Polyamine Functionalized Carbon Nanotubes: Synthesis, Characterization, Cytotoxicity and sirna Binding Prabhpreet Singh a, Cristian Samorì a, Francesca Maria Toma b, Cyrill Bussy c, Antonio Nunes c, Khuloud T. Al-Jamal c, Cécilia Ménard-Moyon a, Maurizio Prato b *, Kostas Kostarelos c *, and Alberto Bianco a * a CNRS, Institut de Biologie Moléculaire et Cellulaire, Laboratoire d Immunologie et Chimie Thérapeutiques, 67000 Strasbourg, France b Dipartimento di Scienze Farmaceutiche, Università di Trieste, 34127 Trieste, Italy c Nanomedicine Laboratory, Centre for Drug Delivery Research, The School of Pharmacy, University of London, London WC1N 1AX, UK ELECTRONIC SUPPORTING INFORMATION Reagents and solvents were purchased from Fluka, Aldrich and used without further purification unless otherwise stated. Moisture-sensitive reactions were performed under argon or N 2 atmosphere. CH 2 Cl 2 was freshly distilled from CaH 2, THF from Na/benzophenone, and DMF dried over 4Ǻ molecular sieves. Chromatographic purification was done with silica gel Merck (Kiesegel 60, 40-60 µm, 230-400 mesh ASTM) in standard columns. TLC was performed on aluminium sheets coated with silica gel 60 F254 (Merck, Darmstadt). LC/MS analyses were performed on ThermoFisher Finnigan LCQ Advantage Max. 1 H and 13 C NMR spectra were recorded on Bruker DPX 300 (operating at 300 and 75 MHz, respectively). The peak values were obtained as ppm (δ) and referenced to the solvent. The protected polyamines 13, 19 and 25 were synthesized through stepwise elongation of the backbone and fully characterized. Protected polyamines 13, 19 and 25 were further modified using reported literature methods to give N-(4- aminobenzoyl)polyamine derivatives 15, 21 and 27, respectively for carrying out addition and amidation reactions on carbon nanotubes. 1
Scheme 1: Synthesis of protected (N-(4-aminobenzoyl)putrescine): (i) di-tert-butyl dicarbonate, CHCl 3, 19 h, rt; (ii) 4-nitrobenzoyl chloride, Et 3 N, toluene, 8 h, 60 o C; (iii) Pd/C, H 2, methanol, 8 h, rt. Synthesis of tert-butyl 4-aminobutylcarbamate (13). To a solution of 1,4-diaminobutane (12.5 g, 0.142 mol) in CHCl 3 (150 ml), di-tert-butyl dicarbonate (3.1 g, 0.0142 mol) previously dissolved in CHCl 3 (75 ml) was added dropwise (3 h) and the reaction was stirred for a further 16 h at room temperature. The organic layer was washed with water (3x100 ml), dried with Na 2 SO 4 and evaporated under vacuum to afford 13, as colourless oil. (2.51 g, quantitative) R f = 0.2 (20% CH 3 OH:CHCl 3 ). 1 H (300 MHz, CDCl 3 ): δ 4.72 (br s, 1 H, NH, NHCO t Bu), 3.10 (q, 2 H, J = 5.4 Hz), 2.68 (t, 2 H, J = 6.6 Hz), 1.55-1.45 (m, 4 H), 1.41 (s, 9 H, t Bu). 13 C (75 MHz, CDCl 3 ): 156.0, 78.7, 41.6, 40.2, 30.6, 28.2, 27.3. Synthesis of N 1 -p-nitrobenzoyl-n 4 -(tert-butyloxycarbonyl)-1,4-diaminobutane (14). 