Inclusion complexes of cationic xanthene dyes in cucurbit[7]uril

Article abstract of DOI:10.1007/s10847-009-9615-9

Improvements in optical properties of organic xanthene fluorophores through molecular encapsulation in cucurbit[7]uril (CB7) should enable better molecular probes and devices to be designed. Although the interactions of several dyes with CB7 have been stu

Full text of DOI:10.1007/s10847-009-9615-9

J Incl Phenom Macrocycl Chem (2010) 66:231–241
DOI 10.1007/s10847-009-9615-9
ORIGINAL ARTICLE
Inclusion complexes of cationic xanthene dyes in cucurbit[7]uril
Ronald L. Halterman Æ Jason L. Moore Æ
Krystle A. Yakshe Æ Julie A. I. Halterman Æ
Kevin A. Woodson
Received: 10 March 2009 / Accepted: 9 June 2009 / Published online: 28 June 2009
Ó Springer Science+Business Media B.V. 2009
Abstract Improvements in optical properties of organic
xanthene fluorophores through molecular encapsulation in
cucurbit[7]uril (CB7) should enable better molecular
probes and devices to be designed. Although the interac-
tions of several dyes with CB7 have been studied, the data
have often been incomplete. Uniformly applied ensemble
Keywords Cucurbit[7]uril Á CB7 Á Rhodamine Á
Pyronin Á Inclusion complex
Introduction
1
spectroscopic studies are presented herein, including H
In recent years much organic dye research has focused on
the development of technologies such as molecular scale
biological probes and sensors [1–7]. However, the use of
organic dyes for these types of applications has been slowed
due to several limitations including undesirable aggrega-
tion, fluorescence blinking and photobleaching, surface
adsorption and relatively poor photostability [8–15]. These
problems can in principle be reduced by supramolecular
encapsulation, especially in terms of minimizing aggrega-
tion and surface adsorption [8, 16, 17]. While the bulk of
this type of research has been performed using b-cyclo-
dextrin as a host, recent efforts have started to examine the
effect of cucurbit[7]uril (abbreviated as CB7 or Q7) [18–21]
on the photophysical and photochemical behavior of fluo-
rophores and in particular common fluorescent dyes [9, 15,
22–28]. Results by Nau and co-workers have shown that the
addition of cucurbit[7]uril to aqueous solutions of rhoda-
mine 6G resulted in several advantageous effects including
enhanced brightness and fluorescence lifetimes and a
reduction of non-specific adsorption and aggregation [16,
29]. In order to better understand these effects as they relate
to cationic organic dyes of interest, we present here
NMR, UV–Vis, and fluorescence titration experiments of
aqueous CB7 complexes with the monocationic xanthene
dyes rhodamine 6G (Rh6G, 1), rhodamine B (RhB, 2),
rhodamine B benzyl ester (RhBBE, 3), pyronin B (PyB, 4)
and pyronin Y (PyY, 5). All of these cationic xanthene
dyes formed 1:1 complexes with cucurbit[7]uril as evi-
denced by NMR data and Job’s plots of fluorescence
changes upon addition of CB7. Non-linear regression
analysis of the fluorescence titration curves gave precise
Ka’s for RhB, RhBBE and PyB between 1.1 9 105 M-1
and 9.1 9 106 M-1. The fluorescence emission intensity of
Rh6G was lowered 0.8-fold in the presence of CB7 while
the other dyes examined showed an increase between 1.3
and 4.7-fold. NMR titration experiments from 0 to 2.0
equivalents of CB7 per equivalent of xanthene gave in only
some cases very clear evidence of inclusion complexation.
Non-specific adsorption of these xanthene dyes onto
borosilicate glass was very pronounced and could be
inhibited by dye inclusion into CB7.
1
Electronic supplementary material The online version of this
article (doi:10.1007/s10847-009-9615-9) contains supplementary
material, which is available to authorized users.
ensemble spectroscopy studies, including H NMR, UV–
Vis, and fluorescence titration experiments of cucur-
bit[7]uril complexes with the monocationic xanthene dyes
rhodamine 6G (Rh6G, 1), rhodamine B (RhB, 2), rhoda-
mine B benzyl ester (RhBBE, 3), pyronin B (PyB, 4) and
pyronin Y (PyY, 5). The formula structures of these com-
pounds are shown in Fig. 1.
R. L. Halterman (&) Á J. L. Moore Á K. A. Yakshe Á
J. A. I. Halterman Á K. A. Woodson
Department of Chemistry and Biochemistry, University of
Oklahoma, 620 Parrington Oval, Norman, OK 73019, USA
e-mail: rhalterman@ou.edu
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J Incl Phenom Macrocycl Chem (2010) 66:231–241
The widely studied fluorescent dye rhodamine 6G was
observed and NMR titration data and Job’s plots were cited
as being consistent with a 1:1 binding model of the dye and
CB7 [15]. Although it is apparent that CB7 interacts with
Rh6G to decrease surface adsorption and to improve the
photophysical properties, the nature of that interaction has
not been conclusively made. In order to establish a solid
basis for comparisons between the dye–CB7 complexes,
we remeasured some of these reported properties and the
data we provide have some differences from the data
summarized in the literature. The absorbance and fluores-
cence emission spectra for aqueous 2.5 lM rhodamine 6G
prepared in polystyrene cuvettes without and in the pres-
ence of 1 mM CB7 are shown in Fig. 2.
investigated to provide calibration between our work and
literature reports on some of the properties of rhodamine
6G—cucurbit[7]uril mixtures [15, 16]. Related confocal
microscopy studies on the fluorescence of single molecules
of rhodamine 6G in CB7 films have also been reported by
our research group [30]. The other xanthene dyes were
studied to probe the changes in binding and photophysical
properties in response to structural changes on the xanthene
core. For each of the dyes, we performed NMR, absorption
and fluorescence titration experiments, as well as Job’s plot
and linear regression analyses to characterize the binding
and photophysical properties of the complexes with CB7.
