Disrupting aggregation of tethered rhodamine B dyads through inclusion in cucurbit[7]uril

Article abstract of DOI:10.1021/jo7026432

(Figure Presented) Hexano- and dodecano-tethered diesters of rhodamine B were prepared. The absorption and fluorescence spectra of these flexibly tethered dyads were compared with those of the rhodamine 3B ethyl ester. Increased J- and H-type dimer format

Full text of DOI:10.1021/jo7026432

chromophores can exhibit potentially undesirable changes in
absorption and fluorescence properties due to dipole-dipole
interactions and additional relaxation pathways.10-12 Applica-
tions involving light harvesting or bright long-lived molecular
probes could benefit from creating tethered packets of chro-
mophores only if these aggregates still exhibit good photosta-
bility and low rates of quenching.13-17 Efforts to control
interactions between tethered chromophores have focused
primarily on the use of rigid linking groups that hold the
chromophoric units at fixed distances.18,19 The approach de-
scribed herein successfully isolates flexibly tethered rhodamine
B ester dyads by binding the cationic chromophoric units in
cucurbit7uril (CB7, shown in Figure 1) hosts resulting in
enhanced fluorescence of these tethered dyads.20
Disrupting Aggregation of Tethered Rhodamine
B Dyads through Inclusion in Cucurbit[7]uril
Ronald L. Halterman,* Jason L. Moore, and Lisa M. Mannel
Department of Chemistry and Biochemistry, UniVersity of
Oklahoma, 620 Parrington OVal, Norman, Oklahoma 73019
rhalterman@ou.edu
ReceiVed December 12, 2007
FIGURE 1. Line drawings of CB7.
Cucurbiturils are pumpkin shaped cyclic oligomers of gly-
coluril that have carbonyl-rimmed portals and a very nonpolar
interior cavity.21,22 Cationic dyes such as alkylviologens,23,24
Hexano- and dodecano-tethered diesters of rhodamine B were
prepared. The absorption and fluorescence spectra of these
flexibly tethered dyads were compared with those of the
rhodamine 3B ethyl ester. Increased J- and H-type dimer
formation and decreased fluorescence emission were ob-
served for the tethered dyads. Complexation of the cationic
chromophoric units in cucurbit[7]uril (CB7) hosts decreased
H-dimer aggregation, especially for the dodecano-tethered
dyad. The monomeric dye and both dye dyads exhibited
enhanced fluorescence upon addition of CB7.
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Xanthenes such as the various rhodamines are important not
only as laser dyes1 but increasingly as components of fluorescent
probes in aqueous media.2,3 Concerns regarding the aggregation
of rhodamines and the effects of aggregation on the photo-
physical properties of these dyes are part of larger questions
regarding interactions between tethered chromophores in mul-
tichromophoric light harvesting systems4-6 and fluorescent
probes.1,7-9 When aggregated or tethered to one another,
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10.1021/jo7026432 CCC: $40.75 © 2008 American Chemical Society
Published on Web 03/27/2008
3266
J. Org. Chem. 2008, 73, 3266-3269

