Fluorescence-on response via CB7 binding to viologen-dye pseudorotaxanes

Article abstract of DOI:10.1021/ol300963c

Fluorescence-on sensors typically rely on disrupting photoinduced electron transfer quenching of the excited state through binding the electron donor. To provide a more general fluorescence-on signaling unit, a quencher-fluorophore dyad has been developed in which quenching by electron transfer to a tethered viologen acceptor can be disrupted through complexation of the viologen by cucurbit[7]uril (CB7). Dyads of benzyl viologen-rhodamine B or a BODIPY fluorophore gave upon CB7 complexation 14- and 30-fold fluorescence enhancement, respectively.

Full text of DOI:10.1021/ol300963c

ORGANIC
LETTERS
2012
Vol. 14, No. 16
4046–4049
Fluorescence-On Response via CB7
Binding to ViologenꢀDye Pseudorotaxanes
Anuradha Singh, Wai-Tak Yip, and Ronald L. Halterman*
Department of Chemistry and Biochemistry, University of Oklahoma, Norman,
Oklahoma 73019, United States
RHalterman@ou.edu
Received April 13, 2012
ABSTRACT
Fluorescence-on sensors typically rely on disrupting photoinduced electron transfer quenching of the excited state through binding the electron
donor. To provide a more general fluorescence-on signaling unit, a quencherꢀfluorophore dyad has been developed in which quenching by
electron transfer to a tethered viologen acceptor can be disrupted through complexation of the viologen by cucurbit[7]uril (CB7). Dyads of benzyl
viologenꢀrhodamine B or a BODIPY fluorophore gave upon CB7 complexation 14- and 30-fold fluorescence enhancement, respectively.
Molecular fluorescence plays a central role in biological
imaging and sensing methods.1 Cellular imaging often re-
lies on tagging a target with a fluorophore such as green
fluorescent protein or small synthetic dyes to enable
visualization of the target using optical microscopy.2 A
drawback of this approach is that the background fluor-
escence of nonspecifically attached fluorophores limits
applications to those cases in which the localized emission
is sufficiently stronger than the background. This disad-
vantage can be overcome by using fluorescence-on binding
sensors in which emission is substantially quenched until a
sensing event takes place to block the quenching. Such
fluorescence-on methods have been most frequently ap-
plied in metal ion sensors in which quenching by photo-
induced electron transfer (PET) from nonbonded electron
pairs to the excited state of the fluorophore is shut off
by coordinating the donor electron pairs with a metal ion
(Figure 1a).1,3 This approach has been most widely fol-
lowed to develop selective binding groups for various
metal ions. While fluorescence-on ion sensing through
disrupting PET from nonbonded electron pairs has been
the focus of extensive development, this approach may not
lend itself to signaling binding events of a more general
nature.
An approach that relies on increasing fluorescence
through disruption of contact between a fluorophore and
a quencher could provide an entry into a more general
signaling method. This fluorescence-on approach has been
much less intensively studied, but notable efforts have
involveddetectingconformational changes inproteins that
move a quenching group away from a fluorophore, or
disrupt aggregation by binding the fluorophore.4 A novel
and more general fluorescence-on approach would entail
disrupting the quenched aggregate through host binding of
a guest quencher. When a host is displaced from an initial
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r
10.1021/ol300963c
Published on Web 08/03/2012
2012 American Chemical Society

