XCage: A Tricyclic Octacationic Receptor for Perylene Diimide with Picomolar Affinity in Water

Article abstract of DOI:10.1021/jacs.9b12982

The rational design of wholly synthetic receptors that bind active substrates with ultrahigh affinities is a challenging goal, especially in water. Here, we report the synthesis of a tricyclic octacationic cyclophane, which exhibits complementary stereoelectronic binding toward a widely used fluorescent dye, perylene diimide, with picomolar affinity in water. The ultrahigh binding affinity is sustained by a large and rigid hydrophobic binding surface, which provides a highly favorable enthalpy and a slightly positive entropy of complexation. The receptor-substrate complex shows significant improvement in optical properties, including red-shifted absorption and emission, turn-on fluorescence, and efficient energy transfer. An unusual single-excitation, dual-emission, imaging study of living cells was performed by taking advantage of a large pseudo-Stokes shift, produced by the efficient energy transfer.

Full text of DOI:10.1021/jacs.9b12982

Subscriber access provided by NEW MEXICO STATE UNIV
Article
XCage: A Tricyclic Octacationic Receptor for
Perylene Diimide with Picomolar Affinity in Water
Wenqi Liu, Sharan Bobbala, Charlotte L. Stern, Jessica Hornick,
Yugang Liu, Alan Enrique Enciso, Evan A. Scott, and J. Fraser Stoddart
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 16 Jan 2020
Downloaded from pubs.acs.org on January 16, 2020
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 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 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.
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 34
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
XCage: A Tricyclic Octacationic Receptor for Perylene Diimide with
Picomolar Affinity in Water
Wenqi Liu,1 Sharan Bobbala,2 Charlotte L. Stern,1 Jessica E. Hornick,3,4 Yugang Liu,2 Alan E.
Enciso,1 Evan A. Scott,2 J. Fraser Stoddart*1,5,6
1. Department of Chemistry, 2145 Sheridan Road, Northwestern University, Evanston, IL 60208, USA
2. Department of Biomedical Engineering, 2145 Sheridan Road, Northwestern University, Evanston, IL 60208, USA
3. Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208
4. Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, USA
5. Institute for Molecular Design and Synthesis, Tianjin University, Tianjin 300072, China
6. School of Chemistry, University of New South Wales, Sydney, NSW 2052, Australia
ABSTRACT
The rational design of wholly synthetic receptors that bind active substrates with ultrahigh
affinities is a challenging goal, especially in water. Here, we report the synthesis of a tricyclic
octacationic cyclophane, which exhibits complementary stereoelectronic binding towards a widely
used fluorescent dye, perylene diimide, with picomolar affinity in water. The ultrahigh binding
affinity is sustained by a large and rigid hydrophobic binding surface, which provides a highly
favorable enthalpy and a slightly positive entropy of complexation. The receptor-substrate
complex shows significant improvement in optical properties, including red-shifted absorption and
emission, turn-on fluorescence, and efficient energy transfer. An unusual single-excitation, dual-
emission, imaging study of living cells was performed by taking advantage of a large pseudo-
Stokes shift, produced by the efficient energy transfer.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 34
1
2
3
4
INTRODUCTION
5
6
7
8
High-affinity receptor-substrate recognition exists ubiquitously in biology where the
concentrations of foreign substrates are maintained below a certain threshold by antibodies.1
Billions of years of evolution have enabled nature to create these biological receptors with binding
cavities that have high stereoelectronic complementarity towards their substrates down to the
atomic level.2 One of the grand challenges in supramolecular chemistry is to develop synthetic
receptors with ultrahigh affinities, especially in water.3–5 The majority of synthetic receptors
described in the literature show6 micromolar affinity or weaker binding. To date, examples of
water compatible high-affinity receptors are rare and mainly limited to cucurbit[n]urils,7–9 with a
sparse distribution of them in pillararenes10 and tetralactam macrocycles11. Exploring new
synthetic receptor-substrate pairs with ultrahigh affinities in water will not only stand the chance
of exposing fundamental insights into the origin of high-affinity interactions, but also deliver
orthogonality towards the existing high-affinity receptor-substrate pairs with emergent properties12
that meet the high demand in the fields of noncovalent click chemistry,13 sensors,14 advanced
materials,15–18 nanotechnology,19 and biomedicine20.
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
Towards this goal, Houk6 has emphasized the importance of constructing large buried surface areas
between receptors and substrates to achieve high-affinity binding. This principle is executed well
by nature but proves challenging when it comes to the design of synthetic receptors capable of
binding substrates in water.12,13,21 Although extra binding surfaces are expected to deliver gains in
binding enthalpy, restriction of conformational flexibility upon binding leads to losses of binding
entropy, resulting in little or no gains in affinity – a concept known as enthalpy-entropy
compensation.22–24 Larger hydrophobic binding surfaces also cause poor water solubility, which
2
ACS Paragon Plus Environment

