Synthesis of Single-walled Carbon Nanotubes Coated with Thiol

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Synthesis of Single-walled Carbon Nanotubes Coated with Thiol-reactive Gel via Emulsion Polymerization Yukiko Nagai, Yusuke Tsutsumi, Naotoshi Nakashima, and Tsuyohiko Fujigaya J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b03873 • Publication Date (Web): 15 Jun 2018 Downloaded from http://pubs.acs.org on June 17, 2018

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Journal of the American Chemical Society

Synthesis of SingleSingle-walled Carbon Nanotubes Coated with ThiThiolol-reactive Gel via Emulsion Polymerization Yukiko Nagai,a Yusuke Tsutsumi,a Naotoshi Nakashima,b and Tsuyohiko Fujigaya*, a, b, c, d a

Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.

b

2

International Institute for Carbon Neutral Energy Research (WPI-I CNER), Kyushu University, Fukuoka 819-0395, Japan c

Japan Science and Technology Agency-Precursory Research for Embryonic Science and Technology (JST-PRESTO), 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan d

Center for Molecular Systems(CMS), Kyushu University, 744

Motooka, Nishi-ku, Fukuoka 819-0395, Japan.

KEYWORDS carbon nanotube, emulsion polymerization, click chemistry, thiol–ene reaction

ABSTRACT: Single-walled carbon nanotubes (SWNTs) have unique near-infrared absorption and photoemission properties that are attractive for in vivo biological applications such as photothermal cancer treatment and bioimaging. Therefore, a smart functionalization strategy for SWNTs to create biocompatible surfaces and introduce various ligands to target active cancer cells without losing the unique optical properties of the SWNTs is strongly desired. This paper reports the design and synthesis of a SWNT/gel hybrid containing maleimide groups, which react with various thiol compounds through Michael addition reactions. In this hybrid, the method called carbon nanotube micelle polymerization was used to non-covalently modify the surface of SWNTs with a cross-linked polymer gel layer. This method can form an extremely stable gel layer on SWNTs; such stability is essential for in vivo biological applications. The monomer used to form the gel layer contained a maleimide group, which was protected with furan in endo-form. The resulting hybrid was treated in water to induce deprotection via retro Diels–Alder reaction and then functionalized with thiol compounds through Michael addition. The functionalization of the hybrid was explored using a thiol-containing fluorescent dye as a model thiol and the formation of the SWNT–dye conjugate was confirmed by energy transfer from the dye to SWNTs. Our strategy offers a promising SWNT-based platform for biological functionalization for cancer targeting, imaging, and treatment.

INTRODUCTION INTRODUCTION Since Iijima discovered single-walled carbon nanotubes (SWNTs) for the first time in 1993,1 their characteristic 1-D structures together with their unique thermal, electrical, physical, and optical properties have been actively investigated for applications in materials science.2 In the field of biotechnology,2 SWNTs are attractive for use in photothermal therapy,3-5 drug delivery,6-7 biosensors,8-9 biomedical imaging,10-11 and tissue engineering.12-13 This is because of the combination of the transparency of biological tissue in the near-infrared (NIR) region14 and unique NIRresponsive properties of SWNTs, such as strong photoabsorption,15-16 photothermal conversion,17 and photoluminescence (PL).18-19 To use SWNTs for such applications, stable modification of hydrophobic SWNT surface to avoid the aggregation of SWNTs in aqueous media without losing their unique optical properties is required. In this regard, covalent modification20 is not suitable be-

cause it destroys the graphitic surface structure and suppresses the unique NIR properties of SWNTs, even though highly stable modification is possible.21 In contrast, non-covalent modification22-24 based on the physical adsorption of the modifying molecules onto the SWNT surface enables both the graphitic structure and optical properties of the SWNTs to be retained; thus, it is more suitable than covalent modification for biotechnology applications. However, the non-covalent approach often suffers from poor modification stability because of the dynamic replacement of the modifying molecules, often called a dispersant, on the SWNT surface.25-26 Recently, based on the previous interfacial engineering techniques of micelle-encapsulated SWNTs,27-29 we developed a unique high-yield non-covalent modification method for SWNTs, in which an ultrathin cross-linked polymer layer (gel layer) was formed by polymerization inside the surfactant micelle formed around SWNTs; this method is denoted as CNT micelle polymerization and is illustrated

