Maged Henary Dissertation

Synthesis and Characterization of NIR Fluorophore

To obtain the final dyes 1428, 3133, and 3638, the precursor salts 610, 30, and 35 (2 mol equiv) were added to a clean, dry round-bottom flask. The compounds were stirred in 10 mL of acetic anhydride, and either sodium acetate or triethyl amine (3 mol equiv) was added along with 1 mol equiv of individual malondialdehyde linker 1113. The reaction was allowed to stir at 40–60 °C for 2 h. The reactions were monitored closely using regular phase thin-layer chromatography with a mobile phase of DCM/MeOH (99:1) as well as UV–vis-NIR spectrophotometer in quartz cuvettes with methanol as a solvent to visualize the absorption band at ~650 nm against that of the starting materials ~400 nm. Upon completion of the reaction, the mixtures were allowed to cool, and the solvent was evaporated. The residue was extracted using DCM and was washed with DI water (3 × 70 mL). The resulting organic layer was dried under magnesium sulfate, gravity filtered, and evaporated to afford crystals that were washed with diethyl ether (2 × 50 mL) and hexanes (50 mL) to yield crystals that were dried under vacuum overnight. The pure product was obtained after dissolving in methanol and precipitating with ether several times, or the compounds were isolated using flash column chromatography and 5% methanol in DCM as the eluting solvent. After purification, the compounds were obtained in the designated yields and were fully characterized.

1,3,3-Trimethyl-2-((1E,3E,5E)-5-(1,3,3-trimethylindolin-2-ylidene)-penta-1,3-dien-1-yl)-3H-indol-1-ium Iodide (14)

Yield 64%, mp > 260 °C; 1H NMR (400 MHz, MeOD-d4) δ: 1.71 (s, 12H), 3.63 (s, 6H), 6.28 (d, J = 16.0 Hz, 2H), 6.65 (t, J = 12.0 Hz, 1H), 7.24 (t, J = 8.0 Hz, 2H), 7.29 (d, J = 8.0 Hz, 2H), 7.39 (t, J = 8.0 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 8.25 (t, J = 12.0 Hz, 2H); 13C NMR (100 MHz, MeOD-d4) δ 27.90, 31.71, 50.50, 104.44, 111.83, 123.31, 126.20, 129.71, 142.56, 144.29, 155.52, 175.28. TOF HRMS m/z [M]+ calcd for [C27H31N2]+ 383.2487, found 383.2474.

2-((1E,3Z,5E)-3-Chloro-5-(1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium Iodide (15)

Yield 64%, mp 230–232 °C; 1H NMR (400 MHz, MeOD-d4) δ 1.76 (s, 12H), 3.71 (s, 6H), 6.435 (d, J = 12 Hz, 2H), 7.32 (t, J = 8 Hz, 2H), 7.39 (d, J = 8 Hz, 2H), 7.45 (t, J = 8 Hz, 2H), 7.54 (d, J = 8 Hz, 2H), 8.345 (d, J = 12 Hz, 2H). 13C NMR (100 MHz, MeOD-d4) δ 25.03, 29.53, 48.55, 98.93, 110.02, 121.00, 121.86, 124.55, 127.43, 140.42, 141.67, 146.72, 174.28. TOF HRMS m/z [M]+ calcd for [C27H30N2Cl]+ 417.2098 found 417.2107.

2-((1E,3E,5E)-3-Bromo-5-(1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium Iodide (16)

Yield 61%, mp 230–232 °C; 1H NMR (400 MHz, CDCl3-d1) δ 1.91 (s, 12H), 3.82 (s, 6H), 6.36 (d, J = 8 Hz, 2H), 7.16 (d, J = 8.0 Hz, 2H, 7.26–7.31 (m, 4H), 7.39–7.44 (m, 4H) 8.93 (d, J = 12 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 27.04, 31.98, 49.85, 102.61, 112.19, 115.98, 122.94, 125.97, 128.93, 141.78, 143.02, 149.64, 175.14. TOF HRMS m/z [M]+ calcd for [C27H30N2Br]+ 461.1592 found 461.1585.