4-Nitro benzoic acid (1.0 g, 0.006 mol) was dissolved in SOCl 2 (0.85 g, 0.52 ml, 0.007 mol) and refluxed for 5h. Few drops of DMF were also added to catalyze the reaction. The excess of SOCl 2 was evaporated under vacuum to obtained quantitative yield of 4-nitrobenzoyl chloride. The 4-nitrobenzoyl chloride was co-evaporated with diethyl ether to remove traces of SOCl 2. The resulting 4-nitrobenzoyl chloride was dissolved in dry toluene (50 ml) and to this solution of 13 (1.1 g, 0.006 mol), in dry toluene (10 ml), Et 3 N (0.97 ml, 0.007 mol) was added. The reaction mixture was stirred for 8 h at 60 o C under inert atmosphere. After completion of the 2
reaction (monitored by TLC), the mixture was cooled to room temperature and Et 2 O was added. The solid precipitate was filtered off, washed with diethyl ether and dried under vacuum to isolate pure 14 (1.8 g, 90%), as a white solid. R f = 0.55 (6% CH 3 OH/CHCl 3 ), LC-MS (337 MW): m/z 338.13 [M+H] +. 1 H (300 MHz, CDCl 3 ) δ 8.26 (d, 2 H, J = 9.0 Hz), 8.02 (d, 2 H, J = 8.7 Hz), 7.20 (br s, 1 H, CONHCH 2 ), 4.72 (br s, 1 H, NH, NHCO t Bu), 3.53 (q, 2 H, J = 5.4 Hz), 3.17 (q, 2 H, J = 5.4 Hz), 1.76-1.58 (m, 4 H), 1.44 (s, 9 H, t Bu). 13 C (75 MHz, CDCl 3 ) δ 165.5, 156.5, 149.4, 140.2, 128.3, 123.6, 79.5, 40.0, 39.7, 28.3, 28.1, 25.6. Synthesis of N 1 -p-aminobenzoyl-n 4 -(tert-butyloxycarbonyl)-1,4-diaminobutane (15). To a solution of 14 (0.5 g, 0.0015 mol), in dry methanol, Pd/C (100 mg) was carefully added; the dissolved oxygen was removed under vacuum; then, a balloon of hydrogen was mounted, and the mixture was stirred for 8 h at room temperature. The catalyst was filtered off on celite pad, and the filtrate was concentrated under vacuum to yield 15 as a white solid (0.40 g, 88%). R f = 0.3 (5% CH 3 OH:CHCl 3 ), LC-MS (307 MW): m/z 308.15 [M+H] +. 1 H (300 MHz, CDCl 3 ) δ 7.94 (t, 1 H, J = 5.7 Hz, CONHCH 2 ), 7.54 (d, 2 H, J = 8.4 Hz, Ar), 6.78 (t, 1 H, J = 5.7 Hz, NH, NHCO t Bu), 6.52 (d, 2 H, J = 8.4 Hz, Ar), 5.55 (s, 2 H, NH 2 ), 3.17 (q, 2 H, J = 6.0 Hz), 2.91 (q, 2 H, J = 6.0 Hz), 1.44-1.40 (m, 4 H), 1.36 (s, 9 H, t Bu). 13 C (75 MHz, CDCl 3 ) δ 166.1, 155.5, 151.3, 128.5, 121.4, 112.4, 77.3, 39.6, 38.6, 28.2, 27.1, 26.8. 3
Scheme 2: Synthesis of protected (N-(4-aminobenzoyl)spermidine): (i) C 6 H 5 CHO, MgSO 4, Et 3 N, methanol, 12 h, rt, followed by addition of NaBH 4, 3 h, 0 o C; (ii) N-(3-bromopropyl)phthalimide, CH 3 CN:CHCl 3 (1:1), 75 o C, 72 h; (iii) Pd/C, H 2, di-tert-butyl dicarbonate, glacial acetic acid, methanol, 24 h, rt; (iv) NH 2 NH 2 H 2 O, toluene:ethanol (1:1), 70 o C, 10 h; (v) 4-nitro benzoylchloride, Et 3 N, toluene, 8 h, 60 o C; (vi) Pd/C, H 2, methanol, 8 h, rt. Synthesis of N 1 -benzyl-n 4 -(tert-butyloxycarbonyl)-1,4 diaminobutane (16). Amine 13 (6.0 g, 0.031 mol), was solubilized in dry methanol (60 ml); benzaldehyde (3.90 ml, 0.038 mol), MgSO 4 (7.68 