Photoresponsive capture and release of lectins in multilamellar complexes

Article abstract of DOI:10.1021/ja3101837

The development of triggered release systems for delivery of peptides and proteins is critical to the success of biological drug therapies. In this paper we describe a dynamic supramolecular system able to capture and release proteins in response to light. The ternary system self-assembles in a dilute aqueous solution of three components: vesicles of amphiphilic cyclodextrin host, noncovalent cross-linkers with an azobenzene and a carbohydrate moiety, and lectins. The cross-linkers form inclusion complexes with the host vesicles, provided the azobenzene is in the trans state. The formation of a ternary complex with lectins requires a high density of cross-linkers on the surface of vesicles. The key innovation in this system is a photoinduced switch from a multivalent, high-affinity state that captures protein to a monovalent, low-affinity state that releases protein. By using isothermal titration calorimetry, dynamic light scattering, UV/vis spectroscopy, and cryogenic transmission electron microscopy, we demonstrate that photoinduced capture and release of lectins in dense multilamellar complexes is highly efficient, highly selective, and fully reversible.

Full text of DOI:10.1021/ja3101837

Article
pubs.acs.org/JACS
Photoresponsive Capture and Release of Lectins in Multilamellar
Complexes
Avik Samanta,† Marc C. A. Stuart,‡ and Bart Jan Ravoo*,†
†Organic Chemistry Institute and Graduate School of Chemistry, Westfalische Wilhelms-Universitat Munster, Corrensstrasse 40,
̈
̈
̈
48149 Munster, Germany
̈
‡Biophysical Chemistry, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747
AG Groningen, The Netherlands
S
* Supporting Information
ABSTRACT: The development of triggered release systems
for delivery of peptides and proteins is critical to the success of
biological drug therapies. In this paper we describe a dynamic
supramolecular system able to capture and release proteins in
response to light. The ternary system self-assembles in a dilute
aqueous solution of three components: vesicles of amphiphilic
cyclodextrin host, noncovalent cross-linkers with an azoben-
zene and a carbohydrate moiety, and lectins. The cross-linkers
form inclusion complexes with the host vesicles, provided the
azobenzene is in the trans state. The formation of a ternary
complex with lectins requires a high density of cross-linkers on the surface of vesicles. The key innovation in this system is a
photoinduced switch from a multivalent, high-affinity state that captures protein to a monovalent, low-affinity state that releases
protein. By using isothermal titration calorimetry, dynamic light scattering, UV/vis spectroscopy, and cryogenic transmission
electron microscopy, we demonstrate that photoinduced capture and release of lectins in dense multilamellar complexes is highly
efficient, highly selective, and fully reversible.
interaction of carbohydrates and lectins. Both recognition
motifs are inherently weak (i.e., binding constant for a single
interaction Ka ≈ 103−104 M−1), so a multivalent display is
essential for the formation of a stable complex in dilute aqueous
solution. The key innovation in this system is a photoinduced
switch from a multivalent, high-affinity state that captures
protein to a monovalent, low-affinity state that releases protein.
The supramolecular system is based on vesicles composed of
amphiphilic CDs (Figure 1).10−12 Vesicles with an average
diameter of 100−150 nm are obtained by extrusion of a
solution of α-CD amphiphiles in water through a 100 nm
polycarbonate membrane. An important advantage of CD
vesicles in comparison with conventional liposomes is the fact
that CD vesicles can selectively bind hydrophobic guest
molecules (such as adamantanes, tert-butylbenzenes, and
azobenzenes) and hence can be decorated with functional
guest molecules such as carbohydrates, peptides, and proteins
simply by mixing the host vesicles with the desired mixture of
guest-appended biomolecules.13−17
INTRODUCTION
■
Peptides and proteins can be powerful drugs to regulate,
restore, or suppress a wide range of physiological processes.1,2
Proteins can also serve as vaccines.2 The breakthrough of
peptide and protein drug therapy has been hampered by two
major obstacles: (1) the large-scale preparation and isolation of
complex peptides and proteins, and (2) the development of an
effective delivery system for peptides and proteins, which are of
course rapidly digested and metabolized in vivo.3 Considerable
progress has recently been made in the area of large-scale
peptide and protein synthesis,4 and also a number of promising
approaches to versatile peptide and protein delivery systems
mostly based on polymer hydrogels5 and polymer nanogels6
have meanwhile been reported. However, stimulus-responsive
supramolecular systems can also play a key role in innovative
delivery systems. Among others, Stoddart and co-workers
reported light-responsive mesoporous silica nanoparticles that
release small molecules,7 Grzybowski and co-workers reported
a redox-responsive nanosponge that can bind and release
nanoparticles,8 and Rotello and co-workers described a
recognition-mediated system to release nanoparticles inside
cells.9
Azobenzenes are particularly interesting photoresponsive
guest molecules for CDs since they can be reversibly isomerized
from trans to cis by irradiation at 350 nm and from cis to trans
by irradiation at 455 nm. The rod-like, apolar trans isomer
forms a stable inclusion complex with α-CD and β-CD, whereas
In this paper we report a dynamic supramolecular system
that is able to capture and release proteins in response to light.
