Fluorescence switching of imidazo[1,5-a]pyridinium ions: PH-sensors with dual emission pathways

Article abstract of DOI:10.1021/ol3012524

Imidazo[1,5-a]pyridinium ions are identified as highly emissive and water-soluble fluorophores accessed by an efficient three-component coupling reaction. Synthetic modifications of groups conjugated to the polyheterocyclic core are shown to profoundly im

Full text of DOI:10.1021/ol3012524

ORGANIC
LETTERS
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Vol. XX, No. XX
000–000
Fluorescence Switching of
Imidazo[1,5-a]pyridinium Ions:
pH-Sensors with Dual Emission Pathways
ꢀ
Johnathon T. Hutt, Junyong Jo, Andras Olasz, Chun-Hsing Chen, Dongwhan Lee,*
and Zachary D. Aron*
Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington,
Indiana 47405, United States
dongwhan@indiana.edu; zaron@indiana.edu
Received May 7, 2012
ABSTRACT
Imidazo[1,5-a]pyridinium ions are identified as highly emissive and water-soluble fluorophores accessed by an efficient three-component
coupling reaction. Synthetic modifications of groups conjugated to the polyheterocyclic core are shown to profoundly impact the emission
properties of these molecules. Notably, two structural isomers of functionalized imidazo[1,5-a]pyridinium ions were found to exhibit distinct
de-excitation pathways, which are responsible for either a fluorescence turn-on or ratiometric response to pH change.
Small-molecule fluorophores are emerging as important
tools for visualizing analytes of biological relevance.1
Quantitative data can readily be obtained for specific
targets by operationally simple detection schemes exploit-
ing binding-induced perturbations of de-excitation path-
ways such as photoinduced electron transfer (PET) or
intramolecular charge transfer (ICT). Changes in the in-
tensity and/or wavelength of light emitted from molecular
probes have been used in the detection of heavy metal ions2
and anionic metabolites;3 in the labeling of amino acids,
peptides, nucleotides, and other macromolecules;4 and for
monitoring reactive oxygen/nitrogen species.5 Although
existing fluorogenic platforms including BODIPY, cou-
marin, naphthalimide, or fluorescein can be functionalized
to display either ratiometric dual-emission (through ICT)
or change in fluorescence intensity (through PET), struc-
tural modifications that control excited-state photophysics
typically entail low-yielding multistep synthetic operations.6
Fluorescent probes operating in biological systems must
also exhibit water solubility, insensitivity to local polarity,
and minimal interference from background emission, re-
quirements which further highlight the ongoing challenges
in this area.
(1) (a) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.;
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In this paper, we disclose the structural and photophy-
sical properties of water-soluble imidazo[1,5-a]pyridinium
ion fluorophores that are readily prepared by a facile three-
component coupling (Scheme 1).7 In both organic and
(2) (a) Chen, X.; Pradhan, T.; Wang, F.; Kim, J. S.; Yoon, J. Chem.
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r
10.1021/ol3012524
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aqueous environments, these molecules exhibit high
quantum-yield emissions whose wavelength can be system-
atically modified through varying substituents on the
polyheterocyclic core. Notably, installation of a proton-
binding amino group to the extended π-system furnished
pH-responsive fluorophores, the de-excitation pathways
of which could be switched between PET and ICT by
conformational control.
studies of neutral imidazo[1,5-a]pyridines have shown that
substitution at the C3 position of the imidazole ring mod-
ulates the emission properties of the molecule.9
Accordingly, we decided to systematically study how the
substitution (1À3) and expansion (4) of the pyridine ring
component of the molecule would influence its optical
properties. As shown in Scheme 1 and Table S1, both 2
and 3 exhibit red-shifted absorption/emission relative to
the benchmark system 1 (Figure S6). Annulation of the
imidazo[1,5-a]pyridinium ion ring system to afford 4 re-
sulted in a significant enhancement in ΦF to 63%.
Scheme 1. Synthesis and Emissive Properties of Functionalized
Imidazo[1,5-a]pyridinium Ionsa
The effect of altering the N-substituent on the imidazole
component of the molecule was studied using compounds
5À9 (Scheme 1; Table S2). Consistent with the trend ob-
served for compounds 1 f 2 f 3, a systematic red shift in the
emission spectrum was observed along the series of 6f7f8
(overall Δλem = 20 nm, Table S2). The UVÀvis and fluo-
rescence spectra of 1 and 5 are essentially superimposable,
presumably resulting from deconjugation of the N-aryl unit
from the imidazo[1,5-a]pyridinium ion core caused by steric
congestion introduced by two ortho-substituents (Figure S7).
