Arylazopyrazoles as Light-Responsive Molecular Switches in Cyclodextrin-Based Supramolecular Systems

Article abstract of DOI:10.1021/jacs.6b00484

A simple and high yield synthesis of water-soluble arylazopyrazoles (AAPs) featuring superior photophysical properties is reported. The introduction of a carboxylic acid allows the diverse functionalization of AAPs. Based on structural modifications of th

Full text of DOI:10.1021/jacs.6b00484

Subscriber access provided by EMORY UNIV
Article
Arylazopyrazoles as light-responsive molecular
switches in cyclodextrin-based supramolecular systems
Lucas Stricker, Eva-Corinna Fritz, Martin Peterlechner, Nikos L. Doltsinis, and Bart Jan Ravoo
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b00484 • Publication Date (Web): 13 Mar 2016
Downloaded from http://pubs.acs.org on March 14, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of the American Chemical Society is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 9
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Arylazopyrazoles as light-responsive molecular switches in
cyclodextrin-based supramolecular systems
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Lucas Stricker,† Eva-Corinna Fritz,† Martin Peterlechner‡ Nikos L. Doltsinis§ and Bart Jan
Ravoo*,†
†Organic Chemistry Institute and Center for Soft Nanoscience, Westfälische Wilhelms-Universität Münster, Cor-
rensstrasse 40, 48149 Münster.
‡Institute of Materials Physics, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm-Strasse 10, 48149
Münster.
§Institute for Solid State Theory and Center for Multiscale Theory & Computation, Westfälische Wilhelms-Universi-
tät Münster, Wilhelm-Klemm-Str. 10, 48149 Münster.
KEYWORDS cyclodextrins, host-guest systems, light-responsive systems, molecular recognition, vesicles, nanoparti-
cles, molecular switches
ABSTRACT: A simple and high yield synthesis of water-soluble arylazopyrazoles (AAPs) featuring superior photophysical
properties is reported. The introduction of a carboxylic acid allows the diverse functionalization of AAPs. Based on struc-
tural modifications of the switching unit the photophysical properties of the AAPs could be tuned to obtain molecular
switches with favorable photostationary states. Furthermore, AAPs form stable and light-responsive host-guest complexes
with β-cyclodextrin (β-CD). Our most efficient AAP shows binding affinities comparable to azobenzenes, but more effective
switching and higher thermal stability of the Z-isomer. As a proof-of-principle, we investigated two CD-based supramolec-
ular systems, containing either cyclodextrin vesicles (CDVs) or cyclodextrin-functionalized gold nanoparticles (CDAuNPs),
which revealed excellent reversible, light-responsive aggregation and dispersion behavior. To conclude, AAPs have great
potential to be incorporated as molecular switches in highly demanding and multivalent photoresponsive systems.
e.g. light-responsive hydrogels,16,17,18 surfaces,19,20 and drug
INTRODUCTION
delivery vehicles.21 Combining such a light-responsive su-
pramolecular system with gold nanoparticles22 (AuNPs) al-
lows to address further fields of application due to their
unique chemical and physical properties.23,24,25 Particularly,
when single AuNPs are densely packed, such as in supra-
molecular aggregates, new properties arise from the in-
terparticular coupling of the surface plasmons which can
be observed in the absorption spectrum.26,27 Furthermore,
our group reported on several examples of supramolecular
systems composed of cyclodextrin vesicles (CDVs) self-as-
sembled from amphiphilic cyclodextrins and azobenzenes
as light-responsive molecular switches.28,29,30
Light-responsive molecular switches are widely used in
different fields of chemistry due to the excellent spatial
control of light exposure. One prominent example are az-
obenzenes which can be readily synthesized and modified,
possess high quantum yields and extinction coefficients al-
lowing reversible light-induced E/Z-isomerization over
several cycles.1 The photoisomerization and the resulting
significant structural, chemical and physical changes of the
thermodynamically stable E-isomer to the metastable Z-
isomer were discovered in 1937 and have been extensively
studied ever since.2 Azobenzenes have been successfully
applied for photo-controlled folding and unfolding of pro-
teins and peptides,3,4,5 as self-assembled monolayers
(SAMs) on gold and silicon for reversible surface wettabil-
ity or adsorption,7,8,9 as light-responsive ligand for nano-
particles10,11,12 or for supramolecular materials that convert
light into mechanical energy.13,14
Despite their extensive use as light-responsive switch,
azobenzenes exhibit considerable disadvantages. For ex-
ample, to trigger the E→Z isomerization UV-light is neces-
sary, which is harmful and can be massively scattered in
biological tissue or in nanomaterials.31,32,33 Additionally, the
thermodynamic stability of the Z-isomer of most azoben-
zenes is low compared to other molecular switches such as
diarylethenes.34 As a result of the overlapping absorbances
of both isomers incomplete photoswitching is observed for
azobenzenes. The photostationary state (PSS) for common
azobenzenes is about 80% for the E→Z and 70% for the
Z→E isomerization.35 In particular in highly multivalent
systems this drawback can limit a complete switching since
Moreover, light-responsive host-guest complexes of
α- or β-cyclodextrin (CD) with azobenzenes are known for
a long time.15 While the linear, non-polar E-isomer forms
an inclusion complex with α- and β-CD, the more polar and
bent Z-isomer does not fit in either cavity. For that reason,
diverse light-responsive supramolecular systems com-
posed of azobenzenes and cyclodextrins were developed,
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 9
a large fraction of remaining E-isomer can still dominate
Syntheses and Analyses. The syntheses of all com-
the properties of the material.35,36,37
pounds and CDAuNPs are described in the supporting in-
formation (SI). Moisture sensitive reactions were carried
out in oven-dried glassware under inert atmosphere. Ana-
lytical thin layer chromatography was performed on Merck
silica gel 60 F254 plates. Visualization of the compounds
was achieved either under UV-light of 254 nm with a Dual
Wavelength UV Lamp (254 nm and 366 nm) (CAMAG,
Muttenz, Switzerland) or by staining with basic permanga-
nate solution. For column chromatography silica gel 60
(230-400 mesh) was used. NMR spectra were recorded on
a Bruker AV300 (300 MHz), Bruker AV400 (400 MHz) or
Agilent DD2 (600 MHz) spectrometer. Chemical shifts
were referenced to internal standards.
