Synlett 2010(9): 1371-1376  
DOI: 10.1055/s-0029-1219924
LETTER
© Georg Thieme Verlag Stuttgart ˙ New York

Synthesis of DNA with Spirobenzopyran as an Internal Covalent Modification

Christoph Beyer, Hans-Achim Wagenknecht*
University of Regensburg, Institute for Organic Chemistry, Universitätsstr. 31, 93053 Regensburg, Germany
Fax: +49(941)9434617; e-Mail: achim.wagenknecht@chemie.uni-regensburg.de;
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Publikationsverlauf

Received 11 February 2010
Publikationsdatum:
10. Mai 2010 (online)

Abstract

The photochromic spirobenzopyran was incorporated as an internal modification into oligonucleotides by two different synthetic strategies: For the first route the spiropyran phosphoramidite was synthesized as a DNA building block, whereas for the second route the postsynthetic ‘click’-type ligation was applied. Photo­induced ring opening of the chromophore could not be achieved in duplex DNA.

25

Synthesis of Compound 2
Under N2, freshly distilled 2,3,3-trimethylindolenine
(1.5 mL, 9.34 mmol) was dissolved in CHCl3 (17 mL) and degassed via freeze-pump-thaw (3 cycles). 3-Iodopropan-1-ol (1.00 g, 10.4 mmol) was added. The mixture was refluxed for 24 h and slowly cooled to r.t. The solvent was evapo-rated. The remaining purple oil was washed with PE and triturated with Et2O to afford 2 as a purple solid (3.22 g, 99%). ¹H NMR (300 MHz, DMSO-d 6): δ = 8.01-7.91 (m, 1 H), 7.90-7.81 (m, 1 H), 7.67-7.57 (m, 2 H), 4.56 (t, 2 H, J = 6.9 Hz), 3.55 (t, 2 H, J = 5.5 Hz), 2.87 (s, 3 H), 2.11-1.97 (m, 2 H), 1.54 (s, 6 H). ¹³C NMR (75 MHz, DMSO-d 6): δ = 196.4, 141.7, 141.0, 129.2, 128.8, 123.4, 115.4, 57.8, 54.1, 45.7, 29.6, 21.8, 14.3, 5.4. ESI-MS (CH2Cl2-MeOH + 10 mmol/L NH4Ac): m/z (%) = 218.0(100) [M+].

26

Synthesis of Compound 3
Compound 2 (3.09 g, 8.97 mmol) was suspended in degassed H2O (53 mL) and finely ground KOH (1.23 g, 21.9 mmol) was added. The reaction mixture was stirred at r.t. under N2 for 2 h. CH2Cl2 (50 mL) was added, and the mixture was stirred for additional 30 min. The aqueous layer was separated and extracted with CH2Cl2 (2 × 45 mL). The combined organic layers were washed with brine and H2O and dried over Na2SO4. The solvent was removed under reduced pressure, and the residue was dried under vacuum. The crude product was purified by gradient flash chromatog-raphy on silica gel (PE-EtOAc, 4:1 to 1:1) to afford 3 as colorless crystals (0.84 g, 43%). ¹H NMR (600 MHz, CDCl3): δ = 7.13 (t, 1 H, J = 7.7 Hz, CH arom.), 7.08 (d, 1 H, J = 7.3 Hz, CH arom.), 6.81 (t, 1 H, J = 7.2 Hz, CH arom.), 6.59 (d, 1 H, J = 7.8 Hz, CH arom.), 4.08 (dt, 1 H, J = 2.6, 12.4 Hz, CH2O), 3.72 (dd, 1 H, J = 5.2, 11.8 Hz, CH2O), 3.66 (dd, 1 H, J = 4.6, 14.6 Hz, CH2N), 3.54 (m, 1 H, CH2N), 2.02-1.92 (m, 1 H), 1.56 (s, 3 H, CH3), 1.30 (s, 3 H, CH3), 1.20 (d, 1 H, J = 13.4 Hz), 1.07 (s, 3 H, CH3). ¹³C NMR (150 MHz, CDCl3): δ = 148.1 (Cquat.), 139.2 (Cq), 127.2 (+, CH), 122.0 (+, CH), 119.2 (+, CH), 108.6 (+, CH), 98.3 (Cq), 61.0 (-, CH2), 48.0 (Cq), 39.1 (-, CH2), 26.7 (+, CH3), 21.7 (-, CH2), 18.6 (+, CH3), 13.0 (+, CH3). HRMS (EI-MS): m/z calcd for C14H19NO [M+ ]: 217.1467; found: 217.1472.