g, 0.063 mol) and Et 3 N (1.33 ml, 0.009 mol) were added. The reaction was stirred overnight at room temperature. Subsequently, the reaction was cooled at 0 C and sodium borohydride (6.0 g, 0.160 mol) was added in portions over 1 h and stirred for an additional 1 h at 0 o C, then warmed at room temperature for a further 1 h. The reaction was evaporated under vacuum and water (250 ml) was added to quench the excess of sodium borohydride. The water layer was extracted with EtOAc (3x200 ml), and the organic phase was dried over Na 2 SO 4 and evaporated under reduced pressure. The crude material was purified by silica gel flash chromatography using 1-3% CH 3 OH/CH 2 Cl 2 to give 16 (7.6 g, 85%) as a colourless oil. R f = 0.5 (10% CH 3 OH:CH 2 Cl 2 ), LC-MS (278 MW): m/z 279.1 [M+H] +. 1 H (300 MHz, CDCl 3 ) δ 7.36-7.22 (m, 5 H, Ar), 4.86 (br s, 1 H, NH NHCO t Bu), 3.77 (s, 2 H), 3.11 (q, 2 H, J = 5.4 Hz), 2.64 (t, 2 H, J = 6.6 Hz), 1.69 (br s, 1 H, NH exchangeable with D 2 O), 1.52 (quintuplet, 4 H, J = 3.6 Hz), 1.43 (s, 9 H, t Bu). 13 C (75 MHz, CDCl 3 ) δ 155.7, 139.7, 127.9, 127.7, 126.5, 78.2, 53.4, 48.4, 39.9, 28.0, 27.4, 26.8. Synthesis of N 1 -phatoloyl-n 4 -Benzyl-N 8 -(tertbutyloxycarbonyl)-1,8 diamino-4-azaoctane (17). Compound 16 (2.00 g, 0.0072 mol) was dissolved in a mixture of CH 3 CN:CHCl 3 (1:1), N- (3-bromopropyl)phthalimide (2.50 g, 0.009 mol) and Na 2 CO 3 (1.14 g, 0.011 mol) were added and the solution was stirred for 3 days at 75 o C. After, the solvents were evaporated under vacuum and residue was purified by silica gel flash chromatography using 1-3% CH 3 OH:CH 2 Cl 2 to yield 17 (2.75 g, 82%) as colourless oil. R f = 0.25 (5% CH 3 OH:CH 2 Cl 2 ), LC-MS (465 MW): m/z 466.16 [M+H] +. 1 H (300 MHz, CDCl 3 ): δ 7.85-7.79 (m, 2 H, Ar), 7.73-7.66 (m, 2 H, Ar), 7.36-7.14 (m, 5 H, Ar), 4.77 (br s, 1 H, NH, NHCO t Bu), 3.68 (t, 2 H, J = 7.5 Hz), 3.52 (s, 2 H), 3.07 (br q, 2 H, J = 5.1 Hz), 2.47 (t, 2 H, J = 6.9 Hz), 2.42 (t, 2 H, J = 6.3 Hz), 1.82 (quintuplet, 2 4
H, J = 7.2 Hz), 1.48 (quintuplet, 4 H, J = 3.3 Hz), 1.43 (s, 9 H, t Bu). 13 C (75 MHz, CDCl 3 ): δ 168.2, 155.9, 139.4, 133.7, 132.0, 128.7, 128.0, 126.6, 123.0, 78.7, 58.4, 53.2, 51.0, 40.3, 36.2, 28.3, 27.6, 25.9, 24.3. Synthesis of N 1 -phatoloyl-n 4,N 8 -di-(tert-butyloxycarbonyl)-1,8 diamino-4-azaoctane (18). To a solution of 17 (1.00 g, 0.0022 mol) in dry methanol (30 ml); Pd/C (150 mg), (Boc) 2 O (0.938 g, 0.0043 mol) were added followed by addition of AcOH (1 ml). The dissolved oxygen was removed under vacuum, then a balloon of hydrogen was mounted, and the mixture was stirred for 24 h at room temperature. The catalyst was removed by filtration through a celite pad, and the filtrate was concentrated under vacuum. The crude material was purified by silica gel flash chromatography (1% CH 3 OH:CH 2 Cl 2 ) to yield 18 (0.78 g, 76%) as colourless oil. R f = 0.7 (3% CH 3 OH:CHCl 3 ), LC-MS (475 MW): m/z 476.1 [M+H] +. 