Thieme Chemistry Journal Awardees - Where Are They Now?Synthesis and Optical Properties of Nile Red Modified 2′-Deoxyuridine and 7-Deaza-2′-deoxyadenosine: Highly Emissive Solvatochromic Nucleosides

Abstract

A general synthetic strategy for the attachment of Nile red through a rigid acetylene linker to 2′-deoxyuridine and 7-deaza-2′-deoxyadenosine using Sonogashira coupling is demonstrated. Protection of either 5′-OH or N-7 of the nucleosides increased the yields of the cross-couplings significantly. Both Nile red modified 2′-deoxyuridine and 7-deaza-2′-deoxyadenosine as well as their derivatives exhibit excellent fluorescence quantum efficiencies and positive solvatochromism. The incorporation of the Nile red modified 2′-deoxyuridine into oligonucleotides yields bright fluorescent probes with maintained canonical base pairing in DNA.

Key words

cross-coupling - dyes - nucleosides - solvent effects - spectroscopy

References and Notes

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General Procedure of Sonogashira Coupling
A mixture of 1 (125 mg, 0.364 mmol), 5-iodo-2′-deoxyuridine, 2 (100 mg, 0.28 mmol), Et₃N (0.6 mL, 4 mmol), Pd(PPh₃)₄ (42 mg, 0.036 mmol), and CuI (15 mg, 0.072 mmol) were dissolved in DMF (5 mL). After the solution was degassed three times via the freeze-pump-thaw method, the mixture was heated at 90 ˚C for 4 h. The reaction solvent was removed under reduced pressure, and the crude product was purified by flash column chromatography (SiO₂, 2-3% MeOH in EtOAc eluent) furnishing the desired product 9 (48 mg, 30%) as a dark green solid. R _f  = 0.4 (SiO₂, 5% MeOH in EtOAc); mp >200 ˚C.

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Analytical Data
Compound 9: ^¹H NMR (300 MHz, CDCl₃): δ = 11.79 (s, 1 H), 8.56 (d, J = 1.7 Hz, 1 H), 8.51 (s, 1 H), 8.13 (d, J = 8 Hz, 1 H), 7.75-7.69 (m, 2 H), 6.87 (dd, J = 9.0, 2.5 Hz, 1 H), 6.70 (d, J = 2.5 Hz, 1 H), 6.31 (s, 1 H), 6.15 (t, J = 6.5 Hz, 1 H), 5.25 (d, J = 4.0 Hz, 1 H), 5.24 (t, J = 5.0 Hz, 1 H), 4.32-4.24 (m, 1 H), 3.83 (q, J = 3.3 Hz, 1 H), 3.73-3.58 (m, 2 H), 3.52 (q, J = 6.7 Hz, 4 H), 2.27-2.15 (m, 2 H), 1.17 (t, J = 7.0 Hz, 6 H) ppm. ^¹³C NMR (75 MHz, DMSO-d ₆): δ = 181.0, 161.3, 151.9, 151.0, 149.5, 146.4, 144.5, 137.0, 134.4, 131.69, 131.53, 131.1, 130.0, 125.5, 125.2, 124.3, 114.2, 110.4, 104.5, 97.6, 95.8, 91.2, 87.5, 85.2, 84.8, 69.8, 60.7, 44.4, 12.3 ppm. ESI-MS: m/z (%) = 569.2 (100) [M + H]⁺. ESI-HRMS: m/z calcd for C₃₁H₂₈N₄O₇ [M + H]⁺: 569.2036; found: 569.2026.

