SUMMARY Recent studies identified cyclic GMP-AMP (cGAMP) as a metazoan second messenger triggering an interferon response. cGAMP is generated from GTP and ATP by cytoplasmic dsDNA sensor cGAMP synthase (cGAS). We combined structural, chemical, biochemical, and cellular assays to demonstrate that this second messenger contains G(2′,5′)pA and A(3′,5′)pG phosphodiester linkages, designated c[G(2′,5′) pA(3′,5′)p]. We show that, upon dsDNA binding, cGAS is activated through conformational transitions, resulting in formation of a catalytically competent and accessible nucleotide-binding pocket for generation of c[G(2′,5′)pA(3′,5′)p]. We demonstrate that cyclization occurs in a stepwise manner through initial generation of 5′-pppG(2′,5′)pA prior to cyclization to c[G(2′,5′)pA(3′,5′)p], with the latter positioned precisely in the catalytic pocket. Mutants of cGAS dsDNA-binding or catalytic pocket residues exhibit reduced or abrogated activity. Our studies have identified c[G(2′,5′)pA(3′,5′)p] as a founding member of a family of metazoan 2′,5′-containing cyclic heterodinucleotide second messengers distinct from bacterial 3′,5′ cyclic dinucleotides.

SUMMARY Binding of dsDNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) triggers formation of the metazoan second messenger c[G(2′,5′)pA(3′,5′)p], which binds the signaling protein STING with subsequent activation of the interferon (IFN) pathway. We show that human hSTINGH232 adopts a ‘‘closed’’ conformation upon binding c[G(2′,5′)pA(3′,5′)p] and its linkage isomer c[G(2′,5′)pA(2′,5′)p], as does mouse mStingR231 on binding c[G(2′,5′)pA(3′,5′)p], c[G(3′,5′)pA(3′,5′)p] and the antiviral agent DMXAA, leading to similar ‘‘closed’’ conformations. Comparing hSTING to mSting, 2′,5′-linkage-containing cGAMP isomers were more specific triggers of the IFN pathway compared to the all-3′,5′-linkage isomer. Guided by structural information, we identified a unique point mutation (S162A) placed within the cyclic-dinucleotide-binding site of hSTING that rendered it sensitive to the otherwise mouse-specific drug DMXAA, a conclusion validated by binding studies. Our structural and functional analysis highlights the unexpected versatility of STING in the recognition of natural and synthetic ligands within a small-molecule pocket created by the dimerization of STING.

Application of the concept of transient protection to the synthesis of protected deoxynucleosides is described. The deoxynucleosides are first treated with trimethylchlorosilane in pyridine for protection of the hydroxyl groups, and then immediately reacted with an acylating agent-benzoyl chloride for la and lb and isobutyric anhydride for ic-to effect N-acylation. Hydrolysis of the trimethylsilyl groups takes a few hours in aqueous pyridine or a few minutes with dilute ammonia. The ammonia also effects selective hydrolysis of the initially formed TV,TV-dibenzoyldeoxyadenosine derivative (3a) to the desired TV-benzoyldeoxyadenosine (4a). This one-flask procedure gives crystalline TV-acyl deoxynucleosides 4a and 4b in 95% yield and 4c in 75% yield, in only a few hours. The 5'-0-dimethoxytrityl deoxynucleosides 8a and 8b are also obtained in a one-flask procedure by initial reaction of the deoxynucleosides with 4,4/-dimethoxytrityl chloride, followed by treatment with trimethylchlorosilane and then benzoyl chloride. Although with deoxycytidine some of the 4-TV,5,-0-bis(dimethoxytrityl) derivative (5c) is formed, benzoyl chloride effects conversion to the 4-TV-benzoyl derivative (7b). After simple purification by flash chromatography 8a and 8b are each obtained in 80-90% overall yield from la or lb.(1) This work has been reported briefly, see "Abstracts of Papers", 181st