Technical and practical aspects of ¹⁹F NMR‐based screening: toward sensitive high‐throughput screening with rapid deconvolution

Abstract

The technical and practical aspects of (19) F NMR-based screening against a macromolecular target are analyzed in detail. A novel method utilizing the relaxation of (19) F homonuclear double quantum coherence is proposed for performing NMR-based binding assays in a direct- or competition-mode format. A combined strategy based on (19) F NMR chemical shift prediction, 2D (19) F NMR DOSY, and 2D (19) F-(1) H NMR long-range COSY experiments is presented for the deconvolution of complex mixtures of fluorinated molecules generated by either addition of single compounds or by chemical synthesis. The approaches presented here allow the screening of complex mixtures, even in the case where the exact composition is not known, and the rapid identification of the binders contained in the mixtures.

19F NMR transverse and longitudinal relaxation filter experiments for screening: a theoretical and experimental analysis

“…The versatility of this methodology allows the screening of complex mixtures for which sometimes the exact composition is not known. A combined approach based on three experiments allows on the fly deconvolution and identification of the active molecules contained in the mixture without the need to separate and purify the molecules . The observable 19 F NMR parameters for the screening that are used in the detection are mainly the transverse relaxation rate and the isotropic chemical shift .…”

19F NMR matrix‐assisted DOSY: a versatile tool for differentiating fluorinated species in mixtures

“…Overlapping signals are very common in 1 H NMR spectra, particularly of mixtures, and by extension in 1 H DOSY. There are many strategies that can be used to avoid signal overlap, such as suppression of multiplet structure (pure shift methods) [26][27][28] , dispersing signals in multiple spectral dimensions [29][30][31][32] , using selective pulses [33] , or using other nuclei such as 13 C [34,35] , 29 Si [36] , 31 P [37,38] , 19 F [39][40][41] 6 Li [42] or 7 Li [43][44][45][46] for DOSY experiments. In DOSY, signals from different compounds are separated according to their diffusion coefficients, which depend on compound size and shape.…”

An improved 19F‐CPMG scheme for detecting binding of polyfluorinated molecules to biological receptors

“…In some cases, the binding of molecules bearing J ‐coupled fluorine nuclei could be assessed through signal broadening in 19 F‐NMR spectra as shown for carbohydrate derivatives containing difluoroacetamide moieties . An alternative is the use of a DQ coherence relaxation filter, as recently proposed for polyfluorinated compounds, although fast DQ relaxation rates could be a limitation for some molecules. Another possibility is to monitor the relaxation of long‐lived states, especially for compounds with J ‐coupled aliphatic fluorine atoms of smaller chemical shift anisotropy …”