TRICYCLIC OCTACATIONIC CYCLOPHANE AND ITS USE IN COMPLEXATION WITH PERLENE DIIMIDE DYES
20220048860 · 2022-02-17
Inventors
Cpc classification
C09B67/0025
CHEMISTRY; METALLURGY
G01N21/6428
PHYSICS
C07D213/22
CHEMISTRY; METALLURGY
C09B5/62
CHEMISTRY; METALLURGY
International classification
C07D213/22
CHEMISTRY; METALLURGY
C09B69/10
CHEMISTRY; METALLURGY
Abstract
Disclosed herein is a tricyclic octacationic cyclophane and complexes comprising the tricyclic octacationic cyclophane and a perylene diimide dye complexed therein and methods of using and making the cyclophane and complexes.
Claims
1. A tricyclic octacationic cyclophane or a salt thereof, the cyclophane comprising a roof, a floor, and four pillars, wherein each of the roof and the floor are composed of a biphenyl unit having four pyridinium units extending therefrom and wherein each of the four pyridinium units of the roof are linked to another pyridinium unit of the floor by one of the four pillars.
2. The cyclophane of claim 1, wherein the cyclophane is ##STR00014##
3. A receptor-substrate complex, the complex comprising the tricyclic octacationic cyclophane according to claim 1 and a perylene diimide dye complexed therein.
4. The complex of claim 3, wherein the cyclophane is ##STR00015##
5. The complex of claim 3, wherein the perylene diimide dye has a formula ##STR00016## wherein R.sup.1 and R.sup.2 are independently selected from hydrogen, a substituted or unsubstituted, branched or unbranched, saturated or unsaturated C.sub.1-C.sub.6 alkyl, a substituted or unsubstituted aryl, or —OCH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.n—OR where R is hydrogen or a substituted or unsubstituted, branched or unbranched, saturated or unsaturated C.sub.1-C.sub.6 alkyl and n is an integer greater than or equal to 0.
6. The complex of claim 5, wherein R is —CH.sub.2CH.sub.2NMe.sub.2 or —CH.sub.2CH.sub.2[OCH.sub.2CH.sub.2].sub.44OMe.
7. A salt comprising the cyclophane of claim 1 and a counter anion.
8. The salt of claim 7, wherein the counter anion is CF.sub.3CO.sub.2.sup.−, PF.sub.6.sup.−, or Cl.sup.−.
9. A crystalline composition comprising the complex of claim 3.
10. The crystalline composition of claim 9, wherein the crystalline composition has a triclinic, space group P-1 (no. 2) crystal parameter and wherein the crystalline composition has unit cell parameters: a=11.0±0.1, b=15.9±0.1, c=19.3±0.1 Å, α=99.2±0.1°, β=99.1±0.1°, and γ=104.4±0.1°.
11. The crystalline composition of claim 9, wherein the crystalline composition has a tetragonal, space group P4.sub.32.sub.12 crystal parameter and wherein the crystalline composition has unit cell parameters: a=40.1±0.1, and c=10.8±0.1.
12. A method for fluorescence spectroscopy, comprising providing the complex of claim 3, irradiating the complex with an irradiation source, and detecting an emission signal from the complex.
13. The method of claim 12 further comprising providing a dye and detecting an emission signal from the dye.
14. The method of claim 13, wherein the complex and the dye are irradiated by the same irradiation source and wherein the emission signal of the complex and the emission signal of the dye are detectably distinct.
15. The method of claim 12, wherein the complex is localized in an aqueous environment.
16. A method for live cell imaging, the method comprising contacting a cell with the complex of claim 3, irradiating the cell with an irradiation source, and detecting an emission signal from the complex.
17. The method of claim 16 further comprising contacting the cell with a dye and detecting an emission signal from the dye.
18. The method of claim 17, wherein the complex and the dye are irradiated by the same irradiation source and wherein the emission signal of the complex and the emission signal of the dye are detectably distinct.
19. The method of claim 16, wherein the emission signal of the complex is localized within the cell.
20. A method for preparing a receptor-substrate complex, the method comprising providing the tricyclic octacationic cyclophane according to claim 1, providing a perylene diimide dye, and contacting the tricyclic octacationic cyclophane and the perylene diimide dye.
21. The method of claim 20, wherein providing the tricyclic octacationic cyclophane comprises contacting ##STR00017## in the presence of a pyrene template and removing the pyrene template.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.
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[0025]
DETAILED DESCRIPTION OF THE INVENTION
[0026] Here, we report the synthesis of a tricyclic octacationic cyclophane, which exhibits complementary stereoelectronic binding towards perylene diimide dyes 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.
[0027] Herein, we report a new synthetic receptor that is tailored to provide a complementary stereoelectronic binding cavity for binding a dye, such as an aromatic dye like perylene diimide (PDI) dye, in water or other solvents. The receptor is a tricyclic octacationic cyclophane receptor featuring a roof-pillar-floor structure. Each of the roof and the floor are composed of a biphenyl unit having four pyridinium units extending therefrom. Pillars connect a pyridinium unit of the roof with another pyridinium unit of the floor. The biphenyl unit provides a large and flat binding surface, and four pillars connecting each of the four pyridinium units of the roof with another pyrinium unit of the floor results in a rigid cavity capable of hosting a dye substrate. The eight cationic pyridinium units provide both sufficient water solubility and complementary electronic binding sites for electron-rich moieties such as carbonyl groups. In some embodiments, the pillars comprise a xylylene unit.
[0028] In some embodiments, the cyclophane is
##STR00003##
This cyclophane is referred to as XCage.sup.8+ in view of its X-shaped structure. 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 aromatic substrate, such as a PDI dye, in its cavity.
[0029] Receptor-substrate complexes may comprise a PDI dye. As used herein, a PDI dye has a perylene core
##STR00004##
and two imide moieties covalently bound thereto to form a seven-membered ring system. In some embodiments, the PDI dye has a formula
##STR00005##
where each R.sup.1 and R.sup.2 are independently selected from hydrogen, a substituted or unsubstituted, branched or unbranched, saturated or unsaturated C.sub.1-C.sub.6 alkyl, a substituted or unsubstituted aryl, or —OCH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.n—OR where R is hydrogen or a substituted or unsubstituted, branched or unbranched, saturated or unsaturated C.sub.1-C.sub.6 alkyl and n is an integer greater than or equal to 0. In some embodiments, n is between 0 and 150. R.sup.1 and R.sup.2 may be optionally substituted with —NRR′, —NRR′R″, —SR, —C(═O)OR, —OR, —C≡R. Each of R, R′, and R″ may be independently selected from hydrogen, a substituted or unsubstituted, branched or unbranched, cyclic or acyclic, saturated or unsaturated C.sub.1-C.sub.6 alkyl, or a substituted or unsubstituted, branched or unbranched, cyclic or acyclic, saturated or unsaturated amine. Exemplary R, R′, and R″ include, without limitation, —H, —CH.sub.3, —CH, —CH.sub.2CCH,
##STR00006##
[0030] Exemplary PDI dyes include, but are not limited to,
##STR00007##
where R.sup.1 and/or R.sup.2 is selected from
##STR00008##
[0031] In some embodiments, the PDI dye includes R selected from —CH.sub.2CH.sub.2NMe.sub.2 or —CH.sub.2CH.sub.2[OCH.sub.2CH.sub.2].sub.44OMe. When R comprises a polyethylene glycol (PEG) chain, the PEG chain may be polydisperse and the notation [OCH.sub.2CH.sub.2]n may indicate an average molecular weight as measured by electrospray ionization mass spectrometry or other suitable measurement technique.