SCHEME 1. Synthesis of Rhodamine B Esters
FIGURE 2. UV-vis absorption spectra: 5 µM Rhodamine B ethyl
ester 1 (A, at 0 mM CB7 and B at 5 mM CB7), 2.5 µM hexano diester
2 (C at 0 mM CB7 and D at 5 mM CB7), and 2.5 µM dodecano diester
3 (E at 0 mM CB7 and F at 5 mM CB7).
xanthenes,25 and brilliant green26 not only bind tightly to CB7
in aqueous solution, but typically exhibit enhanced fluorescence
and photostability due to the unique solvation properties of the
cucurbiturils nonpolarizing interior.25,27-29 We chose to study
rhodamine B ester dyads since rhodamines are widely utilized
chromophores whose ability to form dimeric aggregates in
solution7,30-33 and whose binding to CB7 in solution25 and on
surfaces34 has been established.
Rhodamine 3B, the ethyl ester of rhodamine B, forms dimeric
aggregates in solution at concentrations above 1 mM.7,30 When
adsorbed onto an ionic Montmorillonite surface rhodamine 3B
forms nonassociated species as well as H-type and J-type
aggregates depending upon experimental conditions.35,36 Single
molecule spectroscopy of tethered rhodamine dimers which have
been spin-coated in a PVA matrix indicated two populations of
self-associated species.37 While these studies investigated
changes in aggregation upon solvent changes or matrix isolation,
data on the effects of inclusion are lacking.38 We report here a
comparison of absorbance and emission spectra of aqueous
solutions of the rhodamine 3B monomer 1, the rhodamine B
hexano- and dodecano-diesters 2 and 3 and their enhanced
fluorescence upon CB7 complexation.
initially 9:1 methanol/chloroform to elute rhodamine B, then
90:10:1 methanol/chloroform/acetic acid to elute the monoesters
and finally 5:2:2:1 n-butanol/95% ethanol/water/acetic acid to
elute the diesters. The solvents were removed under vacuum to
provide the products as purple-red waxy solids.
The 1:1 binding of rhodamine B ethyl ester 1 by CB7 in water
was established by absorption and NMR spectroscopy. An
upfield shift of one set of broadened N,N-diethyl signals in the
1H NMR spectrum of 1 (3.9 mM) and one equivalent of CB7
indicated a 1:1 inclusion of one end of the xanthene core into
CB7 and borderline slow exchange on the NMR time scale. In
the presence of two equivalents of CB7 broad averaged signals
were seen for the ethyl groups which indicated borderline fast
exchange at these relatively high concentrations. We speculate
that the excess CB7 can promote faster exchange of the two
N,N-diethyl ends of the xanthane core through an associative
mechanism to form an unstable 2:1 CB7:xanthene intermediate.
Formation of an initial strong 1:1 complex was also indicated
by a continuous variation Job’s plot constructed from absorbance
measurements. A fluorescence titration curve of 1 (5 µM) with
CB7 (0 µM to 12 µM) in water and subsequent nonlinear
regression analysis gave a binding constant (K) of 2.8 × 106
M-1. (Figures and methods provided in supplemental informa-
tion).
The known ethyl ester 1 and the previously unreported
hexano- and dodecano-diesters 2 and 3 were prepared through
DCC-promoted esterifications as shown in Scheme 1. The esters
could be purified as the salts by silica gel chromatography using
The absorption spectra for the ethyl ester 1 and the tethered
dyads 2 and 3 in the absence of and presence of excess CB7 in
water are shown in Figure 2.39 The ethyl ester at 5 µM exhibited
the expected absorption spectrum of the nonaggregated mon-
omeric form showing a λmax around 558 nm and a lower energy
broad monomer shoulder near 510 nm as previously described
for dilute aqueous solutions of rhodamine 3B. An increase in
the concentration of CB7 resulted in only a slight and uniform
increase in absorbance with no indication of altered aggregation.
The minimal effect of CB7 on the wavelength and extinction
coefficient of a nonaggregated rhodamine dye is consistent with
literature data. Rhodamines are known to form face-to-face
H-dimers in which the phenyl side groups are oriented in
opposite directions and thus could only be tethered in such a
head-to-tail conformation by a sufficiently long tether. The short
C6-tether length was chosen to restrict the ability of dyad 2 from
achieving an intramolecular face-to-face H-dimer while still
enabling an oblique edge-to-face J-dimer to form.30,31,40,41 The
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(39) A note of clarificaiton: we refer to the tethered compounds as
“dyads” and to the aggregated chromophores as “dimers.”
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J. Org. Chem, Vol. 73, No. 8, 2008 3267