27

Synthesis of Compound 4
Under strictly degassed conditions, compound 3 (1.642 g, 7.56 mmol) was dissolved in dry EtOH and degassed. 5-Nitrosalicylaldehyde (1.28 g, 7.64 mmol) was added under N2, and the mixture was sonicated at 35 kHz for 1 h. EtOH was removed under reduced pressure. The residue was taken up in CH2Cl2 and washed with H2O. The organic layer was dried over Na2SO4 and evaporated. The product was purified by flash chromatography on silica gel (hexane-EtOAc = 1:1) to yield 4 (2.34 g, 84%) as a purple solid. ¹H NMR (600 MHz, CDCl3): δ = 8.00 (td, 2 H, J = 2.7, 7.9 Hz), 7.19 (dt, 1 H, J = 1.2, 7.7 Hz), 7.09 (dd, 1 H, J = 0.8, 7.2 Hz), 6.91 (d, 1 H, J = 10.3 Hz), 6.88 (dt, 1 H, J = 0.8, 7.5 Hz), 6.75 (d, 1 H, J = 8.9 Hz), 6.65 (d, 1 H, J = 7.8 Hz), 5.88 (d, 1 H, J = 10.4 Hz), 3.71 (t, 2 H, J = 6.0 Hz), 3.40-3.34 (m, 1 H), 3.29-3.23 (m, 1 H), 1.98-1.90 (m, 1 H), 1.84-1.77 (m, 1 H), 1.29 (s, 3 H), 1.19 (s, 3 H). ¹³C NMR (150 MHz, CDCl3): δ = 159.6, 147.0, 141.0, 136.0, 128.2, 127.8, 125.9, 122.7, 121.8, 121.7, 119.6, 118.5, 115.5, 106.9, 106.8, 60.7, 52.6, 40.67, 31.6, 25.9, 19.9. HRMS (EI-MS): m/z calcd for C21H22N2O4 [M+ ]: 366.1580; found: 366.1572.

28

Synthesis of Compound 5
A solution of 4 (1.06 g, 2.89 mmol) in dry CH2Cl2 (22 mL) was cooled to 0 ˚C, and dry DIPEA (2.2 mL, 12.936 mmol) was added under Ar. 4-Nitrophenylchloroformate (1.765 g, 8.757 mmol) was dissolved in dry CH2Cl2 (16.5 mL) and added in small portions over 3 h. The reaction was slowly warmed up to ambient temperature, and the solvent was evaporated. The crude product was dried over night under vacuum and purified by flash chromatography on silica gel (PE-EtOAc = 2:1) to afford a pale pink solid that was repeatedly purified by flash chromatography on silica gel(toluene). The product was triturated with Et2O and obtained as a pale yellow solid (1.44 g, 93%). ¹H NMR (400 MHz, CDCl3): δ = 8.28 (d, 2 H, J = 9.3 Hz), 8.05-7.98 (m, 2 H), 7.34 (d, 2 H, J = 9.3 Hz), 7.20 (dt, 1 H, J = 1.3, 7.7 Hz), 7.11 (dd, 1 H, J = 0.9, 7.3 Hz), 6.91 (t, 2 H, J = 8.6 Hz), 6.77 (d, 1 H, J = 8.8 Hz), 6.61 (d, 1 H, J = 7.7 Hz), 5.88 (d, 1 H, J = 10.3 Hz), 4.34 (dt, 2 H, J = 1.7, 5.9 Hz), 3.44-3.28 (m, 2 H), 2.19-1.98 (m, 2 H), 1.30 (s, 3 H), 1.20 (s, 3 H). ¹³C NMR (100 MHz, CD2Cl2): δ = 162.0 (Cq), 159.9 (Cq), 156.0 (Cq), 152.9 (Cq), 147.3 (Cq), 145.9 (Cq), 141.5 (Cq), 136.6 (Cq), 128.9 (+, CH), 128.2 (+, CH), 126.5 (+, CH), 126.3 (+, CH), 125.7 (+, CH), 123.2 (+, CH), 122.3 (+, CH), 122.3 (+, CH), 122.0 (+, CH), 120.2 (+, CH), 119.1 (Cq), 116.1 (+, CH), 115.9 (+, CH), 107.3 (Cq), 107.0 (+, CH), 67.6 (-, CH2), 40.5 (-, CH2), 28.3 (-, CH2), 26.1 (+, CH3), 20.0 (+, CH3). HRMS (PI-EI): m/z calcd for C28H25N3O8 [M+ ]: 531.1642; found: 531.1639.