[0032] The eight cationic pyridinium units of the cyclophane provide complementary electronic binding sites for the four divergent carbonyl groups in the PDI molecule. The resulting PDI⊂XCage.8CF.sub.3CO.sub.2 complex, which exhibits picomolar binding affinity in water, is 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. PDI-based dyes have superior photophysical properties and wide range of applications in materials science.sup.35 and biotechnology.sup.36. Encapsulations of PDI dyes have been explored.sup.37,38 using cucurbit[8]uril and also a tetracationic cyclophane, known.sup.28 as ExBox.sup.4+; significant improvements in photophysical properties of PDI dyes were observed in water. The affinity constants involving both these receptors remain around 10.sup.5 M.sup.−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.
Synthesis of XCage.8CF.sub.3CO.sub.2
[0033] XCage.8CF.sub.3CO.sub.2 was prepared (
[0034] X-Ray Crystallographic Analysis Numerous attempts to grow the single crystals of XCage.sup.8+ with various counterions did not meet with success. Large (>100 m in diameter) block-like crystals can be obtained by slow diffusion of Et.sub.2O into a MeOH solution of XCage.8CF.sub.3CO.sub.2. These crystals showed poor X-ray diffraction, which prevented further structural determination. Fortunately, a single crystal of Perylene c XCage.8CF.sub.3CO.sub.2 was obtained by slow vapor diffusion of iPr.sub.2O into a MeOH solution of Perylene c XCage.8CF.sub.3CO.sub.2. The solid-state structure of XCage.8CF.sub.3CO.sub.2 (
[0035] A model compound PDI1 (
NMR Spectroscopy and Mass Spectrometry in Solution
[0036] In order to investigate the molecular recognition between XCage.8CF.sub.3CO.sub.2 and PDI in water, a water-soluble PDI2 (
Photophysical Properties
[0037] Encapsulation of PDI2 by XCage.8CF.sub.3CO.sub.2 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.8CF.sub.3CO.sub.2, 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 (
[0038] There is an efficient energy transfer from XCage.8CF.sub.3CO.sub.2 to PDI2 in both MeCN and H.sub.2O. In the excitation spectrum of PDI2⊂XCage.8CF.sub.3CO.sub.2, we observed a strong excitation peak around 300 nm, where XCage.8CF.sub.3CO.sub.2 absorbs light. Remarkably, in MeCN, the fluorescence intensity of PDI2⊂XCage.8CF.sub.3CO.sub.2, 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 (
Binding Kinetics and Thermodynamics
[0039] The changes in optical properties induced on PDI2⊂XCage.8CF.sub.3CO.sub.2 complex formation enable a facile tracking of the recognition process. The kinetics of threading PDI2 into XCage.8CF.sub.3CO.sub.2 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 k.sub.on=(4.8±1.1)×10.sup.5 M.sup.−1s.sup.−1. The half-life at 0.1 μM was calculated to be 21 s. Such rapid complex formation of PDI2⊂XCage.8CF.sub.3CO.sub.2 in water is remarkable when one considers the threading process that involves the chain end of mPEG.sub.2000 polymer finding a “correct” cavity entrance and then exiting at the right opening of the tricyclic cage. This observation agrees with the reported literature.sup.55-57 that threading a PEG polymer through a macrocycle is rapid in water.
[0040] The binding constants (Table 1) were determined by fluorescence titration and isothermal titration calorimetry (ITC). Displacement titration experiments monitored by fluorescence were performed in order to determine the high binding affinity between PDI2 and XCage.8CF.sub.3CO.sub.2. An effort to determine the binding constants between XCage.sup.8+ and PDI2 using the displacement ITC method in both MeCN and water did not meet with success on account of the mutal aggregation among XCage.sup.8+, competitors, PDI2, and their corresponding complexes. In MeCN, binding of XCage.8CF.sub.3CO.sub.2 was tested first of all using Perylene as a substrate. Its binding constant was found to be in the order of 10.sup.6 M.sup.−1, which is similar in magnitude to that of ExCage.sup.6+.6PF.sub.6 and about 86 times higher than that of ExBox.sup.4+.4PF.sub.6..sup.28,29 Next, we performed a competitive experiment, starting with a solution of XCage.8CF.sub.3CO.sub.2 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 10.sup.9 M.sup.−1. The binding affinity between XCage.8CF.sub.3CO.sub.2 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 TΔS value. The formation of PDI2⊂XCage.8CF.sub.3CO.sub.2 in MeCN is mainly driven by favorable enthalpy with a small contribution from positive entropy. Compared with the binding of Perylene towards XCage.8CF.sub.3CO.sub.2, PDI2 shows a similar positive ΔS and also enjoys a more negative ΔH, which originates from the additional [C═O . . . N.sup.+] ion-dipole interactions.
[0041] In order to evaluate the affinity between XCage.8CF.sub.3CO.sub.2 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 that are in the order of 10.sup.5 M.sup.−1. The binding constant between PDI2 and XCage.8CF.sub.3CO.sub.2 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.8CF.sub.3CO.sub.2 was determined to be 7.7×10.sup.10 M.sup.−1, i.e., K.sub.d=13 pM. The formation of PDI2⊂XCage.8CF.sub.3CO.sub.2 in water is enthalpically driven with a small favorable entropic component. The binding enthalpy observed in water is 4 kcal mol.sup.−1 higher when compared with that in MeCN, suggesting that the release of high energy water molecules provides.sup.59 an extra contribution to the stability of PDI2⊂XCage.8CF.sub.3CO.sub.2 in addition to the large area [π-π] stacking and ion-dipole interactions. The small favorable entropy benefits.sup.60 from the release of water into the bulk and the structural rigidity of both XCage.8CF.sub.3CO.sub.2 and PDI2.
