Avance 2 600 mhz

¹H and ¹³C spectra were obtained using the Bruker Avance-II ±600 MHz spectrometer. The ¹H and ¹³C NMR chemical shifts are presented relative to TMS. Chemical shifts are expressed in ppm while the coupling constants are in Hz. The ESI/MS analyses were conducted using the Thermo Finnigan LCQ advantage ion trap mass spectrometer. The purity of the products and the progress of the reaction were checked by TLC on precoated plates of Silica gel 60 F254 (Merck). Spots on the TLC chromatograms were detected by the chlorine/o-tolidine reaction. The melting points were determined using the Kofler apparatus and are uncorrected.

Urine samples (550 μL) were mixed with 55 μL of phosphate buffer (1.5 M NaH₂PO₄/K₂HPO₄; pH 7.4; 100% v/v D₂O) with 0.1% NaN₃ as bacterial growth inhibitor and 5.0 mM 2,2-dimethyl-2-silapentane-5-sulfonate-d₆ (DSS) as chemical shift reference (δ 0.00 ppm). After centrifugation at 4 °C and 12,000 rpm for 10 min, the supernatant was transferred into 5 mm NMR tubes for subsequent NMR analysis. The proton NMR spectra of the urine samples were acquired at 300 K with a Bruker Avance II 600 MHz spectrometer (Bruker Biospin, Rheinstetten, Germany; operating at 600.13 MHz for ¹H) equipped with a broadband-observe probe. A standard water-suppressed one-dimensional NMR spectrum was derived from urine by using the first increment of the gradient-selected NOESY pulse sequence (recycle delay–90°–t₁–90°–t_m–90°–acquire data) with a recycle delay of 2 s, t₁ of 3 μs, mixing time (t_m) of 100 ms, and 90° pulse length of 13.70 μs. A total of 128 transients were collected into 49,178 data points by using a spectral width of 9590 Hz and an acquisition time of 2.56 s. Metabolites were assigned based on chemical shifts, coupling constants, and relative intensities as described in previous reports [38 (link),39 (link),40 (link)] and additional ¹H-¹H correlation spectroscopy and ¹H-¹H total correlation spectroscopy were recorded for selected samples (data not shown).

Serum samples were immediately stored at −80°C after collection. At the time of NMR analysis, samples were thawed on ice. 300 μL of 10% D₂O buffer (5 mM TSP, 140 mM Na₂HPO₄, 0.04% NaN₃, pH 7.4) were added to 300 μL of serum. ¹H-NMR spectra were acquired using a Bruker Avance II 600 MHz spectrometer equipped with triple resonance cryo-probe with a cooled ¹³C preamplifier (TCI) at 310 K (37°C) [49 , 50 (link)]. Metabolites of interest were identified using Amix v 3.9.7 in combination with the Bruker NMR Metabolic Profiling Database BBIOREFCODE 2.0.0 database (Bruker Biospin, Rheinstetten, Germany), as well as other existing public databases and literature reports [12 (link), 22 (link), 51 ]. NMR experiments for each set were independently acquired at two different times.

Reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Merck (Darmstadt, Germany). The commercially available (±)-naringenin 5 (Sigma-Aldrich) was used. The purity of the synthesized products (>95%) was established by HPLC chromatography. Analysis were performed according to the procedure described earlier [33 (link)]. The NMR spectra (¹H-, ¹³C-NMR, ¹³C-DEPT, and correlation spectra: ¹H-¹H-COSY, ¹H-¹³C-HMQC, ¹H-¹³C-HMBC) were recorded in a CDCl₃ solution on a Brüker Avance DRX 300 and Brüker Avance II 600 MHz spectrometers (Brüker, Karlsruhe, Germany). IR spectra (KBr discs) were determined on a Thermo-Nicolet IR300 FT-IR-spectrometer (Madison, WI, USA). UV spectra were run on a Visible Spectrofotometer Cintra 303, GBC, in methanol. Melting points (uncorrected) were determined on a Boetius apparatus. Bioscreen C (Lab system Oy, Helsinki, Finland) has been used in the initial part of the investigations of antimicrobial activity.

The experiments are conducted using 200 μL cell and 550 μL media samples in 103.5 mm x 3 mm and 103.5 mm x 5 mm NMR tubes (Bruker). All the data collection and processing were performed using Topspin 3.6 software (Bruker Analytik, Rheinstetten, Germany). One-dimensional ¹H NMR spectra are acquired on a Bruker Avance II 600 MHz spectrometer using Prodigy BBO cryoprobe at 298 K using the noesygppr1d pulse sequence (Ref1). For a representative sample, two dimensional data ¹H-¹H total correlation spectroscopy (TOCSY, mlevphpr.2) and 2D ¹H-¹³C heteronuclear single quantum coherence (HSQC, hsqcedetgpsisp2.2) were collected for metabolites assignment.

NMR spectra were collected on Bruker Avance II 600 MHz and Bruker Avance III 850 MHz spectrometers (Bruker BioSpin, Rheinstetten, Germany) at 298 K. For backbone assignment, ¹⁵N-,¹³C-labeled DUSP22_155WT was prepared at a final concentration of 1 mM in NMR buffer (40 mM PIPES (pH 7.0), 100 mM NaCl, 10% D₂O (v/v) and 0.16 mM DSS). The collected spectra included HSQC, HNCA, HN(CO)CA, HNCO, HNCACB, and HN(CO)CACB. The assignment of D57N was related to the WT spectra and confirmed by backbone assignment of the Cα spectra (HNCA, HN(CO)CA) as well as ligand titration. For comparison of the mutants, ¹⁵N-labeled proteins were prepared at the concentration of 0.1–0.2 mM to collect ¹H,¹⁵N-heteronuclear single quantum coherence (HSQC) spectra. All spectra were processed by NMRpipe [39 (link)] and analyzed by Sparky [40 (link)]. The CSP values were calculated by the following formula: $CSP \left(ppm\right)=\sqrt{\frac{1}{2}\left({\delta }_{HN}^2+\frac{{\delta }_N^2}{25}\right)}$
The threshold of CSPs was set at two standard deviations above the mean (∆δ + 2σ), which was calculated from 10% trimmed data [30 (link),41 (link)], and the residues with the CSP values above the threshold were labeled on the structure.