Synthesis and Validation of Cyanine-Based Dyes for DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 74

1,369, 1,311, 1,200, 1,130, 996, 757/cm. 1 H NMR (400 MHz,
DMSO- d 6 ): d 9.10 (1H, d, J =14.2 Hz), 7.05-7.70 (9H, m),
5.34 (1H, d, J = 14.2 Hz), 4.06 (2H, t, J = 7.1 Hz), 2.05 (3H,
s), 1.70 (6H, s), 1.60 (2H, m), 0.67 (3H, t, J = 7.4 Hz). 13 C
NMR (100 MHz CDCl 3 ): d 162.80, 154.24, 142.85, 139.56,
129.48, 128.41, 125.42, 123.32, 121.96, 119.42, 109.13,
94.35, 47.84, 45.19, 29.26, 27.86, 20.15, 11.45.
C. 1-(5-Carboxypentyl)-2,3,3-trimethyl-3H-indolium bromide
(11) . A mixture of 2,3,3-trimethyl-3 H -indole (8) (Aldrich,
0.2 g, 1.25 mmol) and 6-bromohexanoic acid (Aldrich, 0.36 g,
1.80 mmol) in 1,2-dichlorobenzene was heated at 110°C for
12 h. The solution was cooled to room temperature, and the
residue obtained was fi ltered and washed with a mixture of
acetonitrile/diethyl ether (1/1). The solid obtained was dried
under vacuum to give the product (11) as a light red powder
(2.3 g, 67%) ( 2, 18-20 ). IR (neat): 3,405, 2,936, 1,724,
1,624, 1,460, 1,392, 1,168, 767/cm. 1 H NMR (400 MHz,
DMSO- d 6 ): d 7.93-7.97 (1H, m), 7.79-7.87 (1H, m), 7.56-
7.64 (2H, m), 4.43 (2H, t, J =7.7 Hz), 2.82 (3H, s), 2.19
(2H, t, J = 7.2 Hz), 1.81 (2H, m), 1.52 (2H, m), 1.50 (6H, s),
1.35 (2H, m). 13 C NMR (100 MHz, DMSO- d 6 ): d 1998,
174.77, 142.33, 141.51, 129.85, 129.40, 123.99, 115.97,
54.62, 47.90, 33.83, 27.41, 25.87, 24.48, 22.47, 14.51.
D. 2-(3-(1-(5-Carboxypentyl)-1,3-dihydro-3,3-dimethyl-2H-indol-
2-ylidene)-1-propenyl)-3,3-dimethyl-1-propyl-3H-indolium, pro-
pyl Cy3 (12) . A mixture of the salt (11) (0.08 g, 0.28 mmol),
enamide (10) (0.10 g, 0.28 mmol), and dry triethylamine
(0.1 mL) in absolute ethanol (10 mL) was refl uxed for 30 min.
The solvent was removed under reduced pressure, and the
crude residue was purifi ed by fl ash chromatography on silica
gel (dichloromethane/hexane/methanol=5:1:1) to give the
dye propyl Cy3 (12) as a deep red powder (0.12 g, 85%) ( 16, 17 ) .
IR (neat): 3,407, 2,969, 2,934, 2,876, 2,734, 2,673, 1,557,
1,453, 1,427, 1,242, 1,192, 1,130, 1,030, 930, 754/cm. 1 H
NMR (400 MHz, DMSO- d 6 ): d 8.31 (1H, dd, J = 13.4,
13.5 Hz), 7.61 (2H, d, J = 7.4 Hz), 7.36-7.47 (4H, m), 7.26
(2H, t, J =7.1 Hz), 54 (1H, d, J =13.5 Hz), 53 (1H, d,
J = 13.4 Hz), 4.08 (2H, t, J = 7.2 Hz), 3.43 (2H, t, J = 7.2 Hz),
2.14 (2H, t, J = 7.2 Hz), 1.75 (2H, m), 1.66 (12H, s), 1.52 (2H,
m), 1.40 (2H, m), 1.19 (2H, m), 0.84 (3H, t, J = 7.4 Hz). 13 C
NMR (100 MHz DMSO- d 6 ): d 174.20, 161.01, 1445, 143.11,
141.37, 137.32, 129.11, 127.92, 1213, 122.98, 122.21, 118.45,
105.73, 102.72, 74.11, 49.36, 44.06, 43.30, 30.26, 27.94,
27.90, 226, 20.89, 19.44, 11.79, 11.4.
E. 2-(3-(1,3-Dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)-1-
propenyl)-1-(6-((2,5-dioxo-1-pyrrolidinyl)oxy)-6-oxohexyl)-3,3-
dimethyl-3H-indolium, propyl Cy3-NHS ester (13) . Anhydrous