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Synlett 2006(15): 2411-2414  
DOI: 10.1055/s-2006-950411
LETTER
© Georg Thieme Verlag Stuttgart · New York

First Synthesis of C8-Arylamine Adducts of 2′-Deoxyadenosine and Incorporation of the Phosphoramidite into an Oligonucleotide

Maike I. Jacobsen, Chris Meier*
Department of Chemistry, Organic Chemistry, Faculty of Mathematics, Informatics and Natural Sciences, University of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany
Fax: +49(40)428382495; e-Mail: chris.meier@chemie.uni-hamburg.de;
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Publikationsverlauf

Received 7 July 2006
Publikationsdatum:
08. September 2006 (online)

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Abstract

The synthesis of C8-adducts of 2′-deoxyadenosine 7a,b with carcinogenic arylamines p-anisidine 4c and 4-aminobiphenyl 4b, their conversion into the phosphoramidites and successful incorporation into oligonucleotides are described.

Key words

Pd-cross coupling - amination - nucleoside adducts - phosphoramidites - oligonucleotides

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N 6-Benzoyl-8-N-(4-methoxyphenylamino)-3′,5′-O-[1,1,3,3-tetrakis(isopropyl)-1,3-disiloxanediyl]-2′-deoxyadenosine (7a) and N 6-benzoyl-8-N-(4-aminobiphenyl)-3′,5′-O-[1,1,3,3-tetrakis(isopropyl)-1,3-disiloxanediyl]-2′-deoxy-adenosine (7b): N 6-Bz-8-bromo-3′,5′-O-(TIPDS)-2′-dA 6 (500 mg, 0.87 mmol), Cs2CO3 (426 mg, 1.31 mmol), tris(di-benzylideneacetone)dipalladium(0) [Pd2(dba)3; 80.0 mg, 87.0 µmol], racemic-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP; 163 mg, 0.26 mmol) and p-anisidine (4c; 215 mg, 1.74 mmol) or 4-aminobiphenyl (4b; 295 mg, 1.74 mmol) were solubilized in anhyd 1,2-dimethoxyethane (30 mL) in an inert gas atmosphere and stirred at 90 °C until the reaction was complete (TLC analysis). After cooling to r.t., sat. NaHCO3 solution (1 mL) was added. After addition of brine (10 mL) the layers were separated and the aqueous layer was extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine (3 × 10 mL) and once with a mixture of brine (10 mL) and H2O (2 mL). The organic layer was dried (Na2SO4) and the solvent was removed in vacuo. Purification by chromatography on silica gel, eluting with 20% EtOAc in hexane afforded 7a (374 mg, 0.61 mmol; 70%) and 7b (345 mg, 0.52 mmol; 60%) as light-yellow foams.

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7a: 1H NMR (400 MHz, CDCl3): δ = 8.16 (s, 1 H), 7.48 (m, 2 H), 7.41 (br s, 1 H), 6.88 (m, 2 H), 6.31 (dd, J = 3.8, 7.8 Hz, 1 H), 5.34 (br s, 2 H), 4.90 (dd, J = 7.9, 15.2 Hz, 1 H), 4.16 (dd, J = 3.4, 9.2 Hz, 1 H), 3.98 (dd, J = 5.0, 12.5 Hz, 1 H), 3.90 (ddd, J = 3.4, 4.9, 7.0 Hz, 1 H), 3.79 (s, 3 H), 3.06 (ddd, J = 3.6, 8.1, 13.4 Hz, 1 H), 2.59 (ddd, J = 7.9, 13.4 Hz, 1 H), 0.89-1.13 (m, 28 H). 13C NMR (101 MHz, CDCl3): δ = 155.8, 152.5, 149.4, 132.5, 121.5, 117.4, 114.5, 85.3, 83.8, 70.5, 61.9, 55.7, 38.8, 17.0-17.6, 12.6-13.5. HRMS (FAB): m/z [M + H]+ calcd for C29H46N6O5Si2: 615.3147; found: 615.3155. 7b: 1H NMR (400 MHz, CDCl3): δ = 8.19 (s, 1 H), 7.78 (br s, 1 H), 7.69 (m, 2 H), 7.58 (m, 4 H), 7.44 (m, 2 H), 7.33 (m, 1 H), 6.35 (dd, J = 3.8, 7.5 Hz, 1 H), 5.59 (s, 2 H), 4.88 (dd, J = 7.9, 15.2 Hz, 1 H), 4.16 (dd, J = 3.6, 12.6 Hz, 1 H), 4.04 (dd, J = 4.8, 12.5 Hz, 1 H), 3.95 (ddd, J = 3.0, 4.4, 7.3 Hz, 1 H), 3.07 (ddd, J = 3.6, 8.0, 13.4 Hz), 2.59 (ddd, J = 7.8, 13.4 Hz, 1 H), 0.91-1.12 (m, 28 H). 13C NMR (101 MHz, CDCl3): δ = 156.7, 152.4, 149.7, 148.3, 140.9, 132.5, 121.5, 117.4, 114.5, 85.3, 83.8, 70.5, 61.9, 55.7, 38.8, 17.0-17.6, 12.6-13.5. HRMS (FAB): m/z [M + H]+ calcd for C34H48N6O4Si2: 661.3354; found: 661.3377.