As detailed herein we did find that xanthenes without the
9-aryl substituent generally gave stronger binding to CB7.
Greater perturbations in the chemical shifts in NMR
spectra and smoother fluorescent titration curves were seen
with the xanthenes containing N,N-diethylamino groups.
Essentially no shift in the wavelength and a modest
decrease in the absorbance (0.9-fold) and fluorescence
intensity (0.8-fold) were noted in the presence of CB7.
A change in the absorbance shoulder near 490 nm could
indicate a change in dye–dye aggregation [31]. For this dye
and the other cationic xanthene dyes in this study we found
it difficult to use the absorbance data to generate titration
curves. Changes in the known H- and J-type aggregation
upon CB7 complexation often led to significant changes in
the absorbance spectra. Since the absorbance changes often
decreased then increased with increasing CB7 concentra-
tion, we chose the smoother changes obtained in the fluo-
rescence spectra for our CB7 titration curves. The complete
titration spectra are provided in the supplementary data
(Fig. S3). In contrast, Nau reported a modest increase in
both the absorbance and fluorescence intensity upon
Results and discussion
Rhodamine 6G (Rh6G, 1)
Some steady state and time resolved photophysical prop-
erties of aqueous rhodamine 6G in the presence of CB7
have been reported, usually as summaries of unpublished
raw data [15, 16]. Enhanced absorbance and fluorescence
lifetimes and intensity in the presence of CB7 were
Fig. 1 Structures of CB[7] and
xanthene dyes used in this study
O
O
O
O
O
O
O
O
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
CH2
CH2
N
N
N
N
N
N
N
N
N
N
N
N
N
O
O
7
O
O
O
O
O
O
O
Cl
Cl
Cl
HN
O
NH
N
N
N
O
N
O
O
O
O
O
OH
1
2
3
Rhodamine 6G (Rh6G)
N
Rhodamine B (RhB)
Rhodamine B benzyl ester (RhBBE)
FeCl4
Cl
N
O
N
O
N
4
5
Pyronin Y (PyY)
Pyronin B (PyB)
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J Incl Phenom Macrocycl Chem (2010) 66:231–241
233
600
500
400
300
200
100
0
0.3
Fluorescence Titration Curve of R6G : CB7
550
500
450
400
350
300
A
C
0.25
B
D
0.2
2.5 uM Rhodamine 6G
0.15
0.1
0.05
0
1
2
3
4
0
420 440 460 480 500 520 540 560 580 600 620 640
[CB7] mM
Wavelength (nm)
Fig. 3 Titration curve of fluorescence emission signal intensity of
aqueous rhodamine 6G (2.5 lM) and CB7 (0–4 mM) prepared in
polystyrene cuvettes upon excitation at 527 nm
Fig. 2 UV–VIS absorption spectra (a at 0 mM CB7 and b at 1 mM
CB7) and fluorescence emission spectra (c at 0 mM CB7 and d at
1 mM CB7) of aqueous rhodamine 6G (2.5 lM) in polystyrene
cuvettes
binding constant. Although the continuous variation Job’s
plot of absorbance provided in Fig. S6 was most consistent
with a strong 1:1 binding of rhodamine 6G and CB7 [32,
33], a shoulder in the Job’s plot curve between 0.5 and 0.6
could be due to subsequent, weaker interactions at higher
CB7 concentrations. The nature of the additional interac-
tions between Rh6G and CB7 could not be extracted from
the data.
addition of a 1 mM solution of CB7 to a 1 lM solution of
Rh6G [15]. When we prepared aqueous samples of 3 lM
Rh6G without and in the presence of up to 4 mM CB7 in
borosilicate glass vials and sequentially transferred the
samples to a quartz cuvette for each absorbance and then
fluorescence measurement, we also observed a 2.1-fold
increase in absorbance and a 2.5-fold increase in fluores-
cence intensity in the presence of CB7 (Figs. S4 and S5).
Given Nau’s solid evidence for non-specific adsorption of
Rh6G onto borosilicate glass [15, 16], the enhancement
factors we observed under these conditions were less likely
to be primarily due to a photophysical enhancement but
rather due to differential non-specific adsorption of the
Rh6G onto the walls of the vials during the 1-2 h between
sample preparation and completing the spectroscopic
measurements. When insufficient CB7 was present the
Rh6G non-specifically adsorbed to the borosilicate glass.
When the solution was transferred to a cuvette for optical
measurements, a lower concentration of total dye (bound to
CB7 and free) was present. When a sufficient amount of
CB7 was present the dye–CB7 complex remained in
solution at maximum concentration.
The fluorescence titration curve of aqueous Rh6G
(3 lM) prepared in borosilicate glass is shown in Fig. 4.
Rather than showing a decrease in signal intensity as was
observed for samples prepared in polystyrene cuvettes, this
series shows an initial increase in signal intensity, followed
by a small decrease in signal strength at higher concentra-
tions of CB7. In this borosilicate glass case, the increase in
signal intensity was likely due to decreasing the amount of
non-specific adsorption of Rh6G as the CB7 concentration
increased and a corresponding increase in the concentration
of total dye in solution. With no CB7 present much of
the Rh6G could adsorb onto the borosilicate glass, reducing
the dye concentration in solution. Samples containing
600
550
500
450
400
350
300
250
200
The titration curve for changes in fluorescence intensity
of 2.5 lM R6G and 0 to 4 mM CB7 prepared in polysty-
rene cuvettes (Fig. 3) appears to indicate an initial 1:1
binding with saturation occurring at less than 0.1 mM CB7.