In contrast to the gradual spectral shift observed with
methoxy and cyano substituents, a dramatic red shift
(Δλem = 140 nm) was observed upon incorporating an
amino substituent (6 f 9, Figure S7). FMO analysis based
on a density functional theory (DFT) energy-minimized
model of 9 (Figure 1) revealed that its HOMO is domi-
nated by the electron-donating N-aryl unit whereas its
LUMO resides predominantly on the ring-fused cationic
core. The calculated electronic structure is thus consistent
with an ICT-type transition to access excited-states having
a significant charge-separated character, the de-excitation
from which gives rise to the observed longer-wavelength
emission.13 In support of this hypothesis, protonation of 9
resulted in a large (Δλem = 140 nm) blue shift in its
emission (Figure S9a).14 With the disengagement of nitro-
gen lone-pair electrons from the extended π-conjugation,
access to the ICT state is effectively suppressed. Conse-
quently, protonated 9 (λem = 375 nm) “behaves” like
simple aryl-substituted analogue 6.
a For each compound, the maximum emission wavelength (λmax,em) in
MeCN is provided along with the fluorescence quantum yield (ΦF) in
parentheses. bSelect compounds (see Supporting Information) were purified
by anion metathesis to replace ClÀ with PF6À or BPh4À; variation of anions
resulted in negligible changes in emission properties (see Supporting Informa-
tion, Figures S1ÀS6). cBroad emission with multiple vibronic features.
In our investigations of imidazo[1,5-a]pyridinium ions
as synthetic precursors to N-heterocyclic carbene (NHC)
ligands,7 we observed that these polyheterocyclic cations
were highly fluorescent. As shown in Scheme 1, the simple
derivative 1 has a high emission quantum yield of ΦF =
27% in MeCN. Although the efficient radiative decay of
selected imidazo[1,5-a]pyridinium ions and related struc-
tures have previously been reported,8 the general structureÀ
property relationships and potential utility of these fluoro-
phores have remained largely unexplored. Additionally,
In order to introduce structural variations at the C1 posi-
tion of the imidazo[1,5-a]pyridinium ion core, compounds
10À14 were prepared (Scheme 1). Installation of aryl
groups (11À14) in place of a simple methyl group (10) at
C1 resulted in a significant improvement in both emission
quantum yields and spectral red shifts (Table S3;
Figure S10). Notably, incorporation of a p-(N,N-
diethylamino)phenyl substituent in compound 14 re-
sulted in an intense emission at λem = 550 nm. Its large
(Δλem = 145 nm) bathochromic shift relative to the
(8) (a) Campbell, E. C.; Glover, E. E.; Trenholm, G. J. Chem. Soc. C
1969, 15, 1987–1990. (b) Mukherjee, A.; Dhar, S.; Nethaji, M.;
Chakravarty, A. R. Dalton Trans. 2005, 349–353. (c) Boydston, A. J.;
Pecinovsky, C. S.; Chao, S. T.; Bielawski, C. W. J. Am. Chem. Soc. 2007,
129, 14550–14551. (d) Boydston, A. J.; Vu, P. D.; Dykhno, O. L.; Chang,
V.; Wyatt, A. R.; Stockett, A. S.; Ritschdorff, E. T.; Shear, J. B.;
Bielawski, C. W. J. Am. Chem. Soc. 2008, 130, 3143–3156. (e) Roy,
M.; Chakravarthi, B. V. S. K.; Jayabaskaran, C.; Anajali, A. K.;
Chakravarty, A. R. Dalton Trans. 2011, 40, 4855–4864.
(9) (a) Shibahara, F.; Sugiura, R.; Yamaguchi, E.; Kitagawa, A.;
Murai, T. J. Org. Chem. 2009, 74, 3566–3568. (b) Yamaguchi, E.;
Shibahara, F.; Murai, T. J. Org. Chem. 2011, 76, 6146–6158.
(10) Bissell, R. A.; de Silva, A. P.; Gunaratne, H. Q. N.; Lynch,
P. L. M.; Maguire, G. E. M.; Sandanayake, K. R. A. S. Chem. Soc. Rev.
1992, 21, 187–195.
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Rulliere, C. J. Phys. Chem. A 1998, 102, 7393–7405. (b) Maus, M.;
Rettig, W.; Bonafoux, D.; Lapouyade, R. J. Phys. Chem. A 1999, 103,
3388–3401. (c) Maus, M.; Rurack, K. New J. Chem. 2000, 24, 677–686.
(d) Rurack, K.; Resch-Genger, U. Chem. Soc. Rev. 2002, 31, 116–127.
(12) Pankratov, A. N.; Uchaeva, I. M.; Doronin, S. Y.; Chernova,
R. K. J. Struct. Chem 2001, 42, 739–746.
(13) In contrast, 6 lacking such an electron-donor group has both its
HOMO and LUMO localized at the imidazopyridinium core (Figure S8).
