The Chemistry of Small-Molecule Fluorogenic Probes - Progress in Molecular Biology and Translational Science - page 25

fluorophores. 138,139 Appropriate structural modification of these dyes through
the techniques discussed previously can yield fluorogenic derivatives. Another
unique dye scaffold is 2-(2 0 -hydroxy-5 0 -chlorophenyl)-6-chloro-4( 3 H )-
quinazolinone (HPQ, 91 ). This compound is insoluble in water and highly
fluorescent in the solid state (

375/510 nm). Derivatization of
the phenolic oxygen blocks the formation of the internal hydrogen bond
and gives soluble nonfluorescent substrates. 140 Finally, hybrid structures
between two dye classes can yield fluorescent molecules with nuanced
properties. These include the “rhodols” such as compound 92 ,whichshare
characteristics of both fluorescein and rhodamine dyes. 141 Another fusion of
dye classes are the rhodamine-cyanine hybrids

l max / l em ¼

such as compound 93 ,

¼

which absorb at long wavelengths ( 720/750 nm), but can be

modulated using similar strategies to fluorogenic rhodamines. 142

l max / l em

11.2. Fluorogenic enzyme substrates
Like the more common fluorophores, attachment of enzyme-labile moieties
to these fluorescent compounds can yield substrates for various enzymes.
In particular, the HPQ scaffold has been used to prepare a variety of fluo-
rogenic substrates by derivatization of the phenolic oxygen. 140 The unique
precipitating HPQ product links the fluorescence signal to the position of
the enzyme, which is useful for immunohistochemistry, mRNA in situ hy-
bridization, and microarray experiments. This nondiffusible substrate can
also be used to map enzyme activity and location in living cells. The most
widely used HPQ substrate is phosphate 94 , which is highly soluble and an
excellent substrate for alkaline phosphatase. 143,144 Another example is amine
95 , which has been used to illuminate the activity of cellular monoamine
oxygenase. 145
The rhodols can also serve as useful scaffolds for enzyme substrates. Anal-
ogous to the Tokyo Green substrate 43 ( Fig. 1.4 ), compound 96 is non-
fluorescent when acylated. Removal of the peptide by protease activity
affords a large increase in fluorescence; the oxygen substituent in 96 can
be used to anchor substrates to a solid support. 146 Another rhodol-based
substrate is b -galactoside 97 . Here, the open-closed equilibrium of the
rhodol is modified by reduction of the ortho -carboxyl group to the alcohol.
Upon alkylation of the rhodol phenol, the compound adopts a closed, non-
fluorescent form around pH 7. Glycolysis elicits a large increase in fluores-
cence; this compound can be used to measure b -galactosidase activity in
living cells. 147