Donor-acceptor-donor fluorene derivatives for two-photon fluorescence lysosomal imaging.

General 9,9-Didecyl-9 H -fluorene-2,7-dicarbaldehyde ( 9 ), 29 (4-(dibutylamino)benzyl) triphenylphosphonium iodide ( 11 ), 30 diethyl 4-(benzo[ d ]thiazol-2-yl)benzylphosphonate ( 12 ), 31 7-bromo-9,9-didecyl-9 H -fluorene-2-carbaldehyde ( 13 ), 32 4-vinyltriphenylamine ( 14 ), 33 1-(bromomethyl)-4-(phenylsulfonyl)benzene ( 16 ), 22 and N,N-di- n -butyl-4-vinylaniline ( 20 ), 34 were prepared according to literature methods. A new route was used for the synthesis of 3 which was different from that previously reported, 35 and the 1 H NMR spectrum was consistent with the previously reported spectrum. 2, 5-Diaminobenzene-1, 4-dithiol ( 22 ) was a gift from the Air Force Research Laboratory/Polymer Branch at Wright-Patterson Air Force Base. 4-(Chloromethyl)benzoyl chloride ( 17 ) was purchased from Acros Organics. Pluronic™ F 108NF was obtained from BASF. LysoTracker® Red was purchased from Invitrogen. All other reagents and solvents were used as received from commercial suppliers. Melting points are uncorrected. 1 H NMR and 13 C NMR spectra were recorded at either 300 or 500 MHz and at 75 or 125 MHz, respectively. Absorption spectra were measured with an UV-visible spectrophotometer. Steady state fluorescence spectra were obtained at room temperature with a spectrofluorimeter using 10 mm quartz cuvettes. Fluorescence anisotropy spectra were measured using three polarizers in the L-format method, with correction for background signals, in the high viscosity solvent polytetrahydrofuran (pTHF) at room temperature. Experimental details of anisotropy measurements were previously reported. 36 , 37 Fluorescence quantum yields were measured for all compounds by a standard method, 38 relative to Rhodamine 6G in ethanol at room temperature. Preparation of 9, 9–didecyl-4, 5–dinitro-9 H –fluorene-2, 7-dicarbaldehyde (10) A mixture of fuming HNO 3 (8 mL, d = 1.42 g/cm 3 ) and 98% H 2 SO 4 was cooled to 0 °C. 9, 9-Didecyl-9 H -fluorene-2, 7-dicarbaldehyde ( 9 , 2.0 g, 3.4 mmol) was then added slowly and the resulting mixture was stirred at 0 °C for 1.5 h. The reaction was terminated by addition of 20 g ice. The organic phase was separated, and the aqueous phase was extracted with hexane. The combined organic phase was then washed with water and dried over MgSO 4 . Solvent was removed under reduced pressure, and the crude product was purification via column chromatography (4:3 hexanes/CH 2 Cl 2 ), affording 0.96 g of off-white solid (41% yield) m.p. 57–58 °C. 1 H NMR (300 MHz, CDCl 3 ) δ 10.09 (s, 2H), 8.36 (s, 2H), 8.09 (s, 2H), 2.10 (m, 4H), 1.16-0.95 (m, 28H), 0.76 (t, J = 6.8 Hz, 6H), 0.46 (m, 4H). 