Optimization of the Coupling of Target Recognition and Signal Generation - Advanced Fluorescence Reporters in Chemistry and Biology - page 54

function such as certain sugar acids (19, Fig. 7 )[ 79 ]. The signaling mechanisms are
very similar in these cases and will not be discussed in detail here. Besides the well-
known receptor sites for anions and cations, boronic acid groups for example
complement the functional entities when targeting sugar derivatives.
A special case of the single binding site-single fluorophore architectures are
compounds like anthracene cryptands, i.e., anthracenes substituted through both
their 9,10 positions with the two nitrogen atoms of a diaza-polyoxa macrocycle so
that the fluorophore literally “sits” on top of the receptor and forms a macrobicyclus
with it [ 80 ]. In such a case, the interaction between fluorophore and nitrogen atoms
of the receptor does not proceed through PET, but through exciplex formation.
Exciplex formation, i.e., the generation of an excited complex between excited
fluorophore and for instance, an amino group in the ground state does not lead to
unspecific quenching of the (isolated) fluorophore's emission, but to the appearance
of a red-shifted, broad, and structureless emission band - the so-called exciplex
band. The reporter dyes are now designed in such a way that binding of the analyte
either interacts with one of the partners (e.g., a metal ion with a crown nitrogen
atom), rendering exciplex formation energetically unfavorable (related to the PET
case) or the guest detaches both partners from each other, making the distance-
dependent process less likely. The result of both effects is an increase of the
fluorophore's typical emission band at the expense of the exciplex band. Such an
intrinsic two-band system again harbors all the advantages of ratiometric indica-
tion. However, because few fluorophores and few receptors constitute ideal exci-
plex partners (mainly anthracene and pyrene, paired with amino nitrogens) and the
synthetic combination is challenging for more complex systems, these probes do
not play a significant role in fluorescent reporter research today. The interested
reader is referred to more specialized review literature [ 81 ].
2.2.2 Single Binding Site-Double or Multi Fluorophore Architectures
Closely related yet more important than exciplex systems are the so-called excimer
probes. These probes also exhibit two distinct emission bands, very similar to the
exciplex type. The short-wavelength band is again connected to emission from an
excited (isolated) fluorophore whereas the red-shifted band stems from an excited
dimer , involving a fluorophore in the ground and a second one in the excited state.
Accordingly, the main characteristic of such probes is that they usually consist of
one receptor site and two fluorophores. These two fluorophores usually do not form
a ground-state dimer, and therefore the absorption spectrum of such bifluorophoric
molecules very closely resembles that of an isolated (or monomeric) fluorophore.
Thus, under conventional irradiation conditions, only one of the two fluorophores is
excited. The entire probe molecule then has to be designed in such a way that
nonexcited and excited fluorophore can encounter each other during the lifetime of
the excited fluorophore, forming the excited dimer. Alignment of the chromophores
is crucial, because the formation of an excimer relies on configurational mixing of
exciton- and charge-resonance states [ 82 ]. In general, better alignment leads to