Page 3 of 34
Journal of the American Chemical Society
1
2
3
4
5
6
further hinders the structural design of synthetic receptors to achieve high-affinity binding in
water.25,26
7
8
9
Recently, we have developed27–29 a series of extended cationic cyclophanes characterized by large
and rigid binding surfaces. These positively charged cyclophanes exhibit remarkable affinities
towards polycyclic aromatic substrates with little conformational changes in organic solvents. The
solubilities of these cyclophanes are highly dependent on their counterions. Generally speaking,
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
–
–
the PF6 salts are soluble in MeCN whereas the Cl– or CF3CO2 salts are soluble in water. The
ability to manipulate large and rigid binding surfaces, without jeopardizing their solubilities,
makes these cyclophanes ideal candidates for the design and synthesis of high affinity receptors in
water.30–34
We began our investigation by using perylene diimide (PDI)-based dyes as the representative
substrates, considering their superior photophysical properties and wide range of applications in
materials science35 and biotechnology36. Encapsulations of PDI dyes have been explored37,38 using
cucurbit[8]uril and also a tetracationic cyclophane, known28 as ExBox4+; significant improvements
in photophysical properties of PDI dyes were observed in water. The affinity constants involving
both these receptors remainaround 105 M–1 or lower in water. A closer inspection of these receptor-
substrate pairs reveals that neither synthetic receptor is able to encapsulate completely the PDI
molecule since the relatively large size of PDI imposes a particular challenge in identifying
suitable receptors.
Herein, we report a new synthetic receptor that is tailored to provide a complementary
stereoelectronic binding cavity for the PDI in water. We have chosen to name this tricyclic
octacationic cyclophane receptor, XCage8+, in view of its X-shaped structure. XCage8+ features a
roof-pillar-floor structure, where the roof and floor are each composed of a biphenyl unit, offering
3
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 34
1
2
3
4
5
6
7
8
9
a large and flat binding surface. Four p-xylylene units serve as pillars with the ideal lengths (7.0
Å) to support aromatic [π···π] stacking interactions (2 × 3.5 Å) with an included PDI molecule.
The eight cationic pyridinium units provide both sufficient water solubility and complementary
electronic binding sites for the four divergent carbonyl groups in the PDI molecule. The resulting
PDI ⊂ XCage•8CF3CO2 complex, which exhibits picomolar binding affinity in water, is
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
enthalpically driven along with a small favorable entropic component. Moreover, the strong
binding is accompanied by a significant improvement in optical properties, including turn-on
fluorescence, red-shifted absorption and emission, in addition to efficient energy transfer, which
remains effective under cell-imaging conditions. The energy transfer results in a large pseudo-
Stokes shift that is utilized in achieving a dual color imaging study of living cells using a single
light excitation.
RESULTS AND DISCUSSION
Synthesis of XCage•8CF3CO2
XCage•8CF3CO2 was prepared (Figure 1a) in three steps from commercially available starting
materials. The key building block TB•4PF6 can be easily accessed by Suzuki coupling, followed
by alkylation of pyridine units in 91% overall yield without the need of column chromatography.
XCage•8CF3CO2 was obtained by a template-assisted synthesis with the help of TBAI as a
catalyst.39 Pyrene was used as a template and was subsequently removed40 by continuous liquid-
liquid extraction. The crude product was isolated by precipitation with TBACl and further purified
by reverse-phase column chromatography. After anion exchange using TFA, XCage8 was
+
–
obtained as its CF3CO2 salt in 26% isolated yield. XCage•8CF3CO2 is highly soluble in water and
MeOH, and slightly soluble in MeCN. It emits a blue fluorescence (10% quantum yield) in the
4
ACS Paragon Plus Environment

Page 5 of 34
Journal of the American Chemical Society
1
2
3
4
range of 350–550 nm in water.41 The results of cyclic voltammetry experiments (Figure S26)
5
6
reveal42 a nonreversible redox process in Me2SO.
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
X-Ray Crystallographic Analysis
Numerous attempts43 to grow the single crystals of XCage8+ with various counterions did not meet
with success. Fortunately, a single crystal of Perylene  XCage•8CF3CO2 was obtained by slow
vapor diffusion of iPr2O into a MeOH solution of Perylene  XCage•8CF3CO2. The solid-state
structure of XCage•8CF3CO2 (Figure 1b) reveals that it has a box-like cavity with dimensions of
12.5 × 11.1 × 7.0 Å. The cavity volume (Figure S34) is estimated to be around 384 Å3, which is
comparable44 with the cavity volume of cucurbit[8]uril. The roof and floor are parallel and
supported by four p-xylylene pillars with an ideal distance (7.0 Å) for aromatic [π···π] stacking
interactions. The large binding surface (Figure S33) of XCage8+ covers 92% of the van der Waals
surface of Perylene, which is sandwiched symmetrically between the roof and the floor. Moreover,
the p-xylylene pillars provide further [CH···π] interactions with Perylene as revealed45,46 by an
independent gradient model (IGM) analysis (Figure S35).
A model compound PDI1 (Figure 2) was synthesized in order to obtain the single-crystal
superstructure of its 1:1 complex with XCage8+. Like other PDI dyes, PDI1 is poorly soluble in
most solvents. In the presence of XCage•8CF3CO2, however, PDI1 can be dissolved readily in
solvents such as H2O, MeOH, MeCN, DMF and Me2SO. A single crystal of PDI1 ⊂
XCage•7PF6•OH was obtained by slow diffusion of Et2O into a solution of PDI1 ⊂ XCage•8PF6
in MeCN. The solid-state superstructure (Figure 3d) reveals that each carbonyl group in PDI1 is
sandwiched between two pyridinium units with an average [C=O···N+] distance of 4.4 Å, which
5
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 34
1
2
3
4
5
6
is the same [C=O···N+] distance as that observed47 in the diamantane ⊂ cucurbit[7]uril complex.
There are in total eight [C=O···N+] ion-dipole interactions within the complex, PDI1 ⊂ XCage8+.
7
8
9
The core of PDI1 is sandwiched between the two biphenyl units as a result of aromatic [π···π]
stacking interactions and separated by a distance about 3.7 Å from the roof and the floor. A surface-
area overlay analysis shows that 80% of the van der Waals surface of the PDI1 core overlaps with
XCage8+. A similar analysis performed on a single crystal of a catenane shows48 that only 40% of
the PDI core overlaps with the ExBox4+ component.49 Thus, XCage8+ provides twice the binding
surface area compared to that of the ExBox4+ component in the catenane. IGM analysis reveals
that PDI is enveloped by favorable van der Waals interactions with the biphenyl-containing roof
and floor, as well as from the pyridinium and p-xylylene units. Moreover, the carbonyl groups on
PDI1 are involved in polar [C=O···HC] interactions with p-xylylene protons on XCage8+.
Interestingly, the planar surface of PDI1 was found to be slightly twisted (12˚) in the bay region
as a result of its aromatic [π···π] stacking interactions with the biphenyl-containing binding
surfaces, which also exhibits a slightly twisted plane with a dihedral angle of 19˚. Such induced-
fit binding is a well-established phenomenon50 in receptor-substrate binding pairs in biological
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
NMR Spectroscopy and Mass Spectrometry in Solution
In order to investigate the molecular recognition between XCage•8CF3CO2 and PDI in water, a
water-soluble PDI2 (Figure 2) flanked with two mPEG2000 chains was synthesized.51 A 1:1 mixture
of PDI2 and XCage•8CF3CO2 in D2O produced PDI2 ⊂ XCage•8CF3CO2 instantly in quantitative
yield. Diagnostic changes in chemical shift, indicating complex formation, were revealed by
1
comparison (Figure 4a) of the H NMR spectra of PDI2, PDI2 ⊂ XCage•8CF3CO2 and
6
ACS Paragon Plus Environment

