Covalent Modification of 2′-Deoxyuridine with Two Different Molecular Switches

Abstract

Two different molecular switches, a spiropyran and a diarylethene, were attached synthetically to the 5-position of 2′-de­oxyuridine. The diarylethene-modified nucleoside can be incorporated synthetically into DNA while preserving its characteristic photochromism.

Key words

oligonucleotide - palladium - DNA - photochromism - phosphoramidite

References and Notes

• For reviews, see:
• 1a Raymo FM. Tomasulo M. Chem. Eur. J.  2006,  12:  3186 
• 1b Pischel U. Angew. Chem. Int. Ed.  2007,  46:  4026 
• 2 For a review, see: Cusido J. Deniz E. Raymo FM. Eur. J. Org. Chem.  2009,  2031 
• 3 For a review, see: Berkovic G. Krongauz V. Weiss V. Chem. Rev.  2000,  100:  1741 
• 4 For a review, see: Mayer G. Heckel A. Angew. Chem. Int. Ed.  2006,  45:  4900 
• 5 For a review, see: Moroder L. Renner C. ChemBioChem  2006,  7:  868 
• 6a Asanuma H. Liang X. Komiyama M. ChemBioChem  2001,  2:  39 
• 6b Nishioka H. Liang X. Asanuma H. Chem. Commun.  2007,  4354 
• 6c Liang X. Takenaka N. Nishioka H. Asanuma H. Chem. Asian J.  2008,  3:  553 
• 7 Yamazawa A. Liang X. Asanuma H. Komiyama M. Angew. Chem. Int. Ed.  2000,  39:  2356 
• 8 Liang X. Asanuma H. Komiyama M. J. Am. Chem. Soc.  2002,  124:  1877 
• 9 Sakata T. Yan Y. Marriott G. J. Org. Chem.  2005,  70:  2009 
• 10 Angelini N. Corrias B. Fissi A. Pieroni O. Lenci F. Biophys. J.  1998,  74:  2601 
• 11 Kocer A. Walko M. Meijberg W. Feringa BL. Science  2005,  309:  755 
• 12 Asanuma H. Shirasuka K. Yoshida T. Takarada T. Liang X. Komiyama M. Chem. Lett.  2001,  30:  108 
• 13 Zhang P. Meng JB. Matsuura T. Wang YM. Chin. Chem. Lett.  2002,  13:  299 
• 14 Andreasson J. Andersson J. Li S. Lincoln P. J. Am. Chem. Soc.  2008,  130:  11836 
• 15 Young D. Deiters A. ChemBioChem  2008,  9:  1225 
• 16 Beyer C. Wagenknecht H.-A. Synlett  2010,  1371 
• 17 Vomasta D. Hogner C. Branda N. König B. Angew. Chem.  2008,  47:  7644 
• 18 Singer M. Jäschke A. J. Am. Chem. Soc.  2010,  132:  8372 
• 19 Beyer C. Wagenknecht H.-A. J. Org. Chem.  2010,  75:  2752 
• 22 Chung-Shan Y. Oberdorfer F. Synlett  2010,  86 
• 24 Stafforst T. Hilvert D. Chem. Commun.  2009,  287 

20

Compound 5: Compound 4 ^¹9 (490 mg, 3.35 mmol) was dissolved in anhyd EtOH (50 mL), freshly distilled 3 ^¹9 (0.59 mL, 3.33 mmol) was added in one portion, and the reaction mixture was sonicated at 35 kHz under Ar. After 53 min the solvent was removed under reduced pressure. The crude product was purified by gradient flash chromatography on silica gel (hexane-THF, 70:1 → 50:1) yielding 5 (762 mg, 76%) as a pale blue foam; R _f 0.36 (hexane-THF, 50:1). ^¹H NMR (600 MHz, CDCl₃): δ = 7.15-7.27 (m, 2 H, ArH), 7.20 (dt, 1 H, J = 1.2, 7.7 Hz, ArH), 7.09 (d, 1 H, J = 7.2 Hz, ArH), 6.87 (t, 1 H, J = 7.4 Hz, ArH), 6.83 (d, 1 H, J = 10.3 Hz, ArH), 6.67 (d, 1 H, J = 8.3 Hz, ArH), 6.55 (d, 1 H, J = 7.7 Hz, ArH), 5.74 (d, 1 H, J = 10.2 Hz, ArH), 2.98 (s, 1 H, CºCH), 2.74 (s, 3 H, NMe), 1.31 (s, 3 H, Me), 1.18 (s, 3 H, Me). ^¹³C NMR (150 MHz, CDCl₃): δ = 155.0, 148.1, 136.6, 133.7, 130.5, 128.8, 127.6, 121.5, 120.3, 119.3, 118.8, 115.2, 113.5, 106.9, 104.8, 83.6, 75.5, 51.9, 28.9, 25.9, 20.1. MS (CI, NH₃): m/z (%) = 302.1 (100) [MH⁺]. HRMS (EI-MS): m/z [M⁺] calcd for C₂₁H₁₉NO: 301.1467; found: 301.1465.

