Biology Reference
In-Depth Information
fluorescence upon Ca
2
þ
binding (
522/536). Indicator
34
can be
further derivatized with a SNAP-Tag ligand to allow labeling of specific
proteins in living cells for spatially restricted calcium imaging.
51
¼
l
max
/
l
em
6. FLUORESCEINS
6.1. Overview
Fluorescein (
2
) was first synthesized in 1871 by Baeyer
52
and has played
a pivotal role in fluorescence technology. Fluorescein was one of the first
labels used in immunofluorescence microscopy experiments
13
and is still
widely employed in many different biological and biochemical applica-
tions.
3
This persistence can be attributed to its ease of synthesis, the excellent
fluorescence properties of the dye (
10
4
M
1
cm
1
, and F
¼
0.95), and the many modes of modulation available
with this dye scaffold.
3,4
Fluorescein is most fluorescent as the phenolate
and bears a phenolic p
K
a
of 6.4.
53
The proximity of this p
K
a
to
physiologically relevant pH values has led to the development of
halogenated derivatives of fluorescein such as 2
0
,7
0
-difluorofluorescein
(Oregon Green;
36
). Oregon Green demonstrates
l
max
/
l
¼
490/514 nm,
e
¼
9.3
em
similar optical
10
4
M
1
properties
to fluorescein (
l
max
/
l
¼
492/516 nm,
e
¼
8.6
em
cm
1
, and
0.92) but displays a lower phenolic p
K
a
of 4.6.
54
Likewise, 2
0
,7
0
-dichlorofluorescein (
36
) exhibits a drop in p
K
a
value to
5.0 and shows a modest bathochromic shift with
F
¼
l
max
/l
em
¼
502/
0.88.
55
Halogenation with
bromine or iodine causes even larger bathochromic shifts but severely
reduces the quantum yield.
56
As mentioned in
Section 3
, a key property of fluoresceins is the equili-
brium between an “open” fluorescent quinoid form and a “closed” non-
fluorescent lactone. The structures of these molecular forms are shown in
Fig. 1.4
(
2-open
and
2-closed
). While the open fluorescent form predom-
inates in aqueous solution, acylation or alkylation of the phenolic oxygens
can lock the molecule in the nonfluorescent lactone. Thus, many of the
blocking group strategies used for hydroxycoumarins can be applied to fluo-
resceins. Fluorogenic compounds based on fluorescein are superior to the
coumarin dyes, however, as the open-closed equilibrium completely inter-
rupts the extended conjugation of the dye and thus endows fluorogenic
fluoresceins with extremely high contrast. Fluorescein is also efficiently
modulated by PeT; attachment of various groups on the pendant phenyl ring
of fluorescein can yield useful sensor molecules and ion indicators.
10
4
M
1
cm
1
, and
525 nm,
e
¼
10.1
F
¼
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