Molecular Recognition Principles and Stationary-Phase Characteristics of Topoisomer-Selective Chemoaffinity Materials for Chromatographic Separation of Circular Plasmid DNA Topoisomers

Abstract

We recently discovered the molecular recognition capability of a quinine carbamate ligand attached to silica as a powerful chemoaffinity material for the chromatographic separation of circular plasmid topoisomers of different linking numbers. In this paper we develop structure-selectivity relationship studies to figure out the essential structural features for topoisomer recognition. By varying different moieties of the original cinchonan-derived selector, it was shown that intercalation by the quinoline moiety of the ligand as assumed initially as the working hypothesis is not an essential feature for topoisomer recognition during chromatography. We found that the key elements for topoisomer selectivity are the presence of a rigid weak anion-exchange site and a H-donor site separated from each other in a defined distance by a 4-atom spacer. Additionally, incorporation of the weak anion-exchange site into a cyclic ring structure provides greater rigidity of the ligand molecule and turned out to be advantageous, if not mandatory, for (close to) baseline separation.

Chemoaffinity Material for Plasmid DNA Analysis by High-Performance Liquid Chromatography with Condition-Dependent Switching between Isoform and Topoisomer Selectivity

“…20,21 Interestingly, the ligand features some structural similarities to minor groove binders (e.g., netropsin), such as a H-donor group allowing bifurcated specific hydrogen bonding with AT-rich sequences and geometrically constrained positioning in the groove supported by H-bond mediated ionic interaction (of quinuclidinium moiety) with the phosphate backbone. 17 The results of above thermodynamic study and ionic strength effects are well in agreement with this hypothesis. Supercoiling makes the minor groove better accessible for binding of low molecular ligands, 15 which explains why the supercoiled species are stronger retained than oc and lin isoforms.…”

“…This persistence of elution order is due to specific interaction of pDNA isoforms via simultaneous multiple contacts with the chemoaffinity ligand allowing selective geometrically constrained recognition of individual pDNA forms (see Figure 1c for binding sites). 17 For comparison, the elution order varies with the gradient slope and plasmid size on a commercial and frequently used DNA-NPR column (Tosoh Bioscience, see the Supporting Information for chromatograms), containing a DEAE ligand. 13 On the quinine carbamate phase, the elution order remains unchanged even if the elution conditions (except for temperature) and the separation mode are changed.…”

“…However, it is likely that the same chemoaffinity principle, which was derived to be responsible for topoisomer recognition (see colored recognition element in Figure 1c), distinguishes between different isoforms. An indication for this assumption is that ligands which exhibit good topoisomer selectivity 17 show also reasonable isoform selectivity, while ligands with lack of topoisomer recognition also fail to separate isoforms. Concerning the isoform separation, Figure 2b represents a chromatogram recorded under optimized conditions using isopropanol as the organic modifier.…”

“…13 In preparative chromatography, several small-molecule ligands were investigated 14 that were able to separate the supercoiled from the oc isoform, which is a very challenging impurity, 15 or nucleic acids in general. 16 We chose to employ a quinine carbamate ligand on the analytical scale (see Figure 1c), which is able to form defined interactions with the analytes, 17 leading to predictable elution properties (Figure 1c). Recently, we have employed such a column for separation of topoisomers of the ccc pDNA, i.e., differently supercoiled plasmids.…”

Design and synthesis of naphthalene‐based chiral strong cation exchangers and their application for chiral separation of basic drugs

“…The versatility of this type of chiral weak anion exchangers (cWAXs) based on Cinchona alkaloids (quinine and quinidine) has been verified in a great number of studies and summarized in several reviews [2,13]. During the two decades on the market, the application field of cWAXs extended from chiral separation of N-protected α-amino acids to various classes of organic molecules, including hydroxy acids [14], various amino acids [15,16], and biomolecules [17].…”