Biology Reference
In-Depth Information
Aminocoumarins serve as another type of scaffold for enzyme substrates.
Amidation of aminocoumarins with peptide chains, as in compound
21
, can
yield valuable protease substrates.
37
This strategy has been expanded to per-
form screening of protease activities against substrate libraries.
38
The low pH
sensitivity of aminocoumarins makes it desirable to use this scaffold for other
enzymes. Installation of a self-immolative linker between the substrate moi-
ety and the fluorophore increases the diversity of enzyme substrates that can
be built upon a given scaffold. For example, esterase substrate
22
is prepared
by inclusion of a “trimethyl lock” unit
39
between an acetate and the
7-aminocoumarin fluorophore
11
.
27
Hydrolysis of the acetate yields an
intermediate that undergoes a rapid N
O acyl shift, releasing the
fluorophore. Attachment of blocking groups at other positions can also
modulate
!
fluorescence. Compound
23
exhibits
low fluorescence
(
F
<
0.1), presumably due to the electron-withdrawing nature of the
ketone. Dehydrogenase-catalyzed reduction of the ketone yields a highly
fluorescent coumarin alcohol.
40
4.3. Photoactivatable probes
In addition to enzyme activity, light can be used to turn on fluorescent spe-
cies. Analogous to the coumarin-based enzyme substrates, attachment of
photolabile groups to the hydroxyl groups of hydroxycoumarins quenches
fluorescence, yielding “caged” fluorophores. Compounds such as
24
bear an
ortho
-nitrobenzyl cage as a blocking group on the phenol. These caged cou-
marins exhibit unexpected and desirable properties including high two-
photon cross sections and extraordinary photochemical quantum yields.
41
These unexpected properties are likely caused by the quenched coumarin
moiety acting as an antenna for the cage. Absorption of light by the couma-
rin moiety, followed by energy transfer to the cage, causes the desirable
properties observed. These properties have enabled sophisticated experi-
ments to trace gap junctions connectivity in developing animals.
42
As mentioned in
Section 3
, another method to modulate fluorescence
using light is to prepare
trans
-cinnamic acid derivatives such as compound
6
(see also
Fig. 1.1C
). Illumination of such molecules with UV light causes
trans
cis
isomerization.
17,18
The cis form undergoes rapid lactonization
with cleavage of the ester bond. This strategy is unique to the coumarins
and has the added benefit of being able to release a molecule, such as an
alcohol, concomitantly with photoactivation. This raises the possibility of
releasing two fluorophores with a single photon.
!
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