21

Compound 8: Compound 6 ^¹9 (335 mg, 1.11 mmol) was dissolved in freshly distilled EtOH (10 mL) and anhyd Et₃N (200 µL, 1.43 mmol). Compound 7 ^¹9 (220 mg, 1.09 mmol) was added and the reaction mixture was sonicated at 35 kHz under Ar. After 2 h the solvent was removed under reduced pressure, the residue was dissolved in CH₂Cl₂ and dried over anhyd Na₂SO₄. The solvent was evaporated under reduced pressure, and the residue was dried in vacuo and purified by gradient flash chromatography on silica gel (hexane-EtOAc, 30:1 → 15:1) to yield 8 (300 mg, 77%) as a pale pink solid; R _f 0.49 (hexane-EtOAc, 19:1). ^¹H NMR (300 MHz, CDCl₃): δ = 7.15-7.27 (m, 3 H, ArH), 7.09 (d, 1 H, J = 7.2 Hz, ArH), 6.87 (t, 1 H, J = 7.4 Hz, ArH), 6.80 (d, 1 H, J = 10.3 Hz, ArH), 6.61 (d, 1 H, J = 9.2 Hz, ArH), 6.55 (d, 1 H, J = 7.7 Hz, ArH), 5.74 (d, 1 H, J = 10.2 Hz, ArH), 2.74 (s, 3 H, NMe), 1.32 (s, 3 H, 3-Me), 1.18 (s, 3 H, 3-Me). ^¹³C NMR (75 MHz, CDCl₃): δ = 153.6 (C_quat), 148.0 (C_quat), 136.5 (C_quat), 132.2 (+, CH), 129.1 (+, CH), 128.4 (+, CH), 127.7 (+, CH), 121.5 (+, CH), 120.7 (+, CH), 120.6 (C_quat), 119.3 (+, CH), 116.9 (+, CH), 111.8 (C_quat), 106.9 (+, CH), 104.5 (C_quat), 51.9 (C_quat), 28.9 (+, Me), 25.9 (+, Me), 20.1 (+, Me). MS (EI, 70 eV): m/z (%) = 357.0(100) [M⁺˙], 339.9 (47) [M⁺ - Me^.]. HRMS (EI-MS): m/z [M⁺] calcd for C₁₉H₁₈BrNO: 355.0572; found: 355.0570.

