Fluorescent Environment-Sensitive Dyes as Reporters of Biomolecular Interactions - Progress in Molecular Biology and Translational Science - page 43

red-shifted absorption (close to 400 nm)( Fig. 2.4 ), with twice as large an ab-
sorption coefficient and a manifold larger two-photon absorption cross sec-
tion (400 Goeppert-Mayer) compared to Prodan. Moreover, studies in
organic solvents have revealed a much stronger dependence of its emission
maximum on solvent polarity ( Fig. 2.4 ), which is connected with its twice as
large change in the dipole moment (14 D).
2.4. 4-Aminophthalimide analogues
The other important family of solvatochromic dyes, which attracted atten-
tion only recently, is 4-aminophthalimide analogues. The simplest of them,
4- N , N -dimethylaminophthalimide (4DMP), can be considered as one of
the first chromophores for polarity sensing. This relatively small and rigid
molecule presents a very strong solvent sensitivity. 21 However, similar to
Prodan, it shows absorption in the UV region (380-390 nm) and a very
low absorption coefficient. To improve its properties, the 4DMP
fluorophore was extended, giving a new environment-sensitive dye
6- N , N -dimethylamino-2,3-naphthalimide (6DMN) 19 ( Fig. 2.3 ). This
fluorophore exhibits interesting fluorescence properties with emission in
the 500-600 nm range and combined (fluorescence intensity and position
of the maximum) response to changes in the environment polarity, though
its absorption properties are not considerably improved. In this respect, a
recently developed analogue of 4DMP, 4- N , N -dimethylamino-
1,8-naphthalimide (4DMN), constitutes a significant improvement, show-
ing a shifted absorption maximum at 440 nm. 36 It should be noted that,
similar to 1,8-ANS, 4DMP and all its analogues are nearly nonfluorescent
in water but become highly fluorescent in aprotic media. These properties
are important for intensiometric detection of molecular interactions. 36
However, their poor fluorescence in water makes them inefficient in the
cases where the label shows significant water exposure at all steps of
interaction.
To apply the 4DMP fluorophore for protein studies, Fmoc-protected
amino acid bearing this fluorophore was synthesized and introduced into
an octapeptide using standard solid-phase synthesis. The label was able to
sense phosphorylation-dependent binding of the synthesized peptide to
the 14-3-3bp protein. 37 Peptides labeled with a 6DMN-based amino acid
were used for monitoring protein-protein interactions, as exemplified in
studies with the SH2 phosphotyrosine binding domains. 19 The same labeled
amino acid was used for sensing peptide binding to proteins of a major his-
tocompatibility complex at the cell surface. 38 Furthermore, an amino acid