However, the fluorescence intensity then continues to vary
as the CB7 concentration is increased up to 4 mM. A non-
linear regression analysis of the low CB7 concentration
data up to 0.1 mM gave a tentative 1:1 binding constant of
1.2 0.4 9 106 M-1, a value which is fraught with
potential error since no data was included for CB7 at higher
concentrations where what could be an inflection point near
0.2 mM CB7 was seen (Figs. S7 and S8). Analyses using
the full data set up to 4 mM CB7 failed to give a valid
0
1
2
3
4
[CB7] mM
Fig. 4 Fluorescence emission titration curve of aqueous rhodamine
6G (3 lM) and CB7 (0–4 mM). Samples were prepared in borosil-
icate glass vials and the fluorescence emission spectra were obtained
in quartz cuvettes
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J Incl Phenom Macrocycl Chem (2010) 66:231–241
increasing amounts of CB7 resulted in increased amounts of
dye–CB7 complex in solution and a higher total dye con-
centration. Once enough CB7 was present to minimize non-
specific binding of the Rh6G to the glass surface, the
gradual decrease in signal strength was similar to the
samples prepared in polystyrene. An initial binding value
can be calculated for the increase in signal strength
(1 9 104 M-1) due primarily to increased total dye con-
centration as a result of decreased non-specific adsorption.
This binding value approached that obtained for the R6G-
CB7 titration in polystyrene. Titration curves in both
polystyrene and borosilicate glass were essentially mea-
suring parallel effects of the same CB7 binding—either
photophysical changes upon binding and/or protection from
non-specific adsorption. The similarity of these outcomes
could easily lead to misinterpretation of titration data.
NMR titration experiments resulted in only minor per-
turbations in the spectra of aqueous rhodamine 6G as CB7
was added (Fig. S9). At the high 4.2 mM concentration of
Rh6G, changes in non-specific binding with increasing
CB7 concentration were unlikely to be as significant as at
the micromolar concentrations used for the optical mea-
surements. In stable inclusion complexes, signals for
hydrogen atoms well inside the CB7 cavity are usually
strongly shifted to higher field, while those near the car-
bonyl portals show little perturbation or a downfield shift
[27, 28, 34]. While the photophysical and stability prop-
erties indicate binding and probably inclusion of rhoda-
mine 6G into CB7, the NMR spectra did not exhibit the
expected strong shifts for a stable inclusion complex hav-
ing hydrogen atoms well within the CB7 cavity. Based on
molecular models it is likely that the 9-aryl group pre-
vented the cationic xanthene core from inserting deeply
into the CB7 cavity, leaving even the included portions of
the guest near the CB7 portal. Furthermore, since only one
set of signals was present throughout the NMR titration, an
exchange of CB7 from one end of the xanthene core to the
other could be likely and would average out and further
minimize any chemical shift changes. Alternatively, the
interaction may be a symmetric exclusion complex having
the xanthene dye lying across the portal of the CB7. Given
the importance of Rh6G as a laser dye and the established
benefits upon adding CB7 to aqueous Rh6G [16], it was
disappointing that a clearer binding model was not sup-
ported by our data.
and it contains 3,6-bis(N,N-diethylamino) groups in place
of the N-ethylamino groups. These structural changes were
reflected in the binding and photophysical properties of
rhodamine B with CB7.
The absorbance and fluorescence emission spectra for
aqueous rhodamine B without and in the presence of CB7
were measured on samples directly prepared in polystyrene
cuvettes. A modest 1.3-fold increase in absorbance as a
function of an increase in the molar absorption coefficient
(e) from 8.0 9 104 M-1 cm-1 to 1.0 9 105 M-1 cm-1, a
slight bathochromic shift from 575 nm to 582 nm and a
1.6-fold increase in fluorescence intensity were noted upon
addition of CB7. A symmetric Job’s plot supports the
formation of a 1:1 rhodamine B and CB7 complex (Fig.
S11). The complete titration spectra are provided in Fig.
S10 and a non-linear regression analysis of the fluorescence
data gave a 1:1 binding constant of 1.6 0.4 9 105 M-1
(Figs. S12 and S13).
1
The H NMR titration spectra in which the concentra-
tion of cucurbit[7]uril was varied and the concentration of
rhodamine B was held constant are provided in Fig. 5.
Unlike rhodamine 6G, there were very significant changes
in the signals of both the xanthene hydrogens and the N,N-
diethyl groups of rhodamine B upon addition of cucur-
bit[7]uril. Most noticeably, there was a broadening and an
upfield shift of both the methylene and methyl signals of
the N,N-diethyl hydrogens. It is most informative to follow
the changes in the methyl signals. At the 1:1 and 1.5:1
ratios of CB7 to rhodamine B, the methyl signal was split
into a broad pair of signals as would be consistent with a
1:1 complex between rhodamine B and cucurbit[7]uril in
which the N,N-diethylamino groups were only slowly
exchanging between included and free chemical environ-
ments. If the exchange between free and complexed methyl
groups were fast, then a sharper averaged methyl signal
would be expected. At the 1:1 ratio a new signal for what is
likely an included methyl group was growing in near
0.7 ppm. When 1.5 equivalents of CB7 were present
broadened signals for both bound (0.7 ppm) and free
(1.2 ppm) methyl signals were observed. The exchange
between free and bound was slow enough for these two
signals to be observed separately. At 0.5 equivalents of
CB7 the methyl signal was only broadened through what
was likely an intermediate exchange process. If a stable,
slowly exchanging 1:1 or even 2:1 RhB–CB7 complex
were formed, then two sets of methyl signals would be
expected (for free and bound methyls). At 2 equivalents of
CB7, a single set of broad signals was observed as expected
for intermediately exchanging sites. If a stable 1:2 RhB–
CB7 complex were present, both methyl groups should be
included and appear as a single sharp signal (likely near
0.7 ppm). Since the single signal near 0.9 ppm was
broadened, some intermediate exchange between bound
Rhodamine B (RhB, 2)
Rhodamine B is a commercially available, widely studied
dye [10, 31, 35, 36] that structurally differs from rhoda-
mine 6G in three ways: its xanthene core lacks the 2,7-
dimethyl groups, it contains a 2-carboxylic acid group on
the 9-phenyl moiety in place of the 2-ethoxycarbonyl group
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J Incl Phenom Macrocycl Chem (2010) 66:231–241
235
and free methyl groups was likely taking place. These
results support an associative exchange mechanism in
which a second CB7 binds the non-included end of the
xanthene core, but as the 2:1 complex is apparently not
stable, the initial CB7 could depart as shown in Fig. 6.
