ABSTRACT
An efficient and high-yielding strategy to prepare "unsymmetrical" 4-aryl-isoxazol-3,5-dicarboxylic acid derivatives from nitroacetic esters and aromatic aldehydes has been developed. The strategy is based on the isolation and usage of the previously missed intermediate of the Dornow reaction-5-hydroxy-6-oxo-4-aryl-6H-1,2-oxazine-3-carboxylates. In addition, the mechanism of the Dornow reaction was partially revised.
ABSTRACT
A genetically encoded fluorescent tag for live cell microscopy is presented. This tag is composed of previously published fluorogen-activating protein FAST and a novel fluorogenic derivative of green fluorescent protein (GFP)-like chromophore with red fluorescence. The reversible binding of the novel fluorogen and FAST is accompanied by three orders of magnitude increase in red fluorescence (580-650â nm). The proposed dye instantly stains target cellular proteins fused with FAST, washes out in a minute timescale, and exhibits higher photostability of the fluorescence signal in confocal and widefield microscopy, in contrast with previously published fluorogen:FAST complexes.
Subject(s)
Fluorescent Dyes/chemistry , Green Fluorescent Proteins/chemistry , Rhodanine/analogs & derivatives , Cell Nucleus/ultrastructure , Fluorescence , HEK293 Cells , HeLa Cells , Humans , Microscopy, Confocal , Microscopy, Fluorescence , Optical ImagingABSTRACT
We design a novel class of excited-state locked GFP chromophores by introducing an amine group at the double exo-bond and a difluoroboryl bridge. We show that these chromophores intrinsically exhibit a very large Stokes shift of 1 eV. Further tuning through chemical modifications of their aryl substituents makes them environmentally sensitive.
