A Dual-Responsive Bola-Type Supra-Amphiphile Constructed from a Water-Soluble Calix[4]pyrrole and a Tetraphenylethene-Containing Pyridine Bis-N-oxide

Article abstract of DOI:10.1021/jacs.6b03159

Complexation between a water-soluble calix[4]pyrrole and a ditopic pyridine N-oxide derivative in aqueous media produces a bola-Type supra-Amphiphile that self-Assembles to produce higher order morphologies, including multilamellar vesicles and micelles d

Full text of DOI:10.1021/jacs.6b03159

Communication
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A Dual-Responsive Bola-Type Supra-amphiphile Constructed from a
Water-Soluble Calix[4]pyrrole and a Tetraphenylethene-Containing
Pyridine Bis‑N‑oxide
Xiaodong Chi, Huacheng Zhang, Gabriela I. Vargas-Zuniga, Gretchen M. Peters, and Jonathan L. Sessler*
́
̃
Department of Chemistry, The University of Texas at Austin, 105 East 24th Street, Stop A5300, Austin, Texas 78712-1224, United
States
S
* Supporting Information
Scheme 1. Structures of WC4P, 1, Model Guests 2, 3, and
Model Host 4
ABSTRACT: Complexation between a water-soluble
calix[4]pyrrole and a ditopic pyridine N-oxide derivative
in aqueous media produces a bola-type supra-amphiphile
that self-assembles to produce higher order morphologies,
including multilamellar vesicles and micelles depending on
the pH. The present bola-type supra-amphiphile exhibits
strong fluorescence due to structural changes and
aggregation induced by host−guest complexation. The
resulting structures may be used to recognize, encapsulate,
and release non-fluorescent, water-soluble small molecules.
acrocyclic receptors that can be used to assemble supra-
M
amphiphiles through guest encapsulation in aqueous
milieus are of interest because they can be used to create a wide
variety of higher order morphologies, including vesicles,
micelles, nanotubes, and nanoribbons.1 The resulting self-
assembled constructs are attractive for use in a wide variety of
fields, such as drug/gene delivery, cell imaging, and fuel
storage.2 To date, a variety of receptor systems, including
crown ethers, cucubiturils, cyclodextrins, calixarenes, and
pillarenes, have been used to create supra-amphiphiles.3−7
Calix[4]pyrroles are another well-recognized class of receptors.
Calix[4]pyrroles are macrocycles consisting of four pyrrole
units linked by fully substituted sp3 hybridized meso-carbon
atoms.8 To date, calix[4]pyrroles have been used to create
supramolecular polymers,9 ion-pair receptors,10 anion transport
agents,11 and sensors,12 albeit in noncompetitive solvent
environments.13 Recently, Ballester et al. reported a water-
soluble, aryl-extended calix[4]pyrrole system. In this case,
complexation of pyridine N-oxide derivatives was achieved
under aqueous conditions via a combination of hydrophobic
and hydrogen bond interactions that stabilize guest binding
deep within the aromatic cavity of the calix[4]pyrrole receptor.
The result was formation of a capsule.8d,14 Here we report the
use of water-solubilized calix[4]pyrroles in conjunction with
appropriately designed ditopic pyridine N-oxide guests to create
bola-type amphiphiles.
nature of the host−guest interaction between WC4P and guest
1 allows for the reversible interconversion between the self-
assembled vesicles and small solid nanoparticles as a function of
pH.
1
Prior to studying the proposed self-assembly processes, H
NMR spectroscopic studies were carried out to characterize the
pyridine N-oxide binding properties of WC4P. Due to the
relatively poor water-solubility of 1, the monofunctional
pyridine N-oxide 2 was used for these experiments. As shown
in Figure 1, when 1.0 equiv of 2 was added to a solution of
WC4P in D2O, substantial shifts in the signals for both the host
and the guest were observed. These changes were consistent
with the N-oxide functionality in 2 lying deep within the cavity
formed by WC4P.
To characterize the supramolecular complex WC4P⊃2 in
greater detail, 2D nuclear Overhauser effect spectroscopy
(NOESY) experiments were carried out (Figure S11). Strong
nuclear Overhauser effect (NOE) correlations were observed
between the signals corresponding to protons Hb and Hd on
guest 2 and protons H1 and H2 of WC4P (For peak
The present bola-type amphiphile relies on the recognition of
a tetraphenylethene (TPE) pyridine N-oxide derivative (1) by a
water-soluble aryl extended calix[4]pyrrole (WC4P) (Scheme
1). This complexation occurs in water and is pH dependent.
