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1 Electronic Supplementary Information B-Z Transition of (da-t) n Duplex Induced by Spermine Porphyrin-Conjugate via an Intermediate DNA Conformation Hideaki Sasaki and Shigeki Sasaki* Graduate School of Pharmaceutical Sciences, Kyushu University, Maidashi, Higashi-ku, Fukuoka , Japan, Fax &Tel: ; saski@phar.kyushu-u.ac.jp. CONTENTS 1. Figure S1 Polyamine-porphyrin conjugates (1 and 2) and their control 2 compounds (3,4,5). 2. Scheme S1 Synthetic route 2 3. Experimental: Synthesis 3 4. Figure S2. HPLC analysis of the purified 1, 2 and Figure S3. CD Titration by spermine-por (1) with poly[(da-t) 7 ] Figure S4. CD Titration Data with [d(at) 7 ] Figure S5. CD Titration Data with [(dg-dc) 7 ] Figure S6. CD changes of [d(at) 7 ] 2 or [(dg-dc) 7 ] 2 by the Zn 2+ or Cu 2+ complex of Figure S7. UV spectrum changed by the binding with DNA Figure S8. ITC results for the binding of 2 with [d(at) 7 ] Table S1. Thermodynamic parameters for the binding of 1 with [(da-t) 7 ] Table S2. Thermodynamic parameters for the binding of 2 with [(da-t) 7 ] Figure S9. The ITC analysis of the binding of spermine-por (1) and triamine-por 14 (2) with [(dg-dc) 7 ] Table S3. Thermodynamic parameters for binding of 1 with [(dg-dc) 7 ] Table S4. Thermodynamic parameters for binding of 2 with [(dg-dc) 7 ] Figure S10. The ITC analysis of the binding of trimine-por (2) with 15 poly[(da-t)] Table S5. Thermodynamic parameters for binding of 2 with poly[(da-t)] References H-NMR of H-NMR of

2 Figure S1 Polyamine-Porphyrin conjugates (1 and 2) and their control compounds (3,4,5). (a) 1) Ethyl trifluoroacetate, MeOH, -78 C to 0 C, 1 h, 2) (Boc) 2 O, stirred at 0 C to r.t. 3 h. 3) NsCl, Et 3 N, CH 2 Cl 2, 0 C, 2 h, 4) 1,3-dibromopropane, K 2 CO 3, DMF, stirred at 0 C to r.t. 13 h. (b) 1) K 2 CO 3, DMF, r.t. 24 h, 2) thiophenol, K 2 CO 3, DMF, r.t. 3 h, 3) (Boc) 2 O, CH 2 Cl 2, 0 C to r.t. 12 h, 4) CH 3 I, CH 2 Cl 2, r.t. 14 h, 5) 5%HCl, MeOH, r.t. 5 h. (c) 1) K 2 CO 3, DMF, r.t. 24 h, 2) thiophenol, K 2 CO 3, DMF, r.t. 3 h, 3) 5%HCl, MeOH, r.t. 5 h. (d) 1) K 2 CO 3, DMF, r.t. 24 h, 2) CH 3 I, CH 2 Cl 2, r.t. 14 h. (e) CH 3 I, CH 2 Cl 2, r.t. 14 h. 2