[0042] In order to illustrate the effect of the extended receptor surface on the affinity enhancement, we investigated the binding thermodynamics of ExBox.4Cl towards PDI2 in water. ExBox.4Cl shows a binding affinity of 2.0×10.sup.6 M.sup.−1 and a binding enthalpy of −7.4 kcal mol.sup.−1. Compared with ExBox.4Cl, XCage.8CF.sub.3CO.sub.2 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 XCage.sup.8+ provides twice the binding surface area towards the PDI binding core compared to that of ExBox.sup.4+. The extended binding surface results in significant gains in binding enthalpy (ΔΔH=−6.7 kcal mol.sup.−1), while the loss of binding entropy (TΔΔδ=−0.5 kcal mol.sup.−1) is trivial, leading to an obvious deviation.sup.23 from the enthalpy-entropy compensation plots for cyclodextrin-guest complexation (
Fluorescence Imaging Studies
[0043] 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.8CF.sub.3CO.sub.2 is non-toxic to MCF-7 cells and shows >95% cell viability at all concentrations tested (2.5-50 μM) (
[0044] Brightfield-merged images show (
[0045] PDI2⊂XCage.8CF.sub.3CO.sub.2 produces (
Conclusions
[0046] An octacationic tricyclic cyclophane XCage.8CF.sub.3CO.sub.2 has been designed and synthesized.
[0047] XCage.sup.8+ 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 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..sup.13
[0048] 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.8CF.sub.3CO.sub.2 and Hoechst stain. While this research illustrates the bioimaging application of PDI2⊂XCage.8CF.sub.3CO.sub.2, 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.8CF.sub.3CO.sub.2 provides for the manipulation of PDI dyes with an eye to a wide range of applications in the fields of single-molecule electronics,.sup.67-69 photonic device,.sup.70 materials science,.sup.35 and molecular biology..sup.36
Miscellaneous
[0049] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”
[0050] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ≤10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.
[0051] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
[0052] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
[0053] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
[0054] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Examples
General Information
[0055] Commercially available solvents and chemicals were purchased from Sigma-Aldrich and Fisher Scientific and used without further purification unless otherwise stated. Water was deionized, and micro filtered using Milli-Q water filtration station. Compounds PDI1.sup.S1 and ExBox.4Cl.sup.S2 were prepared using previously reported procedures. Thin layer chromatography (TLC) was performed on silica gel 60 F254 (E. Merck). Flash column chromatography was performed by Combiflash Rf 200 purification system. Reverse phase column chromatography was performed on Combiflash NEXTGEN 300+ system with SNAP ULTRA C18 cartridges which were purchased from Biotage. UV/Vis Absorption spectra were recorded in a glass cuvette using a UV-3600 Shimadzu spectrophotometer. Steady-state emission spectra were acquired in a quartz cuvette with an optical path-length of 10 mm containing the solution of interest using HORIBA Fluoromax4 spectrofluorometer, which was equipped with an integrating sphere for absolute photoluminescence quantum yield determination and time-correlated single-photon counting (TCSPC) module for emission decays. Nuclear magnetic resonance (NMR) spectra were recorded on Bruker AVANCE III 500 MHz spectrometer equipped with DCH CryoProbe, with working frequencies of 500 MHz for .sup.1H and 125 MHz for .sup.13C nuclei. Chemical shifts were reported in ppm relative to the signals corresponding to the residual non-deuterated solvents (CDCl.sub.3: δ=7.26 ppm, CD.sub.3CN: δ=1.94 ppm, CD.sub.3OH: δ=4.74 ppm, D.sub.2O: δ=4.74 ppm). High-resolution mass spectra were measured on an Agilent 6210 Time of Flight (TOF) LC-MS, using an ESI source, coupled with Agilent 1100 HPLC stack, using direct infusion (0.6 mL/min). Single crystal data were obtained on a Bruker Kappa APEX2 CCD diffractometer using Cu-Kα radiation. Detailed experimental procedures are provided below in the appropriate sections.
[0056] Synthetic Protocols
##STR00009##
[0057] TPBP: Dioxane was added to a solution of K.sub.2CO.sub.3 (5.2 g, 37.7 mmol) in H.sub.2O (15 mL). The resulting solution was degassed by bubbling N.sub.2 gas for 10 min. 3,3′,5,5′-Tetrabromo-1,1′-biphenyl (1.2 g, 2.6 mmol), 4-pyridinylboronic acid (2.0 g, 16.3 mmol), and Pd(PPh.sub.3).sub.4 (160 mg, 0.13 mmol) were added to the degassed solution. The reaction mixture was degassed further using vacuum, followed by a N.sub.2 flow cycle repeated three times before it was heated at 130° C. under reflux for 24 h. After cooling to room temperature, H.sub.2O (100 mL) was added to the reaction mixture, and the resulting solution was extracted by CHCl.sub.3 (3×150 mL). The organic layers were combined and washed with H.sub.2O until no black suspension was observed in aqueous layer. After drying (Na.sub.2SO.sub.4), the organic solvents were removed under vacuum. The resulting yellow solid was washed with MeOH to obtain the product TPBP as an off-white solid (1.1 g, 92% yield). .sup.1H NMR (500 MHz, CDCl.sub.3) δ 8.73 (d, J=5.0 Hz, 8H), 7.97-7.92 (m, 4H), 7.90 (s, 2H), 7.61 (d, J=5.1 Hz, 8H). .sup.13C NMR (125 MHz, CDCl.sub.3) δ 150.6, 150.6, 147.5, 142.3, 140.3, 126.7, 125.5, 121.9. HRMS-ESI (m/z) for TPBP: Calcd for C.sub.32H.sub.23N.sub.4.sup.+: m/z=463.1917 [M+H].sup.+; found 463.1918 [M+H].sup.+.
##STR00010##
[0058] TB.4PF.sub.6: TPBP (500 mg, 1.1 mmol) and 1,4-bis(bromomethyl)benzene (6.7 g, 25.4 mmol) were dissolved in anhydrous DMF (500 mL). The reaction mixture was stirred at room temperature for 48 h. CH.sub.2Cl.sub.2 (1.5 L) was added and the resulting precipitate was collected by filtration. The precipitate was dissolved in DMF (100 mL), and the insoluble materials were filtrated off. NH.sub.4PF.sub.6 (2.0 g) was added to the filtrate, followed by addition of H.sub.2O (500 mL) to precipitate out the product TB.4PF.sub.6, which was collected by filtration as a white solid (1.9 g, 99% yield). .sup.1H NMR (500 MHz, CD.sub.3CN) δ 8.91-8.84 (m, 8H), 8.53 (d, J=1.7 Hz, 4H), 8.52-8.48 (m, 8H), 8.41 (s, 2H), 7.55 (d, J=8.2 Hz, 8H), 7.49 (d, J=8.2 Hz, 8H), 5.77 (s, 8H), 4.61 (s, 8H). .sup.13C NMR (125 MHz, CD.sub.3CN) δ 155.9, 145.3, 141.9, 140.8, 136.9, 133.8, 131.1, 130.8, 130.1, 128.7, 126.9, 64.2, 33.2. HRMS-ESI (m/z) for TB.4PF.sub.6: Calcd for C.sub.64H.sub.54Br.sub.4F.sub.12N.sub.4P.sub.2.sup.2+: m/z=744.0167 [M-2PF.sub.6].sup.2+; found 744.0170 [M-2PF.sub.6].sup.2+.