The binding constant for rhodamine B is lower than the
tentative one found for rhodamine 6G. Apparently any
advantages that rhodamine B may have had through the
thinner xanthene core without the 2,7-dimethyl groups or
through the additional hydrophobic alkyl substitution on
the 1,6-amino groups were outweighed by the advantages
of the potential hydrogen bond donor on the N-ethylamino
groups or the additional hydrophobic 2,7-dimethyl groups.
The effect of having a 2-carboxylic acid or 2-carboxylic
acid ester on the 9-aryl group will be discussed below. In
the NMR spectra, however, the presence of the N,N-
diethylamino groups on rhodamine B did lead to splitting
of the ethyl signals into what are likely included and free
environments.
Rhodamine B Benzyl Ester (RhBBE, 3)
In order to more directly probe a potential factor for CB7-
rhodamine binding strength, we converted the 2-carboxylic
acid group on the 9-phenyl substituent of rhodamine B (2)
into an ester. Benzyl ester 3 was readily prepared by a DCC-
promoted coupling reaction [37] as shown in Scheme 1 and
separated from unreacted rhodamine B through column
Fig. 5 1H NMR spectra
(300 MHz, D2O) of aqueous
rhodamine B (4.2 mM) in the
presence of 2.0 equivalent (a),
1.5 equivalent (b), 1.0
equivalent (c), 0.5 equivalent
(d) and the absence (e) of CB7
(0–8.4 mM)
Fig. 6 Associative exchange of CB7 binding site on PyB
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Cl–
Scheme 1
Cl–
N
O
N
N
O
N
Ph
OH
O
O
O
DCC, DMAP
HO
3
2
chromatography (SiO2, 9:1 methanol/chloroform to elute
unreacted RhB followed by 90:10:1 methanol/chloroform/
glacial acetic acid to elute RhBBE) [31].
hydrogen signals was observed. In contrast to the splitting
seen for the N,N-diethylamino groups in the more strongly
binding rhodamine B, the lower binding constant between
rhodamine B benzyl ester and CB7 appeared to allow
faster exchange and only gave averaged N,N-diethylamino
signals.
The absorbance and fluorescence emission spectra for
aqueous rhodamine B benzyl ester (3) without and in the
presence of CB7 were measured on samples prepared
directly in polystyrene cuvettes and are shown in Fig. 7.
A modest 1.6-fold increase in the absorbance as a func-
tion of an increase in the molar absorption coefficient (e)
from 2.4 9 104 M-1 cm-1 and 3.8 9 104 M-1 cm-1 upon
addition of cucurbit[7]uril. In contrast to rhodamine B, a
very sizable 4.7-fold increase in fluorescence intensity was
noted upon addition of CB7 to the benzyl ester 3. A sym-
metric Job’s plot supports the formation of a 1:1 rhodamine
B benzyl ester and CB7 complex (Fig. S15). The complete
titration spectra are provided in Fig. S14 and a non-linear
regression analysis gave a 1:1 binding constant of
1.1 0.16 9 105 M-1 (Figs. S16 and S17). The binding
constant for rhodamine B benzyl ester was found to be
lower than for rhodamine B by a 5-fold factor, indicating
that the hydrogen bonding carboxylic acid group on the
9-aryl ring was more beneficial to binding than ester groups.
The 1H NMR titration spectra in which the concentration
of CB7 was varied from 0 to 2 equivalents per molecule of
rhodamine B benzyl ester (3.5 mM) are provided in Fig.
S18. Consistent with the formation of an inclusion complex,
broadening of the N,N-diethylamino and xanthenyl
Pyronin B (PyB, 4)
Pyronin B is a flat cationic dye chosen for investigation due
to its anticipated ability to be more completely encapsulated
by cucurbit[7]uril [15]. It contains the same 3,6-bis(N,N-
diethylamino) groups as rhodamine B, but lacks the 9-aryl
substituent. Some photophysical properties of CB7 com-
plexes of PyB and the 3,6-bis(N,N-dimethylamino) deriva-
tive PyY (5) have been reported by Nau, although the
binding constants were not reported [15]. Absorbance and
fluorescence emission spectra of aqueous PyB (2.5 lM) in
the absence and presence of 0.64 mM CB7 were measured
on samples directly prepared in polystyrene cuvettes and are
shown in Fig. 8. No shift in the absorbance wavelength
maximum (553 nm) for PyB was observed in the presence
of cucurbit[7]uril. Essentially no change in the absorbance
was observed at 0.64 mM CB7. In the fluorescence emis-
sion spectra a slight bathochromic shift in the fluorescence
emission maximum from 569 nm to 570 nm for PyB and
0.22
0.2
400
350
300
250
200
150
100
50
275
250
225
200
175
150
125
100
75
0.25
0.2
0.18
0.16
0.14
0.12
0.1
0.15
0.1
0.08
0.06
0.04
0.02
0
C
D
C
B
D
A
0.05
0
50
B
25
A
0
0
450 475 500 525 550 575 600 625 650 675 700
450 475 500 525 550 575 600 625 650 675 700
Wavelength (nm)
Wavelength (nm)
Fig. 7 UV–VIS absorption spectra (a at 0 mM CB7 and b at
0.64 mM CB7) and fluorescence emission spectra (c at 0 mM CB7
and d at 0.64 mM CB7) of aqueous rhodamine B benzyl ester
(2.5 lM) in polystyrene cuvettes
Fig. 8 UV–VIS absorption spectra (a at 0 mM CB7 and b at
0.64 mM CB7) and fluorescence emission spectra (c at 0 mM CB7
and d at 0.64 mM CB7) of aqueous pyronin B (2.5 lM). Samples
prepared and spectra obtained in polystyrene cuvettes
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J Incl Phenom Macrocycl Chem (2010) 66:231–241
237
2.3-fold increase in the fluorescence emission intensity
were observed in the presence of 1.0 mM cucurbit[7]uril. A
Job’s plot of PyB and CB7 indicated the formation of a 1:1
complex (Fig. S20). Full titration plots for the absorbance
and fluorescence emission spectra of PyB with increasing
CB7 concentration are also given in Fig. S19. A non-linear
regression analysis of the fluorescence titration gave a 1:1
binding constant of 9.1 1.6 9 106 M-1 (Figs. S21 and
S22). The binding constant for pyronin B-CB7 was found to
be significantly higher than for rhodamine B, indicating that
the 9-aryl substituent appears to be detrimental to stronger
binding.
quite broad methyl signal was observed, indicating an
intermediate exchange of CB7 from one end of PyB to
the other end as was the case with the RhB sample.
