Fluorescent Environment-Sensitive Dyes as Reporters of Biomolecular Interactions - Progress in Molecular Biology and Translational Science - page 45

than that of NBD, but smaller than that of the UV/blue analogues described
above ( Table 2.1 ). In the recent years, a variety of Nile Red derivatives for
biomolecule labeling have been reported. 41,42 Thiol-reactive Nile Red
derivatives were successfully applied for labeling a galactose/glucose
binding protein. The obtained conjugate was validated as a fluorescence
biosensor for glucose detection. 43 Phenoxazine derivatives, which are more
compact analogues of Nile Red, were also used for designing the thiol-
reactive probe, aminophenoxazone maleimide (APM), which was used for
reporting protein conformational changes. 44 APM has a short linker
between the probe and the protein, ensuring that it can closely follow the
motions the water-exposed domain of the b 2 adrenergic receptor during
its interactions with ligands. In addition, APM was shown to sense the
conformational changes underlying voltage sensing in the Shaker potassium
channel. 44
2.9. New advanced solvatochromic dyes
This overview on the existing environment-sensitive dyes shows that there
is a strong need for new solvatochromic fluorescent dyes presenting both
strong solvatochromism and good fluorescence properties. Most of the dyes
described above (except Nile Red and NBD analogues) absorb in the UV
range ( Table 2.1 ), which is generally not suitable for biological applications.
Excitation at longer wavelengths would decrease significantly the photo-
damage of the biological samples and decrease their autofluorescence.
The photostability of most of environment-sensitive dyes is also limited
so that they are not compatible with some modern techniques, such as
single-molecule fluorescence detection. 45 Moreover, the low fluorescence
quantum yield in aqueous media of most of the described dyes limits their
application for the investigation of small peptides, where the label is exposed
to water. Finally, sensitivity to solvent polarity of these dyes is frequently not
enough to detect subtle changes in the environment of the biomolecule of
interest. Therefore, the research in this field is now focused on improving
the solvent sensitivity of the dyes, shifting their emission wavelength
to the red and increasing fluorescence brightness and photostability.
For example, the 2-dicyanomethylene-3-cyano-2,5-dihydrofuran family
of fluorophores (see DCDHF in Fig. 2.3 ), featuring red-shifted absorption
and good solvatochromism, appears promising for single-molecule experi-
ments because of their very high photostability. 46,47 The recently
introduced 3-methoxychromones (3MC-2) appear also of interest, as in
addition to good photostability and fluorescence quantum yields, they