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in Scheme 1.30 The gel layer formed around the SWNT stably protects the SWNT surface, so the SWNT optical properties are maintained. In addition, this method is applicable to a wide range of vinyl monomer structures,31 including polyethylene glycol methacrylate (PEGMA) and N-isopropylacrylamide, which is one of the most studied polymers in biological fields. Furthermore, this method enables us to prepare size-regulated SWNTs (~100–200 nm) using only ultracentrifugation,32 which is expected to provide SWNTs with an enhanced permeability and retention effect.33

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ture than the exo form44-45 without suffering from the hydrolysis of the maleimide even in water. By realizing the deprotection at low temperature without hydrolysis of the maleimide in water, the subsequent functionalization with thiol compounds via Michael addition becomes possible.

Scheme 2. Synthesis of endo- and exo-FpMMA

Scheme 1. Schematic of CNT Micelle Polymerization

RESULTS RESULTS AND DISCUSSION

In this study, by taking the above advantages of CNT micelle polymerization, we design a vinyl monomer contains a maleimide group that can conjugate with various ligands and biocompatible units for use as active targeting (endo form of furan-protected maleimide methacrylate; endo-FpMMA in Scheme 2). A maleimide group is used because of its high reactivity with thiol-containing molecules via Michael addition under ambient conditions in aqueous systems without the use of a catalyst.34 However, maleimide-containing vinyl monomers have been involved in the radical addition reaction to the maleimide double bond.35 Therefore, furan is introduced via Diels– Alder reaction to protect the maleimide. Recently, Sanyal and co-workers reported that polymers carrying furanprotected maleimide incorporated various thiol materials after deprotection via heat-mediated retro Diels–Alder reactions. However, the heating was conducted either in organic solvent (>100 °C in toluene)36-39 or under vacuum (400 °C),40 probably to avoid the hydrolysis of maleimide in an aqueous system at high temperature.41 Regarding the chemistry of the stereoisomers of furan-protected maleimide, the typically used thermodynamically controlled exo form was synthesized at high temperature (>90 °C) and deprotected at high temperature (>100 °C) because the Diels–Alder and retro Diels–Alder reactions are in equilibrium.42-43 Therefore, we expect that the thermodynamically unstable endo form synthesized at room temperature can be deprotected at lower tempera-

Synthesis of endo-FpMMA. Synthesis of the monomer (endo-form of furan-protected maleimide methacrylate; denoted as endo-FpMMA) was carried out as shown in Scheme 2. First, the maleimide group was protected by furan via Diels–Alder reaction at room temperature, which gave the endo form as a precipitate in 38% yield.46 The exo form of the furan-protected maleimide was also synthesized by reacting furan and maleimide at 90 °C (45% yield).46 In these conditions, endo and exo forms were selectively synthesized and their purity was clearly confirmed by 1H NMR, in which the characteristic proton peak due to bridgehead hydrogen appeared at 3.56-3.58 and 2.89 ppm for endo and exo form, respectively (Supporting Information, Figure S1a). In thermogravimetric analysis (TGA) of these materials, the large weight loss attributed to the deprotection of the endo form (ca. 122 °C) occurred at lower temperature than that of the exo form (ca. 150 °C) synthesized at higher temperature (Supporting Information, Figure S1b). In the second step, we obtained endo- and exo-FpMMA by coupling the endoand exo- form protected maleimide with PEGMA in anhydrous tetrahydrofuran (THF) via Mitsunobu reaction, respectively.46 1H NMR of endo- and exo-FpMMA clearly indicated the absence of their isomer by the absence of the characteristic isomer peaks at 3.54 (Figure 1) and 3.14 ppm (Supporting Information, Figure S12), respectively. To investigate the deprotection of endo-FpMMA via retro Diels−Alder reaction, endo-FpMMA was heated at 80 °C47 in D2O for 90 min and the reaction was monitored by 1 H NMR spectroscopy (Figure 1).37 In the NMR spectrum, the peaks from the furan cycloadduct at 6.49, 5.40, and 3.55 ppm (indicated by light green, pink, and light blue dots in Figure 1a, respectively) disappeared, and a peak at 6.89 ppm caused by the maleimide double bond (indicated by a red dot in Figure 1b) appeared. Other peaks consistent with the occurrence of side reactions such as hydrolysis of maleimide were not observed. The reaction was also supported by IR spectra, showing the clear ap-


