Conspectus
Molecular fluorescent probes
are an essential experimental tool
in many fields, ranging from biology to chemistry and materials science,
to study the localization and other environmental properties surrounding
the fluorescent probe. Thousands of different molecular fluorescent
probes can be grouped into different families according to their photophysical
properties. This Account focuses on a unique class of fluorescent
probes that distinguishes itself from all other probes. This class
is termed photoacids, which are molecules exhibiting a change in their
acid–base transition between the ground and excited states,
resulting in a large change in their p
K
a
values between these two states, which is thermodynamically described
using the Förster cycle. While there are many different photoacids,
we focus only on pyranine, which is the most used photoacid, with
p
K
a
values of ∼7.4 and ∼0.4
for its ground and excited states, respectively. Such a difference
between the p
K
a
values is the basis for
the dual use of the pyranine fluorescent probe. Furthermore, the protonated
and deprotonated states of pyranine absorb and emit at different wavelengths,
making it easy to focus on a specific state. Pyranine has been used
for decades as a fluorescent pH indicator for physiological pH values,
which is based on its acid–base equilibrium in the ground state.
While the unique excited-state proton transfer (ESPT) properties of
photoacids have been explored for more than a half-century, it is
only recently that photoacids and especially pyranine have been used
as fluorescent probes for the local environment of the probe, especially
the hydration layer surrounding it and related proton diffusion properties.
Such use of photoacids is based on their capability for ESPT from
the photoacid to a nearby proton acceptor, which is usually, but not
necessarily, water. In this Account, we detail the photophysical properties
of pyranine, distinguishing between the processes in the ground state
and the ones in the excited state. We further review the different
utilization of pyranine for probing different properties of the environment.
Our main perspective is on the emerging use of the ESPT process for
deciphering the hydration layer around the probe and other parameters
related to proton diffusion taking place while the molecule is in
the excited state, focusing primarily on bio-related materials. Special
attention is given to how to perform the experiments and, most importantly,
how to interpret their results. We also briefly discuss the breadth
of possibilities in making pyranine derivatives and the use of pyranine
for controlling dynamic reactions.