Page 7 of 34
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
XCage•8CF3O2. Large upfield shifts of the signal for protons A (Δ훿 = –0.41 ppm) and protons B
(Δ훿 = –0.61 ppm) on XCage•8CF3CO2, together with a significant upfield shift of the signal for
protons 2 (Δ훿 = –1.98 ppm) on PDI2, indicate the presence of aromatic [π···π] stacking
interactions between PDI2 and the biphenyl units. Meanwhile, large downfield shifts of the signal
for protons D (Δ훿 = +0.29 ppm) and protons F (Δ훿 = +0.37 ppm) are a good indication of polar
interactions between the imide carbonyl groups on PDI2 and XCage•8CF3CO2 protons from both
the pyridinium and xylylene moieties. A NOESY spectrum (Figure 4b) confirmed52 the threaded
structure with through-space corrections between protons 2 on PDI2 and protons B, C and D on
XCage•8CF3CO2. ESI-MS (Figure S25) confirmed the formation of PDI2 ⊂ XCage•8CF3CO2 in
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
water by revealing several peaks clustered around m/z 2000, 1500, 1200 and 1000, corresponding
to different charged states of PDI2 ⊂ XCage•8CF3CO2 along with the loss of between three and
–
1
six CF3CO2 counterions. An analogous H NMR spectroscopic experiment, designed to follow
the formation of PDI2 ⊂ XCage•8CF3CO2 in MeCN, is described in the Supporting Information.
Photophysical Properties
Encapsulation of PDI2 by XCage•8CF3CO2 induces several distinctive changes in photophysical
properties. In the UV-Vis spectra in MeCN, PDI2 shows three sharp absorption peaks at 456, 484,
and 520 nm, corresponding to the non-aggregated state of PDI2. This compound emits a bright
yellow fluorescence with a 66% fluorescence quantum yield. In the presence of XCage•8CF3CO2,
both the absorption and emission maxima of PDI2 are red shifted (23-25 nm), whereas the
fluorescence quantum yield remains unchanged. On the other hand, PDI2 is highly aggregated in
water, as indicated (Figure 5a) by the broad absorption peaks53 and a low fluorescence quantum
yield (4%).54 Upon the addition of one molar equivalent of XCage•8CF3CO2, the color of the PDI2
7
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 34
1
2
3
4
5
6
7
8
9
solution changes instantly from dark red to bright orange; three distinctive absorption peaks at 472,
504, and 542 nm, the characteristic signature of monomeric PDI in solution, were observed.
Meanwhile, the fluorescence quantum yield of PDI2 ⊂ XCage•8CF3CO2 in water increases up to
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
63%, which is close to the brightness of the complex in MeCN. Furthermore, the excitation and
emission maxima of PDI2 ⊂ XCage•8CF3CO2 are red shifted (9–15 nm), and its fluorescence
lifetime increases from 4.7 to 7.3 ns when compared with PDI2. Notably, the fluorescence of PDI2
⊂ XCage•8CF3CO2 remains bright, even at high concentrations (>1 mM) in water, i.e., the
condensed charges on PDI2 ⊂ XCage•8CF3CO2 prevent it from aggregating.
There is an efficient energy transfer from XCage•8CF3CO2 to PDI2 in both MeCN and H2O. In
the excitation spectrum of PDI2 ⊂ XCage•8CF3CO2, we observed a strong excitation peak around
300 nm, where XCage•8CF3CO2 absorbs light. Remarkably, in MeCN, the fluorescence intensity
of PDI2 ⊂ XCage•8CF3CO2, as a result of energy transfer, is 150% higher than that of the complex
under direct excitation at 542 nm, suggesting a superior antenna effect. In water, the energy transfer
process (Figure 5d) produces 86% of its original fluorescence intensity. By comparing the
fluorescent emission of XCage•8CF3CO2 and PDI2 ⊂ XCage•8CF3CO2 at 350–525 nm, the
energy transfer efficiencies are determined to be quantitative in MeCN and 90% in water.
Binding Kinetics and Thermodynamics
The changes in optical properties induced on PDI2 ⊂ XCage•8CF3CO2 complex formation enable
a facile tracking of the recognition process. The kinetics of threading PDI2 into XCage•8CF3CO2
in water was tracked by turn-on fluorescence as a function of time. The kinetic profile was fitted
to a second order kinetic model and revealed kon = (4.8 ± 1.1) × 105 M–1s–1. The half-life at 0.1 µM
was calculated to be 21 s. Such rapid complex formation of PDI2 ⊂ XCage•8CF3CO2 in water is
8
ACS Paragon Plus Environment

Page 9 of 34
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
remarkable when one considers the threading process that involves the chain end of mPEG2000
polymer finding a “correct” cavity entrance and then exiting at the right opening of the tricyclic
cage. This observation agrees with the reported literature55–57 that threading a PEG polymer
through a macrocycle is rapid in water.
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
The binding constants (Table 1) were determined by fluorescence titration and isothermal titration
calorimetry (ITC). Displacement titration experiments monitored by fluorescence58 were
performed in order to determine the high binding affinity between PDI2 and XCage•8CF3CO2. In
MeCN, binding of XCage•8CF3CO2 was tested first of all using Perylene as a substrate. Its
binding constant was found to be in the order of 106 M–1, which is similar in magnitude to that of
ExCage6+•6PF6 and about 86 times higher than that of ExBox4+•4PF6.28,29 Next, we performed a
competitive experiment, starting with a solution of XCage•8CF3CO2 and 50 molar equivalents of
Perylene. PDI2 was titrated into the MeCN solution to displace Perylene from the cavity. The
displacement titration was monitored by turn-on fluorescence and yielded a binding constant in
the vicinity of 109 M–1. The binding affinity between XCage•8CF3CO2 and PDI2 in MeCN is too
high to be evaluated by ITC, and only the binding enthalpy could be extracted from the isotherm.
The Gibbs free energy was estimated from fluorescent titrations which also provide a T∆S value.
The formation of PDI2 ⊂ XCage•8CF3CO2 in MeCN is mainly driven by favorable enthalpy with
a small contribution from positive entropy. Compared with the binding of Perylene towards
XCage•8CF3CO2, PDI2 shows a similar positive ΔS and also enjoys a more negative ΔH, which
originates from the additional [C=O···N+] ion-dipole interactions.
In order to evaluate the affinity between XCage•8CF3CO2 and PDI2 in water, we selected
Caffeine as a competitor, considering its good solubility and structural similarity to PDI. The
binding constants determined from both the fluorescence titration and ITC yielded similar results
9
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 34
1
2
3
4
5
6
7
8
9
that are in the order of 105 M-1. The binding constant between PDI2 and XCage•8CF3CO2 in water
was subsequently measured in the presence of 1000 molar equivalents of Caffeine. As evaluated
by the fluorescence titration, the binding constant between PDI2 and XCage•8CF3CO2 was
determined to be 7.7 ×1010 M–1, i.e., Kd = 13 pM. The formation of PDI2 ⊂ XCage•8CF3CO2 in
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
water is enthalpically driven with a small favorable entropic component. The binding enthalpy
observed in water is 4 kcal mol–1 higher when compared with that in MeCN, suggesting that the
release of high energy water molecules provides59 an extra contribution to the stability of PDI2 ⊂
XCage•8CF3CO2 in addition to the large area [π-π] stacking and ion-dipole interactions. The small
favorable entropy benefits60 from the release of water into the bulk and the structural rigidity of
both XCage•8CF3CO2 and PDI2.
In order to illustrate the effect of the extended receptor surface on the affinity enhancement, we
investigated the binding thermodynamics of ExBox•4Cl –– a structure analogue of
XCage•8CF3CO2 –– towards PDI2 in water. ExBox•4Cl shows (Figure S48) a binding affinity of
2.0 × 106 M–1 and a binding enthalpy of –7.4 kcal mol–1. Compared with ExBox•4Cl,
XCage•8CF3CO2 enhances the binding affinity by a factor of 38,000 and provides twice amount
of binding enthalpy. This observation is in line with the results of surface area overlap analysis,
which reveals that XCage8+ provides twice the binding surface area towards the PDI binding core
compared to that of ExBox4+. The extended binding surface results in significant gains in binding
enthalpy (ΔΔH = –6.7 kcal mol–1), while the loss of binding entropy (TΔΔS = –0.5 kcal mol–1) is
trivial, leading to an obvious deviation23 from the enthalpy-entropy compensation plots for
cyclodextrin-guest complexation (Figure S53) and significantly enhanced affinity. These results
prove that cationic cyclophanes with large and rigid binding surface are promising candidates to
achieve high binding affinities in water.
10
ACS Paragon Plus Environment

