Practical Considerations and Guidelines for Spectral Referencing for Fluorine NMR Ligand Screening

[Image: see text] Fluorine ((19)F) NMR strategies are increasingly being employed for evaluating ligand binding to macromolecules, among many other uses. (19)F NMR offers many advantages as a result of its sensitive spin 1/2 nucleus, 100% natural abundance, and wide chemical shift range. Moreover, because of its absence from biological samples, one can directly monitor ligand binding without background interference from the macromolecule. Therefore, all these aforementioned features make it an attractive approach for screening compounds. However, the detection of ligand binding, especially those with weak affinities, can require interpretations of minor changes in chemical shifts. Thus, chemical shift referencing is critical for accurate measurements and interpretations. Unfortunately, one cannot rely on spectrometer indirect referencing alone, and internal chemical references have sample-dependent issues. Here, we evaluated 10 potential candidate compounds that could serve as (19)F NMR chemical references. Multiple factors were systematically evaluated for each candidate to monitor the suitability for (19)F NMR screening purposes. These factors include aqueous solubility, buffer compatibility, salt compatibility, aqueous stability, tolerability to pH changes, temperature changes, and compound pooling. It was concluded that there was no ideal candidate, but five compounds had properties that met the screening requirements.