Neuroactive Polyamine Wasp Toxins: Nuclear Magnetic Resonance Spectroscopic Analysis of the Protolytic Properties of Philanthotoxin-343

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Abstract
Acid-base properties (pKa values and proton distribution patterns) of philanthotoxin-343(PhTX-343) were investigated by 1H and 13C NMR titration. Chemical shift data and the total ionization shifts were used to assign carbon atoms of the polyamine chain. Nonlinear analysis of the 13C NMR titration curves gave four pKa values (pK1 8.5, pK2 9.5, pK3 10.4, pK4 11.4) and the intrinsic chemical shifts of the non-, mono-, di-, tri-, and tetraprotonated forms. The changes of intrinsic chemical shifts enabled analysis of the deprotonation sequence of fully protonated PhTX-343. The results of analysis of the 13C NMR titration curves were supported by 1H NMR data obtained from two-dimensional 1H, 13C chemical shift correlation experiments. Thus, the first deprotonation mainly takes place at the inner amino group. The phenol group is deprotonated in the second and third deprotonation steps. The preferential deprotonation of the inner amino group is also apparent in the deprotonated form. The monoprotonated form carries a practically fully ionized phenol group and the proton shared between the three amino groups. This characteristic is in agreement with existing data on polyamines. At physiological pH, the tetraprotonated form of PhTX-343 predominates, but the proportion of the triprotonated form becomes significant at low ionic strength. The terminal, primary amino group, which has been shown to be essential for biological activity, remains practically fully protonated at biologically relevant pH values, and this charge is likely to participate in the receptor-binding event. Protonation of the central amino group does not appear to be necessary for biological activity.