23

Compound 1: Compound 5 (53 mg, 0.176 mmol), 9 (50 mg, 0.141 mmol), CuI (6 mg, 0.0315 mmol) and Pd(dppf)Cl₂ (20 mg, 0.0245 mmol) were dissolved in anhyd DMF (2.0 mL) and anhyd Et₃N (100 µL, 0.717 mmol). The mixture was degassed and stirred at r.t. for 26 h. Then all was poured into an EtOAc-H₂O mixture (20 mL, 1:1). After separation, the aqueous phase was extracted with CH₂Cl₂, and the combined organic layers were dried over anhyd Na₂SO₄ and the solvent was evaporated under reduced pressure. The residue was dried in vacuo and purified by gradient flash chromatography on silica gel (CH₂Cl₂-MeOH, 10:1 → 5.1) to yield 1 (50 mg, 67%) as glistening green crystals; R _f 0.29 (CH₂Cl₂-MeOH, 10:1). ^¹H NMR (600 MHz, MeOD): δ = 8.36 (s, 1 H, H-6), 7.27 (d, 1 H, J = 2.0 Hz), 7.23 (dd, 1 H,
J = 8.4 Hz), 7.10 (dt, 1 H, J = 7.6 Hz), 7.03 (d, 1 H, J = 7.2 Hz), 6.92 (d, 1 H, J = 10.3 Hz), 6.78 (t, 1 H, J = 7.1 Hz), 6.61 (d, 1 H, J = 8.4 Hz), 6.52 (d, 1 H, J = 7.8 Hz), 6.26 (t, 1 H,
J = 6.6 Hz, H-1′), 5.80 (d, 1 H, J = 10.3 Hz), 4.40-4.43 (m, 1 H, H-3′), 3.94 (q, 1 H, J = 3.3 Hz, H-4′), 3.83 (dd, 1 H, J = 3.0, 12.0 Hz, H-5′), 3.75 (dd, 1 H, J = 3.4, 12.0 Hz, H-5′), 2.70 (s, 3 H, NMe), 2.29-2.34 (m, 1 H, H-2′), 2.22-2.28 (m, 1 H, H-2′), 1.26 (s, 3 H, MeC), 1.14 (s, 3 H, Me). ^¹³C NMR (150 MHz, MeOD): δ = 164.4, 156.2, 151.2, 149.5, 144.5 (C6), 137.8, 134.2, 131.1, 130.0, 128.6, 122.4, 121.4, 120.5, 120.4, 116.0, 107.9, 106.3, 100.9, 93.9, 89.1 (C-4′), 87.0 (C-1′), 80.1, 72.0 (C-3′), 62.6 (C-5′), 52.9, 41.8 (C-2′), 29.2 (NMe), 26.3 (Me), 20.4 (Me), 9.3. MS (ESI): m/z (%) = 528.2 (100) [MH⁺].

25

Compound 12: Compound 11 (2.50 g, 11.7 mmol) was dissolved under argon atmosphere in anhyd THF (30 mL) and cooled to -78 ˚C. A 2 M lithium diisopropylamide solution in THF-heptane-ethylbenzene (8.79 mL, 17.6 mmol) was added dropwise and the mixture was stirred for 1 h at 0 ˚C. After cooling down to -78 ˚C, MeI (1.10 mL, 17.6 mmol) was added dropwise. The mixture was stirred for 2 additional hours. Then H₂O (30 mL) was added, the phases were separated and the aqueous phase was extracted with EtOAc. The combined organic layers were dried over anhyd Na₂SO₄ and the solvent was evaporated under reduced pressure. The residue was dried in vacuo and purified by flash chromatography on silica gel(hexanes) to yield 12 (2.66 g, 99%) as a white solid; R _f 0.57 (hexanes). ^¹H NMR (300 MHz, CDCl₃): δ = 7.78 (d, 1 H, J = 1.9 Hz, ArH), 7.59 (d, 1 H, J = 8.5 Hz, ArH), 7.34 (dd, 1 H, J = 8.5, 1.9 Hz, ArH), 6.90 (s, 1 H, ArH), 2.59 (d, 3 H, J = 1.2 Hz, Me). ^¹³C NMR (75 MHz, CDCl₃): δ = 143.1 (C_quat), 142.2 (C_quat), 138.4 (C_quat), 126.5 (C_arom), 125.3 (C_arom), 123.4 (C_arom), 121.0 (C_arom), 118.2 (C_arom), 16.3 (Me). MS (EI, 70 eV): m/z (%) = 228.0(100) [M⁺], 147.1 (24) [M - Me].