Two orthogonal molecular recognition motifs operate simulta-
neously in this system: the host−guest interaction of
cyclodextrins (CDs) and azobenzenes, and the ligand−receptor
Received: October 15, 2012
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ja3101837 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
Syntheses and Analyses. The syntheses of G1 and G2 were
carried out by coupling of trichloroimidates of the peracetylated
disaccharides lactose and maltose with 2-(2-(2-(2-(4-(phenyldiazenyl)-
phenoxy)ethoxy)ethoxy)ethoxy)ethanol. After deprotection with
NaOCH3, the target products were obtained. Details of the syntheses
are reported in the Supporting Information. The spectroscopic and
analytical data for G1 and G2 are consistent with their molecular
structure. The synthesis of CDV was performed as described in the
literature.12 All reactions were carried out in oven-dried glassware
under an inert gas atmosphere. Analytical thin-layer chromatography
was performed on Merck silica gel 60 F254 plates. All compounds
were visualized by dipping in basic permanganate solution. Column
chromatography was carried out by using Kieselgel 60 (230−400
1
mesh). H and 13C NMR spectroscopic measurements were carried
out by using a Bruker ARX 300 MHz or Varian 500 MHz INOVA
spectrometer. Chemical shifts were referenced to internal standards
CDCl3 (δ = 7.26 ppm for 1H and 77.0 ppm for 13C) or TMS (d = 0.00
ppm for 1H and 13C). High-resolution mass spectrometry was
performed by using a Bruker MicroTOF instrument.
Preparation of Cyclodextrin Vesicles. Unilamellar bilayer CDV
were prepared by extrusion.12 In brief, several milligrams of CDV in 1
mL of chloroform was dried by slow rotary evaporation to yield a thin
film of CDV in a glass vial. Residual solvent was removed under high
vacuum. Aqueous buffer (10 mL, 20 mM HEPES, 1.0 mM MnCl2, 1.0
mM CaCl2, and 0.15 M NaCl, pH 7.4) was added and stirred
overnight. The resulting suspension was repeatedly passed through a
polycarbonate membrane with 100 nm pore size in a Liposofast
manual extruder.
Isothermal Titration Calorimetry (ITC). ITC was performed by
using a Nano-Isothermal Titration Calorimeter III (model CSC 5300;
Calorimetry Sciences Corp., London, UT). ITC measurements were
performed in 20 mM HEPES buffer. A 10 mM solution of α-CD was
titrated into a 1 mM solution of trans-G1 or trans-G2. Twenty-five
injections (10 μL) were performed with an interval of 400 s. The
stirring rate was 300 rpm.
Figure 1. Schematic representation of a cyclodextrin bilayer vesicle
and molecular structures of amphiphilic α-CD and azobenzene−
carbohydrate cross-linkers.
the bent, polar cis isomer does not fit into the cavity of either
CD. In recent years, the photoresponsive molecular recognition
of CDs with azobenzenes has been used to develop light-
responsive hydrogels,18−21 molecular shuttles,22,23 micelles and
vesicles,24,25 surfaces,26,27 and mesoporous nanoparticles.7 Our
group has reported the photoreversible adhesion of CD vesicles
using a divalent azobenzene cross-linker,28 and we have also
described the self-assembly of a supramolecular system of CD
vesicles and an azobenzene−spermine conjugate which can
photoreversibly capture and release DNA.29
Here we demonstrate that the surface of CD vesicles can be
decorated with bifunctional azobenzene−carbohydrate con-
jugates G1 and G2, which can bind to CD vesicles (CDV)
through inclusion of the hydrophobic trans-azobenzene group.