1
2
3
4
5
6
7
8
For that reason, a lot of research has been carried out to
improve the azobenzene photoswitch. The development of
azobenzenes that isomerize upon irradiation with visible
light by varying the substitution patterns already resulted
in impressive progress.38 In this approach, the aim is either
to shift the π→π* band to longer wavelength or to obtain a
splitting of the n→π* band of the E- and Z-isomer, which
usually overlap in the region of 400-500 nm.39 Beside the
first reported visible-light responsive azobenzene reported
by Temps and co-workers40 other important work has been
published by the groups of Hecht39,41 and Woolley.42,43 They
reported tetra-o-substituted azobenzenes, which showed
splitting of the n→π* band, resulting in good switching
properties. o-Methoxy substituted azobenzenes were re-
cently employed in a cyclodextrin-based hydrogel by Wang
and co-workers.20 Another approach that enhances the
photoswitching and the thermal stability of the Z-isomer
was reported by Fuchter and co-workers.44 They intro-
duced arylazopyrazoles as new light-responsive molecular
switches. Substitution of one benzene by a pyrazole re-
sulted in a significant red shift of the n→π* band of the
Z-isomer, allowing nearly quantitative isomerization by ir-
radiation with UV-(E→Z) or green light (Z→E). Due to de-
creased steric repulsion within the Z-isomer arylazopyra-
zoles feature half-life times up to 1000 days. In general, het-
erocyclic azo-compounds are far less studied but offer a
huge potential in the development of superior molecular
switches.44 Until now, most of the above mentioned molec-
ular switches are not suitable for CD-incorporated supra-
molecular systems since they either bear no functional
group for modifying the switching unit, are poorly water
soluble, undergo no host-guest complexation with CDs or
can be recognized in both isomeric forms.45
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In this article we address these limitations and report
water-soluble arylazopyrazoles (AAPs) that can be utilized
in CD-based supramolecular systems featuring superior
photoswitching characteristics compared to the standard
azobenzenes (Figure 1). The synthesized AAPs showed ex-
cellent photophysical properties and selective host-guest
complexation with CDs of the E-isomer whereas the Z-iso-
mer does not show any interactions. As a proof-of-princi-
ple we successfully incorporated our AAPs in supramolec-
ular systems of CDVs and CD-functionalized gold nano-
particles (CDAuNPs) and demonstrated the light-respon-
sive aggregation and dispersion of these systems over sev-
eral cycles.
Figure 1. a) Molecular structure of AAP1-3 and schematic rep-
resentation of AAP- and CD-based supramolecular systems in-
cluding b) CDV and c) CDAuNP.
Preparation of Cyclodextrin Vesicles (CDVs). The
synthesis of amphiphilic cyclodextrin (aCD) and the prep-
aration of unilamellar bilayer CDVs was carried out accord-
ing to literature.46 Several milligrams of aCD dissolved in a
minimum amount of CHCl3 were dried with a slight argon
stream yielding a thin film in a small flask. Residual solvent
was removed under high vacuum. Aqueous HEPES buffer
(20 mM, pH = 7.2) was added to yield the desired concen-
tration. After stirring over night the result suspension was
repeatedly extruded for a minimum of 15 times through a
polycarbonate membrane (diameter: 100 nm) in a Liposo-
fast manual extruder.
EXPERIMENTAL SECTION
Materials. All chemicals used in this study were pur-
chased from Alfa Aesar (Karlsruhe, Germany), Sigma-Al-
drich Chemie (Taufkirchen, Germany) or TCI Europe
(Zwijndrecht, Belgium) and used without further purifica-
tion. β-CD was kindly donated by Wacker Chemie
(Burghausen, Germany). Solvents were dried according to
conventional methods before use.
Isothermal Titration Calorimetry (ITC). ITC was car-
ried out using a TA Instruments Nano ITC Low Volume
ACS Paragon Plus Environment

Page 3 of 9
Journal of the American Chemical Society
(Waters Corp., Milford, Massachusetts, USA) with a cell
volume of 170 μL using ITCRun Version 2.1.7.0 Firmware
HighPower-LED emitting at 520 nm for Z→E-isomeriza-
tion.