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pearance of the =C-H vibration from maleimide at 695.94 cm-1 (blue line in Supporting Information, Figure S2a).48 In addition, in the ESI-MS profile, the periodic peaks due to the molecular distribution of PEG repeating unit (n=10 in average) exhibited identical shifts by m/z =68.0295 for all the peaks, corresponding the deprotected furan (m/z = 68.03) (Supporting Information, Figure S2b). In contrast, we found that exo-FpMMA showed no obvious changes at 80 °C (Supporting Information, Figure S3) and, at 100 °C, the hydrolysis of maleimide41 was accompanied by the deprotection of exo-FpMMA (Supporting Information, Figure S4). The successful protection/deprotection of endoFpMMA in aqueous media without hydrolysis enabled us to use endo-FpMMA for Michael addition with thiolcontaining compounds in water. We demonstrated the Michael addition reaction of maleimide using glutathione as a model thiol compound.37, 49-50 In the corresponding 1H NMR spectrum (Figure 1c), the peak at 6.89 ppm (indicated by a red dot) disappeared after adding glutathione and the characteristic methylene peaks of maleimide (indicated by an orange dot) and glutathione (indicated by a yellow dot) appeared,37 clearly revealing the successful conjugation of glutathione.

system.30 In CNT micelle polymerization, monomer(s) or oligomers together with a cross-linker penetrate into the surfactant-encapsulated SWNT surface upon radical polymerization to form a gel layer on the SWNT surface. Various monomers suitable for conventional emulsion polymerization can be used in CNT micelle polymerization.31 In our previous reports, polymerization was performed at 70 °C for 7 h using ammonium peroxodisulfate (APS) as an initiator. This time, we chose the APS/tetramethylethylenediamine (TMEDA) system as a redox initiator51-52 to initiate the radical polymerization at lower temperature (25 °C) than used previously to avoid furan deprotection. However, the SWNTs coated with an endo-FpMMA gel layer (endo-FpM/SWNTs) readily aggregated after dispersion by sonication, probably because of the poor hydrophilicity of the endo-FpMMA gel layer (Supporting Information, Figure S5). Therefore, we chose PEGMA as a co-monomer to improve the hydrophilicity53 of the gel layer. Figure 2 shows a photograph of SWNTs coated with an endo-FpMMA and PEGMA gel layer (endoFpM/PEG/SWNTs) after CNT micelle polymerization. The endo-FpM/PEG/SWNT solution remained clear even after the removal of surfactant (sodium dodecyl sulfate; SDS) because of the stable coating of the SWNTs by a gel layer with sufficient hydrophilicity to prevent aggregation.

Figure 2. CNT micelle polymerization using endo-FpMMA and PEGMA (left). Photograph of endo-FpM/PEG/SWNT solution (right).

1

Figure 1. H NMR spectra of endo-FpMMA (a) dissolved in D2O, (b) after heating at 80 °C for 90 min in D2O, and (c) after addition of glutathione in D2O.

