Water-soluble host–guest complexes between fullerenes and a sugar-functionalized tribenzotriquinacene assembling to microspheres

A sugar-functionalized water-soluble tribenzotriquinacene derivative bearing six glucose residues, TBTQ-(OG)6, was synthesized and its interaction with C60 and C70-fullerene in co-organic solvents and aqueous solution was investigated by fluorescence spectroscopy and ultraviolet-visible spectroscopy. The association stoichiometry of the complexes TBTQ-(OG)6 with C60 and TBTQ-(OG)6 with C70 was found to be 1:1 with binding constants of Ka = (1.50 ± 0.10) × 105 M−1 and Ka = (2.20 ± 0.16) × 105 M−1, respectively. The binding affinity between TBTQ-(OG)6 and C60 was further verified by Raman spectroscopy. The geometry of the complex of TBTQ-(OG)6 with C60 deduced from DFT calculations indicates that the driving force of the complexation is mainly due to the hydrophobic effect and to host–guest π–π interactions. Hydrophobic surface simulations showed that TBTQ-(OG)6 and C60 forms an amphiphilic supramolecular host–guest complex, which further assembles to microspheres with diameters of 0.3–3.5 μm, as determined by scanning electron microscopy.

In the past years, the recognition of the biological and pharmaceutical relevance of fullerenes, such as their photodynamic activity, phototoxicity, HIV-1 protease inhibitor ability, and oxidative stability, has promoted the exploration of water-soluble fullerenes for biological use [34,35]. The extremely hydrophobic nature of fullerenes requires a strongly hydrophilic supramolecular host to achieve water solubility. It is important to note that host-guest research on TBTQ derivatives with fullerenes has been limited to organic media so far because of the poor solubility of their complexes in aqueous media. Therefore, the design, synthesis, and exploration of water-soluble TBTQ derivatives offer attractive possibilities for broadening the applications of the TBTQ structural motif. In the work presented here, we have introduced sugar motifs that possess good water solubility and biocompatibility [36][37][38] at the six outer peripheral positions of the TBTQ framework to provide, for the first time, a water-soluble TBTQ-based host bearing an extended cavity. The complexation of this host with C 60 and C 70 -fullerene was investigated in co-organic solvents and in aqueous solution. The hydrophobic surface simulation of this host indicated the formation of a supra-amphiphilic system, which, as shown by scanning electron microscopy, further self-assembles into microspheres in the solid state. It is shown that the inclusion of fullerenes into the water-soluble TBTQ-based host greatly compensates for their water-repulsive nature and results in the formation of self-assembled microspheres that may have some potential for biological and pharmaceutical applications.
Structural characterization. All synthesized compounds were fully characterized by 1 H and 13 C NMR spectroscopy and mass spectrometry (see Supporting Information File 1) and the data were found to be consistent with the proposed structures. For the hexakis-propargyl ether TBTQ-(OP) 6 , the signals at δ = 2.53 ppm in the 1 H NMR spectrum and at δ = 78.98 and 76.07 ppm in the 13 C NMR spectrum were attributed to the acetylene protons and carbons, respectively. The electrospray ionization (ESI) mass spectrum showed the most intense peak at m/z 641.1923, which corresponds to the [M + Na] + molecular adduct ion and matches well with the calculated value (m/z 641.1935, Δ = −1.8 ppm). In addition, the corresponding [M + K] + ion appears at m/z 657.1709 (Δ = + 5.3 ppm). The 1 H NMR and 13 C NMR spectra of compound TBTQ-(OAcG) 6 dissolved in DMSO-d 6 are noteworthy because they reflect the diastereotopic environment of the different molecular building blocks. For example, the 1 H NMR spectrum of TBTQ-(OAcG) 6 exhibits two singlet resonances at δ = 8.62 and δ = 8.58 ppm, each of which indicates three equivalent protons of the six triazole rings. Likewise, two singlets at δ = 7.33 and δ = 7.30 ppm are due to two sets of three equivalent arene protons of the TBTQ core [40]. The acetyl protons of the protected glucose residues appear as eight distinct resonances. The 13 C NMR spectrum shows a similar splitting. The triazole carbons resonate at δ = 123.71 and δ = 123.69 ppm and at δ = 143.88 and δ = 143.79 ppm, indicating two sets of magnetically nonequivalent linkers. This is clearly a consequence of the prochiral nature of the TBTQ core. We also note that such splitting phenomenon was reported for neither six-fold sugar-functionalized cyclotriveratrylenes [41], which are directly comparable to TBTQ-(OAcG) 6 , nor for six-fold sugar-functionalized triptycene [42], and ten-fold sugar-functionalized pillar [5]arene [43]. Temperature-dependent 1 H NMR spectroscopy of TBTQ-(OAcG) 6 in DMSO-d 6 was carried out in the range of 20-70 °C and revealed a slight decrease of the splitting of the aromatic proton resonances with increasing temperature, but no coalescence. While most of the resonances did not shift significantly, the triazole pair of singlets was shifted to higher field by Δδ = −0.14 ppm (see Figure S2 in Supporting Information File 1). All these observations reflect the molecular C 3 -symmetry of TBTQ-(OAcG) 6 in solution and the presence of two diastereotopic sets of three magnetically equivalent tentacles at the rigid, prochiral TBTQ skeleton.