guest complex in response to external stimuli as shown
in Figure 1b, a fluorosecence-on signal would result from
the host binding the new guest to disrupt the flourescence
quenching. Thus, we would have an unprecedented sensor
that has one site for interacting with an analyte and a
second site that produces the fluorescence signal. Since the
fluorescence on the signaling unit is structurally indepen-
dent of the binding event, variation in the binding unit can
be explored without needing to reconfigure the signaling
unit in each case.
dications when the two components are coentrained in a
polymer matrix.7 Given the established binding of CB7
with viologens,8 we sought to establish an ability to gen-
erate a fluorescence-on signal upon addition of CB7 to
tethered viologenꢀrhodamine and viologenꢀBODIPY9
dyads.
The preparation of benzylviologenꢀrhodamine B (RhB)
dyad 3bis shown in Scheme 1. Alkylation of resorcinol with
2-chloroethanol followed by alkylation with 1,2-dibro-
moethane provided the tethering anchor 1 that has the
desired orthogonal hydroxy and alkyl halide reactive sites.
Carbodiimide-promoted esterification of the hydroxy-ter-
minated arm with rhodamine B gave RhB ester 2.
Alkylation on the bromo-terminated arm by excess 4,40-
dipyridine under mild conditions gave a monosubstituted
dipyridyl complex that upon a second pyridine alkylation
by methyl iodide or benzyl bromide gave the benzyl
viologenꢀrhodamine B dyads 3a and 3b. An analogous
set of reactions was used to form benzyl viologenꢀBODIPY
dyad 4. More forcing conditions in these transformations
led to undesired amounts of ester and/or benzylpyridinium
cleavage. Isolation of the polycationic complexes was
achieved using silica gel chromatography using a methanolꢀ
nitromethaneꢀaqueous ammonium chloride eluent followed
by dissolving the desired complex away from the excess
inorganic salts using dichloromethane.10
The aggregation of the viologenꢀdye dyads and their
interaction with CB7 could be modeled by gas phase
semiempirical calculations (Spartan 08, PM3) as shown
in Figure 2 for the BODIPY fluorophore dyad 4. The
contact needed between the viologen and BODIPY moi-
eties for effective electron transfer was observed in the
absence of CB7 and disrupted in the presence of CB7.
The fluorescence brightness of dyads 3a and 3b was
noticeably lower than that of the corresponding free dye
(using RhB ester 2 without the tethered dipyridinium for
comparison). As illustrated in Figure 3, CB7 increased the
fluorescence intensity of RhB ester 2 about 2-fold as
expected due to disruption of RhB aggregation.6a Tether-
ing the RhB moiety to either the methyl or benzyl viologen
quencher in 3a or 3b did result in nearly a 10-fold decrease
in the fluorescence intensity of the fluorophore. Addition
of CB7 to the methyl viologenꢀRhB dyad 3a did not result
in much recovery of fluorescence. In contrast, addition of
CB7 to benzyl viologenꢀRhB dyad 3b gave a much larger,
14-fold increase in fluorescence intensity over 3b alone.
While CB7 is known to bind both methyl viologen and
benzylviologen, the largerhydrophobicgroupon the latter
provides for a stronger association. Thus, known binding
Figure 1. Mechanisms to disrupt fluorescence quenching.
It is well-documented that fluorescence quenched through
homoaggregation of fluorophores can be rescued by dis-
rupting cationic fluorophore aggregation through binding
with cucurbit[7]uril (CB7).5 We have reported a 2-fold
enhancement of fluorescence of a tethered rhodamine
B dyad upon CB7 binding to disrupt a less fluorescent
H-dimeraggregate.6 Inorderto extend thismethod toward
a more useful, larger change in fluorescence intensity, we
have now investigated the disruption of electron transfer
quenching upon CB7 binding to a benzyl viologen pseu-
dorotaxane and report our preliminary findings herein.
These results show that binding a quencher as shown in
Figure 1b can provide a strong fluorescence-on response
and provide impetus to extend the method to the shuttle
mechanism depicted.
Fluorescence from organic fluorophores such as rhoda-
mine B is known to be quenched by electron transfer from
the excited state of the dye to 1,4-dipyridinium viologen
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2008, 73, 3266–3269. (b) Halterman, R. L.; Moore, J. L.; Yakshe, K. A.;
Halterman, J. A. I.; Woodson, K. A. J. Incl. Phenom. Macro. Chem. 2010,
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2012, 21, 1467–1478. (d) Martyn, T. A.; Moore, J. L.; Halterman, R. L.;
(7) Fischer, A. B.; Bronstein-Bonte, I. J. Photochem. 1985, 30, 475–485.
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Org. Lett., Vol. 14, No. 16, 2012
4047

