Improved non-covalent biofunctionalization of multi-walled carbon nanotubes using carbohydrate amphiphiles with a butterfly-like polyaromatic tail

Nano Res. 2010, 3(11): 764–778 764 Improved Non-Covalent Biofunctionalization of Multi-Walled Carbon Nanotubes Using Carbohydrate Amphiphiles with a Butterfly-Like Polyaromatic Tail Mohyeddin Assali1, Manuel Pernía Leal1, Inmaculada Fernández2, Pablo Romero-Gomez3, Rachid Baati4, and Noureddine Khiar1 (  ) 1 Instituto de Investigaciones Químicas, C.S.I.C-Universidad de Sevilla, c/. Américo Vespucio, 49, Isla de la Cartuja, Sevilla 41092, Spain 2 Departamento de Química Orgánica y Farmacéutica, Facultad de Farmacia, Universidad de Sevilla, Sevilla 41012, Spain 3 Instituto de Ciencias de Materiales de Sevilla, C.S.I.C-Universidad de Sevilla, c/. Américo Vespucio, 49, Isla de la Cartuja, Sevilla 41092, Spain 4 Université de Strasbourg Faculté de Pharmacie CNRS/UMR 7199, Laboratoire des Systèmes Chimiques Fonctionnels BP 60024, 74 route du Rhin, 67400 Illkirch, France Received: 27 July 2010 / Revised: 9 September 2010 / Accepted: 9 September 2010 © The Author(s) 2010. This article is published with open access at Springerlink.com ABSTRACT We have developed an efficient strategy for the non-covalent functionalization of multi-walled carbon nanotubes (MWCNTs) which allows a biomimetic presentation of carbohydrates on their surface by π–π stacking interactions. The strategy is based on the use of sugar-based amphiphiles functionalized with tetrabenzo[a,c,g,i]fluorene (Tbf), a polyaromatic compound with a topology that resembles a butterfly with open wings. The new carbohydrate-tethered Tbf amphiphiles have been synthesized in a straightforward manner using click chemistry. The reported method has been developed in order to improve the rather low ability of pyrene-based systems to exfoliate MWCNTs in water. By means of thermogravimetric analysis (TGA), ultraviolet (UV), infrared (IR), and fluorescence spectroscopies the interaction between MWCNTs and the Tbf group has been found to be stronger than those involving pyrene-based amphiphilic carbohydrates. The resulting aggregates with a multivalent sugar exposition on their surface are able to engage in specific ligand–lectin interactions similar to glycoconjugates on a cell membrane. KEYWORDS Carbon nanotubes, non-covalent functionalization, tetrabenzo[a,c,g,i]fluorene, carbohydrates, click chemistry, biocompatible system 1. Introduction Since their discovery in 1991 [1], carbon nanotubes (CNTs) have received an unrivalled interest as a consequence of their unique structural, mechanical, electrical, and optical properties [2, 3]. To date, CNTs are being actively investigated for a wide range of biomedical applications [4, 5], including biosensing [6], imaging [7–9], drug delivery [10–16], and specific targeting and killing of cancer cells [17–19]. However, these and other potential biomedical applications have been hampered by the insolubility of CNTs in Nano Res. 2010, 3(11): 764–778 ISSN 1998-012 4 DOI 10.1007/s12274-010-0044-2 CN 11-5974/O 4 Research Article Address correspondence to [email protected] Nano Res. 2010, 3(11): 764–778 765 most solvents, most importantly in water where they exist as ropes and large bundles. To overcome the drawbacks of insolubility and poor dispersibility, surface functionalization of the CNT sidewalls has emerged as a powerful and viable strategy to exfoliate CNTs, resulting in the preparation of stable aqueous/ organic suspensions with moderate to low cytotoxicity [20–22]. Such surface modification allows