gpgp and bis(3--5-)-cyclic-diguanylic-acid

Commercial N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(propargyl)guanosine is converted to its 3'-O-levulinyl ester in a yield of 91%. The reaction of commercial N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-3'-O-[(2-cyanoethyl)-N,N-diisopropylaminophosphinyl]guanosine with N(2)-isobutyryl-2'-O-propargyl-3'-O-(levulinyl)guanosine provides, after P(III) oxidation, 3'-/5'-deprotection, and purification, the 2'-O-propargylated guanylyl(3'-5')guanosine 2-cyanoethyl phosphate triester in a yield of 88%. Phosphitylation of this dinucleoside phosphate triester with 2-cyanoethyl tetraisopropylphosphordiamidite and 1H-tetrazole, followed by an in situ intramolecular cyclization, gives the propargylated cyclic dinucleoside phosphate triester, which is isolated in a yield of 40% after P(III) oxidation and purification. Complete removal of the nucleobases, phosphates, and 2'-O-tert-butyldimethylsilyl protecting groups leads to the desired propargylated c-di-GMP diester. Cycloaddition of a biotinylated azide with the propargylated c-di-GMP diester under click conditions provides the biotinylated c-di-GMP conjugate in an isolated yield of 62%. Replacement of the 6-oxo function of N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-3'-O-levulinyl-2'-O-(propargyl)guanosine with a 2-cyanoethylthio group is effected by treatment with 2,4,6-triisopropybenzenesulfonyl chloride and triethylamine to give a 6-(2,4,6-triisopropylbenzenesulfonic acid) ester intermediate. Reaction of this key intermediate with 3-mercaptoproprionitrile and triethylamine, followed by 5'-dedimethoxytritylation, affords the 6-(2-cyanoethylthio)guanosine derivative in a yield of 70%. The 5'-hydroxy function of this derivative is reacted with commercial N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-3'-O-[(2-cyanoethyl)-N,N-diisopropylaminophosphinyl]guanosine. The reaction product is then converted to the mono-6-thioated c-di- GMP biotinylated conjugate under conditions highly similar to those described above for the preparation of the biotinylated c-di-GMP conjugate, and isolated in similar yields.

Topics: Azides; Biotinylation; Click Chemistry; Cyclic GMP; Dinucleoside Phosphates; Guanosine; Guanosine Monophosphate; Tetrazoles

Cyclic ribodiguanylic acid, c-(GpGp), is the endogenous effector regulator of cellulose synthase. Its three-dimensional structure from two different crystal forms (tetragonal and trigonal) has been determined by X-ray diffraction analysis at 1 A resolution. In both crystal forms, two independent c-(GpGp) molecules associate with each other to form a self-intercalated dimer. A hydrated cobalt ion is found to coordinate to two N7 atoms of adjacent guanines, forcing these two guanines to destack with a large dihedral angle (32 degrees), in the dimer of the tetragonal form. This metal coordination mechanism may be relevant to that of the anticancer drug cisplatin. Moreover, c-(GpGp) exhibits unusual spectral properties not seen in any other cyclic dinucleotide. It interacts with planar organic intercalator molecules in ways similar to double helical DNA. We propose a cage-like model consisting of a tetrameric c-(GpGp) aggregate in which a large cavity ('host') is generated to afford a binding site for certain planar intercalators ('guests').

Topics: Cyclic GMP; Guanine Nucleotides; Guanosine Monophosphate; Intercalating Agents; Molecular Structure; Spectrophotometry, Ultraviolet; X-Ray Diffraction