The Chemistry of Small-Molecule Fluorogenic Probes - Progress in Molecular Biology and Translational Science - page 26

11.3. Photoactivatable probes
HPTS ( 89 ) is most fluorescent in the phenolate form and has found use as a
pH sensor with a p K a 7.3. 139 Thus, like other phenolic fluorophores, al-

kylation of the phenol is a useful strategy to control fluorescence. This has
been used to prepare highly soluble caged fluorophores such as compound
98 . 148 Caged HPTS has been used to measure diffusion in neuronal spines 149
and for calibration during photo-uncaging experiments. 150

11.4. Indicators
In addition to being useful labels and fixable polar tracers, 3,138 the amino-
napthalimides are modulated by attachment of labile moieties to the
aniline nitrogen, making them useful scaffolds for fluorogenic molecules.
Acylation of this group causes a sharp decrease in fluorescence intensity.
Compound 99 is a sensor for fluoride ion, where F causes cleavage of
the TIPS group and extrusion of quinone methide and CO 2 , leading to
the release of the free amino-naphthalimide (

421/508 nm). 151
Similar naphthalimide sensors have been built for other analytes. 152,153
Finally, the rhodamine-cyanine hybrids can serve as turn-on fluorescent
sensors. Because this dye system is imitative of rhodamine dyes, these
compounds exist in equilibrium between a fluorescent, open form and a
nonfluorescent, closed form. Akin to the rhodamine dyes, lactam
derivatives, including molecule 100 , adopt the closed, nonfluorescent
form. Oxidation of the thiosemicarbazide by hypochlorate recapitulates
the fluorescent structure. 142

l max / l em ¼

12. CONCLUSIONS AND FUTURE DIRECTIONS
In this chapter, we described the major classes of fluorescent dyes and
the chemistry involved in controlling the optical properties of these dyes.
We note some general design principles. Oxygen-containing fluorophores,
such as methylumbelliferone, fluorescein, resorufin, and DDAO, are typi-
cally most fluorescent in the deprotonated state. Thus, O -alkylation or
O- acylation of these fluorophores can be used to suppress fluorescence.
Likewise, N -acylation of nitrogen-containing fluorophores, such as rhoda-
mines, significantly decreases fluorescence intensity. Fluorogenic molecules
based on fluoresceins, rhodamines, and some rhodols have the added benefit
of adopting a colorless form upon derivatization, yielding fluorogenic com-
pounds with extremely high contrast. Rhodamines can also be masked by