the fluorophore. Herein, we describe a new series of rhodamine-
based analogues designed for the optical detection of pH.
Starting with commercially available rhodamine B, we
synthesized various benzylamine structures (see the Supporting
Information for synthesis details) where the amine moiety is
primary, secondary, and tertiary (Figure 1). The rhodamine-
Figure 2. Fluorescence response to pH for compounds 1-4
measured using 0.37 µM solutions in 15% EtOH/H2O (v/v).
Excitation wavelength was 550-560 nm. Fluorescence intensities
are relative to a 0.37 µM solution of rhodamine B in H2O.
Figure 1. Structures of pyronin B, rhodamine B, and benzylamine
moiety substituted pyronin B derivatives 1-4.
for probes 1-3 were as expected, with each compound showing
a completely nonfluorescent form under basic conditions, which
gradually increases in fluorescence intensity by 4-fold when
exposed to acidic conditions. Probe 1 is the most sensitive to
proton concentration, likely due to better solvation and a higher
pKa than the other three. Probe 4, however, had a very
interesting response. The tertiary amine moiety is incapable of
undergoing intramolecular cyclization; therefore, it was surpris-
ing that this compound had a response to pH. The relative
fluorescence intensity remains above 0.1 at all times and triples
as the pH changes from ∼7 to 4. Of the four analogues used to
detect pH, probe 3 in Figure 2 has the most responsive curve
in the “acidic window”, which is regarded to be of great interest
in diagnosis and understanding for some of the aforementioned
diseases.
Given that spiro-cyclic structures have been thoroughly
investigated in other rhodamine systems, and their existence is
proven by X-ray crystallography,7c,8a,b we thought an NMR
study should be sufficient in distinguishing between a spiro-
cyclic and an open structure for our series. Specifically, we
examined the 13C and DEPT spectra for insight on our
various derivatives.
As shown in Figure 3, probes 1-3 all have a quaternary
carbon atom in the alkyl region of their 13C spectra. This signal
corresponds to the carbon atom at position 9, indicating that
probes 1-3 exist in a spiro-cyclic structure. Probe 4, however,
has no such quaternary carbon in the alkyl region; therefore,
carbon atom 9 must lie in the aromatic region. Further NMR
experiments are needed to confirm the exact chemical shift of
carbon atom 9 in the aromatic region. This is consistent with
13C spectra of commercial rhodamine ester derivatives.9
Further evidence obtained by UV-vis titrations verifies a
spirocyclic structure for probes 1-3 and proposes a mode
based literature is rife with fluorescent probes that are regulated
by a spirocyclic structure. These spirocyclic compounds lack
absorbance and fluorescence, in the visible spectrum, when
engaged in the spirocyclic form but will open upon binding to
specific analytes to restore the fluorophore and fluorescence
intensity of that compound. Typically, these compounds are in
the form of a rhodamine lactone- or lactam-type derivative,8
which have been mostly applied in the detection of heavy metal
cations. However, Nagano et al. recently published work on a
probe for hypochlorous acid involving a spiro-thiophene
derivative.7c We envisioned that our series of structurally related
analogues should interact with the pyronin B fluorophore in a
similar fashion but differ in their response to pH on the basis
of the nucleophilicity and steric hindrance of the amine moiety.
Specifically, probes 1-3 should take on a spirocyclic structure
in the absence of protons at high pH, with 3 maintaining the
most spiro-like character. Compound 4, however, contains a
tertiary amine, which will avoid intramolecular cyclization, and
should thus maintain its fluorescence. This is analogous to
rhodamine derivatives bearing a tertiary amide or an ester
functionality at the 2′ position, e.g., rhodamine 6g.
Upon titrating the fluorescence response to pH for probes
1-4 (see the Supporting Information for fluorescence spectra),
we obtained the following curves shown in Figure 2. The results
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Org. Lett., Vol. 12, No. 14, 2010
Best, Quinn A.