26

Compound 13: Compound 12 (969 mg, 4.3 mmol) and glutaryl chloride (0.27 mL, 2.1 mmol) were dissolved under argon atmosphere in anhyd CH₂Cl₂ (20 mL) and the solution was cooled to 0 ˚C. Then AlCl₃ (626 mg 4.7 mmol) was added and the reaction mixture was stirred for 3 h at r.t. After completion of the reaction, a mixture of ice and 1 M aq HCl was added. The solution was let to be warmed to r.t., the phases were separated and the aqueous phase was extracted with EtOAc. The combined organic layers were dried over anhyd Na₂SO₄ and the solvent was evaporated under reduced pressure. The residue was dried in vacuo and purified by flash chromatography on silica gel (hexanes-EtOAc, 10:1) to yield 13 (896 mg, 74%) as a white solid; R _f 0.31 (hexanes-EtOAc, 10:1). ^¹H NMR (400 MHz, CDCl₃): δ = 8.36 (d, 2 H, J = 1.8 Hz, ArH), 7.57 (d, 2 H, J = 8.5 Hz, ArH), 7.42 (dd, 2 H, J = 8.5, 1.8 Hz, ArH), 3.06 (t, 4 H, J = 6.9 Hz, CH₂), 2.77 (s, 6 H, Me), 2.27 (quin, 2 H, J = 7.0 Hz, CH₂). ^¹³C NMR (75 MHz, CDCl₃): δ = 168.6 (CO), 146.1 (C_quat), 142.9 (C_quat), 140.5 (C_quat), 136.5 (C_quat), 127.2 (C_arom), 125.0 (C_arom), 123.4 (C_arom), 118.8 (C_arom), 28.6 (CH₂), 19.6 (CH₂), 15.3 (Me). MS (EI, 70 eV): m/z (%) = 549.9 (14) [M⁺].

27

Compound 14: To a suspension of zinc dust (301 mg, 4.6 mmol, stabilized, grading <63 µm) in anhyd THF (50 mL) TiCl₄ (0.26 mL, 2.3 mmol) was added under argon atmosphere dropwise at 0 ˚C. The suspension was heated to 40 ˚C and stirred for 1 h. Compound 13 (631 mg, 1.1 mmol) was added and the mixture was stirred for 18 h. After cooling to r.t. the reaction mixture was poured through a silica gel pallet and rinsed with hexanes. The solvent was removed under reduced pressure and the residue was dried in vacuo and purified by flash chromatography on silica gel(hexanes) to yield 14 (328 mg, 55%) as a white solid; R _f 0.25 (hexanes). ^¹H NMR (300 MHz, CDCl₃): δ = 7.64 (s, 2 H, ArH), 7.46 (d, 2 H, J = 8.4 Hz, ArH), 7.24 (s, 2 H, ArH), 2.88-3.02 (m, 4 H, CH₂), 2.28 (quin, 2 H, J = 7.22 Hz, CH₂), 2.12 (s, 6 H, Me). ^¹³C NMR (75 MHz, CDCl₃): δ = 140.9 (C_quat), 137.0 (C_quat), 136.8 (C_quat), 129.72 (C_arom), 126.5 (C_arom), 125.1 (C_arom), 123.3 (C_arom), 118.1 (C_arom), 37.8 (CH₂), 24.1 (CH₂), 15.2 (Me). MS (EI, 70 eV): m/z (%) = 517.9 (100) [M⁺].

28

Compound 15: Compound 14 (131 mg, 0.25 mmol), trimethylsilylacetylene (72 µL, 0.51 mmol), anhyd Et₃N (41 µL, 0.51 mmol), Pd(PPh₃)₄ (30 mg, 0.03 mmol) and CuI (5 mg, 0.03 mmol) were dissolved in anhyd THF (3 mL) under argon. The mixture was degassed and refluxed for 19 h. After cooling to r.t. the solvent was removed under reduced pressure, the residue was dried in vacuo and purified by flash chromatography on silica gel(hexanes) to yield 15 (58 mg, 43%) as a white solid; R _f 0.26 (hexanes). ^¹H NMR (300 MHz, CDCl₃): δ = 7.67 (s, 2 H, ArH), 7.54 (d, 1 H, J = 8.2 Hz, ArH), 7.47 (d, 1 H, J = 8.0 Hz, ArH), 7.29 (s, 2 H, ArH), 2.83-3.12 (m, 4 H, CH₂), 2.28 (quin, 2 H, J = 7.8 Hz, CH₂), 2.04 (s, 6 H, Me), 0.29 (s, 9 H, SiMe₃). ^¹³C NMR (75 MHz, CDCl₃): δ = 139.1 (C_quat), 138.6 (C_quat), 137.3 (C_quat), 136.7 (C_quat), 127.1 (C_arom), 126.4 (C_arom), 125.9 (C_arom), 125.1 (C_arom), 123.3 (C_arom), 121.9 (C_arom), 118.0 (C_arom), 105.9 (CºC), 37.9 (CH₂), 29.9 (CºC), 24.2 (CH₂), 15.3 (Me), 0.3 (SiMe₃). MS (EI, 70 eV): m/z (%) = 536.0(100) [M⁺].

