PH-responsive molecular tweezers

Article abstract of DOI:10.1021/ja103153t

Molecular tweezers are dynamic devices that are able to switch from one conformation to another upon stimulation by an external trigger. In this work, we report a new water-soluble macromolecular carrier bearing a pH-responsive molecular tweezer, whose affinity for a substrate depends on the external pH. The conformational change of the switching unit was evidenced by ¹H NMR spectroscopy, and fluorescence studies conducted in aqueous media demonstrated the ability of the carrier to bind to substrates in a pH-dependent fashion.

Full text of DOI:10.1021/ja103153t

Published on Web 06/04/2010
pH-Responsive Molecular Tweezers
Jeanne Leblond,† Hui Gao,† Anne Petitjean,*,‡ and Jean-Christophe Leroux*,†,§
Faculty of Pharmacy, UniVersite´ de Montre´al, Montre´al, H3C 3J7, QC, Canada, Department of Chemistry, Queen’s
UniVersity, Kingston, ON, K7L 3N6, Canada, and Institute of Pharmaceutical Sciences, Department of Chemistry
and Applied Biosciences, ETH Zu¨rich, Zu¨rich, Switzerland
Received April 14, 2010; E-mail: jleroux@ethz.ch; petitjea@chem.queensu.ca
Over the past three decades, significant progress has been made
in the field of drug targeting.1a Although active compounds can be
successfully delivered to some specific sites in the body, drug
release from the carrier at the target site still remains a challenging
step.1b Drug release is commonly achieved through the use of
stimuli-responsive systems such as redox active functions or acid-
sensitive linkage.2a The latter labile bonds undergo cleavage under
a mildly acidic environment, such as those of the endosomes or
the tumoral tissues.2b-d These systems, relying on pH-dependent
chemical hydrolysis, are unfortunately often either too stable or
too labile to offer adequate control over the release rate.3 Alter-
natively, noncovalent interactions offer the possibility to form
supramolecular synthetic receptors with tunable affinities and
responsive properties.4 In particular, dynamic molecular tweezers5
are devices capable of switching from one conformation to another
upon stimulation by an external trigger (light, pH, ions).6 Although
this characteristic has been largely exploited in molecular recogni-
tion and sensing,7 it has yet to be explored for its potential use in
drug delivery.
dibromopyridine and 2-bromoanisole by a Kumada-Corriu-Tamao
coupling.8 At this step, the pH-triggered conformational change of
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ortho-Py (Figure 2A) was investigated by H NMR spectroscopy
and compared to its para counterpart para-Py (Figure 2B), which
presented a single conformational state. Although, in both systems,
protonation influenced the pyridine protons to the same extent, the
behavior of the anisole protons differed. In the para-Py derivative,
the p-Ho proton, facing the nitrogen atom in the neutral state,
became shielded by the NH+ group introduced in its close
environment upon protonation (Figure 2C). Interestingly, this effect
was not obvious for ortho-Py, suggesting a rotation around the
CPy-CPh bond moving o-Ho away from the NH+ proton. These
results, together with 2D NOESY experiments, confirmed the
expected conformational change. The pKa of the system was
estimated by 1H NMR titration to be ∼6.3 (Supporting Information),
which falls in the range of pH encountered in acidic cellular
organelles.2d
In this work, we report a pH-sensitive molecular tweezer
prototype that could serve as a fast responding unit to manipulate
the release rate of a substrate (Figure 1A) by a macromolecular
carrier. The structure comprises three elements: (i) a poly(ethylene
glycol) (PEG) backbone to provide water solubility to the carrier,
(ii) two naphthalene arms to ensure binding to the substrate, and
(iii) a switching unit, i.e. a methoxyphenylpyridinemethoxyphenyl
triad, ortho-Py, to allow the tweezer to switch from the U- to
W-shape conformations at acidic pH, thereby decreasing its affinity
for the substrate (Figure 1B).
Figure 2. Representation of the influence of protonation of the pyridine
ring on ortho-Py (A) and para-Py (B). (C) 400 MHz 1H NMR experimental
chemical shifts of significative protons of ortho-Py (10.9 mM, black) and
para-Py (10.9 mM, white) upon addition of trifluoroacetic acid (TFA) in
1:1 CDCl3/CD3OD.
Figure 1. (A) Schematic representation of the concept. (B) Structure of
the prototype pH-sensitive tweezer and impact of pyridine protonation on
its conformation.
To validate the design of the switching unit itself, a simple,
unsubstituted, ortho-Py triad was readily obtained from 2,6-
To function as a molecular tweezer with binding and release
properties, the ortho-Py system was then flanked with two
hydrophobic binding units. Naphthalene groups were chosen
because of their flat aromatic structure and lower toxicity as
† Universite´ de Montre´al.
‡ Queen’s University.
§ ETH Zu¨rich.
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8544 J. AM. CHEM. SOC. 2010, 132, 8544–8545
10.1021/ja103153t  2010 American Chemical Society

