In this work, several computational techniques and protocols have been developed and applied in an attempt to resolve the longstanding controversy in literature as to the preferred protonation site i.e. primary (HLp) or secondary (HLs) nitrogen atom in singly protonated aliphatic linear polyamines. To achieve this aim, a dedicated conformational search protocol (CSP) for identifying relevant low energy conformers (LECs) of any given aliphatic linear polyamine from a large set of conformers initially generated using MMFF(aq) force field and Monte Carlo algorithm, was developed. Using representative LECs identified and a hybrid solvation model, a mixture of HLp (84%) and HLs (16%) was predicted for the first protonation of triethylenetetramine in excellent agreement with results obtained from the most consistent cluster analysis method. In addition, preliminary theoretical 13C NMR–pH titration study also suggests that a mixture of both monoprotonated forms would exist in solution at thermodynamic equilibrium. Hence contrary to various opposing arguments in literature in favour of either the primary or secondary nitrogen atom, these results suggests strongly that both monoprotonated forms might be present in solution even though the species in which the primary nitrogen atom is protonated will be predominant due to its better solvation. Also, quantum topological methods have been utilized to investigate and understand factors responsible for conformational preference in aliphatic linear polyamines. In addition to NH•••N interactions which were mainly responsible for the conformational preference of polyamines, CH•••HC interactions were uncovered for the first time in low energy conformers of protonated triethylenetetramine (2,2,2-tet).
Furthermore, the CSP developed initially was refined to a 5-step EEBGB- conformer selection protocol which in principle could effectively and in shortest time possible identify low energy conformers for any given aliphatic linear polyamine (E, B and G stands for electronic-energy-, Boltzmann-distribution- and Gibbs-free-energy-based stepwise selection of conformers). This EEBGB-protocol (i) reduced (by 94%) the number of conformers subjected to the frequency calculations (to obtain G-values) from 420 MM-selected to 25 used to compute four stepwise protonation constants of triethylenetetramine and (ii) is of general-purpose as it is applicable to any flexible and poly-charged molecules. Combination of the 5-step EEBGB- conformer selection protocol and competition reaction methodology enabled us to theoretically predict for the first time, the four stepwise macroscopic protonation constants of trien within 0.1(-0.8) log unit of experimental values. This work opens up the gateway for predicting protonation constants of yet-to-be synthesized aliphatic linear polyamines and a comparative determination of their usability as an anticancer drug template by medicinal chemists.