STING; interferon-β (IFNβ)[1]
2',3'-cGAMP sodium targets STING (stimulator of interferon genes) with high affinity, with Kd values of 3.79 nM for human STING and 120 nM for rat STING. By binding to STING, it activates the innate immune response and induces the production of type I interferons and other cytokines.

There are two different phosphodiester connections in 2',3'-cGAMP sodium (2'-3'-cyclic GMP-AMP sodium): one connects 2'-OH of GMP and 5'-phosphate of AMP, while the other connects 3'-OH of AMP and 5′-phosphate of GMP(1–2).[1]

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2′3′-cGAMP is an endogenous second messenger produced by mammalian cells.
2′3′-cGAMP is a high affinity ligand for STING.
2′3′-cGAMP is a potent inducer of type-I interferons.
2′3′-cGAMP binding induces conformational changes of STING.[1]

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In vitro, 2',3'-cGAMP sodium enhances the proinflammatory activation of cultured wild-type macrophages. It induces repolarization of M2 macrophages in vitro. Unlike in macrophages, it displays anti-inflammatory effects in both WT primary mouse hepatocytes and differentiated 3T3-L1 adipocytes. It is a potent inducer of interferon-β.

In vivo, 2',3'-cGAMP sodium binds to STING with high affinity and is a potent inducer of interferon-β. It induces antigen-specific antibodies and T cell responses in mice. As a second messenger in the cGAS-STING pathway, it plays a critical role in the innate immune response to DNA pathogens and cellular stress.

Isothermal titration calorimetry (ITC)[1]
Isothermal titration calorimetry (ITC) was employed to measure the binding affinities between STING and cGAMP isomers or c-di-GAMP using a VP-ITC microcalorimeter (GE Healthcare). The protein and the ligand concentrations are shown in Figure 2D. The titrations were performed at 20°C in the buffer containing 25 mM Hepes, pH 7.8, 150 mM NaCl. 32 injections were performed with 4 minutes spacing time. The titration traces were integrated by NITPIC(Keller et al., 2012) and then the curves were fitted by SEDFIT(Houtman et al., 2007). The figures were prepared using GUSSI (http://biophysics.swmed.edu/MBR/software.html).

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Enzymatic Synthesis and Purification of cGAMP[1]
To generate natural cGAMP using the enzyme cGAS, a reaction containing 20mM Tris-Cl, pH7.5, 5mM MgCl2, 10mM CoCl2, 0.01mg/ml herring testis DNA, 1mM ATP, 1mM GTP, and 0.1μM recombinant SUMO-tagged human cGAS (aa147–522) was incubated at 37°C for 1hr. The mixture was fractionated on a Hitrap Q column using a linear 0–0.5M NaCl gradient; a UV peak corresponding to cGAMP was collected and loaded onto a C18 column (201TP510, 1cmX25cm), and eluted with a linear 0–100% methanol gradient.

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In vitro binding assays for 2',3'-cGAMP sodium involve incubating the compound with STING protein in buffer at physiological pH. Binding affinity (Kd) is measured using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC). For functional assays, STING-mediated signaling is assessed by measuring IFN-β production in STING-expressing cells.

Preparation of Endogenous cGAMP[1]
Endogenous cGAMP was prepared from DNA transfected L929 and THP-1 cells, respectively. After HT-DNA transfection for 4 hours, about 3× 10~7 cells were lysed in hypotonic buffer [10mM Tris-HCl, pH7.4, 10mM KCl, 1.5mM MgCl2]. The lysates were heated at 95°C for 5 min and centrifuged again at 17,000g for 10 min to remove denatured proteins. The heat-resistant supernatant was fractionated on a C-18 column (Eclipse Plus 4.6×30 mm, 3.5μm, Agilent Technologies) equilibrated with 0.1% formic acid and eluted with a linear gradient of 0–100% methanol. The presence of cGAMP in each fraction was monitored by activity assay (Wu et al., 2013), and the fraction with peak activity was used for further MS and MS/MS analysis.

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For in vitro cell assays, macrophages, hepatocytes, or adipocytes are cultured and treated with 2',3'-cGAMP sodium at concentrations ranging from 0.1-100 µM. Cytokine production (IFN-β, IL-6, TNF-α) is measured by ELISA or qPCR. Macrophage polarization is assessed by measuring M1/M2 marker expression. Cell viability is assessed by MTT assay.

For in vivo animal studies, 2',3'-cGAMP sodium is typically administered to mice via intravenous injection or intraperitoneal injection at doses of 0.1-10 mg/kg. Antigen-specific antibody and T cell responses are measured. Cytokine levels in serum are quantified by ELISA. The compound's ability to activate the STING pathway and induce innate immune responses is assessed.

2',3'-cGAMP sodium (MW 718.37) has the molecular formula C20H22N10Na2O13P2. The compound is stable as a powder at -20°C for up to 3 years. It is a sodium salt form of the endogenous second messenger 2',3'-cGAMP. It is soluble in water and physiological buffers.