##STR00011##
[0059] XCage.8CF.sub.3CO.sub.2: 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 CHCl.sub.3 (20 mL) was added to a solution of TB.4PF.sub.6 (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 CHCl.sub.3 (300 mL) were added to the reaction mixture, the yellow precipitate was isolated by filtration and then dispersed in MeOH (100 mL). Celite (5 g) and TFA (2 mL) were added and the solvent was removed by vacuum. The remaining solid was loaded onto a Combiflash flash chromatography system and purified by reverse Cis columns using 0-25% MeCN/H.sub.2O 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. H.sub.2O was removed and the residue was purified further with reverse Cis chromatography using 0-15% MeCN/H.sub.2O with 0.1% TFA as additive to obtain the product XCage.8CF.sub.3CO.sub.2 as a white solid (150 mg, 26% yield).
[0060] .sup.1H NMR (500 MHz, CD.sub.3OD) 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). .sup.13C NMR (125 MHz, CD.sub.3OD) δ 159.9, 159.6, 154.4, 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.8CF.sub.3CO.sub.2: Calcd for C.sub.108H.sub.76F.sub.18N.sub.8O.sub.12.sup.2+: m/z=1009.7659 [M-2CF.sub.3CO.sub.2].sup.2+; found 1009.7688 [M-2CF.sub.3CO.sub.2].sup.2+.
##STR00012##
[0061] TM.4PF.sub.6: TPBP (200 mg, 0.43 mmol) and MeI (1.2 g, 8.64 mmol) were suspended in anhydrous MeCN (25 mL). The reaction mixture was heated at 80° C. for 18 h. After cooling to room temperature, CH.sub.2Cl.sub.2 (100 mL) was added and the resulting precipitate was collected by filtration. H.sub.2O (100 mL) was added to dissolve the precipitate, and NH.sub.4PF.sub.6 (2.0 g) was added to precipitate out the product TM.4PF.sub.6 as a yellow solid (420 mg, 89% yield). .sup.1H NMR (500 MHz, CD.sub.3CN) δ 8.82-8.76 (m, 8H), 8.60 (d, J=1.7 Hz, 4H), 8.57-8.52 (m, 8H), 8.49 (t, J=1.7 Hz, 2H), 4.40 (s, 12H). .sup.13C NMR (125 MHz, CD.sub.3CN) δ 155.1, 146.1, 142.0, 136.9, 130.9, 128.4, 126.2, 48.4. HRMS-ESI (m/z) for TM.4PF.sub.6: Calcd for C.sub.36H.sub.34F.sub.18N.sub.4P.sub.3.sup.+: m/z=957.1703 [M-PF.sub.6].sup.+; found 957.1716 [M-PF.sub.6].sup.+.
##STR00013##
[0062] PDI2: Perylene-3,4,9,10-tetracarboxylic dianhydride (46.3 mg, 0.118 mmol) and mPEG.sub.2000-NH.sub.2 (500 mg, 0.248 mmol) were added to a mixture of imidazole (2.5 g) and Zn(AcO).sub.2. The reaction mixture was heated at 140° C. under N.sub.2 protection for 3 days. After cooling to room temperature, CHCl.sub.3 (200 mL) was added to the reaction mixture. The solution was washed with 1 N HCl solution (5×100 mL) and brine (2×100 mL). The organic layer was dried (Na.sub.2SO.sub.4). The organic solvent was removed, and the residue was purified by column chromatography using 0-10% MeOH/CH.sub.2Cl.sub.2 with 0.1% NH.sub.4OH as additive to obtain the product PDI2 as a dark red solid (120 mg, 23% yield). .sup.1H NMR (500 MHz, CDCl.sub.3) δ 8.70 (d, J=8.0 Hz, 4H), 8.65 (d, J=8.1 Hz, 4H), 4.47 (s, 4H), 3.86 (t, J=6.1 Hz, 4H), 3.78 (dd, J=5.8, 4.0 Hz, 2H), 3.72 (dd, J=5.8, 3.7 Hz, 4H), 3.64 (d, J=3.3 Hz, 346H), 3.38 (s, 6H). HRMS-ESI (m/z) for PDI2: A cluster peak around 2033 was observed as [M+2Na].sup.2+ on account of the polydispersity of PEG chains.
Cyclic Voltammetry
[0063] Cyclic voltammetry (CV) was performed at 298 K under a N.sub.2 atmosphere with a Gamry Multipurpose instrument (Reference 600) interfaced to a PC. All CV experiments were carried out using a glassy carbon working electrode (0.071 cm.sup.2). The electrode surface was polished routinely with 0.05 m alumina-water slurry on a felt surface immediately before use. The counter electrode was a Pt coil, and the reference electrode was the saturated Ag/AgCl electrode. The concentration of the sample and tetrabutylammonium hexafluoro-phosphate (TBAPF.sub.6), were 1.0 mM and 0.1 M, respectively. CV experiments of TM.4PF.sub.6 and XCage.8CF.sub.3CO.sub.2 were conducted at a scan rate of 50 mV/s. The results showed similar behavior with two reduction peaks. Both redox processes are nonreversible, and precipitation developed on electrode after formation of neutral species.
Crystallographic Analysis
Crystal Structure of TPBP (CCDC: 1952500)
[0064] (a) Method: TPBP (2 mg) was dissolved in CHCl.sub.3 (1 mL), and the solution was passed through a 0.45 μm PTFE filter. The solution was allocated into three culture tubes (each tube containing 250 μL solution). Each cultural tube was then placed in a scintillation vial that contains EtOAc (˜3 mL). Slow vapor diffusion of EtOAc into TPBP solution for 4 days yielded colorless crystals of TPBP. A suitable crystal was selected, and the crystal was mounted on a MITIGEN holder with Paratone oil on a Bruker APEX-II CCD diffractometer. The crystal was kept at 100.03 K during data collection. Using Olex2.sup.S3, the structure was solved with the ShelXD.sup.S4 structure solution program using Dual Space and refined with the XL.sup.S5 refinement package using Least Squares minimization.
[0065] (b) Crystal Parameters: Empirical formula=C.sub.32H.sub.22N.sub.4, Formula weight=462.53, monoclinic, space group P2.sub.1/c (no. 14), a=14.0428(12), b=10.8941(9), c=16.0875(13) Å, β=113.291(5°), V=2260.6(3) Å.sup.3, Z=4, T=100.03 K, μ(CuKα)=0.634 mm.sup.−1, D.sub.calc=1.359 g/mm.sup.3, 12620 reflections measured (6.852≤2Θ≤127.35), 3705 unique (R.sub.int=0.0335, R.sub.sigma=0.0339) which were used in all calculations. The final R.sub.1 was 0.0634 (I>2σ(I)) and wR.sub.2 was 0.1751 (all data).
[0066] (c) Refinement Details: No special refinement necessary.
