Biology Reference
In-Depth Information
diaphorase, inside living cells yields the fluorescent resorufin (
62
), making
this molecule a valuable reagent for the determination of cell viability.
111
Resorufin can also be reduced to a nonfluorescent form as in compound
70
. This molecule is oxidized by H
2
O
2
in a horseradish peroxidase-catalyzed
reaction to yield resorufin and is widely used in the enzyme-linked immu-
nosorbent assay (ELISA).
3,100
The amine-containing phenoxazoles are also useful scaffolds for enzyme
assays. Analogous to the rhodamine-based protease substrates, acylation of
Cresyl Violet and its derivatives with amino acids gives valuable fluorogenic
compounds.
27,104,112,113
One example is compound
71
, which releases a far-
red fluorophore (
10
4
M
1
cm
1
, and
l
max
/
l
¼
633/675 nm,
e
¼
3.6
em
0.10) upon cleavage of the peptide.
114
In a similar vein, the Cresyl
Violet sulfonamide
72
has been described as a fluorogenic glutathione
transferase substrate.
91
F
¼
8.3. Photoactivatable probes
Photolabile “caged” resorufin was developed by Mitchison for use in im-
aging assays to determine the dynamic behavior of actin filaments.
99
A benefit of this dye is the relatively long wavelengths (
572/
585 nm) when compared to the caged fluoresceins and rhodamines. How-
ever, like the other alkyl resorufins, the quenching of the fluorescence is
incomplete, limiting the utility of this dye in experiments that require
high-contrast dyes.
l
max
/
l
¼
em
8.4. Indicators
The long wavelength of resorufin makes it an attractive platform for sensors.
One example is the reduced boronic ester
74
, which reacts spontaneously
with H
2
O
2
to yield the fluorescent resorufin. Other phenolic fluorophores,
such as fluorescein, can be masked in a similar fashion.
115
Resorufin can also
be appended with arsenical substituents to give the resorufin arsenical hair-
pin binder (ReAsH;
75
). This compound is nonfluorescent in solution but
becomes fluorescent upon binding to an engineered tetracysteine motif on a
protein.
116
Yet another sensor molecule based on the phenoxazines is Nile
Red (
8
). As highlighted in
Section 3
, this compound becomes fluorescent in
a lipophilic environment. In addition to staining lipid droplets in cells,
20
this
fluorogenic phenomenon has also been used to perform super-resolution
microscopy on cellular membranes.
117
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