The compound is for research use only. No specific toxicity data are available. As an endogenous second messenger, it is expected to have low toxicity at physiological concentrations. However, activation of the STING pathway may induce inflammatory responses at higher doses. Standard laboratory safety precautions should be followed.

[1]. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Mol Cell. 2013 Jul 25;51(2):226-35.

The presence of microbial or autologous DNA in the cytoplasm of mammalian cells is a danger signal that can be detected by the DNA sensor cyclic GMP-AMP (cGAMP) synthase (cGAS). cGAS catalyzes the production of cGAMP, which then acts as a second messenger to activate the innate immune response. This study shows that endogenous cGAMP in mammalian cells contains two different phosphodiester bonds: one linking the 2'-OH of GMP to the 5'-phosphate of AMP, and the other linking the 3'-OH of AMP to the 5'-phosphate of GMP. This molecule, called 2'3'-cGAMP, possesses unique properties, exhibiting a significantly higher affinity for the adaptor protein STING than cGAMP molecules containing other phosphodiester bond combinations. The crystal structure of STING bound to 2'3'-cGAMP reveals the structural basis of this high-affinity binding and the ligand-induced conformational change in STING, which may be the mechanism of its activation. [1]

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Although 2'3'-cGAMP has a much higher affinity for STING than cGAMP isomers containing other phosphodiester bonds, all four cGAMP isomers showed similar EC50 values for inducing IFNβ, and were much lower than c-di-GMP. Therefore, all cGAMP isoforms are effective IFNβ inducers, suggesting that cGAMPs with different phosphodiester bonds may exist in nature, perhaps in some lower organisms. In fact, Vibrio cholerae contains a cyclase that synthesizes 3'3'-cGAMP, which is involved in bacterial chemotaxis and colonization (Davies et al., 2012). It is currently unclear why mammals evolved to produce 2'3'-cGAMP as an endogenous second messenger to trigger innate immune responses. [1]

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In summary, our findings indicate that: 1) 2′3′-cGAMP is an endogenous second messenger produced by mammalian cells in response to cytoplasmic DNA stimulation; 2) 2′3′-cGAMP is a high-affinity ligand for STING; 3) 2′3′-cGAMP is a potent inducer of IFNβ in mammalian cells; 4) 2′3′-cGAMP induces conformational rearrangement of STING, which may be the basis for its activation; 5) The extensive interactions between 2′3′-cGAMP and STING observed in the crystal structure of the complex explain their specific and high-affinity binding. [1]

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2',3'-cGAMP sodium (CAS# 2734858-36-5) is an endogenous high-affinity STING agonist and second messenger in the cGAS-STING innate immune pathway. It has not been approved as a therapeutic drug but is widely used in immunology research to study innate immunity, STING activation, and interferon responses. It is a valuable tool for studying antiviral and antitumor immune responses.

C20H25N10NAO13P2

698.408995389938

718.063

2734858-36-5

2',3'-cGAMP;1441190-66-4

137120248

White to off-white solid powder

5

19

2

47

1290

8

OC1([H])[C@@]2([H])COP(O[C@@]3([H])[C@@H](O)[C@H](N4C=NC5=C(N=CN=C45)N)O[C@]3([H])COP(O)(=O)O[C@@]1([H])[C@H](N1C=NC3C(N=C(N)NC1=3)=O)O2)(O)=O.[NaH]

CNVCOPPPOWRJAV-DQNSRKNCSA-L

InChI=1S/C20H24N10O13P2.2Na/c21-14-8-15(24-3-23-14)29(4-25-8)18-11(32)12-7(41-18)2-39-45(36,37)43-13-10(31)6(1-38-44(34,35)42-12)40-19(13)30-5-26-9-16(30)27-20(22)28-17(9)33;;/h3-7,10-13,18-19,31-32H,1-2H2,(H,34,35)(H,36,37)(H2,21,23,24)(H3,22,27,28,33);;/q;2*+1/p-2/t6-,7-,10-,11-,12-,13-,18-,19-;;/m1../s1

disodium;2-amino-9-[(1R,6R,8R,9R,10S,15R,17R,18R)-8-(6-aminopurin-9-yl)-9,18-dihydroxy-3,12-dioxido-3,12-dioxo-2,4,7,11,13,16-hexaoxa-3λ5,12λ5-diphosphatricyclo[13.2.1.06,10]octadecan-17-yl]-1H-purin-6-one

2',3'-cGAMP sodium salt; 2734858-36-5; JX6B238JSL; 2'3'-cGAMP (sodium salt); 2'-3'-cyclic GMP-AMP sodium; PD077435; adenylyl-(3'-->5')-2'-guanylic acid, cyclic nucleotide, disodium salt; 2'3'-CYCLIC GUANOSINE MONOPHOSPHATE-ADENOSINE MONOPHOSPHATE DISODIUM SALT

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: 50 mg/mL (69.60 mM)
DMSO: < 1 mg/mL

Solubility in Formulation 1: 18.33 mg/mL (25.52 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)