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Synlett 2008(7): 1066-1070  
DOI: 10.1055/s-2008-1072659
LETTER
© Georg Thieme Verlag Stuttgart · New York

First Route to Phosphoramidites of N 2-Hydrazinoaryl- and N 2-Azoaryl-dG Adducts and Their Site-Selective Incorporation into DNA Oligonucleotides

Nicolas Böge, Marcus Schröder, Chris Meier*
Institute of Organic Chemistry, Department of Chemistry, Faculty of Science, University of Hamburg, Martin-Luther-King Platz 6, 20146 Hamburg, Germany
Fax: +49(40)428382495; e-Mail: chris.meier@chemie.uni-hamburg.de;
Further Information

Publication History

Received 22 January 2008
Publication Date:
31 March 2008 (online)

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Abstract

N 2-Hydrazinoaryl and N 2-azoaryl-dG adducts of borderline carcinogens and the bladder and breast carcinogen 4-aminobiphenyl were prepared using Pd-catalyzed cross-coupling chemistry. After conversion into the phosphoramidites site-specifically damaged oligonucleotides were prepared by automated DNA synthesis. DNA Hybrids were studied with respect to their thermal stability (UV melting-temperature analysis) and circular dichroism.

Key words

DNA damage - chemical carcinogenesis - aromatic amines - N 2-adducts - cross-coupling

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General Procedure for the Synthesis of the N ² -Arylhydrazino Adducts (7a,b)
Under nitrogen atmosphere 5 (1 equiv), racemic BINAP (30 mol%), Pd2dba3 (10 mol%), K3PO4 (2 equiv), triethylammonium chloride (0.1 equiv), and arylhydrazine (2 equiv) were suspended in dry DME and stirred at 80 °C. After the reaction was completed, sat. NaHCO3 and NaCl solution were added. The layers were separated and the aqueous layer was extracted three times with EtOAc. The combined organic layers were dried over Na2SO4 and the solvent was removed in vacuo. The product was isolated by column chromatography using PE-EtOAc (10% → 35%).

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Compound 7a: The general procedure was conducted with 3.05 g of 5 (4.40 mmol) and 1 mL of 6a (8.8 mmol); yield 2.52 g (3.52 mmol, 80%) of 7a as a slightly brown solid. 1H NMR (400 MHz, DMSO-d 6): δ = 8.46 (s, 2 H, H-8), 7.43-6.82 (m, 9 H, HAr), 6.67 (dd, 3 J H,H = 7.3 Hz, 3 J H,H = 7.2 Hz, 1 H, H-1′), 5.34 (s, 2 H, NH), 4.42-4.19 (m, 2 H, H-a), 3.72-3.71 (m, 3 H, H4′, H5′a, H5′b), 3.06 (t, 3 J H,H = 6.9 Hz, 3 J H,H = 6.9 Hz, 2 H, Hb), 2.97-2.91 (m, 1 H, H2′a), 1.91 (s, 1 H, H2′b), 0.75 [s, 9 H, Si(CH3)3 at C3′], 0.80 [s, 9 H, Si(CH3)3 at C5′], 0.00 [s, 6 H, Si(CH3)2], -0.15 [2 × s, 2 × 3 H, Si(CH3)2] ppm. 13C NMR (101 MHz, DMSO-d 6): δ = 159.1 (C-6), 156.7 (C-2), 152.4 (C-D), 145.7 (C-A), 144.4 (C-f), 139.6 (C-8), 130.0 (C-C), 129.5 (C-c), 127.6 (C-d), 127.5 (C-e), 123.6 (C-4), 119.0 (C-B), 118.6 (CN), 114.8 (C-5), 87.1 (C-4′), 84.0 (C-1′ 71.6 (C-3′), 67.8 (C-a), 62.8 (C-5′), 39.7 (C-2′), 34.7 [2 × SiC(CH3)3], 34.3 (C-b) ppm. Mp 84-87 °C; [α]546 20 -3 (c 1.0, CHCl3). IR (KBr): 3744, 3037, 1737, 1695, 1612, 1507, 1110, 838 cm-1. HRMS-FAB: m/z calcd: 715.3698; found: 716.3736 [M + H+].
Compound 7b: The general procedure was conducted with 2.36 g of 5 (3.42 mmol) and 1.42 g of 6b (7.60 mmol); yield 1.35 g (1.71 mmol, 50%) of 7b as a yellow solid. 1H NMR (400 MHz, DMSO-d 6): δ = 8.19 (s, 1 H, H8), 7.77-7.33 (m, 13 H, Har), 6.31 (dd, 3 J H,H = 6.7 Hz, 1 H, H1′), 5.37, 5.20
(2 × s, 2 × 1 H, NH), 4.70 (t, 3 J H,H = 6.9 Hz, 2 H, Ha), 4.54-4.51 (m, 2 H, H3′), 3.85-3.82 (m, 1 H, H4′), 3.72-3.62 (m, 2 H, H5′a, H5′b), 3.17 (t, 3 J H,H = 6.9 Hz, 2 H, Hb), 2.97-2.90 (m, 1 H, H2′a), 2.30-2.24 (m, 1 H, H2′b), 0.88, 0.84 [2 × s, 2 × 9 H, Si(CH3)3], 0.10, 0.00 [2 × s, 2 × 6 H, Si(CH3)2] ppm. 13C NMR (101 MHz, DMSO-d 6): δ = 159.2 (C6), 157.5, 153.1 (C4), 148.2 (C2), 144.1, 139.7 (C8), 132.0, 128.7, 126.1, 118.7 (CN), 115.1 (C5), 87.1 (C4′), 83.2 (C1′), 72.3 (C3′), 65.8 (Ca), 62.8 (C5′ 38.3 (C2′), 34.1 (Cb), 25.6, 25.5 [2 × SiC(CH3)3], -5.6, -5.1 [2 × Si(CH3)2] ppm. Mp 75-78 °C; [α]546 20 +25 (c 0.4, CHCl3). IR (KBr): 2954, 2928, 2857, 1687, 1109, 776, 740, 482 cm-1. MS (HRFAB): m/z calcd: 791.4011; found: 791.4035 [M + H]+.

