Qci f cryoprobe

K562 cells were maintained in IMDM media supplemented with 10% FBS. For NMR measurements cells were harvested by centrifugation, washed, and resuspended in Opti-MEM serum-free media to obtain a suspension with 65% v/v cell content. 10% D₂O and 500 µM prodrug in DMSO-d6 were added (0.5% final DMSO concentration) and incubated on a spinning wheel for 5 min. A 200 µl suspension was transferred into a 3 mm Shigemi tube (without insert) matched to the magnetic susceptibility of water. 1D ¹⁹F spectra with inverse-gated decoupling of protons were collected at 37 °C with 2048 scans (experiment time 37 min) on a Bruker AVANCE III spectrometer with an 11.7 T magnet using a QCI-F cryoprobe at 470 MHz Lamour frequency. After 4 h the sample was lysed by freeze-thawing and a further spectrum of the lysate was collected. To confirm the identity of the observed species, the lysate was sequentially spiked with 500 µM MN551 and prodrug and measured again. For comparison, 1D ¹⁹F spectra of pure MN551 and prodrugs were collected in Opti-MEM media with 10% D₂O and 0.5% DMSO-d6. All spectra were processed and analyzed using Topspin 4.1.1.

Samples for transmission electron microscopy (TEM) were resuspended at a PA concentration of 10 mg/mL and then diluted 10-fold–1 mg/mL immediately before 10 µL of sample solution was transferred to plasma-cleaned 300-mesh copper grids with lacey carbon support (Electron Microscopy Science). Samples were stained with 2% uranyl acetate. Imaging was performed using a FEI Spirit G2 TEM working at 120 kV accelerating voltage.
Nuclear magnetic resonance (NMR) spectroscopy was performed on a Bruker Neo 600 MHz system with QCI-F cryoprobe at 298 K. The lyophilized powders from the sample preparation protocol above were resuspended in D₂O at 10 mg/mL PA concentration and 5 mg/mL losartan concentration. This corresponds to 8.7 mM E-PA, 8.7 mM K-PA, and 10.8 mM losartan.

Nuclear magnetic resonance (NMR) spectroscopy was used to analyse metabolite extracts. All experiments were carried out at 298 K on a Bruker Avance 3 600 MHz spectrometer, equipped with a QCI-F cryoprobe. Datasets were acquired with 64,000 points and a proton window size of 16 ppm. Spectra were referenced against an internal standard of DSS. The excitation sculpting method was used to suppress the water peak using pulsed field gradients.
Bruker TopSpin™ and AMIX data analysis software were used to analyse the NMR spectroscopy spectrum for each sample. The Madison Metabolomics Consortium Database was also used for the identification and quantification of metabolites using NMR spectroscopy (www.mmcd.nmrfam.wisc.edu/).

NMR experiments were performed on a Bruker 500 MHz Avance III spectrometer using a QCI-F cryoprobe with cooled ¹H and ¹⁹F channels and sample temperature control unit. Spectra were initially processed and analysed using Topspin 4.1 and Dynamics Centre 2.7 (both Bruker), and plotted in GraphPad Prism 9.

STD NMR samples were prepared with a ligand to protein ratio of 50:1 (500 μM ligand, 10 μM FcRn_ECD) in 500 μL phosphate buffered saline, pH 7.4 (90% H₂O, 10% D₂O) with 5% d6-DMSO to help solubilize the ligand. STD NMR spectra were recorded using a Bruker Avance III HD 600 MHz spectrometer equipped with a 5 mm QCI-F Cryoprobe. Data were acquired and processed using the standard Bruker software and were collected at 298 K. The protein was saturated in the methyl region of the spectrum at 0 ppm, and off-resonance saturation was performed at 33 ppm. A series of 120 EBurp2 pulses (50 ms each) were applied with a 4-μs delay between each pulse, resulting in total saturation time of 6 s. Protein signals were removed by applying a spinlock of 100 ms. Interleaved on- and off-resonance data were recorded, processed separately, and then the difference spectra obtained by subtracting the on- from the off-resonance spectra. Data were zero filled once and an exponential multiplication window function applied (LB 2 Hz).