Preparation of endo-FpMMA-coated SWNTs via CNT Micelle Polymerization. The surface of SWNTs was modified with endo-FpMMA through CNT micelle polymerization based on our previous report.30 In this method, SWNTs were non-covalently modified with a gel layer and the resulting SWNT/gel hybrid showed remarkable stability in the absence of a surfactant in an aqueous

The absorption spectrum of the endo-FpM/PEG/SWNT solution (red line in Figure 3a) contained several sharp peaks characteristic of the interband transition of semiconducting SWNTs54 in the visible-to-NIR region, suggesting that the SWNTs remained isolated even after the removal of SDS. Notably, the absorption intensity was comparable to that of the dispersion before CNT micelle polymerization (blue line), indicating that most of the SWNTs participated in the CNT micelle polymerization. The absorption spectrum of the endo-FpM/PEG/SWNT solution shifted to longer wavelength compared with that of SWNTs in 0.2 wt% SDS aqueous solution (blue line in Figure 3a). Such a clear shift after the polymerization indicated the replacement of hydrophobic SDS with a hydrophilic gel layer on the SWNT surface.30 Figure 3b displays a PL 2D map of the endo-FpM/PEG/SWNT solution. Clear PL spots from the various semiconducting SWNTs further confirmed the presence of isolated SWNTs because such PL signals are observed only when SWNTs are individually isolated.19 Such characteristic optical properties of the SWNTs were maintained because the addition reaction of polymer radical on the SWNT



sidewall was negligible and the gel was formed noncovalently around SWNTs. As the matter of fact, Raman D-band intensity at 1295 cm−1 with respective to that of the G-band at 1595 cm−1 (G/D ratio), practical indicator of the defect content in a SWNT surface,55-56 was almost unchanged during the polymerization (Supporting Information, Figure S6b). To explore the structure of the gel coating in endoFpM/PEG/SWNTs, atomic force microscopy (AFM) was carried out. Prior to the AFM measurements, the endoFpM/PEG/SWNT solution was ultracentrifuged at 600,000 ×g for 1 h to selectively remove the polymer particles from the SWNTs as a supernatant (Supporting Information, Figure S7).30 Figure 3c shows AFM images of the endo-FpM/PEG/SWNTs cast from the sediment layer. Clear needle-like structures were observed. Together with the major component (70%) with an average height of 3.2 ± 0.7 nm, thicker structures with the height over 5 nm were also observed (Figure 3d). We consider the thicker component to the presence of the bundled SWNT inside endo-FpM/PEG/SWNTs. Indeed, the absorption peaks were slightly broadened (Figure 3a) and the PL intensity was decreased compared with the SWNT dispersion before the polymerization (Figure S6a), both of which can be originated from the small bundle. Since such a thicker components were not observed for SWNT coated by poly(N-isopropropyl acrylamide) (PNIPAM) in our previous report30 but were observed when the PEG unit was incorporated as observed in PNIPAM/PEG/SWNT30, we considered that the formation of thicker components occurred especially when the PEG was incorporated in the gel layer. Further optimization in the polymerization condition such as surfactant type and concentration, and monomer concentration will be necessary when the PEGMA was used as a co-monomer. Considering the average SWNT diameter in this study (~1.0 nm), the thickness of the gel layer was only about 1.1 nm. According to dynamic light scattering (DLS) measurements, the average size of endo-FpM/PEG/SWNTs was 199 nm, which is suitable for biological applications32 (Supporting Information, Figure S7b).

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Figure 3. (a) Absorption spectra of endo-FpM/PEG/SWNT solution (red line) and SWNTs in 0.2 wt% SDS aqueous solution (blue line). (b) PL 2D map of endo-FpM/PEG/SWNT solution. (c) AFM image of endo-FpM/PEG/SWNTs (scale bar: 2 μm). (d) Height histogram of endo-FpM/PEG/SWNTs obtained from AFM images.