CNTs to engage in specific binding events with various biological systems [23]. So far many strategies have been developed to exfoliate CNTs into solution, including covalent and non-covalent functionalization [24–26]. The non-covalent approach is more desirable as it conserves the nanotube structure, while the covalent approach has been shown to disrupt their π-network, leading to possible loss of their mechanical, electrical, and biosensing properties. Following the pioneering work of Dai et al. [27], the supramolecular non-covalent π−π stacking strategy has shown itself to be an efficient way to introduce a large number of functionalities onto the surface of carbon nanotubes [28–31], and has been recently used to load polyaromatic chemotherapy agents directly onto the surface [32, 33]. Yet, despite the significant advances achieved, the attachment of functional aromatic scaffolds onto the nanotube surface generally depends on the use of pyrene (Py) as the anchor [34]. Taking into account the large heterogeneity of the surface curvature and diameter of carbon nanotubes, the pyrene moiety with its specific size and shape is far from being a general anchor for all kind of nanotubes [35]. Thus, the design of a new aromatic anchor, ideally complementary to pyrene, to non-covalently tailor the surface of CNTs is highly desirable. On the other hand, recent investigations have confirmed the prime role of carbohydrates in critical biological events, including cell adhesion, inflammation, tumour cell metastasis, and pathogen infections [36–38]. Interestingly, it has been demonstrated that the weak interaction between an individual ligand and the corresponding specific lectin is compensated by multivalent presentation (display) of carbohydrates through the so called cluster effect [39, 40]. Thus, biocompatible model systems able to display a large number of glycoligands on their surface are of interest in glycobiology to study carbohydrate functions and to interfere with native binding events. Consequently, owing to their high aspect ratio and surface area — which enable the display of a large number of carbohydrate motifs — and to their semi flexible nature — which facilitates their binding to specific receptors — CNTs constitute an ideal molecular platform for the multivalent display of carbohydrates [41, 42]. Following our interest in the utilization of CNTs as a molecular jig for the preparation of sugar-based bionanomaterials [43, 44], and in order to improve the rather low ability of pyrene-based systems to exfoliate multi-walled carbon nanotubes (MWCNTs) in water [45], herein we report the synthesis and use of new carbohydrates-based amphiphiles I with a polyaromatic tetrabenzo[a,c,g,i]fluorene (Tbf) tail, designed to optimize the π−π interactions with the sidewalls of MWCNTs. This work has resulted in the development of an efficient strategy for the functionalization of MWCNTs, which allows the synthesis of novel robust water soluble nanoglycoarrays with a biomimetic presentation of carbohydrates on their surface (Scheme 1). 