29

Synthesis of Compound 7
A solution of 5 (25 mg, 0.047 mmol) in dry DMF (5 mL) was cooled to 0 ˚C. Dry DIPEA (50 µL, 0.30 mmol) was added under N2 and stirred for 5 min. Compound 6 (37 mg, 0.09 mmol) was added. The reaction mixtures was stirred at 0 ˚C over 5 h, was slowly warmed to r.t., concentrated in vacuo, and purified by flash chromatography on silica gel (PE-EtOAc = 1:1 + 0.1% DIPEA). Compound 7 (36 mg, 97%) was obtained as a pale pink solid. ¹H NMR (400 MHz, CDCl3): δ = 8.05-7.93 (m, 2 H), 7.41 (d, 2 H, J = 7.3 Hz), 7.33-7.27 (m, 6 H), 7.24-7.15 (m, 2 H), 7.09 (dd, 1 H, J = 0.8, 7.2 Hz), 6.90-6.81 (m, 6 H), 6.74 (d, 1 H, J = 8.9 Hz), 6.57 (d, 1 H, J = 7.7 Hz), 5.84 (d, 1 H, J = 10.4 Hz), 4.96 (s, 1 H, OH), 4.15-4.01 (m, 3 H), 3.90-3.83 (m, 1 H), 3.78 (s, 6 H, 2 × OMe), 3.39 (s, 1 H, NH), 3.32-3.25 (m, 1 H), 3.25-3.12 (m, 4 H), 2.01-1.92 (m, 1H), 1.90-1.83 (m, 1 H), 1.28 (s, 3 H), 1.18 (s, 3 H). ¹³C NMR (100 MHz, CDCl3): δ = 159.5, 158.6, 146.9, 144.5, 141.0, 136.0, 135.7, 130.0, 128.2, 128.0, 128.0, 127.8, 126.9, 125.9, 122.7, 121.7, 119.6, 118.5, 115.5, 113.2, 106.7, 106.7, 86.3, 70.3, 70.3, 65.0, 64.9, 62.5, 60.4, 55.2, 52.6, 44.1, 40.3, 31.5, 28.2, 28.2, 25.9, 25.5, 21.0, 19.8, 14.2. HRMS (PI-MS): m/z calcd for C46H47N3O9 [M+ ]: 785.3312; found: 785.3325.

30

Synthesis of Compound 8
Compound 7 (100 mg, 0.13 mmol) was dissolved in dry CH2Cl2 (5 mL). Et3N (250 µL, 1.794 mmol) and 2-cyano-ethyl-N,N-diisopropylchlorophosphoramidite (56.8 µL, 0.26 mmol) were added and the solution stirred for 11 h at r.t. The solvents were evaporated in vacuo, CH2Cl2 was added, the solution was poured into aq sat. NaHCO3, and extracted with CH2Cl2 (2×). The combined organic layers were dried over Na2SO4, the solvent was evaporated, and the remaining solid was purified by flash chromatography on silica gel (PE-EtOAc, 2:1 + 1% Et3N). The product was resolved in benzene (3 mL) and lyophilized yielding 8 (119 mg, 95%) as pale purple foam, which was dissolved in dry MeCN and applied directly for oligonucleotide synthesis. ³¹P NMR(121 MHz, CDCl3): δ = 149.9, 149.8. ESI-MS (CH2Cl2-MeOH + 10 mmol/L NH4Ac): m/z (%) = 986.5 (62) [MH+], 303.2 (100) [DMT+].

31

The oligonucleotide DNA1 was prepared using an Expedite 8909 DNA synthesizer (Applied Biosystems) via standard phosphoramidite protocols using CPGs (1 µmol) with a longer coupling time of 6.3 min and a higher concentration of 8 (0.1 M). After preparation, the trityl-off oligonucleotide was cleaved off the resin and deprotected by treatment with concd NH4OH at r.t. for 18 h, dried and purified by RP HPLC using the following conditions: A = NH4OAc buffer (50 mM), pH 6.8; B = MeCN; gradient = 20% B over 45 min, flow rate 2.5 mL/min. ESI-MS: DNA1: m/z calcd: 5408.0; found: 1802.7 [M - 3 H]³-, 1351.6 [M - 4 H]4-.