13 C NMR (75MHz, DMSO-D 6 ) δ 187.7, 155.3, 145.9, 135.9, 133.0, 124.5, 124.4, 55.7, 38.7, 30.7, 28.5, 28.3, 28.1, 28.0, 22.7, 21.5, 13.0. Anal. Calcd. for C 35 H 48 N 2 O 6 (592.77): C, 70.92; H, 8.16; N, 4.73. Found: C, 71.20; H, 8.12; N, 4.83. General procedure for preparation of dyes 1-5 9,9-Didecyl-9 H -fluorene-2,7-dicarbaldehyde ( 9 ) or 9,9-didecyl-4,5-dinitro-9 H -fluorene-2,7-dicarbaldehyde ( 10 , 0.5 mmol), 4-( N,N -dibutylaminobenzyl) triphenylphosphonium iodide ( 11 ) and/or diethyl 4-(benzo[ d ]thiazol-2-yl) benzylphosphonate ( 12 , 1.0 mmol) in 10 mL of dry DMF was degassed with Ar for 30 min. NaH (0.24 g, 10.0 mmol) was then added, and the mixture was stirred at room temperature under Ar for 20–24 h. The crude product precipitated upon slow addition of water, and was collected by filtration, followed by column chromatographic purification to afford pure compounds 1 - 4 and 7 (characterization details below). 4,4′-(1E,1′E)-2, 2′-(9, 9-Didecyl-9 H -fluorene-2, 7-diyl)bis(ethane-2, 1-diyl)bis( N , N -dibutylaniline) (1) 3:1 Hexanes/CH 2 Cl 2 as eluent (62% yield) m.p. 95 – 96 °C. 1 H NMR (300 MHz, CDCl 3 ) δ 7.56 (d, J = 8.1 Hz, 2H), 7.41–7.36 (m, 8H), 7.06 (d, J = 16.2 Hz, 2H), 6.94 (d, J = 15.9 Hz, 2H), 6.61 (d, J = 9.0 Hz, 4H), 3.27 (t, J = 7.5 Hz, 8H), 1.97 (m, 4H), 1.58 (m, 8H), 1.35 (m, 8H), 1.25–1.05 (m, 28H), 0.95 (t, J = 7.2 Hz, 12H), 0.82 (t, J = 6.6 Hz, 6H), 0.69 (m, 4H). 13 C NMR (75MHz, CDCl 3 ) δ 151.3, 147.6, 139.8, 137.0, 128.0, 127.7, 125.0, 124.8, 124.4, 120.1, 119.6, 111.7, 55.1, 51.0, 40.9, 32.2, 30.4, 30.0, 29.9, 29.8, 29.6, 24.1, 23.0, 20.7, 14.5, 14.4. Anal. Calcd. for C 65 H 96 N 2 (905.47): C, 86.22; H, 10.69; N, 3.09. Found: C, 86.18; H, 10.83; N, 3.16. 4, 4′-(1 E , 1′ E) -2, 2′-(9, 9-didecyl-4, 5-dinitro-9 H -fluorene-2, 7-diyl)bis(ethene-2, 1-diyl) bis( N , N -dibutylaniline) (2) 2:1 Hexanes/CH 2 Cl 2 as eluent (55% yield) m.p. 68 – 69 °C. 1 H NMR (300 MHz, CDCl 3 ) δ 7.98 (s, 2H), 7.56 (s, 2H), 7.42 (d, 4H, J = 8.7 Hz), 7.18 (d, J = 15.9 Hz, 2H), 6.94 (d, J = 16.2 Hz, 2H), 6.65 (d, J = 9.0 Hz, 4H), 3.32 (t, J = 7.2 Hz, 8H), 2.07 (m, 4H), 1.61 (m, 8H), 1.36 (m, 8H), 1.25–1.00 (m, 28H), 0.97 (t, J = 7.4 Hz, 12H), 0.82 (t, J = 6.8 Hz, 6H), 0.63 (m, 4H). 13 C NMR (75 MHz, CDCl 3 ) δ 154.8, 148.6, 146.5, 139.8, 132.5, 129.6, 128.5, 128.5, 123.4, 123.2, 120.9, 111.7, 56.1, 51.2, 40.9, 32.3, 32.1, 30.2, 30.0, 29.9, 29.7, 29.6, 24.0, 23.1, 20.8, 14.6, 14.5. Anal. Calcd. for C 65 H 94 N 4 O 4 (995.47): C, 78.43; H, 9.52; N, 5.63. Found: C, 78.06; H, 9.57; N, 5.44. 2, 2′-(4, 4′-(1 E , 1′ E )-2, 2′-(9, 9-Didecyl-9 H -fluorene-2, 7-diyl)bis(ethene-2, 1-diyl)bis(4, 1-phenylene)) dibenzo[ d ]thiazole 35 (3) 1:2 Hexanes/CH 2 Cl 2 as eluent (81% yield) m.p. 154 – 155 °C (lit. m.p 143 – 144 °C). 