Synthesis of DNA with Spirobenzopyran
as an Internal Covalent Modification

Christoph Beyer; Hans-Achim Wagenknecht

doi:10.1055/s-0029-1219924

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Synlett 2010(9): 1371-1376
DOI: 10.1055/s-0029-1219924

LETTER

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;

Further Information

Publication History

Received 11 February 2010
Publication Date:
10 May 2010 (online)

Abstract
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References
Supplementary Material

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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.

Key words

oligonucleotide - molecular switch - click ligation - photochromism - phosphoramidite

Supporting Information

References and Notes

For reviews, see:
1a Raymo FM. Tomasulo M. Chem. Eur. J. 2006, 12: 3186

Google Scholar
1b Pischel U. Angew. Chem. Int. Ed. 2007, 46: 4026

Google Scholar
2 For review, see: Cusido J. Deniz E. Raymo FM. Eur. J. Org. Chem. 2009, 2031

Crossref Google Scholar
3 For review, see: Mayer G. Heckel A. Angew. Chem. Int. Ed. 2006, 45: 4900

Crossref PubMed Google Scholar
4 For review, see: Moroder L. Renner C. ChemBioChem 2006, 7: 868

Crossref Google Scholar
5a Asanuma H. Liang X. Komiyama M. ChemBioChem 2001, 2: 39

Google Scholar
5b Nishioka H. Liang X. Asanuma H. Chem. Commun. 2007, 4354

Google Scholar
5c Liang X. Takenaka N. Nishioka H. Asanuma H. Chem. Asian J. 2008, 3: 553

Google Scholar
6 Yamazawa A. Liang X. Asanuma H. Komiyama M. Angew. Chem. Int. Ed. 2000, 39: 2356

Crossref Google Scholar
7 Liang X. Asanuma H. Komiyama M. J. Am. Chem. Soc. 2002, 124: 1877

Crossref Google Scholar
8 For review, see: Berkovic G. Krongauz V. Weiss V. Chem. Rev. 2000, 100: 1741

Crossref Google Scholar
9 Andersson J. Andersson J. Li S. Lincoln P. J. Am. Chem. Soc. 2008, 130: 11836

Crossref Google Scholar
10 Young D. Deiters A. ChemBioChem 2008, 9: 1225

Crossref Google Scholar
11 Sakata T. Yan Y. Marriott G. J. Org. Chem. 2005, 70: 2009

Crossref Google Scholar
12 Angelini N. Corrias B. Fissi A. Pieroni O. Lenci F. Biophys. J. 1998, 74: 2601

Crossref Google Scholar
13 Kocer A. Walko M. Meijberg W. Feringa BL. Science 2005, 309: 755

Crossref Google Scholar
14 Asanuma H. Shirasuka K. Yoshida T. Takarada T. Liang X. Komiyama M. Chem. Lett. 2001, 30: 108

Google Scholar
15 Zhang P. Meng JB. Matsuura T. Wang YM. Chin. Chem. Lett. 2002, 13: 299

Google Scholar
16a Huber R. Amann N. Wagenknecht H.-A. J. Org. Chem. 2004, 69: 744

Google Scholar
16b Amann N. Huber R. Wagenknecht H.-A. Angew. Chem. Int. Ed. 2004, 43: 1845

Google Scholar
17a Wagner C. Wagenknecht H.-A. Org. Lett. 2006, 8: 4191

Google Scholar
17b Baumstark D. Wagenknecht H.-A. Angew. Chem. Int. Ed. 2008, 47: 2652

Google Scholar
18 Wagner C. Wagenknecht H.-A. Org. Biomol. Chem. 2008, 6: 48

Crossref Google Scholar
19a Menacher F. Rubner M. Berndl S. Wagenknecht H.-A. J. Org. Chem. 2008, 73: 4263

Google Scholar
19b Berndl S. Wagenknecht H.-A. Angew. Chem. Int. Ed. 2009, 48: 2418

Google Scholar
20 Amann N. Wagenknecht H.-A. Tetrahedron Lett. 2003, 44: 1685

Crossref Google Scholar
21a Zhang L. Peritz A. Meggers E. J. Am. Chem. Soc. 2005, 127: 4174

