Broadening the scope of 19F NMR

Over the past decade, fluorine NMR has established itself as a powerful fragment-finding method due to the advantages Teddy laid out in his classic “fluorine fetish” post. One feature of ¹⁹F NMR is that the chemical shifts of organofluorine molecules span a very wide range, in theory allowing large mixtures to be screened. However, existing NMR methods do not work across such large spectral windows, thereby requiring multiple experiments to screen an entire library. This limitation has now been overcome as described in a paper just published in Angew. Chem. by Andreas Lingel, Andreas Frank, and collaborators at Novartis and Karlsruhe Institute of Technology.

The researchers developed an experiment based on “broadband universal rotation by optimized pulses” (BURBOP). I confess that the details evade me (though they are all there in the supporting information if you wish to try it at home), but the upshot is a type of CPMG experiment in which fluorine-containing fragments bound to a protein show decreased peak intensities. Crucially, a single experiment can cover the full frequency range of pharmacologically relevant fluorine-containing molecules, spanning about 210 ppm. Previously, this required four two separate experiments.

Such increased throughput led the researchers to revamp their library, increasing the size from 1600 to 4000 fragments in an augmented library dubbed LEF4000. The paper has a nice, broadly applicable description of their curation process. Candidate members were brought in from both commercial and in-house sources and chosen to complement existing library members in terms of diversity. A modified rule of three was applied, with trifluoromethyl-containing fragments allowed to go up to 350 Da.

An in-house analysis of 25,000 fragments revealed that only about half of those with a clogD_7.4 greater than 3 were soluble above 0.5 mM, so this was applied as an upper limit. Fragment solubilities were experimentally measured, and only compounds with solubilities above 0.2 mM were kept. (Although fluorine NMR is often done at low concentrations, complementary biophysical experiments are not.) Additional quality control measures included NMR and LC-MS purity assessments and removal of compounds that formed soluble aggregates as assessed by CPMG. Ultimately, 3969 of 5600 candidate molecules passed the gauntlet, and were combined in 131 mixtures of about 30 compounds each.

Having built their library, the researchers screened it against the antibacterial target CoaD, which is involved in coenzyme A synthesis. The screen took just two days, and automated hit identification took only a few hours on a standard laptop. The overall hit rate was ~6%, and some of the hits were confirmed using two-dimensional protein-observed NMR methods, revealing that they bind in the enzyme active site with affinities in the mid micromolar to low millimolar range.

Pushing the technique further, the researchers built a “Supermixture” of 152 compounds, including five of the hits spanning a wide range of chemical shifts, from -50 to -220 ppm. Even under these conditions the binders were readily identifiable, and the paper states that libraries exceeding 20,000 fragments could in principle be screened in a few days.

In 2009 I wondered why ¹⁹F NMR was not used more widely. How things change! At Novartis the LEF4000 library has been screened against “a wide variety of disease-related targets” and identified “tractable hits for each of the screened targets, among them many considered undruggable by small molecules such as transcription factors, a cytokine, a nuclear receptor, and a repeat RNA.” Practical Fragments looks forward to seeing some of these appear in the growing list of FBDD-derived clinical candidates.