The high density of carbohydrate ligands on the vesicle surface
leads to high-affinity binding of lectins [peanut agglutinin
(PNA) in the case of azobenzene−lactose conjugate (G1) and
concanavalin A (ConA) in the case of azobenzene−maltose
conjugate (G2)] in a dense multilamellar complex. The most
remarkable feature of this ternary complex is a fully reversible
photoinduced transition from a high-affinity, multivalent state
to a low-affinity, monovalent state, which effectively results in
the light-controlled capture and release of protein. The
supramolecular system operates in dilute aqueous solution
under ambient conditions at physiological pH.
UV/Vis Spectroscopy. Optical density measurements were carried
out in 1.5 mL disposable cuvettes with dimensions 12.5 × 12.5 × 45
mm and 10 mm path length using a Uvikon 923 double-beam
spectrophotometer. The optical density was measured at λ = 600 nm
(OD600), which is far from the absorption of the azobenzene
chromophore. Measurements were performed for 30−40 min, unless
otherwise noted, with data points collected every 12 s. Freshly
prepared vesicles and lectin solutions were used for each measurement,
and the measurement procedure was as follows. A 1 mL solution of
CDV (0.1 mM) in aqueous buffer (20 mM HEPES, 1.0 mM MnCl2,
1.0 mM CaCl2, and 0.15 M NaCl, pH 7.4) was injected in a cuvette,
and OD600 was measured for 2 min. After 2 min, 20 μL of conjugate
trans-G1 or trans-G2 (2.5 mM, prepared in Millipore water) was
added to the solution in the cuvette to make the resultant conjugate
concentration 50 μM (this addition was done with slight mixing within
one interval of 12 s), and OD600 was measured for 2 min. After 2 min,
25 μL of PNA or ConA (1 mg mL−1 in Millipore water) was added to
the above solution, and OD600 was measured for at least 30 min. The
same measurements were performed with the other samples by
following the above procedure. Typical concentrations: [CDV] = 0.1
mM, [trans-G1] = [trans-G2] = 0.05 mM, and [PNA] = [ConA] = 1
mg mL−1.
Dynamic Light Scattering (DLS). DLS measurements were
performed by using a Malvern Nano-ZS instrument (Malvern
Instruments) with low-volume disposable cuvettes kept at 25 °C.
The average sizes of the free CDV, the binary mixture of CDV and
bifunctional conjugate trans-G1 (or trans-G2), and the ternary
complex of CDV, bifunctional conjugate trans-G1 (or trans-G2), and
PNA (or ConA) were measured after mixing the corresponding
components. Immediately after alternate UV (350 nm, 30 min) and
visible light (455 nm, 30 min) irradiations, the corresponding average
size of the ternary complex was measured. Typical concentrations:
[CDV] = 0.1 mM, [trans-G1] = [trans-G2] = 0.05 mM, and [PNA] =
[ConA] = 1 mg mL−1 in aqueous buffer (20 mM HEPES, 1.0 mM
MnCl2, 1.0 mM CaCl2, and 0.15 M NaCl, pH 7.4).
EXPERIMENTAL SECTION
■
Materials. All chemicals and lectins used in this study were
purchased from Acros Organics (Schwerte, Germany) or Sigma-
Aldrich Chemie (Taufkirchen, Germany) and were used without
further purification. α-CD was kindly donated by Wacker Chemie
(Burghausen, Germany). All solvents were dried according to
conventional methods before use.