To characterize the chemical structure of the gel layer after CNT micelle polymerization together with the copolymerization ratio of endo-FpMMA and PEGMA, the 1H NMR spectrum of the polymer gel particles obtained after the polymerization (before any purification such as filtration or centrifugation) prepared in the absence of SWNTs was measured instead of that of endo-FpM/PEG/SWNTs. This is because the 1H NMR signals of endoFpM/PEG/SWNTs were hardly observed, which was probably caused by the low mobility of the gel layer. As shown in Figure 4, monomer vinyl peaks at 6.17 and 5.74 ppm (indicated by black dots) caused by endo-FpMMA and PEGMA were not observed and a broad peak at around 1.3 ppm (indicated by a gray dot) originating from the polymer backbone appeared, indicating the polymerization conversion was quantitative and the copolymerization ratio was equal to the monomer feed ratio; i.e., endoFpMMA : PEGMA = 1 : 1. The presence of the furan cycloadduct peaks at 6.51 and 5.44 ppm (indicated by light green and pink dots, respectively) revealed that the protecting furan group was not removed during the polymerization. These results strongly supported that the gel layer of endo-FpM/PEG/SWNTs consisted of a 1 : 1 ratio of endo-FpMMA to PEGMA without any hydrolysis. In addition, to confirm that endo-FpMMA was included in the gel layer of endo-FpM/PEG/SWNTs, the 1H NMR spectrum of the polymer gel particles obtained as a supernatant after the polymerization of endo-FpM/PEG/SWNTs was measured. The presence of the furan cycloadduct peaks confirmed that the protecting furan group was not removed upon CNT micelle polymerization (Supporting Information, Figure S8a). Furthermore, the deprotection of the polymer gel particles induced by heating at 80 °C (Supporting Information, Figure S8b) and subsequent Michael addition reaction with glutathione (Supporting Information, Figure S8c) was also demonstrated. These promising results motivated us to functionalize endoFpM/PEG/SWNTs in aqueous media.


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Journal of the American Chemical Society Therefore, we assumed that the difference of the intensity between the two spectra was caused by Coumarin-SH covalently functionalized onto endo-FpM/PEG/SWNTs via thiol–maleimide Michael addition reaction.

1

Figure 4. H NMR spectra of (a) a mixture of monomers (endo-FpMMA and PEGMA), cross-linker (BIS), and surfactant (SDS) and (b) endo-FpM/PEG free polymer gel particles.

In Situ Functionalization of endoFpM/PEG/SWNTs. In situ functionalization of endoFpM/PEG/SWNTs in aqueous media was carried out using a thiol-containing fluorescent dye (Coumarin-SH, Figure 5) as a model thiol compound. A dispersion of endo-FpM/PEG/SWNTs in water was heated at 80 °C for 90 min and then Coumarin-SH saturated aqueous solution was added and reacted for 24 h.

To study endo-FpM/PEG/SWNTs after reaction with Coumarin-SH in more detail, the NIR PL spectrum of the sample excited at 365 nm was measured (Figure 6). Clear NIR emission was observed for endo-FpM/PEG/SWNTs with deprotection treatment (red line), whereas endoFpM/PEG/SWNTs without deprotection treatment (black line) and without adding Coumarin-SH (Supporting Information, Figure S9) showed no detectable PL signal even when the same concentration of endoFpM/PEG/SWNTs was used. It was apparent that the PL emission from the SWNTs was observed because of the energy transfer from Coumarin-SH to the SWNTs. In the presence of the covalently functionalized Coumarin-SH, there was clear PL emission only from those SWNTs whose absorption maxima overlapped with the emission spectrum of Coumarin-SH,54 judging from the PL 2D map in Figure 3. This finding confirmed that the functionalization of Coumarin-SH occurred within the range59 to allow energy transfer from Coumarin-SH to endoFpM/PEG/SWNTs. Similar energy transfer from the dye to SWNTs was reported for a SWNT/Nile blue A complex.60 The absence of a similar PL signal from endoFpM/PEG/SWNTs without deprotection treatment clearly indicated that covalently bonded Coumarin-SH was responsible for the energy transfer and the physically adsorbed Coumarin-SH, if present, was either not responsible for the energy transfer or its contribution was negligible. These results strongly suggest that the SWNT surface was functionalized by Coumarin-SH via Michael addition reaction. Therefore, based on the present findings, we consider that endo-FpM/PEG/SWNTs is a promising platform for functionalization with various thiol compounds. In particular, bioactive functionalization using thiolmodified ligands such as antibodies for active cancer cell targeting is a potential application of endoFpM/PEG/SWNTs. Furthermore, the thin gel coating of endo-FpM/PEG/SWNTs also makes it attractive for use as a functionalized electrode in sensing and electrochemical catalysis.

