Optimization of the Coupling of Target Recognition and Signal Generation - Advanced Fluorescence Reporters in Chemistry and Biology - page 51

seminal works of de Silva's and Czarnik's groups [ 60 , 61 ]. In contrast to ICT
probes, both functional units are not -conjugated, i.e., they are both characterized

by their own characteristic molecular orbitals, absorption, and emission bands. The
absorption spectrum of the supramolecule is a linear combination of the individual
fragments' bands. These molecules are designed in such a way that either the
HOMO or the LUMO of the receptor (HOMO R , LUMO R ) is energetically situated
between the HOMO and LUMO of the fluorophore (HOMO F , LUMO F ). In the first
case, absorption of a photon by the fluorophore promotes an electron from HOMO F
to LUMO F , making room for an electron to relax from the intermediately lying
HOMO R into HOMO F . This ET is a nonradiative process and is then followed by a
second nonradiative process, the decay of the electron from LUMO F into HOMO R
upon return of the molecule to S 0 . For the second configuration, the sequence is as
follows. Photoexcitation again transfers an electron from HOMO F to LUMO F .
However, when now LUMO R is situated between the fluorophore-centered MOs,
the electron relaxes from LUMO F into the empty LUMO R and finally decays from
there back to HOMO F , again through two nonradiative processes. Because two ET
processes are triggered in these probes by the absorption of a photon, these probes
are commonly referred to as photoinduced ET or PET probes. The net effect in both
cases is the quenching of the fluorescence of the fluorophore. The degree of
quenching depends on the relative energetic positions of HOMO F , LUMO F and
HOMO R (or LUMO R ) and the distance between fluorophore and receptor. To
provide these supramolecules with signaling capabilities, the receptor unit has to
be chosen or designed in such a way that the binding of the analyte at the receptor
dramatically alters the energy levels of the receptor-centered MOs, shifting them at
best out of the HOMO F -LUMO F window and thus rendering PET energetically
unfavorable. Since the fluorescence transition is only fluorophore-localized, the
effect of target binding is an enhancement of the typical fluorophore emission,
without any pronounced spectral shifts (cf. traces 2 and DMA in Fig. 6c ). A detailed
account on PET signaling can be found in [ 64 ] and earlier reviews cited therein.
With respect to signal enhancement, the PET mechanism harbors an enormous
potential and already the very first PET probes reported set benchmarks compara-
tively high (Fig. 6 ). Fluorescence enhancement factor (FEF) of several hundred or
higher are even today reached in only a few cases. Moreover, compared with the
integrative ICT probes, system design is rather simple in the PET case. Knowledge
of the redox potentials, absorption and emission data of common fluorophores, and
receptor mimics, together with the knowledge of binding constants and coordina-
tion features for a certain host-guest pair (in a particular solvent), 11

p

allows to

largely predict the achievable enhancement, employing the Rehm-Weller

11

The solvent has to be taken into account when comparing absolute FEF values (in particular of
amine-containing probes such as 13 and 14) because the quenched “off” state of a PET probe is
often higher fluorescent in protic organic and especially aqueous solvents (or solvent mixtures)
than in nonprotic solvents due to hydrogen bonding interactions between solvent and nitrogen
atom. For example, whereas 14 shows a 2,260-fold weaker fluorescence than its parent 9,10-
dimethylanthracene in nonrotic acetonitrile, the maximum FEF in water amounts to ca. 360.