Page 11 of 34
Journal of the American Chemical Society
1
2
3
4
Fluorescence Imaging Studies
5
6
7
8
The potential application of these emergent properties was illustrated by fluorescence imaging and
flow cytometry studies with MCF-7 cells, i.e., a human breast adenocarcinoma cell line. PDI2 ⊂
XCage•8CF3CO2 is non-toxic to MCF-7 cells and shows >95% cell viability at all concentrations
tested (2.5–50 µM) (Figure S54). Live-cell confocal microscopic images of MCF-7 cells were
collected after incubation with 10 µM PDI2 or PDI2 ⊂ XCage•8CF3CO2 for 6 h. Brightfield-
merged images show (Figure 6a) no fluorescence after treatment with PDI2 and a strong
fluorescence signal after PDI2 ⊂ XCage•8CF3CO2 treatment. The punctate signal of PDI2 ⊂
XCage•8CF3CO2 co-localizes with the lysotracker signal (Figure S58), indicating the lysosomal
localization of PDI2 ⊂ XCage•8CF3CO2 inside the cells. The concentration-dependent uptake,
observed by confocal microscopic analysis and flow cytometry studies, shows (Figure 6b and
Figure S57) that the PDI2 ⊂ XCage•8CF3CO2 could be detected inside MCF-7 cells at incubation
concentrations as low as 0.1 µM. These results confirmed the superior fluorescence properties and
high stability of PDI2 ⊂ XCage•8CF3CO2 complex, even at low concentrations under cell-
imaging conditions.
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
PDI2 ⊂ XCage•8CF3CO2 produces (Figure 5d) a similar fluorescence intensity as a result of
efficient energy transfer when compared with the fluorescence by a direct excitation at 540 nm.
Meanwhile, such an energy transfer process endows61 the complex with a large pseudo-Stokes
shift (239 nm). The bright fluorescence and large pseudo-Stokes shift are highly desirable for dual
color imaging investigations62 where a single light excitation can be used to excite two
fluorophores that emit simultaneously in different wavelength regions. For this purpose, we first
of all tested the effectiveness of cell imaging using energy transfer. MCF-7 cells were incubated
with 20 µM PDI2 ⊂ XCage•8CF3CO2 for 6 h and the live-cell fluorescence imaging was
11
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 12 of 34
1
2
3
4
5
6
7
8
9
performed by widefield microscopy. When PDI2 ⊂ XCage•8CF3CO2 treated cells were excited
using a DAPI excitation filter (ex: 381–399 nm), a bright fluorescence signal was detected (Figure
6c) with a TRITC emission filter (em: 571–617 nm). As a control, 20 µM PDI2 treated cells did
not show any fluorescence signal. Next, we tested the dual-color imaging, following incubation of
MCF-7 cells with PDI2 ⊂ XCage•8CF3CO2 and Hoechst 33342 stain (Hoechst), a widely used
nucleus stain with excitation and emission peaks at 350 and 461 nm, respectively. The micrograph
(Figure 6d) was obtained with a single DAPI excitation filter (ex: 381–399 nm) and two emission
filters: DAPI (em: 411–459 nm) for Hoechst, and TRITC (em: 571–617 nm) for PDI2 ⊂
XCage•8CF3CO2. The Hoechst stain was localized in the nucleus and visualized as a blue color.
Meanwhile, PDI2 ⊂ XCage•8CF3CO2 was visualized as the red punctate signals. These results
demonstrate that the large pseudo-Stokes shift produced by efficient energy transfer can be utilized
to achieve two-color channels imaging by a single light excitation, a procedure which has been
explored previously with mutated fluorescent proteins62 and synthetic dyes63 that have large Stokes
shifts. This property is highly desirable for the simultaneous study of two biological processes with
advanced microscopic techniques, such as dual-color, single-laser fluorescence, cross-correlation
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
spectroscopy64 and multicolor stimulated emission depletion microscopy65,66
.
CONCLUSIONS
An octacationic tricyclic cyclophane XCage•8CF3CO2 has been designed and synthesized.
XCage8+ shows high complementary stereoelectronic binding towards PDI in water with
picomolar affinity. The ultrahigh affinity of the complex is sustained by a blend of the hydrophobic
effect as well as aromatic [π···π] stacking and ion-dipole interactions. This investigation proves
that cationic cyclophanes with large and rigid surfaces are promising receptors for achieving high
12
ACS Paragon Plus Environment