The increasing concentration of CB7 could facilitate the
increase in exchange rate from slow to intermediate. The
broad nature of the signals do not support a single stable 1:2
PyB–CB7 complex since such a complex should have given
a sharper set of equivalent signals. These shifts and the
sharpness of the more central hydrogen atoms are consistent
with a partial insertion of PyY into the CB7 pocket as shown
for the two minimized structures in Fig. 10. Semiempirical
(PM3) calculations gave two lower energy gas phase
structures with an energy difference of only 0.4 kcal/mol
and aid us in visualizing favored structures that could
contribute to a solution structure of the complex. The two
broad sets of signals for the N,N-diethylamino groups
indicate a slow exchange of the CB7 from one amino end of
the xanthene to the other end. When a second equivalent
of CB7 is present, the exchange is faster and a single set of
broadened signals are observed. Even in the presence
of excess CB7 the data do not fit a stable 2:1 binding of
CB7 and PyY since broadened signals are present rather
than the sharp signals expected for a stable symmetric
complex.
The NMR titration spectra are shown in Fig. 9. As was
seen for rhodamine B, the N,N-diethylamino groups gave a
splitting of both the methylene and methyl groups at a 1:1
CB7:PyY ratio. We also observed a downfield shift of H-9
and H-1/H-8 on the xanthene core. The hydrogens adjacent
to the amino group were strongly broadened and split into
two sets of signals at the 1:1 ratio. At one equivalent of CB7
two signals for the methyl groups were clearly located near
0.7 ppm (bound) and 1.2 ppm (free) which indicates slow
exchange of CB7 from one end of the PyB group to the
other end. The onset of coalescence was likely at 1.5
equivalents of CB7. At 2.0 equivalents of CB7 a single but
Fig. 9 1H NMR spectra
(300 MHz, D2O) of pyronin B
in the presence of 2.0 equivalent
(a), 1.5 equivalent (b), 1.0
equivalent (c), 0.5 equivalent
(d) and the absence (e) of CB7
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J Incl Phenom Macrocycl Chem (2010) 66:231–241
Fig. 10 PM3-calculated
(Wavefunction MacSpartan
program) gas-phase structures
of PyB-CB7. a and b Lower
energy with PyB non-
symmetrically inserted in CB7.
c Higher energy with PyB
centered in CB7
Pyronin Y (PyY, 5)
concentration in the solutions which were transferred into
quartz cuvettes for optical measurements. When the PyY
samples were prepared in the presence of CB7, much less
dye was apparently lost to non-specific adsorption and the
increase in total dye concentration (mostly as a CB7
complex) lead to a stronger observed signal. As discussed
below, PyY samples prepared in polystyrene cuvettes gave
markedly different results.
Pyronin Y is a flat cationic dye chosen for investigation due
to its anticipated ability to be more completely encapsu-
lated by cucurbit[7]uril and to provide comparison data on
the effects of N,N-diethylamino versus N,N-dimethylamino
groups on the binding of cationic xanthene dyes to CB7. As
was initially done with Rh6G, dilute solutions of PyY were
prepared in borosilicate glass vials which were then
sequentially transferred to quartz cuvettes for optical
absorption and fluorescence emission measurements. A
very unusual 50-fold enhancement in the fluorescence
intensity was observed in going from the 15 lM PyY
aqueous solution with no CB7 to one containing 70 lM
CB7 (Fig. 11). Consistent with Nau’s report on the sus-
ceptibility of pyronins towards non-specific adsorption
onto glass substrates [15], we were apparently not mea-
suring photophysical effects of CB7–PyY complexation,
but rather a decrease in non-specific adsorption which led
to a large increase in signal. With no or little CB7 present,
the low concentration of the PyY could non-specifically
adsorb onto the borosilicate vials, lowering its
The optical measurements were repeated with dilute
solutions of PyY with varying amounts of CB7 prepared
directly in polystyrene cuvettes. In this case, we observed
only a small 1.1-fold decrease in the absorbance and
modest 1.3-fold increase in fluorescence intensity for the
solution containing 0.64 mM CB7 (Fig. 12). A Job’s plot
of PyY and CB7 indicated the formation of a 1:1 complex
(Fig. S24). Full titration plots for the absorbance and
fluorescence emission spectra of PyY with increasing CB7
concentration are also given in Fig. S23. A non-linear
regression analysis of the fluorescence titration gave a 1:1
binding constant of 9.7 0.5 9 107 M-1 (Figs. S26 and
S27). The binding constant for pyronin Y-CB7 was found
to be 10-fold higher than for pyronin B, indicating that the
N,N-dimethylamino groups significantly improve binding
to CB7.