1
2
3
4
5
6
7
8
version 1.31 (TA Instruments, WatersCorp., Milford, Mas-
sachusetts, USA). All titrations were performed using a
50 μL syringe and 20 injections of 2.5 μL at a temperature
of 25 °C with a stirring rate of 350 rpm while titrating the
CD to the AAP solution. All samples were prepared in
ddwater and degassed for 10 min before use. The data were
analyzed using NanoAnalyse Data Analysis version 2.36
(TA Instruments, Waters Corp., Milford, Massachusetts,
USA), Microsoft® Excel version 14.07113.5005 as part of Mi-
crosoft® Office Professional Plus 2010 (Microsoft Corp.,
Redmond, Washington, USA) and OriginPro 9.1.G
(OriginLab Corp., Northampton, Massachusetts, USA). Be-
fore analysis, all data were corrected by subtraction of a
blank titration of the CD into pure solvent.
RESULTS AND DISCUSSION
To obtain water-soluble and effectively photoswitchable
AAPs that can undergo host-guest complexation, different
structural modifications were investigated. By introducing
a carboxylic acid group, AAPs can be easily further func-
tionalized. To obtain an optimal photoisomerization three
different structural modifications were designed and syn-
thesized (Scheme 1). mAAP1 and mAAP3 differ in the posi-
tion of the carboxylic acid group which should result in
host-guest complexation occurring from the benzene side
(mAAP1) or pyrazole side (mAAP3). In contrast to mAAP1,
fluorine atoms were introduced at the o-positions of the
benzene ring giving mAAP2. The synthesis of mAAP1-3 was
performed according to a recently reported procedure in
excellent yields (Scheme 1).44 Starting from commercially
available anilines diazo coupling with 2,4-pentadienone
followed by condensation using either hydrazine or methyl
hydrazine yielded AAPs. The carboxylic acid functionality
was introduced via Williamson ether synthesis using me-
thyl bromoacetate followed by ester hydrolysis giving
AAP1-3. With regard to the light-responsive aggregation
experiments AAP1-3 were functionalized either with a
monovalent polyamine or a divalent amine yielding water-
soluble mAAP1-3 or dAAP1-3, respectively. Details of the
synthesis and analysis of all AAPs can be found in the SI.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
UV/vis Spectroscopy. UV/vis- and OD600 measure-
ments were conducted using a JASCO V-650 double-beam
spectrophotometer (JASCO Labor- und Datentechnik
GmbH, Gross-Umstadt) at 25 °C using 1 mL low-volume
disposable PMMA cuvettes (Brand GmbH & CO KG,
Wertheim). The spectrometer was controlled by Spectra
Manager version 2.08.04 (Jasco Labor- und Datentechnik
GmbH, Gross-Umstadt). The samples were dissolved in an
appropriate solvent and measured against the same sol-
vent. Data analysis was carried out using OriginPro 9.1G
(OriginLab Corp., Northampton, Massachusetts, USA).
Optical density measurements were performed at λ =
600 nm where AAPs show no absorbance. Measurements
were conducted for 60 min and data points collected every
minute. For all measurements 1 mL of CDV (50 µM) dis-
solved in HEPES buffer (10 mM, pH = 7.2) was placed in a
cuvette and measured. Addition of the desired amount of
dAAP (2 mM stock solution in DMSO) occurred after
4 min. Spectra of the aggregation of CDAuNP were rec-
orded in the range of 400-800 nm. Details about the con-
centration are described in the SI.
Scheme 1. General synthetic strategy towards water-
soluble monovalent mAAP1-3 and divalent dAAP1-3.
Dynamic Light Scattering (DLS). For DLS measure-
ments a Malvern Nano-ZS instrument (Malvern Instru-
ments) with low-volume disposable cuvettes kept at 25 °C
was used. The average sizes of CDV, CDAuNP and binary
mixtures of AAPs with CDV or CDAuNP were measured
after mixing the corresponding components.
Transmission Electron Microscopy (TEM). TEM in-
vestigations were performed using a Zeiss Libra 200FE with
in-column energy filter (Carl Zeiss AG, Oberkochen, Ger-
many) equipped with a Gatan US 4000 CCD camera (Gatan
GmbH, München, Germany). For size measurements Im-
ageJ 1.45s (National Institutes of Health, USA) was used.
TEM samples were prepared by immersing the copper grid
in the nanoparticle solution for 20 s. Grids were dried un-
der ambient conditions overnight.
Reaction conditions: i) NaNO2, HCl, AcOH, 2,4 pentadione,
NaOAc, 85-90%; ii) hydrazine × hydrate or methyl hydrazine,
quant.; iii) 1) methyl bromoacetate, K2CO3; 2) LiOH, 80-95%;
iv) 1) Boc-protected polyamine, PyBOP, DIPEA, 98-100%; 2)
acetylchloride, quant. ; v) divalent amine linker, EDCl, HOBt,
NMM, 67-74%.
Photoswitching Experiments. Two different light
sources were utilized for photoswitching experiments, a
Rayonet photochemical reactor (The Southern New Eng-
land Ultraviolet Company) equipped with 16 RPR-3500
lamps (365 nm) for E→Z- isomerization and a LSC-G
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 9
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Experimental normalized and calculated UV/vis spectra of the photoisomerization of mAAP1-3 in water, a) mAAP1, b)
mAAP2 and c) mAAP3 (each 50 μM; irradiation time 10 min); insets: Zoom-in at the 500 nm area.
