500 mhz spectrophotometer

Lyophilized R67 DHFR (100–750 μM tetramer) was dissolved in 10 mM perdeuterated Tris‐DCl (Tris‐d11) buffer, pH 8.0 in D₂O. ¹⁹F NMR measurements were acquired on a Varian 500 MHz spectrophotometer equipped with a OneNMR probe operating at 470 MHz. ¹⁹F spectra were obtained with 512 scans, a spectral width of 230 ppm, relaxation delay of 1 second, and 87° pulse angle. The ¹⁹F chemical shifts were referenced to 10 mM trifluoroacetic acid. All NMR data were processed using MestReNova (Mestrelab) applying a 20 Hz line‐broadening. Fluorine incorporation levels were measured by integrating the area of a known concentration of labeled apo R67 DHFR and comparing to an internal 10 mM trifluoroacetic acid standard.
Incomplete labeling of 5F and 6F R67 DHFR was explored using the pH‐induced tetramer‐to‐dimer dissociation of R67 DHFR.¹⁶ The NMR spectra for 5F or 6F R67 DHFR were obtained at pH 8. Next, the fluorine‐labeled R67 DHFR was mixed with a molar equivalent of unlabeled R67 DHFR, equilibrated for 5 min. Subsequently, the pH of the mixture of labeled and unlabeled R67 DHFR was lowered to pH 5 to form dimeric R67 DHFR. Finally, the pH of the solution was adjusted back to pH 8 to reform the tetramer, which should be a mixture of fully labeled, fully unlabeled, and partially labeled R67 DHFR tetramers. NMR spectra were recorded at each step.

All solvents and reagents were purchased from Sigma-Aldrich and used without further purification. THF was dried using an MB-SPS-800 solvent dryer (MBraun) and pyridine was dried over 4 Å activated molecular sieves. Thin layer chromatography was performed on EM Science Kieselgel 60 F254 (1 mm) plates and flash chromatography on Silicycle 40–63 μm (230–400 mesh) silica gel. ¹H-NMR, ¹³C-NMR, ¹⁹F-NMR and ³¹P-NMR spectra were recorded on a Varian 500 MHz spectrophotometer with chemical shift values reported in ppm. ¹H-NMR and ¹³C-NMR spectra were referenced to residual solvent. ³¹P-NMR were measured from 85% H₃PO₄ as an external standard.