(14) Under similar conditions, 1 shows no significant change in
emission (see Figure S11 in the Supporting Information).
B
Org. Lett., Vol. XX, No. XX, XXXX

the intensity of this band was diminished, with a new
emission band emerging at λem = 400 nm (Figure 2a).
Subsequent addition ofDBU resultedin the disappearance
of the emission at λem = 400 nm and reappearance of the
emission at λem = 565 nm, demonstrating the reversibility
of this process. In contrast to this ICT behavior seen with 15,
its regioisomer 16 displayed a broad and negligible (ΦF <
0.2%) emission (Figure 2b). Addition of TFA, however,
resulted in the appearance of an intense (ΦF = 57%) band
at λem = 450 nm. Subsequent addition of DBU quenched
this fluorescence, demonstrating the reversible nature of this
protonation-driven fluorescence turn-on/off switching.
The strikingly different emission properties of the two
imidazo[1,5-a]pyridinium ion isomers 15 and 16 suggest
that the p-dialkylaminophenyl group plays distinctively
different roles in each case. For 15, the aniline unit acts in a
manner analogous to the corresponding substituent in
9 or 14, producing an ICT-type emission. Protonation
suppresses charge separation in the excited state, so that
only local emission (LE) at higher energy is observed. For
16, the aniline substituent participates in a PET-type de-
excitation to quench fluorescence.10 Upon protonation,
however, its lone-pair electrons are no longer available for
internal ET in the excited state. As a consequence, the
inherent imidazo[1,5-a]pyridinium ion emission is re-
stored. A question that immediately arises is what struc-
tural factorsdictate the choice between ICT and PET made
by the regioisomeric 15 and 16.
Figure 1. (a) ORTEP diagram of 9 with thermal ellipsoids at
50% probability. The diethylamino groups are disordered over
two positions, for which only one model is shown. (b) FMO
isosurface plots (cutoff = 0.05) of the DFT model of 9.
reference system 11 is reminiscent of the behavior of 9
(vide infra) and implicates the involvement of charge-
separated excited states. This postulation was confirmed
by a protonation-induced blue shift of 14 (Figure S9b),
which parallels the chemistry of 9.
The intense ICT-type long-wavelength emissions ob-
served for 9 and 14, and the ability to modulate their optical
properties, inspired us to prepare compounds 15 and 16 as
structural analogues containing extended π-conjugation
(Figure 2). It should be noted that initial attempts to access
desmethoxy 15 were impaired by the competing copolymer-
ization of aniline and formaldehyde,15 whereas incorpora-
tion of the para substituent onto the aniline precursor
suppressed this side reaction. As a regioisomeric pair of
molecules, 15 and 16 differ only in the relative positioning of
the two aryl groups that are attached to the common
imidazo[1,5-a]pyridinium ion core. This seemingly minor
structural variation, however, resulted in profoundly differ-
ent luminescent properties as detailed below.
Unlike their simpler analogues 9 and 14, the presence of
two aryl substituents in 15 and 16 introduces severe steric
constraints that should prevent coplanar arrangement of
the entire π-system. To investigate the effects of such
structural distortion on the molecular photophysics, we
have prepared a model system 17 (Figure 3) as an electro-
nically equivalent but sterically distorted analogue of 9.
Figure 3. Emission spectra of (a) 9 (= 10 μM; as PF6À salt) and
(b) 17 (= 5 μM; as BPh4À salt) in H2O at pH = 7.5 (red) and
pH = 2.5 (black). T = 298 K.
Figure 2. Emission spectra of (a) 15 and (b) 16 (= 30 μM) (as ClÀ
salt) in MeCN, (i) prior to and (ii) after addition of CF3CO2H
(60 mM), and (iii) after addition of DBU (90 mM) to the sample
(ii). T = 298 K.
(16) Compound 16 crystallizes in monoclinic space group P21/c with
˚
In MeCN, 15 showed an intense (ΦF = 59%) yellow
fluorescence at λem = 565 nm, which is further red-shifted
than its simpler analogue 14 (Scheme 1). Upon acidification,
˚
˚
a = 17.387(8) A, b = 6.257(3) A, c = 19.289(8) A, β = 102.012(10)°,
V = 2052.6(16) A , and Z = 4. The structure of 16 was not fully refined
3
˚
due to poor crystal quality and low parameter to reflection ratio.
Although detailed structural information cannot be provided, the
chemical connectivity and twisting of the N-aryl group away from
coplanarity with the imidazopyridinium ring portion of the molecule
were unambiguously confirmed.
(15) Alger, M. Polymer Science Dictionary, 2nd ed.; Chapman & Hall:
London, 1997; p 23.