1 H (300 MHz, CDCl 3 ): δ 7.83-7.79 (m, 2 H, Ar), 7.71-7.67 (m, 2 H, Ar), 4.67 (br s, 2 H, NH, NHCO t Bu), 3.66 (t, 2 H, J = 7.5 Hz), 3.20-3.07 (m, 6 H), 1.88 (quintuplet, 2 H, J = 7.2 Hz), 1.57-1.45 (m, 4 H), 1.40 (br s, 18 H, 2x t Bu). 13 C (75 MHz, CDCl 3 ): δ 168.2, 155.9, 155.3, 133.8, 131.9, 128.3, 126.8, 123.1, 79.4, 78.9, 64.9, 46.7, 44.9, 40.1, 35.7, 28.3, 27.2, 25.6. Synthesis of N 1 -p-nitrobenzoyl-n 4,N 8 -di-(tert-butyloxycarbonyl)-1,8 diamino-4-azaoctane (20). Compound 18 (3.00 g, 0.0063 mol) was dissolved in a mixture of toluene-ethanol (1:1) (100 ml) and heated at 70 o C. Subsequently, hydrazine monohydrate (0.614 ml, 0.0126 mol) was added and the reaction was stirred for 10 h. The reaction mixture was cooled and the white precipitate was filtered off and the excess of solvents were evaporated under reduced pressure to afford 19 sufficiently pure for the following step. This compound was solubilized in dry DMF (20 ml) and Et 3 N (1.05 ml, 0.0075 mol) was added. 4-Nitro benzoyl chloride (1.17 g, 0.0063 mol), was dissolved in dry toluene (75 ml) and added dropwise. The reaction mixture was stirred for 8 h at 60 o C. After, the mixture was cooled to room temperature and then the solvents were evaporated under reduced pressure and the residue was purified by silica gel flash chromatography using 1-3% CH 3 OH/CH 2 Cl 2 to afford 20, (2.4 g, 77%), as a colourless oil. R f = 0.55 (5% CH 3 OH:CHCl 3 ), LC-MS (494 MW): m/z 495.19 [M+H] +. 1 H (300 MHz, CDCl 3 ): δ 8.24-8.29 (m, 3 H), 8.07 (d, 2 H; J = 8.4 Hz), 4.59 (s, 1 H), 3.42 (q, 2 H, J = 6.0 Hz), 3.35 (t, 2 H, J = 5.7 Hz), 3.09-3.19 (m, 4 H), 1.75 (m, 2 H), 1.50-1.51 (m, 4 H), 1.46 (s, 9 H), 1.42 (s, 9 H). 5
13 C (75 MHz, CDCl 3 ): δ 164.7, 156.8, 155.9, 149.2, 140.9, 128.2, 123.5, 80.1, 79.0, 53.3, 46.5, 43.0, 39.9, 35.8, 28.3, 27.3, 25.5. Synthesis of N 1 -p-aminobenzoyl-n 4,N 8 -di-(tert-butyloxycarbonyl)-1,8 diamino-4-azaoctane (21). To a solution of 20 (0.5 g, 0.001 mol) in dry methanol (10 ml), Pd/C (110 mg) was carefully added to the reaction mixture and the dissolved oxygen was removed under vacuum. Then, a balloon of hydrogen was mounted, and the mixture was stirred for 8 h at room temperature. The catalyst was removed by filtration through a celite pad and the filtrate was concentrated under vacuum to afford 21 (0.41 g, 87%) as a white solid. R f = 0.3 (10% CH 3 OH:CH 2 Cl 2 ), LC-MS (464 MW), m/z 465.23 [M+H] +. 1 H (300 MHz, CDCl 3 ): δ 7.95 (t, 1 H, J = 4.8 Hz, NHCO), 7.55 (dd, 2 H, J 1 = 6.9 Hz, J 2 = 1.8 Hz), 6.80 (t, 1 H, J = 4.2 Hz), 6.52 (dd, 2 H, J 1 = 6.9 Hz, J 2 = 1.8 Hz), 5.56 (s, 2 H), 3.20-3.09 (m, 6 H), 2.93-2.88 (m, 2 H), 1.67 (br quintuplet, 2 H, J = 5.7 Hz), 1.40-1.30 (m, 22 H). 13 C (75 MHz, CDCl 3 ): δ 166.6, 156.1, 151.9, 129.0, 121.8, 113.0, 78.7, 77.8, 46.7, 45.1, 44.7, 37.2, 28.7, 28.5, 27.4, 26.1, 25.7. Scheme 3: Synthesis of protected (N-(4-aminobenzoyl)spermine): (i) CH 2 =CHCN, MeOH, 0 C, 20 h; (ii) (Boc) 2 O, Et 3 N, MeOH, 24 h; (iii) Ni/Raney, Pd/C, LiOH. H 2 O, H 2, dioxane/h 2 O (iv) EtCO 2 CF 3, 6
