Cyclic-di-GMP diammonium is a STING agonist and bacterial second messenger that coordinates different aspects of bacterial growth and behavior such as motility, virulence, biofilm formation, and cell cycle progression. Cyclic-di-GMP diammonium has anti-cancer cell proliferation/growth activity and can also induce increased CD4 receptor expression and cell cycle arrest. Cyclic-di-GMP diammonium may be utilized in cancer research.

Physicochemical Properties

Molecular Formula	C20H27N11O14P2
Molecular Weight	707.44
Exact Mass	724.147
CAS #	609343-82-0
Related CAS #	Cyclic-di-GMP disodium;2222132-40-1;Cyclic-di-GMP;61093-23-0;Cyclic-di-GMP sodium
PubChem CID	162640464
Appearance	White to off-white solid powder
Hydrogen Bond Donor Count	8
Hydrogen Bond Acceptor Count	18
Rotatable Bond Count	2
Heavy Atom Count	48
Complexity	1320
Defined Atom Stereocenter Count	8
SMILES	O=C1N=C(N)NC2N([C@@H]3O[C@]4([H])COP(O[C@@]5([H])[C@@H](O)[C@H](N6C=NC7C(N=C(N)NC6=7)=O)O[C@]5([H])COP(O)(=O)O[C@@]4([H])[C@H]3O)(O)=O)C=NC1=2.N
InChi Key	ORSUSHQNAQSNEL-XDODWTFASA-N
InChi Code	InChI=1S/C20H24N10O14P2.2H3N/c21-19-25-13-7(15(33)27-19)23-3-29(13)17-9(31)11-5(41-17)1-39-45(35,36)44-12-6(2-40-46(37,38)43-11)42-18(10(12)32)30-4-24-8-14(30)26-20(22)28-16(8)34;;/h3-6,9-12,17-18,31-32H,1-2H2,(H,35,36)(H,37,38)(H3,21,25,27,33)(H3,22,26,28,34);2*1H3/t5-,6-,9-,10-,11-,12-,17-,18-;;/m1../s1
Chemical Name	diazanium;2-amino-9-[(1S,6R,8R,9R,10S,15R,17R,18R)-17-(2-amino-6-oxo-1H-purin-9-yl)-9,18-dihydroxy-3,12-dioxido-3,12-dioxo-2,4,7,11,13,16-hexaoxa-3λ5,12λ5-diphosphatricyclo[13.3.0.06,10]octadecan-8-yl]-1H-purin-6-one
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	STING[1][2][3][4].
ln Vitro	Human colon cancer cells cannot proliferate when exposed to cyclic-di-GMP diammonium (0.5-50 μM; 5 days) [1]. In Jurkat cells, cyclic-di-GMP diammonium (0.5-50 μM; 5 days) selectively upregulates CD4 expression [2]. In S phase cells, cyclic-di-GMP diammonium (0.5-50 μM; 5 days) causes Jurkat cell cycle arrest [2].
ln Vivo	TriVax stimulates an immunological response against melanoma in mice when administered intravenously with cyclic-di-GMP diammonium (100 μg/time); this further amplifies TriVax's anti-tumor impact[3].
Cell Assay	Cell Proliferation Assay[1] Cell Types: H508 cells Tested Concentrations: 0.5-50 µM Incubation Duration: 5 days Experimental Results: diminished basal H508 cell proliferation by approx 15%, even inhibited acetylcholine- and EGF-induced cell proliferation. Cell Viability Assay[2] Cell Types: Jurkat cells Tested Concentrations: 50 µM Incubation Duration: 24 h Experimental Results: Specifically induced of CD4 (no effect on the expression of CD8), with a 6.3-fold upregulation over control and in a dose-dependent manner. Cell Cycle Analysis [2] Cell Types: Jurkat cells Tested Concentrations: 50 µM Incubation Duration: 24 h Experimental Results: Increased the percentage of cells in S-phase by 79%, with almost complete disappearance of G2/M-phase cells which diminished by 93%.
Animal Protocol	Animal/Disease Models: C57BL/6 (B6) mice (8- to 10weeks old)[3]. Doses: 100 µg/per Route of Administration: intravenous (iv) injection; two sequential vaccinations 9 days apart; combine with TriVax. Experimental Results: Dramatically higher numbers of antigen-specific CD8 T cells when combined with TriVax. (TriVax consisted of a mixture of 120 μg Pam-hgp100, 100 μg hgp100 or 100 μg Ova, 50 or 25 μg anti-CD40 antibody, and 25 μg Poly-IC). Enhanced the anti-tumor activity of TriVax.
References	[1]. 3',5'-Cyclic diguanylic acid (c-di-GMP) inhibits basal and growth factor-stimulated human colon cancer cell proliferation. Biochem Biophys Res Commun. 2005 Apr 1;329(1):40-5. [2]. Elevated expression of the CD4 receptor and cell cycle arrest are induced in Jurkat cells by treatment with the novel cyclic dinucleotide 3',5'-cyclic diguanylic acid. FEBS Lett. 1999 Feb 5;444(1):125-9. [3]. STING activator c-di-GMP enhances the anti-tumor effects of peptide vaccines in melanoma-bearing mice. Cancer Immunol Immunother. 2015 Aug;64(8):1057-66. [4]. Cyclic di-GMP: second messenger extraordinaire. Nat Rev Microbiol. 2017 May;15(5):271-284.

Solubility Data

Solubility (In Vitro)	H2O: 50 mg/mL (69.02 mM)
Solubility (In Vivo)	Solubility in Formulation 1: 100 mg/mL (138.03 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	1.4135 mL	7.0677 mL	14.1355 mL
	5 mM	0.2827 mL	1.4135 mL	2.8271 mL
	10 mM	0.1414 mL	0.7068 mL	1.4135 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.