The Chemistry of Small-Molecule Fluorogenic Probes - Progress in Molecular Biology and Translational Science - page 13

substrates for glycosylases, such as the b -galactosidase substrate 40 . 60,63
Substrates for b -lactamase, such as 41 , can be synthesized based on
this scaffold. 64 Despite the utility of fluorescein diether substrates, the
synthesis of such compounds can be difficult. Treatment of fluoresceins
with alkylating agents typically gives an ether-ester mixture, such as
observed with dibenzylfluorescein 42 . 65 While still useful for some
assays—compound 42 is a cytochrome P450 substrate with modest
utility 66 —the ether-ester cannot adopt the colorless lactone form and
displays a much higher fluorescence background than a diether substrate.
To overcome this synthetic issue, fluorescein diethers such as 41 can be
prepared using a reduced fluorescein intermediate, which allows efficient
alkylation of both phenolic oxygens. 64,67 Another strategy to circumvent
the diether problem is to tune the electronics of the dye such that only
one masking group is required to quench fluorescence. Fluorescein
derivative 43 bears an electron-rich pendant ring that quenches the
fluorescence of the dye when it is alkylated. Glycolysis catalyzed by
b -galactosidase yields the bright Tokyo Green fluorophore. 68 Finally,
fluorescein diethers can be used to probe esterase activity. Fluorescein
diacetate 1 is notoriously unstable in aqueous solution, making it of
limited utility for the sensitive measurement of esterase activity in
complex biological environments. Insertion of an oxygen-methylene
moiety into the ester bond yields fluorescein di(acetoxymethyl ether)
substrates such as 44 . These compounds are chemically stable, allowing
whole-cell analysis of endogenous esterase activity and discovery of
selective esterase-ester pairs. 58,69
6.3. Photoactivatable probes
Like the dialkylfluorescein enzyme substrates, attachment of photolabile
groups to the oxygens of fluorescein can yield “caged” fluorescein. These
photoactivatable dyes have proved instrumental in the elucidation of
different dynamic cellular processes. 70 As mentioned above, dialkylation
of fluorescein is hampered by the competing reactivity of the carboxylate
on the bottom ring. Use of reduced fluorescein intermediates has enabled
the synthesis of new photoactivatable dyes such as the caged
difluorofluorescein 45 bearing a carboxyl group for bioconjugation. 67 This
general strategy should allow the synthesis of many caged fluorescein
derivatives with diverse photolabile groups.