3 Experimental General: The 1 H NMR (400 MHz) spectra were recorded by Varian UNITY-400 and INOVA-500 spectrometers. The UV-vis spectra measurement was obtained using a Beckman-Coulter DU-800. The fluorescent spectrum was obtained using a JASCO FP-750. The ESI mass spectra were recorded by an Applied Biosystems Mariner System 5299 spectrometer. The MALD-TOF/MS spectra were recorded by a Brucker Daltonics Microflex instrument. DNA oligomers were synthesized by the automated DNA synthesizer using the conventional amidite chemistry. The DMTr-protected DNA was cleaved from the resin with NH 4 OH solution and purified by HPLC equipped with a ODS column using 0.1 M TEAA-CH 3 CN linear gradient system. The DMTr protecting group of the purified ODN was cleaved in 10 % aqueous AcOH, and the mixture was washed with ether. The purity of the ODN was confirmed by MADI-TOF MS and UV measurements. The concentration of the stock solution was estimated by the UV absorbance at 260 nm, which was further confirmed to be less than 5% error by HPLC analysis of the component nucleosides after enzymatic hydrolysis using BAP and VPDE. Poly[(dA-T)] 2 was purchased from Sigma (P UN). Bromopropyl derivative of TriBoc and Ns-protected spermine (S1) A solution of ethyl CF3COOEt (0.88 ml, 7.41 mmol) in dry MeOH (7 ml) was added to a solution of spermine (1.5 g, 7.41 mmol) in dry MeOH (13 ml) during 30 min at -78 C under Ar, and the mixture was stirred at the same temperature for 1h. The mixture was raised to 0 C, followed by the addition of di(tert-bu)carbonate (6.8 ml, mmol) in dry MeOH (10 ml) during 30 min, then the whole was stirred for 3 h at room temperature. The solvent was evaporated to half volume, and diluted with 28 % aqueous NH4OH (20 ml), and the mixture was stirred for 15 h at room temperature. The reaction mixture was diluted with water (50 ml), extracted with CH2Cl2 (70 ml 2), and the organic layers were washed with aqueous saturated NaCl (100 ml 2), dried over Na2SO4, evaporated to dryness. The residue was column chromatographed (silica gel, 28 % NH4OH:MeOH:CH2Cl2=1:10:100) to give the tri-boc derivative of spermine (1.65 g, 3.28 mmol) as a colorless oil in 44% yield. A solution of 2-nitorobenzensulfonylchloride (1.09 g, 4.92 mmol) in dry CH2Cl2 (22.8 ml) was added into a solution of tri-boc derivative of spermine obtained above (1.65 g, 3.28 mmol) and triethylamine (2.29 ml, 16.4 mmol) in dry CH2Cl2 (10mL) at 0 C, and the mixture was stirred for 2 h at 0 C. The reaction mixture was diluted with CH2Cl2 (40mL), washed successively with 1M KHSO4 (80 ml), saturated NaHCO3 (80 ml), saturated NaCl (100 ml 2), dried over Na2SO4, evaporated to dryness. The residue was column chromatographed (silica gel, CH2Cl2: MeOH =1:10:00) to give the Nosyl derivative of tri-boc-spermine (2.24 g, 3.26 mmol) as a colorless oil in 66% yield. The Nosyl derivative of tri-boc-spermine (2.02 g, 2.95 mmol) obtained as above was azeotropically dried with dry acetonitrile (20 ml 2), and dissolved in dry DMF (15 ml), followed by the addition of dried K2CO3(2.04 g, mmol), and the mixture was stirred at 3

