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

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.

Protolytic properties of polyamine wasp toxin analogues studied by 13C NMR spectroscopy

“…Very similar pK a values of 2 were previously obtained in D 2 O. 23 The amide carbonyl groups, the aliphatic carbon atoms of the tyrosine residue (C-A and C-B, Fig. 1 However, the shapes of the 13 C NMR titration curves were different; in the case of PhTX-3nm they were roughly sigmoid, as expected, whereas in the case of PhTX-4nm a maximum was observed, as shown in Fig.…”

“…), respectively, and pK a1 , pK a2 , pK a3 , and pK a4 are the four equilibrium constants (acidity constants) governing the acid-base equilibria for each deprotonation step. 23 Thus, each carbon atom in a philanthotoxin molecule gives a 13 C NMR titration curve described by the above equation, and simultaneous nonlinear fitting of the entire set of curves to the experimental data gives pK a values as well as intrinsic chemical shifts (υ Table 1, together with pK a values determined previously for related compounds. Although the acidity constants of phenols and protonated amines are similar, the high information content of the 13 C NMR titration curves and the fact that all carbon atoms are simultaneously taken into account result in highly reliable macroscopic pK a values for each compound.…”

“…On the other hand, C-1 is appreciably affected only by the deprotonation of N-a ( -or υ-deprotonation shift). Since˛-deprotonation shifts in amines are considerably larger than the -or υ-shifts, 23,24 the carbon experiencing the smallest deprotonation shift is assigned to C-1 (Fig. 5).…”

“…These signals belong to three categories: carbon atoms in thě -position to an amino group with υ values around 26-28, carbon atoms in the˛-position to a primary amino group or the amide nitrogen with υ values around 40, and carbon atoms in the˛-position to a secondary amino group with υ values about 47-50 (values for fully protonated polyamines at pH about 5). 23,24 The resonances of the polyamine moiety show a complex titration behavior owing to the close proximity of three acid/base sites, as exemplified in Fig. 5.…”

Contrasting Actions of Philanthotoxin-343 and Philanthotoxin-(12) on Human Muscle Nicotinic Acetylcholine Receptors

“…Voltage-dependent antagonism of neuronal-type nAChR by PhTX-343 has also been demonstrated . PhTX-343 is trivalent at physiological pH with all three of its amino groups positively charged (Jaroszewski et al, 1996). It follows that if PhTX-343 blocks the ion channel pore of nAChR, then its activity should be influenced by the transmembrane electric field.…”