35 1 H NMR (500 MHz, CDCl 3 ) δ 8.12 (d, J = 8.0 Hz, 4H), 8.09 (d, J = 8.0 Hz, 2H), 7.93 (d, J = 8.0 Hz, 2H), 7.69 (d + d, 6H), 7.56 (d, J = 8.0 Hz, 2H), 7.52 (m, 4H), 7.41 (t, J = 8.3 Hz, 2H), 7.35 (d, J = 16.0 Hz, 2H), 7.23 (d, J = 16.0 Hz, 2H), 2.05 (t, J = 7.5 Hz, 4H), 1.22–1.08 (m, 28H), 0.82 (t, J = 7.0 Hz, 6H), 0.68 (m, 4H). 2, 2′-(4, 4′-(1 E , 1′ E )-2, 2′-(9, 9-Didecyl-4, 5-dinitro-9 H -fluorene-2, 7-diyl) bis(ethene-2, 1-diyl) bis(4, 1-phenylene)) dibenzo[ d ]thiazole (4) 1:3 Hexanes/CH 2 Cl 2 as eluent (82% yield) m.p. 171 – 172 °C. 1 H NMR (300 MHz, CDCl 3 ) 8.15 – 8.06 (m, 8H), 7.91 (d, 2H, J = 8.4 Hz), 7.69 (m, 6H), 7.50 (d, J = 7.5 Hz, 2H), 7.39 (d, J = 7.5 Hz, 2H), 7.32 (s, 4H), 2.10 (m, 4H), 1.15–1.07 (m, 28H), 0.81 (t, J = 6.6 Hz, 6H), 0.62 (m, 4H). 13 C NMR (75 MHz, CDCl 3 ) δ 167.3, 155.2, 154.2, 146.8, 138.9, 138.6, 135.2, 133.7, 131.4, 129.8, 128.2, 127.8, 127.6, 126.6, 125.5, 124.3, 123.4, 121.8, 56.4, 40.8, 32.3, 30.2, 29.9, 29.7, 29.6, 24.0, 23.1, 14.6. Anal. Calcd. for C 63 H 66 N 4 O 4 S 2 (1007.35): C, 75.11; H, 6.60; N, 5.56. Found: C, 75.24; H, 6.78; N, 5.42. [4-(2-{7-[2-(4-Benzothiazol-2-yl-phenyl)-vinyl]-9, 9-didecyl-9 H -fluoren-2-yl}-vinyl)-phenyl]-dibutyl-amine (7) 1:3 Hexanes/CH 2 Cl 2 as eluent, (43% yield) m.p. 95 – 96 °C. 1 H NMR (300 MHz, CDCl 3 ) δ 8.07 (m, 3H), 7.89 (d, J = 7.8 Hz, 1H), 7.63 (m, 4H), 7.51–7.38 (m, 8H), 7.31 (d, J = 16.8 Hz, 2H), 7.17 (d, J =16.2 Hz, 2H), 7.08 (d, J =16.2 Hz, 2H), 6.95 (d, J =16.2 Hz, 2H), 6.62 (d, J =8.1 Hz, 2H), 3.29 (m, 4H), 2.02 (m, 4H), 1.58 (m, 4H), 1.37 (m, 4H), 1.15–1.06 (m, 28H), 0.97 (t, J = 7.1 Hz, 6H), 0.82 (t, J = 5.9 Hz, 6H), 0.68 (m, 4H). 13 C NMR (75 MHz, CDCl 3 ) δ 154.3, 151.6, 147.9, 141.6, 140.5, 139.5, 137.7, 135.5, 135.1, 132.4, 131.3, 128.5, 128.1, 127.9, 127.0, 126.7, 126.5, 126.1, 125.3, 124.8, 124.4, 123.3, 121.8, 121.1, 120.3, 120.1, 119.9, 111.8, 55.3, 51.2, 41.0, 32.3, 32.1, 30.5, 30.1, 30.0, 30.0, 29.7, 24.3, 23.1, 20.9, 14.6, 14.5. Anal. Calcd. for C 64 H 82 N 2 S (911.42): C, 84.34; H, 9.07; N, 3.07. Found: C, 84.34; H, 9.28; N, 3.08. Preparation of ( E )-9, 9-didecyl-7-(4-(diphenylamino)styryl)-9 H -fluorene-2-carbaldehyde 15 7-Bromo-9,9-didecyl-9 H -fluorene-2-carbaldehyde ( 13 ) (1.384 g, 2.5 mmol), 4-vinyltriphenylamine ( 14 , 0.678 g, 2.5 mmol), PPh 3 (65.56 mg, 0.25 mmol), and K 2 CO 3 (6.25 mmol, 0.864g) in DMF were degassed with Ar for 30 min, then Pd(OAc) 2 (84.20 mg, 0.125 mmol) and n -Bu 4 NCl (0.695 g, 2.5 mmol) were added, followed by refluxing the mixture under Ar for 20 h. Water and hexanes were added and the organic phase was concentrated. The crude product was purified by column chromatography (20:1 hexanes/EtOAc), affording 1.31g of 15 as yellow sticky oil (70% yield). 1 H NMR (500 MHz, CDCl 3 ) δ 10.05 (s, 1H), 7.86 (m, 2H), 7.80 (d, J = 7.5 Hz, 1H), 7.74 (d, J = 7.5 Hz, 1H), 7.48–7.53 (m, 2H), 7.42 (d, J = 9.0 Hz, 2H), 7.25–7.29 (m, 4H), 7.11–7.18 (m, 6H), 7.03–7.08 (m, 4H), 2.03 (m, 4H), 1.03–1.25 (m, 28H), 0.83 (t, J = 7.0 Hz, 6H), 0.62 (m, 4H). 