Dynamic titration: determination of dissociation constants for noncovalent complexes in multiplexed format using...

With recent growth in fields such as life sciences and supramolecular chemistry, there has been an ever increasing need for high-throughput methods that would permit determination of binding affinities for noncovalent complexes of various host-guest systems. These are traditionally measured by titration experiments where concentration-dependent signals of species participating in solution-based binding equilibria are monitored by methods such as UV-vis spectrophotometry, calorimetry, or nuclear magnetic resonance spectrometry. Here we present a new titration technique that unifies and allows chromatographic separation of guests with determination of dissociation constants by electrospray mass spectrometry in a multiplexed format. A theoretical model has been derived that describes the complex formation for the guests eluted from a chromatographic column when hosts are admixed postcolumn. The model takes possible competition equilibria into account; i.e., it can deal with unresolved peaks of guests with the possible addition of multiple hosts in one experiment. This on-line workflow makes determination of binding affinities for large libraries of compounds possible. The potential of the method is demonstrated on the determination of dissociation constants for complexes of beta- and gamma-cyclodextrins with nonsteroidal antiinflammatory drugs ibuprofen, naproxen, and flurbiprofen.