B
dx.doi.org/10.1021/ja3101837 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
Table 1. Thermodynamic Data for the Host−Guest Interaction of α-CD with trans-G1 and trans-G2
host
guest
Ka (M−1
)
ΔH (kJ mol−1
)
ΔG (kJ mol−1
)
ΔS (J K−1 mol−1
)
α-CD
α-CD
trans-G1
trans-G2
8.42 × 103
8.43 × 103
−15.8
−15.5
−22.4
−24.5
21.8
30.0
Irradiation Experiments. Two different light sources were
utilized for irradiation experiments. One source was a Rayonet
photochemical reactor (The Southern New England Ultraviolet Co.)
equipped with 16 RPR-3500 lamps used to generate UV light (350
nm) to isomerize azobenzene from trans to cis. The other source was a
Philips Lumileds royal blue LUXEON K2 emitter (LXK2-PR14-Q00)
used to generate visible light (455 nm) to isomerize azobenzene from
cis to trans.
Cryogenic Transmission Electron Microscopy (Cryo-TEM).
Samples for cryo-TEM were prepared by deposition of a few
microliters of free vesicles or a ternary complex (15 min after the
addition of the corresponding components when OD600 of the
ternary complex is ca. 1.0) on glow-discharged holey carbon-coated
grids (Quantifoil 3.5/1, Quantifoil Micro Tools, Jena, Germany). After
the excess liquid was blotted at 100% humidity and 22 °C, the grids
were vitrified in liquid ethane (Vitrobot, FEI, Eindhoven, The
Netherlands). The vitrified specimens were mounted in a liquid-
nitrogen-cooled Gatan 626 cryo-holder (Gatan Inc., Pleasanton, CA)
and inserted in the electron microscope. Low-dose images were
recorded with a Gatan 4K slow-scan CCD camera (Pleasanton, CA)
on a Philips CM 120 electron microscope (FEI, Eindhoven, The
Netherlands) equipped with a LaB6 tip operated at 120 kV. Typical
concentrations: [CDV] = 1 mM, [trans-G1] = [trans-G2] = 0.5 mM,
and [PNA] = [ConA] = 3 mg mL−1 in 20 mM HEPES buffer (pH
7.4).
complex of a hydrophobic guest molecule and α-CD. The
association constants Ka = 8.42 × 103 for trans-G1 and 8.43 ×
103 for trans-G2 are typical for nonionic azobenzene derivatives
and α-CD.31 It can be seen from Figures S1 and S2 that the
ratio of host to guest is slightly higher than 1:1, which indicates
that an inclusion complex of α-CD and azobenzene with some
secondary interaction (pseudorotaxane inclusion complex) of
the α-CD and the tetra(ethyleneglycol) spacer is formed. The
quality of the titration is somewhat affected by the amphiphilic
character of G1 and G2, which have the tendency to form
micelles at higher concentrations in aqueous media. These
findings are fully consistent with our earlier work on
azobenzene−CD inclusion complexes on vesicles.28,29
Having verified the formation of an inclusion complex
between conjugates trans-G1 and trans-G2 and α-CD, we
investigated the characteristics of CD vesicles decorated with
azobenzene−carbohydrate conjugates. To this end, a 0.05 mM
solution of either trans-G1 or trans-G2 was added to a 0.1 mM
solution of CDV. It can be assumed that trans-G1 and trans-G2
do not (or only very slowly) permeate the vesicle membrane
due to the hydrophilicity of the carbohydrates. Hence, at these
concentrations most of the CD cavities on the outside surface
of the vesicles will be occupied with trans-G1 (or trans-G2), so
that the carbohydrates are displayed in high density at the
vesicle surface, while relatively little carbohydrate is available in
solution. We have previously coined the term “artificial
glycocalix” for this self-assembled multivalent arrangement of
carbohydrate ligands.14 CDV decorated with G1 or G2 should
respond strongly to the addition of lectins that bind to the
carbohydrates. To investigate this phenomenon we selected
two lectins: PNA and ConA. It is known that both PNA and
ConA form tetramers at neutral pH, and each lectin has four
carbohydrate binding sites.33 PNA binds exclusively to β-
galactose and lactose and their derivatives and shows no affinity
toward other carbohydrates (such as α-glucose and maltose).