UV/vis spectroscopy was carried out to investigate the
photophysical properties of the prepared mAAPs. UV/vis
spectra shown in Figure 2 clearly indicate the high poten-
tial of mAAP1-3 as molecular switches. Regarding the E→Z-
isomerization upon irradiation with UV-light (365 nm), all
mAAPs show the characteristic changes in the absorbance
which is a significant decrease and a slight blue shift of the
π→π* band from 300-350 nm to ca. 300 nm and an increase
of the n→π* band around 430 nm. Additionally, a slight
red shift of the n→π* band is observed for mAAP1 and
mAAP3. Upon irradiation with green light (520 nm) the E-
isomer is reformed. Remarkably, the re-obtained intensity
of mAAP1 and mAAP3 amounts to 94% which is almost
quantitative. However, mAAP2 only returns to 71% of the
initial intensity. The efficient photoisomerization of
mAAP1 and mAAP3 can be explained by the observed red
shift of the n→π* bands for the Z-isomers resulting in
higher extinction coefficients at wavelengths above 500 nm
and the consequent splitting of the n →π* absorbances for
the Z- and E-isomers of mAAP1 and mAAP3 which allows a
more specific excitation. For mAAP2 no red-shift of the n
→π* absorbance is observed resulting in a lower switching
efficiency (s. insets Figure 2). Calculated spectra of all AAPs
(CONH2-terminated) were obtained by time-dependent
density functional theory (TDDFT), revealing good agree-
ment with the measured spectra. In general a slight red-
shift of the absorbances are observed for the calculated
spectra (Figure 2, dashed curves).
irradiating for 60 min. Exemplarily for all mAAPs, the aro-
matic region of the NMR spectra of mAAP1 is shown in Fig-
ure 3 (full spectra of mAAP1-3 can be found in the SI). Com-
paring the spectra of the E-and the Z-isomer, the signals
for the E-isomer almost vanished completely whereas three
new signals of the Z-isomer arose shifted to lower fields.
Upon the second irradiation with green light, the initial
signals of the E-isomer re-appeared, whereas the signals of
the Z-isomer disappeared. Based on signal integration, the
PSS of mAAP1 amounts to ca. 90% for both isomerization
processes. The PSS of mAAP2 and mAAP3 as well as the
corresponding half-life times are listed in Table 1 support-
ing the UV/vis spectroscopic findings. Details about the
determination of the half-life time can be found in the SI.
The PSSs of mAAP1-3 rating the efficiency of the pho-
toisomerization were determined by 1H-NMR spectroscopy
(Figure 3). To this end, NMR spectra of the E-, Z- and re-
formed E-isomer of mAAP1-3 were recorded directly after
Figure 3. Aromatic region of the 1H-NMR spectra of the E-iso-
mer, PSSE Z and PSSZ E of mAAP1 recorded in D2O (0.5 mM;
→
→
irradiation time 60 min).
ACS Paragon Plus Environment

Page 5 of 9
Journal of the American Chemical Society
In summary, the photophysical properties clearly indi-
binding constants of each interaction. ITC results of each
titration is summarized in Table 2 (corresponding titration
curves can be found in the SI). As comparison, E-azoben-
zene with a comparable substituent shows 1:1 interactions
with α- and β-CD with binding constants of K ~ 103 M-1.35
All three E-isomers form stable 1:1 host-guest inclusion
complexes with β-CD. In the case of E-mAAP3 an interac-
tion of K = 18.3 × 102 M-1 was quite unexpected due to the
fact that the pyrazole unit is similar to an imidazole which
is no suitable guest because of its high polarity.47 There-
fore, we assume that binding occurs via the substituted
benzene unit resulting in a threading-like complexation
placing the benzene unit in the CD cavity. In contrast, the
benzene unit is easily accessible for mAAP1 and mAAP2
and can be placed inside the cavity without threading (Fig.
5c). ITC measurements of E-dAAP3 confirmed our hypoth-
esis concerning the binding mechanism since no interac-
tion of E-dAAP3 with β-CD can be detected (Figure S10). In
the dimer, both ends of the molecule are blocked by the
bulky methylated pyrazole prohibiting the threading. As a
consequence there is no possibility to place the benzene
inside the CD cavity. E-mAAP2 revealed a rather low bind-
ing constant of K = 5.05 × 102 M-1 which can be explained by
the fact that in general fluorinated aromatic compounds
show less affinity to CDs.48 The highest binding constant is
found for the inclusion complex of β-CD and E-mAAP1
which amounts to K = 23.8 × 102 M-1 and is comparable to
the interaction between β-CD and azobenzene (Figure 5).
Regarding α-CD, ITC titrations reveal that the smaller
sized CD shows only a weak interaction with E-mAAP1. No
significant host-guest complexation was detected with E-
mAAP2 and E-mAAP3. For the determination of the bind-
ing constant of the dimer of AAP1 we used dAAP1*, in
which the AAP units are linked by a polyamine instead of
a tetraethylene glycol chain. dAAP1* has significantly
higher water solubility than dAAP1. In earlier work our
group has already shown that also these dimers are suitable
cross-linkers for CDV.49 In further experiments we only
used dAAP1 for cross-linking of CDV or CDAuNP to avoid
electrostatic interactions of the polyamine linker with the
negative surface of AuNPs.