Page 13 of 34
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
binding affinities in water. Meanwhile, the strong-affinity binding pair reported here offers an
orthogonality to existed high-affinity binding pairs that can be used in noncovalent click
chemistry.13
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
The encapsulated PDI dye results in improved optical properties, increased solubility and efficient
energy transfer. The potential application of these emergent properties was demonstrated by a
single-excitation dual-emission imaging of living cells with PDI2 ⊂ XCage•8CF3CO2 and
Hoechst stain. While this research illustrates the bioimaging application of PDI2 ⊂
XCage•8CF3CO2, it is worth emphasizing that there is a multitude of applications of PDI in
various other scientific fields as well. The high affinity and exceptional optical properties of PDI2
⊂ XCage•8CF3CO2 provide a new starting point for the manipulation of PDI dyes with an eye to
a wide range of applications in the fields of single-molecule electronics,67–69 photonic device,70
materials science,35 and molecular biology.36
EXPERIMENTAL SECTION
Synthesis of XCage•8CF3CO2
A solution composed of TPBP (120 mg, 0.26 mmol), pyrene (315 mg, 1.60 mmol) and
tetrabutylammonium iodide (20 mg, 0.05 mmol) in CHCl3 (20 mL) was added to a solution of
TB•4PF6 (450 mg, 0.26 mmol) in MeCN (250 mL). The reaction mixture was heated at 85 ˚C for
3 days. After cooling to room temperature, tetrabutylammonium chloride (500 mg, 1.8 mmol) and
CHCl3 (300 mL) were added to the reaction mixture, the yellow precipitate was isolated by
filtration and then dispersed in MeOH (100 mL). Celite (5g) and TFA (2 mL) were added, and the
solvent was removed by vacuum. The remaining solid was loaded onto Combiflash flash
13
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 14 of 34
1
2
3
4
5
6
7
8
9
chromatography system and purified by reverse C18 columns using 0–25% MeCN/H2O with 0.1%
TFA as additive. Fractions containing the product were combined and MeCN was removed by
vacuum. The remaining aqueous solution was extracted by continuous liquid-liquid extraction for
48 h until the yellow solution became colorless. Water was removed and the residue was further
purified by reverse C18 chromatography using 0–15% MeCN/H2O with 0.1% TFA as additive to
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
1
obtain XCage•8CF3CO2 as a white solid (150 mg, 26% yield). H NMR (500 MHz, CD3OD) δ
9.15 – 9.09 (m, 16H), 8.81 (t, J = 2.0 Hz, 8H), 8.66 – 8.61 (m, 16H), 8.56 (q, J = 1.4 Hz, 4H), 7.75
(d, J = 2.5 Hz, 16H), 6.00 – 5.83 (m, 16H). 13C NMR (125 MHz, CD3OD) δ 159.9, 159.6, 154.5,
144.1, 138.9, 136.4, 135.3, 130.2, 128.6, 127.5, 125.5, 117.1, 114.8, 63.4. HRMS-ESI (m/z) for
XCage•8CF3CO2: Calcd for C108H76F18N8O122+: m/z = 1009.7659 [M–2CF3CO2]2+; found
1009.7688 [M–2CF3CO2]2+.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/
Chemical synthesis and characterization, mass spectral data, NMR spectra, X-ray crystal data,
computational analysis, photophysical data, binding studies, and cell imaging data (PDF)
Crystallographic data for TPBP (CIF)
Crystallographic data for Perylene ⊂ XCage•8CF3CO2 (CIF)
Crystallographic data for PDI1 ⊂ XCage•7PF6•OH (CIF)
Crystallographic data for PDI1 (CIF)
14
ACS Paragon Plus Environment

Page 15 of 34
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
AUTHOR INFORMATION
Corresponding Author
* stoddart@northwestern.edu
ORCID
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
Wenqi Liu: 0000-0001-6408-0204
Sharan Bobbala: 0000-0002-0740-330X
Jessica E. Hornick: 0000-0002-2180-5399
Evan A. Scott: 0000-0002-8945-2892
J. Fraser Stoddart: 0000-0003-3161-3697
Notes
The authors declare no competing financial interest
ACKNOWLEDGEMENTS
The authors thank Professor Bradley D. Smith and Professor Douglas Philp for their insightful
comments and suggestions on the manuscript. The authors also thank Dr. Margaret Schott for
helping to polish the language in the manuscript. This research is part of the Joint Center of
Excellence in Integrated Nano-Systems (JCIN) at King Abdulaziz City for Science and
Technology (KACST) and Northwestern University (NU). The authors would like to thank both
KACST and NU for their continued support of this research.
15
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 16 of 34
1
2
3
4
REFERENCES
5
6
7
8
(1)
(2)
(3)
Szwajkajzer, D.; Carey, J. Molecular and Biological Constraints on Ligand-Binding
Affinity and Specificity. Biopolymers 1997, 44, 181–198.
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
Persch, E.; Dumele, O.; Diederich, F. Molecular Recognition in Chemical and Biological
Systems. Angew. Chem. Int. Ed. 2015, 54, 3290–3327.
Cremer, P. S.; Flood, A. H.; Gibb, B. C.; Mobley, D. L. Collaborative Routes to
Clarifying The Murky Waters of Aqueous Supramolecular Chemistry. Nat. Chem. 2017,
10, 8–16.
(4)
(5)
Liu, Y.; Zhao, W.; Chen, C.-H.; Flood, A. H. Chloride Capture Using a C-H Hydrogen
Bonding Cage. Science. 2019, 365, 159–161.
Tromans, R. A.; Carter, T. S.; Chabanne, L.; Crump, M. P.; Li, H.; Matlock, J. V.;
Orchard, M. G.; Davis, A. P. A Biomimetic Receptor for Glucose. Nat. Chem. 2019, 11,
52–56.
(6)
(7)
Houk, K. N.; Leach, A. G.; Kim, S. P.; Zhang, X. Binding Affinities of Host–Guest,
Protein–Ligand, and Protein–Transition‐state Complexes. Angew. Chem. Int. Ed. 2003,
42, 4872–4897.
Shetty, D.; Khedkar, J. K.; Park, K. M.; Kim, K. Can We Beat The Biotin–Avidin Pair?:
Cucurbit[7]uril-Based Ultrahigh Affinity Host–Guest Complexes and Their Applications.
Chem. Soc. Rev. 2015, 44, 8747–8761.
(8)
(9)
Assaf, K. I.; Nau, W. M. Cucurbiturils: From Synthesis to High-Affinity Binding and
Catalysis. Chem. Soc. Rev. 2015, 44, 394–418.
Barrow, S. J.; Kasera, S.; Rowland, M. J.; Del Barrio, J.; Scherman, O. A. Cucurbituril-
Based Molecular Recognition. Chem. Rev. 2015, 115, 12320–12406.
16
ACS Paragon Plus Environment

