The Chemistry of Small-Molecule Fluorogenic Probes - Progress in Molecular Biology and Translational Science - page 16

sensors. 9,84,85 Finally, like the related fluoresceins, addition of different
functionality on the bottom ring of rhodamines can also be used to
modulate fluorescence, usually through PeT.
7.2. Enzyme substrates
Similar to fluorescein, acylation of rhodamine nitrogens locks the mole-
cule into a closed lactone form. This allows the construction of a variety
of fluorogenic substrates based on N , N 0 -diacylrhodamines. The classic
rhodamine-based enzyme substrates are dipeptidyl rhodamines (e.g., 53 ),
which serve as important protease substrates for many enzymes 86 including
the caspases. 87 Rhodamines can also be reduced to nonfluorescent leuco
forms. Compound 54 (i.e., Mitotracker Orange) is oxidized within the
mitochondria; the electrophilic chloromethyl group serves to retain the
compound in that organelle for imaging. 3 Other enzyme activities can
be probed with rhodamine-based probes. As with the aminocoumarins,
insertion of a self-immolative linker between the substrate moiety and
the dye can improve stability and expand the substrate scope of the dye
scaffold. Asymmetric rhodamine 55 bears a trimethyl lock acetate on
one of the nitrogen substituents and a urea on the other. The trimethyl
lock acetate makes this compound a highly stable esterase substrate; the
nonhydrolyzable urea functionality preserves significant fluorescence while
dramatically improving enzyme kinetics. 78 The trimethyl lock strategy has
been expanded to create rhodamine-based substrates for phosphatases, 88
reductases, 89 and cytochrome P450 enzymes. 90 Rhodamine sulfonamides,
such as compound 56 , have also been used to assay glutathione transferase
activity. Enzyme-mediated attack of glutathione on the dinitrophenyl ring
results in extrusion of SO 2 and release of a fluorescent rhodamine
species. 91
7.3. Photoactivatable probes
Rhodamines can serve as valuable photoactivatable dyes. The most versatile
method to cage rhodamines is via acylation of the nitrogens, as in caged
Q-rhodamine 57 , which locks the molecule in the nonfluorescent lactone
form. 67,70,81 Unfortunately, the installation of caging groups is hampered
by the poor nucleophilicity of the rhodamine nitrogens. Like fluoresceins,
the use of reduced leuco-rhodamine derivatives as intermediates can
significantly improve the synthesis of caged rhodamine dyes. 67 Alternatively,