2. Experimental 2.1 General methods All reactions were performed under an atmosphere of dry argon using oven-dried glassware and freshly distilled and dried solvents. Tetrahydrofuran (THF) and diethyl ether were distilled from sodium benzophenone ketyl. CH2Cl2 was distilled from calcium hydride. Thin layer chromatography (TLC) was performed on silica gel GF254 (Merck) with detection by charring with phosphomolybdic acid/EtOH and sulphuric acid/EtOH. Reagents were obtained from commercial suppliers and used without further purification. For flash chromatography, silica gel (Merck 230–400 mesh) was used. The organic extracts were dried over anhydrous sodium sulfate and concentrated under vacuum. Columns were eluted with positive air pressure. Chromatographic eluents are given as volume to volume ratios (v/v). Nuclear magnetic resonance (NMR) spectra were recorded with a Bruker AMX500 (1H, 500 MHz) and Bruker Avance DRX500 (1H, 500 MHz) Nano Res. 2010, 3(11): 764–778 766 spectrometers. Chemical shifts are reported in ppm, and coupling constants are reported in Hz. Routine spectra were referenced to the residual proton or carbon signals of the solvent. High-resolution mass spectra (HRMS) were recorded on a Kratos MS-80RFA 241-MC apparatus. Optical rotations were determined with a PerkinElmer 341 polarimeter. Transmission electron microscopy (TEM) images of glycolipid-coated CNTs were obtained on a Philips CM-200 microscope operating at an electron energy of 200 keV and magnification of 10,000–300,000 times. Samples were prepared by depositing 15 μL of a suspension onto grids and allowing the grids to absorb the material for 2 min. Scanning electron microscope (SEM) images of glycolipid-coated CNTs were obtained on a JEOL JSM-5400 microscope with a magnification of 15,000 times. Samples were prepared by depositing 15 μL of a suspension onto grids and allowing the grids to absorb the material for 2 min. Infrared (IR) spectra were obtained with a Bruker Vector 22 spectrometer as KBr disks. Ultraviolet–visible (UV–vis) spectra were obtained with a PerkinElmer Lambda 12 spectrometer. Raman spectra were recorded on a LabRAM HR800 high resolution UV confocal Raman microscope using a green laser (He–Ne 532.14 nm), 600 line/mm, 20X objective, 20 mW, pinhole 100 μm. Thermogravimetric analysis (TGA) was carried out with a TA instruments TGA Q600 thermal analyzer in the temperature range 0–600 °C, with a heating rate of 20 °C/min rate, under N2 (100 mL/min). Fluorescence measurements were performed using a HORIBA Jobin Yvon Fluorolog FL3-11 fluorimeter equipped with two monochromators (excitation wavelength 345 nm). MWCNTs (Aldrich) were produced by chemical vapor deposition (external diameter 10–15 nm; internal diameter 2–6 nm; length 0.1–10 μm) and were used as received. 2.2 Synthesis Flow charts of the synthesis process are shown in Schemes 2 and 3. Synthesis of 17-(prop-2-yn-1-yl)-tetrabenzo[a,c,g,i] fluorene (5) To 8bH-tetrabenzo[a,c,g,i]fluorene (4) (1 g, 2.73 mmol), in degassed dioxane (50 mL) heated at reflux, was added tetrabutylammonium hydroxide (40% w/w in H2O, 644 mg, 2.48 mmol) as a solution in degassed dioxane (10 mL), resulting in the immediate formation of a yellow precipitate. This was filtered under N2 and washed with warm dioxane (100 mL) and ether (100 mL). The salt was resuspended in dioxane (100 mL), treated with propargyl bromide (214 μL, 2.48 mmol), and the mixture heated to reflux for 2 h. The solvent was removed from the darkened solution, Scheme 1 Schematic representation of the synthesis of the Tbf-linked carbohydrate-MWCNTs nanoglycoarrays used in this work Nano Res. 2010, 3(11): 764–778 76 7 forming a residue which was dissolved in ether (50 mL), and the supernatant removed to leave a yellow slurry which was washed with ether (2 × 30 mL). The extract was dried (Na2SO4), and the solvent removed under reduced pressure, forming a residue which was purified by silica gel chromatography using (hexane/ CH2Cl2 3:1) to give 400 mg of an amorphous yellow solid (40% yield): Rf = 0.44 (hexane/CH2Cl2 3:1). 