The mass spectrometric characterization of TBTQ-(OAcG) 6 turned out to be quite difficult. The MALDI-(+) mass spectrum (see Figure S10 and Table S1 in Supporting Information File 1), recorded with α-cyano-4-hydroxycinnamic acid (CHCA) as a matrix, exhibits the base peak at m/z 2879 along with an adjacent intense peak at m/z 2896, which are assigned to the [M + Na] + and [M + K] + molecular adduct ions, respectively. Unfortunately, attempts to perform accurate mass measurements were unsuccessful. However, the MALDI mass spectrum also shows the characteristic losses of up to at least three tentacle residues from both the [M + Na] + and [M + K] + molecular ions [44][45][46]. In contrast to the MALDI mass spectrum, the high-resolution ESI-(+) mass spectrum of TBTQ-(OAcG) 6 (see Figure S11 and Table S2) exhibits a sole peak group with the maximum component at m/z 1505.9690 and Δ(m/z) = 0.5, indicating the presence of doubly charged ions. The most intense peak is assigned to the doubly charged [M + 1] isotopolog of the molecular adduct [M + 6H 2 O + 2Na] 2+ , the theoretical value of which is calculated to be m/z 1505.9611 (Δ = + 5.2 ppm). The surprising presence of six equivalents of water points to the formation of strong hydrogen bonds between the sugar ester bonds of TBTQ-(OAcG) 6 and the water molecules as guests. However, the origin of this intriguing observation associated with the attachment of just two sodium cations requires further studies. Despite the unusual mass spectrometric behavior of the compound, the combined spectroscopic evidence strongly supports the identity of TBTQ-(OAcG) 6 .
After deprotection of the glucose units, the acetyl signals disappeared in the 1 H and 13 C NMR spectra of the target compound TBTQ-(OG) 6 . The 1 H NMR spectrum showed characteristic resonances for the TBTQ core, the six triazole rings and the six glucose units but also some broadened signals. In contrast to the acetylated precursor, splitting of the proton signals is almost completely absent. At first glance, the 13 C NMR spectrum exhibited only 14 of the 15 resonances expected for a C 3 -symmetric structure. However, a closer inspection revealed again a tiny splitting of several resonances, e.g., of those of the triazole rings at δ = 124.21 and δ = 124.24 ppm and at δ = 142.80 and δ = 142.82 ppm, but also for those of the benzene rings of the TBTQ core. Even the six resonances of the glucose carbons appear to split into two signals (e.g., at δ = 79.96 and δ = 79.98 ppm. Temperature-dependent 1 H NMR spectroscopy of TBTQ-(OG) 6 again revealed a similar upfield shift of the triazole resonance as that observed for the precursor (Δδ = −0.13 ppm). Even more significant upfield shifts were found for the resonances of the glucose protons with exception of the doublet of the glycosidic protons.
The MALDI-(+) mass spectrum of TBTQ-(OG) 6 (see Figure  S14 and Table S3 1871 and m/z 1887, respectively, in analogy to the spectrum of the precursor compound. Also, characteristic fragment ions peaks of minor intensity appear m/z 1709 and m/z 1628, indicating the elimination of a glucose unit as C 6 H 10 O 5 (162 u) and, respectively, the loss of the entire tentacle as an C 9 H 14 N 3 O 5 · radical (244 u) from the [M + Na] + ion followed by H atom transfer. Again, accurate mass measurements were not obtained. As another surprise, the high-resolution ESI-(−) mass spectrum of TBTQ-(OG) 6 (see Figure S15 and Table S4 Complexation of TBTQ-(OG) 6 with fullerenes. In order to investigate the host-guest relationship between TBTQ-(OG) 6 and fullerenes, fluorescence titration experiments were performed in toluene/DMSO 1:1 (v/v), in which both the host and the guest components could be dissolved, instead of in water. As shown in Figure 2, the spectrum of TBTQ-(OG) 6 exhibits an emission maximum peak at 329 nm upon excitation at 294 nm. This emission band is probably due to the formation of the groundstate dimer by interaction between the benzene rings upon excitation or excimer emission caused by the interaction between the aromatic rings [41,43]. As the concentration of the fullerenes C 60 and C 70 increase, the emission is significantly quenched, indicating the photoinduced energy transfer from TBTQ-(OG) 6 to the fullerenes [47,48]. Molar ratio plots (see Figure S16, Supporting Information File 1) on the basis of the fluorescence titration experiments suggested a 1:1 stoichiometric ratio of both TBTQ-(OG) 6 C 60 and TBTQ-(OG) 6 C 70 , and the association constants were calculated to be K a = (1.50 ± 0.10) × 10 5 M −1 and K a = (2.20 ± 0.16) × 10 5 M −1 , respectively, using a global nonlinear curve fitting method ( Figure 2) [49]. The slightly stronger association affinity of C 70 -fullerene may be attributed to its larger size and surface area as compared to C 60 -fullerene [50].