Crystal Structure of Perylene⊂XCage.8CF.sub.3CO.sub.2 (CCDC: 1952501)
[0067] (a) Method: XCage.8CF.sub.3CO.sub.2 (2.2 mg, 1 mmol) and perylene (0.5 mg, 2 mmol) was dissolved in MeOH (1 mL), and the solution was passed through a 0.45 μm PTFE filter to obtain Perylene c XCage.8CF.sub.3CO.sub.2 solution. Slow vapor diffusion of isopropyl ether into a Perylene c XCage.8CF.sub.3CO.sub.2 solution for a week yielded yellow crystals of Perylene c XCage.8CF.sub.3CO.sub.2. A suitable crystal was selected, and the crystal was mounted on a MITIGEN holder with Paratone oil on a Bruker APEX-IT CCD diffractometer. The crystal was kept at 99.99 K during data collection. Using Olex2.sup.S3, the structure was solved with the ShelXT.sup.S4 structure solution program using Intrinsic Phasing and refined with the XL.sup.S5 refinement package using Least Squares minimization.
[0068] (b) Crystal Parameters: Empirical formula=C.sub.136H.sub.104F.sub.24N.sub.8O.sub.20, Formula weight=2626.27, triclinic, space group P-1 (no. 2), a=11.0308(14), b=15.926(2), c=19.347(3) Å, a=99.202(6), β=99.067(6), γ=104.442(6°), V=3179.0(7) Å3, Z=1, T=99.99 K, μ(CuKα)=0.998 mm.sup.−1, D.sub.calc=1.372 g/mm.sup.3, 36937 reflections measured (4.728≤2Θ≤127.758), 10415 unique (R.sub.int=0.0399, R.sub.sigma=0.0384) which were used in all calculations. The final R.sub.1 was 0.0536 (I>2σ(I)) and wR.sub.2 was 0.1503 (all data).
[0069] (c) Refinement Details: The enhanced rigid-bond restraint (SHELX keyword RIGU) was applied on the disordered trifluoroacetate and perylene molecules..sup.S6 The solvent masking procedure as implemented in Olex2 was used to remove the electronic contribution of solvent molecules from the refinement. As the exact solvent content is not known, only the atoms used in the refinement model are reported in the formula here. Total solvent accessible volume/cell=321.3 Å.sup.3 [10.1%] Total electron count/cell=92.7.
[0070] (d) Solvent Treatment Details: No special treatment necessary. (d) Solvent Treatment Details: Not applicable.
Crystal Structure of PDI1⊂XCage.7PF.SUB.6.—OHJ (CCDC: 1952502)
[0071] (a) Method: a mixture of XCage.8CF.sub.3CO.sub.2 (3 mg) and access amounts of PDI1 (20 mg) were mixed in DMF, and the suspension was stirred at 50° C. for 30 min. After cooling to room temperature, the solution was filtrated through a 0.45 μm PTFE filter to remove insoluble PDI1. DMF was then removed by vacuum, and the residue was dissolved in H.sub.2O. NH.sub.4PF.sub.6 was added, and the precipitation was collected by centrifuge to obtain PDI1⊂XCage.8PF.sub.6 as a red solid, which was subsequently dissolved in MeCN (1 mL). Slow vapor diffusion of isopropyl ether into a MeCN solution of PDI1⊂XCage.8PF.sub.6 for a week yielded pink crystals of PDI1⊂XCage.7PF.sub.6.OH. Meanwhile, single crystal of PDI1.2HPF.sub.6 was also obtained in the same sample. A suitable crystal was selected, and the crystal was mounted on a MITIGEN holder on a Bruker APEX-IT CCD diffractometer. The crystal was kept at 100.0 K during data collection. Using Olex2.sup.S3, the structure was solved with the ShelXD.sup.S4 structure solution program using Dual Space and refined with the XL.sup.S5 refinement package using Least Squares minimization.
[0072] (b) Crystal Parameters: Empirical formula=C.sub.136H.sub.118F.sub.42N.sub.16O.sub.6P.sub.7, Formula weight=3087.25, tetragonal, space group P4.sub.32.sub.12 (no. 96), a=40.109(4), c=10.8259(13) Å, V=17416(4) Å.sup.3, Z=4, T=100.0 K, μ(CuKα)=1.492 mm.sup.−1, D.sub.calc=1.177 g/mm.sup.3, 37061 reflections measured (3.116≤2Θ≤108.522), 10132 unique (R.sub.int=0.0658, R.sub.sigma=0.0634) which were used in all calculations. The final R.sub.1 was 0.1051 (I>2σ(I)) and wR.sub.2 was 0.2963 (all data).
[0073] (c) Refinement Details: The enhanced rigid-bond restraint (SHELX keyword RIGU) was applied globally..sup.S6 Additionally, isotropic restraints (ISOR) were applied to several ill-behaved atoms on the disordered chains of the substrate molecule. Distance restraints were imposed on the disordered atoms as well as the some of the PF.sub.6 anions.
[0074] (d) Solvent Treatment Details: The solvent masking procedure as implemented in Olex2 was used to remove the electronic contribution of solvent molecules from the refinement. As the exact solvent content is not known, only the atoms used in the refinement model are reported in the formula here. Total solvent accessible volume/cell=4074.5 Å.sup.3 [23.4%]. Total electron count/cell=1026.1.
Crystal Structure of PDI1 (CCDC: 1952502)
[0075] (a) Method: Single crystal of PDI1 was co-obtained with PDI1⊂XCage.sup.8+ crystal sample. A suitable crystal was selected, and the crystal was mounted on a MITIGEN holder with Paratone oil on a Bruker APEX-II CCD diffractometer. The crystal was kept at 100.0 K during data collection. Using Olex2.sup.S3, the structure was solved with the ShelXT.sup.S4 structure solution program using Intrinsic Phasing and refined with the XL.sup.S5 refinement package using Least Squares minimization.
[0076] (b) Crystal Parameters: Empirical formula=C.sub.32H.sub.30F.sub.12N.sub.4O.sub.4P.sub.2, Formula weight=824.54, triclinic, space group P-1 (no. 2), a=6.009(2), b=8.492(3), c=16.817(6), α=81.867(7), β=86.681(8), γ=71.757(7°), V=806.7(5) Å.sup.3, Z=1, T=100.0 K, μ(MoKα)=0.252 mm.sup.−1, D.sub.calc=1.697 g/mm.sup.3, 9396 reflections measured (2.446≤2Θ≤52.588), 3201 unique (R.sub.int=0.0583, R.sub.sigma=0.0781) which were used in all calculations. The final R.sub.1 was 0.0471 (I>2σ(I)) and wR.sub.2 was 0.1135 (all data).
[0077] (c) Refinement Details: No special refinement necessary.
[0078] (d) Solvent Treatment Details: Not applicable.