(Boc) 2 O, K 2 CO 3, MeOH, H 2 O; (v) 4-nitrobenzoylchloride, toluene, 8 h, 60 o C; (vi) Pd/C, H 2, MeOH, 8 h, rt. Synthesis of 3-[4-(2-cyano-ethylamino)-butylamino] propionitrile (22). A solution of 1,4- butane diamine (4.385 g, 49.7 mmol) in absolute MeOH (10 ml) was cooled in an ice bath, and acrylonitrile (5.279 g, 99.5 mmol), previously dissolved in absolute MeOH (100 ml), was added dropwise at 0 C. The nearly colorless solution was allowed to warm to room temperature and stirred for additional 20 h. Subsequently, the solvent was removed at reduced pressure, and the crude material 22 was used for the next step without further purification. Yield: quantitative. R f = 0.36 (15% MeOH-CHCl 3 ), LC/MS (MW 194): m/z 217 [M+Na + ], 195 [M+H + ]. 1 H (300 MHz, CDCl 3 ) δ 2.86 (t, 4 H, J = 6.6 Hz), 2.61-2.57 (m, 4H), 2.46 (t, 4 H, J = 6.6 Hz), 1.49-1.44 (m, 4 H), 1.30-1.20 (br s, 2 H). 13 C (75 MHz, CDCl 3 ) δ 118.8, 48.9, 45.0, 27.6, 18.4. Synthesis of N 4,N 9 -di(tert-butyloxycarbonyl)-3-(4-[(2-cyanoethyl)-amino]butylamino) propionitrile (23). A solution of 22 (4.0 g, 20.6 mmol) in MeOH (85 ml) was cooled to 0 C, and Et 3 N (6.32 ml, 45.4 mmol) was added. After, (Boc) 2 O (43.3 g, 9.45 mmol) dissolved previously in MeOH (90 ml) was added dropwise. The mixture was stirred at room temperature for 24 h, then the solvent was removed under reduced pressure, and the residue was purified by silica gel flash chromatography (5% MeOH-CH 2 Cl 2 ) to afford 23 as colorless oil, which crystallized on standing at room temperature. Yield: quantitative. R f = 0.57 (5% MeOH-CH 2 Cl 2 ), LC/MS (MW 264): m/z 265.2 [M+H] +. 1 H (300 MHz, CDCl 3 ) δ 3.41 (t, 4 H, J = 6.6 Hz), 3.24 (br t, 4 H, J = 6.3 Hz), 2.70-2.45 (m, 4 H), 1.49-1.39 (m, 11 H). 13 C (75 MHz, CDCl 3 ) δ 155.0, 118.1, 80.4, 47.7, 43.5, 28.3, 25.6, 17.3. Synthesis of N 1,N 4 -di(tert-butyloxycarbonyl)-n 1,N 4 -di(3-aminopropyl)-1,4-butanediamine (24). Compound 23 (5.5 g, 14.0 mmol) was dissolved in a mixture of dioxane/water 4:1 (350 ml) and treated with Raney nickel (9.8 g) (50% suspension in water), Pd/C (300 mg,) and lithium hydroxide monohydrate (1.2 g, 29.3 mmol). The reaction mixture was stirred under hydrogen atmosphere at room temperature until disappearance of the starting material. The catalyst was filtered off, and the solvents were removed in vacuo. The residue was dissolved in CH 2 Cl 2 and the organic layer was washed with water (150 ml). After phase separation, the 7