4 0 C. Into the above mixture was added slowly 1,3-dibromopropane (1.5 ml, 14.6 mmol), and the mixture was stirred for 13 h at room temperature. The reaction mixture was diluted with water (40 ml), extracted with CH2Cl2 (40 ml 2), and the organic layers were washed with aqueous saturated NaCl (100 ml 2), dried over Na2SO4, evaporated to dryness. The residue was column chromatographed (silica gel, CH2Cl2:MeOH =70:1) to give the bromopropyl derivative of triboc and Ns-protected spermine (S1) (2.12 g, 2.63 mmol) as a pale oil in 89% yield. IR(cm -1 ) 2974, 1678, H-NMR(400MHz, CDCl3) ppm (m, 1H), (m, 2H), (m, 1H), (m, 2H), (m, 2H), (m, 2H), 3.14 (br, 12H), 2.09 (br, 2H), 1.80 (br, 2H), 1.61 (br, 2H), 1.43 (s, 27H). ESI/MS (m/z): (100.0%), (99.7%), calculated for C34H58BrN5O10S, found [M+H] +, [M+H] +. Bromopropyl derivative of diboc and Ns-protected triamine (S2) A solution of ethyl CF3COOEt (0.23 ml, 1.0 mmol) in dry MeOH (7 ml) was added to a solution of triamine (1.07 g, 7.63 mmol) in dry MeOH (13 ml) during 30 min at -78 C under Ar, and the mixture was stirred at the same temperature for 1 h. The mixture was raised to 0 C, followed by the addition of di(tert-bu)carbonate (6.8 ml, mmol) in dry MeOH (10 ml) during 30 min, then the whole was stirred for 3 h at room temperature. The solvent was evaporated to dryness. The residue was column chromatographed (silica gel, 28 % NH4OH:MeOH:CH2Cl2=1:10:100) to give the diboc derivative of triamine (864 mg, 3.74 mmol) as a colorless oil in 49% yield. A solution of 2-nitorobenzensulfonylchloride (2.5 g, 11.2 mmol) in dry CH2Cl2 (20 ml) was added into a solution of di-boc derivative of triamine obtained above (864 mg, 3.74 mmol) and triethylamine (1.0 ml, 7.48 mmol) in dry CH2Cl2 (17 ml) at 0 C, and the mixture was stirred for 2 h at 0 C. The reaction mixture was diluted with CH2Cl2 (40mL), washed successively with 1M KHSO4 (80 ml), saturated NaHCO3 (80 ml), saturated NaCl (100 ml 2), dried over Na2SO4, evaporated to dryness. The residue was column chromatographed (silica gel, CH2Cl2: MeOH =1:10:00) to give the Nosyl derivative of di-boc-triamine (2.1 g, 3.51 mmol) as a colorless oil in 94% yield. The Nosyl derivative of di-boc-triamine (2.1 g, 3.51 mmol) obtained as above was azeotropically dried with dry acetonitrile (20 ml 2), and dissolved in dry DMF (18 ml), followed by the addition of dried K 2 CO 3 (2.04 g, 17.6 mmol), and the mixture was stirred at 0 C. Into the above mixture was added slowly 1,3-dibromopropane (1.8 ml, 17.6 mmol), and the mixture was stirred for 13 h at room temperature. The reaction mixture was diluted with water (40 ml), extracted with CH 2 Cl 2 (40 ml 2), and the organic layers were washed with aqueous saturated NaCl (100 ml 2), dried over Na 2 SO 4, evaporated to dryness. The residue was column chromatographed (silica gel, CH 2 Cl 2 :MeOH =60:1) to give the bromopropyl derivative of diboc and Ns-protected triamine (S2) (2.3 g, 3.2 mmol) as a pale oil in 91% yield. IR (cm -1 ) 2972, 1706, 1544, 1369, H-NMR (400MHz, CDCl 3 ) ppm (m, 2H), (m, 4H), (m, 2H), 3.39 (t, 4H, J=7.32Hz), (m, 6H), (m, 4

5 2H), 2.07 (quint, 2H, J=6.79Hz), 1.86(quint, 2H, J=7.52Hz), 1.71(quint, 2H, J=6.56Hz), 1.41(s, 9H). ESI-MS(m/z) (100.0%), (99.6%) calculated for C 26 H 36 BrN 5 O10S 2, found [M+H] +, [M+H] +. Synthesis of Spermin-por (1) 5,15-Di(4-hydroxylphenyl)-10,20-di(pyridin-4-yl)porphyrin (DHPhPyP) was prepared according to the literature. 1 A mixture of DHPhPyP (20 mg, 0.03 mmol), bromopropyl derivative of triboc and Ns-protected spermine (S1) (72.7 mg, 0.09 mmol) and K 2 CO 3 (33.2 mg, 0.24 mmol) in DMF (1.5 ml) was stirred at room temperature for 24 h under Ar. The mixture was diluted with CHCl 3 (10 ml) and washed with saturated NaCl (5 ml 2) and dried over Na 2 SO 4, then evaporated to dryness. The residue was column chromatographed (silica gel, CHCl 3 :MeOH =70:1) to give the non-methylated derivative of the Boc and Ns-protected spermine-por (31 mg, mmol) as purple solids in 50 %. A solution of the above product (34.3 mg, mmol), thiophenol (4.9 L, mmol) and K 2 CO 3 (12.2 mg, mmol) in dry DMF (1.6 ml) was stirred for 3 h at room temperature, then the solvents were evaporated to dryness. The residue was column chromatographed (silica gel, CHCl 3 only to CHCl 3 :MeOH =90:1) to give the Ns deprotected Boc-spermine-por (22 mg, mmol) as a purple oil in 81 %. Di-tert-butyl dicarbonate (12 L, mmol) was added into a solution of the Ns deprotected Boc-spermine-por (30 mg, mmol) obtained above in dry CH 2 Cl 2 (1.7 ml) at 0 C, and the mixture was stirred for 12 h at room temperature, then the solvents were evaporated to dryness. The residue was column chromatographed (silica gel, CHCl 3 :MeOH =45:1) to give the triboc protected-spermine-por (27.2 mg, mmol) as a purple oil in 27 %. A solution of MeI (0.55 ml, 8.8 mmol) and the above triboc protected-spermine-por (42 mg, mmol) in dry CH 2 Cl 2 (0.2 ml) was stirred for 13 h at room temperature, and the solvents were evaporated to dryness.. The residue was column chromatographed (silica gel, CHCl 3 :MeOH =10:1) to give the methylated triboc protected-spermine-por (39.5 mg, 0.02 mmol) as a purple oil in 91 %. The above product (39.5 mg, 0.02 mmol) was dissolved in MeOH-5% aqueous HCl (1 ml-1.5 ml), and the mixture was stirred for 14 h at room temperature, then the solvents were evaporated to dryness. The residue was purified by HPLC and the purified compound as green solids (23.2 mg, mmol, 95% yield) was treated with 5% aqueous HCl to produce the HCl salt of Spermin-por (1). mp>300 C, UV max =423nm (3uM, H 2 O), IR(cm -1 ) 3323, 2926, H-NMR (400MHz, D 2 O) ppm 9.41 (d, 4H, J=6.1Hz), 8.96 (s, 12H), 8.52 (d, 4H, J=6.7 Hz), 7.65 (d, 4H, J=8.5 Hz), 4.78 (s, 6H), 4.51 (br, 4H), 3.47 (t, 4H, J=7.6 Hz), 3.28 (t, 4H, J=7.9 Hz), 3.20 (t, 4H, J=7.9 Hz), (m, 24H), 2.40 (br, 4H), 2.20 (t, 4H, J=7.9 Hz). ESI-MS(m/z) calculated for [C 70 H 96 N 14 O 2 ] 4+, found [M] 4+. The concentration of a stock solution of spermin-por (1) was determined by measuring integration of signals in 1 H-NMR in the presence of maleic acid as the internal standard. 5