1H NMR (500 MHz, CDCl3) δ : 8.78–8.75 (m, 4H), 8.67 (d, J = 8.2, 2H), 8.16 (d, J = 7.8, 2H), 7.70–7.60 (m,8H), 4.79 (t, J = 4.5, 1H), 3.25–3.24 (m, 2H), 1.51 (t, J = 2.5, 1H). 13C NMR (125.7 MHz, CDCl3) δ : 143.3, 137.1, 131.6, 130.6, 128.4, 128.0, 127.7, 127.0, 126.1, 125.9, 125.1, 124.4, 123.6, 80.2, 71.1, 53.5, 45.9, 24.4. HRMS calcd. for C32H21 [M]+: m/z 405.1643, found: 405.1671. Synthesis of Tbf-PEG-Lac-8 To a solution of the azide (6) (82 mg, 0.09 mmol) and the alkyne (5) (45 mg, 0.11 mmol) in anhydrous CH2Cl2 (5 mL), were added N,N-diisopropylethylamine (DIPEA) (48 μL, 0.27 mmol) and copper(I) iodide (6 mg, 0.027 mmol). The resulting solution was stirred under argon overnight at room temperature. After dilution with CH2Cl2 (100 mL), washing with 1 mol/L HCl (15 mL), saturated aqueous NaHCO3 (30 mL), and brine (15 mL), the organic phase was dried over Na2SO4 and evaporated under reduced pressure. The crude product was purified by silica gel chromatography, eluting with ethyl acetate/hexane/isopropanol (4:2:1) to give the per-acetylated Tbf-PEG-Lac-8 (81 mg, 70% yield) as a yellow oil: Rf = 0.51 (ethyl acetate/hexane/ isopropanol 4:2:1). [α]D = –4.0 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) δ : 8.80 (t, J = 8.3, 4H), 8.57 (d, J = 8.3, 2H), 8.23 (d, J = 7.3, 2H), 7.74–7.68 (m, 6H), 7.61 (t, J = 7.7, 2H), 7.09 (t, J = 6.2, 1H), 5.96 (s, 1H), 5.36–5.34 (m, 2H), 5.19 (t, J = 9.2, 1H), 5.11 (dd, J = 10.2, J = 2.2, 1H), 4.97 (dd, J = 10.4, J = 3.4, 1H), 4.91 (t, J = 9.5, 1H), 4.49–4.47 (m, 3H), 4.14–4.03 (m, 4H), 3.94 (t, J = 5.0, 2H), 3.88–3.84 (m, 5H), 3.78 (t, J = 9.5, 1H), 3.63–3.13 (m, 12H), 2.84–2.64 (m, 2H), 2.17 (s, 3H), 2.08 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 1.99 (s, 3H). 13C NMR (125.7 MHz, CDCl3) δ : 170.3, 170.2, 170.1, 169.8, 169.6, 169.1, 143.9, 142.8, 136.7, 131.4, 130.4, 128.6, 128.0, 127.3, 127.1, 126.2, 125.9, 125.1, 124.9, 123.7, 123.5, 122.1, 101.1, 83.6, 76.1, 73.7, 71.0, 70.7, 70.4, 70.2, 70.1, 70.0, 69.1, 69.0, 66.6, 62.1, 60.8, 49.5, 46.9, 38.9, 31.2, 30.4, 20.8, 20.7, 20.6, 20.5. HRMS calcd. for C68H75N4O21S [M]+: m/z 1315.4645, found: 1315.4656. To a solution of per-acetylated Tbf-PEG-Lac-8 (40 mg, 0.031 mmol) in dry MeOH (3 mL) was added NaOMe (1.1 mg, 0.02 mmol). The reaction was allowed to proceed at room temperature for 1 h at which time the reaction was judged complete by TLC analysis. A sufficient quantity of Amberlyst Ir-120 (plus) (H+ form) resin was added to the mixture to render the pH of the solution neutral. The resin was removed by filtration and the solvent removed under vacuum. The crude product was purified by size-exclusion chromatography (Sephadex® 20), eluting with MeCN/H2O/NH4OH 6:1:1. Lyophylization of the solvent gave Tbf-PEG-Lac-8 (30 mg, 94%) as a yellowish solid: Rf = 0.60 (MeCN/H2O/NH4OH 6:1:1); [α]D = –7.9 (c 1.0, MeOH). 1H NMR (500 MHz, MeOD) δ : 8.91 (d, J = 8.2, 4H), 8.50 (d, J = 8.3, 2H), 8.41 (d, J = 7.9, 2H), 7.95 (t, J = 6.5, 1H), 7.83–7.71 (m, 6H), 7.62 (t, J = 7.7, 2H), 5.95 (s, 1H), 5.39 (t, J = 4.5, 1H), 4.40–4.35 (m, 2H), 3.97–3.38 (m, 24H), 3.16–3.10 (m, 4H), 3.07– 3.03 (m, 2H), 2.85–2.66 (m, 2H). 