF (0.5 ml), then a DMF (0.5 ml) solution of bromomethyl acetate 51.9 mg (34 µmol) and diisopropylethylamine 58.4 mg (45 µmol) was added dropwise. The mixture was stirred under an Ar atmosphere at room temperature for 1 hour. CH 2 Cl 2 (100 ml) was poured into the reaction mixture, and the organic solution was washed with H 2 (100 ml x 4). The organic layer was dried over anhydrous Na 2 S 4, and filtered. The filtrate was added to 1 g of silica gel and evaporated to dryness. The residue was purified on a silica gel column (CH 2 Cl 2 /MeH=95/5) to afford TG-NPE AM 14.5 mg (24 µmol, 74%, orange solid). 1 H-NMR (CDCl 3, 300 MHz) δ: 1.76 (d, 3H, J = 6.2 Hz), 2.01 (s, 3H), 2.14 (s, 3H), 4.76 (s, 2H), 5.88 (s, 2H), 6.19 (q, 1H, J = 6.1 Hz), 6.40 (s, 1H), 6.55 (dd, 1H, J = 9.7 Hz, 1.5 Hz), (m, 1H), (m, 5H), 7.04 (d, 1H, J = 8.3 Hz), 7.47 (t, 1H, J = 7.6 Hz), 7.63 (t, 1H, J = 7.5 Hz), 7.72 (d, S32

33 1H, J = 7.7 Hz), 8.07 (d, 1H, J = 8.1 Hz). 13 C-NMR (CDCl 3, 75 MHz) δ: 19.9, 20.6, 23.4, 64.9, 72.3, 79.5, 102.5, 102.7, 105.9, 112.1, 113.4, 113.7, 115.0, 116.9, 119.0, 125.1, 125.9, 127.1, 128.8, 129.6, 130.2, 130.4, 130.5, 134.3, 137.7, 138.2, 147.3, 148.6, 154.3, 158.3, 158.7, 161.7, 167.5, 169.4, HRMS (ESI + ): m/z calcd for M+H; found; (-4.71 mmu). Synthesis of bis-cmnb-caged fluorescein AM ester (BisCMNB-FL AM) BisCMNB-FL 8.0 mg (9.7 µmol) was dissolved in DMF (2 ml), and a DMF (0.5 ml) solution of bromomethyl acetate 17.2 mg (112 µmol) and diisopropylethylamine 18.1 mg (140 µmol) was added dropwise. The mixture was stirred under an Ar atmosphere at room temperature for 1 hour. CH 2 Cl 2 (60 ml) was poured into the reaction mixture, and the organic solution was washed with H 2 (100 ml x 4). The organic layer was dried over anhydrous Na 2 S 4, filtered and evaporated to afford a light yellow oil. This oil was dissolved in a small amount of CH 2 Cl 2 and subjected to preparative TLC (CH 2 Cl 2 /MeH = 99/1) to afford BisCMNB-FL AM 6.5 mg (7.3 µmol, 75%, colorless solid). 1 H-NMR (300 MHz, CDCl 3 ) δ: 2.11 (s, 6H), 4.77 (s, 4H), 5.54 (s, 4H), 5.80 (s, 4H), 6.74 (d, 4H, J = 1.1 Hz), 6.87 (s, 2H), 6.93 (dd, 2H, J = 9.2 Hz, 2.9 Hz), 7.20 (d, 1H, J = 7.2 Hz), 7.36 (d, 2H, J = 2.8 Hz), (m, 2H), 8.04 (d, 1H, J = 7.0 Hz), 8.27 (d, 2H, J = 9.2 Hz). 13 C-NMR (75 MHz, CDCl 3 ) δ: 20.6, 64.9, 67.2, 79.6, 102.2, 111.9, 112.2, 112.3, 113.6, 113.8, 124.0, 125.1, 126.7, 128.0, 129.4, S33

34 129.8, 135.1, 136.7, 140.5, 152.5, 152.9, 159.6, 161.9, 166.5, 169.3, HRMS (ESI + ); m/z calcd for M+Na; found; (+2.63 mmu). Synthesis of TG-NPE Succinimidyl ester (TG-NPE SE) TG-NPE 28.6 mg (0.054 mmol) and N-hydroxysuccinmide 12.7 mg (0.11 mmol) were dissolved in DMF (1 ml) in a 10 ml round-bottomed flask, and WSCD HCl 21.3 mg (0.11 mmol) was added at once. The mixture was stirred at room temperature for 19 hours under an Ar atmosphere. After confirmation of disappearance of starting materials, the reaction mixture was poured into CH 2 Cl 2 (200 ml). The organic layer was washed with 10% citric acid aqueous solution (100 ml x 3), and brine (100 ml), dried over anhydrous Na 2 S 4, filtered and evaporated to dryness. The residue was purified with silica gel column chromatography (CH 2 Cl 2 100% AcEt100%) to afford TG-NPE SE (7.44 mg, 22%, yellow solid). 1 H-NMR (300 MHz, CDCl 3 ) δ: 1.76 (d, 3H, J = 6.2 Hz), 2.03 (s, 3H), 2.90 (s, 4H), 5.06 (s, 2H), 6.20 (q, J = 6.4 Hz), 6.45 (dd, 1H, J = 1.9 Hz, 0.6 Hz), 6.59 (dd, 1H, J = 9.7 Hz, 2.0 Hz), 6.73 (dt, 1H, J = 8.7 Hz, 2.5 Hz), 6.83 (dd, 1H, J = 6.8 Hz, 2.4 Hz), (m, 4H), 7.07 (d, 1H, J = 8.4 Hz), 7.48 (t, 1H, J = 7.7 Hz), 7.63 (t, 1H, J = 7.6 Hz), 7.72 (d, 1H, J = 8.1 Hz), 8.08 (dt, 1H, J = 8.1 Hz, 1.7 Hz). S34

35 13 C-NMR (75 MHz, CDCl 3 ) δ: 19.9, 23.4, 25.4, 25.6, 63.4, 72.4, 102.6, 102.7, 105.7, 112.5, 113.7, 113.9, 115.0, 116.9, 118.8, 125.1, 126.3, 127.1, 128.9, 129.8, 130.0, 130.6, 130.7, 134.3, 137.6, 137.7, 138.4, 147.3, 149.4, 154.4, 158.0, 159.0, 161.9, 164.5, 168.5, 171.8, HRMS (ESI + ); m/z calcd for M+H; found; (+1.92 mmu). (SR1) Papadopoulos, N. G.; Dedoussis, G. V. Z.; Spanakos, G.; Gritzapis, A. D.; Baxevanis, C. N.; Papamichail, M. J. Immunol. Methods 1994, 177, (SR2) Ahern, G. P., Hsu, S. F., Jackson, M. B., J. Physiol. 1999, 520 (Pt. 1), S35