title: Biotinylation of a propargylated cyclic (3'-5') diguanylic acid and of its mono-6-thioated analog under "click" conditions.
abstract: 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.
authors.affiliation: Food and Drug Administration, Bethesda, Maryland.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Grajkowski
authors.affiliation: Food and Drug Administration, Bethesda, Maryland.
authors.forename: Jacek
authors.initials: J
authors.lastname: Cieślak
authors.affiliation: Department of Microbiology & Immunology and Department of Medicine, Columbia University, New York, New York.
authors.forename: Christian
authors.initials: C
authors.lastname: Schindler
authors.affiliation: Food and Drug Administration, Bethesda, Maryland.
authors.forename: Serge L
authors.initials: SL
authors.lastname: Beaucage
mesh_terms: D001386:Azides / Q000737:chemistry; D019921:Biotinylation / Q000379:methods*; D057930:Click Chemistry / Q000379:methods*; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000737:chemistry; D015226:Dinucleoside Phosphates / Q000737:chemistry; D006151:Guanosine / Q000737:chemistry; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D013777:Tetrazoles / Q000737:chemistry
chemical_list: D001386:Azides; D015226:Dinucleoside Phosphates; D013777:Tetrazoles; D006151:Guanosine; C024662:GpGp; C045574:1H-tetrazole; C062025:bis(3',5')-cyclic diguanylic acid; D006157:Guanosine Monophosphate; D006152:Cyclic GMP