Electrostatic Potential Map Calculation
[0079] The Cartesian coordinates of single crystal structure of PDI were modified as initial input and optimized by Gaussian 16 at B3LYP/6-31G* level..sup.S7 The out-put files were used further to calculate electron static potential maps by GaussView..sup.S8
Surface-Area Overlap (SAO) Analysis
[0080] Surface-area overlap analysis was performed by Chimera and Image J software. Single crystal structures of the receptor-substrate complex were truncated by removing the top half of XCage.sup.8+ and visualized by Chimera..sup.S9 The substrates and XCage.sup.8+ receptor were colored to show the bridging units (2), the binding cavity of the receptor (1), the area of substrate (3), and the overlapping portion between the receptor and substrate (4). ImageJ 1.49 software was used to measure the percent of SAO in each receptor-substrate complex..sup.S10 Values were calculated for the SAO-XCage (the overlapping portion between the receptor and substrate divided by the total area of the receptor) and SAO-substrate (the overlapping portion between the receptor and substrate divided by the total area of the substrate)..sup.S11
[0081] The Cartesian coordinates of the single crystal structure of Perylene c XCage.sup.8+ was modified by removing perylene substrate, and the resulting empty XCage.sup.8+ was then analyzed by Multiwfn program 3.6 program.sup.S11 to calculate the cavity volume through the domain analysis function.
Independent Gradient Model Analysis
[0082] Independent gradient model (IGM) analysis is an approach based on pro-molecular density to identify and isolate intermolecular interactions..sup.S12 Hydrogen bonds and van der Waals contacts are visualized as an iso-surface with blue and green color respectively. Single crystal structures of the receptor-substrate complexes were used as input file. The binding surface was calculated by Multiwfn 3.6 program.sup.S11 through function 20 (visual study of weak interaction) and visualized by Chimera program.
Photophysical Characterization
[0083] The fluorescence quantum yield of PDI dyes were measured by using rhodamine 6G in EtOH (Φ.sub.f=0.95) as standard..sup.S2 The concentrations of rhodamine 6G and PDI dyes were adjusted to the absorption value 0.08 at 450 nm. The fluorescence spectrum of each solution was obtained with excitation at 450 nm, and the integrated area was used in the fluorescence quantum yield calculation. The estimated error for this method.sup.S13 is ±10%. The fluorescent life-time was measured by Horiba Fluoromax-4 fluorometer equipped with TCSPC. Samples were excited by a laser at 374 nm, and the fluorescence decay over time was monitored at 560 nm. The fluorescent decay profiles were analyzed by a DAS6 software and matched either by a single exponential decay or double exponential decay model through mathematic fittings. Tables 2 and 3 summarize the photophysical properties in H.sub.2O and MeCN.
Binding Studies by Fluorescence
[0084] In all experiments shown below, ExBox.4Cl and XCage.8CF.sub.3CO.sub.2 were used in titration studies. Here we use ExBox.sup.4+ and XCage.sup.8+ for short. All of the solutions were prepared in spectroscopic grade solvents and equilibrated for 24 h at room temperature before use. All of the studies were independently duplicated and the corresponding isotherms were fitted to calculate the average K.sub.a or k.sub.on values with the relevant standard errors. All of the titrations were performed at 25° C., and the corresponding Gibbs free energy was determined from K.sub.a.
Determination of Binding Constants
[0085] Direct fluorescence titration experiments: Since the association between PDI2 and ExBox.sup.4+ in H.sub.2O produces turn-on fluorescence, we tracked the increase of fluorescence (ex: 545 nm, em: 555 nm) of PDI2 by varying equivalents of ExBox.sup.4+. Caffeine is not fluorescent in H.sub.2O and its association with XCage.sup.8+ quenches the fluorescence of XCage.sup.8+. Thus, the fluorescence quenching of XCage.sup.8+ was tracked by the addition of Caffeine. In MeCN, the fluorescence of perylene is quenched by XCage.sup.8+ and thus the titration was performed by tracking the fluorescence quenching of Perylene. A plot of fluorescent intensity versus receptor concentration [R].sub.0 or substrate concentration [S].sub.0 was fitted with a nonlinear least-squares fitting equation for 1:1 binding model to calculate the binding constant K.sub.a using Origin Lab 8.6 software..sup.S14, S15
[0086] Displacement fluorescence titration experiments in H.sub.2O: A large access of the competitor caffeine (10 mM) was pre-mixed with XCage.sup.8+ (10 μM), and the solution was equilibrated for 30 min before titration. The substrate solution of PDI2 was injected in aliquots. For each injection, the guest displacement process was relatively slow and took about 3-10 min to reach the equilibrium as monitored by the change of fluorescence (ex: 560 nm, em: 660 nm) over time. At this excitation wavelength, PDI2⊂XCage.sup.8+ is fluorescent; XCage.sup.8+, PDI2 and Caffeine are nonfluorescent. The association constants were calculated by fitting the titration data with a nonlinear least-squares fitting equation for displacement binding using Origin Lab 8.6 software..sup.S14
[0087] Displacement fluorescence titration experiments in MeCN: Perylene (250 μM) was premixed with XCage.sup.8+ (5 μM) in MeCN, and the solution was equilibrated for 30 min before titration. The substrate solution of PDI2 was injected in aliquot amount. Between each injection, the change of fluorescence intensity was monitored over time until the fluorescence (ex: 550 nm, em: 660 nm) intensity reached a steady state. At this excitation wavelength, PDI2 (XCage.sup.8+ is fluorescent while XCage.sup.8+ and PDI2 are nonfluorescent. The association constants were calculated by fitting the titration data with a nonlinear least-squares fitting equation for displacement binding using Origin Lab 8.6 software..sup.S14
Determination of Binding Kinetics
[0088] Equal volume of PDI2 (1 μL, 100 μM) and XCage.sup.8+ (1 μL, 100 μM) were mixed in H.sub.2O (1 mL) and the change of fluorescence (ex: 540 nm, em: 554 nm) was monitored over time. The resulting threading kinetics profiles were fitted using a second order kinetics model by Origin Lab 8.6 software. In MeCN, the change of fluorescence over time upon the formation of PDI2⊂XCage.sup.8+ is too small to produce a kinetics profile as a result of the similar quantum yield between PDI2⊂XCage.sup.8+ and PDI2.
Isothermal Titration Calorimetry (ITC)
[0089] In all ITC experiments, ExBox.4Cl and XCage.8CF.sub.3CO.sub.2 were used in titrations. Here we use ExBox.sup.4+ and XCage.sup.8+ for short. All of the solutions were prepared in spectroscopic grade solvents and equilibrated for 24 h at room temperature before use. All of the titrations were independently duplicated—shown below is one set of titration isotherms—and all isotherm fittings were used to calculate the average K.sub.a and ΔH with relevant standard errors.
[0090] Isothermal titration was performed by TA Nano Isothermal Titration Calorimeter at 25° C. A hastelloy cell was used with an active cell volume 190 μL. The stirring speed was set at 150 rpm. Receptor and substrate solutions were prepared in Milli-Q water or MeCN and allowed to equilibrate overnight if necessary. In each titration experiment, 20-25 injections were performed with gradually decreased titration peaks until saturation is reached, at which point only heat of dilution was measured. After subtracting the heat of dilution, the resulting data were analyzed with NanoAnalyze software using a 1:1 binding model and plotted by Origin Lab 8.6 software.
Cell Imaging Studies
[0091] For all cell imaging experiments, XCage.8CF.sub.3CO.sub.2 was supplied for cell study; we use XCage.sup.8+ for short.