Page 17 of 34
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
(10) Ogoshi, T.; Yamagishi, T. A.; Nakamoto, Y. Pillar-Shaped Macrocyclic Hosts
Pillar[n]arenes: New Key Players for Supramolecular Chemistry. Chem.Rev 2016, 116,
7937–8002.
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
(11) Li, D.-H.; Smith, B. D. Molecular Recognition Using Tetralactam Macrocycles with
Parallel Aromatic Sidewalls. Beilstein J. Org. Chem. 2019, 15, 1086–1095.
(12) Liu, W.; Samanta, S. K.; Smith, B. D.; Isaacs, L. Synthetic Mimics of
Biotin/(Strept)Avidin. Chem. Soc. Rev. 2017, 46, 2391–2403.
(13) Schreiber, C. L.; Smith, B. D. Molecular Conjugation Using Non-Covalent Click
Chemistry. Nat. Rev. Chem. 2019, 3, 393–400.
(14) Mako, T. L.; Racicot, J. M.; Levine, M. Supramolecular Luminescent Sensors. Chem. Rev.
2019, 119, 322–477.
(15) Heinzmann, C.; Weder, C.; de Espinosa, L. M. Supramolecular Polymer Adhesives:
Advanced Materials Inspired by Nature. Chem. Soc. Rev. 2015, 342, 342–358.
(16) Wang, H.; Ji, X.; Li, Z.; Huang, F. Fluorescent Supramolecular Polymeric Materials. Adv.
Mater. 2017, 29, 1606117.
(17) Ariga, K.; Li, J.; Fei, J.; Ji, Q.; Hill, J. P. Nanoarchitectonics for Dynamic Functional
Materials from Atomic-/Molecular-Level Manipulation to Macroscopic Action. Adv.
Mater. 2016, 28, 1251–1286.
(18) Pan, J.; Chen, W.; Ma, Y.; Pan, G. Molecularly Imprinted Polymers as Receptor Mimics
for Selective Cell Recognition. Chem. Soc. Rev. 2018, 47, 5574–5587.
(19) Sarikaya, M.; Tamerler, C.; Jen, A. K.-Y.; Schulten, K.; Baneyx, F. Molecular
Biomimetics: Nanotechnology through Biology. Nat. Mater. 2003, 2, 577–585.
17
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 18 of 34
1
2
3
4
5
6
(20) Webber, M. J.; Langer, R. Drug Delivery by Supramolecular Design. Chem. Soc. Rev.
2017, 46, 6600–6620.
7
8
9
(21) Murray, J.; Kim, K.; Ogoshi, T.; Yao, W.; Gibb, B. C. The Aqueous Supramolecular
Chemistry of Cucurbit[n]urils, Pillar[n]Arenes and Deep-Cavity Cavitands. Chem. Soc.
Rev. 2017, 46, 2479–2496.
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
(22) Chodera, J. D.; Mobley, D. L. Entropy-Enthalpy Compensation: Role and Ramifications
in Biomolecular Ligand Recognition and Design. Annu. Rev. Biophys. 2013, 42, 121–142.
(23) Rekharsky, M. V; Mori, T.; Yang, C.; Ko, Y. H.; Selvapalam, N.; Kim, H.; Sobransingh,
D.; Kaifer, A. E.; Liu, S.; Isaacs, L.; et al. A Synthetic Host-Guest System Achieves
Avidin-Biotin Affinity by Overcoming Enthalpy-Entropy Compensation. Proc. Natl.
Acad. Sci. U. S. A. 2007, 104, 20737–20742.
(24) Liu, W.; Johnson, A.; Smith, B. D. Guest Back-Folding: A Molecular Design Strategy
That Produces a Deep-Red Fluorescent Host/Guest Pair with Picomolar Affinity in Water.
J. Am. Chem. Soc. 2018, 140, 3361–3370.
(25) Spenst, P.; Würthner, F. A Perylene Bisimide Cyclophane as a “Turn-On” and “Turn-Off”
Fluorescence Probe. Angew. Chem. Int. Ed. 2015, 54, 10165–10168.
(26) Sapotta, M.; Hofmann, A.; Bialas, D.; Würthner, F. A Water-Soluble Perylene Bisimide
Cyclophane as A Molecular Probe for The Recognition of Aromatic Alkaloids. Angew.
Chem. Int. Ed. 2019, 58, 3516–3520.
(27) Dale, E. J.; Vermeulen, N. A.; Juríček, M.; Barnes, J. C.; Young, R. M.; Wasielewski, M.
R.; Stoddart, J. F. Supramolecular Explorations: Exhibiting The Extent of Extended
Cationic Cyclophanes. Acc. Chem. Res. 2016, 49, 262–273.
18
ACS Paragon Plus Environment

Page 19 of 34
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
(28) Barnes, J. C.; Juríček, M.; Strutt, N. L.; Frasconi, M.; Sampath, S.; Giesener, M. A.;
McGrier, P. L.; Bruns, C. J.; Stern, C. L.; Sarjeant, A. A.; et al. ExBox: A Polycyclic
Aromatic Hydrocarbon Scavenger. J. Am. Chem. Soc. 2013, 135, 183–192.
(29) Dale, E. J.; Vermeulen, N. A.; Thomas, A. A.; Barnes, J. C.; Juríček, M.; Blackburn, A.
K.; Strutt, N. L.; Sarjeant, A. A.; Stern, C. L.; Denmark, S. E.; et al. ExCage. J. Am.
Chem. Soc. 2014, 136, 10669–10682.
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
(30) Tsutsui, T.; Kusaba, S.; Yamashina, M.; Akita, M.; Yoshizawa, M. Open Versus Closed
Polyaromatic Nanocavity: Enhanced Host Abilities toward Large Dyes and Pigments.
Chem. Eur. J 2019, 25, 4320–4324.
(31) Yoshizawa, M.; Catti, L. Bent Anthracene Dimers as Versatile Building Blocks for
Supramolecular Capsules. Acc. Chem. Res. 2019, 52, 2392–2404.
(32) Yamashina, M.; Tsutsui, T.; Sei, Y.; Akita, M.; Yoshizawa, M. A Polyaromatic Receptor
with High Androgen Affinity. Sci. Adv. 2019, 5, 1–8.
(33) Jono, K.; Suzuki, A.; Akita, M.; Albrecht, K.; Yamamoto, K.; Yoshizawa, M. A
Polyaromatic Molecular Clip That Enables the Binding of Planar, Tubular, and Dendritic
Compounds. Angew. Chem. Int. Ed. 2017, 56, 3570–3574.
(34) Yamauchi, Y.; Yoshizawa, M.; Akita, M.; Fujita, M. Engineering Double to Quintuple
Stacks of a Polarized Aromatic in Confined Cavities. J. Am. Chem. Soc. 2010, 132, 960–
966.
(35) Würthner, F.; Saha-Möller, C. R.; Fimmel, B.; Ogi, S.; Leowanawat, P.; Schmidt, D.
Perylene Bisimide Dye Assemblies as Archetype Functional Supramolecular Materials.
Chem. Rev. 2016, 116, 962–1052.
19
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 20 of 34
1
2
3
4
5
6
(36) Sun, M.; Müllen, K.; Yin, M. Water-Soluble Perylenediimides: Design Concepts and
Biological Applications. Chem. Soc. Rev. 2016, 45, 1513–1528.
7
8
9
(37) Ryan, S. T. J.; Del Barrio, J.; Ghosh, I.; Biedermann, F.; Lazar, A. I.; Lan, Y.; Coulston,
R. J.; Nau, W. M.; Scherman, O. A. Efficient Host-Guest Energy Transfer in Polycationic
Cyclophane-Perylene Diimide Complexes in Water. J. Am. Chem. Soc. 2014, 136, 9053–
9060.
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
(38) Biedermann, F.; Elmalem, E.; Ghosh, I.; Nau, W. M.; Scherman, O. A. Strongly
Fluorescent, Switchable Perylene Bis(Diimide) Host-Guest Complexes with
Cucurbit[8]uril in Water. Angew. Chem. Int. Ed. 2012, 51, 7739–7743.
(39) Barnes, J. C.; Juríček, M.; Vermeulen, N. A.; Dale, E. J.; Stoddart, J. F. Synthesis of
ExnBox Cyclophanes. J. Org. Chem. 2013, 78, 11962–11969.
(40) In the absence of template, no product could be isolated on account of the structural
flexibilty of TB•4PF6. The presence of pyrene template helps preorganize and rigidify the
precusor and faciliate XCage8+ formation. Moreover, the template also helps visualize the
formation of XCage8+, which forms a yellow precipitate from the reaction mixture after its
complexation with pyrene.
(41) The absolute fluorescent quantum yield of XCage•8CF3CO2 in water was measured using
an integrating sphere detector.
(42) The nonreversible redox process of XCage•8CF3CO2 in Me2SO is a result of the poor
solubilites of the neutral species upon redution. After the first redox cycle, precipitations
were observed on electrodes, and no redox signal could be further detected.
20
ACS Paragon Plus Environment

