Convenient Synthesis of a Propargylated Cyclic (3'-5') Diguanylic Acid and Its Click Conjugation to a Biotinylated Azide

The ribonucleoside building block, N2-isobutyryl-2′-O-propargyl-3′-O-levulinyl guanosine, was prepared from commercial N2-isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-O-propargyl guanosine in a yield of 91%. The propargylated guanylyl(3′-5′)guanosine phosphotriester was synthesized from the reaction of N2-isobutyryl-2′-O-propargyl-3′-O-levulinyl guanosine with N2-isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-O-tert-butyldimethylsilyl-3′-O-[(2-cyanoethyl)-N,N-diisopropylaminophosphinyl] guanosine and isolated in a yield of 88% after P(III) oxidation, 3′-/5′-deprotection, and purification. The propargylated guanylyl(3′-5′)guanosine phosphotriester was phosphitylated using 2-cyanoethyl tetraisopropylphosphordiamidite and 1H-tetrazole and was followed by an in situ intramolecular cyclization to give a propargylated c-di-GMP triester, which was isolated in a yield of 40% after P(III) oxidation and purification. Complete N-deacylation of the guanine bases and removal of the 2-cyanoethyl phosphate protecting groups fro...