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N 6-Benzoyl-8-N-(4-methoxyphenylamino)-5′-O-dimeth-oxytrityl-2′-deoxyadenosine-3′-yl-β-cyanoethyl-N,N′-diisopropylphosphoramidite (10a) and N 6-benzoyl-8-N-(4-aminobiphenyl)-5′-O-dimethoxytrityl-2′-deoxy-adenosine-3′-yl-β-cyanoethyl-N,N′-diisopropylphosphor-amidite (10b): N 6-Bz-8-N-(4-methoxyphenylamino)-5′-O-DMTr-2′-dA (150 mg, 0.19 mmol) and N 6-Bz-8-N-(4-aminobi-phenyl)-5′-O-DMTr-2′-dA (150 mg, 0.18 mmol), respec-tively, were dissolved in anhydrous CH2Cl2 (3 mL) and anhydrous MeCN (3 mL) in an inert gas atmosphere and treated subsequently with a solution of 4,5-dicyano-imidazole in MeCN (0.25 M, 1.5 mL, 0.38 mmol) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite (86 mg, 0.29 mmol). After stirring for 30 min at r.t., the reaction was stopped by adding of MeOH (0.5 mL). The solution was diluted with CH2Cl2 (50 mL) and washed with 5% aq NaHCO3 followed by brine. The organic layer was dried and concentrated to dryness. The residue was purified by chromatography on silica gel, eluting with CH2Cl2, 2% Et3N and 0-3% MeOH. The products were redissolved in CH2Cl2 and further washed with H2O to give 10a (130 mg, 0.13 mmol; 69%) and 10b (116 mg, 0.11 mmol; 62%) as light-yellow solids.