Page 21 of 34
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
(43) Large (>100 μm in diameter) block-like crystals can be obtained by slow diffusion of Et2O
into a MeOH solution of XCage•8CF3CO2. These crystals showed poor X-ray diffraction,
which prevented further structural determination.
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
(44) Nau, W. M.; Florea, M.; Assaf, K. I. Deep Inside Cucurbiturils: Physical Properties and
Volumes of Their Inner Cavity Determine The Hydrophobic Driving Force for Host-Guest
Complexation. Isr. J. Chem. 2011, 51, 559–577.
(45) Lefebvre, C.; Rubez, G.; Khartabil, H.; Boisson, J. C.; Contreras-García, J.; Hénon, E.
Accurately Extracting The Signature of Intermolecular Interactions Present in The NCI
Plot of The Reduced Density Gradient Versus Electron Density. Phys. Chem. Chem. Phys.
2017, 19, 17928–17936.
(46) Lu, T.; Chen, F. Multiwfn: A Multifunctional Wavefunction Analyzer. J. Comput. Chem.
2012, 33, 580–592.
(47) Cao, L.; Sekutor, M.; Zavalij, P. Y.; Mlinaric-Majerski, K.; Glaser, R.; Isaacs, L.
Cucurbit[7]uril.Guest Pair with an Attomolar Dissociation Constant. Angew. Chem., Int.
Ed. 2014, 53, 988–993.
(48) Ryan, S. T. J.; Young, R. M.; Henkelis, J. J.; Hafezi, N.; Vermeulen, N. A.; Hennig, A.;
Dale, E. J.; Wu, Y.; Krzyaniak, M. D.; Fox, A.; et al. Energy and Electron Transfer
Dynamics within a Series of Perylene Diimide/Cyclophane Systems. J. Am. Chem. Soc.
2015, 137, 15299–15307.
(49) A bolded descriptor denotes a compound, be it free or complexed, and an unbolded
descriptor refers to either (i) a component within a compound or (ii) a component part of a
mechanically interlocked molecule.
21
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 22 of 34
1
2
3
4
5
6
7
8
9
(50) Ringe, D.; Petsko, G. A. How Enzymes Work. Science. 2008, 320, 1428–1429.
(51) PDI1 is polydispersed with an average molecular weight about 4000 according to
electrospray ionization mass spectrometry.
(52) The NOESY spectrum was obtained using 0.2 s mixing time. The 1H NMR spectrum of
PEG chains were found to split into several peaks between 3.05 ppm and 4.20 ppm; these
peaks also reveals through-space correlations with XCage8+ in NOESY experiments.
(53) Heek, T.; Fasting, C.; Rest, C.; Zhang, X.; Würthner, F.; Haag, R. Highly Fluorescent
Water-Soluble Polyglycerol-Dendronized Perylene Bisimide Dyes. Chem. Commun. 2010,
46, 1884–1886.
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
(54) The observed weak fluorescence of PDI2 originates from residual monomeric species,
judging from its excitation spectrum.
(55) Ishida, T.; Morisaki, Y.; Chujo, Y. Synthesis of Covalently Bonded Nanostructure from
Two Porphyrin Molecular Wires Leading to a Molecular Tube. Tetrahedron Lett. 2006,
47, 5265–5268.
(56) Yamashina, M.; Kusaba, S.; Akita, M.; Kikuchi, T.; Yoshizawa, M. Cramming Versus
Threading of Long Amphiphilic Oligomers into a Polyaromatic Capsule. Nat. Commun.
2018, 9, 3–9.
(57) Deutman, A. B. C.; Monnereau, C.; Elemans, J. a a W.; Ercolani, G.; Nolte, R. J. M.;
Rowan, A. E. Mechanism of Threading a Polymer through a Macrocyclic Ring. Science.
2008, 322, 1668–1671.
22
ACS Paragon Plus Environment