600
500
400
300
200
100
0
F, G, H
0.15
0.12
0.09
0.06
0.03
0
550
500
450
400
350
300
250
200
150
100
50
E
D
A
D
C
CB7
C
B
B
A
560
520
540
580
600
620
0
440
465
490
515
540
565
590
615
640
Wavelength (nm)
Wavelength (nm)
Fig. 11 Steady-state fluorescence emission spectra of aqueous pyro-
nin Y (15 lM) at different concentrations of CB7. Samples were
prepared in borosilicate glass vials and spectra were obtained using
quartz cuvettes. a 0 lM CB7. b 10 lM CB7. c 20 lM CB7. d 30 lM
CB7. e 40 lM CB7. f 50 lM CB7. g 60 lM CB7. h 70 lM CB7
Fig. 12 UV–VIS absorption spectra (a at 0 mM CB7 and b at
0.64 mM CB7) and fluorescence emission spectra (c at 0 mM CB7
and d at 0.64 mM CB7) of aqueous pyronin Y (2.5 lM). Samples
prepared and spectra obtained in polystyrene cuvettes
123

J Incl Phenom Macrocycl Chem (2010) 66:231–241
239
The NMR titration spectra are shown in Fig. S28. cence emission measured before transferring back to the
Unlike the PyB where strong shifts were seen for the glass, polypropylene or polystyrene vessels. The absor-
N,N-diethylamino groups, the N,N-dimethylamino groups bance and fluorescence emission spectra for PyY with and
in PyY were not much perturbed by the addition of the CB7. without 0.64 mM CB7 after 0, 1, 12 and 24 h are given for
A broadening and downfield shift for H-9 was seen as was a polystyrene (Fig. S29), polypropylene (Fig. S30) and
broadening and slight upfield shift for H-1/8 and H-2/7. The borosilicate glass (Fig. S31). A summary of the data at 0
signal for H-4/5 broadened and did not reappear. Although and 24 h is shown in Fig. 13. After 24 h for solutions with
it was apparent that the CB7 caused significant changes in no CB7 present, we observed a dramatic decrease in the
the spectrum of PyY, firm insight into the nature of the signal for the solution stored in borosilicate glass to just
association could not be extracted from the data.
26% of the initial signal intensity, a minor decrease in
Substrate-promoted loss of signal intensity due to non- polypropylene to 95% of the original signal and essentially
specific adsorption of PyY onto borosilicate glass was no change for the sample stored in polystyrene. For the
further confirmed through a set of direct control experi- samples containing 0.64 mM CB7, no changes in signal
ments. Dilute aqueous solutions of 2.5 lM PyY with 0 mM strength were found. These results confirm the statements
and 0.64 mM CB7 were prepared in polystyrene cuvettes made by Nau regarding non-specific adsorption of pyronins
and their initial emission intensity was measured. Two to borosilicate glass and the protective value of CB7 [15].
samples were then transferred to a borosilicate glass vial or We did not, however, see the reported strong non-specific
a polypropylene tube; the third sample was left in a poly- adsorption of pyronins to polypropylene.
styrene cuvette. Periodically, all three samples were
transferred to fresh polystyrene cuvettes and their fluores-
Conclusions
We have provided spectroscopy studies, including 1H
700
NMR, UV–Vis, and fluorescence titration experiments of
cucurbit[7]uril complexes with the monocationic xanthene
dyes rhodamine 6G (Rh6G, 1), rhodamine B (RhB, 2),
rhodamine B benzyl ester (RhBBE, 3), pyronin B (PyB, 4)
and pyronin Y (PyY, 5). A summary of the binding con-
stants and changes in optical properties are in Table 1. All
of these cationic xanthene dyes bind strongly with cucur-
bit[7]uril and in all cases except for Rh6G exhibit more
intense fluorescence in the presence of CB7. The binding to
the linear PyB dye with its more hydrophobic N,N-diethyl
substituents was strongest. These xanthene dyes non-spe-
cifically adsorb onto borosilicate glass but not onto poly-
styrene surfaces. CB7 protects against this adsorption. The
data set provided here enables clearer comparisons and
understanding of interactions between CB7 and these
xanthene dyes; this information should be useful in the
design of molecular-scale probes and photonic devices.
600
500
400
300
200
100
0
F, G, H
E
A
B
C
D
520 530 540 550 560 570 580 590 600 610 620 630
Wavelength (nm)
Fig. 13 Temporal evolution of the fluorescence emission intensity of
aqueous pyronin Y (2.5 lM) in different sample containers with and
without CB7. a Polystyrene, 0 h, no CB7. b Polystyrene, 24 h, no
CB7. c Polypropylene, 24 h, no CB7. d Borosilicate glass, 24 h, no
CB7. e Polystyrene, 0 h, 0.64 mM CB7. f Polystyrene, 24 h,
0.64 mM CB7. g Polypropylene, 24 h, 0.64 mM CB7. h Borosilicate
glass, 24 h, 0.64 mM CB7
Table 1 Summary of association constants and changes in optical absorbance and fluorescence emission intensity of aqueous xanthene dyes
with CB7
Xanthene (aqueous conc.)
Ka with CB7 (M-1
)
Ratio of absorbance with/without CB7
Ratio of fluorescence with/without CB7
Rh6G, 1 (2.5 lM)
RhB, 2 (2.5 lM)
RhBBE, 3 (2.5 lM)
PyB, 4 (2.5 lM)
PyY, 5 (2.5 lM)
a
a
0.9 (1.0 mM CB7)
1.3 (1.0 mM CB7)
1.6 (0.64 mM CB7)
1.0 (0.64 mM CB7)
0.9 (0.64 mM CB7)
0.8 (1.0 mM CB7)
1.6 (1.0 mM CB7)
4.7 (0.64 mM CB7)
2.3 (0.64 mM CB7)
1.3 (0.64 mM CB7)
1.6 0.4 9 105
1.1 0.16 9 105
9.1 1.6 9 106
a
Values were tentative and could be fit only using CB7 concentrations less than 0.1 mM
123

240
J Incl Phenom Macrocycl Chem (2010) 66:231–241
Experimental section
and concentrated at room temperature under reduced
pressure. A portion of the crude product (0.106 g) was
purified via silica gel column chromatography (methanol/
chloroform, 9:1), (methanol/chloroform/glacial acetic acid,
90:10:1), respectively, to give 0.056 g (0.10 mmol, 53%)
Sample preparation for absorbance and fluorescence
measurements
1
Unless otherwise noted, 1 mM aqueous solutions of each
cationic dye were prepared in borosilicate glass vials using
Millipore water. The 1 mM aqueous solutions were
immediately diluted to the appropriate concentration in
polystyrene cuvettes. For spectra obtained in quartz cuv-
ettes, 1 mM aqueous solutions were prepared in borosilicate
glass vials using Millipore water followed by subsequent
dilution to the appropriate concentration in additional
borosilicate glass vials. For the optical measurements, each
solution was sequentially transferred to and from the quartz
cuvettes.