13C NMR (125.7 MHz, MeOD) δ : 172.7, 144.8, 143.4, 137.4, 132.9, 131.8, 129.9, 129.0, 128.4, 128.3, 127.6, 127.2, 126.2, 125.9, 124.9, 124.8, 123.4, 105.1, 86.9, 80.6, 80.4, 77.9, 77.1, 74.8, 74.1 72.5, 71.7, 71.4, 71.1, 71.0, 70.9, 70.3, 69.9, 62.5, 62.1, 50.6, 47.8, 40.4, 31.7, 30.7, 30.5. HRMS calcd. for C54H60N4O14SNa [M + Na]+: m/z 1043.3724, found: 1043.3770. Synthesis of Tbf-PEG-Man-9 To a solution of the azide (7) (90 mg, 0.14 mmol) and the alkyne (5) (70 mg, 0.17 mmol) in anhydrous CH2Cl2 (7 mL), were added DIPEA (76 μL, 0.43 mmol) and copper(I) iodide (9 mg, 0.043 mmol). The resulting solution was stirred under argon overnight at room temperature. After dilution with CH2Cl2 (100 mL), washing with 1 mol/L HCl (15 mL), saturated aqueous NaHCO3 (30 mL), and brine (15 mL), the organic phase was dried over Na2SO4 and evaporated under reduced pressure. The crude product was purified by silica gel chromatography eluting (with ethyl acetate/ether (9:1) to ethyl acetate/isopropanol (9:1)) to give the per-acetylated Tbf-PEG-Man-9 (98 mg, 68% yield) as a yellowish oil: Rf = 0.33 (CH2Cl2/MeOH 20:1). [α]D = +36.8 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) δ : 8.76 Nano Res. 2010, 3(11): 764–778 768 (t, J = 7.9, 4H), 8.52 (d, J = 8.4, 2H), 8.18 (d, J = 7.4, 2H), 7.69–7.63 (m, 6H), 7.56 (t, J = 7.4, 2H), 7.08 (t, J = 5.7, 1H), 5.92 (s, 1H), 5.30–5.22 (m, 4H), 5.18 (dd, J = 9.94, J = 3.2, 1H), 4.32–4.25 (m, 2H), 4.06 (dd, J = 10.2, J = 1.8, 1H), 3.89 (t, J = 5.32, 2H), 3.83–3.81 (m, 4H), 3.42–3.35 (m, 4H), 3.25 (t, J = 5.1, 2H), 3.21–3.19 (m, 2H), 3.13– 3.09 (m, 4H), 2.73–2.62 (m, 2H), 2.10 (s, 3H), 2.03 (s, 3H), 2.00 (s, 3H), 1.94 (s, 3H). 13C NMR (125.7 MHz, CDCl3) δ : 170.6, 170.0, 169.9, 169.8, 169.7, 143.9, 137.0, 131.4, 130.4, 128.6, 127.9, 127.3, 127.1, 126.2, 125.9, 125.1, 124.9, 123.7, 123.5, 122.0, 82.4, 70.9, 70.7, 70.3, 70.1, 69.9, 69.4, 69.2, 69.0, 66.2, 62.4, 49.7, 46.9, 38.0, 31.3, 31.1, 20.9, 20.7, 20.6. HRMS calcd. for C56H59N4O13S [M]+: m/z 1027.3799, found: 1027.3820. To a solution of per-acetylated Tbf-PEG-Man-9 (50 mg, 0.048 mmol) in dry MeOH (3 mL) was added NaOMe (1.1 mg, 0.02 mmol). The reaction was allowed to proceed at room temperature for 1 h at which time the reaction was judged complete by TLC analysis. A sufficient quantity of Amberlyst Ir-120 (plus) (H+ form) resin was added to the mixture to render the pH of the solution neutral. The resin was removed by filtration and the solvent removed under vacuum. The crude product was purified by size-exclusion chromatography (Sephadex® G20), eluting with CH2Cl2/MeOH 9:1. Lyophylization of the solvent gave Tbf-PEG-Man-9 (38 mg, 90%) as a yellow solid: Rf = 0.33 (CH2Cl2/MeOH 9:1); [α]D = +29.5 (c 1.0, MeOH). 1H NMR (500 MHz, MeOD) δ : 8.78–8.75 (m, 4H), 8.33 (d, J = 8.1, 2H), 8.20 (d, J = 7.8, 2H), 7.70–7.61 (m, 6H), 7.49 (t, J = 7.3, 2H), 5.79 (s, 1H), 5.24 (s, 1H), 5.00 (t, J = 4.5, 1H), 3.89–3.84 (m, 3H), 3.83 (s, 2H), 3.78 (d, J = 4.7, 2H), 3.75–3.69 (m, 3H), 3.66–3.60 (m, 2H), 3.43–3.36 (m, 4H), 3.23–3.21 (m, 2H), 3.06–3.03 (m, 4H), 2.96– 2.94 (m, 2H), 2.77–2.57 (m, 2H). 13C NMR (125.7 MHz, MeOD) δ : 173.6, 145.6, 138.7, 133.6, 132.6, 130.6, 129.8, 129.6, 129.1, 128.5, 128.3, 128.0, 126.9, 126.7, 125.7, 125.6, 124.2, 87.4, 76.1, 74.5, 74.0, 72.6, 72.0, 71.9, 71.8, 70.7, 69.8, 63.7, 51.4, 50.4, 49.4, 48.5, 40.5, 32.4, 32.2. HRMS calcd. for C48H50N4O9SNa [M + Na]+: m/z 881.3196, found: 881.3250. 2.3 Functionalization of MWCNTs with the neoglycolipids In a typical preparation, 4 mg of MWCNTs were suspended in 5 mL of aqueous solution containing 4 mg of the neoglycolipid. The mixture was sonicated using a water-bath sonicator for 1 h, incubated overnight, and then sonicated for another 30 min. Insoluble materials and impurities (amorphous carbon and catalyst) were then removed by low-speed centrifugation at 3000 r/min for 5 min. A second high-speed centrifugation at 14,000 r/min for 12 min was then performed to sediment the stable aggregates, and eliminate the excess neoglycolipids that remained in the supernatant solution. After lyophilization, stable black solutions free of any visible particulates were obtained, indicating the absence of large bundles, with solubility values of 3.4 mg/mL in the case of MWCNTTbf-PEG-Lac-8, and 1.4 mg/mL in the case of MWCNTPy-PEG-Lac-10. The absence of aggregation is also supported by TEM images, which typically show mainly individual nanotubes (see Fig. S-1 in the Electronic Supplementary Material (ESM)). 