Page 23 of 34
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
(58) An effort to determine the binding constants between XCage8+ and PDI2 using the
displacement ITC method in both MeCN and water did not meet with success on account
of the mutal aggregation among XCage8+, competitors, PDI2, and their corresponding
complexes.
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
(59) Biedermann, F.; Nau, W. M.; Schneider, H. J. The Hydrophobic Effect Revisited - Studies
with Supramolecular Complexes Imply High-Energy Water as a Noncovalent Driving
Force. Angew. Chem. Int. Ed. 2014, 53, 11158–11171.
(60) Gibb, B. C. Supramolecular Assembly and Binding in Aqueous Solution: Useful Tips
Regarding The Hofmeister and Hydrophobic Effects. Isr. J. Chem. 2011, 51, 798–806.
(61) Fan, J.; Hu, M.; Zhan, P.; Peng, X. Energy Transfer Cassettes Based on Organic
Fluorophores: Construction and Applications in Ratiometric Sensing. Chem. Soc. Rev.
2013, 42, 29–43.
(62) Chu, J.; Oh, Y.; Sens, A.; Ataie, N.; Dana, H.; Macklin, J. J.; Laviv, T.; Welf, E. S.; Dean,
K. M.; Zhang, F.; et al. A Bright Cyan-Excitable Orange Fluorescent Protein Facilitates
Dual-Emission Microscopy and Enhances Bioluminescence Imaging in Vivo. Nat.
Biotechnol. 2016, 34, 760–767.
(63) Garwin, S. A.; Kelley, M. S. J.; Sue, A. C.; Que, E. L.; Schatz, G. C.; Woodruff, T. K.;
O’Halloran, T. V. Interrogating Intracellular Zinc Chemistry with a Long Stokes Shift
Zinc Probe ZincBY-4. J. Am. Chem. Soc. 2019, 141, 16696–16705.
(64) Kogure, T.; Karasawa, S.; Araki, T.; Saito, K.; Kinjo, M.; Miyawaki, A. A Fluorescent
Variant of a Protein from The Stony Coral Montipora Facilitates Dual-Color Single-Laser
Fluorescence Cross-Correlation Spectroscopy. Nat. Biotechnol. 2006, 24, 577–581.
23
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 24 of 34
1
2
3
4
5
6
7
8
9
(65) Butkevich, A. N.; Lukinavičius, G.; D’Este, E.; Hell, S. W. Cell-Permeant Large Stokes
Shift Dyes for Transfection-Free Multicolor Nanoscopy. J. Am. Chem. Soc. 2017, 139,
12378–12381.
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
(66) Sednev, M. V.; Belov, V. N.; Hell, S. W. Fluorescent Dyes with Large Stokes Shifts for
Super-Resolution Optical Microscopy of Biological Objects: A Review. Methods Appl.
Fluoresc. 2015, 3, 042004.
(67) Weil, T.; Vosch, T.; Hofkens, J.; Peneva, K.; Müllen, K. The Rylene Colorant Family-
Tailored Nanoemitters for Photonics Research and Applications. Angew. Chem. Int. Ed.
2010, 49, 9068–9093.
(68) Li, X.; Hihath, J.; Chen, F.; Masuda, T.; Zang, L.; Tao, N. Thermally Activated Electron
Transport in Single Redox Molecules. J. Am. Chem. Soc. 2007, 129, 11535–11542.
(69) Xu, B.; Xiao, X.; Yang, X.; Zang, L.; Tao, N. Large Gate Modulation in The Current of a
Room Temperature Single Molecule Transistor. J. Am. Chem. Soc. 2005, 127, 2386–2387.
(70) Xin, N.; Guan, J.; Zhou, C.; Chen, X.; Gu, C.; Li, Y.; Ratner, M. A.; Nitzan, A.; Stoddart,
J. F.; Guo, X. Concepts in The Design and Engineering of Single-Molecule Electronic
Devices. Nat. Rev. Phys. 2019, 1, 211–230.
24
ACS Paragon Plus Environment

Page 25 of 34
Journal of the American Chemical Society
1
2
3
4
Captions for Figures
5
6
Figure 1. (a) Synthesis of XCage•8CF3CO2. (b) Solid-state superstructures of Perylene ⊂
7
8
9
XCage8+ obtained from single-crystal X-ray crystallography. Counterions and solvents are omitted
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
for the sake of clarity
Figure 2. Substrates molecules evaluated in the present study. Perylene and caffeine are used as
competitive substrates for displacement studies. PDI2 has been modified with polydispersed PEG
chains to enhance its solubility in water
Figure 3. (a) Plan and (b) side-on views of the solid-state superstructure of PDI1 ⊂ XCage8+
obtained from single-crystal X-ray crystallography. (c) Surface-area overlap analysis of PDI1 ⊂
XCage8+. Half of XCage8+ is deleted for the sake of clarity. Green: roof or floor; yellow: pillar
units; Red: total area of the PDI core; Dark red: overlapping area. (d) [N+···O=C] ion-dipole
interaction distances in PDI1 ⊂ XCage8+. (e) Electrostatic potential map of the PDI core
Figure 4. (a) 1H NMR (500 MHz, D2O, 25 ˚C) spectra of PDI2 (top), PDI2 ⊂ XCage•8CF3CO2
(middle), and XCage•8CF3CO2 (bottom). (b) 1H-1H NOESY (500 MHz, D2O, 25 ˚C) of PDI2 ⊂
XCage•8CF3CO2. Protons labels are shown in Figure 1 and Figure 2. All samples were measured
at 1.5 mM concentration
25
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 26 of 34
1
2
3
4
5
6
7
8
9
Figure 5. (a) Absorption and (b) emission (ex: 440 nm) spectra of PDI2 (blue) and PDI2 ⊂
XCage•8CF3CO2 (red) in water: inserts show aqueous solutions of PDI2 (left) and PDI2 ⊂
XCage•8CF3CO2 (right) under day light and UV light (ex: 365 nm). (c) Emission spectra (ex: 290
nm) of PDI2 (blue) and PDI2 ⊂ XCage•8CF3CO2 (red) in water. (d) Excitation spectra (em: 600
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
nm) of PDI2 (blue) and PDI2 ⊂ XCage•8CF3CO2 (red) in water
Figure 6. (a) Brightfield-merged micrograph of MCF-7 cells treated with PDI2 and PDI2 ⊂
XCage8+. Images were obtained using a 514 nm confocal laser with an emission window in the
range of 530–580 nm. Scale bar: 20 µm. (b) Concentration dependent uptake of PDI2 ⊂ XCage8+
by MCF-7 cells analyzed by flow cytometry (ex: 552 nm) in the PE-Cy5 channel (em: 656–684
nm); MFI represents mean fluorescence intensity. (c) Fluorescence micrograph of MCF-7 cells
with PDI2 and PDI2 ⊂ XCage8+. Images were obtained using a DAPI excitation filter (ex: 381–
399 nm) and a TRITC emission filter (em: 571–617 nm). (d) Dual-color micrograph of MCF-7
8
+
cells with Hoechst and PDI2 ⊂ XCage . Images were obtained using a single DAPI excitation
filter (ex: 381–399 nm) and two emission filters: DAPI emission filter (em: 411–459 nm) and
TRITC emission filter (em: 571–617 nm). Scale bar: 25 µm
26
ACS Paragon Plus Environment

Page 27 of 34
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
Figure 1
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 28 of 34
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
ACS Paragon Plus Environment

Page 29 of 34
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
a)
c)
b)
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
4.2 Å
4.0 Å
4.0 Å
e)
d)
–12 kcal mol-1
+12 kcal mol-1
4.2 Å
Figure 3
ACS Paragon Plus Environment