5-Fluoro-2-((1E,3E,5E)-5-(5-fluoro-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium Iodide (17)

Yield 78%, mp > 260 °C; 1H NMR(400 MHz, DMSO-d6) δ 1.68 (s, 12H), 3.59 (s, 6H), 6.23 (d, J = 12.0 Hz, 2H), 6.52 (s, 1H), 7.24 (s, 2H), 7.39 (s, 2H), 7.62 (s, 2H), 8.29 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 27.27, 31.85, 49.54, 103.64, 103.76, 110.82, 111.07, 112.43, 112.49, 112.59, 112.66, 115.15, 125.54, 139.55, 143.60, 143.69, 154.27, 154.29, 159.32, 161.72, 173.64. TOF HR-MS ESI m/z [M]+ calcd for [C27H29F2N2]+ 419.2299, found 419.2296.

2-((1E,3E,5E)-3-Chloro-5-(5-fluoro-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-5-fluoro-1,3,3-trimethyl-3H-indol-1-ium (18)

Yield 37%, mp > 260 °C; 1H NMR(400 MHz, DMSO-d6) δ: 1.72 (s, 12H), 3.67 (s, 6H), 6.26 (d, J = 14 Hz, 2H), 7.29,7.31 (t, J = 6.8 Hz, 8.8 Hz, 2H), 7.52–7.54 (m, 2H), 7.70 (d, J = 6 Hz, 2H), 8.41 (d, J = 14 Hz, 2H). 19F NMR (375 MHz, DMSO-d6) δ: −116.67 (s, 2F). TOF HR-MS ESI m/z [M]+ calcd for [C27H29F2ClN2]+ 453.1909 found 453.1914.

2-((1E,3E,5E)-3-Bromo-5-(5-fluoro-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-5-fluoro-1,3,3-trimethyl-3H-indol-1-ium (19)

Yield 52%, 1H NMR(400 MHz, DMSO-d6) δ: 1.72 (s, 12H), 3.67 (s, 6H), 6.26 (d, J = 13.2 Hz, 2H), 7.32 (t, J = 8.8 Hz, 2H), 7.55–7.52 (m, 2H), 7.70 (d, J = 7.6, 2H), 8.46 (d, J = 13.6, 2H). 19F NMR (375 MHz, DMSO-d6) δ: −116.67 (s, 2F). TOF HR-MS ESI m/z [M]+ calcd [C27H29F2N2Br]+ was 497.1404 found 497.1394.

5-Chloro-2-((1E,3E,5E)-5-(5-chloro-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium Iodide (20)

Yield 59%, mp > 260 °C; 1H NMR(400 MHz, DMSO-d6) δ: 1.69 (s, 12H), 3.59 (s, 6H), 6.27 (d, J = 14 Hz, 2H), 6.551 (t, J = 12.4, 12.0 Hz, 1H), 7.39 (d, J = 8.8 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.80 (s, 2H), 8.32 (t, J = 13.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 27.30, 31.84, 49.51, 104.09, 112.87, 123.29, 126.16, 128.67, 129.50, 142.23, 143.54, 154.72, 173.65. TOF HR-MS ESI m/z [M]+ calcd for [C27H29Cl2N2]+ 451.1708, found 451.1722.

5-Chloro-2-((1E,3E,5E)-3-chloro-5-(5-chloro-1,3,3-trimethylindo-lin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium (21)

Yield 53%, mp > 260 °C; 1H NMR(400 MHz, DMSO-d6) δ: 1.72 (s, 12H), 3.67 (s, 6H), 6.26 (d, J = 14 Hz, 2H), 7.52 (m, 4H), 7.89 (s, 2H), 8.43 (d, J = 13.6, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 26.81, 32.15, 49.98, 100.72, 113.62, 123.06, 123.49, 128.80, 130.31, 142.02, 143.83, 147.96, 174.97. TOF HR-MS ESI m/z [M]+ calcd for [C27H29Cl3N2]+ 485.1318 found 485.1317.