aqueous phase was extracted twice with CH 2 Cl 2, the combined organic layers were dried over Na 2 SO 4, and the solvent was removed under reduced pressure. Amine 24 was obtained as yellow, highly viscous oil. Yield: quantitative. R f = 0.36 (35% aq NH 3 /MeOH 1:10), LC/MS (MW 402): m/z 403.2 [M+H] +, 303.2 [M+H-Boc] +, 203.2 [M+H-2xBoc] +. 1 H (300 MHz, CDCl 3 ) δ 3.30-2.90 (m, 8 H), 2.66 (t, 4 H, J = 6.9 Hz), 1.72-1.55 (m, 8 H), 1.48-1.27 (m, 22 H). 13 C (75 MHz, CDCl 3 ) δ 155.8, 79.4, 46.6, 44.0, 39.2, 31.8, 28.6, 25.5. Synthesis of (N 1,N 4,N 9 -tri-tert-butyloxycarbonyl)-1,12-diamino-4,9-diazadodecane (25). To a solution of 24 (10.0 g, 24.9 mmol) in methanol (400 ml), at -78 C under nitrogen, ethyl trifluoroacetate (3.5 g, 24.9 mmol) was added dropwise over 30 min. Stirring was continued for a further 30 min, then the temperature was increased to 0 C to afford predominantly the monotrifluoroacetamide. Without isolation, the remaining amino functional group was quantitatively protected by dropwise addition of an excess of (Boc) 2 O (8.15 g, 37.3 mmol) in MeOH (100 ml) over 10 min. The reaction was then warmed at room temperature and stirred for a further 15 h to afford the fully protected polyamine. R f = 0.6 (EtOAc). The trifluoroacetate protecting group was removed in situ by adding K 2 CO 3 (17.2 g, 124.4 mmol) and H 2 O (30 ml). The reaction was heated at reflux for 3 h, and subsequently the solvent was evaporated, water was added and the residue was extracted with CHCl 3, (3 100 ml). The combined organic layers were dried over Na 2 SO 4 concentrated under reduced pressure and the crude was purified by column chromatography over silica gel (CH 2 Cl 2 /MeOH/NH 3 70:10:1 to 50:10:1) to afford 25 as a colourless homogeneous oil. Yield: 70%. R f = 0.5 (CH 2 Cl 2 /MeOH/NH 3 50:10:1) LC/MS (MW 502): m/z 503.2 [M+H] +. 1 H (300 MHz, CDCl 3 ) δ 5.40-4.70 (br s, 1 H), 3.40-3.00 (m, 10 H), 2.64 (t, 2 H, J = 6.9 Hz), 1.98 (s, 2 H), 1.60-1.48 (m, 4 H), 1.42-1.30 (m, 31 H). 13 C (75 MHz, CDCl 3 ) (Relevant peaks, rotamers are present) δ 156.0, 156.0, 79.5, 79.3, 46.7, 46.6, 43.8, 43.7, 43.7, 38.9, 37.3, 37.3, 32.5, 28.4, 25.9, 25.6. Synthesis of N 1 -p-nitrobenzoyl-n 4,N 9,N 12 -tri-(tert-butyloxycarbonyl)-1,12-diamino-4,9- diazadodecane (26). To a solution of 25 (6 g, 11.95 mmol), in toluene (200 ml), Et 3 N (5.83 ml, 41.8 mmol) was added, followed by addition dropwise of p-nitrobenzoyl chloride (3.32 g, 17.9 mmol), which was before dissolved in toluene (75 ml). The reaction was stirred at 60 C for 8 h, then the solvents were evaporated under reduced pressure and the residue was purified by silica 8