29

Compound 16: Compound 15 (35 mg, 0.07 mmol) was dissolved in anhyd MeOH (3 mL) and K₂CO₃ (27 mg, 0.19 mmol) was added. The reaction mixture was stirred for 18 h at 40 ˚C. After cooling to r.t. MeOH was removed under reduced pressure. The residue was dried in vacuo and purified by flash chromatography on silica gel (hexanes) to yield 16 (30 mg, 99%) as a white solid; R _f 0.65 (hexanes-EtOAc, 20:1) ^¹H NMR (300 MHz, CDCl₃): δ = 7.71 (s, 2 H, ArH), 7.57 (d, 2 H, J = 8.0 Hz, ArH), 7.46 (d, 1 H, J = 7.5 Hz, ArH), 7.28 (s, 1 H, ArH), 3.07 (s, 1 H, CºCH), 2.84-3.01 (m, 4 H, CH₂), 2.28 (quin, 2 H, J = 8.0 Hz, CH₂), 2.02 (s, 6 H, Me). ^¹³C NMR (75 MHz, CDCl₃): δ = 146.8 (C_quat), 139.2 (C_quat), 137.2 (C_quat), 136.9 (C_quat), 126.9 (C_arom), 126.4 (C_arom), 125.1 (C_arom), 123.3 (C_arom), 122.0 (C_arom), 118.1 (C_arom), 117.6 (C_arom), 76.5 (CºC), 37.8 (CH₂), 29.9 (CºC), 24.1 (CH₂), 15.2 (Me). MS (EI, 70 eV): m/z (%) = 461.9 (100) [M⁺].

30

Compound 2: Compound 16 (126 mg, 0.27 mmol), 9 (97 mg, 0.27 mmol), anhyd Et₃N (45 µL, 0.54 mmol), Pd(PPh₃)₄ (63 mg, 0.05 mmol) und CuI (11 mg, 0.05 mmol) were dissolved in anhyd DMF (10 mL). The mixture was degassed and stirred for 18 h at 60 ˚C. After cooling to r.t. the solvent was removed under reduced pressure, the residue was dried in vacuo and purified by flash chromatography on silica gel (CH₂Cl₂-MeOH, 25:1) to yield 2 (121 mg, 65%) as a light yellow foam; R _f 0.40 (CH₂Cl₂-MeOH, 10:1). ^¹H NMR (300 MHz, MeOD): δ = 8.37 (s, 1 H, NH), 7.72 (s, 2 H, ArH), 7.58-7.68 (m, 1 H, C=CH), 7.55 (d, 1 H, J = 8.2 Hz, ArH), 7.46 (d, 1 H, J = 6.8 Hz, ArH), 6.95-7.29 (m, 2 H, ArH), 6.26 (t, 1 H, J = 6.2 Hz, 1′-H), 4.28-4.50 (m, 1 H, 3′-H), 3.88-4.03 (m, 1 H, 4′-H), 3.78-3.87 (m, 1 H, 5′-H), 3.66-3.78 (m, 1 H, 5′-H), 2.19-2.61 (m, 4 H, CH₂), 2.00-2.48 (m, 9 H, 2′-H, CH₂, Me), 1.95 (s, 1 H, 2′-H). ^¹³C NMR (75 MHz, MeOD): δ = 151.2 (CO), 144.7 (C=C), 140.4 (CO), 139.8 (C_quat), 138.6 (C_quat), 138.5 (C_quat), 138.1 (C_quat), 131.3 (C_quat), 127.3 (C_arom), 126.0 (C_arom), 124.3 (C_arom), 122.9 (C_arom), 118.9 (C_arom), 94.5 (CºC), 89.2 (4′-C), 87.1 (1′-C), 72.1 (3′-C), 62.6 (5′-C), 41.8 (2′-C), 38.6 (CH₂), 25.0 (CH₂), 15.1 (Me). MS (FAB): m/z (%) = 689.1 (6) [M⁺]. HRMS (ESI): m/z [M + H⁺] calcd for C₃₄H₃₀BrN₂O₅S₂: 691.0759; found: 691.0766.

