Synthesis and Validation of Cyanine-Based Dyes for DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 76

earlier (see Subheading 3.1.2 , step C and Fig. 2 ) in ethanol in the
presence of sodium acetate afforded the desired dye (18) in 69%
yield as a blue powder. The dye was easily converted into the NHS
ester by treatment with commercially available DSC in the pres-
ence of pyridine to give the activated dye (19) in 92% yield. All
spectroscopic data, especially high-fi eld NMR and mass spectrom-
etry, were in agreement with the structures assigned (see Note 1).
A. N-((1E)-3-(Phenylimino)prop-1-enyl)benzenamine hydrochloride
(15) . A solution of distilled water (140 mL), HCl (10 mL), and
aniline (Alfa Aesar, 7.4 mL, 0.08 mol) was added dropwise to a
solution of distilled water (171 mL), HCl (8.5 mL), and malon-
dialdehyde bis(dimethyl acetal) (14) (Aldrich, 10.5 mL,
0.06 mol) with stirring at 50°C. The precipitate was isolated by
fi ltration to give malondialdehyde dianil hydrochloride (15)
(12 g, 85%) as an orange powder ( 2, 21 ). IR (neat): 3,425,
1,642, 1,620, 1,580, 1,492, 1,343, 1,273, 1,194, 749, 683/
cm. 1 H NMR (400 MHz, DMSO- d6 ): d 12.72 (2H, d,
J =13.2 Hz), 8.92 (2H, t, J =12.4 Hz), 7.40 (8H, m), 7.20
(2H, m), 50 (1H, t, J = 11.5 Hz). 13 C NMR (100 MHz,
DMSO- d6 ): d 158.89, 139.18, 130.33, 1231, 117.86, 99.09.
B. 2-(4-Phenylamino-1E,3E-butadien-1-yl)-1,3,3-trimethy-lindolium
chloride (17) . A mixture of malondialdehyde dianil hydrochlo-
ride (15) (1 g, 4.49 mmol) and 1,3,3-trimethyl-2-methyle-
neindoline (16) (Aldrich, 0.93 mL, 4.49 mmol) in glacial acetic
acid (10 mL) was refl uxed for 4 h. The solution was cooled to
room temperature, the acetic acid was removed under reduced
pressure, and the residue was purifi ed by fl ash chromatography
on silica gel (dichloromethane/hexane/methanol=5:1:1) to
give the product (16) as a red powder (0.9 g, 66%) ( 22 ). 1 H
NMR (400 MHz, DMSO- d6 ): d 8.18 (1H, d, J = 9.8 Hz), 7.52
(1H, t, J = 13.3 Hz), 74-7.36 (9H, m), 48 (1H, dd, J = 14.1,
9.9 Hz), 5.55 (1H, d, J = 12.5 Hz), 3.24 (3H, s), 1.55 (6H, s).
13 C NMR (100 MHz, DMSO- d6 ): d 129.41, 129.17, 128.04,
125.24, 121.71, 121.36, 120.41, 118.18, 107.27, 468, 28.38.
C. 2-(5-(1-(5-Carboxypentyl)-1,3-dihydro-3,3-dimethyl-2H-indol-
2-ylidene]-1,3-pentadienyl]-1,3,3-trimethyl-3H-indolium,
methyl Cy5 (18) . A solution of the anil (16) (0.20 g,
0.65 mmol), the acid (11) synthesized earlier in Subhead-
ing 3.1.2 , step C (0.18 g, 0.65 mmol), and anhydrous sodium
acetate (0.11 g, 0.79 mmol) in absolute ethanol (50 mL) under
nitrogen was refl uxed for 4 h. The solid was purifi ed by fl ash
chromatography on silica gel (dichloromethane/metha-
nol = 5:1) to give the methyl Cy5 dye (18) (0.22 g, 69%) as a
blue powder ( 2 ). IR (neat): 3,406, 2,925, 1,716, 1,575, 1,470,
1,425, 1,371, 1,335, 1,217, 1,146, 1,016, 1,040, 923, 796,
750, 708/cm. 1 H NMR (400 MHz, DMSO- d6 ): d 8.30