46707-B4
An Investigation of Chiral Recognition in Electrokinetic Chromatography by means of NMR Spectroscopy
NMR spectroscopy was used to study the association of four β-blocker drugs with chiral molecular micelles (MM) of varying hydrocarbon chain length. β-blockers efficiently hinder the effects of adrenaline and reduce the force with which the heart contracts. They are used to treat conditions such as angina pectoris, tremors, and glaucoma. The molecular micelles used in this investigation contained covalently linked monomers with a chiral leucine headgroup and a carbamate linkage to a hydrocarbon chain containing eight to eleven carbons. These polymers were provided by Shahab Shamsi from Georgia State and are polymerized forms of the monomeric surfactant Enantioselect (Waters, Inc.). These MM are used as chiral selectors in electrokineitc chromatography (EKC), therefore, understanding the interactions between the polymers and chiral molecules is needed to design efficient chiral separations of drug mixtures. (R) and (S) enantiomers of the β-blockers atenolol and propranolol and racemic mixtures of metoprolol and pindolol were used in this investigation.
NMR diffusion experiments were used to establish association constants for each chiral drug binding to MM with eight (C8), nine (C9), ten (C10) and eleven (C11) carbon atoms in their hydrocarbon chains. The diffusion results showed that propranolol had the strongest interactions with each of the polymers, while atenolol had the weakest. Strong positive correlations were also seen between retention times from chromatography and NMR association constants in that the magnitudes of the association constants were consistent with the elution order from EKC. NMR diffusion experiments were also used to correlate the differences in the free energies of binding (Δ(ΔG)) for the enantiomers of atenonol with chiral selectivities from chromatography. The NMR results showed that the Δ(ΔG) values increased when moving from C8 to C9 to C10 polymers and then decreased slightly for C11. An identical trend was observed for the chiral selectivities.
Two-dimensional NMR experiments are underway to identify the intermolecular interactions responsible for chiral recognition in this system. Preliminary results suggest that the β-blockers experience both stereoselective interactions with the MM headgroup and nonstereoselective hydrophobic interactions with the molecular micelle hydrocarbon chains. In more hydrophobic β-blockers, the later interaction dominates resulting in poorer chiral resolution in EKC and smaller NMR Δ(ΔG) values.
NMR relaxation experiments were also used to study the motional dynamics of chiral molecules bound to the chiral MM poly(sodium N-undecanoyl-L-leucylvalinate) (poly(SULV)). The chiral molecules chosen for this study were 1,1-binaphthyl-2,2-diyl hydrogen phosphate (BNP) and 1,1-bi-2-naphthol (BOH). These molecules were chosen based upon their structural similarity to chiral drugs that have been enantioseparated in EKC using poly(SULV) as a chiral selector. Spin-lattice and spin-spin relaxation times for the protons of (R) and (S) enantiomers of BNP and BOH were measured in free solution and in the presence of the MM. These data were then used to compute each proton’s correlation time or the average time required for the proton to move through one radian. The results showed increases in correlation times when both BNP and BOH bound to the polymer. In addition, significant differences were seen between the proton correlation times within the same molecule with the longest correlation times observed for protons closest to the chiral molecules’ hydrogen bond donor/acceptor atoms. These results are consistent with a chiral recognition model developed using two-dimensional NOESY experiments in which the MM forms a chiral pocket into with molecules insert. The bound molecules simultaneously experience hydrogen bonding interactions with the MM headgoup and hydrophobic interactions with the hydrocarbon chain. In the bound state the motion of the chiral molecule protons nearest the hydrogen bonds is most restricted resulting in the longer correlation times observed here. The motions of the atoms interacting with the hydrocarbon chain in contrast are less restricted, thus these protons have more freedom of motion and thus shorter correlation times.
In the last project, a modified Diffusion Ordered NMR (DOSY) experiment was developed that used perdeuterated surfactant micelles to enhance resolution in the diffusion dimension. Diffusion-Ordered NMR Spectroscopy resolves mixture components based upon differences in their diffusion coefficients or molecular sizes. However, when components have near-identical diffusion coefficients, they are not resolved in the diffusion dimension of a DOSY spectrum. Adding surfactant micelles to these mixtures has been shown to enhance resolution when the component molecules interact differentially with the micelles. This approach is similar to EKC where MM are used to enhance the separation of mixture components. In this project, perdeuterated surfactants were added to mixtures studied with the DOSY technique. Since no micelle resonances appear in the mixture spectra, the error associated with performing biexponential analyses in spectral regions where analyte and surfactant resonances overlap is avoided. The approach was demonstrated using mixtures of peptides with near-identical diffusion coefficients.