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Compound 10a: 1H NMR (400 MHz, benzene-d 6): δ = 7.84 [s, 1 H, H-8 (I)], 7.81 [s, 1 H, H-8 (II)], 7.71-7.41 [m, 44 H, HAr (I + II), DMTr-H (I + II)], 6.25 [dd, 3 J H,H = 6.4 Hz, 3 J H,H = 6.3 Hz, 2 H, H-1′ (I + II)], 4.74-4.58 [m, 2 H, NH (I)], 4.47-4.37 [m, 2 H, NH (II)], 4.16-4.05 [m, 4 H, H-a (I + II)], 3.58-3.35 (m, 12 H, OCH3, H-5′a/b (I + II), H-b(I)] 3.15-3.02 [m, 2 H, H-b (II)], 2.54-2.29 (m, 12 H, i-PrH (I + II), H-α (I + II), H-2′a/b (I + II)], 1.81 [t, 2 H, H-β (I)], 1.73 [m, 2 H, H-β (II)], 1.15-1.01 [m, 24 H, CH3 i-Pr (I + II)] ppm. 13C NMR (101 MHz, benzene-d 6): δ = 160.1, 159.3, 158.7, 148.2, 145.8, 143.3, 138.0, 137.4, 136.1, 132.0, 131.4, 130.6, 130.1, 129.8, 119.0, 113.7, 113.0, 111.0, 87.1, 74.4, 74.2, 66.0, 54.8, 45.3, 43.6, 35,1, 24.7, 24.6, 22.8 ppm. 31P NMR (162 MHz, benzene-d 6): δ = 149.35, 149.19 ppm. Mp 68-71 °C. [α]546 20 +183 (c 1.0, CHCl3). IR KBr): 3870, 3816, 3752, 3744, 3676, 3447, 2966, 1654, 1609, 1581, 1508, 1465, 1383, 1250, 1179, 1034, 829 cm-1. MS-FAB: m/z calcd: 989.4353; found: 990.5 [M + H]+.
Compound 11a: 1H NMR (400 MHz, benzene-d 6): δ = 7.88 [s, 1 H, H-8 (I)], 7.85 [s, 1 H, H-8 (II)], 7.62-6.58 [m, 49 H, DMTr-H (I + II), HAr (I + II)], 6.27-6.18 [m, 2 H, H-1′ (I + II)], 5.07-4.95 [m, 2 H, H-3′ (I + II)], 4.73-4.67 [m, 4 H, H-a (I + II)], 4.13-4.06 [m, 2 H, H-4′ (I + II)], 3.54-3.26 [m, 20 H, OCH3 (I + II), H-b (I + II), H-5′ (I + II)], 3.00-2.82 [m, 2 H, H-2′a (I + II)], 2.72-2.61 [m, 8 H, i-PrH (I + II), H-a (I + II)], 2.56-2.47 [m, 1 H, H-2′b (I)], 2.41-2.29 [m, 1 H, H-2′b (II)], 1.73-1.67 (m, 4 H, H-b (I + II)], 1.13-1.05 [m, 24 H, CH3 i-Pr (I + II)] ppm. 13C NMR (101 MHz, benzene-d 6):
δ = 190.6, 186.5, 185.4, 177.7, 169.4, 168.0, 163.6, 159.3, 153.4, 146.1, 145.8, 143.3, 136.3, 132.1, 130.7, 129.9, 129.4, 124.0, 113.8, 87.6, 87.0, 54.8, 47.5, 43.6, 35.2, 24.6 ppm. 31P NMR (162 MHz, benzene-d 6): δ = 149.46, 149.16 ppm. Mp 86-89 °C; [a]546 20 +39 (c 1.0, CHCl3). IR (KBr): 3854, 3744, 3676, 3432, 2965, 2227, 1607, 1508, 1445, 1341, 1251, 1178, 1034 cm-1. MS-FAB: m/z calcd: 987.4197; found: 988.7 [M + H]+.
