Compound 5

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InChIKey IMSQQBIHJHSRIY-OJRKMGBJSA-N

Synthetic procedure: See article for the definitive version of this procedure and for full experimental details.

A 6.1 mM RNA aptamer solution (sequences: 5'-GGA CUG GGC GAG AAG UUU AGU CC-3' (apt1) or 5'-CUG CAG UCC GAA AAG GGC CAG-3' (apt2)) (816 µL, 4.98 µmol) in 10 mM sodium phosphate buffer (pH 6.8) was heated to 85°C for 10 min and was afterwards kept at room temperature for 15 min. Then a 4.8 mM solution of the neomycin B 1 (684 µL, 3.28 µmol) in 10 mM sodium phosphate buffer (pH 7.4) was added and the mixture was allowed to stand for 30 min at room temperature. Afterwards 30 equiv. NHS ester 3b (18.2 mg, 98.5 µmol) dissolved in DMF (108 µL) was added and the reaction mixture was allowed to react for 24 hours at room temperature. After addition of a 7 wt. % ethylamine water solution (126 µL) and further incubation for 30 min at room temperature, the crude mixture was heated to 99°C for 10 min. To the hot solution a 53 mM aqueous solution of didodecyldimethylammonium bromide (DDDMABr) (3 mL) was added to precipitate the RNA. After incubation for 15 min at room temperature and centrifugation for 30 min at 6°C (16.1 u/s) the supernatant was freeze-dried and dissolved in water (400 µL). Each fraction (30 µL) was purified by HPLC using a Waters Spherisorb ODS-2C₁₈ analytic column (water/acetone 1 : 0.9 containing 12.1 mM HFBA) at a flow rate of 1 ml/min at 40°C to yield 5 (R_t = 9.65 min). TLC (Chloroform/MeOH/17% NH₄OH 2:1:1 v/v/v) R_f = 0.50. ¹H-NMR (D₂O, 500 MHz) δ (p.p.m.) 5.98 (s, 1H, 1-H^I), 5.38 (s, 1H, 1-H^II), 5.15 (s, 1H, 1-H^III), 4.39 (d, J = 6.0 Hz, 1H, 3-H^II), 4.37 (m, 1H, 2-H^II), 4.21 (m, 1H, 3-H^III), 4.17 (m, 1H, 4-H^II), 4.06 – 4.01 (m, 2H, 5-H^III, 4-H), 3.96 (t, J = 10.3 Hz, 1H, 5-H^I), 3.92 – 3.88 (m, 3H, 5-H^II, 5-H, 3-H^I), 3.70 (dd, 1H, J = 14 Hz, J = 5.8 Hz, 5-H^II), 3.65- 3.61 (m, 2H, 4-H^III, 6-H), 3.54-3.50 (m, 3H, 6a-H^III, 3-H, 2-H^III), 3.46 (m, 1H, 4-H^I), 3.44-3.38 (m, 3H, 6a-H^I, 2-H^I, 6b-H^III), 3.56-3.33 (m, 1H, 1-H), 3.28 (dd,J = 13.5 Hz, J = 6 Hz, 1H, 6b-H^I), 2.47 (m, 2H, CH(CH₃)₂, 2-H_e), 1.86 (dd, J = 12.2 Hz, 1H, 2-H_a), 1.09 (s, J = 6.5 Hz, 6H, CH₃). ¹³C-signals based on HSQC (D₂O, 500 MHz) δ (p.p.m.) 110.31 (C-1^II), 95.48 (C-1^I), 95.18 (C-1^III), 84.72 (C-5), 81.54 (C-4^II), 75.23 (C-4), 74.90 (C-3^II), 73.49 (C-2^II), 72.47 (C-5^III), 72.39 (C-6), 70.42 (C-4^I), 69.23 (C-5^I), 67.87 (C-3^I), 67.48 (C-3^III), 66.08 (C-4^III), 60.04 (C-5^II), 53.33 (C-2^I), 50.87 (C-2^III), 49.59 (C-1), 48.22 (C-3), 39.94 (C-6^I), 39.03 (C-6^III), 34.93 (CH(CH₃)₂), 27.95 (CH(CH₃)₂), 27.89 (C-2). MS (EI+) (m/z): found 685.36163 [M+H]⁺, 707.34369 [M+Na]⁺; calculated 685.36143 [M+H]⁺, 707.34337 [M+Na]⁺.