The high-affinity, multivalent interaction of PNA with lactose
G1 at the surface of CDV should result in agglutination
(aggregation) of the vesicles due to the formation of numerous
noncovalent cross-links between the CD vesicles. On the other
hand, ConA binds exclusively to α-glucose and α-mannose and
their derivatives (including maltose) and shows no significant
affinity toward other carbohydrates (such as galactose and
lactose). Because ConA binding at the CDV surface is also
mediated by a multivalent interaction, the addition of ConA to
CDV decorated with maltose G2 should also result in
agglutination.
RESULTS AND DISCUSSION
■
Amphiphilic α-CD was synthesized as described.12 Unilamellar
bilayer CDV were prepared by extrusion in 20 mM HEPES
buffer at pH 7.4. The diameter of the vesicles was around 100
nm according to DLS. The syntheses of G1 and G2 were
carried out using trichloroimidate glycoside donors. Details of
the syntheses are included in the Supporting Information. The
spectroscopic and analytic data for G1 and G2 are consistent
with their molecular structure.
The guest molecules G1 and G2 each carry two orthogonal
molecular recognition sites. The azobenzene group forms an
inclusion complex with α-CD and β-CD. The azobenzene unit
on conjugate G1 and G2 is known to be a good inclusion guest
for CDV,28,29 even though the binding of guests to CDV is
typically somewhat weaker than that to unmodified CDs in
solution due to presence of the oligo(ethyleneglycol) residues
on the vesicle surface.12 The azobenzene−carbohydrate
conjugates have a tetra(ethyleneglycol) spacer between the
azobenzene and the carbohydrate. It is anticipated that this
spacer enhances the tendency of G1 and G2 to form
pseudorotaxane inclusion complexes with both CDs.30,31
Formation of the host−guest complex of G1 and G2 with
CDs should be photoresponsive because the rod-like and apolar
trans-azobenzene isomer favors complexation with CD, whereas
the bent and polar cis-azobenzene does not.
The interaction between host α-CD and guests trans-G1 and
trans-G2 was measured by using ITC (see Table 1 as well as
Figures S1 and S2 in the Supporting Information). We
performed an “inverse titration” in which a 10 mM solution
of α-CD host was titrated into a 1 mM solution of azobenzene
guest. It can be seen from Table 1 that the thermodynamic
parameters for the titration are very similar for trans-G1 and
trans-G2 and characteristic for the formation of an inclusion
Optical density measurements at 600 nm (Figure 2A)
indicate that indeed spontaneous aggregation of vesicles occurs
in the ternary systems of CDV, conjugate trans-G1, and PNA.
The OD600 of a solution of CDV at a concentration of 0.1 mM
is less than 0.05. When trans-G1 is added (after 2 min, denoted
by first arrow in Figure 2A) to CDV, there is no change in
OD600. When PNA is added (after 4 min, denoted by second
arrow in Figure 2A) to the binary mixture of CDV and
conjugate trans-G1, the OD600 increases from approximately
0.05 to 1.0 within 30 min. This observation indicates the
spontaneous formation of a ternary complex of CDV, trans-G1,
C
dx.doi.org/10.1021/ja3101837 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
Figure 2. Formation of a ternary complex of CD vesicles,
azobenzene−carbohydrate linkers, and lectins. Time-dependent
optical density measurement at λ = 600 nm. Conditions: [CDV] =
0.1 mM, [trans-G1] = [trans-G2] = 0.05 mM, [PNA] = [ConA] = 0.1
mg mL−1; 20 mM HEPES buffer (1.0 mM MnCl2, 1.0 mM CaCl2, 0.15
M NaCl, pH 7.4); T = 23 °C.