31

Compound 17: Compound 2 (50 mg, 0.07 mmol), dimethoxytrityl chloride (37 mg, 0.11 mmol) and anhyd Et₃N (10 µL, 0.12 mmol) were dissolved in anhyd pyridine (2 mL) under argon atmosphere. The solution was stirred for 18 h at 40 ˚C. The solvent was removed, the residue was dried in vacuo and purified by flash chromatography on silica gel (CH₂Cl₂-MeOH, 100:3 + 0.1% Et₃N) to yield 17 (62 mg, 90%) as a light yellow foam; R _f 0.61 (CH₂Cl₂-MeOH, 10:1). ^¹H NMR (300 MHz, DMSO): δ = 8.05 (s, 1 H, NH), 7.70 (d, 2 H, J = 8.2 Hz, ArH_bt), 7.63 (s, 1 H, C=CH), 7.43 (d, 2 H, J =7.4 Hz, ArH_bt), 7.18-7.37 (m, 8 H, ArH_DMT), 7.14 (d, 2 H, J = 7.3 Hz, ArH_bt), 6.75-6.96 (m, 5 H, ArH_DMT), 6.16 (t, 1 H, J = 6.7 Hz, 1′-H), 4.26-4.34 (m, 1 H, 3′-H), 3.93-398 (m, 1 H, 4′-H), 3.66-3.80 (m, 2 H, 5′-H), 3.61 (d, 6 H, J = 2.5 Hz, CH_3,DMT), 2.79-2.93 (m, 4 H, CH₂), 2.06-2.37 (m, 9 H, 2′-H, CH₂, Me), 1.95-2.03 (m, 1 H, 2′-H). ^¹³C NMR (75 MHz, acetone): δ = 163.9 (C_quat), 161.9 (C_quat), 159.6 (C_quat), 157.7 (C_quat), 150.4 (CO), 146.0 (C_quat), 143.1 (C=C), 140.3 (CO), 139.0 (C_quat), 138.1 (C_quat), 137.9 (C_quat), 136.6 (C_quat), 131.0 (C_arom), 128.9 (C_arom), 127.6 (C_arom), 127.1 (C_arom), 126.0 (C_arom), 122.7 (C_arom), 114.1 (CºC), 87.7 (4′-C), 86.6 (1′-C), 64.8 (5′-C), 58.4 (3′-C), 55.4 (2′-C), 46.4 (5′-C), 41.9 (s), 38.3 (CH₂), 24.4 (CH₂), 15.4 (Me), 8.9 (Me). MS (ESI): m/z (%) = 1015.2 (4)[(M + Na)⁺].

32

Compound 18: Compound 17 (62 mg, 0.06 mmol), diisopropylethylamine (36 µL, 0.16 mmol) and 2-cyano-ethyldiisopropylphosphoramidite (27 µL, 0.16 mmol) were dissolved in anhyd CH₂Cl₂ (3 mL). The solution was stirred at r.t. for 40 min. The product was purified by flash chromatography on silica gel (CH₂Cl₂-MeOH, 100:4 + 0.1% Et₃N) and additionally lyophilized out of benzene to yield 18 (74 mg, 99%) as a light yellow foam; R _f 0.85, 0.91 (CH₂Cl₂-MeOH, 25:1). ^³¹P NMR (101 MHz, DMSO): δ = 150.6 [P(III), 37%], 16.3 [P(V), 63%]. MS (MALDI): m/z (%) = 1191.3 (28) [M⁺].

33

DNA 1: MS (MALDI): m/z (%) = 5950.0(100) [(M + DMT)⁺], 5647.9 (38) [M⁺], 2974.6 (17) [(M + DMT)^²+/2], 2823.6 (7) [M^²+/2].