Synthesis and Validation of Cyanine-Based Dyes for DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 72

and DSC (Aldrich, 21 mg, 0.08 mmol) were added to a stirred
solution of the dye (6) (25 mg, 0.05 mmol) in dry DMF
(Fisher, 2 mL) under nitrogen. The reaction mixture was
stirred at 60°C for 1.5 h. After evaporation of the solvent, the
deep yellow residue was purifi ed by column chromatography
on silica gel (dichloromethane/hexane/methanol = 5:1:1) to
give the pure Cy2-NHS ester (7) (26 mg, 86%) as an orange
powder. IR (neat): 2,924, 2,851, 1,736, 1,565, 1,507, 1,461,
1,395, 1,348, 1,280, 1,201, 1,115, 1,082, 747/cm. 1 H NMR
(400 MHz, CDCl 3 ): d 8.40 (1H, t, J = 13.2 Hz), 7.22-7.49
(12H, m), 52 (1H, d, J = 13.2 Hz), 40 (1H, d, J = 13.2 Hz),
5.49 (2H, s), 4.31 (2H, br q, J = 7.1 Hz), 3.85 (2H, s), 2.78
(4H, s), 1.46 (3H, t, J = 7.1 Hz). 13 C NMR (100 MHz,
CDCl 3 ): d 168.99, 1651, 162.18, 161.73, 1495, 1482,
132.87, 132.02, 131.37, 130.82, 130.13, 128.36, 1209,
125.28, 125.08, 110.98, 110.90, 110.76, 110.66, 87.15,
849, 37.19, 25.60, 13.34. MS (EI): m/z (%) = 550 (100), 453
(6), 304 (6).

The synthesis of the propyl Cy3 dye and its NHS ester (see Fig. 2 )
began with the alkylation of the commercially available 2,3,3-trim-
ethyl-3 H -indole (8) with propyl bromide to give the 1-propyl-2-
methyleneindoline (9) (

3.1.2. Cy3 Synthesis

16, 17 ) in an unoptimized yield of 44%.
Condensation with commercially available N , N ¢-diphenylform-
amidine in the presence of excess acetic anhydride as solvent
afforded the acetanilidylvinyl indolium salt (10) in 87% yield. The
second component of the dye was prepared by alkylation of trim-
ethylindole (8) with 6-bromohexanoic acid in dichlorobenzene at
110°C for 12 h to give 67% yield of the methylindolium salt (11)
( 2, 18-20 ). This compound was then reacted with the acetanili-
dylvinyl indolium salt (10) in ethanol in the presence of trieth-
ylamine to give the desired dye (12) in 85% yield as a red powder
( 14, 15 ). The dye was easily converted into the NHS ester by treat-
ment with DSC in the presence of pyridine to give the activated
dye (13) in 96% yield. Again, all the pertinent spectroscopic data,
especially high-fi eld NMR and mass spectrometry, were in agreement
with the structures assigned (see Note 1).

A. 3,3-Dimethyl-2-methylene-1-propylindoline (9) . A mixture of
2,3,3-trimethyl-3 H -indole (8) (Aldrich, 0.2 g, 1.25 mmol)
and 1-bromopropane (Aldrich, 2.28 mL, 0.025 mol) in
1,2-dichlorobenzene (Aldrich) was heated at 110°C for 24 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 vac-
uum to give the 3,3-dimethyl-2-methylene-1-propylindoline
(9) as a light red powder (0.11 g, 44%) (see Note 1). IR (neat):
2,966, 2,925, 1,617, 1,601, 1,474, 1,454, 1,356, 1,290,