42

Synthesis of Compound 9
Under strictly degassed conditions freshly distilled 2,3,3-trimethylindolenine (30 mL, 186.9 mmol) was dissolved MeCN (40 mL) and degassed using freeze-pump-thaw method (3 cycles). Freshly distilled 1,3-diiodopropane (75 mL, 653 mmol) was added, and the reaction mixture was refluxed for 48 h. After cooling, the remaining solid was filtered off, washed with MeCN and CHCl3 (2×) and dried under vacuum yielding 9 (65.7 g, 77%) as a pale grey-yellow solid. ¹H NMR (400 MHz, DMSO-d 6): δ = 8.02-7.94 (m, 1 H, arom. C8-H), 7.88-7.80 (m, 1 H, arom. C5-H), 7.69-7.59 (m, 2 H, arom. C6/7-H), 4.48 (t, 2 H, N-CH2), 3.43 (t, 2 H, ICH2), 2.86 (s, 3 H, C2Me), 2.41 (m, 2 H, CH2-propyl), 1.55 (s, 6 H, C3-Me). ¹³C NMR (100 MHz, DMSO-d 6): δ = 197.3, 141.8, 141.1, 129.4 (Carom), 129.3, 128.9 (Carom), 123.5 (Carom), 115.2 (Carom), 54.2, 48.4 (NCH2), 31.0 (CH2-propyl), 22.0, 14.3 (C2Me), 2.2 (ICH2). ESI-MS (CH2Cl2-MeOH + 10 mmol/L NH4Ac): m/z (%) = 327.8 (100) [M+].

43

Synthesis of Compound 10
Under N2 9 (212 mg, 0.466 mmol) was suspended in degassed H2O (56 mL) and finely ground NaOH (559 mg, 13.98 mmol) was added in one portion. The mixture was heated at 80 ˚C for 15 min, cooled to r.t., Et2O (60 mL) was added, stirred for additional 1 h, and extracted with Et2O and CH2Cl2 (2 × 20 mL). The organic layers were washed with H2O, combined, dried over Na2SO4, and evaporated to give 10 (139 mg, 91%) as a pale pink solid. ¹H NMR (300 MHz): δ = 7.18-7.08 (m, 2 H), 6.83-6.61 (m, 2 H), 3.93 (dd, 2 H, J = 1.9, 22.2 Hz), 3.63 (t, 2 H, J = 6.8 Hz), 3.23 (t, 2 H, J = 6.7 Hz), 2.21 (quint, 2 H, J = 6.8 Hz), 1.35 (s, 6 H).
¹³C NMR (75 MHz): δ = 161.5 (Cq), 145.7 (Cq), 137.5 (Cq), 127.6 (+, CH), 122.0 (+, CH), 118.7 (+, CH), 105.3 (+, CH), 104.1 (+, CH3), 87.5 (+, CH3), 73.8 (-, CH2), 44.3 (Cq), 42.6 (-, CH2), 30.1 (-, CH2), 3.6 (-, CH2). MS (EI, 70 eV): m/z (%) = 184.0(100) [(M - HI - CH3)+ ], 198.9 (42) [(M - HI)+ ], 327.0(9) [M+ ].

44

Synthesis of Compound 11
Freshly prepared 10 (2.78 g, 8.49 mmol) was dissolved in dry EtOH (85 mL) and degassed via freeze-pump-thaw (3 cycles). 5-Nitrosalicylaldehyde (1.43 g, 8.53 mmol) was added. The mixture was sonicated at 35 kHz for 1 h. The solvent was removed under reduced pressure, the residue was taken up in CH2Cl2, and washed with H2O. The organic layer was dried over MgSO4 and evoporated. The raw product was dried under vacuum and purified by flash chormatography on silica gel (CH2Cl2) yielding 11 (7.97 mmol, 94%) as golden foam. ¹H NMR (400 MHz, CDCl3): δ = 8.06-8.01 (m, 2 H), 7.23 (dt, 1 H, J = 1.3, 7.7 Hz), 7.14 (dd, 1 H, J = 0.9, 7.3 Hz), 6.99 (d, 1 H, J = 10.2 Hz), 6.93 (dt, 1 H, J = 0.8, 7.5 Hz), 6.76 (d, 1 H, J = 8.5 Hz), 6.69 (d, 1 H, J = 7.7 Hz), 5.93 (d, 1 H, J = 10.3 Hz), 3.41-3.17 (m, 4 H), 2.34-2.21 (m, 1 H), 2.17-2.06 (m, 1 H), 1.34 (s, 3 H), 1.23 (s, 3 H). ¹³C NMR (100 MHz, CDCl3): δ = 159.2, 146.6, 140.8, 135.8, 128.4, 127.6, 125.7, 122.6, 121.6, 121.6, 119.6, 118.3, 115.3, 106.6, 106.4, 52.4, 43.9, 32.3, 25.8, 19.7, 3.3. HRMS (EI-MS): m/z calcd for C21H21N2O3I [M+ ]: 476.0597; found: 476.0598.