13 C NMR (125 MHz, CDCl 3 ) δ 14.1, 22.7, 23.8, 29.3, 29.5, 29.6, 30.0, 31.9, 40.3, 55.2, 119.8, 120.7, 121.2, 123.0, 123.1, 123.4, 124.6, 125.7, 127.1, 127.4, 128.6, 129.3, 130.7, 131.3, 135.1, 138.4, 138.9, 147.4, 147.5, 147.5, 151.7, 152.8, 192.4. HRMS-(APPI) for C 54 H 65 NO: theoretical m/z [M+H] + = 744.5139, found [M+H] + = 744.5157. Preparation of bis(4-(bromomethyl) phenyl) sulfane 18 Phenyl sulfide 17 (1.86 g, 0.01 mmol) and paraformaldehyde (1.2 g, 0.04 mmol) in 33% HBr/HOAc (10 mL) were heated at 70 °C for 48 h. Water and 2:1 hexanes/EtOAc were added. White solid precipitated and was collected by filtration to afford 2.86 g of 18 (77% yield), m. p. 129 – 130.5 °C (lit. 135.3 – 136.3 °C). 39 1 H NMR (500 MHz, CDCl 3 ) δ 7.29 –7.34 (m, 8H), 4.47 (s, 4H). 13 C NMR (125 MHz, CDCl 3 ) δ 32.9, 129.9, 131.3, 135.8, 136.8. HRMS(DIP-CI) for C 14 H 12 Br 2 S: theoretical m/z [M] + = 369.9026, found [M] + = 369.9008. Preparation of 4, 4′-sulfonylbis((bromomethyl)benzene) 19 Bis(4-(bromomethyl) phenyl)sulfane 18 (0.5 g, 1.34 mmol) was dissolved in HOAc (4 mL) at 70 °C. 30 % H 2 O 2 (1 mL) was slowly added and the mixture was heated at 90 °C for 30 min. The product precipitated after cooling and was collected by filtration, washed with water, and dried, providing 0.37 g of 19 (68% yield), m. p. 145 – 146 °C (lit. 134 – 135 °C). 40 1 H NMR (500 MHz, CDCl 3 ) δ 7.92 (d, J = 5.0 Hz, 4H), 7.53 (d, J = 5.0 Hz, 4H), 4.46 (s, 4H). 13 C NMR (125 MHz, CDCl 3 ) δ 31.3, 128.3, 130.0, 141.1, 143.3. HRMS(DIP-CI) for C 14 H 12 Br 2 O 2 S: theoretical m/z [M+H] + = 402.9003, found [M+H] + = 402.9010. Preparation of dye 5 1-(Bromomethyl)-4-(phenylsulfonyl) benzene ( 16 , 36.2 mg, 0.116 mmol) was refluxed with triethyl phosphite (1 mL) for 2 h. Excess triethyl phosphite was evaporated and the residue was dried in vacuo . ( E )-9,9-didecyl-7-(4-(diphenylamino)styryl)-9 H -fluorene-2-carbaldehyde ( 15 , 86.5 mg, 0.116 mmol) was then mixed with above product in dry DMF (2 mL) under Ar. NaH (27.8 mg, 1.16 mmol) was added and the reaction was stirred at room temperature for 20 h. Water was added to terminate the reaction, and the product was extracted with EtOAc. Solvent was removed and the crude product was purified by column chromatography (1:1 hexanes/CH 2 Cl 2 ), producing 58.6 mg of 5 (53% yield) as a soft glassy material, which turned into viscous liquid upon heating. No defined melting point was observed. 1 NMR (500 MHz, CDCl 3 ) δ 7.88 (m, 4H), 7.57 (m, 4H), 7.52–7.33 (m, 8H), 7.23–7.17 (m, 6H), 7.07–6.95 (m, 11H), 1.93 (m, 4H), 1.18–0.97 (m, 28H), 0.73 (t, J = 5.0 Hz, 6H), 0.58 (m, 4H). 