2.4 Lectin binding assay A 1 mL solution of peanut agglutin labeled with fluorescein isothiocyanate (PNA–FITC) (100 μg/mL) in buffer (0.10 mol/L Tris, and 0.15 mol/L NaCl, pH 8.0) was added to a suspension of amphiphile-coated nanotubes in water (0.3 mg/mL). An additional 0.5 mL of buffer solution was added and the reaction was incubated overnight at room temperature in the dark. Lactose inhibition of PNA binding to the MWCNT aggregates was tested by preincubating 1 mL of PNA– FITC solution in buffer (100 μg/mL) with 0.5 mL of lactose in buffer (500 mg/mL) for 1 h at room temperature in the dark. This preincubated solution was added to the suspension of MWCNT aggregates in water (1 mL), and the resulting solution was incubated overnight at room temperature in the dark, as described above. After incubation, the solutions were centrifuged three times at 14,000 r/min for 10 min to remove the unreacted lectins. The centrifuged solutions were dissolved in 100 μL of buffer and their fluorescence intensities at 510–550 nm were determined using a fluorescence microplate reader (excitation wavelength 492 nm). Nano Res. 2010, 3(11): 764–778 769 3. Results and discussion 3.1 Chemistry Experimental [26, 46, 47], and theoretical studies [48] have shown that the strength of the π−π interactions between aromatic molecules and surface of carbon nanotubes increases with the size of the aromatic species and depends on the substituents in the aromatic ring. Additionally, based on work with the planar sheets of graphene, the physiosorption of aromatics onto the nanotube sidewalls through π−π stacking is assumed to take place in a coplanar geometry [49]. Nevertheless, due to the intrinsic curvature of the rolled nanotube surface, the shape of the aromatic moiety can also influence the affinity [46, 50]. Hence, sugars functionalized with Tbf, a polyaromatic compound that exhibits a high affinity for porous graphitized carbon [51–53], and possesses a topology that resembles a butterfly with open wings (Scheme 1) are expected to solvate MWCNTs through π−π interactions better than those functionalized with the widely used pyrene tail (Scheme 1) [31, 44, 45]. This hypothesis is shown in Scheme 1, where the sugarbased Tbf amphiphilic compound I that contains a hydrophilic spacer interacts with the CNT surface leading to robust nanoglycoarrays with a multivalent presentation of sugar epitopes. In order to develop a general and efficient bioconjugation of the Tbf polyaromatic tail to a given biomolecule in general and to glycoligands in particular, we made use of the unsurpassed copper-catalyzed azide–alkyne cycloaddition (CuAAC), the paradigmatic example of “click chemistry”, which offers the advantages of good yield, mild reaction conditions and broad tolerance towards functional groups [54, 55]. To achieve this goal, we installed the azido function on the sugar part of the amphiphile, and developed a three-step synthesis of a novel alkyne-functionalized Tbf (5). The 8bH-tetrabenzo[a,c,g,i]fluorene (Tbf-H) (3) is easily synthesized on a multigram scale starting from commercially available 9-bromophenanthrene (1) using a reported procedure, via the symmetrical alcohol (2) [56]. Then the formation of the tetrabutylammonium salt of Tbf (4), by treatment of (3) with NBu4OH, followed by immediate reaction with propargyl bromide at room temperature provided the desired Tbf alkyne (5) in excellent isolated yield (Scheme 2). The targeted Tbf-linked carbohydrate compounds Tbf-PEG-Lac-8 and Tbf-PEG-Man-9, were then obtained in a straightforward manner by copper(I)- catalyzed azide–alkyne cycloaddition reactions of our recently reported azido sugars (6) and (7), with the alkyne-derivatized fluorene (5). Finally, Zemplen deacetylation, followed by purification of the free sugars on Sephadex G-20 yielded the water-soluble neoglycoconjugates shown in Scheme 3. 3.2 Functionalization of MWCNTs We next studied the ability of these Tbf-tethered sugar molecules to exfoliate MWCNTs and to form robust water soluble bioactive nanoglycoarrays. By merely mixing either Tbf-PEG-Lac-8 or Tbf-PEG-Man-9 with MWCNTs in pure water without any additive, followed by sonication for 1 h, a characteristic black stable suspension was obtained, indicating the formation of stable MWCNT-Tbf-PEG-Lac-8 and MWCNT-TbfPEG-Man-9 soluble aggregates, respectively (Fig. 1, vials (b) and (c)). Scheme 2 Synthesis of the Tbf-tethered alkyne (5) Nano Res. 2010, 3(11): 764–778 770 The resulting suspension remained stable for several months whereas, in contrast, the sonication of MWCNTs in water without the sugar amphiphiles, led to unstable suspensions resulting in the rapid precipitation of the black CNTs after a few minutes (Fig. 1, vial (a)). The observation of a stable black suspension when Tbf-PEG-Lac-8 or Tbf-PEG-Man-9 are used, shows the change in the native hydrophobic characteristics of the nanotube side wall, which can become more hydrophilic after functionalization of the MWCNTs surface with glycolipids and the exposition of the sugar epitopes towards the polar water phase. The MWCNT-nanoglycoarrays were characterized by TEM, SEM, and UV–vis, IR, fluorescence, and Raman spectroscopies. Furthermore, in order to determine the nature of the supramolecular interaction Figure 1 Photographs of vials with aqueous solutions of: (a) as produced MWCNTs, (b) MWCNT-Tbf-PEG-Lac-8, (c) MWCNTTbf-PEG-Man-9 between the Tbf-functionalized amphiphiles and the surface of the CNTs, and to establish the strength and efficiency of these interactions — which are responsible for the solubilisation of MWCNTs — the results were compared with those obtained for related compounds functionalized with a pyrene tail. Therefore, amphiphiles Py-PEG-Lac-10 and Py-PEG-Man-11, with the same sugar epitopes as molecules (8) and (9) (lactose and mannose respectively) were synthesized following a similar approach to that described earlier, in which the key step is the [2+3] Huisgen cycloaddition using as alkyne the pyrene-derived alkyne 12 (Fig. 2). Details are given in the ESM. 3.3 Characterization of the aggregates Initial evidence for the formation of stable exfoliated MWCNT-Tbf-PEG-sugar aggregates came from TEM and SEM analysis, obtained by analyzing 15 μL of diluted aqueous stable solutions on a copper-coated holey carbon grid. As a consequence of the hydrophobic interactions, van der Waals forces, and π-stacking of individual nanotubes, as produced CNTs usually exist as dense bundles or ropes that are deeply interconnected as shown in Figs. 3(a) and 3(c). Quite remarkably, functionalization with the neoglycolipids Tbf-PEG-Lac-8 and Tbf-PEG-Man-9 in pure water under strictly neutral conditions, allowed