Photoactivatable Hemicyanine Chromophores as Fluorescent Lables

Abstract

A series of photoactivatable hemicyanine chromophores containing azido group instead of the usual amino donor group has been designed and synthesized. Before photolysis, these compounds have negligible fluorescence. Upon photolysis, a molecule of nitrogen is lost from the azido group leaving behind a highly reactive nitrene moiety, which can react with and crosslink neighboring molecules to produce an amine. This fully restores the “push-pull” environmentally sensitive hemicyanine chromophore. If the molecule is in a cell membrane it will covalently crosslink lipid or protein molecules and remain relatively immobile. The photoactivity of the dyes was tested on neuroblastoma cells by selectively shining near UV or visible light on a small region of the cell and then observing the whole cell's fluorescence using a confocal microscope. This experiment shows strong fluorescence primarily in the illuminated area, which proves that we can selectively and stably stain a portion of the cell membrane using these photoactivatable hemicyanine dyes. The photoreleased dyes are fluorescent in the membrane because the amino-chromophore is similar to our voltage sensitive ASP and ANEP chromophores. These compounds are promising as targetable sensors of membrane potential. (This work is supported by Human Frontier Science Program grant RGP0027/2009 and NIH grant R01EB001963.)