600 mhz nmr spectrometer

¹H-NMR spectra were acquired using a Varian 600 MHz NMR spectrometer equipped with a 1H{13C/15N} 5mm PFG Automatable Triple Resonance probe at room temperature (25 °C). The CPMG pulse sequence with presaturation for water suppression was applied. In total, 128 transients were collected with 64 K data points, a relaxation delay of 5 s and an acquisition time of 4 s, Receiver gain was kept constant for all acquisitions. ¹H NMR spectra were referenced at TSP chemical shift (0.00 ppm) and processed with 0.3 exponential line broadening (17 (link)).

¹H and ¹³C nuclear magnetic resonance (NMR) spectra of KCFP were acquired using a 600 MHz NMR spectrometer (Varian, United States). Briefly, after drying the KCFP at 50 °C to constant weight, approximately 15 mg of sample was dissolved in 0.6 mL of 99.98% D₂O, and the ¹H NMR spectrum was collected using the PRESAT water signal suppression method described by the manufacturer. In addition, approximately 30 mg of KCFP was dissolved in 0.6 mL of 99.98% D₂O for ¹³C NMR spectroscopy. Each sample was scanned 1024 times for ¹H NMR analysis and 70000 times for ¹³C NMR analysis.

The hydrophilic metabolites were reconstituted in 500 µl of D₂O containing 0.05 mM 4, 4-dimethyl-4-silapentane-1-sulfonic acid (DSS) as chemical shift reference and internal concentration standard, and 0.2 % sodium azide as bacteriostatic agent to prevent biodegradation. About 450 µl of the prepared sample was loaded into a standard 5 mm NMR tube (Wilmad, Vineland, NJ, USA). All ¹H NMR spectra were acquired on a Varian 600 MHz NMR spectrometer equipped with a Z axis-gradient 5mm HCN probe at 20°C. One-dimension ¹H NMR spectra were acquired from each sample using the standard Varian PRESAT pulse sequence with a single pulse excitation and 1s low power presaturation at the water peak position to suppress the residual water signal. The acquisition time and recycle delay (RD) of a single scan was 3 s and 1 s, respectively, and the spectral width was 7200 Hz. A total of 20 k transients were accumulated, corresponding a total measuring time of about 22 hours for each sample, to ensure that a high quality ¹H spectrum was obtained with sufficient signal to noise ratio for metabolites with concentration as low as about 0.5 µM or even lower in the NMR tube.

Hypromellose (HPMC; 1.5 g) was dissolved in N‐methylpyrrolidone (NMP; 60 mL) by stirring at 80°C for 1 h. Once the polymer had completely dissolved, the solution was heated to 50°C. A solution of 1‐dodecylisocyanate (0.5 mmol, 10% dodecyl modification by weight) was dissolved in NMP (5 mL) and added to the reaction mixture followed by 150 μL of N,N‐diisopropylethylamine as a catalyst. The solution was then stirred at room temperature for 16 h. This solution was then precipitated from acetone and the HPMC‐C₁₂ polymer was recovered by filtration, dialyzed within a 3.5 kDa cut‐off dialysis bag for 3 days in water, and lyophilized, yielding a white amorphous material. HPMC was found to have a molecular weight of Mn(Đ) = 272,900 (1.37) by aqueous size exclusion chromatography (SEC; full GPC trace shown in Figure S2). Aqueous SEC‐RI traces were obtained on a Optilab rEX refractive index detector (Wyatt) after passing through a column (Superose 6 Increase 10/300 GL column, Mw range of 5000–5,000,000 g/mol (GE healthcare)). 1H‐NMR was used to confirm modification (Figure S3). 1H‐NMR spectra were obtained and recorded on a Varian 600 MHz NMR spectrometer at 298 K, and chemical shifts are given in parts per million. 1H‐NMR spectra were referenced to residual proton resonances in the deuterated solvents (DMSO‐d₆ shift = 2.50).