2-((1E,3E,5E)-3-Bromo-5-(5-chloro-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-5-chloro-1,3,3-trimethyl-3H-indol-1-ium Iodide (22)

Yield 69%, mp 252–254 °C 1H NMR(400 MHz, DMSO-d6) δ: 1.73 (s, 12H), 3.67 (s, 6H), 6.28 (d, J = 13.2 Hz, 2H), 7.52 (m, 4H), 7.89 (s, 2H), 8.47 (d, J = 13.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 26.79, 32.16, 50.03, 102.96, 113.65, 116.40, 123.51, 128.82, 130.35, 141.99, 143.83, 149.98, 175.15. TOF HR-MS ESI m/z [M]+ calcd [C27H28BrN2Cl2]+ was 529.0813 found 529.0810.

5-Bromo-2-((1E,3E,5E)-5-(5-bromo-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium Iodide (23)

Yield 62%, mp > 260 °C; 1H NMR(400 MHz, DMSO-d6) δ: 1.69 (s, 12H), 3.58 (s, 6H), 6.27 (d, J = 14 Hz, 2H), 6.56 (t, J = 12.4, 12 Hz, 1H), 7.35 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.4, 2H), 7.93 (s, 2H), 8.33 (t, J = 13.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 27.25, 31.71, 49.48, 104.07, 113.33, 117.45, 126.09, 131.51, 142.63, 143.85, 154.86, 173.45. TOF HR-MS ESI m/z [M]+ calcd for [C27H29Br2N2]+ 539.0697, found 539.0695.

5-Bromo-2-((1E,3E,5E)-5-(5-bromo-1,3,3-trimethylindolin-2-yli-dene)-3-chloropenta-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium (24)

Yield 72%, mp > 260 °C; 1H NMR(400 MHz, DMSO-d6) δ: 1.72 (s, 12H), 3.67 (s, 6H), 6.28 (d, J = 12.0 Hz, 2H), 7.47 (d, J = 8.0 Hz, 2H), 7.63 (d, J = 8.0 Hz, 2H), 8.01 (s, 2H), 8.43 (d, J = 12.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 26.81, 32.17, 49.96, 100.69, 114.05, 118.37, 123.13, 126.26, 131.63, 142.40, 144.11, 147.96, 174.78. TOF HR-MS ESI m/z [M]+ calcd for [C27H28Br2N2Cl]+ 573.0308, found 573.0316.

5-Bromo-2-((1E,3E,5E)-3-bromo-5-(5-bromo-1,3,3-trimethylindo-lin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium Iodide (25)

Yield 46%, mp 231–232 °C; 1H NMR(400 MHz, DMSO-d6) δ: 1.72 (s, 12H), 3.66 (s, 6H), 6.28 (d, J = 13.2 Hz, 2H), 7.47 (d, J = 8.0 Hz, 2H), 6.64 (d, J = 8.4 Hz, 2H), 8.01 (s, 2H), 8.48 (d, J = 13.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 26.79, 32.11, 50.03, 102.97, 114.07, 116.47, 118.42, 126.27, 131.65, 142.40, 144.13, 150.03, 175.01. TOF HR-MS ESI m/z [M]+ calcd [C27H28Br3N2]+ was 616.9803, found 616.9814.

1,3,3-Trimethyl-5-(trifluoromethyl)-2-((1E,3E)-5-((E)-1,3,3-tri-methyl-5-(trifluoromethyl)indolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium Iodide (26)

Yield 55%, mp 218–221 °C; 1H NMR (400 MHz, CDCl3): δ 1.81 (s, 12H), 3.79 (s, 6H), 6.53 (d, J = 13.6 Hz, 2H), 7.11 (t, J = 13.6 Hz, 1H), 7.21(d, J = 8.4 Hz, 2H), 7.60 (s, 2H), 7.70 (d, J = 7.6 Hz, 2H), 8.02 (t, J = 13.6, 2H). 19FNMR: (375 MHz, DMSO-d6): δ 62.00. TOF HRMS m/z [M]+ calcd for [C29H29N2F6]+ 519.2297, found 519.2393.

