Fluorescent Environment-Sensitive Dyes as Reporters of Biomolecular Interactions - Progress in Molecular Biology and Translational Science - page 44

analogue of 4DMN, featuring improved spectroscopic properties and
chemical stability, was introduced into a peptide that is recognized by
calmodulin. Remarkably, the interaction event between these two mole-
cules yielded > 900-fold increase in the fluorescence intensity of the dye. 36
2.5. Fluoroprobe
In the search for advanced environment-sensitive dyes, a bichromophoric
dye Fluoroprobe ( Fig. 2.3 ) was developed. 14 This dye exhibits a charge
transfer through space, which generates an exceptional change in the dipole
moment (27 D) and, thus, solvent sensitivity. For the moment, Fluoroprobe
remains the most solvatochromic fluorescent dye. However, it has found no
applications in biology, because of the extremely strong quenching of its
fluorescence in polar media, UV absorption (308 nm), and very low extinc-
tion coefficient ( Table 2.1 ).
2.6. Dapoxyl Ò derivatives
It is also worth mentioning a Dapoxyl Ò dye ( Fig. 2.3 ) showing remarkable
fluorescent solvatochromism (up to 200 nm red shift from hexane to water/
acetonitrile (4/1) mixture), as well as high fluorescence quantum yield and
extinction coefficient. 16 However, its absorption in the UV range remains a
disadvantage. Moreover, its applications are limited so far to only a few
examples, such as biosensor development 39

and FRET-based assays in

peptides. 40
2.7. NBD
An important example of red-shifted environment-sensitive dye is NBD. Its
small-sized fluorophore and absorption around 480 nm are very convenient
for biological applications. However, its solvatochromism is very small,
which limits its applications to the cases where the changes in the environ-
ment are drastic. NBD was one of the first environment-sensitive dyes
(together with Dansyl) used as a protein label. NBD helped revealing a
mechanism for the transduction of ligand-induced protein conformational
changes 24 through the distinct maltose-dependent fluorescence response
observed with three individual cysteine mutants of Escherichia coli maltose-
binding protein, covalently labeled with NBD. The results provided insights
for designing fluorescent biosensors.

2.8. Phenoxazine derivatives
A representative example of phenoxazine-based dyes is Nile Red. 23 It com-
bines red-shifted absorption (around 530 nm) and emission together with
high brightness. Nevertheless, its solvatochromism is moderate, being larger