Google Scholar
21b Zhang L. Peritz AE. Carroll PJ. Meggers E. Synthesis 2006, 645

Google Scholar
21c Schlegel MK. Essen L.-O. Meggers E. J. Am. Chem. Soc. 2008, 130: 8158

Google Scholar
22a Filichev VV. Christensen UB. Pedersen EB. Babu BR. Wengel J. ChemBioChem 2004, 5: 1673

Google Scholar
22b Filichev VV. Pedersen EB. J. Am. Chem. Soc. 2005, 127: 14849

Google Scholar
22c Geci I. Filichev VV. Pedersen EB. Chem. Eur. J. 2007, 13: 6379

Google Scholar
23 Gierlich PME. Warncke S. Gierlich J. Carell T. Angew. Chem. Int. Ed. 2008, 47: 3442

Crossref Google Scholar
24 Berndl S. Herzig N. Kele P. Lachmann D. Li X. Wolfbeis OS. Wagenknecht H.-A. Bioconjugate Chem. 2009, 20: 558

Crossref Google Scholar
32 Weisbrod SH. Marx A. Chem. Commun. 2008, 5675

Crossref Google Scholar
33 Sletten EM. Bertozzi CR. Angew. Chem. Int. Ed. 2009, 48: 6974

Article in Thieme Connect PubMed Google Scholar
34 Amblard F. Cho JH. Schinazi RF. Chem. Rev. 2009, 109: 4207

Crossref PubMed Google Scholar
35 Gramlich PM. Wirges CT. Manetto A. Carell T. Angew. Chem. Int. Ed. 2008, 47: 8350

Crossref Google Scholar
36 Huisgen R. Angew. Chem., Int. Ed. Engl. 1962, 2: 565

Crossref Google Scholar
37 Tornoe CW. Christensen C. Meldal M. J. Org. Chem. 2002, 67: 3057

Crossref PubMed Google Scholar
38 Lewis WG. Green LG. Grynszpan F. Radic Z. Carlier PR. Taylor P. Finn MG. Sharpless KB. Angew. Chem. Int. Ed. 2002, 41: 1053

Google Scholar
39 Berndl S. Herzig N. Kele P. Lachmann D. Li X. Wolfbeis OS. Wagenknecht H.-A. Bioconjugate Chem. 2009, 20: 558

Crossref Google Scholar
40a Binder WH. Sachsenhofer R. Straif CJ. Zirbs R. J. Mater. Chem. 2007, 17: 2125

Google Scholar
40b Zhang Q. Ning Z. Yan Y. Qian S. Tian H. Macromol. Rapid Commun. 2008, 29: 193

Google Scholar
41 Guo H. Zou WX. Ji Q. Meng JB. Chin. Chem. Lett. 2005, 16: 751

Google Scholar
47a Görner H. Chem. Phys. 1997, 222: 315

Google Scholar
47b Chibisov AK. Görner H. J. Phys. Chem. A 1997, 101: 4305

Google Scholar

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 (C_quat.), 139.2 (C_q), 127.2 (+, CH), 122.0 (+, CH), 119.2 (+, CH), 108.6 (+, CH), 98.3 (C_q), 61.0 (-, CH₂), 48.0 (C_q), 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 (C_q), 159.9 (C_q), 156.0 (C_q), 152.9 (C_q), 147.3 (C_q), 145.9 (C_q), 141.5 (C_q), 136.6 (C_q), 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 (C_q), 116.1 (+, CH), 115.9 (+, CH), 107.3 (C_q), 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 (C_arom), 129.3, 128.9 (C_arom), 123.5 (C_arom), 115.2 (C_arom), 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 (C_q), 145.7 (C_q), 137.5 (C_q), 127.6 (+, CH), 122.0 (+, CH), 118.7 (+, CH), 105.3 (+, CH), 104.1 (+, CH₃), 87.5 (+, CH₃), 73.8 (-, CH₂), 44.3 (C_q), 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-.

Supplementary Material

Supporting Information