Synthesis and Validation of Cyanine-Based Dyes for DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 77

(2H, t, J = 13.1 Hz), 7.58 (2H, d, J = 7.4 Hz), 7.34 (4H, m),
7.21 (2H, m), 53 (1H, t, J = 12.3 Hz), 25 (2H, dd, J = 13.8,
13.9 Hz), 4.06 (2H, t, J = 7.2 Hz), 3.56 (3H, s), 2.15 (2H, t,
J = 7.2 Hz), 1.69 (2H, m), 1.64 (12H, s), 1.52 (2H, m), 1.32
(2H, m). 13 C NMR (100 MHz DMSO- d6 ): d 175.03, 173.69,
172.95, 154.47, 143.21, 142.47, 141.54, 141.47, 128.80,
125.83, 125.16, 125.05, 122.89, 122.76, 111.49, 103.77,
103.51, 55.39, 49.30, 43.67, 34.16, 31.56, 27.61, 27.44,
27.15, 212, 24.73.

D. 2-(5-(1,3-Dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)-1,3-

pentadienyl]-1-(6-((2,5-dioxo-1-pyrrolidi-nyl)oxy]-6-oxohexyl]-
3,3-dimethyl-3H-indolium, methyl Cy5-NHS ester (19) .
Anhydrous pyridine (0.1 mL) and DSC (23.6 mg, 0.09 mmol)
were added to a stirred solution of the acid (18) (0.36 g,
0.06 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 blue residue was purifi ed by column
chromatography on silica gel (dichloromethane/hexane/
methanol = 5:1:1) to give the pure NHS ester of the dye (19)
(0.33 g, 92%) as a blue powder ( 2, 6 ) . IR (neat): 2,926, 1,733,

1,496, 1,481, 1,456, 1,372, 1,336, 1,217, 1,182, 1,149, 1,097,
1,040, 1,016, 924, 796, 756, 708, 668/cm. 1 H NMR
(400 MHz, DMSO- d6 ): d 8.30 (2H, t, J = 13.1 Hz), 7.58 (2H,
d, J = 7.4 Hz), 7.36 (4H, m), 7.20 (2H, m), 53 (1H, t, J = 12.4 Hz),
25 (2H, t, J = 14.2 Hz), 4.05 (2H, t, J = 7.2 Hz), 3.56 (3H, s),
2.77 (4H, s), 2.64 (2H, t, J = 7.2 Hz), 1.68 (2H, m), 1.65 (2H,
m), 1.64 (12H, s), 1.44 (2H, m). 13 C NMR (100 MHz,
DMSO- d6 ): d 173.74, 173.26, 172.93, 170.71, 169.35, 154.49,

151.10, 143.21, 142.44, 141.54, 141.47, 129.11, 128.81,
125.83, 125.19, 125.05, 122.87, 122.76, 111.50, 103.44, 49.31,
49.03, 43.63, 31.54, 30.44, 27.61, 27.43, 284, 25.90, 25.66,
24.37. MS (EI): m/z (%) = 580 (100).

3.2. Validation
of Newly Synthesized
Dyes in DIGE
Experiments

To validate the newly synthesized dyes, which will be called LAB
dyes further, we applied them in a regular DIGE experiment and
compared them to commercially available dyes from GE Healthcare,
which further will be described as COM dyes.
For the experimental model, we chose a comparison between
two strains of Escherichia coli K-12 MG1655: a wild-type (WT)
and a Fis - mutant strain (both strains were kindly provided by
Daniel Yoo and Reid Johnson, Department of Biological Chemistry,
UCLA School of Medicine). Fis is a small nucleoid-associated pro-
tein, the expression levels of which undergo dramatic changes dur-
ing cell growth: it has been shown that if stationary phase cells are
transferred into a fresh rich medium such as LB (1% Tryptone,
0.5% yeast extract, 1% NaCl, pH 7.3), in about 1 h Fis levels
increase from about 100 copies per cell to tens of thousands of