cGAMP (Cyclic GMP-AMPP) diammonium functions as an endogenous second messenger and triggers the production of interferons in response to cytosolic DNA. cGAMP diammonium activates stimulator of interferon genes (STING), activating signaling cascades leading to the production of type I interferons and other immune mediators.

Physicochemical Properties

Molecular Formula	C20H30N12O13P2
Molecular Weight	708.47
Related CAS #	cGAMP disodium;2407516-83-8;cGAMP;849214-04-6
Appearance	Typically exists as solid at room temperature
HS Tariff Code	2934.99.9001
Storage	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.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Human Endogenous Metabolite; second messenger; STING/stimulator of interferon genes
ln Vitro	The ability of mouse splenocytes to proliferate in response to specific antigens is enhanced by cGAMP diammonium [2]. In vitro, human and mouse dendritic cells are immediately activated by cGAMP diammonium [2]. Patient fibroblasts exhibited enhanced transcription of IFNB1 upon stimulation with cGAMP diammonium, but not of genes encoding TNF, IL6, or interleukin 1 (IL1) [3]. The endoplasmic reticulum (ER)-resident receptor STING is activated by cGAMP diammonium, which results in the induction of an antiviral state and type I interferon production [4].
ln Vivo	In mice that have received vaccinations, the spleen cells produce more antigen-specific cytokines when exposed to 5 μg of nasal mucosal adjuvant diammonium (cGAMP) [2]. The recently discovered mammalian enzyme cyclic GMP-AMP synthase produces cyclic GMP-AMP (cGAMP) after being activated by pathogen-derived cytosolic double stranded DNA. The product can stimulate STING-dependent interferon type I signaling. Here, we explore the efficacy of cGAMP as a mucosal adjuvant in mice. In this study, researchers show that cGAMP can enhance the adaptive immune response to the model antigen ovalbumin. It promotes antigen specific IgG and a balanced Th1/Th2 lymphocyte response in immunized mice. A characteristic of the cGAMP-induced immune response is the slightly reduced induction of interleukin-17 as a hallmark of Th17 activity – a distinct feature that is not observed with other cyclic di-nucleotide adjuvants. We further characterize the innate immune stimulation activity in vitro on murine bone marrow-derived dendritic cells and human dendritic cells. The observed results suggest the consideration of cGAMP as a candidate mucosal adjuvant for human vaccines[2].
Enzyme Assay	Cytosolic DNA induces type I interferons and other cytokines that are important for antimicrobial defense but can also result in autoimmunity. This DNA signaling pathway requires the adaptor protein STING and the transcription factor IRF3, but the mechanism of DNA sensing is unclear. We found that mammalian cytosolic extracts synthesized cyclic guanosine monophosphate-adenosine monophosphate (cyclic GMP-AMP, or cGAMP) in vitro from adenosine triphosphate and guanosine triphosphate in the presence of DNA but not RNA. DNA transfection or DNA virus infection of mammalian cells also triggered cGAMP production. cGAMP bound to STING, leading to the activation of IRF3 and induction of interferon-β. Thus, cGAMP functions as an endogenous second messenger in metazoans and triggers interferon production in response to cytosolic DNA[1].
Cell Assay	In vitro stimulation of primary cells[2] The culture medium of primary cells was supplemented with 5 µg/ml (murine cells) or 60 µg/ml (human cells) of c-di-AMP or cGAMP or left without additive. Cells were incubated for 24 h at 37°C. Scrape loading[3] HEK STING cells were seeded at a density of 2.5 × 105 cells ml−1 in 96-well plates. After 16 h cGAMP(2′-5′) was added to the medium to a final concentration of 50 μg ml−1. Monolayers of cells were manually wounded by six scratches per well using an 18G needle. Images were acquired after 4–8 h.
Animal Protocol	Animal/Disease Models: Female C57BL/6 (H-2b) mice 6-8 weeks old[2] Doses: 5 µg Route of Administration: Nostril mucosal adjuvant Experimental Results: Higher titers of ovalbumin (OVA)-specific IgA and total IgG as well as IgG1 and IgG2c in the sera of mice immunized with cGAMP-adjuvanted OVA as compared to sera from OVA-immunized mice. Mouse immunization experiments[2] Five animals per group were immunized i. n. on days 0, 14 and 28. Animals were anesthetized with Isoflurane and treated 10 µl per nostril with 15 µg OVA alone or co-administered with 5 µg per dose of c-di-AMP, cGAMP or cholera toxin B subunit in Ampuwa or with Ampuwa alone in the control group (mock immunization). On day 42 after immunization animals were sacrificed and samples were collected.
References	[1]. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science. 2013 Feb 15;339(6121):826-30. [2]. Cyclic GMP-AMP displays mucosal adjuvant activity in mice. PLoS One. 2014 Oct 8;9(10):e110150. [3]. Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP. Nature. 2013 Nov 28;503(7477):530-4.

Solubility Data

Solubility (In Vitro)	H2O :~100 mg/mL (~141.15 mM)
Solubility (In Vivo)	Solubility in Formulation 1: 33.33 mg/mL (47.05 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.4115 mL	7.0575 mL	14.1149 mL
	5 mM	0.2823 mL	1.4115 mL	2.8230 mL
	10 mM	0.1411 mL	0.7057 mL	1.4115 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.