Figure 3. Formation of a ternary complex of CD vesicles,
azobenzene−carbohydrate linkers, and lectins. Size distribution
according to DLS. Conditions: [CDV] = 0.1 mM, [trans-G1] =
[trans-G2] = 0.05 mM, [PNA] = [ConA] = 0.1 mg mL−1; 20 mM
HEPES buffer (1.0 mM MnCl2, 1.0 mM CaCl2, 0.15 M NaCl, pH 7.4);
T = 23 °C.
and PNA. In every other case (ConA instead of PNA, no
conjugate, no lectin), no aggregation is observed. This
experiment demonstrates two vital points: complexation is
the result of a high density of lactose G1 on the vesicle surface,
and PNA binds lactose-decorated vesicles only. Similarly,
optical density measurements (Figure 2B) show that
agglutination of the vesicles occurs exclusively in the ternary
system of CDV, conjugate trans-G2, and ConA. In every other
case (PNA instead of ConA, no conjugate, no lectin), no
agglutination is observed. Furthermore, agglutination can be
reversed by the addition of a large excess of glucose (see Figure
S3 in the Supporting Information). These experiments
demonstrate that in this case agglutination is the result of a
high density of maltose G2 on the vesicle surface and ConA
binds lactose-decorated vesicles only. We note that the rate of
agglutination is higher for ConA and G2 than for PNA and G1,
which is consistent with observations reported for a comparable
supramolecular system.15
The results of the optical density measurement were
corroborated by DLS measurements before and after formation
of the ternary complex (Figure 3). According to DLS
measurement the average diameter of the CDV is less than
100 nm. There is no change in the average particle size after
addition of trans-G1 or trans-G2. Upon addition of PNA to
vesicles decorated with trans-G1, the average particle size
increases from ca. 100 to ca. 900 nm (Figure 3A). Similarly, the
average particle size increases from ca. 80 to more than 1000
nm after adding ConA to vesicles decorated with trans-G2
(Figure 3B).
trans-G2) and lectins (either PNA or ConA) was confirmed
by cryo-TEM (see Figure 4). It is evident from Figure 4A that
CD vesicles are spherical and unilamellar in the absence of
guest and lectin. However, after sequential addition of guest
and lectin, large aggregates of multilamellar vesicles and lectins
are observed (Figure 4B,C). It appears the lectin−carbohydrate
interaction and host−guest complexation function as a two-
component supramolecular glue that induces tight adhesion,
flattening, and stacking of CD vesicles, which results in dense,
multilamellar complexes in which the lectins are buried
between bilayers of CD amphiphiles. The particle size observed
in cryo-TEM is consistent with the diameters obtained by DLS.
We note that on the basis of the mixing ratio, the multilamellar
complexes consist of approximately 26% protein, 65% CD, and
9% linker by weight.
Most significantly, UV irradiation of the ternary complex of
CDV, conjugate trans-G1, and PNA at 350 nm for 20 min
decreases both the OD600 from roughly 1.0 to 0.12 (Figure 5A,
red trace) and the average particle size from 900 to about 100
nm (Figure 3A, cyan trace). UV irradiation (350 nm) induces
the photoisomerization of trans-G1 to cis-G1, and the
azobenzene−lactose conjugates detach from the vesicle surface,
since only trans-azobenzene forms an inclusion complex with α-
CD. As a consequence, the carbohydrates are no longer
displayed in a high-affinity multivalent arrangement, and they
readily dissociate from lectins since monovalent binding is
insignificant at this concentration. The OD600 and DLS
measurements indicate that the ternary complex dissociates into
its components, CDV, conjugate trans-G1, and PNA (Figures
3A and 5A). Upon subsequent visible light irradiation of the
The formation of ternary complexes of CDV with
azobenzene−carbohydrate conjugates (either trans-G1 or
D
dx.doi.org/10.1021/ja3101837 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
Figure 4. Cryo-TEM images. (A) Unilamellar CDV. (B) Multilamellar complexes of CDV in the presence of trans-G1 and PNA. (C) Multilamellar
complexes of CDV in the presence of trans-G2 and ConA.
formation and dissociation of the ternary complex is reversible
and thus allows the light-responsive capture and release of
lectins. The reversibility of the light-induced formation of the
ternary complex is quantitative over four cycles, provided the
irradiation time is sufficient (40 min at 350 nm and 40 min at
455 nm) (Figure 5). In comparison, the thermal isomerization
of cis-azobenzene to trans-azobenzene is very slow (see Figure
S4 in the Supporting Information).