The complexation between TBTQ-(OG) 6 and fullerenes was also examined by UV-vis spectroscopy in both toluene/DMSO 1:1 (v/v) and water. Figure 3a shows the UV-vis spectra of TBTQ-(OG) 6 , C 60 and TBTQ-(OG) 6 C 60 in toluene/ DMSO. TBTQ-(OG) 6 absorbs at 297 nm and C 60 exhibits two absorption peaks at 298 nm and 333 nm. When C 60 was mixed with TBTQ-(OG) 6 in a 1:1 molar ratio, the absorption of C 60 at 298 nm was slightly shifted to 302 nm. Similarly, the absorption of C 70 at 298 nm was shifted to 301 nm after mixing the fullerenes with TBTQ-(OG) 6 in the same molar ratio (Figure 3b). In aqueous solution, TBTQ-(OG) 6 showed an absorption at 292 nm and, as expected, practically no absorption was observed for C 60 and C 70 due to their poor solubility (Figure 4a and Figure 4b). However, 1:1 molar mixture of TBTQ-(OG) 6 and C 60 in water exhibited an increased absorption at 292 nm and generated a new absorption band at 354 nm ( Figure 4a). This observation is attributed to the increased solubility of C 60 in water due to formation of the host-guest complex with TBTQ-(OG) 6 . A similar absorption behavior was observed for TBTQ-(OG) 6 C 70 (Figure 4b). The dispersibility of higher amounts of fullerenes in water in the presence of TBTQ-(OG) 6 was further assessed at different rest times after sonication for 10 min. As illustrated in Figure S17 (Supporting Information File 1), the pristine C 60 and C 70 -fullerenes precipitated rapidly within 5 min. In contrast, the TBTQ-(OG) 6 C 60 and TBTQ-(OG) 6 C 70 complexes showed improved water dispersibility of the fullerenes, which was maintained even after  20 days. The maximum solubility of C 60 in water was measured to be about 0.14 mg/mL in the presence of ten equivalents of TBTQ-(OG) 6 , while the maximum solubility of C 70 was about 0.17 mg/mL. Raman spectroscopy has proven to be a useful tool for the characterization of carbon nanomaterials [51,52]. The Raman spectra of TBTQ-(OG) 6 , C 60 and TBTQ-(OG) 6 C 60 are displayed in Figure 5. The TBTQ-(OG) 6 shows a weak peak at 1112 cm −1 . The pristine C 60 -fullerene shows characteristic peaks at 494 cm −1 (A g -breathing mode), 1467 cm −1 (A g -pentagonal pinch mode), as well as signals at 272 cm −1 and 1572 cm −1 (H g modes) [53]. The Raman spectrum of TBTQ-(OG) 6 C 60 clearly shows the presence of TBTQ-(OG) 6 and fullerene, and the slight shift of the peaks further indicates the successful complexation of TBTQ-(OG) 6 and C 60 . 6 C 60 in water. In spite of numerous attempts, we failed to obtain good-quality crystals to determine the binding conformation of complex TBTQ-(OG) 6 C 60 by X-ray diffraction. Therefore, the optimized geometry of the 1:1 complex of TBTQ-(OG) 6 C 60 in water was simulated by density functional theory (DFT) calculations at the B3LYP/6-31G(d) level of theory, which was completed with the aid of Molclus, MOPAC, and ORCA 4.1.0 programs [54][55][56]. As shown in Figure 6, C 60 -fullerene can be embedded between the six arms of TBTQ-(OG) 6 and the distance between the center of one benzene ring of the host and the closest five-membered ring of the guest was calculated to be 3.224 Å. The distances between the center of a triazole ring of TBTQ-(OG) 6 and the adjacent five and six-membered rings of C 60 were calculated to be 3.541 and 3.702 Å, respectively. These results suggest significant host-guest π-π interactions be-  tween TBTQ-(OG) 6 and C 60 [57]. In order to intuitively assess the hydrophilicity and hydrophobicity of the complex, the hydrophobic surface diagram was generated and is reproduced in Figure 6c. It is evident that TBTQ-(OG) 6 provides a strongly hydrophobic region at the inner bottom. Thus, the hydrophobic effect is assumed to be an important driving force for the formation of the complex TBTQ-(OG) 6 C 60 .

