High throughput multiplexed method for evaluation of enantioselective performance of chiral selectors by HPLC‐ESI‐MS and dynamic titration: Cinchona alkaloid carbamates discriminating N‐blocked amino acids

Abstract

Because of the generally different interaction of enantiomers with biological systems, there has been an ever increasing demand for artificial highly enantioselective systems that can facilitate separation processes involved in the research and development of enantiomerically pure drugs. Such systems may be discovered by large-scale screening of compound libraries which warrants rapid and cost-efficient screening methods. Here, we demonstrate enantioselectivity determination for systems of cinchona alkaloid carbamates and N-blocked amino acids using HPLC-MS and the recently developed dynamic titration technique (Frycák P, Schug KA. Anal Chem 2008;80:1385-1393). A mixture of nine N-blocked amino acids (either D or L enantiomers) was separated on a reversed-phase column with cinchona alkaloid carbamates added postcolumn. Dissociation constants of the observed noncovalent complexes were determined from the HPLC-MS data. Enantioselectivity was then calculated from the dissociation constants, pointing out the best performing systems. For these systems, apparent dissociation constants were measured for the whole range of enantiomeric composition and were shown to obey a proposed theoretical model.

Controlled band dispersion for quantitative binding determination and analysis with electrospray ionization‐mass spectrometry

“…The most recent report on dynamic titration uses the method to screen the enantioselective performance of four CAC chiral selectors (two pseudoenantiomeric pairs) towards eight chiral N ‐blocked amino acids (NBAA) (Frycak & Schug, 2009). Mixtures of enantiomerically pure NBAA ( D or L ) were separated on a short reverse‐phase column under isocratic conditions.…”

Determination of Noncovalent Binding Using a Continuous Stirred Tank Reactor as a Flow Injection Device Coupled to Electrospray Ionization Mass Spectrometry

“…Using MS, association constants can be obtained by different approaches, such as titration [24][25][26][27][28], competitive binding [1,17], or hydrogen/deuterium exchange [16,29,30] experiments. Among these approaches, titration is the most used method for the determination of binding constants.…”

“…To improve throughput, Fryčák and Schug developed a different titration method that uses a single solution of host and guest and is based on controlled band-dispersion [24,25,32,33]. The controlled band-dispersion concept appeared together with the development of flow injection analysis.…”

“…The product of the reaction is measured in the detector placed downstream. The use of FIA and controlled dispersion for MS-based binding determinations has been termed dynamic titration [24,25,32,33]. This method involves the injection of the guest solution into a flowing stream.…”

“…The host solution, present in the flowing stream at constant concentration, mixes with the guest during band dispersion to create a time-dependent compositional gradient of guest in the presence of host. A binding constant can be determined either by fitting the dispersion profile data using an appropriate (e.g., Gaussian) function combined with a simple binding model [32] or by integrating the degree of association across the observed signal peak [24,25]. The use of these strategies is facilitated by in-house developed software; however, a drawback of the curve fitting procedure is that an appropriate function must be chosen to best match the shape of the dispersed band.…”