Figure 5. PL spectra (365-nm excitation) of Coumarin-SH solution (green line) and supernatant solutions obtained after reacting Coumarin-SH with heat-treated endoFpM/PEG/SWNT solution (red line) or non-heat-treated endo-FpM/PEG/SWNT solution (blue line).

Figure 5 shows the fluorescence spectra of CoumarinSH solution after reaction with endo-FpM/PEG/SWNTs with (red line) and without (blue line) initial deprotection treatment. The decrease of fluorescence intensity was larger for endo-FpM/PEG/SWNTs with deprotection treatment (red line). The signal decrease observed for endo-FpM/PEG/SWNTs without deprotection (blue line) was probably caused by the non-covalent physical adsorption of Coumarin-SH into the gel layer of endoFpM/PEG/SWNTs and/or the polymer gel particles.57-58

Figure 6. NIR PL spectra (365-nm excitation) of (a) heattreated endo-FpM/PEG/SWNTs (b) non-heat-treated endoFpM/PEG/SWNTs after reacting with Coumarin-SH. Both endo-FpM/PEG/SWNTs were obtained as a sediment after the centrifugation as illustrated in Figure 5. The sediments were further washed with water to remove the unbound Coumarin-SH from SWNT surface by repeating the disper-



sion/ultracentrifugation cycles until the Coumarin-SH absorption was not detected in the supernatant.

CONCLUSION In this study, by taking advantage of the monomer flexibility of CNT micelle polymerization developed by our group, we newly fabricated SWNT hybrid containing reactive group on the SWNT surface, which allowed postfunctionalization of SWNT hybrid under mild condition in water. This time, we designed and synthesized FpMMA to introduce a thiol-reactive maleimide group onto SWNT hybrid. It was revealed that the endo form of FpMMA monomer underwent clear deprotection by heating and functionalization with model thiol compound in aqueous media at room temperature, while their exo isomer resulted in the hydrolysis upon the deprotection. To avoid the deprotaction of endo-FpMMA during the polymerization, CNT micelle polymerization was carried out at room temperature using APS/TMEDA as an initiator in the presence of PEGMA as a co-monomer. The resulting endo-FpM/PEG/SWNTs formed a clear dispersion in aqueous solution even after the removal of surfactant because of the stable gel coating and maintained the characteristic optical properties of the SWNTs. After the deprotection of endo-FpM/PEG/SWNTs and subsequent reaction with Coumarin-SH, the resulting hybrid showed an energy transfer from Coumarin to SWNT, indicating the successful functionalization of endo-FpM/PEG/SWNTs with a thiol compound via Michael addition. Based on the present results, we propose that endo-FpM/PEG/SWNTs can be used as a ‘platform’ for various antibody functionalization to enable active cancer cell targeting. Such bioactive functionalization using endo-FpM/PEG/SWNTs and in vivo targeting experiments together with NIR imaging and treatment are now in progress in our group.