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Compound 10: ^¹H NMR (300 MHz, CDCl₃): δ = 8.58 (d, J = 1.4 Hz, 1 H), 8.26 (s, 1 H), 8.17 (br s, 1 H), 7.96 (d, J = 8.0 Hz, 1 H), 7.52 (d, J = 7.4 Hz, 1 H), 7.42 (d, J = 7.4 Hz, 2 H), 7.31 (m, 4 H), 7.25 (s, 1 H), 7.20 (s, 1 H), 7.14-7.06 (m, 1 H), 6.88 (dd, J = 8.2, 1.7 Hz, 1 H), 6.78-6.71 (m, 4 H), 6.64 (dd, J = 9.1, 2.7 Hz, 1 H), 6.42 (d, J = 2.5 Hz, 1 H), 6.34 (m, 1 H), 6.32 (s, 1 H), 4.58 (m, 1 H), 4.07 (m, 1 H), 3.62 (s, 3 H), 3.60 (s, 3 H), 3.49-3.37 (m, 6 H), 3.33 (m, 1 H), 2.53 (m, 1 H), 2.48 (m, 1 H), 1.21 (t, J = 4.7 Hz, 6 H) ppm. ^¹³C NMR (75 MHz, CDCl₃): δ = 183.1, 161.1, 158.5, 152.2, 150.9, 149.1, 146.8, 144.4, 142.9, 138.87, 135.5, 135.4, 133.0, 132.5, 132.1, 132.0, 131.7, 131.6, 129.9, 128.6, 128.4, 128.1, 127.9, 127.0, 125.5, 125.2, 125.1, 113.3, 113.1, 110.0, 100.1, 96.1, 93.3, 87.1, 86.8, 72.2, 63.5, 55.1, 45.1, 12.6 ppm. ESI-MS: m/z (%) = 871.4 (100) [M + H]⁺.

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Compound 11: ^¹H NMR (300 MHz, CDCl₃): δ = 8.41 (s, 1 H), 8.37 (s, 1 H), 7.94 (d, J = 8.2 Hz, 1 H), 7.64-7.35 (m, 2 H), 6.55 (m, 1 H), 6.34-6.25 (m, 3 H), 4.67 (m, 1 H), 4.08-3.85 (m, 4 H), 3.39 (q, J = 7.1 Hz, 4 H), 2.50-2.27 (m, 3 H), 1.63-1.53 (m, 2 H), 1.26-1.16 (m, 24 H), 0.83 (t, J = 7.1 Hz, 3 H) ppm. ^¹³C NMR (75 MHz, CDCl₃): δ = 181.2, 161.2, 151.2, 151.0, 150.1, 146.6, 144.4, 138.5, 135.0, 132.9, 132.1, 132.0, 131.6, 128.6, 128.4, 125.4, 109.0, 104.4, 101.1, 99.5, 96.1, 91.3, 89.5, 87.5, 74.2, 67.6, 46.0, 45.1, 31.9, 29.3, 29.6, 29.5, 27.0, 22.7, 14.1, 12.6 ppm. ESI-HRMS: m/z calcd for C₄₃H₅₂N₄O₇ [M⁺ ]: 736.3836; found: 736.3845.

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Compound 12: ^¹H NMR (300 MHz, CDCl₃): δ = 8.7 (d, J = 1.3 Hz, 1 H), 8.27-8.19 (m, 2 H), 7.97 (s, 1 H), 7.69-7.38 (m, 2 H), 6.65 (dd, J = 9.0, 2.7 Hz, 1 H), 6.43 (d, J = 2.7 Hz, 1 H), 6.35 (s, 1 H), 6.24 (t, J = 6.3 Hz, 1 H), 4.52-4.12 (m, 4 H), 3.44 (q, J = 6.8 Hz, 4 H), 2.56-2.34 (m, 4 H), 2.21 (m, 1 H), 1.59-1.49 (m, 1 H), 1.26-1.04 (m, 22 H), 0.78 (t, J = 6.8 Hz, 3 H) ppm. ^¹³C NMR (75 MHz, CDCl₃): δ = 181.7, 176.5, 161.1, 151.4, 150.5, 149.6, 146.5, 143.6, 138.0, 135.4, 132.9, 132.2, 131.6, 131.0, 128.4, 127.2, 125.1, 108.0, 105.2, 102.1, 100.3, 96.4, 91.3, 86.0, 85.8, 72.7, 67.5, 60.4, 45.2, 35.8, 31.9, 30.9, 29.7, 29.5, 22.6, 21.0, 14.2, 12.6 ppm. ESI-HRMS: m/z calcd for C₄₃H₅₀N₄O₈ [M⁺ ]: 750.3629; found: 750.3604.

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Compound 13: ^¹H NMR (300 MHz, CDCl₃): δ = 8.69 (s, 1 H), 8.24 (br s, 1 H), 8.21 (s, 1 H), 7.62 (s, 1 H), 7.45-7.20 (m, 2 H), 6.79-6.73 (m, 2 H), 6.43 (d, J = 2.4 Hz, 1 H), 6.34 (s, 1 H), 4.56 (m, 1 H), 4.01 (m, 1 H), 3.71-3.61 (m, 2 H), 3.42 (q, J = 7.1 Hz, 4 H), 2.57-2.25 (m, 2 H), 1.23-1.14 (m, 6 H) ppm. ^¹³C NMR (75 MHz, CDCl₃): δ = 180.6, 165.2, 156.8, 151.7, 150.4, 148.8, 146.9, 139.2, 135.9, 135.0, 132.0, 131.9, 130.5, 127.7, 126.5, 124.4, 113.9, 110.7, 103.0, 110.7, 104.6, 103.0, 93.2, 90.3, 87.3, 84.2, 83.4, 69.8, 63.7, 48.3, 45.6, 13.0 ppm. ESI-MS: m/z (%) = 591.2 (100) [M + H]⁺.

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Compound 14: ^¹H NMR (300 MHz, CDCl₃): δ = 8.70 (s, 1 H),8.29-8.21 (m, 2 H), 7.66-7.11 (m, 14 H), 6.82-6.62 (m, 4 H), 6.46 (d, J = 2.4 Hz, 1 H), 6.36 (s, 1 H), 4.57 (m, 1 H), 4.05 (m, 1 H), 3.77-3.63 (m, 8 H), 3.45 (q, J = 7.1 Hz, 4 H), 2.53-2.27 (m, 2 H), 1.26-1.16 (m, 6 H) ppm. ^¹³C NMR (75 MHz, CDCl₃): δ = 182.2, 158.5, 157.4, 155.0, 152.1, 150.8, 149.4, 146.9, 144.6, 142.6, 139.2, 136.5, 135.7, 135.6, 133.7, 132.1, 132.0, 130.2, 130.1, 128.4, 128.2, 127.9, 126.3, 125.1, 113.2, 113.1, 111.7, 109.7, 101.9, 100.3, 96.2, 92.8, 86.6, 85.5, 81.1, 71.6, 69.5, 66.9, 55.2, 53.7, 45.1, 12.6 ppm. ESI-MS: m/z (%) = 893.3 (100) [M + H]⁺.

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Compound 15: ^¹H NMR (300 MHz, CD₃CN): δ = 8.5 (s, 1 H), 8.01-7.98 (m, 2 H), 7.79-7.52 (m, 7 H), 7.46-7.11 (m, 10 H), 6.85-6.74 (m, 6 H), 6.60-6.52 (m, 2 H), 6.45 (m, 1 H), 5.32 (br, 1 H), 4.48 (m, 1 H), 3.93 (m, 1 H), 3.74 (d, J = 2.7 Hz, 6 H), 3.69 (m, 2 H), 3.48 (q, J = 7.1 Hz, 4 H), 2.52 (m, 1 H), 2.28 (m, 1 H), 1.18-1.14 (m, 6 H) ppm. ^¹³C NMR (75 MHz, CD₃CN): δ = 182.2, 169.0, 160.7, 157.0, 155.2, 152.9, 150.8, 146.2, 144.0, 141.0, 139.1, 136.9, 135.9, 135.6, 134.3, 133.9, 133.1, 131.6, 131.1, 130.6, 129.6, 129.3, 128.6, 128.4, 127.7, 127.3, 126.6, 124.7, 114.0, 108.9, 106.5, 110.2, 102.5, 101.4, 97.8, 91.2, 87.6, 85.3, 82.6, 73.2, 64.8, 52.6, 45.7, 38.4, 13.5 ppm. ESI-MS: m/z (%) = 997.4 (100) [M + H]⁺.

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The oligonucleotides were prepared on an Expedite 8909 DNA synthesizer (Applied Biosystems) via standard phosphoramidite protocols using CPGs (1 µmol) with a longer coupling time of 15 min and a higher concentration of the phosphoramidite (0.1 M). After preparation, the trityl-
off oligonucleotide was cleaved off the resin and was deprotected by treatment with concd NH₄OH at r.t. for 10 h. The oligonucleotides were dried and purified by reverse-phase HPLC using the following conditions: A = NH₄OAc buffer (50 mM), pH = 6.5; B = MeCN; gradient = 0-20% B over 50 min. The oligonucleotides were lyophilized and quantified by their absorbance at 260 nm on a Varian Cary Bio 100 spectrometer. Duplexes were prepared by heating of Nile red modified oligonucleotides in the presence of 1 equiv unmodified complementary strand to 90 ˚C (10 min), followed by slow cooling to r.t. ESI-MS: DNA1: m/z calcd 5475.4; found: 5475.1 (100%); DNA2: m/z calcd 5575.4; found = 5575.1 (100%).