45

Synthesis of Compound 12
Compound 11 (1.58 g, 3.31 mmol) was dissolved in dry DMF (62 mL). NaN3 (877 mg, 13.5 mmol) was added in one portion, and the reaction mixture was stirred in the dark at r.t. for 19 h. The solvent was evaporated under reduced pressure, and the crude product was purified by flash chromatography on silica gel (CH2Cl2) yielding 12 (1.08 g, 83%) as a golden foam. ¹H NMR (400 MHz, CDCl3): δ = 8.01 (m, 2 H), 7.20 (dt, 1 H, J = 1.3, 7.7 Hz), 7.10 (dd, 1 H, J = 0.9, 7.3 Hz), 6.94 (d, 1 H, J = 10.3 Hz), 6.90 (dt, 1 H, J = 0.9, 7.5 Hz), 6.75 (d, 1 H, J = 8.4 Hz), 6.60 (d, 1 H, J = 7.8 Hz), 5.86 (d, 1 H, J = 10.4 Hz), 3.39-3.18 (m, 4 H), 2.01-1.90 (m, 1 H), 1.88-1.75 (m, 1 H), 1.29 (s, 3 H), 1.19 (s, 3 H). ¹³C NMR (100 MHz, CDCl3): δ = 159.4, 146.8, 141.1, 136.0, 128.4, 127.8, 125.9, 122.8, 121.8, 121.6, 119.8, 118.4, 115.5, 106.7, 106.6, 52.6, 49.0, 40.8, 28.1, 25.9, 19.9. HRMS (PI-EI): m/z calcd for C21H21N5O3 [M+ ]: 391.1644; found: 391.1644.

46

The concentration of the DNA building block was increased to 0.1 M and the coupling time extended to 6.3 min.³9 The trityl-off oligonucleotides were cleaved from the resin and deprotected by treatment with concd NH4OH at r.t. for 24 h. Compound 12 (1.5 mL, 10 mM), CuI (223.6 mL, 100 mM), TBTA (447 mL, 100 mM), each in DMSO-t-BuOH (3:1), and sodium ascorbate (125 mL, 400 mM) in H2O were added to the oligonucleotide (1 mmol). The vial was vortexed, shaken 22 h at r.t., and then evaporated to dryness using a SpeedVac. NaOAc (100 mL, 0.15 mmol) was added, and the mixture was stored for 1 h r.t. After EtOH precipation, the oligonucleotides were dissolved in H2O (500 mL), desalted by NAP-5 column (GE Healthcare), dried, purified by RP HPLC using the following conditions: A = NH4OAc buffer (50 mM), pH 6.8; B = MeCN; gradient = 15% B over 45 min, flow rate 2.5 mL/min, lyophilized, and quantified by their absorbance at 260 nm. Duplexes were prepared by heating of the modified oligonucleotides in the presence of 1.2 equiv unmodified complementary strand to 90 ˚C (10 min), followed by slow cooling to r.t. ESI-MS: DNA2: m/z calcd 5550.0 [M]-, 1849.0 [M - 3 H]³-; found: 1849.2 [M - 3 H]³-, 1386.8 [M - 4 H]4-; DNA3: m/z calcd 5580.0 [M]-, 1859.0 [M - 3 H]³-; found: 1860.1 [M - 3 H]³-, 1395.0 [M - 4 H]4-; DNA4: m/z calcd 5598.0 [M]-, 1865.0 [M - 3 H]³-; found: 1866.2 [M - 3 H]³-, 1399.3 [M - 4 H]4-; DNA5: m/z calcd 5630.5 [M]-, 1875.8 [M - 3 H]³-; found: 1876.7 [M - 3 H]³-, 1407.5 [M - 4 H]4-.