13 C NMR (125 MHz, CDCl 3 ) δ 151.6, 147.5, 147.3, 141.8, 140.0, 139.5, 133.2, 133.1, 131.6, 129.3, 127.6, 127.5, 127.3, 126.9, 126.2, 125.6, 125.5, 124.5, 123.6, 123.0, 121.1, 120.6, 120.1, 119.9, 55.0, 40.5, 31.9, 29.6, 29.6, 29.3, 29.3, 23.8, 22.7, 14.1. HRMS (MALDI-TOF) for C 67 H 75 NO 2 S: theoretical m/z [M+H] + = 958.5591, found [M+H] + = 958.5587. Preparation of dye 6 4, 4′-Sulfonyl bis((bromomethyl)benzene) ( 19 , 0.1 g, 0.247 mmol) was refluxed with triethyl phosphite for 2 h. Excess triethyl phosphite was evaporated, and the residue was dried in vacuo . ( E )-9,9–didecyl-7-(4-(diphenylamino) styryl)-9 H -fluorene-2-carbaldehyde ( 15 , 0.37 g, 0.5 mmol) was then mixed with the above product in dry DMF (5 mL) under Ar. NaH was added, and the reaction was stirred at room temperature for 20 h. Water was added, and the product was extracted with EtOAc. Solvent was removed, and the crude product was purified by column chromatography (1:1 hexanes/CH 2 Cl 2 ), providing 0.16 g of 6 (37% yield) as a glassy material, which turned into viscous liquid upon heating. No defined melting point was observed. 1 NMR (500 MHz, CDCl 3 ) δ 7.95 (d, J = 10.0 Hz, 4H), 7.65 (m, 8H), 7.41–7.51 (m, 12H), 7.29 (m, 12 H), 7.02–7.13 (m, 20H), 2.01 (m, 8H), 1.04–1.26 (m, 56H), 0.79 (t, J = 5.0 Hz, 12H), 0.65 (m, 8H). 13 C NMR (125 MHz, CDCl 3 ) δ 151.7, 147.6, 147.3, 141.8, 140.0, 139.8, 137.0, 133.1, 131.6, 129.3, 128.1, 127.7, 127.6, 127.3, 126.9, 126.2, 125.7, 125.5, 124.5, 123.6, 123.1, 121.1, 120.6, 120.1, 120.0, 55.0, 40.5, 31.9, 29.6, 29.6, 29.3, 29.3, 23.8, 22.7, 14.1. HRMS(MALDI-TOF) for C 122 H 140 N 2 O 2 S: theoretical m/z [M] + = 1697.0636, found [M] + = 1697.0663. Preparation of (E)-9, 9-didecyl-7-(4-(dibutylamino)styryl)- 9H -fluorene-2-carbaldehyde (21) 7-Bromo-9,9-didecyl-9H-fluorene-2-carbaldehyde 13 (0.554 g, 1.0 mmol), N,N-di- n -butyl-4-vinylaniline ( 20 , 0.231 g, 1.0 mmol), PPh 3 (26.2 mg, 0.10 mmol), and K 2 CO 3 (0.346g, 2.5 mmol) in DMF were degassed for 30 min, then Pd(OAc) 2 (33.7 mg, 0.05 mmol) and n -Bu 4 NCl (0.278 g, 1.0 mmol) were added. The mixture was then refluxed for 20 h under Ar. Water and hexane were added, the organic phase was concentrated, and the crude product was purified by column chromatography using 1.5:1 hexanes/CH 2 Cl 2 as eluent, affording 0.50 g of 21 as yellow oil (71% yield). 1 H NMR (300 MHz, CDCl 3 ) δ 9.95 (s, 1H), 7.77–7.68 (m, 3H), 7.62 (d, 1H), 7.40 (d, J =8.1 Hz, 1H), 7.35–7.32(m, 3H), 7.05(d, J =16.2 Hz, 1H), 6.88(d, J =16.2 Hz, 1H), 6.57 (d, J =8.7 Hz, 2H), 3.23 (t, J =7.5 Hz, 4H), 1.95(t, J =8.0 Hz, 4H), 1.51(m, 4H), 1.29(m, 4H), 1.20–0.88 (m, 28H), 0.83–0.74(m, 12H), 0.54(m, 4H). 