Sequence-dependent separation of trinucleotides by ion-interaction reversed-phase liquid chromatography—A structure-retention study assisted by soft-modelling and molecular dynamics

We studied sequence-dependent retention properties of synthetic 5′-terminal phosphate absent trinucleotides containing adenine, guanine and thymine through reversed-phase liquid chromatography (RPLC) and QSRR modelling. We investigated the influence of separation conditions, namely mobile phase composition (ion interaction agent content, pH and organic constituent content), on sequence-dependent separation by means of ion-interaction RPLC (II-RPLC) using two types of models: experimental design–artificial neural networks (ED-ANN), and linear regression based on molecular dynamics data. The aim was to determine those properties of the above-mentioned analytes responsible for the retention dependence of the sequence.Our results show that there is a deterministic relation between sequence and II-RPLC retention properties of the studied trinucleotides. Further, we can conclude that the higher the content of ion-interaction agent in the mobile phase, the more prominent these properties are. We also show that if we approximate the polar component of solvation energy in QSRR by the electrostatic work in transferring molecules from vacuum to water, and the non-polar component by the solvent accessible surface area, these parameters best describe the retention properties of trinucleotides. There are some exceptions to this finding, namely sequences 5′-NAN-3′, 5′-ANN-3′, 5′-TGN-3′, 5′-NTA-3′and 5′-NGA-3′ (N stands for generic nucleotide). Their role is still unknown, but since linear regression including these specific constellations showed a higher observable variance coverage than the model with only the basic descriptors, we may assume that solvent-analyte interactions are responsible for the exceptional behaviour of 5′-NAN-3′ & 5′-ANN-3′ trinucleotides and some intramolecular interactions of neighbouring nucleobases for 5′-TGN-3′, 5′-NTA-3′and 5′-NGA-3′ trinucleotides.