Compound 10b: 1H NMR (400 MHz, benzene-d 6): δ = 7.88 [s, 1 H, H8 (I)], 7.86 [s, 1 H, H8 (II)], 7.62-6.58 [m, 52 H, DMTr-H (I + II), Har (I + II)], 6.29-6.19 [m, 2 H, H1′ (I + II)], 5.76, 5.37 [2 × s, 2 × 2 H, NH (I + II)], 5.04-5.01 [m, 2 H, H3′ (I + II)], 4.60-4.50 [m, 4 H, Ha (I + II)], 4.13-4.08 [m, 2 H, H4′ (I + II)], 3.52-3.17 [m, 20 H, OCH3 (I + II), Hb (I + II), H5′ (I + II)], 2.98-2.93 [m, 1 H, H2′a (I)], 2.89-2.84 [m, 1 H, H2′a (II)], 2.71-2.68 [m, 8 H, i-PrH (I + II) + a′ (I + II)], 2.56-2.48 [m, 1 H, H2′b (I)], 2.41-2.34 (m, 1 H, H2′b (II)], 1.73-1.67 [m, 4 H, b′ (I + II)], 1.13-1.05 [m, 24 H, CH3 i-Pr (I + II)] ppm. 13C NMR (101 MHz, benzene-d 6):
δ = 159.1, 155.6, 153.2, 144.1, 139.7, 135.6, 134.6, 130.0, 127.8, 122.3, 118.8, 116.2, 109.1, 87.6, 83.1, 70.8, 65.8, 61.7, 39.7, 34.4 ppm. 31P NMR (162 MHz, benzene-d 6): δ = 149.87, 149.09 ppm. Mp 75-78 °C; [a]546 20 +33 (c 0.1, CHCl3). IR (KBr): 3744, 3675, 2966, 1607, 1457, 1200, 1077, 978 cm-1. MS-FAB: m/z calcd 1065.4666; found: 1066.6 [M + H]+.
Compound 11b: 1H NMR (400 MHz, benzene-d 6): δ = 7.88 [s, 1 H, H8 (I)], 7.86 [s, 1 H, H8 (II)], 7.62-6.58 [m, 59 H, DMTr-H (I + II), Har (I + II)], 6.29-6.19 [m, 2 H, H1′ (I + II)], 5.04-5.01 (m, 2 H, H3′ (I + II)), 4.60-4.50 [m, 4 H, Ha (I + II)], 4.13-4.08 [m, 2 H, H4′ (I + II)], 3.52-3.17 [m, 20 H, OCH3 (I + II), Hb (I + II), H5′ (I + II)], 2.98-2.93 [m, 1 H, H2′a (I)], 2.89-2.84 [m, 1 H, H2′a (II)], 2.71-2.68 [m, 8 H, i-PrH (I + II) + α′ (I + II)], 2.56-2.48 [m, 1 H, H2′b(I)], 2.41-2.34 [m, 1 H, H2′b(II)], 1.73-1.67 [m, 4 H, β′ (I + II)], 1.13-1.05 [m, 24 H, CH3 i-Pr (I + II)] ppm. 13C NMR (101 MHz, benzene-d 6): 159.1, 155.6, 153.2, 144.1, 139.7, 135.6, 134.6, 130.0, 127.8, 122.3, 118.8, 116.2, 109.1, 87.6, 83.1, 70.8, 65.8, 61.7, 39.7, 34.4 ppm. 31P NMR (162 MHz, benzol-d 6): δ = 149.29, 149.08 ppm. Mp 63-65 °C; [α]546 20
-13 (c 0.4, CHCl3). IR (KBr): 3821, 3675, 3447, 2966, 1734, 1700, 1653, 1507, 1363, 1179, 1033, 829 cm-1. MS-FAB: m/z calcd: 1063.4510; found: 1064.7 [M + H]+.