Cell Culture
[0092] MCF-7 cells (human breast adenocarcinoma cell line) obtained from American Type Culture Collection (ATCC, Rockville, Md., USA) was utilized for cell culture experiments. MCF-7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU/mL) and streptomycin (100 μg/mL) at 37° C. in the presence of air (95%) and carbon dioxide (5%).
MTT Assay
[0093] MCF-7 cells (2.5×10.sup.5 cells/ml, 100 μL) were seeded in each well of a flat bottomed 96-well plate and adhered overnight. PDI2 or PDI2⊂XCage.sup.8+ or XCage.sup.8+ in PBS (10 μL) was added to each well to achieve working concentrations and incubated for 24 h. After incubation, wells were washed with PBS and incubated with MTT (0.5 mg/ml in DMEM, 100 μL) for 4 h. Following incubation with MTT, media from each well was aspirated, and the resultant formazan crystals deposited on the plate were dissolved in 200 μL of dimethyl sulfoxide. Then the absorbance of each well was measured using a microplate reader at 560 nm. All the samples were analyzed in quadruplicates.
The percentage cell viability was then calculated using the formula
% cell viability=(OD of treated sample/OD of untreated sample)*100.
Cell Uptake Studies
[0094] MCF-7 cells (2×10.sup.5 cells/ml, 500 μL) were seeded in each well of a 24-well plate and adhered overnight. For time dependent uptake studies, each well was treated with 10 μM PDI2, XCage.sup.8+ or PDI2⊂XCage.sup.8+ or for 30 min, 1 h, 2 h, 4 h, or 6 h. For concentration dependent uptake studies, each well was treated 0.1 or 1 or 10 or 20 μM of PDI2⊂XCage.sup.8+ for 6 h. After each incubation, cells were washed twice with 1×PBS, trypsinized, and incubated with 50 μL of 1:100 Zombie Aqua fixable cell viability dye for 20 min at 4° C. Further cells were washed with 600 μL 1×PBS, spun at 400 relative centrifugal forces (rcf) for 5 min and cell pellets were resuspended in 200 μL of 2% paraformaldehyde prior to being analyzed using a BD Fortessa flow cytometer. Data analysis was performed using Cytobank software (Cytobank Inc). Cells were first gated for singlet events using FSC-A vs FSC-H, after which debris was excluded used FSC-A vs SSC-A. Cells gated as Zombie Aqua low were considered live cells, which were then analyzed for their median fluorescence intensity (MFI) in the PE-Cy5 channel for PDI2⊂XCage.sup.8+ fluorescence, representing the amount of PDI2⊂XCage.sup.8+ taken up by each cell.
Live Cell Confocal and Widefield Microscopy
[0095] MCF-7 cells (1×10.sup.5 cells/ml, 300 μL) were plated in each well of an 8-well chamber slide (ThermoFischer Scientific) and adhered overnight. PDI2 or PDI2⊂XCage.sup.8+ was added to each well and incubated for 6 h or 24 h. Cells were then washed with PBS, and stained with LysoTracker green (lysosome stain, 1:1000 dilution) or NucBlue™ Live ReadyProbes™ Reagent (nuclear stain, 1 drop) or Hoechst 33342 nucleic acid stain (1:2000 dilution from 10 mg/mL stock). Plated cells were imaged within a humidified chamber using a 63× oil-immersion objective on a SP5 Leica Confocal Microscope using HyD detectors and lasers or a Deltavision Core Elite with a DAPI excitation filter (381-399 nm), DAPI emission filter (em: 411-459 nm) and TRITC emission filter (em: 571-617 nm) at equivalent light levels and exposure time. Intensity of signal in cells was measured with FIJI/ImageJ software.
Tables
[0096]
TABLE-US-00001 TABLE 1 Binding Constants and Thermodynamic Data at 25° C..sup.a Ka/M.sup.−1 ΔG ΔH TΔS Entry Solvent Guest Fluorescence ITC kcal mo1.sup.−1 1 MeCN Perylene 5.0 × 10.sup.6 3.6 × 10.sup.6 −08.9.sup.c −06.9 2.0 2 MeCN PDI2 3.5 × 10.sup.9 ND.sup.b −13.0.sup.d −10.2 2.8 3 H.sub.2O Caffeine 1.2 × 10.sup.5 1.5 × 10.sup.5 −7.1.sup.c −8.6 −1.5 4 H.sub.2O PDI2 7.7 × 10.sup.10 ND.sup.b −14.8.sup.d −14.1 0.7 .sup.a The standard error is presented in Supporting Information. .sup.b Not determined. .sup.c Directly determined by ITC. .sup.d Estimated from fluorescence titrations
TABLE-US-00002 TABLE 2 Summary of Photophysical Properties in H.sub.2O Compound PDI2 PDI2 ⊂ XCage.sup.8+ λ.sub.abs (nm) 497/534 472/504/542 λ.sub.ex (nm) 461/502/529 470/502/540 λ.sub.em (nm) 542/586/633 554/598/648 logε 4.48 4.65 Φ.sub.f 0.04 0.63 τ (ns) 4.71 7.32
TABLE-US-00003 TABLE 3 Summary of Photophysical Properties in MeCN Compound PDI2 PDI2 ⊂ XCage.sup.8+ λ.sub.abs (nm) 456/484/520 472/504/543 λ.sub.ex (nm) 452/482/518 474/505/542 λ.sub.em (nm) 529/568/617 553/597/649 logε 4.91 4.71 Φ.sub.f 0.66 0.66 τ (ns) 4.47 3.30/8.34
REFERENCES
[0097] (1) Szwajkajzer, D.; Carey, J. Molecular and Biological Constraints on Ligand-Binding Affinity and Specificity. Biopolymers 1997, 44, 181-198. [0098] (2) Persch, E.; Dumele, O.; Diederich, F. Molecular Recognition in Chemical and Biological Systems. Angew. Chem. Int. Ed. 2015, 54, 3290-3327. [0099] (3) 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. [0100] (4) Liu, Y.; Zhao, W.; Chen, C.-H.; Flood, A. H. Chloride Capture Using a C—H Hydrogen Bonding Cage. Science. 2019, 365, 159-161. [0101] (5) 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. [0102] (6) 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. [0103] (7) 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. [0104] (8) Assaf, K. I.; Nau, W. M. Cucurbiturils: From Synthesis to High-Affinity Binding and Catalysis. Chem. Soc. Rev. 2015, 44, 394-418. [0105] (9) Barrow, S. J.; Kasera, S.; Rowland, M. J.; Del Barrio, J.; Scherman, O. A. Cucurbituril-Based Molecular Recognition. Chem. Rev. 2015, 115, 12320-12406. [0106] (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. [0107] (11) Li, D.-H.; Smith, B. D. Molecular Recognition Using Tetralactam Macrocycles with Parallel Aromatic Sidewalls. Beilstein J. Org. Chem. 2019, 15, 1086-1095. [0108] (12) Liu, W.; Samanta, S. K.; Smith, B. D.; Isaacs, L. Synthetic Mimics of Biotin/(Strept)Avidin. Chem. Soc. Rev. 2017, 46, 2391-2403. [0109] (13) Schreiber, C. L.; Smith, B. D. Molecular Conjugation Using Non-Covalent Click Chemistry. Nat. Rev. Chem. 2019, 3, 393-400. [0110] (14) Mako, T. L.; Racicot, J. M.; Levine, M. Supramolecular Luminescent Sensors. Chem. Rev. 2019, 119, 322-477. [0111] (15) Heinzmann, C.; Weder, C.; de Espinosa, L. M. Supramolecular Polymer Adhesives: Advanced Materials Inspired by Nature. Chem. Soc. Rev. 2015, 342, 342-358. [0112] (16) Wang, H.; Ji, X.; Li, Z.; Huang, F. Fluorescent Supramolecular Polymeric Materials. Adv. Mater. 