C O M M U N I C A T I O N S
compared to anthracene or pyrene.9 ortho-PyNaph was therefore
synthesized by two successive Suzuki coupling reactions. Its crystal
structure confirmed the anticipated U-shape (Supporting Informa-
neutral medium (Figure 4A). This overall decrease in intensity at
pH 7.4 therefore results from the competing effects of (i) fluores-
cence enhancement by the PEG portion and (ii) significant
quenching by contact with the tweezing unit. In contrast, at pH
4.5, the PEG-tweezer did not quench quinizarin’s fluorescence and
the signal remained comparable to that obtained with the uncon-
jugated PEG (Figure 4B). A similar phenomenon was also observed
with mitoxantrone (Supporting Information). These findings point
to a greater affinity of both substrates for the PEG-tweezer under
neutral conditions, where it adopts a U-shape, than at acidic pH,
where the W conformation predominates.
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tion). H NMR titration with trifluoroacetic acid showed that the
switching properties were maintained in the presence of naphthalene
groups (Figure 3). As far as the naphthalene protons were
concerned, chemical shifts were spread over the aromatic region
in the neutral form, significantly lower than that in free naphthalene
(Figure 3B, bottom). This observation is in agreement with the
mutual shielding of the aromatic groups, which face each other in
the neutral state. Upon addition of TFA, all the naphthalene protons
became deshielded and reached final chemical shifts in two clusters
(n3+n4+n7 and n1+n2+n5+n6), very similar to those of naphthalene
itself (Figure 3B, top). This effect is consistent with a conforma-
tional change from a U- to a W-shape where each naphthalene unit
no longer feels the electronic effect of its counterpart (Figure 3A).
Figure 4. Emission fluorescence of quinizarin (5 µM, λexc ) 490 nm) upon
the addition of PEG-tweezer (0 to 10 equiv), at pH 7.4 (A) and 4.5 (B).
Inset: structure of the substrates.
Overall, the PEG-tweezer reported herein, due to its water
solubility and pH responsiveness, may have potential to control
the release of drugs.
Acknowledgment. The NSERC (Steacie Fellowship to J.C.L.),
CRC program, Queen’s University, and the Canada Foundation for
Innovation are acknowledged for their financial support.
Supporting Information Available: Supporting figures, experi-
mental procedures, NMR, X-ray crystallographic data and mitoxantrone
binding studies. This material is available free of charge via the Internet
at http://pubs.acs.org.
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Figure 3. (A) Representation of the conformational change of ortho-
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Finally, the tweezer structure was grafted to a branched PEG
through position 4py of the pyridine to obtain a water-soluble
carrier. This was achieved through peptide coupling, resulting in a
50% grafting efficiency (Supporting Information). The drug binding/
release properties of the ortho-PyNaph tweezer were investigated
with two substrates: quinizarin and mitoxantrone, an anticancer
drug.10 Both molecules possess an aromatic moiety that can interact
with the two naphthalene units Via aromatic stacking, donor/
acceptor, as well as hydrophobic interactions (Figure 4). Quinizarin
was selected because of its fluorescence properties and the absence
of ionization in the studied pH range.11 As illustrated in Figure 4,
in aqueous conditions (pH 7.4 and 4.5), the addition of the free
PEG slightly enhanced the fluorescence intensity of quinizarin
(compare Dye alone and PEG), possibly due to the better dispersion
(and hence poorer self-quenching) of the dye. In this context, it is
particularly striking to notice that the PEG-tweezer actually led
to an overall decrease of fluorescence of the dye, and so only in
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J. AM. CHEM. SOC. VOL. 132, NO. 25, 2010 8545