the CNT bundles to exfoliate producing small, mainly Scheme 3 Synthesis of the fluorene-functionalized neoglycolipid derivatives Nano Res. 2010, 3(11): 764–778 771 individual, sugar coated nanotubes, as evidenced by TEM and SEM analysis (Figs. 3(b) and 3(d); see Fig. S-2 in the ESM for TEM and SEM images of MWCNT-Tbf-PEG-Man-9, MWCNT-Py-PEG-Lac-10, and MWCNT-Py-PEG-Man-11). The UV–vis spectra of MWCNT-Tbf-PEG-Lac-8 aggregates (Fig. 4(a), red curve) differ significantly from the free amphiphile Tbf-PEG-Lac-8. Indeed, due probably to the strong interaction of the polyaromatic Tbf group with MWCNTs, the intensity of the absorbance of the aggregates was drastically decreased compared to that observed for the free amphiphile in solution (Fig. 4(a) black curve, see Fig. S-3 in the ESM for further spectra). In contrast, apart from a slight decrease in intensity, the UV–vis spectra for the MWCNT-Py-PEG-Lac-10 aggregates (Fig. 4(b), red curve) were almost identical Figure 3 (a) TEM image of MWCNTs. (b) TEM image of MWCNT-Tbf-PEG-Lac-8. (c) SEM image of MWCNTs. (d) SEM image of MWCNT-Tbf-PEG-Lac-8 to the neoglycolipid Py-PEG-Lac-10 alone (Fig. 4(b), black curve), indicative of the weak interaction of the pyrene moiety with the sidewall of MWCNTs. Figure 4 (a) UV–vis spectra of Tbf-PEG-Lac-8 (black line), and aggregate MWCNT-Tbf-PEG-Lac-8 (red line). (b) UV–vis spectra of Py-PEG-Lac-10 (black line), and aggregate MWCNTPy-PEG-Lac-10 (red line) Figure 2 Structure of pyrene-based neoglycolipids derivatives used in this study Nano Res. 2010, 3(11): 764–778 772 The analysis of the IR spectra of the MWCNTs functionalized with the disaccharide amphiphiles Tbf-PEG-Lac-8 and Py-PEG-Lac-10 afforded more evidence in favour of the π−π stacking interaction of the Tbf and Py tails with the nanotube sidewalls. The intensity of the band at 2923 cm–1 ( ν C sp2–H stretching) was lower in the IR spectra of both aggregates, MWCNT-Tbf-PEG-Lac-8 (Fig. 5(a), red spectrum), and MWCNT-Py-PEG-Lac-10 (Fig. 5(b), red spectrum) compared to the spectra of the free sugar based molecules. The intensity of this band is drastically decreased in the case of the aggregate MWCNT-Tbf-PEG-Lac-8 compared to the free glycolipid Tbf-PEG-Lac-8, due to the strong interaction between the aromatic rings of the fluorene and the MWCNT sidewalls (Fig. 5(a), see also Fig. S-4 in the ESM for further spectra) [57]. Figure 5 (a) IR spectra of Tbf-PEG-Lac-8 (black line), and aggregate MWCNT-Tbf-PEG-Lac-8 (red line). (b) IR spectra of Py-PEG-Lac-10 (black line), and aggregate MWCNT-Py-PEGLac-10 (red line) The aggregates were further characterized by Raman spectroscopy (Fig. 6). Resonance Raman spectroscopy has been extensively used to study CNTs [58]. Important information regarding the degree of defects, the nanotube chirality, and the electronic type can be obtained. Table 1 shows the intensity ratio between the G band (sp2 hybridization, graphene band) and D band (sp3 hybridization, disordered band) of MWCNTs, and the aggregates of the MWCNTs with the neoglycolipids Tbf-PEG-Lac-8, Tbf-PEG-Man-9, Py-PEG-Lac-10, and Py-PEG-Man-11. Interestingly, as shown in Table 1, the intensity ratio (IG/ID) for the aggregates MWCNT-Tbf-PEG-Lac-8 and MWCNTTbf-PEG-Man-9 is significantly increased, when compared with the value for the unmodified MWCNTs, while this ratio remains almost unchanged for Figure 6 (a) Fluorescence spectra of Tbf-PEG-Lac-8 (black line), and aggregate MWCNT-Tbf-PEG-Lac-8 (red line). (b) Fluorescence spectra of Py-PEG-Lac-10 (black line), and aggregate MWCNTPy-PEG-Lac-10 (red line)