bis(3--5-)-cyclic-diguanylic-acid and thiophosphoric-acid

Cyclic dinucleotides (CDNs) are second messengers that bind to the stimulator of interferon genes (STING) and trigger the expression of type I interferons and proinflammatory cytokines. Here we evaluate the activity of 3',3'-c-di(2'F,2'dAMP) and its phosphorothioate analogues against five STING allelic forms in reporter-cell-based assays and rationalize our findings with X-ray crystallography and quantum mechanics/molecular mechanics calculations. We show that the presence of fluorine in the 2' position of 3',3'-c-di(2'F,2'dAMP) improves its activity not only against the wild type (WT) but also against REF and Q STING. Additionally, we describe the synthesis of the acyloxymethyl and isopropyloxycarbonyl phosphoester prodrugs of CDNs. Masking the negative charges of the CDNs results in an up to a 1000-fold improvement of the activities of the prodrugs relative to those of their parent CDNs. Finally, the uptake and intracellular cleavage of pivaloyloxymethyl prodrugs to the parent CDN is rapid, reaching a peak intracellular concentration within 2 h.

Topics: Crystallography, X-Ray; Density Functional Theory; Esters; HEK293 Cells; Humans; Interferon-gamma; Leukocytes, Mononuclear; Magnetic Resonance Spectroscopy; Membrane Proteins; Phosphates; Prodrugs; Tumor Necrosis Factor-alpha

An integrated set of reactions and conditions that allow an eight-step one-flask synthesis of the protected derivatives of c-di-GMP and the [R(p),R(p)] and [R(p),S(p)] thiophosphate analogues is reported. Deprotection is also carried out as a one-flask procedure, with the final products isolated by crystallization from the reaction mixture. Chromatography is only used for separation of the thiophosphate diastereomers.

Topics: Cyclic GMP; Kinetics; Phosphates; Stereoisomerism

Seven phosphorothioate analogs of c-di-GMP (all diastereomers of mono-, di-, and trithiophosphates) were prepared to assess the impact of the thioate substitutions on c-di-GMP polymorphism using 1D (1)H and (31)P NMR, along with 2D NOESY and DOSY, for both the Na(+) and K(+) salts. The K(+) salts display more extensive higher order complex formation than the Na(+) salts, resulting primarily in octamolecular complexes with K(+), but tetramolecular complexes with Na(+). Further, the presence of one or two [S(P)] sulfurs specifically stabilizes anti complexes and/or destabilizes syn complexes, while the presence of two [S(P)] sulfurs promotes extensive aggregation.

Topics: Cyclic GMP; Isomerism; Magnetic Resonance Spectroscopy; Molecular Conformation; Phosphates; Potassium; Salts; Sodium

The synthesis of mono- and bisphosphorothioate analogues of 3',5'-cyclic diguanylic acid (cdiGMP) via the modified H-phosphonate chemistry is reported. The immunostimulatory properties of these analogues were compared with those of cdiGMP.

Topics: Bronchi; Chemistry, Pharmaceutical; Chromatography, High Pressure Liquid; Cyclic GMP; Dose-Response Relationship, Drug; Drug Design; Humans; Immune System; Inflammation; Models, Chemical; Neutrophils; Organophosphonates; Phosphates; Vaccines