Synthesis and Validation of Cyanine-Based Dyes for DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 71

washed with acetonitrile. The solid obtained was dried under
vacuum to give the salt (4) as a light yellow powder (3.20 g,
80%) ( 9 ). IR (neat): 3,014, 1,733, 1,579, 1,456, 1,360, 1,226,
1,164, 753/cm. 1 H NMR (400 MHz, DMSO- d 6 ): d 7.13
(4H, s), 7.01 (1H, br t, J = 7.8 Hz), 95 (1H, br d, J = 8.1 Hz),
85 (1H, br d, J = 7.8 Hz), 68 (1H, br t, J = 7.6 Hz), 5.20 (1H,
d, J = 15.0 Hz), 4.19 (1H, d, J = 15.0 Hz), 3.48 (2H, s), 3.17
(3H, s). 13 C NMR (100 MHz DMSO- d 6 ): d 173.18, 170.49,
153.38, 1375, 133.98, 130.15, 130.04, 129.54, 129.46,
128.38, 119.71, 117.15, 50.79, 40.84, 22.21.
C. 2-(2-Phenylacetamido-E-1-ethenyl)-N-ethylbenzoxazolium salt
(5) . A mixture of N , N ¢-diphenylformamidine (Aldrich, 0.38 g,
1.98 mmol) and the salt (2) (0.26 g, 1.65 mmol) in acetic
anhydride (10 mL) was refl uxed for 30 min. The solution was
cooled to room temperature, the solvent was removed under
reduced pressure, and the residue was purifi ed by fl ash chroma-
tography on silica gel (dichloromethane/hexane/metha-
nol = 5:1:1) to give the salt (5) as a light yellow powder (0.41 g,
93%) ( 9 ) . IR (neat): 3,084, 3,064, 2,978, 1,719, 1,646, 1,613,
1,589, 1,491, 1,466, 1,413, 1,372, 1,319, 1,252, 1,151,
1,004, 755, 702/cm. 1 H NMR (400 MHz, CDCl 3 ): d 9.19
(1H, d, J =13.8 Hz), 7.26-7.83 (9H, m), 5.33 (1H, d,
J = 13.8 Hz), 4.44 (2H, q, J = 7.4 Hz), 2.02 (3H, s), 1.31 (3H,
t, J = 7.4 Hz). 13 C NMR (100 MHz, CDCl 3 ): d 173.25, 169.92,
162.86, 147.68, 1488, 138.12, 131.30, 130.86, 129.62,
128.57, 127.85, 113.93, 112.21, 87.14, 42.36, 23.45, 13.79.
D. 2-(3-(3-(4-(2-carboxyethyl)phenylmethyl)-2(3H)-
benzoxazolylidene)-1-propenyl)-3-ethylbenzoxazolium, Cy2 (6) .
A mixture of the enamide (5) (0.10 g, 0.32 mmol), the salt (4)
(0.92 g, 0.32 mmol), and dry triethylamine (0.1 mL) in abso-
lute 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 (6) as a deep yellow
powder (0.11 g, 75%). IR (neat): 3,408, 2,927, 1,710, 1,609,
1,565, 1,508, 1,461, 1,394, 1,347, 1,280, 1,201, 1,154, 1,116,
1,083, 978, 906, 748/cm. 1 H NMR (400 MHz, CDCl 3 ):
d 8.38 (1H, dd, J = 13.2, 13.3 Hz), 7.16-7.48 (12H, m), 5.90
(1H, d, J = 13.3 Hz), 5.88 (1H, d, J = 13.2 Hz), 5.22 (2H, s),
4.12 (2H, br q, J = 7.2 Hz), 3.51 (2H, s), 1.30 (3H, t, J = 7.2 Hz).
13 C NMR (100 MHz CDCl 3 ): d 174.31, 1637, 162.25, 161.74,
148.00, 1488, 1473, 131.23, 130.93, 130.58, 130.38, 127.17,
1233, 1223, 125.59, 125.38, 111.01, 110.86, 110.76, 110.53,
85.95, 85.26, 47.41, 45.52, 39.60, 12.98.
E. 2-(3-(3-((4-(2-((2,5-Dioxo-1-pyrrolidinyl)oxy)-2-oxoethyl)phe-
nyl)methyl)-2(3H)-benzoxazolylidene)-1-propenyl)-3-ethyl-
benzoxazolium, Cy2-NHS ester (7) . Anhydrous pyridine (0.1 mL)