BPS2026 – Designing relaxation-optimized 19F-13C probes for multidimensional NMR

19 F-bioNMR has re-emerged as a powerful approach for probing conformational dynamics in challenging biomolecular systems, particularly proteins that cannot be recombinantly expressed in E. coli . Site-specific cysteine labeling with thiol-reactive trifluoromethyl probes has provided transformative insights—for example, revealing the conformationally selective nature of G protein-coupled receptor (GPCR) activation. Yet, the impact of this methodology remains constrained: poor chemical shift dispersion and broad linewidths produce overlapped peak shapes that necessitate highly-subjective spectral deconvolution and typically restrict labeling to a single site. These limitations have prevented 19 F-bioNMR from realizing its full potential in studying complex biomolecular dynamics. A breakthrough opportunity arises from the recent discovery that aromatic 19 F- 13 C pairs can exhibit a pronounced TROSY effect, significantly improving spectral resolution and sensitivity. Building on this principle, we employed Bloch-Redfield-Wangsness relaxation theory to design cysteine-reactive aromatic 19 F- 13 C probes optimized for favorable relaxation. Unlike biologically constrained fluorinated amino acids and nucleotides, such as 3-fluorotyrosine or 5-fluorouracil, our synthetic scaffolds are specifically engineered to achieve narrow linewidths in both 19 F and 13 C dimensions. We validated this design strategy by synthesizing a suite of probes and demonstrating efficient, site-selective labeling of a 42 kDa maltose-binding protein double mutant (K34C/R354C). These probes produced strong 19 F- 13 C TROSY effects, enabling acquisition of high-quality 2D HSQC spectra in under two hours. Despite the molecular weight, we measured exceedingly slow R2 transverse relaxation rates of 43.8 s -1 ( 19 F) and 1.7 s -1 ( 13 C). To approximate the relaxation behavior of a 240 kDa system, MBP spectra were recorded in 20% glycerol at 5 °C: while the 19 F linewidth broadened, the 13 C dimension remained nearly unaffected. These results establish cysteine-reactive 19 F- 13 C probes as a promising new class of reporters, extending 19 F-bioNMR to larger systems and enabling development of multidimensional experiments for simultaneously probing structural dynamics at many sites.