EXPERIMENTAL SECT SECTION Materials. SWNTs (HiPco) were purchased from Carbon Nanotechnologies, Inc., USA. N, N’-methylene bisacrylamide (BIS), TMEDA, APS, 40% diethyl azodicarboxylate toluene solution (DEAD), and triphenylphosphine (PPh3) were purchased from Wako Pure Chemical Industries, Ltd., Japan. SDS, PEGMA (Mn = 526), maleimide, and furan were purchased from Sigma Aldrich, Japan. Glutathione was purchased from Tokyo Chemical Industry Co., Ltd., Japan. HS-(CH2)11-NHCO-Coumarin (Coumarin-SH) was purchased from ProChimia, USA. All reagents were used as received unless otherwise stated. Measurements. IR and Raman spectra were measured by an Infrared spectrometer (Spectrum 65, Perkin Elmer) and a Raman spectrophotometer (Raman RXN System, Kaiser Optical Systems) at an excitation wavelength of 785 nm at room temperature, respectively. Mass spectra were obtained using an electrospray ionization time-offlight mass spectrometer (Micro TOF, Bruker) and matrix-assisted laser desorption/ionization–time of flight– mass spectrometer (Autoflex III, Bruker). Thermogravi-

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metric analysis (TGA) curve were obtained by an Thermogravimetric/DTA Analyser (EXSTAR 6000 TG/DTA 6300,SII). UV-vis-NIR spectra at given temperatures were measured by a spectrophotometer (V-670, JASCO). PL spectra were measured using a spectrofluorometer (FluorologR-3, Horiba Jobin Yvon). AFM was conducted using a probe microscope (Agilent 5500). NMR spectra were recorded with Bruker Avance 300- and 400-MHz spectrometers. NMR measurements were performed in deuterated chloroform (CDCl3), water (D2O), and dimethyl sulfoxide (DMSO-d6). Chemical shifts are given relative to tetramethylsilane and 3-(trimethylsilyl)propionic2,2,3,3-d4 acid sodium salt (δ = 0 ppm) as internal standards, and coupling constants J are reported in Hz. DLS was measured using a particle size analysis system (ELSZ2, Otsuka Electronics). Synthesis of endo-form furan-protected maleimide. Maleimide (0.855 g, 8.8 mmol) and furan (1.1 mL, 15 mmol) were stirred in water (10 mL) at room temperature for 5 days. The precipitated white crystalline material was collected by vacuum filtration, rinsed with water, and dried in vacuo overnight (0.56 g, 38%). 1H NMR (300 MHz, THF-d8): δ/ppm 3.41 (dd, J = 1.8 Hz, 2H), 5.16 (m, 2H), 6.41 (s, 2H), 9.68 (s, 1H); 13C NMR (300 MHz, THF-d8): δ 48.29, 79.87, 135.08, 176.05; ESI-MS: m/z: [M-H]− 164.0389, found 164.0349, M.p.: 130–131 °C (Literature: 131 °C).46 Synthesis of endo-FpMMA.46 DEAD (0.65 mL, 1.43 mmol) was added dropwise over a period of 10 min into a mixture of endo-form furan-protected maleimide (206.5 mg, 1.25 mmol), PPh3 (375 mg, 1.43 mmol), and PEGMA (300 μL, 0.625 mmol) in anhydrous THF (5 mL) in an ice bath with stirring. The mixture was stirred for 22 h at room temperature. The solvent was removed by vacuum evaporation. The resulting oily product was dissolved in diethyl ether and then extracted with water twice. The combined aqueous phase was extracted with CHCl3 three times. The combined organic phase was dried with MgSO4. After evaporating the solvent, the orange oily residue was purified by silica-gel flash column chromatography (dichloromethane : methanol = 90 : 10 v/v). The product was obtained as a yellow oil (285 mg, 68%). 1H NMR (400 MHz, D2O): δ/ppm 1.95 (s, 3H), 3.55 (s, 2H), 3.69 (m, 36H), 3.82 (d, J = 4.0 Hz, 2H), 4.33 (d, J = 4.8 Hz, 2H), 5.40 (s, 2H), 5.74 (s, 1H), 6.17 (s, 1H), 6.49 (s, 2H); 13C NMR (CDCl3): δ/ppm 18.33, 37.69, 49.95, 63.88, 67.02, 69.13, 69.89, 70.02, 70.55, 79.44, 125.77, 134.41, 167.37, 174.87. Deprotection of endo-FpMMA by Retro Diels–Alder Reaction. Endo-FpMMA (285 mg) was dissolved in water and heated at 80 °C for 90 min. 1H NMR (D2O): δ/ppm 1.95 (s, 3H), 3.69 (m, 36H), 3.83 (s, 2H), 4.33 (s, 2H), 5.75 (s, 1H), 6.17 (s, 1H), 6.89 (s, 2H). Conjugation of Deprotected endo-FpMMA with Glutathione via Michael Addition Reaction. EndoFpMMA after deprotection (200 mg) was dissolved in D2O (1 mL). Glutathione (10 mg) was added and reacted with the maleimide groups of the copolymer at room temperature for 24 h.


