Chiral cation exchange polymer beads and monoliths

Research project P 13965 Chiral cation exchange polymer beads and monliths Michael LÄMMERHOFER 24.01.2000 Many of biologically active compounds (drugs, agrochemicals, flavors, etc.) are chiral and the enantiomers may differ considerably in their biological activity spectra. Consequently, there is a need for the production of enantiorrierically pure compounds as well as for analytical tools to evaluate the enantiomeric purity and to determine the enantiomeric ratio in plasma, urine, etc. in the course of pharmacological studies. Liquid chromatographic (LC) and electrophoretic (CE) enantioseparation methods fulfill these tasks. These technologies need chiral selectors (SOs), and for LC they are usually grafted onto silica representing a chiral stationary phase (CSP), which interacts intermolecularly with the enantiomers of the analyte (selectand, SA) at different rates leading to enantioseparation. A wide variety of CSPs are already available; however, each of them has its scope of applicability and disadvantages so that there is still a need for novel CSPs, in particular for charged SAs (bases and acids). The general aim of this project is a.) the development of novel CSPs based on organic polymeric support materials that work in the cation exchange (CX) mode for enantioseparation of basic chiral compounds and b.) to adopt these novel CSPs for application in capillary electrochromatography (CEC) with focus on the development of monolithic capillary columns. These promising project strategies are based on own accomplishments in the field of the development of chiral anion exchange type selectors and CSPs, as well as on experience in enantioselective CEC methods with these SOs and CSPs. The first task of the current project focusses on the development and screening of highly enantioselective anionic chiral SOs. Thus a variety of acidic chiral templates, in particular dedicatedly derivatized a/0-amino carboxylic, phosphonic, and sulfonic acids, should be screened for their enantiodiscriniination capability for a set of basic SAs by employing non-aqueous capillary electrophoresis (CE) technology which has proved in our earlier work as a powerful and rapid screening assay. The most promising SO candidates will then be further optimized implementing computational chemistry (3D-QSAR CoMFA, molecular modeling, SO-SA docking) and spectroscopic investigations (NMR, X-ray) paralleled by synthesis. In the second task these SOs will be grafted onto organic polymer type chromatographic support materials for HPLC application. Glycidyl fimetionalized monodisperse methacrylate polymer beads are selected to immobilize the SOs. This way, strong non-(stereo)specific analyte-support interactions, which would result with silica as support material, can be eliminated. Variables related to the grafting chemistry, SO-coverage, pore size and surface area including aspects affecting the thermodynamic and kinetic properties of the CSP will be studied. Third and parallel to these tasks dedicated focus of the proposed project relates to the development of macroporous CX type chiral monolithic capillary columns for application in capillary electrochromatography (CEC). CEC is a new separation technique combining chromatographic and electrophoretic principles with the advantage of higher efficiency and peak capacity compared to HPLC. CEC activities in the field of enantioseparation are still rather rare, although it should be ideal in particular for charged CSPs and SAs, provided that non-stereoselective effects can be suppressed. Monolithic continuous chromatographic beds based on organic polymers and prepared within the confines of a fused silica capillary by in situ copolymerization of finictional organic monomer and cross-linker in presence of porogens, which allow fine-control of the pore structure, should` overcome many technical, but also fundamental limitations. In the course of this project CX type chiral inethacrylate monoliths will be prepared: i.) by a two step approach preparing first macroporous rigid monolithic capillary columns with well-defined pore structure and reactive groups accessible for subsequent inimobilization of the chiral SOs; ii.) by a single step approach via in situ polymerizing the chiral methacrylic SO monomer in presence of suitable cross-linker and porogens. Factors having influence on pore structure, enantioselectivity and efficiency, like composition of porogenic system and polymer mixture as well as surface polarity and SO density, will be studied in detail and optimized. Overall, the interdisciplinary project aim and goal can be summarized as a multi task approach which combines enantioselective molecular recognition concepts, polymer chemistry and separation technology.

Novel highly selective cation-exchange materials based on particulate inorganic silica or monolithic organic polymethacrylate have been developed for the selective binding and separation of the stereoisomers of chiral basic drugs by pressure-driven or electric field-driven chromatography. Many drugs, agrochemicals and toxins are chiral and exist as pair of enantiomers (molecules that are non- superimposable mirror images to each other). The individual enantiomers, since they interact selectively with proteins like receptors or enzymes, may differ considerably in their biological activity profiles i.e. in their pharmacological or toxicological behavior. Thus, from a pharmacological standpoint they should be regarded as different compounds. In fact, however, many of the drugs are still administered as a mixture of the both enantiomers, although side effects or even toxic effects may very often be attributed to only one of the individual enantiomers. Consequently, in the drug development process the individual enantiomers need to be investigated separately nowadays. This inter alia necessitates the production of these compounds as single enantiomers in sufficient amount and high enantiomeric purity. On the other hand, analytical assays are required for the determination of the enantiomeric purity of single enantiomer drugs and intermediates and for the evaluation of the enantiomeric ratio in plasma, urine, etc. in the course of pharmacological studies. In the course of the present project novel separation materials with dedicated synthetic surface structures as molecular selectors and analytical methods implementing these materials, which may fulfill the above tasks, have been developed. The novel separation materials, which can be classified as chiral cation-exchangers, are comprised of a solid support such as silica particles or polymer beads and molecular selectors, being composed of a chiral moiety and at least one acidic group, as well as a spacer connecting selector and support. The new cation-exchange selectors are of low molecular size and have been synthesized from chiral aminosulfonic acids, aminophosphinic acids, and sulfopeptides. They have been structurally designed to specifically recognize and bind enantiomers of basic drugs. The completely new concept of enantioselective cation-exchange was then realized by implementation of the new separation media as stationary phases in capillary electrochromatography, a new microscale separation technique which employs an electric field to drive the mobile phase and the analytes through the capillary column that contains the stationary phase. A wide variety of basic drugs including beta-sympathomimetic bronchospasmolytics, beta-blocking antihypertensive drugs, antimalarial agents and many others could be successfully separated into the individual enantiomers. Moreover, in the studies of the present project it turned out that the cation-exchange materials with strongly acidic sulfonic and phosphonic acids show better enantiomer separation capabilities over analogs based on weakly acidic carboxylic acids. Besides the development of particulate enantioselective cation-exchangers, a strategy for the preparation of corresponding monolithic enantioselective cation-exchangers, which are macroporous materials with interconnected solids and through-pores that allow liquid flow through the material. The monolithic materials have been prepared by a 2-step approach i.e. by covalently binding the selectors onto the surface of poly(glycidyl methacrylate-co-ethylene dimethacrylate) monoliths that have prior been synthesized in situ within capillaries (typically 100 m inner diameter) utilizing simple rinsing procedures. Such obtained enantioselective cation- exchange monolithic materials may face a great future as they are not only highly selective and often possess better kinetic properties in flow-through applications such as chromatography, but can easily be fabricated in the channels of miniaturized analytical systems such as microchips for lab-on-a-chip technologies. The analytical applicability of the new cation-exchange materials could be proven. However, the enantioselectivity of the cation-exchangers need to be optimized by further structural refinement of the selectors that are immobilized on the surface of the materials before they have the potential to become commercialized products. Overall, the novel separation materials and corresponding chromatographic methods are certainly of interest for the pharmaceutical industry and are supposed to become helpful tools in drug development research.