FIGURE 3. Fluorescence emission spectra: 5 µM Rhodamine B ethyl
ester 1 (A, at 0 mM CB7, and B at 5 mM CB7), 2.5 µM hexano diester
2 (C at 0 mM CB7, and D at 5 mM CB7), and 2.5 µM dodecano diester
3 (E at 0 mM CB7, and F at 5 mM CB7).
FIGURE 4. Line drawings and MM2 calculated three dimensional
depictions: (a) C12-Tethered 3 in a face-to-face H-dimer form. (b) C12-
Tethered 3 after complexation with CB7 in an oblique edge-to-face
J-dimer form (CB7 omitted for clarity).
longer C12-tether should enable not only J-dimer formation but
should also allow the two xanthane cores to adopt a face-to-
face H-dimer form. The absorption spectra of high concentration,
aggregated rhodamines, including rhodamine 3B show a new
band at 525 nm that has been assigned to an H-type dimer as
well as a much more intense J-dimer band that is shifted a few
nanometers bathochromically from the 558 nm band in the
nonaggregated form. The three and 4-fold increase in the
absorbance at 560 nm of the dyads 2 and 3 support the presence
of more strongly absorbing J-dimer aggregated forms. The
presence of a pronounced band at 525 nm in the C12-dyad 3
along with a decrease in the band at 560 nm supports the
presence of more H-dimer and less J-dimer form than in the
monoester 1.30 The hexano-tethered dyad 2 exhibited less
H-dimer form than seen for the longer dodecano-tethered 3. The
striking decrease of this 525 nm band and the increase in the
size of the band at 560 nm upon addition of CB7 to the C12-
dyad 3 supports a conversion of the H-dimer into the J-dimer
in this C12-tethered dyad.
These results should facilitate the design of CB7-protected
multichromophoric systems that retain desirable photophysical
properties.
Experimental Section
Rhodamine B Ethyl Ester (1). A mixture of Rhodamine B (0.500
g, 1.04 mmol), methylene chloride (25 mL), 1,3-dicyclohexyl
carbodiimide (0.258 g, 1.3 mmol), absolute ethanol (0.240 g, 5.2
mmol), and 4-dimethylaminopyridine (0.013 g, 0.10 mmol) was
stirred under nitrogen at room temperature for 72 h. The crude dark-
pink filtrate was collected via vacuum filtration and concentrated
at room temperature under reduced pressure. A portion of the crude
product (0.173 g) was purified via silica gel column chromatography
(methanol/chloroform, 9:1), (methanol/chloroform/glacial acetic
acid, 90:10:1), respectively, to give 0.094 g (0.185 mmol, 54%) of
pure 1 as a purple-red waxy solid: 1H NMR (300 MHz, CD3CN):
δ 8.27 (ddd, J ) 0.5, 1.7, 5.4 Hz, 1H), 7.81 (m, 2H), 7.38 (ddd, J
) 0.5, 1.7, 5.4 Hz, 1H), 7.07 (d, J ) 9.5 Hz, 2H), 6.93 (dd, J )
2.5, 9.5 Hz, 2H), 6.84 (d, J ) 2.5 Hz, 2H), 4.00 (q, J ) 7.1 Hz,
2H), 3.61 (q, J ) 7.1 Hz, 8H), 1.24 (t, J ) 7.1 Hz, 12H), 0.96 (t,
J ) 7.1 Hz, 3H). 13C NMR (75 MHz, CD3CN): δ 173.9, 159.3,
158.2, 156.1, 133.9, 133.3, 131.7, 131.4, 130.9, 130.7, 130.6, 114.7,
113.9, 96.3, 61.7, 46.1, 21.3, 13.4, 12.2. MS (ESI): m/z 471.33
As is typical for a rhodamine dye, the fluorescence emission
for the ethyl ester increased 2.4 fold upon addition of excess
CB7 (Figure 3).27 Although the C6- and C12-dyads exhibited
about a 3-fold absorbance enhancement compared to ethyl ester
1, their fluorescence decreased about 1.2-1.5 fold in the
absence of CB7. Upon addition of CB7 dyads 2 and 3 each
exhibited a fluorescence enhancement of about 3.1 fold (Figure
4).
+
[M- Cl]+ (100%), 443.33 (25%), (M ) C30H35N2O3 requires
These results support the conclusion that we observed a
J-dimer form in the C6-dyad and a form of this type was
maintained upon complexation by CB7. Notably in the C12-
dyad in the absence of CB7 we observed a significant amount
of H-dimer but in the presence of CB7 this H-dimer portion
was apparently converted to the J-dimer form. As a visualization
aid locally minimized structures for this conversion were
obtained and are depicted in Figure 4.42
471.26).
Rhodamine B Hexano diester (2). A mixture of Rhodamine B
(2.533 g, 5.3 mmol), methylene chloride (40 mL), 1,3-dicyclohexyl
carbodiimide (1.091 g, 5.3 mmol), 1,6-hexanediol (0.250 g, 2.1
mmol), and 4-dimethylaminopyridine (0.026 g, 0.21 mmol) was
stirred under nitrogen at room temperature for 72 h. The crude dark-
pink filtrate was collected via vacuum filtration and concentrated
at room temperature under reduced pressure. A portion of the crude
product (0.331 g) was purified via silica gel column chromatography
(methanol/chloroform, 9:1), (methanol/chloroform/glacial acetic
acid, 90:10:1) and (n-butanol/95% ethanol/distilled water/glacial
acetic acid, 5:2:2:1), respectively, to give 0.049 g, (0.047 mmol,
15%) of 2 as a purple-red waxy solid: 1H NMR (300 MHz, CD3-
CN): δ 8.26 (dd, J ) 1.1, 7.5 Hz, 2H), 7.81 (m, 4H), 7.37 (dd, J
) 1.1, 7.5 Hz, 2H), 7.05 (d, J ) 9.5 Hz, 4H), 6.89 (dd, J ) 2.4,
9.5 Hz, 4H), 6.77 (d, J ) 2.4 Hz, 4H), 3.87 (t, J ) 6.3 Hz, 4H),
3.56 (q, J ) 7.1 Hz, 16H), 1.20 (m, 32H). 13C NMR (75 MHz,
CD3CN): δ 165.8, 159.1, 158.2, 156.0, 133.8, 133.4, 131.7, 131.6,
130.8, 130.7, 130.6, 114.7, 113.9, 96.3, 65.7, 46.1, 28.3, 25.6, 12.3.
In summary, we have demonstrated the ability to convert a
tethered rhodamine B dyad from an H-dimer form to a J-dimer
form upon addition of CB7. The strong fluorescence enhance-
ment of the monomeric rhodamine B ethyl ester is preserved
in CB7 complexes of C6- and C12-tethered rhodamine dyads.
(41) Burghardt, T. P.; Lyke, J. E.; Ajtai, K. Biophys. Chem. 1996, 59,
119-131.
(42) Molecular mechanics minimization of provided arbitrary conforma-
tions performed using Spartan 02.
3268 J. Org. Chem., Vol. 73, No. 8, 2008