Under neutral or basic conditions, the interaction between 1
and WC4P produces a supra-amphiphile, which undergoes
further self-assembly to form vesicles. The pH-responsive
Received: March 26, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.6b03159
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Journal of the American Chemical Society
Communication
Scheme 2. Schematic Illustration of the Self-Assembly
Processes That Lead to the Formation of Calix[4]pyrrole-
Based Bola-Type Supra-amphiphiles and Their Self-
Assembly into Multilamellar Stacked Vesicles; Also Shown Is
Their pH-Triggered Vesicle-to-Micelle Transition
1
Figure 1. Partial H NMR spectra (400 MHz, D2O, 25 °C) of (a) 2
alone (2.00 mM); (b) 2.00 mM 2 and 2.00 mM WC4P; and (c)
WC4P alone (2.00 mM).
assignments see Figure S11). These observation provide further
support for the conclusion that 2 is indeed deeply embedded
within the cavity defined by WC4P.
WC4P came from fluorescence spectroscopy. As shown in
Figure 2a, the amphiphile 1 (5.00 × 10−5 M) alone showed
almost no fluorescence in water (Figure 2a, red line). This lack
of appreciable emission intensity for the free form of 1 is
A mole ratio plot based on a UV−vis spectroscopic titration
revealed a maximum consistent with the formation of a 1:1
inclusion complex between WC4P and 2 in water (Figure
S14c). Using nonlinear curve-fitting methods, an association
constant, Ka = (1.43 0.12) × 104 M−1, corresponding to the
formation of WC4P⊃2 could be calculated from these titrations
(Figure S14b). The formation of this complex was further
confirmed by electrospray ionization mass spectrometry (ESI-
MS); cf. Figure S10.
It is well-established that neutral carboxylic acid groups and
anionic carboxylate groups can be interconverted reversibly by
adjusting the solution pH.15 Therefore, the pH-dependent
behavior of WC4P⊃2 complex was studied by first lowering the
solution pH (initially at pH 7.4) to 4.0 by adding HCl. This led
to precipitation of the protonated (carboxylic acid) form of
WC4P and release of guest 2. Support for the proton-induced
1
decomplexation came from H NMR spectroscopic analyses,
which revealed the disappearance of the signals corresponding
to H1, H2, and H3 of the WC4P host. Moreover, only signals
corresponding to free 2 were seen in the deuterated
supernatant (Figure S19b). When the pH was adjusted back
to 7.4 by adding sodium hydroxide, the precipitate dissolved.
Presumably, this reflects reformation of WC4P⊃2, a conclusion
1
supported by H NMR spectral studies (cf. Figure S19c).
The release engendered by the addition of HCl to WC4P⊃2
is ascribed to protonation rather than competition from the
chloride anion. As shown in Figure S12, when 3.0 equiv of
TBACl was added to a mixture of WC4P and 2 at pH 7.4, the
proton chemical shift of 2 underwent little change. On the
other hand, when 2.0 equiv of 3 was added to a D2O solution of
2 and WC4P (both 2.0 mM), the signals corresponding 3
underwent an upfield shift while those for 2 shifted back to
those of the free form (cf. Figure S13). Such findings are
consistent with 3 acting as a competitive guest and displacing 2.
The above studies led us to consider that a ditopic pyridine
N-oxide could be used to promote the formation of
supramolecular aggregates. To test this hypothesis, we prepared
the functionalized amphiphile 1 (Scheme 2). Initial support for
the formation of a supramolecular complex between 1 and
Figure 2. (a) Fluorescence spectra of 1 (red line), 4 + 1 (cyan line), a
1:2 mixture of 1 (5.00 × 10−5 M) and WC4P (black line), and the
latter solution + TBACl (blue line)17 λex = 340 nm in H2O, inset:
Photographs of 1 (left) and 1 + WC4P (right) excited using a 365 nm
UV light source. (b) Tyndall effect (left: 1 alone; right: WC4P + 1).