CONCLUSION
■
We have developed a photoresponsive ternary supramolecular
system based on orthogonal host−guest complexation and
carbohydrate−lectin interaction that can selectively capture and
release lectins. OD600, DLS, and cryo-TEM measurements
prove that a ternary complex was formed by sequential addition
of azobenzene−carbohydrate conjugate and lectin to the
cyclodextrin vesicle solution at physiological pH. The formation
and dissociation of the ternary complex is based on the light-
responsive interactions of azobenzene−carbohydrate conju-
gates with vesicles. Light-induced dissociation of the ternary
complex leads to the release of lectins at this dilute
concentration. The complexation is highly selective and fully
reversible over four cycles. In the experiments shown here, the
complex contained more than 25% of protein by weight, which
is a significant loading efficiency for a drug delivery system. We
envisage that the incorporation of selective ligands at the
Figure 5. Photoresponsive assembly and disassembly of a ternary
surface of the vesicles, along with light-responsive conjugates,
complex of CD vesicles, azobenzene-carbohydrate linkers, and lectins.
could yield a versatile modular delivery system for the capture,
Time-dependent increase and decrease of OD600 under alternate
transport, and triggered release of proteins to target cells.
irradiation with UV light (350 nm) and visible light (455 nm). (A)
CDV in the presence of trans-G1 and PNA. (B) CDV in the presence
of trans-G2 and ConA. The inset shows the reversible light-responsive
formation of the ternary complex.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis, NMR spectra, and ITC data. This material is
available free of charge via the Internet at http://pubs.acs.org.
ternary system at 455 nm for 30 min (to obtain trans-G1 from
cis-G1), both OD600 increases from ca. 0.12 to ca. 1.0 (Figure
5A, blue trace) and the average particle size increases from ca.
AUTHOR INFORMATION
90 to ca. 1000 nm (Figure 3A, pink trace). The OD600 and
DLS measurements consistently indicate that the ternary
complex of CDV, conjugate trans-G1, and PNA reassembles
upon visible light irradiation. Identical observations are made
for the complex of CDV, conjugate trans-G2, and ConA upon
UV irradiation (Figures 3B and 5B). The light-induced
■
Corresponding Author
b.j.ravoo@uni-muenster.de
Notes
The authors declare no competing financial interest.
E
dx.doi.org/10.1021/ja3101837 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
(30) Nepogodiev, S. A.; Stoddart, J. F. Chem. Rev. 1998, 98, 1959−
1976.
(31) Harada, A. Acc. Chem. Res. 2001, 34, 456−464.
(32) Rekharsky, M. V.; Inoue, Y. Chem. Rev. 1998, 98, 1875−1917.
(33) Lis, H.; Sharon, N. Chem. Rev. 1998, 98, 637−674.
ACKNOWLEDGMENTS
■
We are grateful to the Graduate School of Chemistry for
financial support of A.S.
REFERENCES
■
(1) Vlieghe, P.; Lisowski, V.; Martinez, J.; Khrestchatisky, M. Drug
Discovery Today 2010, 15, 40−56.
(2) Leader, B.; Baca, Q. J.; Golan, D. E. Nature Rev. Drug Discovery
2008, 7, 21−39.
(3) Latham, P. W. Nat. Biotechnol. 1999, 17, 755−757.
(4) Bray, B. L. Nature Rev. Drug Discovery 2003, 2, 587−593.
(5) Lee, K. Y.; Yuk, S. H. Prog. Polym. Sci. 2007, 32, 669−697.
(6) Nochi, T.; Yuki, Y.; Takahashi, H.; Sawada, S.; Mejima, M.;
Kohda, T.; Harada, N.; Kong, I. G.; Sato, A.; Kataoka, N.; Tokuhara,
D.; Kurokawa, S.; Takahashi, Y.; Tsukada, H.; Kozaki, S.; Akiyoshi, K.;
Kiyono, H. Nat. Mater. 2010, 9, 572−578.
(7) Ferris, D. P.; Zhao, Y. L.; Khashab, N. M.; Khatib, H. A.;
Stoddart, J. F.; Zink, J. I. J. Am. Chem. Soc. 2009, 131, 1686−1688.
(8) Klajn, R.; Olson, M. A.; Wesson, P. J.; Fang, L.; Coskun, A.;
Trabolsi, A.; Soh, S.; Stoddart, J. F.; Grzybowski, B. A. Nature Chem.