MS (ESI): m/z 484.40 [M- 2Cl]2+ (100%), 443.40 (26%), (M )
path. The baseline was recorded with water in both sample and
reference cuvettes. All measurements were performed at ambient
temperature (ca. 20 °C).
Fluorescence Measurements. Steady-state measurements were
performed on a spectrofluorophotometer, equipped with a Xenon
lamp of 150 W as the excitation source using disposable polystyrene
cuvettes of 10.0 mm path. The slit width was adjusted to 1.5 for
all measurements. All measurements were performed at ambient
temperature (ca. 20 °C).
+
C62H72N4O6 requires 968.55).
Rhodamine B Dodecanodiester (3). A mixture of Rhodamine B
(1.480 g, 3.09 mmol), methylene chloride (40 mL), 1,3-dicyclohexyl
carbodiimide (0.6373 g, 3.09 mmol), 1,12-dodecanediol (0.250 g,
1.2 mmol), and 4-dimethylaminopyridine (0.0151 g, 0.12 mmol)
was stirred under nitrogen at room temperature for 72 h. The crude
dark-pink filtrate was collected via vacuum filtration and concen-
trated at room temperature under reduced pressure. A portion of
the crude product (0.2513 g) was purified via silica gel column
chromatography (methanol/chloroform, 9:1), (methanol/chloroform/
glacial acetic acid, 90:10:1) and (n-butanol/95% ethanol/distilled
water/glacial acetic acid, 5:2:2:1), respectively, to give 0.0385 g
(0.034 mmol, 15%) of 3 as a purple-red waxy solid: 1H NMR (300
MHz, CD3CN): δ 8.26 (d, J ) 7.0 Hz, 2H), 7.81 (m, 4H), 7.37 (d,
J ) 7.0 Hz, 2H), 7.07 (d, J ) 9.3 Hz, 4H), 6.93 (d, 9.3 Hz, 4H),
6.83 (s, 4H), 3.92 (t, J ) 6.1 Hz, 4H), 3.60 (q, J ) 6.7 Hz, 16H),
1.19 (m, 44H). 13C NMR (75 MHz, CD3CN): δ 165.9, 159.1, 158.2,
156.1, 133.7, 133.3, 131.7, 131.5, 130.9, 130.8, 130.7, 114.8, 113.9,
96.4, 65.9, 46.1, 29.7, 29.6, 29.374, 28.5, 26.0, 12.3. MS (ESI):
Acknowledgment. Support from the University of Okla-
homa and Oklahoma State Regents for Higher Education is
appreciated. J.L.M. acknowledges the DOEd for a GAANN
Fellowship. L.M.M. thanks the OU Honors College for an
Undergraduate Research Award.
Supporting Information Available: Spectral data (1H NMR
and 13C NMR) for compounds 1-3 and CB7. 1H NMR spectra for
the titration of 1 with CB7. Job’s plot, absorption. and fluorescence
titration spectra, titration curve, nonlinear regression. and Scatchard
analysis and binding constant determination for 1-CB7. Absorption
and fluoresence titration spectra of 2-CB7 and 3-CB7. This
information is available free of charge via the Internet at
http://pubs.acs.org.
m/z 627.47 [M- 2Cl]2+ (100%), 443.40 (29%) (M ) C68H84N4O6
+
requires 1052.64).
Absorption Measurements. Absorption spectra were performed
on a UV-vis scanning spectrophotometer using freshly prepared
aqueous solutions in disposable polystyrene cuvettes of 10.0 mm
JO7026432
J. Org. Chem, Vol. 73, No. 8, 2008 3269