13 C NMR (74.5 MHz, CDCl 3 ) δ 13.0, 13.1, 19.3, 21.6, 22.7, 28.2, 28.4, 28.4, 28.5, 28.9, 30.5, 30.8, 39.2, 49.8, 54.0, 107.2, 110.5, 118.4, 119.0, 119.9, 121.7, 122.6, 124.0, 126.6, 128.1, 129.4, 133.6, 136.8, 137.9, 146.3, 146.5, 150.3, 151.4, 191.0. HRMS (ESI) for C 50 H 73 NO: theoretical m/z [M+H] + = 704.5765. Found [M+H] + = 704.5751. Preparation of 2, 6-bis[4-(chloromethyl)phenyl] benzo[ 1, 2-d:4, 5-d' ]bisthiazole (24) 2, 5-Diaminobenzene-1, 4-dithiol ( 22 , 0.39 g, 2.26 mmol) and 4-(chloromethyl)benzoyl chloride ( 23 , 0.86g, 4.55 mmol) in 5 mL anhydrous NMP was heated at 110 °C for 3 h. Upon cooling, water was added and the precipitate was collected by filtration. Recrystallization from toluene/ o -dichlorobenzene afforded 0.71g of 24 (70% yield), m.p. >500 °C (decomp.). 1 H NMR (300 MHz, DMSO–d 6 ) δ 8.87 (s, 2H), 8.15 (d, J = 7.8 Hz, 4H), 7.66 (d, J = 7.8 Hz, 4H), 4.88 (s, 4H). 13 C NMR could not be collected due to the limited solubility. HRMS (ESI) for C 22 H 14 Cl 2 N 2 S 2 : theoretical m/z [M+H] + = 441.0052. Found [M+H] + = 441.0061. Anal. Calcd. for (C 22 H 14 Cl 2 N 2 S 2 ): C, 59.86; H, 3.20; N, 6.35; S, 14.53. Found: C, 60.01; H, 3.26; N, 6.43; S, 14.59. Preparation of tetraethyl 4, 4′-(benzo[1, 2-d:4, 5-d']bisthiazole-2, 6-diyl) bisbenzylphosphonate (25) A solution of 24 (0.78 g, 1.77 mmol) and triethyl phosphite (1.17 g, 7.08 mmol) in 5 mL o -dichlorobenzene was heated at reflux for 16 h. The product was then precipitated from hexane, collected by filtration, and purified by column chromatography using 20:1 CH 2 Cl 2 /MeOH as eluent, providing 1.06 g of 24 (93% yield), m.p. 289.5 – 290 °C. 1 H NMR (300 MHz, CDCl 3 ) δ 8.52 (s, 2H), 8.06 (d, J = 8.1 Hz, 4H), 7.45 (d, J = 8.1 Hz, 4H), 4.05 (m, 8H), 3.23 (d, J = 22.2 Hz, 4H), 1.27(t, J = 7.1 Hz, 12H). 13 C NMR (75 MHz, CDCl 3 ) δ 16.8, 33.4, 35.2, 62.7, 115.6, 128.0, 130.7, 132.3, 134.6, 135.5, 152.3, 168.7. Anal. Calcd. for (C 30 H 34 N 2 O 6 P 2 S 2 ): C, 55.89; H, 5.32; N, 4.35; S, 9.95. Found: C, 56.11; H, 5.36; N, 4.41; S, 10.04. Preparation of dye 8 A solution of 21 (58 mg, 0.082 mmol) and 25 (26 mg, 0.041 mmol) in 1 mL DMF was degassed by Ar for 30 min. NaH (14 mg, 0.82 mmol) was then added and the mixture was stirred at room temperature under Ar for 20 h. The product was precipitated by addition of water and collected by filtration. The crude product was purified by column chromatography using 1:2 CH 2 Cl 2 /hexanes as eluent, affording 31 mg of 6 (43% yield), m. p. 196 – 197 °C. 1 H NMR (300 MHz, CDCl 3 ) δ 8.37 (s, 2H), 7.99 (d, J = 8.0 Hz, 4H), 7.54–7.40 (m, 18H), 7.33–7.27 (m, 4H), 7.10 (d, J = 16.0 Hz, 2H), 7.09 (d, J = 16.0 Hz, 2H), 7.00 (d, J = 16.0 Hz, 2H), 6.65 (d, J = 16.0 Hz, 4H), 3.32 (t, J = 7.5 Hz, 8H), 2.03 (m, 8H), 1.62 (m, 8H), 1.39 (m, 8H), 1.23–0.98 (m, 68H), 0.83 (m, t, J = 7.2 Hz), 0.73 (m, 8H). 13 C NMR (125 MHz, CDCl 3 ) δ 168.5, 152.3, 151.6, 151.5, 147.8, 141.5, 140.5, 139.4, 137.6, 135.3, 134.5, 132.1, 128.0, 127.9, 127.7, 127.1, 126.9, 124.7, 121.3, 121.0, 120.6, 120.3, 120.0, 119.9, 115.4, 115.2, 111.6, 55.0, 50.8, 40.6, 31.9, 30.1, 29.5, 29.3, 23.9, 22.7, 20.6, 20.4, 20.3, 14.3, 14.1, 14.0. HRMS (ESI–TOF) for C 122 H 158 N 4 S 2 : theoretical m/z [M] 2+ = 871.5958, found [M] 2+ = 871.5959.

