Synthesis and Validation of Cyanine-Based Dyes for DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 75

Fig. 3. Synthesis of the methyl Cy5 dye and its NHS ester.

pyridine (0.1 mL) and DSC (21 mg, 0.08 mmol) were added
to a stirred solution of the dye (12) (27 mg, 0.05 mmol) in dry
DMF (2 mL) under nitrogen. The reaction mixture was stirred
at 60°C for 1.5 h. After evaporation of the solvent, the deep red
residue was purifi ed by column chromatography on silica gel
(dichloromethane/hexane/methanol = 5:1:1) to give the pure
propyl Cy3-NHS ester (13) (31 mg, 96%) as a red powder ( 2, 6 )

.
IR (neat): 2,924, 2,853, 1,737, 1,555, 1,456, 1,428, 1,371,
1,248, 1,196, 1,158, 1,116, 1,019, 930, 796, 579, 680/cm.
1 H NMR (400 MHz, DMSO- d 6 ): d 8.32 (1H, dd, J = 13.4,
13.4 Hz), 7.61 (2H, d, J = 7.6 Hz), 7.38-7.47 (4H, m), 7.26
(2H, t, J = 7.4 Hz), 48 (2H, d, J = 13.5 Hz), 4.09 (4H, m),
2.74 (4H, s), 2.66 (2H, t, J = 7.2 Hz), 1.74 (2H, m), 1.69
(2H, m), 1.66 (12H, s), 1.48 (2H, m), 1.19 (2H, m), 0.95
(3H, t, J = 7.4 Hz). 13 C NMR (100 MHz, DMSO- d 6 ): d 174.20,
173.40, 169.17, 168.61, 151.50, 142.30, 140.52, 129.11,
128.86, 125.04, 122.03, 111.14, 110.96, 105.17, 48.90, 440,
44.20, 30.70, 29.71, 28.20, 27.21, 25.82, 25.62, 24.53, 21.17,
11.30. MS (EI): m/z (%) = 582 (100), 485 (22).

For the synthesis of the methyl Cy5 dye and its NHS ester (see
Fig. 3 ), the three-carbon spacer (15) had to be prepared. Conden-
sation of commercially available malondialdehyde bis(dimethyl
acetal) (14) with aniline under acidic conditions afforded the
anilino anilinium salt (15) in 85% yield ( 2, 21 ). The reaction of
(15) with the commercially available 1,3,3-trimethyl-2-methylenein-
doline (16) in refl uxing acetic acid afforded the anilinobutadienyl
salt (17) in 66% yield ( 2, 22 ). Finally, the reaction of the activated
indolium salt (17) with the methylindolium salt (11) synthesized

3.1.3. Cy5 Synthesis