Determination of protonation sites in thermospermine and in some other polyamines by 15N and 13C nuclear magnetic resonance spectroscopy.

The pK values of thermospermine, a novel asymmetric tetraamine (4,8-diazadodecane-1,12-diamine), have been determined for the first time by the conventional titration method with subsequent data analyses. It was found to be the most basic polyamine among naturally occurring tetraamines. The 15N NMR titration method was applied to determine its protonation site. Natural-abundance 15N magnetic resonance spectra of the polyamine have been recorded as a function of pH from its free to the protonated form. Its apparent pK values, as well as the intrinsic chemical shifts for each stage of protonation, have been calculated from the NMR titration curve by using a nonlinear least-squares method. The four protonation sites of thermospermine have been successfully determined to be N12, N1, N-8, and N-4 in that order from the free to the completely protonated form. In a similar manner the protonation sites of spermidine and two symmetric tetraamines, thermine and spermine, have been established. 13C NMR experiments for the four polyamines were almost consistent with those of 15N NMR. Owing to the much higher concentration of the polyamines furnished for NMR titration experiments, the pK values obtained by NMR were larger than those derived by the conventional titration methods.