gel chromatography (cyclohexane/acoet 2:3) to afforded 26 as yellowness solid. Yield: 87%. (MW 651): m/z 575.0 [M+Na-Boc] +. 1 H NMR (300 MHz, CDCl 3 ) δ 8.26 (br s, 1 H), 8.21 (d, 2 H, J = 9.0 Hz), 8.02 (br d, 2 H, J = 9.0 Hz), 5.01 (br s, 1 H, NH, NHCO t Bu), 3.42-2.92 (m, 10 H), 1.70 (br t, 2 H, J = 5.1 Hz), 1.60 (br t, 2 H, J = 5.1 Hz), 1.48-1.41 (m, 6 H), 1.41 (s, 9 H, t Bu), 1.39 (s, 9 H, t Bu), 1.36 (s, 9 H, t Bu). 13 C (75 MHz, CDCl 3 ) (Relevant peaks) δ 164.8, 156.9, 156.0, 149.4, 140.2, 128.3, 123.6, 80.2, 79.6, 78.9, 46.8, 46.2, 44.2, 43.8, 43.1, 37.6, 37.4, 35.8, 27.3, 25.9, 25.6. Synthesis of N 1 -p-aminobenzoyl-n 4,N 9,N 12 -tri-(tert-butyloxycarbonyl)-1,12-diamino-4,9- diazadodecane (27). To a solution of 26 (6.67 g, 10.4 mmol) in dry methanol (300 ml), Pd/C (500 mg) was carefully added to the reaction mixture, and the dissolved oxygen was removed under vacuum. Then, a balloon of hydrogen was mounted, and the mixture was stirred for 8 h at room temperature. The catalyst was removed by filtration through a celite pad, and the filtrate was concentrated under vacuum to afford the crude material 27 as white solid. Yield: quantitative. LC/MS (MW 621): m/z 622.1 [M+H] +, 522.2 [M+H-Boc] +. 1 H (300 MHz, DMSOd 6 ) δ 7.96 (t, 1 H, J = 5.7 Hz), 7.55 (d, 2 H, J = 8.7 Hz), 6.74 (br s, 1 H), 6.53 (d, 2 H, J = 8.7 Hz), 5.57 (s, 2 H), 3.26-2.99 (m, 10 H), 2.88 (q, 2 H J = 6.6 Hz), 1.74-1.60 (m, 2 H), 1.60-1.49 (m, 2 H), 1.46-1.27 (m, 31 H). 13 C (75 MHz, DMSO-d 6 ) (Relevance peaks) δ 166.6, 156.0, 155.1, 151.9, 129.0, 121.8, 112.9, 78.7, 77.9, 46.9, 44.8, 38.0, 37.2, 34.2, 28.7, 28.5, 26.1, 25.6. 9
Figure SI-1. Cell viability of A549 cell line assessed by the modified LDH assay after 72 h exposure to f- SWCNTs (1-100 µg/ml). LDH assay for f-swcnts 1b, 3b and 5b. Cell viability was not affected by exposure to f-swcnts for 72 h. Ten percent DMSO and 1 mm H 2 O 2 in complete cell culture medium were used as positive controls. The vehicle (5% dextrose) was used as a negative control. Percentage viability was expressed as percentage of control untreated cells (*, p 0.05). Figure SI-2. Cell viability of A549 cell line assessed by the modified LDH assay after 72 h exposure to f- CNTs (1-100 µg/ml). LDH assay of A549 cells treated with f-swcnts 7b, 9b, and 11b, and f- MWCNTs 8b, 10b, and 12b. Cell viability decreased at 100 µg/ml concentration of all f-cnts. Ten percent DMSO and 1 mm H 2 O 2 in complete cell culture medium were used as positive controls. The vehicle (5% dextrose) was used as a negative control. Percentage viability was expressed as percentage of control untreated cells (*, p 0.05). 10
Supplementary Material (ESI) for Journal of Materials Chemistry Figure SI-3. Electrophoretic motility of polyamine-cnts 9b, 10b, 7b and 8b (reconstituted at 1 mg/ml in 5% dextrose) complexed with sirna. Various N/P (+/-) ratios of the four conjugates were complexed to a fixed 0.5 µg of sirna. Figure SI-4. (Top) A549 cells incubated with 5% dextrose, and (Bottom) cells incubated with 10 µg/ml of polyamine-cnt 8b for 24 hours showing some interaction between the cells and the tubes. Red arrows are pointing at the CNTs interacting with the cells. 11