Zhao, Jianwei. Synthesis and characterization of seven thiophosphate analogs of cyclic diguanosine monophosphate. Retrieved from https://doi.org/doi:10.7282/T3J38SSZ
DescriptionCyclic diguanosine monophosphate (c-di-GMP) was first identified as an activator of bacterial cellulose synthase in 1987. Since then, it has been recognized as an important second messenger molecule in many bacterial processes, including cellulose synthesis, biofilm formation, and host-pathogen interactions. More recently, it found to stimulate innate immunity in mammals. The monothiophosphate analog was synthesized some years ago and was shown to be a highly potent activator/second messenger with a high stability to enzymatic degradation.
In the work presented here, synthetic methods have been developed to synthesize mono-, di- and trithiophosphate c-di-GMP analogs. All seven diastereomers of these analogs have been separated and purified. Stereochemistry of the two diastereomers of the monothioate analog was assigned by enzymatic digestion of the linear dimer, as well as direct enzymatic degradation of the cyclic dimers. In addition, the same linear dimer was cyclized to produce a cyclic dimer of known configuration. The correlation between stereochemistry and 31P NMR chemical shifts of the two diastereomers of the monothioate was then used to assign configurations of the di- and trithiophosphate analogs.
1D 1H and 31P NMR, as well as 2D NOSEY and DOSY were used to characterize all seven thiophosphate analogs in both Na+ and K+ forms. It was found that the [S] sulfurs, as well as the counterions, display dramatic effects on the equilibrium among five different complexes that can form. It is concluded that: 1) in all cases, the presence of an [S] sulfur promotes more extensive complex formation than [R]; 2) in both Na+ and K+ forms, two [S] sulfurs promote extensive aggregation, forming large aggregates that cannot be observed in the NMR spectra; 3) K+ promotes more extensive complex formation than Na+, with primarily octamolecular complexes in the K+ forms, but tetramolecular complexes in the Na+ forms; and 4) in all the Na+ forms, but not the K+ forms, the presence of one [S] sulfur stabilizes anti complexes and/or destabilizes syn complexes.