6 Figure S2. HPLC analysis of the purified 1, 2 and 4. HPLC conditions: A: 0.05 % TFA in water, B: 0.05 % TFA in MeOH, B: 39 %/30 min, linear gradient, monitored at 423 nm. Synthesis of Triamine-por (2) The triamine-por (2) was synthesized using DHPhPyP as the intermediate and the similar procedures described for the synthesis of Spermine-por (1) except for the use of bromopropyl derivative of diboc and Ns-protected triamine (S2) as the conjugation partner. The crude product was purified by HPLC, and the purified compound as green solids was treated with 5% aqueous HCl to produce the HCl salt of Triamne-por (2). mp>300 C, UV max =423nm (3uM, H 2 O), IR(cm -1 ) 3430, 2922, H-NMR (400MHz, D 2 O) ppm 9.38 (d, 4H, J=6.0 Hz), 8.99 (d, 4H, J=4.7 Hz), 8.95 (d, 4H, J=4.7 Hz), 8.46 (d, 4H, J=8.2 Hz), 7.62 (d, 2H, J=8.5 Hz), 4.81 (s, 6H), 4.55 (t, 4H, J=5.5 Hz), 3.47 (t, 4H, J=7.7 Hz), (m, 20H), 2.39 (t, 4H, J=6.2 Hz), 2.21 (t, 4H, J=5.6 Hz), 2.10 (t, 4H, J=5.50Hz). ESI-MS(m/z) calculated for [C 62 H 76 N 12 O 2 ] 2+, found [M] 2+. The concentration of a stock solution of triamie-por (2) was determined by measuring integration of signals in 1 H-NMR in the presence of maleic acid as the internal standard. Non methylated derivative (3). The intermediate for the synthesis of 2 was deprotected without methylation and purified by HPLC. Green solids, mp>300 C, UV max =422nm (3uM, H 2 O), IR(cm -1 ) 3418, 2331, 1658, H-NMR (400MHz, D 2 O) ppm 9.44 (br, 4H), 9.25 (br, 4H), 8.98 (br, 4H), 8.94 (br, 4H), 8.59 (br, 4H), 7.69(br, 4H), 4.53 (br, 4H), (m, 20H), 2.41 (br, 4H), 2.21 (br, 4H), 2.09(br, 4H). ESI-MS(m/z) calculated for [C 60 H 70 N 12 O 2 ] +, found [M+H] +. Polyether derivative (4). A mixture of DHPhPyP (20 mg, 0.03 mmol), 1-iodo-3,6,9,12-tetraoxapentadec-14-yne (31.8 mg, mmol) and Na 2 CO 3 (34.6 mg, 0.25 mmol) in DMF (1.5 ml) was stirred at room temperature for 24 h 6