Simulations of complex TBTQ-(OG)
Surface morphologies. The surface morphologies of C 60 , TBTQ-(OG) 6 , and TBTQ-(OG) 6 C 60 were investigated by scanning electron microscopy (SEM). As shown in Figure 7a and 7b, respectively, the SEM image of C 60 -fullerene displays cylindrical nanotubes and that of TBTQ-(OG) 6 does not indicate any definite shape. However, well-dispersed microspheres with diameters of 0.3-3.5 μm were observed for the complex TBTQ-(OG) 6 C 60 . Obviously, these aggregates form by further assembly of the supra-amphiphilic host-guest systems, as suggested in Figure 6c, due to the hydrophobic interactions governing the association behavior of TBTQ-(OG) 6 C 60 in water.

Conclusion
In summary, we have successfully synthesized a six-fold sugarfunctionalized, water soluble tribenzotriquinacene derivative, TBTQ-(OG) 6 , which significantly improves the solubility of C 60 and C 70 -fullerenes in water. The target compound and its peracetylated precursor TBTQ-(OAcG) 6 have been characterized in detail by NMR spectroscopy and mass spectrometry. The host-guest complexation of TBTQ-(OG) 6 with C 60 and C 70 takes place in a 1:1 molar ratio in both cases and with association constants of K a = (1.50 ± 0.10) × 10 5 M −1 and K a = (2.20 ± 0.16) × 10 5 M −1 , respectively. It is suggested that the formation of the host-guest complexes is primarily due to hydrophobic effects and π-π interactions. The TBTQ-(OG) 6 C 60 complex was found to further assemble to microspheres with diameters of 0.3-3.5 μm. The inclusion complexation between TBTQ-(OG) 6 and fullerenes in aqueous solution may shed light on potential future applications of fullerenes in biological and pharmaceutical areas.

Experimental
General information. All commercially available reagents were used as received unless otherwise stated. Anhydrous solvents were collected from a Mikrouna Solv Purer G3 solvent purification system. The 1 H NMR and 13 C NMR spectra were recorded on a 400 MHz Bruker NMR spectrometer and chemical shifts were reported in ppm (δ). The fluorescence spectra were measured on a F97Pro fluorescence spectrophotometer (LengGuang Tech, Shanghai, China) and UV-vis spectra were measured on a UV-3300PC spectrometer (Mapada Instruments Co., Ltd, Shanghai, China). Mass spectra were recorded using either electrospray ionization (ESI) on a LCMS-IT-TOF instrument (Shimadzu, Kyoto, Japan) or a matrix-assisted laser desorption/ionization (MALDI) on a RapifleX MALDI Tissue-typer system (Bruker Daltonik GmbH, Bremen, Germany). Raman spectra were recorded on an inVia Reflex confocal Raman microscope (Renishaw plc, Wotton-under-Edge, UK) by dropping the sample solutions of TBTQ-(OG) 6 and TBTQ-(OG) 6 C 60 onto a slide glass with subsequent air drying. C 60 was tested in powder form on a slide glass. Ultrasonic mixing was performed with a 100 W ultrasonic cleaner. The surface morphology was investigated on a Phenom ProX scanning electron microscopy (SEM, Phenom World, Eindhoven, Netherlands). Samples were prepared from the freeze-dried aqueous solution/suspension of TBTQ-(OG) 6 , C 60 , and TBTQ-(OG) 6 C 60 , and then gold-sputtered prior to imaging. Freeze drying was conducted on a Scientz-18N freeze dryer (Scientz Biotech, Zhejiang, China). 6 . A mixture of TBTQ-(OH) 6 (2.17 g, 5.56 mmol), potassium carbonate (9.23 g, 66.8 mmol), and propargyl bromide (5.40 g, 44.5 mmol, 98 wt %) in acetonitrile (100 mL) was stirred and heated under a nitrogen atmosphere at 70 °C for 12 h. The mixture was then cooled to room temperature and the suspension was filtered, and washed with acetonitrile. The resulting filtrate was evaporated under vacuum to afford the crude product as yellow solid. Further recrystallization from dichloromethane/ethanol provided the pure product as pale-yellow solid ( Synthesis of TBTQ-(OG) 6 . To a solution of TBTQ-(OAcG) 6 (0.95 g, 0.33 mmol) in methanol (60 mL), sodium methoxide (about 1 mL) was added to adjust the pH value to 11. The mixture was stirred at room temperature for 12 h and then the suspension was filtered and washed with methanol. The filtrate was concentrated and purified by a Biogel P 2 column to give TBTQ-(OG) 6

Supporting Information
Supporting Information File 1 1 H NMR, 13 C NMR spectroscopy, and mass spectrometry of all new compounds, and the xyz coordinates (in Å) of the complex of TBTQ-(OG) 6 with C 60 .