Thiophosphate analogs of c-di-GMP: impact on polymorphism

Abstract

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.

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Fig 3

¹H NMR spectra (H8 region) for 5, 6, and 7 at 30 °C (a = anti, s = syn, B = bimolecular, T = tetramolecular, O = octamolecular, * = residual resonances associated with extreme aggregation)

Fig 4

³¹P NMR spectra for 5, 6, and 7 at 30 °C (a = anti, s = syn, B = bimolecular, T = tetramolecular, O = octamolecular, *= residual resonances associated with extreme aggregation)

Fig 5

2D NOESY plots for the H8/H1′ region for 5a K⁺, 5b K⁺, 5a Na⁺, 6a K⁺, 6b K⁺, 6a Na⁺ and 7a K⁺, all at 30 °C, and 7b K⁺ at 45 °C, all showing the H8/H1′ crosspeaks of syn conformations. Not shown: plots for 5b Na⁺, 6b Na⁺, and 7a Na⁺ which displayed good signal/noise ratio at 30 °C, but no H8/H1′ crosspeaks, or plots of 6c Na⁺ and 7b Na⁺ which only displayed good signal/noise ratio at 45 °C because of extensive aggregation, but no H8/H1′ crosspeaks. A plot for 6c K⁺ did not show good signal/noise ratio even at 45 °C.

Fig 6

2D DOSY spectra at 30 °C for all samples, with diffusion coefficients (D) displayed on the vertical axes in m²/sec × 10⁻

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