Biology Reference
In-Depth Information
3. The chelator should be chemically and photolytically stable.
4. Photolysis by a bright flash of light should allow rapid changes in the free
Ca
2
þ
level; this characteristic requires rapid photochemical and subsequent dark
reactions of the chelator.
5. Photolysis should be achievable with biologically appropriate wavelengths,
which requires a high quantum e
ciency and absorbance at wavelengths that
readily penetrate cytoplasm but cause little biological damage, that is, that are
not highly ionizing. For the chelator to be protected from photolysis by light
needed to view the preparation would also be useful.
6. The photoproducts, or postphotolysis bu
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V
er mixture, should continue to
er Ca
2
þ
, and so hold it at the new level in the face of homeostatic pressure
from membrane pumps and transport processes.
7. Neither the unphotolyzed chelator nor its photoproducts should be toxic, but
rather should be inert with respect to all ongoing cellular molecular and physio-
logical processes. Three classes of compounds, the nitr series, DM-nitrophen, and
the diazo series, share enough of these properties to have generated intense interest
and widespread popularity, and form the subjects of this review.
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Numerous more general reviews of photolabile or caged compounds, which
contain some information on photolabile Ca
2
þ
chelators, have appeared (
Adams
and Tsien, 1993; Gurney, 1993; Kao and Adams, 1993; Kaplan and Somlyo, 1989;
McCray and Trentham, 1989; Ogden, 1988; Parker, 1992; Walker, 1991
). Reviews
focused more on photosensitive Ca
2
þ
chelators may also be consulted (
Ashley
et al., 1991a; Ellis-Davies, 2003; Gurney, 1991; Kaplan, 1990
).
II. Nitr Compounds
A. Chemical Properties
The first useful class of photosensitive Ca
2
þ
chelators was developed by Roger
Tsien. This nitr class of compounds relies on the substitution of a photosensitive
nitrobenzyl group on one or both of the aromatic rings of the Ca
2
þ
chelator 1,2-bis
(o-aminophenoxy)ethane-N,N,N
0
,N
0
-tetracetic acid (BAPTA) (
Adams and Tsien,
1993; Adams et al., 1988; Kao and Adams, 1993; Tsien and Zucker, 1986
). Light
absorption results in the abstraction of the benzylic hydrogen atom by the excited
nitro group and oxidation of the alcohol group to a ketone. The resulting nitro-
sobenzoyl group is strongly electron withdrawing, reducing the electron density
around the metal-coordinating nitrogens and reducing the a
nity of the tetracar-
boxylate chelator for Ca
2
þ
. In the first member of this series, nitr-2, methanol is
formed as a by-product of photolysis, but in subsequent members (nitr-5, nitr-7,
and nitr-8) only water is produced. Photolysis of nitr-2 is also slow (200 ms time
constant). For the other nitr chelators, the dominant photolysis pathway is much
faster (nitr-7, 1.8 ms; nitr-5, 0.27 ms; and nitr-8, not reported). For these reasons,
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