(c) SEM image of aggregates formed from a mixture of WC4P and 1
at 25 °C. (d) TEM image of WC4P + 1 aggregates at 25 °C. (e) DLS
data for aggregates formed from a mixture of WC4P and 1 at 25 °C.
(f) Fluorescence microscopic image of the aggregates. ([WC4P] = 3.0
× 10−4 M, [1] = 1.5 × 10−4 M).
B
DOI: 10.1021/jacs.6b03159
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Journal of the American Chemical Society
Communication
ascribed to interactions between the constituent TPE subunits,
which are favored in the polar aqueous medium. However,
when 2.0 equiv of WC4P was added to this solution, the
fluorescence intensity increased dramatically (Figure 2a, black
line). Presumably, this increase is due to the fact that the
rotation of the phenyl rings of 1 becomes restricted upon the
addition of WC4P. Therefore, the fluorescence intensity
increases,16 a finding that is consistent with the results of Liu
and co-workers.16b In a control experiment, compound 4
(corresponding to a substructure of WC4P) was added in
excess to an aqueous solution of 1. No appreciable change in
the fluorescence of 1 was seen (Figure 2a, green line).
By monitoring the concentration-dependent surface tension,
the critical aggregation concentration (CAC) of 1 was
calculated to be ca. 1.2 × 10−4 M (Figure S13). The CAC of
1 recorded in the presence of WC4P was lower by more than a
factor of 20 (CAC of WC4P⊃1 = 5.3 × 10−6 M) (Figure S14).
This decrease in the CAC of 1 in the presence of WC4P is
ascribed to the formation of a stable host−guest complex with
WC4P. Furthermore, a solution of WC4P (1.00 × 10−4 M) and
1 (5.00 × 10−5 M) in water shows a Tyndall effect, something
that is not observed for a 5.00 × 10−5 M aqueous solutions of 1
alone (Figure 2b). Such findings are consistent with self-
assembled aggregates being formed from 1:2 mixtures of 1 and
WC4P, not for the individual components.
Scanning electron microscopy (SEM) and transmission
electron microscopy (TEM) were used to study the presumed
self-assembled aggregates (Figure 2c,d). Both studies were
consistent with the formation of spherical aggregates with an
average diameter of about 160 nm (Figure 2c). From the TEM
studies, the thickness of the vesicle walls was calculated to be
about 30 nm (Figure 2d). Dynamic light scattering (DLS)
analyses revealed that the average size of these self-assembled
ensembles was ∼148 nm (Figure 2e), Confocal fluorescence
images showed that the aggregates formed from WC4P⊃1
under these conditions exhibited strong blue fluorescence
(Figure 2f).
diameter of about 40 nm appeared when the pH of the solution
was adjusted to pH 4.0 by adding HCl. However, when the pH
was adjusted back to 7.4 via the addition of NaOHaq, vesicles
analogous to those obtained originally at near-neutral pH were
again observed (Figure 3b). DLS measurements revealed
concordant size differences as a function of pH (Figure 3c,d).
The chemical responsive behavior seen for WC4P⊃2 when
exposed to competitor 3 were reproduced in the case of the
aggregates formed from WC4P⊃1 (cf. Figure S18).
A graphic model of the supramolecular recognition and self-
assembly processes that occur when 2 equiv of WC4P is mixed
with 1 is shown in Scheme 2. Briefly, we suggest that upon
complexation, the two pyridine N-oxide subunits of 1 bind to
WC4P resulting in the formation a bola-type supra-amphiphile,
which then further self-assembles to form a bilayer structure
that curves back on itself to generate multilamellar spheres.
Acidic conditions induce the precipitation of WC4P from
water, which releases 1 in the form of small solid nanoparticles.
Release can also be engendered via the addition of a
competitive guest (3) that contains a pyridine N-oxide subunit.
To date, stimuli-responsive self-assembled materials have
been used to capture and release various cargoes, including
inherently fluorescent encapsulated drugs.18 However, the
release of non-fluorescent drugs cannot easily be monitored
by these means. Our system involves fluorescent components
whose emission intensity increases as the aggregates it forms
are destroyed by lowering the pH or via the addition of a
competitive guest (i.e., 3).