1
cate that AAPs are highly interesting candidates as molec-
ular switches. mAAP1 and mAAP3 showed excellent light-
responsive isomerization behavior which is superior to the
light-response of commonly used azobenzenes. Therefore,
our data is in good agreement to the work of Fuchter and
co-workers demonstrating quantitative isomerization for
AAPs.44 Regarding our structural modifications, the func-
tionalization of AAPs with carboxylic acid groups resulted
only in a marginal decrease of the PSS. However, the intro-
duction of the o-F substituents increases the half-life time
of the Z-isomer of mAAP2. Similar observations have been
reported by Hecht and co-workers. The efficiency of the
E→Z-isomerization remains unchanged whereas the PSS
of the Z→E-isomerization drops drastically to 55% due to
the complete overlap of the n→π* bands of both isomers.
It follows that mAAP1 features the best photophysical
properties and AAP1 should be a promising molecular
switch for applications in multivalent host-guest systems.
Additionally the quantum yields for the photoisomeriza-
tions of all AAPs were determined by irradiation time de-
pendent UV/vis measurements. The calculated quantum
yields can be found in the SI.
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. Summary of photophysical properties of
AAP1-3 in comparison to azobenzene.
τ1/2 Z-state
5.70 d
PSSE
80%35
PSSZ
E
→
→
Z
azobenzene
mAAP1
70%35
88%
55%
92%
9.49 d
mAAP2
94%
89%
>>11 d
mAAP3
92%
1.32 d
The optimized geometry of all AAPs (terminated by
CONH2) were calculated by the DFT method. In all pre-
dicted structures the E-isomers show a planar structure,
whereas the Z-isomers adopt a twisted geometry to mini-
mize the steric strain between the methyl groups of the py-
razole ring and the phenyl ring (Figure 4 and Figure S9).
These results are similar to the geometries calculated by
Fuchter and co-workers.44 The summarized calculated
changes in relative energy and the rotation barrier of AAPs
are stated in the SI.
Table 2. Binding constants of α-CD and β-CD with
E-AAPs and E-azobenzene determined via ITC.
Guest
E-azobenzene35
E-azobenzene35
E-mAAP1
Host
α-CD
β-CD
α-CD
β-CD
α-CD
β-CD
α-CD
β-CD
β-CD
β-CD
K × 102 M-1
59.0
24.0
4.01
23.8
—
E-mAAP1
E-mAAP2
E-mAAP2
5.05
—
E-mAAP3
E-mAAP3
18.3
Figure 4. DFT-optimized geometry of E/Z-AAP1 terminated
with CONH2.
E-dAAP1*
35.9
—
Next, we investigated if E-mAAP1-3 can form host-guest
complexes with α-and β-CD. To this end, isothermal titra-
tion calorimetry (ITC) was carried out to determine the
E-dAAP3
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 9
irradiation with green light the OD600 value increased to
1
2
3
4
5
6
7
0.65 and the hydrodynamic diameter to 2500 nm. In total,
the light-induced switching between the aggregated and
dispersed state was possible over at least five cycles with-
out decreasing efficiency. These findings demonstrate the
highly reversible and light-responsive character of the
AAP/CD-based supramolecular system.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. a) ITC titration curve; b) corresponding 1:1 fit of
E-mAAP1 and β-CD (E-mAAP 1 mM and β-CD 10 mM in H2O)
and c) Proposed binding mechanism for mAAP1-3.
Based on our preliminary results and with regard to the
incorporation of AAPs into CD-based supramolecular sys-
tems, we focused on investigations of β-CD as host mole-
cule. As guest molecules, we used the divalent dAAP1-3.
The construction of such supramolecular systems shall be
a proof-of-principle demonstrating the versatility of AAPs
as photoswitches. First, we developed a supramolecular
system of dAAP1-3 and cyclodextrin vesicles (CDVs). CDVs
are unilamellar bilayer vesicles composed of amphiphilic
CD with a size of ca. 100 nm. These nanostructures com-
bine the characteristics of vesicles and cyclodextrins and
display β-CD cavities at the vesicle surface.46 To character-
ize the formation of the host-guest inclusion complexes
time-dependent optical density measurements at 600 nm
(OD600) were performed (Figure 5a). Hereafter, the char-
acterization is exemplarily discussed for dAAP1, the results
of dAAP2-3 can be found in the SI. At t = 0 min CDVs show
an OD600 of 0.049 which increases immediately upon the
addition of E-dAAP1 and reaches a plateau after 60 min.
The extent of the increasing OD600 depends on the guest
concentration which is due to the non-covalent cross-link-
ing of the CDVs by the divalent guest and the resulting ves-
icle aggregation. Both control experiments, the addition of
Z-dAAP1 to CDVs and E-dAAP1 measured in the absence of
CDV, did not affect the optical density. Furthermore, the
size of the formed aggregates was determined by dynamic
light scattering (DLS). Upon addition of 10 µM E-dAAP1 to
CDVs the average size of the aggregates changes from
120 nm to 2200 nm, whereas no significant change was ob-
served when the corresponding Z-isomer was added (Fig-
ure 6b). The reversible, light-responsive aggregation and
dispersion of this supramolecular system upon alternating
light irradiation was again followed by DLS and OD600
(Figure 6c). The applied concentration of E-dAAP1 was
10 µM. Starting from the vesicle aggregates, irradiation
with UV-light results in a decrease in the OD600 to 0.07
and of the hydrodynamic diameter to 136 nm. Upon second
Figure 6. a) OD600 of concentration-dependent aggregation
of CDV and E-dAAP1 and reversible aggregation and disper-
sion of CDV and dAAP1 studied by b) DLS and c) OD600 (E-
dAAP1 10 µM, Z-dAAP1 20 µM, aCD 50 µM, irradiation time
10 min).