General procedure for preparation of dyes 1-5 9,9-Didecyl-9 H -fluorene-2,7-dicarbaldehyde ( 9 ) or 9,9-didecyl-4,5-dinitro-9 H -fluorene-2,7-dicarbaldehyde ( 10 , 0.5 mmol), 4-( N,N -dibutylaminobenzyl) triphenylphosphonium iodide ( 11 ) and/or diethyl 4-(benzo[ d ]thiazol-2-yl) benzylphosphonate ( 12 , 1.0 mmol) in 10 mL of dry DMF was degassed with Ar for 30 min. NaH (0.24 g, 10.0 mmol) was then added, and the mixture was stirred at room temperature under Ar for 20–24 h. The crude product precipitated upon slow addition of water, and was collected by filtration, followed by column chromatographic purification to afford pure compounds 1 - 4 and 7 (characterization details below).

General procedure for preparation of dye encapsulated micelles A solution of dye in CH 2 Cl 2 (5 mL) was mixed with Pluronic™ F 108NF (30 mg) in water (5 mL). The resulting mixture was stirred at room temperature for 48 h to slowly evaporate the CH 2 Cl 2 . The dye concentration was approximately 0.5 mM in water, as estimated by absorption spectra.

General procedure for HCT 116 cells incubation with fluorescent probes An epithelial colorectal carcinoma cell line HCT 116, purchased from ATCC (America Type Culture Collection, Manassas, VA, USA), was used. All cells were incubated in Dulbecco's modified Eagle's Medium (DMEM, Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals, Lawrenceville, GA, USA), 100 units/mL penicillin-streptomycin (Atlanta Biologicals, Lawrenceville, GA, USA), and incubated at 37 °C in a 95% humidified atmosphere containing 5% CO 2 . HCT 116 cells were placed onto poly-D-lysine coated coverslips placed into 24-well glass plates (40,000 cells per well) and incubated for 36 h before incubating with the fluorescent probes. A 0.5 mM stock solution of the fluorescent probe in Pluronic™ F 108NF micelles was dissolved in water. Diluted solutions of probe (30 μM) by complete growth medium, Dulbecco's modification of Eagle's medium (DMEM), were then freshly prepared and placed over the cells for a 3 h period. For colocalization experiments, a diluted mixture of dye (30 μM) and LysoTracker® Red (75 nM) in the growth medium was used. After the incubation, cells were washed with PBS (pH=7.2, Fischer Scientific) three to five times, fixed using 3.7% formaldehyde solution at 37 °C for 15 min, and incubated with 0.5 mL/well NaBH 4 (1 mg/mL) solution in PBS (pH=8.0, prepared by adding few drops of 6N NaOH solution into PBS) at 37 °C for 15 min. The last step was then repeated. The plates were washed twice with PBS and once with water. Finally, the glass coverslips were mounted using Prolong Gold mounting media for microscopy imaging.

Supplementary Material 1_si_001