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Synthesis of endo-FpM/PEG Polymer Gel Particles. A mixture of endo-FpMMA (92 mg), BIS (10 mg), and PEGMA (55 μL) in 0.2 wt% SDS aqueous solution (5 mL) was bubbled with N2 gas for 15 min. TMEDA (4.4 μL) and 20 wt % aqueous solution of APS (25 μL) were added to the mixture. The solution was stirred for 24 h at room temperature under N2 atmosphere.

1. 2.

3.

Synthesis of endo-FpM/PEG/SWNTs. SWNTs (6 mg) in 0.2 wt% SDS aqueous solution (60 mL) were dispersed by ultrasonication (Branson 5510) for 1 h, and then centrifuged at 60,000 ×g (Hitachi himac, CS 100 GXL). After endo-FpMMA (92 mg), BIS (10 mg), and PEGMA (55 μL) were added to the supernatant (5 mL), the resulting mixture was bubbled with N2 gas for 15 min. TMEDA (4.4 μL) and 20 wt % aqueous solution of APS (25 μL) were added to the mixture. Polymerization was then carried out at room temperature for 24 h under N2 atmosphere. The resulting precipitate was removed by filtration through cotton. The obtained supernatant was filtered (MWCO: 10000) seven times. For NMR measurement, the solution was treated by ultracentrifugation at 600,000 ×g for 1 h to remove the free polymer gel particles from the SWNTs.

4.

Modification of endo-FpM/PEG/SWNTs. A dispersion of endo-FpM/PEG/SWNTs (100 μg) in water (1 mL) was heated at 80 °C for 90 min. Coumarin-SH saturated aqueous solution (1 mL) was added and then the mixture was reacted at room temperature for 24 h.

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ASSOCIATED CONTENT

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Supporting Information. Supporting Figure S1–S13, TGA 1 characterization of endo- and exo-FpMMA, H NMR spectra of exo-FpMMA heated at 80 °C and 100 °C, photographs of endo-FpM/SWNT solution, Raman spectrum of endoFpM/PEG/SWNTs and SWNTs in 0.2 wt% SDS aqueous solu1 tion, DLS histograms of endo-FpM/PEG/SWNT solution, H NMR spectra of the polymer gel perticles, full characterization of the products NMR spectra,. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION Corresponding Author * E-mail: Tsuyohiko Fujigaya ([email protected])

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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript

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ACKNOWLEDGMENT

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This study was supported in part by the Nanotechnology Platform Project, from the Ministry of Education, Culture, Sports, Science and Technology, Japan, KAKENHI (No. JP15H01002, JP16K14084, and JP16H06056) from Japan Society for the Promotion of Science, and PRESTO (No. JPMJPR15R6) from JST, Japan. We thank Natasha Lundin, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

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