2-((1E,3Z)-3-Chloro-5-((E)-1,3,3-trimethyl-5-(trifluoromethyl)-indolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-5-(trifluoro-methyl)-3H-indol-1-ium Iodide (27)

Yield 71%, mp 226–228 °C; 1H NMR (400 MHz, CDCl3): δ 1.91 (s, 12H), 3.87 (s, 6H), 6.47 (d, J = 13.6 Hz, 2 H), 7.30 (s, 2H), 7.65 (m, 4H), 8.90 (d, J = 10.4 Hz, 2H). 19F NMR (375 MHz, DMSO-d6): δ 62.05. TOF HRMS m/z [M]+ calcd for [C29H28N2F6Cl]+ 553.1840, found 553.1996.

2-((1E,3Z)-3-Bromo-5-((E)-1,3,3-trimethyl-5-(trifluoromethyl)-indolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-5-(trifluoro-methyl)-3H-indol-1-ium Iodide (28)

Yield 71%, mp 226–228 °C; 1H NMR (400 MHz, CDCl3): δ 1.91 (s, 12H), 3.87 (s, 6H), 6.46 (bs, 2H), 7.30 (s, 2H), 7.69 (m, 4H), 8.91 (d, J = 7.2 Hz, 2H). 19F NMR (375 MHz, DMSO-d6), δ: 62.01. TOF HRMS m/z [M]+ calcd for [C29H28N2F6Br]+ 597.1335, found 597.1597.

5-Methoxy-2,3,3-trimethyl-3H-indole (29)

Yield 78%, reddish brown oil; 1H NMR (400 MHz, DMSO-d6) δ 1.17 (s, 6H), 2.16 (s, 3H), 3.68 (s, 3H), 6.74 (m, 2H), 7.38 (s,1H).

5-Methoxy-1,2,3,3-tetramethyl-3H-indol-1-ium Iodide (30)

1H NMR (400 MHz, DMSO-d6) δ 1.51 (s, 6H), 2.71 (s, 3H), 3.86 (s, 3H), 3.94 (s, 3H), 7.14 (d, J = 8.0 Hz, 1H), 7.47 (s, 1H), 7.81 (d, J = 8.0 Hz, 1H).

5-Methoxy-2-((1E,3E,5E)-5-(5-methoxy-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium Iodide (31)

Yield 75%, mp 228–230 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.66 (s, 12H), 3.56 (s, 3H), 3.81 (s, 6H), 6.17 (d, J = 12 Hz, 2H), 6.46 (t, J = 12 Hz, 1H), 6.95 (d, J = 8 Hz, 2H), 7.30–7.28 (m, 4H), 8.23 (t, J = 12 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 26.48, 30.62, 48.45, 55.29, 102.06, 108.43, 111.02, 112.84, 123.73, 135.80, 142.15, 151.94, 156.98, 171.55. TOF HRMS m/z [M]+ calcd for [C29H35N2O2]+ 443.2699, found 443.2692.

2-((1E,3E,5E)-3-Chloro-5-(5-methoxy-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-5-methoxy-1,3,3-trimethyl-3H-indol-1-ium Iodide (32)

Yield 80%, mp 235–237 °C; 1H NMR (400 MHz, MeOD-d4) δ 1.73 (s, 12H), 3.67 (s, 6H), 3.85 (s, 6H), 6.34 (d, J = 12 Hz, 2H), 6.99 (d, J = 8.0 Hz, 2H), 7.14 (s, 2H), 7.29 (d, J = 8.0 Hz, 2H), 8.23 (d, J = 12 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 27.05, 31.96, 49.88, 56.33, 99.74, 109.36, 112.76, 114.07, 121.81, 136.50, 143.49, 146.18, 158.51, 173.76. TOF HRMS m/z [M]+ calcd for [C29H34N2O2Cl]+ 477.2309; found 477.2327.

2-((1E,3E,5E)-3-Bromo-5-(5-methoxy-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-5-methoxy-1,3,3-trimethyl-3H-indol-1-ium Iodide (33)

Yield 55%, mp 214–216 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.69 (s, 12H), 3.63 (s, 6H), 3.81 (s, 6H), 6.18 (d, J = 12 Hz, 2H), 6.97 (d, J = 8.0 Hz, 2H), 7.35 (s, 2H), 7.40 (d, J = 8 Hz, 2H), 8.38 (d, J = 12 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 26.20, 31.16, 49.13, 55.53, 101.20, 108.56, 112.00, 113.28, 114.41, 135.67, 142.68, 147.32, 157.72, 173.09. TOF HRMS m/z [M]+ calcd for [C29H34N2O2Br]+ 521.1804 found 521.1801.