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We anticipated that bulky isopropyl groups installed at
ortho-positions should twist the aniline ring out of con-
jugation from the fluorogenic imidazo[1,5-a]pyridinium
ion core of 17. This effective deconjugation across the
CÀN bond indeed resulted in fluorescence quenching of
17 at pH = 7.5 (Figure 3b), whereas 9 remains emissive
(λem = 550 nm; Figure 3a). At pH = 2.5, both molecules
display strong fluorescence at λem = 360À375 nm. These
findings are consistent with the ICT-type emission of 9 and
PET-type relaxation of 17, which can be controlled rever-
sibly by straightforward acidÀbase chemistry. For twisted
π-conjugated systems, a rapid intersystem crossing (ISC)
and subsequent nonradiative relaxation could also make
the molecule nonemissive.11 This interpretation, however,
cannot explain the recovery of fluorescence upon proto-
nation that we observed.
Figure 4. (a) An overlay of the X-ray structure of 9 (light gray)
with that of 16 (black); selected atoms (1À8) are labeled to define
the interplanar torsional angles τ1 and τ2. (b) PES contour plot
of the DFT model of 16, with a cross mark corresponding to the
crystallographically determined structure of 16.
An overlay of the X-ray structures of 1616 and 9
(Figure 4a) indeed shows that the presence of the
p-methoxyphenyl substituent at C1 significantly increases
the torsional angle between the p-diethylaminophenyl
group and the imidazo[1,5-a]pyridinium ion ring system.
Additionally, the crystallographically determined tor-
sional angles of 16 correspond to a point in the potential
energy surface (PES) contour plot that is only 1.1 kcal
molÀ1 above the DFT minimum energy structure
(Figure 4b). As anticipated, a completely coplanar ar-
rangement of the entire π-system (τ1 = τ1 = 0°) has to
overcome severe steric congestion between two adjacent
aryl groups, which would amount to a barrier ca. 22 kcal
molÀ1 higher in energy relative to the fully relaxed geome-
try. In contrast, adopting a near orthogonal orientation
between the aniline ring and the imidazo[1,5-a]pyridinium
ion core, i.e. τ1 f 90°, is an energetically less costly process.
This conformational preference of 16 might be responsible
for the shift of de-excitation pathways from ICT to
PET. Apparently, the ICT de-excitation pathway of 15
suffers less from this structural distortion, which results in
strikingly different emission properties between the two
regioisomers (Figure 2).
As positively charged imidazo[1,5-a]pyridinium ions,
both 15 and 16 are readily water-soluble, which allowed
us to investigate their pH-dependent emission properties in
buffered aqueous solutions in detail. As shown in Figure 5,
15 displays a ratiometric response to pH, with nicely
correlated increases and decreases in the emission intensity
at λem,max = 400 nm and λem,max = 580 nm within the pH
window of 2.5À7.5. In water at pH = 7.5, 16 remains
essentially nonemissive (ΦF = 0.06%), but lowering the pH
resulted in a ca. 700-fold enhancement in cyan fluorescent
intensity (ΦF = 42% at pH = 2.5) at λem,max = 450 nm to
provide turn-on signaling. Nonlinear regression of the sig-
moidal response of fluorescence intensity vs pH provided
pKa = 5.0 for 15 and pKa = 4.4 for 16 (Figures S12
and S13). These values are significantly lower than that of
N,N-diethylaniline (pKa = 6.5)12 and reflect the inductive
effect of the cationic ring system.
Figure 5. pH-Dependent changes in the emission spectra of (a)
15 and (b) 16 (30 μM) in water. Inset images: aqueous solution
samples under UV-lamp at pH = 2.5 (left) and 7.5 (right).
compounds can be directly accessed through an efficient
three-component coupling reaction, are highly fluores-
cent, and inherently water-soluble, making them ideal for
customization for nearly any imaging need. Specifically,
the relatively low pKa value of these pH-sensitive fluoro-
phores, operating either in a ratiometric fashion for precise
determination of local pH values or in a turn-on/off fashion
for visualization/imaging with high spatial resolution, is
ideal for investigating acidic cellular environments with
minimal interference from background fluorescence. Efforts
are currently underway in our laboratories to develop
target-oriented applications of these and related imidazo-
[1,5-a]pyridinium ion derivatives in vivo.
Acknowledgment. D.L. thanks DTRA/Army Research
Office (W911NF-07-1-0533), and Z.D.A. thanks Indiana
University for financial support of this work.
Supporting Information Available. Experimental pro-
cedures for all compounds generated through this work,
and spectroscopic, X-ray crystallographic, and computa-
tional data. This material is available free of charge via
the Internet at http://pubs.acs.org.
In summary, we have demonstrated the potential
utility of imidazo[1,5-a]pyridinium fluorophores. These
The authors declare no competing financial interest.
D
Org. Lett., Vol. XX, No. XX, XXXX