Ication lies inside the mixed prefunctionalization together with amino (and, in
Ication lies within the mixed prefunctionalization along with amino (and, in principle, also with alkyne) moieties on the same RNA to allow for selective and stepwise attachment of sensitive moieties that cannot be directly integrated into RNA. Efficient generation of complex labeling patterns is, e.g.,EXPERIMENTAL PROCEDURES Common Remarks. 1H and 13C NMR spectra have been recorded on the Bruker DRX 300 MHz or Avance II 600 MHz instrument. The chemical shifts are referenced towards the residual proton signal of the deuterated solvents: CDCl3 (7.26 ppm), d6-DMSO (two.49 ppm) for 1H NMR spectra; CDCl3 (77.0 ppm) or d6-DMSO (39.5 ppm) for 13C NMR spectra (see also Figures S3-S6). 1H- and 13C-assignments were determined by COSY and HSQC experiments. MS experiments had been performed on a Finnigan LCQ Advantage MAX ion trap instrument. Analytical thin-layer chromatography (TLC) was carried out on Marchery-Nagel Polygram SIL GUV254 plates. Flash column chromatography was carried out on silica gel 60 (70-230 mesh). All reactions were carried out under argondx.doi.org10.1021bc400513z | Bioconjugate Chem. 2014, 25, 188-required for multicolor single-molecule FRET studies and it is PAK6 Synonyms presently undertaken in our laboratory.Bioconjugate Chemistry atmosphere. Chemical reagents and solvents have been obtained from industrial suppliers and utilised without the need of additional purification. Natural solvents for reactions had been dried overnight in excess of RGS4 Molecular Weight freshly activated molecular sieves (4 . 2-O-(2-Azidoethyl)uridine (2). 2,2-Anhydrouridine 1 (565 mg, 2.5 mmol) was coevaporated with dry pyridine three instances and stored more than P2O5 within a desiccator for 4 hrs in advance of use. Then, compound 1 was suspended in DMA (4 mL) and BF3 Et2 (785 L, six.25 mmol) was extra underneath argon and heated to 120 . 2-Azidoethanol (1250 mg, 14.3 mmol) was injected to the resolution plus the mixture was refluxed for 16 h. Soon after the response was finished solvents have been eliminated in vacuo, as well as the oily residue was redissolved in methanol and adsorbed on silica gel. Compound 2 was purified by column chromatography on SiO2 with CHCl3CH3OH, 95:5. Yield: 431 mg of 2 as a white solid (55 ). TLC (CH2Cl2CH3OH = 85:15): Rf = 0.51. 1H NMR (300 MHz, DMSO): three.17 (m, 2H, H1-C(2) H2-C(2)); 3.58 (m, 2H; H1-C(5) H2- C(5)); 3.86 (m, 2H, H1-C(one) H2-C(1)); three.88 (m, 1H, H- C(four)); four.04 (m, 1H, H-C(2)); four.60 (dd, J = four.8 Hz, J = 9.eight Hz, 1H, H-C(three)); 5.14 (m, 2H, HO-C(3), HO-C(5)); five.72 (d, J = 8.0 Hz, 1H, H-C(five)); 5.88 (d, J = 4.8 Hz, 1H, H- C(1)); 7.94 (d, J = 8.0 Hz, 1H, H-C(six)); 11.29 (s, 1H, NH) ppm. 13C NMR (150 MHz, DMSO): 49.93 (C(2)); 60.39 (C(5)); 68.two (C(three)); 68.86 (C(1); 81.31 (C(2); 84.93 (C(four)); 86.15 (C(1)); 101.79 (C(five)); 140.32 (C(6)); 150.56; 163.10 ppm. ESI-MS (mz): [M-H]- calcd for C11H15N5O6, 312.eleven; uncovered 312.46. 2-O-(2-Azidoethyl)-5-O-(4,4-dimethoxytrityl)uridine (2a). Compound 2 (372 mg, 1.19 mmol) was coevaporated with dry pyridine three instances and dissolved in pyridine at room temperature and beneath argon ambiance. 4,4-Dimethoxytrityl chloride (443 mg, 1.31 mmol) was extra in two portions above a time period of two h. Stirring was continued overnight, and when TLC showed comprehensive response, methanol was additional as well as the resolution was evaporated in vacuo. The residue was dissolved in CH2Cl2 and extracted with 5 citric acid, water, and saturated NaHCO3. The organic layer was dried more than Na2SO4 and evaporated. The crude merchandise was purified by column chromatography on SiO2 with CH2Cl2CH3OH, a hundred:0 to 98:two. Yield: 549 mg of 2a like a white foam (75.