7 under Ar. The mixture was diluted with CHCl 3 (10 ml) and washed with saturated NaCl (5 ml 2) and dried over Na 2 SO 4, then evaporated to dryness. The residue was column chromatographed (silica gel, CHCl 3 :MeOH =70:1) to give the polyether conjugated porphyrin (12.2 mg, mmol) as purple solids in 38 %. A solution of MeI (0.28 ml, 4.53 mmol) and the above compound (12.2 mg, mmol) in dry CH 2 Cl 2 (0.2 ml) was stirred for 12 h at room temperature, and the solvents were evaporated to dryness. The residue was crystalized in CHCl 3 at 0 C, and purple solids were collected (11.3 mg, 0.01 mmol, 90 % yield). mp>300 C, UV max =423nm (3uM, H 2 O), IR(cm -1 ) 3313, 2922, 1741, 1247, H-NMR (400MHz, D 2 O) ppm 9.44 (d, 4H, J=6.11Hz), 8.99 (s, 12H), 8.12 (d, 4H, J=8.54Hz), 7.43 (d, 4H, J=8.54Hz), 4.69 (s, 6H), 4.41 (br, 4H), 4.14 (s, 4H), 3.95 (br, 4H), 3.73 (t, 4H, J=4.87Hz), 3.65 (t, 4H, J=4.58Hz), (m, 16H), 3.50 (s, 2H), (s, 2H). ESI-MS(m/z) calculated for C 66 H 70 N 6 O 2+ 10, found [M] 2+. Methoxy porphyrin derivative (5) 5,15-Di(4-methoxylphenyl)-10,20-di(pyridin-4-yl)porphyrin 1 was methylated with MeI and the product was purified as above. Black solid, mp>300 C, UV max =425 nm (3uM, H 2 O), IR(cm -1 ) 2974, H-NMR (400MHz, DMSO) ppm 9.43 (s, 4H), 8.99 (m, 12H), 8.13 (d, 4H, J=7.3 Hz), 7.42 (d, 4H, J=7.3 Hz), 4.69 (s, 6H), 4.06 (s, 6H), (s, 2H). ESI-MS(m/z) calculated for C 46 H 38 N 6 O + 22, found [M] 2+. CD Measurement. CD spectra were recorded between 500 nm and 200 nm by a Jasco-J720 spectrometer using a 200 L 2 mm quartz glass cell at the constant temperature at 20 C. A solution of 15 M of DNA in the buffer containing 1 mm Na cacodylate, 100 mm NaCl at ph 7 was used for the CD measurement. After the addition of the ligand solution into the ODN solution, the solution was mixed and kept at the same temperature for 10 min before CD measurement. All CD data were blank corrected, and reported as the average of 7 scans. 0 M 0 M 75 M 75 M Spermine-por (1) Triamine-por (2) Figure S3. CD Titration of [poly(da-t)] 2. CD spectrum became unstable at concentrations higher than 75 M, probably because of small sedimentations. 7

8 5 -ATATATATATATAT-3 3 -TATATATATATATA-5 5 -ATATATATATATAT-3 3 -TATATATATATATA-5 5 -ATATATATATATAT-3 3 -TATATATATATATA-5 5 -ATATATATATATAT-3 3 -TATATATATATATA-5 Figure S4. CD Titration Data with [(da-t) 7 ] 2 8

9 5 -GCGCGCGCGCGCGC-3 3 -CGCGCGCGCGCGCG-5 5 -GCGCGCGCGCGCGC-3 3 -CGCGCGCGCGCGCG-5 5 -GCGCGCGCGCGCGC-3 3 -CGCGCGCGCGCGCG-5 5 -GCGCGCGCGCGCGC-3 3 -CGCGCGCGCGCGCG-5 Figure S5. CD Titration Data with [(dg-dc) 7 ] 2 9