2017, 29, 1606117. [0113] (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. [0114] (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. [0115] (19) Sarikaya, M.; Tamerler, C.; Jen, A. K.-Y.; Schulten, K.; Baneyx, F. Molecular Biomimetics: Nanotechnology through Biology. Nat. Mater. 2003, 2, 577-585. [0116] (20) Webber, M. J.; Langer, R. Drug Delivery by Supramolecular Design. Chem. Soc. Rev. 2017, 46, 6600-6620. [0117] (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. [0118] (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. [0119] (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. [0120] (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. [0121] (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. [0122] (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. [0123] (27) Dale, E. J.; Vermeulen, N. A.; Jurifek, 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. [0124] (28) Barnes, J. C.; Jurifek, 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. [0125] (29) Dale, E. J.; Vermeulen, N. A.; Thomas, A. A.; Barnes, J. C.; Jurifek, 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. [0126] (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. [0127] (31) Yoshizawa, M.; Catti, L. Bent Anthracene Dimers as Versatile Building Blocks for Supramolecular Capsules. Acc. Chem. Res. 2019, 52, 2392-2404. [0128] (32) Yamashina, M.; Tsutsui, T.; Sei, Y.; Akita, M.; Yoshizawa, M. A Polyaromatic Receptor with High Androgen Affinity. Sci. Adv. 2019, 5, 1-8. [0129] (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. [0130] (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. [0131] (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. [0132] (36) Sun, M.; Mullen, K.; Yin, M. Water-Soluble Perylenediimides: Design Concepts and Biological Applications. Chem. Soc. Rev. 2016, 45, 1513-1528. [0133] (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. [0134] (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. [0135] (39) Barnes, J. C.; Jurifek, M.; Vermeulen, N. A.; Dale, E. J.; Stoddart, J. F. Synthesis of Ex.sup.nBox Cyclophanes. J. Org. Chem. 2013, 78, 11962-11969. [0136] (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. [0137] (45) Lefebvre, C.; Rubez, G.; Khartabil, H.; Boisson, J. C.; Contreras-Garcia, 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. [0138] (46) Lu, T.; Chen, F. Multiwfn: A Multifunctional Wavefunction Analyzer. J. Comput. Chem. 2012, 33, 580-592. [0139] (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. [0140] (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. [0141] (50) Ringe, D.; Petsko, G. A. How Enzymes Work. Science. 2008, 320, 1428-1429. [0142] (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. [0143] (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. [0144] (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. [0145] (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. [0146] (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. [0147] (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. [0148] (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. [0149] (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. [0150] (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. [0151] (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. [0152] (65) Butkevich, A. N.; Lukinavicius, 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. [0153] (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. [0154] (67) Weil, T.; Vosch, T.; Hofkens, J.; Peneva, K.; Mullen, K. The Rylene Colorant Family-Tailored Nanoemitters for Photonics Research and Applications. Angew. Chem. Int. Ed. 2010, 49, 9068-9093. [0155] (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. [0156] (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. [0157] (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. [0158] S1. Jiao, Y., Liu, K., Wang, G., Wang, Y., and Zhang, X. Supramolecular Free Radicals: Near-Infrared Organic Materials with Enhanced Photothermal Conversion. Chem. Sci. 2015, 6, 3975-3980. [0159] S2. Ryan, S. T. J., Del Barrio, J., Ghosh, I., Biedermann, F., Lazar, A. I., Lan, Y., Coulston, R. J., Nau, W. M., and Scherman, O. A. Efficient Host-Guest Energy Transfer in Polycationic Cyclophane-Perylene Diimide Complexes in Water. J. Am. Chem. Soc. 2014, 136, 9053-9060. [0160] S3. Dolomanov, O. V, Bourhis, L. J., Gildea, R. J., Howard, J. A. K., and Puschmann, H. OLEX2: A Complete Structure Solution, Refinement and Analysis Program. J. Appl. Crystallogr. 2009, 42, 339-341. [0161] S4. Sheldrick, G. M. SHELXT-Integrated Space-Group and Crystal-Structure Determination. Acta Crystallogr. Sect. A. 2015, 71, 3-8. [0162] S5. Sheldrick, G. M. A Short History of SHELX. Acta Crystallogr. Sect. A. 2008, 64, 112-122. [0163] S6. Thorn, A., Dittrich, B., and Sheldrick, G. M. Enhanced Rigid-Bond Restraints. Acta Crystallogr. Sect. A. 2012, 68, 448-451. [0164] S7. Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A., Cheeseman, J. R., Scalmani, G., Barone, V., Petersson, G. A., Nakatsuji, H., et al. 2016, Gaussian 16. [0165] S8. Dennington, R., Keith, T. A., and Millam, J. M. 2019, GaussView, Version 6.1. [0166] S9. Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., and Ferrin, T. E. UCSF Chimera—A Visualization System for Exploratory Research and Analysis. J. Comput. Chem. 2004, 25, 1605-1612. [0167] S10. Schneider, C. A., Rasband, W. S., and Eliceiri, K. W. NIH Image to ImageJ: 25 Years of Image Analysis. Nat. Methods 2012, 9, 671-675. [0168] S11. Dale, E. J., Vermeulen, N. A., Thomas, A. A., Barnes, J. C., Juricek, 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. [0169] S12. Lefebvre, C., Rubez, G., Khartabil, H., Boisson, J. C., Contreras-Garcia, J., and 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. [0170] S13. Brouwer, A. M. Standards for Photoluminescence Quantum Yield Measurements in Solution (IUPAC Technical Report). Pure Appl. Chem. 2011, 83, 2213-2228. [0171] S14. Hargrove, A. E., Zhong, Z., Sessler, J. L., and Anslyn, E. V. Algorithms for The Determination of Binding Constants and Enantiomeric Excess in Complex Host: Guest Equilibria Using Optical Measurements. New J Chem. 2010, 34, 348-354. [0172] S15. Thordarson, P. Determining Association Constants from Titration Experiments in Supramolecular Chemistry. Chem. Soc. Rev. 2011, 40, 1305-23. [0173] S16. 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. Nat. Acad. Sci. U.S.A. 2007, 104, 20737-20742.