Qnp cryoprobe

Compound 1 (6 mg)
was dissolved in 120 μL of 20% H₂O in DMSO-d₆. H₂O was used
instead of D₂O to prevent hydrogen–deuterium chemical
exchange. All spectra were acquired on a Bruker AVANCE III spectrometer
with a proton frequency of 600 MHz fitted with a QNP cryoprobe. All
spectra were acquired at a nominal probe temperature of 298 K. Proton
spectra were acquired with suppression to reduce the intense water
resonance. The ¹H NMR spectra were referenced to residual
DMSO-d₅ at 2.50 ppm. The proton spectrum
was assigned using standard 1D and 2D methods. Individual assignments
of the diastereotopic protons 19, 29, and 31 and methyl protons 33
and 34 were made based on the distances to adjacent protons determined
from the rotating-frame nuclear Overhauser effect correlation spectroscopy
(ROESY) spectrum. Assignments are shown in Table S2.
Interproton distances were estimated from a jump-symmetrized
ROESY spectrum:^{35 (link)} Bruker pulse sequence:
roesyadjphpr, spinlock time: 250 ms, tilt angle: 45°, recycle
time: 5.3 s, total experiment time: 51 h. Cross-peak intensities were
normalized to the diagonal by applying the PANIC correction.^{36 (link)} Where possible, cross-peaks were taken from
both quadrants and averaged. A reference distance of 1.75 Å for
the geminal H29 proton pair was used to convert the intensities of
cross-peaks into an average distance.

¹H-NMR spectroscopic analysis of chicken serum samples was performed by the NMR Laboratory (nmr@chemistry.mcmaster.ca) in the Department of Chemistry and Chemical Biology, McMaster University (Hamilton, Ontario, Canada). One hundred microlitres of 0.9% saline in D₂O with 1 mg/ml sodium [2,2,3,3-d4]3-trimethylsilyIpropanoate (TSP-d4) was mixed with 500 µl serum in high quality 5-mm NMR tubes. ¹H NMR spectra of serum were recorded on a Bruker Avance III 700 MHz NMR spectrometer (Bruker Biospin, Rheinstetten, Germany) equipped with a 5 mm QNP cryo-probe and SampleJet autosampler, and operating at a proton frequency of 700.17 MHz. A carr-Purcel-Meiboom-Gill (CPMG) spin-echo pulse sequence [recycle delay−90°− (τ–180°–τ)_n–acquisition] was used to emphasize resonances from low molecular-weight metabolites^{14 (link),28 (link),61 (link)}. ¹H NMR data for each sample were acquired using 128 scans (64k data points) with a 2.5 second relaxation delay. Chemical shifts were referenced to the internal reference (TSP-d₄: 0.00 ppm). ¹H NMR data with water suppression using excitation sculpting with gradients were subsequently acquired using the same number of scans and time of relaxation delay as that for ¹H NMR data.

Urine samples were combined with 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS, chemical shift standard) in D₂O, difluoro trimethylsilanyl phosphonic acid (DFTMP, pH standard), and sodium phosphate buffer for analysis. A pooled urine sample was also prepared for NMR and run at regular intervals during data analysis. Urine samples were analyzed on a Bruker Avance NMR spectrometer operating at 600.133 MHz for proton and equipped with a Bruker QNP cryoprobe. Water suppression was achieved using the noesy1dpr pulse sequence. For each sample, 128 scans were collected into 32 K data points over a spectral width of 9615.39 Hz. Raw data was processed in ACD/Labs 1D NMR Manager (ACD/Labs, Toronto, ON, Canada). Processed data were grouped and integrated. The individual spectra exported were as *.jdx files for quantification of select metabolites in Chenomx NMR Suite (Edmonton, AB, Canada). The metabolite concentrations were normalized to the urine creatinine concentration in each sample.