Determination of Protonation Sites in Thermospermine and in Some Other Polyamines by ¹⁵N and ¹³C Nuclear Magnetic Resonance Spectroscopy

Abstract

The pK values of thermospermine, a novel asymmetric tetraamine (4,8-diazadodecane-l,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 I5N N M R titration method was applied to determine its protonation site. Natural-abundance I5N 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 N M R titration curve by using a nonlinear least-squares method. The four protonation sites of thermospermine have been successfully determined to be Nt2, N', N-8, and N-4 in chat 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. I3C N M R experiments for the four polyamines were almost consistent with those of I5N NMR. Owing to the much higher concentration of the polyamines furnished for N M R titration experiments, the pK values obtained by NMR were larger than those derived by the conventional titration methods.A considerable number of observations show a correlation of enhanced synthesis of polyamines with rapid growth or cell proliferation [1,2]. From the point of view of the physiological role of polyamines in cells, polyamines were implicated as regulators of cell proliferative activity [3-S]. Thus it is tempting to speculate that association as well as dissociation of polyamines with biological macromolecules may be controlled dynamically. In fact, it has been shown that polyamines can bind with nucleic acids [S -81 and that they exert a remarkable stabilization of a teritary structure of the nucleic acids [9 -121. This led us to determine the protonation sites in polyamines and the corresponding pK values, and to examine their effect on the interaction with biological macromolecules.The pK values of several naturally occurring and synthetic polyamines have been determined by means of potentiometric titration methods [I 3 -IS]. However, the pK values of thermospermine, i.e. 4,s-diazadodecane-l , 12-diamine have remained undetermined. This polyamine is a new asymmetric tetraamine which was found in an extremely thermophilic microorganism, Thermus tl~rrmnphilus, and its structure has been determined [16]. Thus we synthesized it and measured the p K values by potentiometric titration.Determination of the protonation sites in polyamines has been attempted by calorimetric measurements [I 7,181 and the calculation of protonation enthalpy by applying quantum chemical methods [I 91. These attempts were almost successfull, but they are indirect methods. NMR titration, on the other hand, provides pK values and information about the protonation pattern, since chemical shifts of the nuclei at the basic sites are often markedly influenced by the ionization o...

The Spermine Phosphate-Bound Cyclooctaoxygen Sodium Epigenetic Shell of Euchromatin DNA is Destroyed by the Epigenetic Poison Glyphosate

“…This original phenol extraction, although variously modified for nowadays use [27], consistently precipitates the spermine as sperminediium (terminal NH3 + ) di(phenolate) at pH 7.9-8.0 [27], since the pKa value of phenol is 9.97 [potentiometric titration in H2O, 25 °C, ionic strength (NaCl) 0.1] [28], and the four pKa values of spermine are: pKa1 = 10.86 (terminal [29] NH3 + ), pKa2 = 10.05 (terminal NH3 + ), pKa3 = 8.82 (inner [29] NH2 + ), pKa4 = 7.95 (inner NH2 + ) [potentiometric titration in H2O, 25 °C, ionic strength (NaCl) 0.1] [30]. The cyclo-O8-Na + could react with alkaline (pH 7.9-8.0) buffered phenol [27] to disodium rhodizonate (C6Na2O6), a known [31] oxidation product of p-benzoquinone, which in turn is an oxidation product of phenol [32].…”

Protonation sites on unsymmetrical 1,3-diamines: a nuclear magnetic resonance study of 3-aminotropanes

“…Many nitrogen-containing natural products are aliphatic amines, which usually are protonated under physiological conditions and are, therefore, thought of as cationic species. Polyamines are usually considered to be polycationic; based on the published pK values, spermine (1) 1,2 will be 77-81% tetraprotonated and 19-23% triprotonated at physiological pH. Since it is entirely possible that some of the physiology of spermine is elicited by the triprotonated species, this raises the interesting question of its nature.…”

“…Since it is entirely possible that some of the physiology of spermine is elicited by the triprotonated species, this raises the interesting question of its nature. Using NMR methods, it has been concluded that in spermine the secondary (internal) amino groups are protonated last 1 and, therefore, the triprotonated species must have an unprotonated (neutral) secondary amino group. Superficially this seems contrary to expectation since, based on inductive contribution, secondary amines should be more basic than their primary analogs.…”

“…To resolve this issue, we followed literature reports of experimental determinations of relative basicities of amino groups based on the premise that charged groups have a significant effect on the NMR chemical shifts of neighboring nuclei. 1,3 This paper describes the use of 1 H NMR spectroscopy to determine whether, in isomeric 3-aminotropanes 2 and 3, the primary or the tertiary amino group is more basic.…”

Novel Occurrence of Uncommon Polyamines in Higher Plants

“…In bacterial systems, tetraamines protect membranes against osmotic lysis. The physiochemical characteristics of the polyamines support macromolecular interactions with various modes of complex formation and various formation constants (19). These interactions are affected kinetically by small changes in the microenvironment.…”