Nuclear magnetic resonance (NMR) spectroscopy is a gold standard for fragment-based screening and drug design. Photochemically induced dynamic nuclear polarization (photo-CIDNP) enables to overcome sensitivity limitations of non-hyperpolarized NMR, enabling fragment screening at low micromolar concentrations in single-scan experiments of a few seconds. For ¹H NMR, we established a 212-compound photo-CIDNP fragment library and screened it against cancer target Pin1 in a proof-of-concept study¹. Combined with a fully automated NMR platform, the library can be screened in only half a day on a high-field (600 MHz) NMR spectrometer².

Benchtop NMR spectrometers provide a significantly cheaper, low-maintenance, cryogen-free platform for screening experiments since they operate with permanent low-field instead of superconducting high-field magnets³. We show that photo-CIDNP fragment screening can be performed on a benchtop NMR (80 MHz) with a measurement time of only 3 minutes per sample in a screening of 30 compounds against target Pin1 at 500 μM fragment and 10 μM protein⁴. Binding could still be observed at 50 μM fragment and 1 μM protein. Obtaining signal enhancements up to 3200-fold, the detection limit was pushed to 250 nM for a 3-minute experiment. Photo-CIDNP provides a higher binding contrast at low magnetic field due to higher ligand polarization. This reduces the amount of protein required for the screening.

Further optimization of protein consumption and screening throughput can be achieved with ¹⁹F photo-CIDNP. Fluorine NMR is the gold standard for NMR screenings at high field and allows screening in larger compound mixtures due to its broad chemical shift dispersion. Our first dedicated ¹⁹F photo-CIDNP library comprises 70 fragments that can be screened in mixtures of up to 10 compounds. The library was screened at 600 MHz against the two cancer targets Kras and Pin1 at 20 μM fragment and 10 μM protein in single-scan experiments, resulting in a total screening time of only 40 minutes in fully automated fashion⁵. Due to the high binding contrast of Fluorine photo-CIDNP, nanomolar protein concentrations can be used when screening in multi-scan experiments at high field. In the future, ¹⁹F-photo-CIDNP will also enable screenings in low micromolar fragment mixtures on the benchtop NMR in only a few hours.

In conclusion, photo-CIDNP presents a tremendous boost to NMR fragment screening by overcoming sensitivity and cost limitations. The use of benchtop NMR will facilitate the accessibility of NMR for a diverse life science community. Fluorine photo-CIDNP will allow to screen several thousand compounds per day by pooling fragments and will facilitate measurements in complex samples due to elimination of background signals. The high binding contrast will also enable photo-CIDNP NMR to tackle challenging screening targets like membrane proteins and intrinsically disordered proteins.

[1] F. Torres, M. Bütikofer, G. R. Stadler, R. Riek et al., J. Am. Chem. Soc., 2023, 145, 22, 12066-12080.
[2] W. Wüster, P. Gebbers, A. Renn, M. Bütikofer, S. Rüdiger, R. P. Riek, F. Torres, MR, 2024, 5, 61–67.
[3] M. Bütikofer^#, G. R. Stadler^#, F. Torres, Chem.–Methods, 2024, 4, e202400009. ^#equal contribution
[4] G. R. Stadler, F. Torres, R. Riek et al., Angew. Chem. Int. Ed., 2023, 62, 40, e20230869.
[5] G. R. Stadler, F. Torres, R. Riek et al., 2025, in preparation.