Scheme 1. Preparation of ViologenꢀFluorophore Dyads
Figure 3. Fluorescence spectra of RhB ester 2, methyl viologen-
RhB 3a, and benzyl viologen-RhB 3b (each 5 μM) without and
with 8 mM CB7 (excitation 560 nm).
we examined the dyad 4 containing the neutral BODIPY
fluorophore. Upon titration of benzyl viologenꢀBODIPY
dyad 4 (5 μM) with CB7 (0 ꢀ2 mM) in water, the fluo-
rescence intensity increased a remarkable 30-fold to give a
verysizable fluorescence-on response. The titration data fit
well to a one-site binding model, which gives Ka = 1.2 ꢁ
104 Mꢀ1 for binding CB7 to dyad 4. Although no associa-
tion constant between CB7 and BODIPY is available from
the literature, we presume that CB7 will bind more effec-
tively to the cationic viologen moiety in preference to the
neutral BODIPY group. Our association value with 4 is
about 102 weaker than typical complexation of CB7 to
isolated viologen;8a,c,11 this difference in binding can be
attributed to the penalty required to disrupt the energeti-
cally favorable aggregation of the BODIPY with the
viologen. If CB7 was indeed bound to the viologen moiety
as expected, this would imply that the association constant
between BODIPY and viologen should be on the order of
102. While solubility limitations hindered titrations with
BODIPY dyad 4 in the required aqueous medium, chemi-
cal shift changes in 1H NMR spectra of 3b•CB7 supported
inclusion of the benzyl viologen moiety by CB7.
An important aspect of sensor design is illustrated by
these results. The complexation of CB7 onto various dyads
to disrupt the contact of a quenching unit with the fluor-
ophore is dependent on the different association constants
of the various quenching moieties with CB7. Given the
similar association constant of CB7 with the benzyl violo-
gen in either the BODIPY 4 or rhodamine B 3b dyads, we
can utilize a common benzyl viologen binding element
while still producing a different emission frequency from
Figure 2. (a) PM3-optimized structure/electrostatic surface po-
tential of 4 before and (b) after binding with CB7.
of the CB7 to cationic RhB would better compete with the
methyl viologen than with the benzyl viologen. Binding
of CB7 over one diethylamino group of the RhB moiety in
the presence of the methyl viologen could still allow the
nonincluded portion of the rhodamine chromophore to
contact the methyl viologen quencher. Fluorescence titra-
1
tion curves of 2, 3aꢀb, and 4 with CB7 and H NMR
titration spectra for 3aꢀb are provided in the Supporting
Information. Curve fitting analysis gave a good one-site
binding model fit with the benzyl viologen and supported
one preferential binding site thatwas effective at disrupting
quenching. The methyl viologen data were more consistent
with two competing sites.
In order to minimize competitive binding of the CB7
between the viologen and the cationic RhB fluorophore,
(11) Liu, S. M.; Ruspic, C.; Mukhopadhyay, P.; Chakrabarti, S.;
Zavalij, P. Y.; Isaacs, L. J. Am. Chem. Soc. 2005, 127, 15959–15967.
4048
Org. Lett., Vol. 14, No. 16, 2012

the various fluorophores. The crucial design element would
be that the fluorophore be initially quenched through contact
with the viologen and that binding of the CB7 to the viologen
is roughly independent of the tethered fluorophore
Acknowledgment. Support through NSF (DMR-
0805233 toR.L.H.) andhelpful discussions withV. Rotello
(UMass, Amherst) are appreciated.
The results presented here demonstrate a novel approach
to a fluorescence-on system that relies on shuttling a CB7
onto and disrupting contact in a viologenꢀfluorophore
dyad. While the demonstration of a sensor involved dimin-
ishing the binding affinity of CB7 for its initial guest through
a deprotonation, one could in principle lower the binding
affinity of the CB7 for the initial site through other stimuli
while maintaining a common fluorescence-on signaling unit.
Supporting Information Available. Experimental pro-
cedures and characterization of all synthesized com-
pounds. Absorbance and fluorescence titrations of 2, 3a,
3b, 4; 1H NMR spectral titrations of 3a, 3b. This material
is available free of charge via the Internet at http://pubs.
acs.org.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 16, 2012
4049