In comparison, the divalent fluorinated guest E-dAAP2
is indeed able to induce the aggregation of CDVs although
a much higher guest concentration is necessary to achieve
the same increase in OD600 compared to E-dAAP1 (Figure
S17). To obtain an OD600 of 0.3 a fivefold higher concen-
tration of 30 µM had to be added. The corresponding DLS
measurements showed the expected signal at 1200 nm but
also a second signal indicating remaining non-aggregated
vesicles (Figure S17). An explanation for these results might
be the lower binding affinity of E-mAAP2 to β-CD (see Ta-
ble 2). Nevertheless, a UV-light-induced dispersion of the
aggregated CDVs could be observed whereby OD600 and
DLS values of 0.08 and 140 nm are comparable to the re-
sults of E-dAAP1. A significant re-aggregation upon a sec-
ond irradiation with green light cannot be observed which
is likely due to the combination of the incomplete pho-
toswitching (PSS of 55%) and the rather low binding con-
stant (K = 505 M-1) of E-mAAP2. Longer irradiation times
of 60 min only result in an increase to 0.12 in the OD600,
aggregates could not be detected by DLS. A second cycle of
photoswitching revealed no significant difference between
the E- and Z-isomer. We conclude that dAAP2 is an inferior
molecular switch compared to dAAP1.
The addition of E-dAAP3 as a guest molecule resulted in no
aggregation of CDVs (Figure S18). These findings support
the proposed host-guest complexation of β-CD being
threaded from the polyamine end to the benzene for the
monomeric guest (E-mAAP3) which is not possible for the
divalent structure. We conclude that dAAP3 is not a suita-
ble divalent cross-linker for CDVs.
ACS Paragon Plus Environment

Page 7 of 9
Journal of the American Chemical Society
that increasing concentrations of E-dAAP1 can be corre-
1
2
3
4
5
6
7
lated to an increasing size of the formed aggregates (Figure
7b). Furthermore, it is possible to control and adjust the
size of the aggregates in the range of ca. 30 and 300 nm. As
a control experiment Z-dAAP1 (0.16 mM) was added which
did not result in an aggregation of CDAuNPs (Figure 7b) or
a shift in the SPR band (Figure S21).
Transmission electron microscopy (TEM) was carried out
to obtain further information of the shape and the macro-
scopic structure of the CDAuNP aggregates. TEM samples
were taken from highly diluted aggregate solution to pre-
vent aggregation effects simply caused by the drying of the
TEM grid. In Figure 7c aggregates induced by an E-dAAP1
concentration of 0.16 mM are shown. Remarkably, all im-
aged CDAuNPs are clustered in aggregates. CDAuNPs are
aggregated in a high density which is increasing in a radial
fashion towards the center of the aggregates indicating a
spherical shape. The light-responsive and reversible aggre-
gation and dispersion upon alternating light irradiation
was investigated by the position of the SPR band as well as
the OD600. First, CDAuNP aggregates using an E-dAAP1
concentration of 0.16 mM were formed upon irradiating
with green light for 3 min (Figure 7d, green gradients). The
SPR band is located at 536 nm and the OD600 value
amounts to 0.49. Focusing on the n→π* band around
430 nm of dAAP1, the isomerization to Z-dAAP1 within the
CDAuNP aggregates is completed after irradiating with
UV-light for 3 min (Figure 7d, red gradients). More inter-
estingly, the position of the SPR band is blue-shifted to
533 nm and the intensity at 600 nm is decreased to 0.45
both strongly indicating a dispersion of the aggregates.
Upon irradiating the dispersed sample again with green
light, re-aggregation can be observed since both the posi-
tion of the SPR band as well as the OD600 value are recov-
ered (Figure 7e). Altogether, the light-responsive switch-
ing between the aggregated and dispersed state is fully re-
versible over five cycles.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 7. Investigations of the concentration-dependent ag-
gregation of CDAuNP and E-dAAP1 by a) UV/vis spectroscopy,
b) DLS (E-dAAP1 0.08-0.25 mM (green gradient), Z-dAAP1
0.16 mM (red curve) and c) TEM (0.16 mM) and the reversible
aggregation and dispersion of CDAuNP and dAAP1 monitored
by c) UV/vis spectroscopy, e) the SPR band (top) and the
OD600 value (bottom)(E-dAAP1 0.16 mM, CDAuNP 0.05 mg,
irradiation time 3 min (520 nm), 10 min (365 nm).
