A Screening Method for Chiral Selectors that Does Not Require Covalent Attachment

Abstract

A high throughput screening protocol is proposed for chiral selector discovery. It is modeled after the protocol for biological screening of candidate drugs from chemical libraries. The procecure works based on target distribution between an aqueous phase and an organic phase. The target may be a racemate or separate enantiomers. Screening for noncovalent intermolecular association between target and candidate selectors is carried out by partitioning experiments in the presence and absence of the candidate chiral selectors in the organic phase (plasticized poly(vinylchloride)).The partition ratio measurement uses 96-well plates for high throughput. The feasibility of this approach is validated by working with a known target/chiral selector pair, N-(3,5-dinitrobenzoyl)-α-phenylglycine and 2,2,2-trifluoro-1-(9-anthryl)ethanol. The validated protocol is applied to a small library of 12 cyclopropyl dipeptide isosteres. Eight bind the racemic target, econazole. Among them, one has measurable chiral selectivity. The advantage of the method is that it does not require the covalent attachment of either the analyte or the selector, and the required amount of the potential chiral selector is about 100 micrograms.The most widely used technique for chiral separations at the analytical and semipreparative scale is liquid chromatography (LC) on chiral stationary phases (CSPs) 1 . Numerous CSPs exist 1,2 . The desire for more generality, better selectivity, more robustness and predictability drives the search for new CSPs 3 . While understanding the mechanism of chiral selectivity will certainly advance the search 4 for better selectors, screening of libraries is showing promise for CSP discovery 5 . We note that all the current screening methods require the immobilization of either the target or the selector to a stationary phase; some of them also require packing and using the CSP in a column. These steps require time, labor and material. It would be extremely useful to have a library screening protocol for chiral selector discovery that would function Email: sweber@pitt.edu. Supporting Information Available: Protocol for assays, capillary electrophoresis separation of econazole enantiomers, chemical structure of the 12 library members, and a detailed comparison of this screening method to others. Here we introduce such a method based on target distribution between an aqueous phase and an organic (film) phase in a microtiter plate 6, 7 . Partitioning experiments are performed in the presence and the absence of a candidate selector in the organic phase. The difference in the observed distribution of the target reports on the binding of the target to the selector. Since ordinary organic solvents are difficult to work with, especially at low volume, we prefer thin polymer films as the organic phase. Plasticized polyvinyl chloride (PVC) films have been used to study molecular recognition 8 , so it is naturally a good choice for chiral recognition. Figure 1 gives the sequence of operations for the screening procedure. Th...

Determination of binding constants by affinity capillary electrophoresis, electrospray ionization mass spectrometry and phase-distribution methods

“…The binding equilibrium can be studied in either phase. This method has been applied to measure binding constants for the complex formation of caffeine/benzoic acid [18], solute/cyclodextrin [2,96-103], metal ion/anion [104], and drug/chiral selector [105]. …”

“…Advantages of this method include relatively low sample requirement, availability to measure binding constants under physiological conditions (e.g., pH, temperature and ionic strength) and the potential to utilize high-throughput screening technologies [105,106]. …”

High-Throughput Method for Lipophilicity Measurement

“…Recently, our group developed a high-throughput screening method for chiral selectors, which was modeled after the protocol for biological screening of combinatorial libraries. 28 This method was based on target partitioning between a selector-containing polymer-film phase and an aqueous phase. The polymer used was plasticized poly(vinyl chloride) (PVC).…”

A multichannel electrophoresis microchip platform for rapid chiral selector screening

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