2009, 1, 733−738.
(9) Kim, C.; Agasti, S. S.; Zhu, Z.; Isaacs, L.; Rotello, V. M. Nature
Chem. 2010, 2, 962−966.
(10) Ravoo, B. J.; Darcy, R. Angew. Chem., Int. Ed. 2000, 39, 4324−
4326.
(11) Mazzaglia, A.; Donohue, R.; Ravoo, B. J.; Darcy, R. Eur. J. Org.
Chem. 2001, 1715−1721.
(12) Falvey, P.; Lim, C. W.; Darcy, R.; Revermann, T.; Karst, U.;
Giesbers, M.; Marcelis, A. T. M.; Lazar, A.; Coleman, A. W.;
Reinhoudt, D. N.; Ravoo, B. J. Chem.Eur. J. 2005, 11, 1171−1180.
(13) Versluis, F.; Tomatsu, I.; Seda, K.; Fregonese, C.; Tepper, A. W.
J. W.; Stuart, M. C. A.; Ravoo, B. J.; Koning, R. I.; Kros, A. J. Am.
Chem. Soc. 2009, 131, 13186−13187.
(14) Voskuhl, J.; Stuart, M. C. A.; Ravoo, B. J. Chem.Eur. J. 2010,
16, 2790−2796.
(15) Vico, R. V.; Voskuhl, J.; Ravoo, B. J. Langmuir 2011, 27, 1391−
1397.
(16) Uhlenheuer, D. A.; Wasserberg, D.; Haase, C.; Nguyen, H. D.;
Schenkel, J. H.; Huskens, J.; Ravoo, B. J.; Jonkheijm, P.; Brunsveld, L.
Chem.Eur. J. 2012, 18, 6788−6794.
(17) Kauscher, U.; Ravoo, B. J. Beilstein J. Org. Chem. 2012, 8, 1543−
1551.
(18) Tomatsu, I.; Hashidzume, A.; Harada, A. J. Am. Chem. Soc. 2006,
128, 2226−2227.
(19) Liao, X.; Chen, G.; Liu, X.; Chen, W.; Chen, F.; Jiang, M. Angew.
Chem., Int. Ed. 2010, 49, 4409−4413.
(20) Yamaguchi, H.; Kobayashi, Y.; Kobayashi, R.; Takashima, Y.;
Hashidzume, A.; Harada, A. Nature Commun. 2012, 3, 603.
(21) Harada, A.; Kobayashi, R.; Takashima, Y.; Hashidzume, A.;
Yamaguchi, H. Nature Chem. 2011, 3, 34−37.
(22) Murakami, H.; Kawabuchi, A.; Kotoo, K.; Kunitake, M.;
Nakashima, N. J. Am. Chem. Soc. 1997, 119, 7605−7606.
(23) Wenz, G.; Han, B. H.; Muller, A. Chem. Rev. 2006, 106, 782−
̈
817.
(24) Wang, Y. P.; Ma, N.; Wang, Z. Q.; Zhang, X. Angew. Chem., Int.
Ed. 2007, 46, 2823−2826.
(25) Zou, J.; Guan, B.; Liao, X. J.; Jiang, M.; Tao, F. G.
Macromolecules 2009, 42, 7465−7473.
(26) Yabe, A.; Kawabata, Y.; Niino, H.; Tanaka, M.; Ouchi, A.;
Takahashi, H.; Tamura, S.; Tagaki, W.; Nakahara, H.; Fukuda, K.
Chem. Lett. 1988, 1, 1−4.
(27) Wan, P. B.; Jiang, Y. G.; Wang, Y. P.; Wang, Z. Q.; Zhang, X.
Chem. Commun. 2008, 5710−5712.
(28) Nalluri, S. K. M.; Ravoo, B. J. Angew. Chem., Int. Ed. 2010, 49,
5371−5374.
(29) Nalluri, S. K. M.; Voskuhl, J.; Bultema, J. L.; Boekema, E. J.;
Ravoo, B. J. Angew. Chem., Int. Ed. 2011, 50, 9747−9751.
F
dx.doi.org/10.1021/ja3101837 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX