Direct Resolution of Enantiomers in High-Performance Immunoaffinity Chromatography under Isocratic Conditions

ArticleinAnalytical Chemistry 74(9):2119-25 · June 2002with6 Reads
Impact Factor: 5.64 · DOI: 10.1021/ac0157369 · Source: PubMed

This paper describes the application of stereoselective antibodies as tailor-made chiral selectors for the separation of enantiomers in HPLC under isocratic conditions. Stereoselective monoclonal antibodies to D- and L-alpha-amino acids, raised against protein conjugates of p-amino-D- and L-phenylalanine, were immobilized on a synthetic high-flow-through support material and used for rapid enantiomer separation of a number of amino acids at flow rates between 0.1 and 10 mL/min. Since separations could be performed in a mild buffer, column lifetime considerably exceeded that of classical immunoaffinity systems. Using an anti-D-amino acid antibody as chiral selector, the L-enantiomers eluted with the void volume, while the D-enantiomers eluted second. Inverted elution orders were obtained on chiral stationary phases prepared from an anti-L-amino acid antibody. These results demonstrate, for the first time, that antibody-based chiral stationary phases are useful for routine enantiomer separation under true high-performance chromatographic conditions.

    • "WAC is usually performed under mild and isocratic conditions. Useful applications of WAC and closely related techniques have been found in many fields, such as, in chiral separations (Hofstetter et al., 2002; Ravelet et al., 2004), and in the evaluation of protein-analyte interactions (Moaddel & Wainer, 2006; Bergström et al., 2009; Yoo & Hage, 2009). WAC has also been characterized under overloaded conditions to predict its behaviors in non-ideal conditions (Hubble, 2001). "
    [Show abstract] [Hide abstract] ABSTRACT: Fragment-based drug discovery is an emerging process that has gained popularity in recent years. The process starts from small molecules called fragments. One major step in fragment-based drug discovery is fragment screening, which is a strategy to screen libraries of small molecules to find hits. The strategy in theory is more efficient than traditional high-throughput screening that works with larger molecules. As fragments intrinsically possess weak affinity to a target, detection techniques of high sensitivity to affinity are required for fragment screening. Furthermore, the use of different screening methods is necessary to improve the likelihood of success in finding suitable fragments. Since no single method can work for all types of screening, there is a demand for new techniques. The aim of this thesis is to introduce weak affinity chromatography (WAC) as a novel technique for fragment screening. WAC is, as the name suggests, an affinity-based liquid chromatographic technique that separates compounds based on their different weak affinities to an immobilized target. The higher affinity a compound has towards the target, the longer it remains in the separation unit, and this will be expressed as a longer retention time. The affinity measure and ranking of affinity can be achieved by processing the obtained retention times of analyzed compounds. In this thesis, WAC is studied for fragment screening on two platforms. The first system comprised a 24-channel affinity cartridge that works in cooperation with an eight-needle autosampler and 24 parallel UV detector units. The second system was a standard analytical LC-MS platform that is connected to an affinity column, generally called WAC-MS or affinity LC-MS. The evaluation criteria in studying WAC for fragment screening using these platforms were throughput, affinity determination and ranking, specificity, operational platform characteristics and consumption of target protein and sample. The model target proteins were bovine serum albumin for the first platform, thrombin and trypsin for the latter. Screened fragments were either small molecule drugs, a thrombin-directed collection of compounds, or a general-purpose fragment library. To evaluate WAC for early stages of fragment elaboration, diastereomeric mixtures from a thrombin-directed synthesis project were screened. Although both analytical platforms can be used for fragment screening, WAC-MS shows more useful features due to easy access to the screening platform, higher throughput and ability to analyze mixtures. Affinity data from WAC are in good correlation with IC50 values from enzyme assay experiments. The possibility to distinguish specific from non- specific interactions plays an important role in the interpretation of WAC results. In this thesis, this was achieved by inhibiting the active site of the target protein to measure off-site interactions. WAC proves to be a sensitive, robust, moderate in cost and easy to access technique for fragment screening, and can also be useful in the early stages of fragment evolution. In conclusion, this thesis has demonstrated the proof of principle of using WAC as a new tool to monitor affinity and to select hits in fragment-based drug discovery. This thesis has indicated the primary possibilities, advantages as well as the limitations of WAC in fragment screening procedures. In the future, WAC should be evaluated on other targets and fragment libraries in order to realize more fully the potential of the technology. Link:
    Full-text · Thesis · Apr 2013
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    • "Sixth, because of the exquisite flexibility and virtually unlimited nature of Ab-Ag interactions, it is possible to make antibodies against epitopes that do not occur naturally on Earth but could conceivably be incorporated by other life forms (Hofstetter et al., 1999). For example, while non-synthetic proteins consist of L-enantiomers of amino acids (Bada, 1985 ), antibodies can be raised against peptides consisting of D-enantiomers (Hofstetter et al., 2002; Vandenabeele-Trambouze et al., 2002), to test whether life on other planets exhibits varied chemical " handedness . " (Some organisms possess isomerase enzymes that, after synthesis, will racemize some amino acids to the D-isomer.) "
    [Show abstract] [Hide abstract] ABSTRACT: We propose a three-phase approach to test for evidence of life in extraterrestrial samples. The approach capitalizes on the flexibility, sensitivity, and specificity of antibody-antigen interactions. Data are presented to support the first phase, in which various extraction protocols are compared for efficiency, and in which a preliminary suite of antibodies are tested against various antigens. The antigens and antibodies were chosen on the basis of criteria designed to optimize the detection of extraterrestrial biomarkers unique to living or once-living organisms.
    Full-text · Article · Mar 2005 · Astrobiology
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    • "Reusability of an antibody support has been improved by crosslinking immobilised antibody fragments with glutaraldehyde (Vuolanto et al., 2004). Enantioselective antibodies have been used to fractionate chiral synthesis products (Mertens et al., 1982, Knox and Galfre, 1986) and other mixtures of enantiomers (Hofstetter et al., 1998Hofstetter et al., , 2002) hitherto in a relatively small scale, probably due to the use of poly-and monoclonal antibodies. Enantiomers from biological samples such as sera, plasma and urine have been extracted by specific antibody supports for analytical purposes. "
    [Show abstract] [Hide abstract] ABSTRACT: Thesis (doctoral)--University of Helsinki, 2004. Includes bibliographical references.
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