of pure 3 as a purple/red waxy solid: H NMR (300 MHz,
CD3CN): d 8.29 (m, 1H), 7.80 (m, 2H), 7.34 (m, 1H), 7.21
(m, 3H), 7.01 (d, J = 9.5 Hz, 2H), 6.87 (dd, J = 2.5,
9.5 Hz, 4H), 6.67 (d, J = 2.5 Hz, 2H), 4.90 (s, 2H), 3.61
(q, J = 7.1 Hz, 8H), 1.26 (t, J = 7.1 Hz, 12H). 13C NMR
(75 MHz, CD3CN): d 173.7, 165.3, 158.1, 157.6, 155.6,
134.8, 133.3, 132.9, 131.2, 131.1, 130.3, 130.2, 130.1,
128.5, 128.3, 128.1, 114.2, 113.4, 95.9, 67.3, 45.6, 11.9.
MS (ESI): m/z 533.33 [M-Cl]? (100%), 443.27 (5%),
?
(M = C35H37N2O3 requires 533.28).
Acknowledgements Support through NSF (DMR-0805233) and
from the University of Oklahoma and Oklahoma State Regents for
Higher Education is appreciated. JLM acknowledges the DOEd for a
GAANN Fellowship. KY, JAIH, KAW acknowledge support through
the Undergraduate Research Program.
Absorbance measurements
Absorbance measurements were performed on a UV–Vis
scanning spectrophotometer. Disposable polystyrene cuv-
ettes of 10.0 mm path were used for the measurements
unless otherwise noted, in which case quartz cuvettes of
10.0 mm path were used instead. The baseline was recorded
with water in both sample and reference cuvettes. All
measurements were performed at ambient temperature (ca.
25 °C).
References
1. Aslan, K., Gryczynski, I., Malicka, J., Matveeva, E., Lakowicz,
J.R., Geddes, C.D.: Metal-enhanced fluorescence: an emerging
tool in biotechnology. Curr. Opin. Biotechnol. 16, 55–62 (2005)
2. Dooley, C.T., Dore, T.M., Hansom, G.T., Jackson, W.C., Rem-
ington, S.J., Tsien, R.Y.: Imaging dynamic redox changes in
mammalian cells with green fluorescent protein indicators.
J. Biol. Chem. 279, 22284–22293 (2004)
3. Kettling, U., Koltermann, A., Schwille, P., Eigen, M.: Real-time
enzyme kinetics monitored by dual-color fluorescence cross-
correlation spectroscopy. Proc. Natl. Acad. Sci. USA. 95, 1416–
1420 (1998)
Fluorescence measurements
Steady-state measurements were performed on a spectro-
fluorophotometer equipped with a Xenon lamp of 150 W as
the excitation source.
Disposable polystyrene cuvettes of 10.0 mm path were
used for the measurements unless otherwise noted, in
which case quartz cuvettes of 10.0 mm path were used
instead. The slit width was adjusted to the minimum setting
for all measurements. Measurements were performed at
ambient temperature. A 0.5 neutral density filter was used
for all measurements except those obtained for aqueous
solutions of Pyronin Y at 2.5 lM, in which case no filter
was required. Although duplicate measurements were
made, single point data from selected single measurements
were plotted and used for the binding analyses.
4. Yan, P., Holman, M.W., Robustelli, P., Chowdhury, A., Ishak,
F.I., Adams, D.M.: Molecular switch based on a biologically
important redox reaction. J. Phys. Chem. B 109, 130–137 (2005)
5. Zhang, Y.B., Kanungo, M., Ho, A.J., Freimuth, P., van der Lelie,
D., Chen, M., Khamis, S.M., Datta, S.S., Johnson, A.T.C.,
Misewich, J.A., Wong, S.S.: Functionalized carbon nanotubes for
detecting viral proteins. Nano Lett. 7, 3086–3091 (2007)
6. Palacios, R.E., Fan, F.-R.F., Bard, A.J., Barbara, P.F.: Single-
molecule spectroelectrochemistry (SMS-EC). J. Am. Chem. Soc.
128, 9028–9029 (2006)
7. Fabbrizzi, L., Poggi, A.: Sensors and switches from supramo-
lecular chemistry. Chem. Soc. Rev. 25, 197–202 (1995)
8. Arunkumar, E., Forbes, C.C., Smith, B.D.: Improving the prop-
erties of organic dyes by molecular encapsulation. Eur. J. Org.
Chem. 2005, 4051–4059 (2005)
Rhodamine B benzyl ester (3)
9. Mohanty, J., Bhasikuttan, A.C., Nau, W.M., Pal, H.: Host-guest
complexation of neutral red with macrocyclic host molecules:
contrasting pK(sub a) shifts and binding affinities for cucur-
bit[7]uril and b-cyclodextrin. J. Phys. Chem. B 110, 5132–5138
(2006)
10. Rohatgi, K.K., Singhal, G.S.: Nature of bonding in dye aggre-
gates. J. Phys. Chem. 70, 1695–1701 (1966)
11. Selwyn, J.E., Steinfeld, J.I.: Aggregation equilibria of xanthene
dyes. J. Phys. Chem. 76, 762–774 (1972)
A mixture of rhodamine B (1.0 g, 2.0 mmol), methylene
chloride (40 mL), 1,3-dicyclohexyl carbodiimide (0.474 g,
2.3 mmol), benzyl alcohol (0.249 g, 2.3 mmol), and
4-(N,N-dimethylamino)pyridine (0.026 g, 0.2 mmol) was
stirred under nitrogen at room temperature for 19 h. The
crude dark pink filtrate was collected via vacuum filtration
123

J Incl Phenom Macrocycl Chem (2010) 66:231–241
241
¨
12. Vosch, T., Hofkens, J., Cotlet, M., Kohn, F., Fujiwara, H.,
25. Schiller, R.L., Lincoln, S.F., Coates, J.H.: The inclusion of
pyronine Y by b- and g-cyclodextrin. J. Chem. Soc. Faraday
Trans. 83, 3237–3248 (1987)
Gronheid, R., van der Biest, K., Weil, T., Herrmann, A., Mu¨llen,
K., Mukamel, S., van der Auweraer, M., de Schryver, F.C.:
Influence of structural and rotational isomerism on the triplet
blinking of individual dendrimer molecules. Angew. Chem. Int.
