Synthesis of zwitterionic cyanine dyes for use in proteomics

Abstract

The CyDye family of fluorescent dyes are the tools currently in use today for applications in two dimensional difference gel electrophoresis (2D-DIGE) techniques. The lysine labeling CyDyes are limited by problems with over labeling resulting in protein precipitation and isoelectric point (pI) drift at high pH's. These limitations have been addressed by a family of highly water soluble and pI balancing zwitterionic BODIPY dyes, which were previously synthesized in the Grieco group. The absorbance maxima of the BODIPY fluorophores were tuned through extension of the pi system to produce a three color, spectrally resolved dye set. However the fluorescence of the green emitting BOPIDY suffered at pH's less than 3.5 and greater than 11, while the red emitting BODIPY was susceptible to Michael addition changing its emission profile. To address the limitations of the BODIPY family of dyes, a new family of zwitterionic 2DDIGE dyes based on the established CyDye fluorophores have been synthesized. A complete three dye zwitterionic minimal labeling set which features a cysteic acid motif, titratable amine functionality and an NHS activated ester group reactive towards lysine residues has been synthesized: Z-Cy2 (QY= 6.8% ± 0.1, epsilon= 155,000), Z-Cy3 (QY= 11.1% ± 0.4, epsilon= 124,500), Z-Cy5 (QY= 43.3% ± 0.6, epsilon= 217,600). In addition, a complete three dye zwitterionic saturation labeling set which incorporates a cysteic acid motif and maleimide functionality reactive towards cysteine residues has also been synthesized: Z-Cy2-Mal (QY= 6.6 % ± 0.1, epsilon= 104,500), Z-Cy3-Mal (QY= 12.4 % ± 0.5, epsilon= 127,700), Z-Cy5-Mal (QY= 40.2 % ± 0.4, epsilon= 217,400).