Cryogenic probe

Virstatin was solubilized in DMSO-d₆ and compounds 5a and 3b in DMSO, at working stock concentrations of 35.5 mM, 394 mM, and 50.5 mM, respectively. Purified ToxT in protein buffer (20 mM Tris, 1 mM EDTA, 320 mM NaCl, pH 7.5) was used at a final concentration of 20 μM. All samples contained 50 μM 3-(Trimethylsilyl)-1-propanesulfonic acid sodium salt (TSP) as an internal standard and 5% D₂O. All NMR experiments were carried out on a Bruker Avance 600 MHz or 700 MHz spectrometer equipped with a TCI cryogenic probe. Samples were stored at 4 °C prior to acquisition.

¹⁵N spin-lattice (R₁), transverse (R₂) relaxation, and ¹H-¹⁵N NOE experiments for Xt3a were recorded at 25 °C on a 900 MHz spectrometer equipped with cryogenic probe (Bruker, Billerica, MA). The sample contained 500 μM of ¹⁵N/¹³C labeled Xt3a (as above). The relaxation delay was sampled at 10, 20, 60, 100, 200, 400, 600 and 1200 ms for the R₁ experiments and 16, 33, 67, 135, 169, 203, 237 and 271 ms for the R₂ measurements. Spectra were processed using Topspin (v4.1.3, Bruker) and the signal decay was analysed and plotted using CcpNmr (v2.4.1). The time constant error (TC in CcpNmr) from the exponential fit is used in the the R₁ and the R₂ plots. The noise in the spectrum (relative to the peak height) was used to estimate the uncertainty (error bars) in the heteronuclear NOE plots.

Conducted as described before [29 (link)]. Briefly: liver tissues (about 50 mg) were homogenized in cold methanol and water (v/v = 2:1) using a Qiagen TissueLyser (Retsch GmBH, Germany). All plasma NMR spectra were acquired at 298 K on a Bruker Avance III 600 MHz NMR spectrometer (600.13 MHz for ¹H frequency) equipped with a cryogenic probe (Bruker Biospin, Germany). One-dimensional ¹H NMR spectra were acquired with the Carr–Purcell–Meiboom–Gill pulse train [35 (link)]. The NMR spectroscopic analysis, NMR data processing, and multivariate data analysis were performed as previously described [36 (link)]. The cutoff value (|r| > 0.602) was based on the significance threshold value of P < 0.05, which was determined according to the test for significance of the Pearson’s product-moment correlation coefficient (n = 10, P < 0.05).

Nuclear Magnetic Resonance (NMR) analysis was carried out to provide detailed structural information about the EPS [34 (link)]. The NMR analyses were performed on a Bruker 600 MHz equipped with a cryogenic probe, and spectra were recorded at 298 K. Acetone was used as internal standard (1 H 2.225 ppm, 13 C 31.45 ppm) and 2D spectra (1 H–1 H DQF-COSY, 1 H–1 H NOESY, 1 H–1 H TOCSY, 1–13 C HSQC and 1–13 C HMBC) were acquired by using Bruker software (TopSpin 2.0). 31P NMR experiments were carried out with a Bruker DRX-400 spectrometer; aqueous 85% phosphoric acid was used as an external reference (δ = 0.00 ppm). Homonuclear experiments were recorded using 512 FIDs of 2048 complex with 32 scans per FID; mixing times of 100 and 200 ms were used for TOCSY and NOESY spectra acquisition, respectively. HSQC and HMBC spectra were acquired with 512 FIDs of 2048 complex points, accumulating 40 and 80 scans, respectively. Spectra were processed and analyzed using a Bruker TopSpin 3 program.

Freeze-dried intracellular metabolites extracts were dissolved in 600 μl phosphate buffer 0.1 m (pH 7.4, 99.9% D₂O) containing 0.001% sodium 3-trimethylsilyl-1-[2,2,3,3-²H4] propionate as previously described [48 (link)]. All samples were centrifuged (12 000 g/4 °C, 10 min) after short vortexing and supernatants transferred into the 5 mm NMR tubes for NMR detection. All one-dimensional ¹H-NMR spectra were acquired on a Bruker AVIII 600 MHz NMR spectrometer equipped with a cryogenic probe (BrukerBiospin, Rheinstetten, Germany) at 298 K. The first increment of NOESY pulse sequence was employed with continuous wave irradiation on the water peak during recycle delay and mixing time for water suppression. Recycle delay of 2 s and mixing time of 100 ms were set. The 90° pulse was adjusted to 10 μs approximately and 64 scans were collected into 32 k data points with the spectral width of 20 p.p.m. For metabolite assignments, two-dimensional NMR spectra including ¹H–¹H COSY, ¹H–¹H TOCSY, ¹H J-resolved, ¹H–¹³C HSQC and ¹H–¹³C HMBC for typical samples were acquired and processed as described previously [49 (link)].