Prodigy cryoprobe

Silica gel plates were purchased from E. Merck (Darmstadt, Germany). Compounds on TLC plates were visualized under UV light. Ethyl acetate extracts were screened by thin layer chromatography (TLC) technique. Pure Compounds were recovered by preparative TLC procedure (system solvent Chloroform/Methanol 8:2 by vol).
Samples for NMR analysis were dissolved in Methanol-d₄. Chemical shifts are reported with the residual MeOD (δ_H 3.32 ppm) as the internal standard for ¹H NMR spectrometry, and MeOD (δ_C 49.0 ppm) for ¹³C NMR spectrometry. 1D- and 2D-NMR spectra were recorded at 600.13 MHz on a Bruker Avance III-600 spectrometer equipped with a TCI Cryo ProbeTM fitted with a gradient along the Z-axis, at a probe temperature of 27°C. ¹³C NMR spectra were recorded on a Bruker avance 400 spectrometer equipped with a Cryo Probe Prodigy (100.62 MHz). ¹H,¹³C NMR, COSY, TOCSY, HSQC, HSQC-EDITED, HMBC (³J: 10 Hz) experiments were used for structural determinations. Mass spectra were acquired on a MicroQ-Tof mass spectrometer coupled with an Alliance HPLC (Waters, Milford, MA, United States) equipped with an ESI positive and negative source.

To compare metabolic profiles of AU containing S. pseudintermedius after crystallization in AU at day 3 and the initial time-point (day 0), SPMAP09 was suspended in AU to a concentration of 10⁵ CFU/mL at 37°C for day 0 and day 3. Each AU sample included five replicates. The AU samples were centrifuged at 13,000 rpm for 10 min. A total of 540 μL of supernatant was mixed with 60 μL urine buffer containing 1.5M KH₂PO₄, 2mM NaN₃ and 1% TSP as internal standard. Then, the mixture was centrifuged at 12,000 rpm at 4°C for 19 min and 580 μL of supernatant was transferred into a NMR tube with 5 mm diameter for metabolic profiling. ¹H NMR spectra were acquired using a 400 MHz NMR spectrometer (Bruker, USA) with CryoProbe Prodigy and the Carr−Purcell−Meiboom−Gill (CPMG) pulse sequence [RD−90˚−(τ−180˚−τ)n−acquisition] was applied to analyze the AU samples at 310 K in 64 scans [10 (link)].

The NMR spectra were recorded on a Bruker 600 MHz spectrometer, equipped with a 5 mm broadband CryoProbe Prodigy. The acquisition parameters are: 90° pulse calibrated at 12 μs, 1.3 s acquisition time, 2 s time of relaxation, no spinning, 300 K, and 2048 scans. The proton NMR spectra were obtained at Forschungszentrum Jülich (Juelich, Germany). The FT-IR spectrum was recorded using FT-IR spectrometry, Nicolet iS5, iD3 with ATR (Thermo Scientific, Japan). Thermogravimetric measurements were carried out using the TGA, Q50 V20.10, Build 36 instrument at a heating rate of 10 °C/min under N₂ gas. The polymer morphologies were investigated using scanning electron microscopy (SEM, Tubney Woods, JEOL 7400F Oxford Instruments Inca, Abingdon, Oxon OX13 5QX, UK).
High Performance Liquid Chromatography (HPLC) was carried out on Waters 1525 Binary HPLC Pump with Photodiode Array Detector PAD (Waters 2998, USA). Data were analyzed using Breeze QS software with the X TERRA C18 column, 5 μm, 250 mm × 4.6 mm, at a flow rate of 1 m/min. The mobile phase was a combination of 36% distilled water and 64% acetonitrile.

Each replicate was weighed out and ground thoroughly in methanol:chloroform:water (1:1:0.7, v/v/v). The volume of the extraction solvent was adjusted according to weight of the sample (1 mL of solvent / 100 mg of sample). The upper aqueous phase was collected after centrifugation at 1000 g at 4 °C for 15 min and further evaporated using a speed vacuum concentrator (Labconco, MO, USA) at 40 °C until dry. The crude extracts were stored at − 80 °C prior to analysis. A total of 600 μl buffer containing 100 mM sodium phosphate, pH 7.4 in D₂O, 0.1 mM 3-trimethysilypropionic acid (TSP) (Cambridge Isotype Laboratories, Tewksbury, MA, USA) as a chemical shift reference (δ¹H = 0 ppm) and optionally 0.2% NaN₃ was added to dissolve the sample. Mixture was sonicated using ultrasonicator (JeKen, China) for 10 min and filtered through 0.20 μm filter (Corning, USA) before centrifugation at 12,000 g at 4 °C for 5 min. An equal amount of 30 μl was aliquoted from all samples and pooled for the QC. Then, a total of 550 μl of supernatant was transferred into NMR tube for metabolic profiling. Proton NMR spectra were acquired using a 400 MHz NMR spectrometer (Bruker, USA) with CryoProbe Prodigy and Carr−Purcell−Meiboom−Gill (CPMG) pulse sequence [RD−90˚−(τ−180˚−τ)n−acquisition] was applied to analyse the samples at 310 K in 64 scans.

Unless otherwise noted, NMR experiments
were recorded on a Bruker Avance III 600 HD spectrometer operating
at 600.13 MHz for ¹H and 150.92 MHz for ¹³C,
equipped with 5 mm CryoProbe Prodigy and pulse gradient units, capable
of producing magnetic field pulsed gradients in the z-direction of 50 G cm^–1. The NMR measurements have
been done with standard BRUKER pulse sequences, in deuterium oxide
(D₂O), at 300 K and at pD 1.4. ¹H NMR experiments
were performed with water suppression using excitation sculpting with
gradients (Hwang and Shaka, 1995), the acquisition time of 1.36 s,
relaxation delay of 2 s, and 128 or 256 transients of the spectral
width of 10 000 Hz collected into 32 K time domain points.