10 Metal complexes of 1 were prepared according to the reported procedure, 2,3 and CD spectrum was measured as described above. [d(at) 7 ] 2 [d(gc) 7 ] 2 [d(at) 7 ] 2 [d(gc) 7 ] 2 Figure S6. CD changes of [d(at) 7 ] 2 or [d(gc) 7 ] 2 by the Zn 2+ - or Cu 2+ complex of 1. 10

11 Measurement of UV spectrum. A DNA solution was added into a ligand solution containing 3 M spermin-por (1) in the buffer containing 1 mm Na cacodylate, 100 mm NaCl at 25 C and ph 7.0. DNA concentration increased from 0 to 90 M. Spermine-por (1) (A) [d(at) 7 ] 2 (B) [d(gc) 7 ] 2 (C) Poly[d(AT)] 2 /poly d(ta) Triamine-por (2) (D) [d(at) 7 ] 2 (E) [d(gc) 7 ] 2 (F) Poly[d(AT)] 2 /poly d(ta) Figure S7. UV spectrum changed by the binding with DNA. Larger effects on both red-shift and hypochromicity (B) and (E) indicate interaction binding mode. 11

12 Isothermal titration calorimetry (ITC). The ligand solution was prepared by dissolution in the final dialysate obtained from the ODN dialysis, and was titrated into 15 μm DNA in 1 mm Na cacodylate buffer containing 100 mm NaCl at ph 7.0 and 20.0 C. The first injection was 2 L and then 5 L each of the ligand stock was added to the ODN solution. The data obtained by the first injection was omitted from the analysis. The ODN concentrations are reported as molar concentrations of double strands. All samples were degassed prior to use by application of vacuum with simultaneous stirring on a magnetic stir plate. A NanoITC SV (TA Instruments) was used for the calorimetric measurements. The calorimetric dilution heats were subtracted from raw the binding heats, and binding isotherm was analyzed by the combination of independent curve fitting method. Process A Process C Process B Figure S8. ITC results for the binding of 2 with [d(at) 7 ] 2 Table S1. Thermodynamic parameters for the binding of 1 to [(da-t) 7 ] 2 Process Ka(M -1 ) n G (kj/mol) H (kj/mol) G (kj/mol) A 2.49 x B 7.74 x C 2.90 x Table S2. Thermodynamic parameters for the binding of 2 with [(da-t) 7 ] 2. Process Ka(M -1 ) n G (kj/mol) H (kj/mol) G (kj/mol) A 6.1 x B 2.45 x C 6.3 x

13 Figure S9. The ITC analysis of the binding of spermine-por (1) and triamine-por (2) with [(dg-dc) 7 ] 2. The ligand solution was prepared by dissolution in the final dialysate obtained from the ODN dialysis, and was titrated into 15 μm DNA in 1 mm Na cacodylate buffer containing 100 mm NaCl at ph 7.0 and 20.0 C. The first injection was 2 L and then 10 L of stock solution of the ligand (200 M) was added each time. Table S3. Thermodynamic parameters for binding of 1 with [(dg-dc) 7 ] 2. Ka(M -1 ) n G (kj/mol) H (kj/mol) G (kj/mol) x x Table S4. Thermodynamic parameters for binding of 2 with [(dg-dc) 7 ] 2. Ka(M -1 ) n G (kj/mol) H (kj/mol) G (kj/mol) x x

14 Figure S10. The ITC analysis of the binding of amine-por (2) with poly[(da-t)] 2 The ligand solution was prepared by dissolution in the final dialysate obtained from the ODN dialysis, and was titrated into 15 μm DNA in 1 mm Na cacodylate buffer containing 100 mm NaCl at ph 7.0 and 20.0 C. The first injection was 2 L and then 10 L each of the ligand stock solution (400 mol) was added to the ODN solution. Table S5. Thermodynamic parameters for binding of 2 with poly[(da-t)] 2. Ka(M -1 ) n G (kj/mol) H (kj/mol) G (kj/mol) x x References 1. Tsuchiya, Y.; Arimura, T.; Tachiya, M. J. Photopolym. Sci. Technol. 2006, 19, Pasternack, R. F.; Francesconi, L.; Raff, D.; Spiro, E. Inorg. Chem., 1973, 12, B ütje, K.; Nakamoto, K. Inorg. Chim. Acta 1990,167,

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