Ed 40, 4643–4648 (2001)
26. Reija, B., Al-Soufi, W., Novo, M., Tato, J.V.: Specific interac-
tions in the inclusion complexes of pyronines Y and B with b-
cyclodextrin. J. Phys. Chem. B 109, 1364–1370 (2005)
27. Wang, R., Yuan, L., Macartney, D.H.: A green to blue fluores-
cence switch of protonated 2-aminoanthracene upon inclusion in
cucurbit[7]uril. Chem. Commun. 5867–5869 (2005)
28. Wagner, B.D., Stojanovic, N., Day, A.I., Blanch, R.J.: Host
properties of cucurbit[7]uril: fluorescence enhancement of anili-
nonaphthalene sulfonates. J. Phys. Chem. B 107, 10741–10746
(2003)
13. Tinnefeld, P., Buschmann, V., Weston, K.D., Sauer, M.: Direct
observation of collective blinking and energy transfer in a
bichromophoric system. J. Phys. Chem. A 107, 323–327 (2003)
14. Lee, T.-H., Gonzalez, J.I., Zheng, J., Dickson, R.M.: Single
molecule optoelectronics. Acc. Chem. Res. 38, 534–541 (2005)
15. Nau, W.M., Mohanty, J.: Taming fluorescent dyes with cucur-
bit[7]uril. Inter. J. Photoenergy 7, 717–726 (2005)
16. Mohanty, J., Nau, W.M.: Ultrastable rhodamine with cucurbituril.
Angew. Chem. Int. Ed 44, 3750–3754 (2005)
29. Mohanty, J., Pal, H., Ray, A.K., Kumar, S., Nau, W.M.: Supra-
molecular dye laser with cucurbit[7]uril in water. Chem. Phys.
Chem. 8, 54–56 (2007)
17. Buschmann, H.-J., Schollmeyer, E.: Stabilization of dyes against
hydrolytic decomposition by the formation of inclusion com-
pounds. J. Incl. Phenom. Macro. Chem. 7, 133–141 (1992)
18. Freeman, W.A., Mock, W.L., Shih, N.Y.: Cucurbituril. J. Am.
Chem. Soc. 103, 7367–7368 (1981)
19. Day, A., Arnold, A.P., Blanch, R.J., Snushall, B.: Controlling
factors in the synthesis of cucurbituril and its homologues. J. Org.
Chem. 66, 8094–8100 (2001)
20. Kim, J., Jung, I., Kim, S., Lee, E., Kang, J., Sakamoto, S., Yam-
aguchi, K., Kim, K.: New cucurbituril homologues: synthesis,
isolation characterization and X-ray crystal structures of cucur-
bit[n]uril (n = 5, 7 and 8). J. Am. Chem. Soc. 122, 540–541
(2000)
21. Lagona, J., Mukhopadhyay, P., Chakrabarti, S., Isaacs, L.: The
cucurbit[n]uril family. Angew. Chem. Int. Ed 44, 4844–4870
(2005)
22. Saleh, N., Al-Rawashdeh, N.A.F.: Fluorescence enhancement of
cabendazim fungicide in cucurbit[6]uril. J. Fluoresc. 16, 487–493
(2006)
23. Maafi, M., Laassis, B., Aaron, J.-J.: Photochemically induced
fluorescence investigation of b-cyclodextrin; azure A inclusion
complex and determination of analytical parameters. J. Incl.
Phenom. Macro. Chem. 22, 235–247 (1995)
30. Martyn, T.A., Moore, J.L., Halterman, R.L., Yip, W.T.: Cucur-
bit[7]uril induces superior probe performance for single-molecule
detection. J. Am. Chem. Soc. 129, 10338–10339 (2007)
31. Halterman, R.L., Moore, J.L., Mannel, L.M.: Disrupting aggre-
gation of tethered rhodamine B dyads through inclusion in
cucurbit[7]uril. J. Org. Chem. 73, 3266–3269 (2008)
32. Ingham, K.C.: On the application of job’s method of continuous
variation to the stoichiometry of protein-ligand complexes. Anal.
Biochem. 68, 660–663 (1975)
33. Likussar, W., Boltz, D.F.: Theory of continuous variations plots
and a new method for spectrophotometric determination of
extraction and formation constants. Anal. Chem. 43, 1265–1271
(1971)
34. Ong, W., Gomez-Kaifer, M., Kaifer, A.E.: Cucurbit[7]uril: a very
effective host for viologens and their cation radicals. Org. Lett. 4,
1791–1794 (2002)
35. Gutierrez, M.C., Hortiguela, M.J., Ferrer, M.L., delMonte, F.:
Highly fluorescent rhodamine B nanoparticles entrapped in
hybrid glasses. Langmuir 23, 2175–2179 (2007)
36. Klika, Z., Weissmannova, H., Capkova, P., Pospisil, M.: The
rhodamine B intercalation of montmorillonite. J. Coll. Interface
Sci. 275, 243–250 (2004)
24. Singh, M.K., Pal, H., Koti, A.S.R., Sapre, A.V.: Photophysical
properties and rotational relaxation dynamics of neutral red bound
to b-cyclodextrin. J. Phys. Chem. A 108, 1465–1474 (2004)
37. Hassner, A., Alexanian, V.: Direct room temperature esterifica-
tion of carboxylic acids. Tetrahedron Lett. 46, 4475–4478
(1978)
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