Based on the results of the E-dAAP-induced aggregation
of CDVs which clearly show that dAAP1 is the most effi-
cient light-responsive guest, we focused on this guest mol-
ecule for the incorporation in the second proof-of-princi-
ple supramolecular system based on CDAuNPs. First, wa-
ter-soluble CDAuNPs were synthesized by reducing Au3+ in
the presence of per-thiolated β-cyclodextrin (tCD). The
successful immobilization of tCD on the AuNPs can be ob-
CONCLUSION
In summary, we have presented water-soluble AAPs and
their huge potential as efficient molecular switches in a va-
riety of CD-based supramolecular systems. Our key struc-
tural modification of the AAP unit is the introduction of a
carboxylic acid group which can be easily further function-
alized. The synthesis of the mono- and divalent AAPs is
straightforward and proceeds in high yields and the final
compounds have excellent water solubility. Comparing
mAAP1-3, we showed how structural modifications of the
switching unit affect the photophysical properties. All
monovalent mAAPs showed excellent E→Z-isomerization
and favorable photostationary states. mAAP1 features the
most attractive photophysical properties and forms a sta-
ble and light-responsive 1:1 host-guest complexation with
β-CD. Light-responsive switching of this inclusion com-
plex is more efficient and the thermal half-life time of the
Z-isomer is superior compared to commonly used azoben-
zenes. On the basis of two examples of CD-based supramo-
lecular systems, we demonstrated the diversity of possible
1
served from the H-NMR spectra showing characteristic
signals for tCD that are significantly broadened compared
to the unbound ligand (NMR spectra and further charac-
terization of CDAuNPs can be found in the SI). To investi-
gate the host-guest complexation of CDAuNPs and
E-dAAP1 and the resulting aggregation-induced plasmon
coupling UV/vis spectroscopy was carried out (Figure 7a).
To this end, different concentrations of E-dAAP1 (0-
0.25 mM) were added to CDAuNPs and allowed to equili-
brate for 2 h. The resulting spectra show a red shift and
broadening of the SPR band which is more pronounced
with increasing E-dAAP1 concentration indicating the ex-
pected particle aggregation. Exemplarily, an E-dAAP1 con-
centration of 0.25 mM results in the significant shift from
529 nm (0 mM) to 628 nm. DLS measurements confirm
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 9
(15) Ueno, A.; Yoshimura, H.; Saka R.; Osa, T.; J. Am. Chem. Soc.
1979, 101, 2779.
(16) Tomatsu, I.; Hashidzume, A.; Harada, A. Macromolecules
applications of AAPs as molecular switches. Both the in-
1
2
3
4
5
6
7
8
corporation of dAAPs in a system containing CDVs and
CDAuNPs revealed excellent reversible, light-responsive
aggregation and dispersion. Since heterocyclic azo-com-
pounds such as AAPs are rather unknown so far but the
research on molecular switches and light-responsive sys-
tems is recently very popular, our findings offer great po-
tential to improve existing and to develop new supramo-
lecular materials.
2005, 38, 5223.
(17) Zhao, Y. L.; Stoddart, J. F. Langmuir 2009, 25, 8442.
(18) Wang, D.; Wagner, M.; Butt, H.-J.; Wu S. Soft Matter 2015,
11, 7656.
(19) Klajn, R. Pure Appl. Chem. 2010, 82, 2247.
(20) Voskuhl, J.; Sankaran, S.; Jonkheijm, P. Chem.Commun.
2014, 50, 15144.
(21) 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.
(22) Peng, L.; You, M.; Wu, C.; Han, D.; Öçsoy, I.; Chen, T.;
Chen, Z.; Tan, W. ACS Nano 2014, 8, 2555.
(23) Fritz, E.-C.; Nimphius, C.; Goez, A.; Würtz, S.; Peterlech-
ner, M.; Neugebauer, J.; Glorius, F.; Ravoo, B. J. Chem. Eur. J. 2015,
21, 4541.
(24) Klajn, R.; Stoddart, J. F.; Grzybowski, B. A. Chem. Soc. Rev.
2010,39, 2203.
(25) Liu, Z.; Jiang, M. J. Mater. Chem. 2007, 17, 4249.
(26) Ghosh, S. K.; Pal, T. Chem. Rev. 2007, 107, 4797.
(27) Saha, K.; Agasti, S. S.; Kim, C.; Li, X.; Rotello, V. M. Chem.
Rev., 2012, 112, 2739.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ASSOCIATED CONTENT
Supporting Information. Experimental details including
synthesis and further experimental data. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*b.j.ravoo@uni-muenster.de
Funding Sources
This work was funded by the Volkswagen foundation and the
Deutsche Forschungsgemeinschaft (SFB 858).
(28) Nalluri, S. K. M.; Ravoo, B. J. Angew. Chem. Int. Ed. 2010,
49, 5371.
(29) Samanta, A. Stuart, M. C. A.; Ravoo, B. J. J. Am. Chem. Soc.
2012, 134, 19909.
(30) Schenkel, J. H.; Samanta, A.; Ravoo, B. J. Adv. Mater. 2014,
ACKNOWLEDGMENT
We thank Prof. Jens Voskuhl for supplying the per-thiolated
β-cyclodextrin. Julian Simke is acknowledged for the synthesis
of the reference azobenzene.
26, 1076.
(31) Molecular Switches, 2nd ed.; Feringa, B; Browne, W. R.;
Eds.; Wiley-VCH, Weinheim, 2011.
(32) Yang, Y.; Hughes, R. P.; Aprahamian, I. J. Am. Chem. Soc.,
2012, 134, 15221.
(33) Browne, W. R.; Feringa, B. L. Nature Nanotechnol. 2006, 1,
25.
(34) Yanga, S.; Tian, H. Chem. Soc. Rev. 2004, 33, 85.
(35) Nalluri, S. K. M.; Voskuhl, J.; Bultema, J. B.; Boekema, E. J.;
Ravoo, B. J. Angew. Chem. Int. Ed. 2011, 50, 9747.
(36) Moratz, J.; Samanta, A.; Voskuhl, J.; Nalluri, S. K. M.;
Ravoo, B. J. Chem. Eur. J. 2015, 21, 3271.