To test whether our system could be used with non-
fluorescent payloads, we chose gemcitabine (a widely used anti-
cancer chemotherapeutic agent) as a model non-fluorescent
drug. As the pH of the solution containing gemcitabine and
WC4P⊃1 was decreased from 7.4 to 4.0, the fluorescence
intensity of the vesicles decreased (Figure 4a, cyan blue line).
The morphological changes of the aggregates formed from
WC4P + 1 were analyzed at two different pH values using
TEM. As shown in Figure 3a, solid nanoparticles with a
Figure 4. (a) Fluorescence emission spectra (WC4P⊃1 with
gemcitabine inside the vesicle), λex = 340 nm; (b) UV−vis spectra
of the supramolecular vesicle solution (with gemcitabine inside) upon
the addition of acid. The concentration of the WC4P⊃1 is 8.0 × 10−5
M.
Concurrently, the absorbance intensity of the gemcitabine
increased (Figure 4b, black line). A similar fluorescence
intensity decrease was seen upon adding the competitive
guest 3. These changes are consistent with the gemcitabine,
initially encapsulated in the vesicles, being released into the
aqueous medium.19
In summary, we have successfully created a water-soluble
pH-responsive calix[4]pyrrole-based molecular recognition
motif, WC4P. In the presence of the ditopic pyridine bis-N-
oxide 1, WC4P forms a bola-type supra-amphiphile
(WC4P⊃1), which self-assembles to form fluorescent vesicles
in water. This system is responsive both to pH and chemical
competitors (e.g., 3). Breakup of the supramolecular vesicles
Figure 3. TEM images of (a) the aggregates formed from WC4P + 1
when the pH of the solution is 4.0 and 7.4 (b). Results of DLS studies
involving (c) the aggregates formed from WC4P + 1 when the pH of
the solution is 4.0 and 7.4 (d). ([WC4P] = 3.0 × 10−4 M, [1] = 1.5 ×
10−4 M).
C
DOI: 10.1021/jacs.6b03159
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Journal of the American Chemical Society
Communication
(10) (a) Ciardi, M.; Galan
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, A.; Ballester, P. J. Am. Chem. Soc. 2015,
generated from WC4P⊃1 releases 1 in the form of small
nanoparticles and represents a responsive switch effect that has
been successfully applied to the controlled release of
gemcitabine. An advantage of the present system is that it
allows the uptake and release of non-fluorescence payload to be
followed by optical means. The present work also serves to
show that calix[4]pyrroles can be used to create self-assembled
materials in aqueous environments.
137, 2047. (b) Kim, S. K.; Sessler, J. L. Acc. Chem. Res. 2014, 47, 2525.
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Soc. 2000, 122, 9350. (b) Liu, Y.; Minami, T.; Nishiyabu, R.; Wang, Z.;
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(13) (a) Allen, W. E.; Gale, P. A.; Brown, C. T.; Lynch, V. M.;
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.6b03159.
(15) Duan, Q.; Cao, Y.; Li, Y.; Hu, X.; Xiao, T.; Lin, C.; Pan, Y.;
Wang, L. J. Am. Chem. Soc. 2013, 135, 10542.
Experimental details and characterization data (PDF)
(16) (a) Hong, Y.; Lam, J. W. Y.; Tang, B. Z. Chem. Commun. 2009,
4332. (b) Jiang, B.-P.; Guo, D.-S.; Liu, Y.-C.; Wang, K.-P.; Liu, Y. ACS
Nano 2014, 8, 1609. (c) Wang, P.; Yan, X.; Huang, F. Chem. Commun.
2014, 50, 5017.
(17) Almost no change in the fluorescence signature was seen when
TBACl was added to the host−guest complex WC4P⊃2.
(18) Reynhout, I. C.; Cornelissen, J. J. L. M.; Nolte, R. J. M. Acc.
Chem. Res. 2009, 42, 681.
AUTHOR INFORMATION
Corresponding Author
*sessler@cm.utexas.edu
■
Notes
The authors declare no competing financial interest.
(19) Zhu, S.; Lansakara-P, D. S. P.; Li, X.; Cui, Z. Bioconjugate Chem.
2012, 23, 966.
ACKNOWLEDGMENTS
■
The work was supported by the U.S. National Science
Foundation (grant CHE-1402004) and the R. A. Welch
Foundation (Grant F-1018).
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DOI: 10.1021/jacs.6b03159
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX