Studies on the Chemical Biology of Natural and Chemical Ribonucleotide Modifications

This work is centered on the synthesis and chemical recognition of modified RNA. Phosphoramidites of m5U, Ψ, m1G, m2G and m22G were synthesized and were incorporated into RNA oligomers by standard SPS. The synthesized oligomers were used for two purposes: (i) the total construction of tRNA via enzyme catalyzed ligation, and (ii) investigations of modifications in siRNAs. The construction of human mitochondrial tRNAIle (i) with and without modification was achieved in a one-pot 3 fragments ligation using T4-DNA-ligase with a DNA-splint. The yield for the ligation was 20-30 % and the products were further investigated for the stability of the tertiary structure via UV-melting curve analysis. The unmodified tRNA was found to be significantly less stable than the fully modified tRNA. In the second case (ii) RNAi efficiency and the immunostimulation in cells were investigated with 22 nucleotide long double-stranded siRNAs that were synthesized containing the modifications m1G, m2G, m22G, rT and in varying stochiometry. The investigations showed differential effects in knock-down as well as immunostimulation. In a related perspective, compounds for the chemical recognition of modifications in RNA were synthesized. Since the recognition of pseudouridine by 4-bromomethyl-coumarin derivates is of particular interest, 7-azido-4-bromomethyl-coumarin was obtained with an overall yield of 44 % in a 5-step synthesis. Another derivative, the biotinylated 4-bromomethyl-coumarin spaced with jeffamine-148 was obtained in a 6-step synthesis with an overall yield of 17 %, to allow the use of strepavidin for affinity separation. In the course of further investigations of coumarine conjugates, mild and bio-compatible conditions for the syntheses of rhodols and rhodamines were discovered. The conversion of fluorescein into the 3’-mesylated or 3’-6’-dimesylated derivate and the subsequent displacement of the mesylate with a nitrogenous nucleophile gave rise to rhodols or rhodamines, respectively. As these reaction conditions are compatible with biomolecular labeling, the tagging of polypeptides was investigated. Tri-and pentapeptides were synthesized, and dye-labeled via 3’/6’-xanthene substitution, and the product identity confirmed by MALDI-TOF-MS. Further syntheses via 3’/6’-xanthene substitution lead to moderate yields of 3',6'-bis(dimethylamino)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one, 3',6'-di(piperidin-1-yl)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one and the 3',6'-bis(2-(2-(2-aminoethoxy)ethoxy)ethyl amino)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one