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9a: 2 diastereomers + 2 rotamers. 1H NMR (400 MHz, C6D6): δ = 9.55 (m, 2 H), 9.18 (s, 1 H), 9.16 (s, 1 H), 8.90 (m, 2 H), 8.67 (s, 1 H), 8.66 (s, 1 H), 8.29 (s, 1 H), 8.27 (s, 1 H), 7.91 (m, 2 H), 7.82 (m, 5 H), 7.75 (m, 3 H), 7.64-7.70 (m, 4 H), 7.54-7.62 (m, 6 H), 7.43-7.53 (m, 8 H), 7.32 (m, 12 H), 7.32 (m, 5 H), 7.08-7.16 (m, 8 H), 6.87-7.06 (m, 22 H), 6.64-6.80 (m, 24 H), 5.99-6.13 (m, 2 H), 5.91 (m, 2 H), 5.25 (m, 2 H), 5.07 (m, 4 H), 4.50-4.64 (m, 2 H), 4.41 (m, 2 H), 3.81-3.93 (m, 4 H), 3.61-3.75 (m, 4 H), 3.44-3.62 (m, 16 H), 3.22-3.43 (m, 36 H), 2.28-2.53 (m, 4 H), 1.68-2.00 (m, 12 H), 1.11-1.23 (m, 48 H). 13C NMR (101 MHz, C6D6): δ = 165.9, 165.1, 159.2, 159.2, 159.9, 154.1, 152.6, 151.1, 150.1, 149.4, 146. 1, 145.9, 145.3, 144.2, 142.0, 136.9, 136.8, 136.0, 135.8, 135.0, 132.7, 132.6, 130.9, 130.8, 130.5, 128.6, 127.1, 127.0, 124.0, 123.4, 121.6, 121.5, 117.8, 117.6, 115.4, 114.6, 113.6, 113.5, 111.7, 86.9, 86.8, 86.0, 85.4, 82.5, 82.3, 64.9, 63.4, 63.2, 59.2, 59.1, 59.0, 58.8, 58.6, 55.0, 54.9, 54.9, 43.6, 43.6, 37.5, 37.2, 30.5, 30.2, 24.8, 24.8, 24.7, 20.2, 20.1, 20.1. 31P NMR (202 MHz, C6D6): δ = 149.20, 148.95, 148.84, 148.20 (0.30:0.33:0.20:0.17). HRMS (ESI): m/z [M + Na]+ calcd for C24H24N6O5: 1001.4093; found: 1001.4120. 9b: 2 diastereomers + 2 rotamers. 1H NMR: (400 MHz, C6D6): δ = 9.87 (m, 2 H), 8.94 (s, 1 H), 8.92 (s, 1 H), 8.85 (s, 1 H), 8.84 (s, 1 H), 8.60 (m, 2 H), 8.44 (s, 1 H), 8.42 (s, 1 H), 8.24 (s, 1 H), 8.21 (s, 1 H), 8.03 (m, 4 H), 7.92 (m, 4 H), 7.86 (m, 4 H), 7.69 (m, 12 H), 7.49-7.63 (m, 20 H), 7.40 (m, 12 H), 7.32 (m, 12 H), 7.22 (m, 10 H), 7.04-7.18 (m, 12 H), 6.97 (m, 2 H), 6.85 (m, 8 H), 6.77 (m, 10 H), 5.87-6.03 (m, 2 H), 5.17-5.34 (m, 2 H), 4.99-5.15 (m, 2 H), 4.54-4.66 (m, 2 H), 4.41 (m, 2 H), 3.88 (m, 4 H), 3.74 (m, 2 H), 3.57-3.66 (m, 10 H), 3.31 (m, 12 H), 3.30 (m, 12 H), 3.00-3.16 (m, 8 H), 2.28-2.62 (m, 4 H), 1.70-1.97 (m, 10 H), 1.01-1.10 (m, 48 H). 13C NMR (101 MHz, C6D6): δ = 159.2, 149.7, 141.1, 136.0, 135.9, 134.9, 132.6, 131.9, 130.8, 130.6, 129.0, 129.0, 128.7, 128.6, 127.9, 127.1, 127.0, 119.7, 117.6, 117.0, 116.3, 113.6, 113.5, 87.0, 86.8, 85.8, 59.2, 59.0, 58.0, 58.0 54.8, 45.4, 45.3, 43.7, 43.6, 24.8, 24.8, 24.7, 24.6, 23.7, 22.9, 22.8, 22.8, 22.8, 20.6, 20.1, 20.1, 19.5, 19.4. 31P NMR (202 MHz, C6D6): δ = 149.37, 149.11, 148.69, 148.38 (0.31:0.37:0.17:0.15). MS (FAB): m/z [M + H]+ calcd for C29H26N6O4: 1025.4480; found: 1025.4.

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Böge, N.; Gräsl, S.; Meier, C. J. Org. Chem. 2006, submitted.

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Enzyme degradation: Oligonucleotide (3 µg) was dissolved in a NaOAc buffer (100 µL, pH 5.3, 30 mM). ZnSO4 solution (5 µL, 20 mM) and the nuclease P1 (3 units, from peni-cillium citrinum; Roche Nr. 91095923/30) were added and incubated for 4 h at 37 °C. Then a tris(hydroxy-methyl)aminomethane-HCl buffer (20 µL; pH 8.7; 50 mM) and 4 units of the alkaline phosphatase (Roche Nr. 49048126/15) were added and incubated overnight at 37 °C.