REFERENCES
(1) Bandara, H. M. D.; Burdette, S. C. Chem. Soc. Rev. 2012, 41,
1809.
(2) Yagai, S.; Karatsu, T.; Kitamura, A. Chem. Eur. J. 2005, 11,
4054.
(3) Beharry, A. A.; Wong, L.; Tropepe, V.; Woolley, G. A. Angew.
Chem., Int. Ed. 2011, 50, 1325.
(4) Schrader, T. E.; Schreier, W. J.; Cordes, T.; Koller, F. O.;
Babitzki, G.; Denschlag, R.; Renner, C.; Loweneck, M.; Dong, S.-
L.; Moroder, L.; Tavan P.; Zinth W. Proc. Natl.Acad. Sci. U. S. A.
2007, 104, 15729.
(5) Schierling, B.; Noel, A.-J.; Wende, W.; Hien, L. T.; Volkov,
E.; Kubareva, E.; Oretskaya, T.; Kokkinidis, M.; Roempp, A.;
Spengler, B.; Pingoud, A.; Proc. Natl. Acad. Sci. U. S. A. 2010, 107,
1361.
(6) Han, M.; Ishikawa, D.; Honda, T.; Ito, E.; Hara, M. Chem.
Commun. 2010,46, 3598.
(7) Siewierski, L. M.; Brittain, W. J.; Petrash S.; Foster, M.D.
Langmuir 1996, 12, 5838.
(8) Dietrich, P.; Michalik, F.; Schmidt, R.; Gahl, C.; Mao, G.;
Breusing, M.; Raschke, M. B.; Priewisch, B.; Elsaesser, T.; Men-
delsohn, R.; Weinelt, M.; Rueck-Braun, K. Appl.Phys. A: Mater.
Sci. Process. 2008, 93, 285.
(9) Min, M.; Bang, G. S.; Lee, H.;Yu, B.-C. Chem. Commun. 2010,
46, 5232.
(10) Lee, J.-W.; Klajn, R. Chem. Commun. 2015, 51, 2036.
(11) Manna, D.; Udayabhaskararao, T.; Zhao, H.; Klajn, R. An-
gew. Chem. Int. Ed. 2015, 54, 12394.
(37) Roling, O.; Stricker, L.; Voskuhl, J.; Lamping, S.; Ravoo, B.
J. Chem. Commun., 2016, 52, 1964-1966.
(38) For a review see: Dong, M.; Babalhavaeji, A.; Samanta, S.;
Beharry, A. A.; Woolley, G. A. Acc. Chem. Res. 2015, 48, 2662.
(39) Knie, C.; Utecht, M.; Zhao, F. Kulla, H.; Kovalenko, S.;
Brouwer, A. M.; Saalfrank, P.; Hecht, S.; Bléger, D. Chem. Eur. J.
2014, 20, 16492.
(40) Siewertsen, R.; Neumann, H.; Buchheim-Stehn, B.; Herges,
R.; Näther, C.; Renth, F.; Temps, F. J. Am. Chem. Soc., 2009, 131,
15594.
(41) Bléger, D.; Schwarz, J.; Brouwer, A. M.; Hecht, S. J. Am.
Chem. Soc. 2012, 134, 20597.
(42) Samanta, S.; Beharry, A. A.; Sadovski, O.; McCormick, T.
M.; Babalhavaeji, A. Tropepe, V. Woolley, G. A. J. Am. Chem. Soc.
2013, 135, 9777.
(43) Samanta, S.; McCormick, T. M.; Schmidt, S. K.; Seferosa D.
S.; Woolley, G. A. Chem. Commun. 2013, 49, 10314.
(44) Weston, C. E.; Richardson, R. D.; Haycock, P. R.; White, A.
J. P.; Fuchter, M. J. J. Am. Chem. Soc. 2014, 136, 11878.
(45) Zhang, L.; Zhang, H.; Gao, F.; Peng, H.; Ruan, Y.; Xua, Y.;
Weng, W.; RSC Adv., 2015, 5, 12007.
(46) Falvey, P.; Lim, C. W.; Darcy, R.; Revermann, T.; Karst, U.;
Giesbers, M.; Marcelis, A. T. M.; Lazar, A.; Coleman, A. W.; Rein-
houdt, D. N.; Ravoo, B. J. Chem. Eur. J. 2005, 11, 1171.
(47) Rekharsky M. V.; Inoue Y. Chem. Rev. 1998, 98, 1875.
(48) Becker, M. M.; Ravoo, B. J. Chem. Commun., 2010, 46, 4369.
(49) Samanta, A.; Ravoo, B.J. Chem. Eur. J. 2014, 20, 4966–4973.
(12) Köhntopp, A.; Dabrowski, A.; Malickia, M.; Temps, F.
Chem. Commun. 2014, 50, 10105.
(13)Iamsaard, S.; Aßhoff, S. J.; Matt, B.; Kudernac, T.; Cornelis-
sen, J. J. L. M.; Fletcher, S. P.; Katsonis, N. Nature Chem. 2014, 6,
229.
(14) Bushuyev, O. S.; Tomberg, A.; Friščić, T.; Barrett, C. J.; J.
Am. Chem. Soc. 2013, 135, 12556.
ACS Paragon Plus Environment

Page 9 of 9
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment