M6L12 Nanospheres with Multiple C70 Binding Sites for 1O2 Formation in Organic and Aqueous Media

Singlet oxygen is a potent oxidant with major applications in organic synthesis and medicinal treatment. An efficient way to produce singlet oxygen is the photochemical generation by fullerenes which exhibit ideal thermal and photochemical stability. In this contribution we describe readily accessible M6L12 nanospheres with unique binding sites for fullerenes located at the windows of the nanospheres. Up to four C70 can be associated with a single nanosphere, presenting an efficient method for fullerene extraction and application. Depending on the functionality located on the outside of the sphere, they act as vehicles for 1O2 generation in organic or in aqueous media using white LED light. Excellent productivity in 1O2 generation and consecutive oxidation of 1O2 acceptors using C70⊂[Pd6L12], C60⊂[Pd6L12] or fullerene soot extract was observed. The methodological design principles allow preparation and application of highly effective multifullerene binding spheres.


■ INTRODUCTION
Singlet oxygen, an electronically excited form of oxygen, has numerous applications in synthetic chemistry, 1,2 purification, 3 and pharmacology 4−6 due to its strong oxidizing properties. 7uring the last decades, many synthetic protocols were developed based on the reactivity of 1 O 2 with C−H bonds, C�C double bonds, aromatic systems, and heteroatoms (Figure 1). 1 Singlet oxygen finds major application in the clinical photodynamic therapy treatment (PDT) of tumors in which oxidative stress caused by 1 O 2 leads to cell damage or cell death (Figure 1). 6Generation of singlet oxygen can be achieved via different methods.Apart from stoichiometric chemical reactions, photochemical excitation of an endogenous photosensitizer and transfer of its excitation energy describe one of the most common methods of 1 O 2 generation (Figure 1).Classically, organic dyes, such as rose bengal or methylene blue, are applied as a photosensitizer. 8,9Clinical trials using these photosensitizers in PDT are currently pursued; 10 however, these conventional dyes are prone to chemical, photoinduced or enzymatic degradation, limiting their application in vivo and lowering their overall efficiency in synthetic chemistry. 11These major challenges related to PDT can be circumvented using fullerenes, which exhibit ideal stability and good absorbance in the visible light, for photochemical generation of 1 O 2 (Figure 1). 12,13However, application of fullerenes for in vivo 1 O 2 generation or for oxidation reactions for synthetic purposes is often hampered by their poor solubility in most solvents, including water.Therefore, there is an interest in structures that bind fullerene to allow fullerene application in a wide variety of media.Common design features of supramolecular structures that bind fullerene include the use of π surfaces that allow good interaction with the aromatic surface of the fullerene.With that in mind, coordination-based self-assemblies with fullerene binding capability can be separated into three different design types (Figure 2).−17 These tweezers typically consist of two aromatic surfaces which are connected by either coordination chemistry or by covalent bonds.
A second type are those with a sandwich type arrangement.−34 Binding multiple fullerenes to a single sphere can not only enhance the fullerene extraction efficiency of the spheres, but can also lead to useful electronic and spectroscopic properties for catalytic applications or preparation of functional materials (such as electron storage devices). 33,35o further boost the widespread application of fullerenes, easily accessible and robust structures that effectively bind fullerenes are highly desirable.Here, we present a straightforward strategy to prepare cubic M 6 L 12 nanospheres that have four independent binding sites for fullerene, which can be readily prepared from commercial materials (Figure 2).We introduce a new design in which fullerene binding occurs at the windows of the self-assembled structure, leading to efficient binding under various conditions.Depending on the structure of the applied building blocks, high binding affinities for fullerenes are realized, leading to novel materials which bear up to four C 70 bound to a single nanosphere.The application of functionalized building blocks used for the self-assembly result in nanospheres with various exo-functionalization, enabling the binding of fullerene in various organic solvents and even in water.An exploration of their ability to produce 1 O 2 in a variety of media and subsequent oxidation of 1 O 2 acceptors revealed high productivity using C 70 ⊂[M 6 L 12 ] or materials in which fullerenes were directly extracted from fullerene soot using [M 6 L 12 ].The availability of the herein reported nanospheres together with their ability in 1 O 2 generation in various media allow for a more efficient, sustainable application in organic synthesis.The general design principles provide a useful strategy for the construction of novel water-soluble fullerene-binding cages, which are potentially suitable for PDT.With the general simple design principles, we hope to inspire further development of multiple-fullerene binding structures and their widespread applications.

■ RESULTS AND DISCUSSION
Inspired by a fullerene binding system developed by Mukherjee and Stang 36 and by square shaped Pd 6 L 12 nanospheres developed by Fujita, 37 we designed four different building blocks with similar dibenzofuran/carbazole cores.Two of the building blocks L acetylO and L O were chosen in order to study the influence of the rigidity and sphere size on fullerene binding properties (Figure 3).Both are easily obtained in a one-step procedure via Sonogashira or Suzuki cross-coupling from 2,6-dibromo-dibenzofurane in excellent yields (section S1).Two other types of building blocks were derived from carbazole L N and L PEGPy (Figure 3).−40 L N has an extra benzene moiety to potentially increase the π−π interactions between the host and the guest and to provide better solubility in organic solvents.L PEGPy has a hydrophilic group attached to the ligand, making it suitable for the preparation of water-soluble nanospheres.All herein presented ligands have a dihedral angle of ∼90°between the pyridine donors and should therefore form Pd 6 L 12 spheres upon coordination with palladium, as has been shown before for L acetylO and L O . 38,42phere formation was performed by mixing 1 equiv of L N with 0.6 equiv [Pd(BF 4 ) 2 (MeCN) 4 ] and 5 mol % PdCl 2 (MeCN) 2 as catalyst in dimethyl sulfoxide (DMSO) at 100 °C for 24 h according to a previously reported procedure 41 (Figure 4A).After this period, one clear set of protons was observed in the 1 H NMR spectrum of this solution, implying the formation of a highly symmetrical structure (Figure 4C).A downfield shift of the pyridyl protons was observed in accordance to coordination to palladium (signal a and b, Figure 4C).Diffusion ordered NMR (DOSY) displayed one signal corresponding to a hydrodynamic radius of 2 nm in line  , which is the nanosphere based on a ditopic ligand with only aromatic rings, leads to a color change of the solution from colorless to red-brown.In line with this, an additional absorption was observed in the UV−vis spectrum between 400 and 500 nm, which is characteristic for C 70 (Figure S33).12 ] giving the most pronounced signal with a distribution around this stoichiometry (Figure S46 and Figure 5C).Interestingly, the highest peaks in the MS spectra are those of the host−guest complex with a stoichiometry of 1:4, with only small peaks corresponding to (C 70 ) 5 ⊂[Pd 6 L N 12 ].The nanosphere has in total eight pockets which are available for fullerene binding (Figure 4A, discussion on MS distribution can be found in the Supporting Information, section S8).However, fullerene binding to a pocket withdraws electron density from the adjacent aromatic linkers of the nanosphere and possibly bends the linker framework toward the bound fullerene.As a result, the empty pockets adjacent to those that bind a fullerene may therefore bind with lower affinity.Therefore, while the sphere consists of eight binding pockets, it contains only four independent binding pockets (Figure 5A).Our MS experiments show that four binding pockets are occupied by C 70 in the [Pd 6 L N 12 ] nanosphere (as displayed in Figure 5A, the found 1:5 will be discussed later).Interestingly, also mixtures of C 60 and [Pd 6 L N 12 ] displayed a color change to brown. 13C NMR displayed a signal which can be attributed to C 60 (Figure 5B).Also, ESI-MS analysis of the solution displayed multiple species with (C 60 ) 1 ⊂[Pd 6 L N 12 ] being the most present species (Figure S43 5C).These experiments suggest that both the higher electron density and extra aromatic rings on the ditopic ligand building block (L N ) located at the building block contribute significantly to better binding of the fullerene guest.
Next to qualitative analysis of the fullerene-sphere host guest complexes using MS analysis, their binding constants were determined by UV−vis titrations (Figure 5E; for details and elaborate discussion see section S3).Due to the solubility limitation of fullerenes in DMSO, stock solutions of fullerene in toluene were used for these titrations.While the binding may be affected by the presence of toluene, the binding constants obtained provide a relative binding affinity and a lower limit of the binding constant.Upon addition of the C 70 (or C 60 ) fullerene (in toluene) to a solution of the sphere (in DMSO), changes in the UV−vis spectra are observed.The main absorption corresponding to the spheres (374 nm for [Pd 6 L N 12 ]/[Pd 6 L PEGPy 12 ] and 320 nm for [Pd 6 L O 12 ]) decreased, whereas signals associated with the fullerene increased (Figure 5D).As discussed previously, Pd 6 L 12 nanospheres are multivalent receptors for fullerenes with four independent binding pockets (Figure 5A).As a starting point, we fitted the obtained binding curves of the titration of C 70 to [Pd 6 L N 12 ] using a noncooperative 1:4 or 1:3 model.This gave a binding curve with a large error (20%), a sigmoidal shaped curve and large covariances (Figures S19−S21), implying that a noncooperative 1:4 model is not a good description of the system under diluted UV−vis conditions. 42As the binding in the presence of toluene as cosolvent may be weaker, we anticipated low contributions of the third and fourth binding at the low concentrations typically used for UV−vis.When we fitted the binding curve in a noncooperative 1:2 model in order to determine the binding strength between C 70 and [Pd 6 L N 12 ], a better fit was obtained with a lower error (6%) and lower covariances (Figure S23, for elaborate discussion see section S3).Therefore, we employed a 1:2 binding model instead of the 1:4 model for a rough estimation of all binding constants.All binding constants were obtained in good accuracy (error <10%).In agreement with our MS distribution analysis, [Pd 6 L N 12 ] showed the highest binding constant for C 70 (  .In summary, dibenzofurane and carbazole moieties as part of sphere forming building blocks generate nanospheres that allow fullerene binding.Fully aromatic building blocks show better binding than elongated (acetylene linked) ones.Their binding ability can easily be improved by increasing the electron density of the aromatic group at the building block (carbazole > dibenzofurane).The binding can be further increased by the introduction of extra aromatic moieties on the carbazole nitrogen (L N > L PEGPy ).
Computational Investigation of Binding.To get further structural insights into the binding stoichiometry of C 70 to [Pd 6 L N 12 ], we studied the complex in silico using molecular dynamics (MD).Our MD models were parametrized following our previously developed protocols. 43odel environments were constructed to feature Pd 6 L N 12 and 0−8 C 70 positioned randomly within the cage using ProFit. 44These structures were annealed in explicitly solvated MD simulations (2000 molecules DMSO, 12 molecules BF 4 − ) for 50 ns at 300 K. Annealed structures were then optimized, and association enthalpies (ΔH) were estimated by a MMGBSA approach (a technique for estimating the energy of association from energy differences due to host/guest interaction) (Figure 6A, black trace). 45These simulations showed that C 70 bound preferentially in the windows of [Pd 6 L N 12 ] (Figure 6B) due to the fitting size.While the first C 70 binding is enthalpically unfavorable (ΔH 1 = 1.30kcal• mol −1 ), associations of up 2−6 C 70 guests is enthalpically favored with an optimum of four guest molecules per cage (ΔH 4 = −2.48kcal•mol −1 ) in line with our HRMS results (Figure 6A, red trace).This preference for multiple guest binding (2−6 C 70 ) arises from favorable guest−guest interactions (π−π stacking) within the capsule.When 3−4 fullerenes are associated with the windows of a sphere, a π-rich binding site is created on the interior space of the sphere, facilitating the further association of a fifth C 70 (Figure 6C, Figure S45).We anticipate this π-rich environment may facilitate the encapsulation of guest substrate molecules as a biomimetic active site, benefiting photocatalytic applications (see discussion S10).These calculations provide a good explanation why we observe mostly a 4:1 complex by ESI-MS from samples in which the fullerene was extracted using nanosphere solutions in DMSO.As the binding constants were obtained from titration experiments carried out in toluene− DMSO mixtures, quantitative comparison of these data is difficult.
Photocatalytic Formation of 1 O 2 .Although fullerenes have ideal photostability and efficiency in 1 O 2 generation, their broad applicability in singlet oxygen generation is limited due to their limited solubility (Table 1, right).Typically, only rather apolar solvents such as benzene and chloroform allow for sufficient concentrations of fullerene.Therefore, substrates which do not dissolve in these rather apolar solvents cannot be efficiently oxidized using fullerene-mediated photogenerated 1 O 2 .To extend the application of fullerenes to water and polar solvents, which are generally suitable for many organic compounds and materials, fullerene-binding spheres can act as vehicles which allow solubility in these solvents.

Figure 1 .
Figure 1.Schematic picture of the mechanism of photochemical generation of singlet oxygen by fullerene.

Figure 2 .
Figure 2. Illustration of fullerene binding hosts based on coordination driven self-assembly (top).Design strategy for a multiple-fullerene binding assembly (bottom).

Figure 3 .
Figure 3. Structure of the herein investigated ditopic ligand building blocks used for the preparation of Pd 6 L 12 nanospheres.

13 C
NMR displayed one new set of signals which can be attributed to C 70 , indicating the presence of C 70 in solution, as a result of binding to [Pd 6 L O 12 ] (Figure S26).ESI-MS analysis of solutions containing [Pd 6 L O 12 ] and C 70 displayed a range of signals corresponding to host−guest complexes (Figure S31).The most dominant species was attributed to (C 70 ) 1 ⊂[Pd 6 L O 12 ] with a distribution around this main species (Figure 5C).For C 60 and [Pd 6 L O 12 ], a slight color change was observed (with a weak absorption above 350 nm). 13C NMR showed the presence of C 60 in solution (Figure S26).Furthermore, ESI-MS analysis displayed a range of signals corresponding to C 60 ⊂[Pd 6 L O 12 ] (Figure S28).Compared to a reaction mixture with C 70 and [Pd 6 L O 12 ], the spectroscopic features and the peaks in the MS spectra attributed to C 60 bound to [Pd 6 L O 12 ] were less intense, indicating a weaker affinity of the sphere for fullerene C 60 than for C 70 .On the basis of these initial results that suggest stronger binding of fullerenes to nanospheres based on ligand building blocks containing aromatic rings only, that is, the absence of the acetylene bridge between the aromatic units in the building block (L acetylO ), we next investigated the binding to the nanosphere based on the carbazole building block without any acetylene linkers.Stirring a mixture of solid C 70 and a DMSO solution of [Pd 6 L N 12 ] resulted in a color change from light yellow to dark brown/red.An additional absorption between 400 and 500 nm appeared in the UV−vis spectrum indicative of C 70 binding (Figure S50). 13C NMR displayed all signals corresponding to the [Pd 6 L N 12 ] nanosphere and signals which can be attributed to C 70 (Figure 5B).ESI-MS analysis of the solution displayed multiple species with (C 70 ) 4 ⊂[Pd 6 L N

Figure 4 .
Figure 4. Characterization of Pd 6 L N 12 .(A) Reaction conditions for formation of nanospheres.Molecular structure of the displayed sphere was minimized at the PM3 level.Carbon is displayed in yellow, nitrogen in blue, palladium as orange spheres.(B) Overlayed DOSY NMR of the [Pd 6 L N 12 ] sphere (blue) and the building block (red).(C) 1 H NMR spectra of [Pd 6 L N 12 ] sphere and the corresponding building block.(D) ESI-MS spectrum of [Pd 6 L N 12 ].
and Figure 5C).The lower amount of C 60 associated with [Pd 6 L N 12 ] (according to ESI-MS analysis, Figure 5C) than C 70 indicates a stronger binding for C 70 over C 60 .Similar studies using the [Pd 6 L PEGPy 12 ] nanosphere showed that a mixture of host−guest complexes formed, with a different number of C 70 bound to the sphere, as judged by the MS data (Figure S56).The species with 1 or 2 C 70 per nanosphere were dominant as indicated by ESI-MS distribution analysis (Figure 5C).The average number of fullerenes C 70 bound to a single [Pd 6 L PEGPy 12 ] sphere is 1.5 C 70 and is in between the average number of C 70 bound to [Pd 6 L N 12 ] (3.5 C 70 ) and to [Pd 6 L O 12 ] (1 C 70 ) (Figure 2.6 ± 0.16 × 10 6 M −1 ) and [Pd 6 L O 12 ] binds C 70 the weakest (3.4 ±

Figure 5 .
Figure 5. Fullerene binding assay of Pd 6 L 12 nanospheres.(A) Reaction conditions for formation of host−guest complexes.Molecular structure of the displayed assembly was minimized at the PM3 level.Carbon is displayed in yellow, nitrogen in blue, palladium as orange spheres, and fullerene C 70 as white spheres.(B) 13 C NMR spectra of [Pd 6 L N 12 ] nanosphere and the corresponding fullerene adducts.(C) Distribution of fullerenes bound to different types of nanospheres based on ESI-MS analysis.(D) Example of an UV−vis titration of C 70 to a solution of [Pd 6 L N 12 ].Inset: 1:2, H/G binding fit on changes of two different wavelengths.(E) Binding constant of fullerene to different types of spheres obtained by UV−vis titrations.
Given the strong binding between the fully aromatic spheres [Pd 6 L N 12 ], [Pd 6 L O 12 ], and [Pd 6 L PEGPy 12 ] with fullerenes, their application in singlet oxygen generation in different solvents and consecutive oxidation of model substrates was studied.First, the conversion of anthracene (which is a well-known aromatic

Figure 6 .
Figure 6.Computational investigation on C 70 binding of [Pd 6 L N 12 ] using molecular dynamics (MD).(A) Display of averaged total association enthalpies for different amount of C 70 bound to a single sphere and the obtained distribution of C 70 associated with [Pd 6 L N 12 ] using the MS analysis.(B) Optimized structure of four C 70 associated with a single sphere, displaying the window binding motif.(C) Optimized structure of 5 C 70 associated with a single sphere, displaying the creation of a hydrophobic interior binding site for the fifth C 70 .

Table 1 .
Oxidation of Organic 1 O 2 Acceptors by Light Induced Singlet Oxygen Formation in Different Media a Standard condition: sphere, 4.16 nmol, substrate 20 μmol in 1 mL solvent, 4 h, room temperature; reactions performed in quartz containers located 2 cm away from a white LED light source.b Conversion and turnover number (TON) based on nanosphere amount was determined by 1 H NMR using mesitylene as internal standard.c N-(tert-Butoxycarbonyl)-L-methionine (20 μmol) was used as substrate.d C 70 16.6 nmol dissolved in 10 μL of toluene and 1 mL of cosolvent (described in the table).e Free C 70 was added as a solid.