Synthesis and Validation of Cyanine-Based Dyes for DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 69

3. Methods

General notes
All reactions were carried out under an atmosphere of nitrogen,
and all commercial reagents were used as provided by the manufac-
turers. 1 H and 13 C NMR data were obtained on a Bruker 400 MHz
spectrometer. 1 H NMR and 13 C NMR data are reported in parts
per million (d) downfi eld from tetramethylsilane. The following
abbreviations are used: s (singlet), d (doublet), t (triplet), q (quar-
tet), m (multiplet), and br (broad). Infrared spectra were recorded
on a Thermo Nicolet Avatar 370 FT infrared spectrophotometer as
a liquid fi lm or as a thin crystalline fi lm. All IR data are reported in
wavenumbers (per cm). Thin-layer chromatography (TLC) was
performed using Merck silica gel 60F254 0.2 mm alumina-backed
plates. Visualization was accomplished using ultraviolet light or
one of the following stains: anisaldehyde, phosphomolybdic acid,
and potassium permanganate. Flash chromatography was carried
out using ICN Biomedicals silica gel 60 (230-400 mesh). Mass
spectrometry analysis was carried out using electrospray ionization
(ESI) on an IonSpec FT mass spectrometer.

3.1. Dye Synthesis

The synthesis of the Cy2, the benzoxazolium dye, and its NHS
ester (see Fig. 1 ) began with alkylation of the commercially avail-
able 2-methylbenzoxazole (1) with two different alkyl halides:
alkylation with ethyl bromide gave the ethyl salt (2) ( 8 ) in 22%
yield (this compound is also commercially available), while alkyla-
tion with the commercially available 4-bromomethylphenylacetic
acid (3) gave an 80% yield of the salt (4) ( 9 ). Condensation of the
ethyl salt (2) with diphenylformamidine in the presence of excess
acetic anhydride as solvent afforded the acetanilidylvinyl indolium
salt (5) in 93% yield ( 10-13 ). This compound was then reacted
with the other benzoxazolium salt (4) in ethanol in the presence of
triethylamine to give the desired dye (6) in 75% yield as a deep yellow
powder. The dye was easily converted into the NHS ester by treat-
ment with DSC in the presence of pyridine to give the activated
dye (7) in 86% yield. The spectroscopic data were in agreement
with the structures assigned (see Note 1).

3.1.1. Cy2 Synthesis

A. N-Ethyl-2-methylbenzoxazolium bromide (2) . A mixture of
2-methylbenzoxazole (1) (Aldrich, 2.24 g, 0.01 mol) and bro-
moethane (Aldrich, 2.5 mL, 0.03 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 l-
tered and washed with diethyl ether. The solid obtained was
dried under vacuum to give the salt (2) as a white powder
(0.60 g, 22%) (

14, 15 ) . This compound is also commercially
available from various suppliers (Aldrich, Alfa Aesar, TCI, etc.).
IR (neat): 3,084, 3,047, 2,974, 2,929, 2,859, 2,729, 1,593,