Biology Reference
In-Depth Information
6.4. Indicators
Attachment of disparate groups to fluorescein can endow this dye with sen-
sitivity to environmental changes; numerous indicator molecules have been
built from this scaffold. Three main strategies of sensing are exemplified by
the following examples. Diaminofluorescein
46
is relatively nonfluorescent
(F
¼
0.004) as a result of quenching by PeT. Irreversible reaction with nitric
oxide (NO) yields a triazine, which shows a significant increase in fluores-
cence intensity (
0.78), allowing sensing of NO in a biological context.
71
Reactive groups on the phenolic oxygens can also quench fluorescence.
Allyl ether
47
is relatively nonfluorescent but can be activated through
deallylation with Pd. This could allow sensitive detection of catalyst contam-
inants in pharmaceuticals.
72
Perhaps the most important fluorescein-based
indicators are the BAPTA-based “Fluo” calcium ion sensors originally de-
veloped by Tsien and coworkers. The dichlorofluorescein derivative
48
(Fluo-3) shows
>
100-fold increase in fluorescence intensity upon binding
to Ca
2
þ
.
3,21
Importantly, this and related molecules can be delivered to the
interior of cells and have been instrumental for high-throughput screening
in living cells
73
and functional imaging in intact brain tissue.
74
F
¼
7. RHODAMINES
7.1. Overview
The amino isologs of fluorescein, namely, the rhodamines, were first described
in the 1880s.
75-77
The simplest rhodamine, rhodamine 110 (
49
;
Fig. 1.5
), has
similar
optical
properties
to
fluorescein
(
l
max
/
l
¼
496/517 nm,
em
10
4
M
1
cm
1
,and
e
¼
0.92) but, like aminocoumarins, is pH
insensitive.
3,78
Also analogous to fluorescein, acylation of the rhodamine
nitrogens in dye
49
locks the molecule into a closed lactone form that is
colorless and nonfluorescent. This enables a variety of fluorogenic
compounds to be built on
N
,
N
0
-diacylrhodamines.
67,78,79
However, unlike
fluoresceins, alkylation of the aniline nitrogens does not enforce the closed
form but instead maintains high fluorescence and elicits a bathochromic
shift. For example, tetramethylrhodamine (
50
) displays
7.4
F
¼
l
max
/l
em
¼
540/
10
4
M
1
cm
1
albeit with a lower quantum yield of
0.68.
2
Fully alkylated rhodamines with more rigid structures impose even
larger spectral shifts, as in the julolidine derivative rhodamine 101 (
51
;
565 nm and
e
¼
9.5
l
max
/
l
575/596 nm). Rhodamine 101 also displays high quantum efficiency
approaching unity due to the rigid structure of the dye.
80
Although this
¼
em
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