5,7,7-Trimethyl-6-((1E,3E,5Z)-5-(5,7,7-trimethyl-5H-[1,3]dioxolo-[4,5-f ]indol-6(7H)-ylidene)penta-1,3-dien-1-yl)-7H-[1,3]dioxolo[4,5-f ]indol-5-ium Iodide (36)

Yield 38%, 1H NMR (400 MHz, DMSO-d6) δ: 1.63 (s, 12H), 3.54 (s, 6H), 6.06 (s, 4H), 6.18 (d, J = 16.0 Hz, 2H), 6.46 (t, J = 16.0 Hz, 1H), 7.13 (s, 2H), 7.28 (s, 2H), 8.16 (t, J = 15 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 27.59, 31.89, 49.29, 94.35, 102.18, 103.40, 104.03, 125.11, 134.93, 137.52, 145.55, 148.02, 152.68, 173.32. TOF HRMS m/z [M]+ calcd for [C29H31O4N2]+ 471.2278 found 471.2268.

6-((1E,3E,5E)-3-Chloro-5-(5,7,7-trimethyl-5,7-dihydro-6H-[1,3]-dioxolo[4,5-f ]indol-6-ylidene)penta-1,3-dien-1-yl)-5,7,7-trimethyl-7H-[1,3]dioxolo[4,5-f ]indol-5-ium (37)

Yield 39%, 1H NMR (400 MHz, DMSO-d6) δ: 1.66 (s, 12H), 3.63 (s, 6H), 6.10 (s, 4H), 6.21 (d, J = 13.6 Hz, 2H), 7.29 (s, 2H), 7.38 (s, 2H), 8.30 (d, J = 13.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 27.10, 32.20, 49.70, 94.92, 100.10, 102.34, 104.12, 135.49, 137.25, 146.04, 146.12, 148.10, 174.41. TOF HRMS m/z [M]+ calcd for [C29H30N2O4Cl]+ 505.1894 found 505.1871.

6-((1E,3E,5E)-3-Bromo-5-(5,7,7-trimethyl-5,7-dihydro-6H-[1,3]-dioxolo[4,5-f ]indol-6-ylidene)penta-1,3-dien-1-yl)-5,7,7-trimethyl-7H-[1,3]dioxolo[4,5-f ]indol-5-ium (38)

Yield 27%, 1H NMR (400 MHz, DMSO-d6) δ: 1.66 (s, 12H), 3.62 (s, 6H), 6.10 (s, 4H), 6.21 (d, J = 13.6 Hz, 2H), 7.29 (s, 2H), 7.39 (s, 2H), 8.36 (d, J = 13.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 27.08, 32.20, 49.76, 94.94, 102.34, 104.14, 113.95, 135.50, 137.22, 146.16, 146.16, 148.02, 174.59. TOF HRMS m/z [M]+ calcd for [C29H30N2O4Br]+ 549.1389 found 549.1368.

Introduction

Section:

Cyanine dyes are a class of conjugated, fluorescent molecules with polymethine chromophores composed of an odd number of carbon atoms. These dyes exhibit unusually long-wavelength absorbance and fluorescence relative to the size of their chromophores, typically absorbing light in the visible to near infrared (NIR) region.1 These compounds were originally utilized as sensitizing additives to photographic emulsions, but their unique structural and photophysical characteristics have since proven useful for a wide variety of other applications requiring photosensitive materials, such as optical recording media and solar cells.2–4 Additionally, cyanine dyes can be used as fluorescent labels of both proteins and DNA, thereby greatly enhancing the sensitivity of fluorescence detection for these types of biomolecules.5–7 NIR-absorbing cyanine dyes are particularly well-suited for use as fluorescent labels of proteins and nucleic acids, as there is no interfering autofluorescence from biomolecules at these long wavelengths, and have been applied to both in vitro analytical studies and in vivo biomedical imaging.5–9 Due to the tremendous utility and versatility of this class of compounds, significant research efforts are being directed at developing new cyanine dyes functionalized for specific applications and optimizing the properties of these dyes.

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