Avance 2 500 mhz spectrometer

For ethanol extractions, the Na₂-β-glycerophosphate in the CDM growth medium was replaced by 15.4 g l^-1 MES (2-(N-morpholino) ethanesulfonic acid), to avoid the intense buffer resonance in the phosphomonoester (PME) region. The ethanol extracts were prepared as described previously by Carvalho et al. [9 (link)]. Cells were harvested (20,980 x g, 4ºC, 5 min) during exponential growth and the pellet suspended in milliQ water, pH 6.5. Cell suspensions were transferred to the appropriate volume of cold ethanol 70% (vol/vol) in an ice bath, and extraction was performed for 30 min with vigorous agitation. Cell debris was removed by centrifugation (39,191 x g, 4ºC, 20 min). The ethanol in the supernatant was removed via a rotavap and the extract frozen in liquid nitrogen, and lyophilized overnight. The dried extract was dissolved in 1 ml of deuterated water containing 5 mM EDTA. The pH was set to 6.5 and the extract was stored at -20ºC until analysis by ³¹P-NMR. Resonances were assigned by addition of pure compounds to the extracts or on basis of comparison with previous studies [9 (link)]. ³¹P-NMR spectra were recorded using a selective probe head (³¹P-SEX) at 30ºC on a Bruker AVANCE II 500 MHz spectrometer (Bruker BioSpin GmbH) by using standard Bruker pulse programs. Spectra were referenced to the resonance of external 85% H₃PO₄, designated at 0 ppm.

¹H NMR (500 MHz) and ¹³C NMR (126 MHz) were measured on a Bruker Avance II 500 MHz spectrometer using chloroform (CDCl₃) and methanol (CD₃OD) as solvents. HREIMS was carried out using Thermo Scientific Q Exactive (Waltham, USA). UV-visible spectra were obtained using UV-2600 spectrophotometer (Shimadzu, Japan) scanning from 240 to 450 nm. Melting points were determined using a X-4 digital display melting point apparatus (Shanghai, China) and are uncorrected.

Cells were grown on galactose-CDM, glucose-CDM and in the nasal synthetic medium to an OD₆₀₀ = 0.4 and treated with 250 μM spermine NONOate (or left untreated, the control sample), and harvested 1 and 3 h later. After centrifugation (11800 ×g for 2 min) and filtering (0.22 μm Pall filters), the supernatants were stored at -20°C and later used for the ¹H-NMR studies. Extracellular metabolites were quantified on a Bruker AVANCE II500 MHz spectrometer (Bruker BioSpin GmbH) operated by TOPSPIN software using a 5 mm BBIXYZ high resolution probe head, at 16°C, and standard Bruker pulse programs. Spectra were referenced to the resonance of externally added trimethylsilyl propionate (TSP), designated at 0 ppm.
Resonances were assigned by addition to the supernatants of pure compounds and comparison with data available in the literature (Carvalho et al., 2011 (link)). Concentrations were calculated from the areas of the resonances of the ¹H-NMR spectra considering the TSP resonance area, and using an appropriate resonance saturation correction factor (Carvalho et al., 2011 (link)). The metabolites concentration was normalized to the OD₆₀₀, measured for each condition.

The compounds ES1, ES2 and ESR35 were synthesized as previously described (McKibbin et al., 2012 (link)).ES24 and ES47 were prepared in good yields by condensation of acyl hydrazides 1 and 2 with the appropriate conjugated aldehyde. Phenylacetic hydrazide 1 is commercially available and the synthesis of 2 (ESR35) has been reported previously (Cross et al., 2009a (link)). The double bond geometry in both compounds was confirmed by the magnitude of the corresponding vicinal ¹H-¹H coupling constant (15.9 Hz for ES24 and 14.8 Hz for ES47).
General experimental synthetic reagents and solvents were purchased from Sigma-Aldrich or Alfa Aesar and used as supplied. IR spectra were recorded on a Perkin Elmer 881 spectrometer, an AT1-Matson Genesis Series FTIR spectrometer or a Perkin Elmer Spectrum BX FTIR spectrometer. ¹H NMR spectra were recorded on a Bruker Avance II 500MHz spectrometer. Chemical shifts are referenced to the residual solvent peak. Mass spectra were recorded on a Micromass Platform II (electrospray) spectrometer. Melting points were recorded using a Sanyo Gallenkamp MPD350 heater and are uncorrected.

All NMR experiments were performed on a Bruker Avance III 700 MHz spectrometer equipped with a TCI inverse cryoprobe and on a Bruker Avance II 500 MHz spectrometer equipped with a 5 mm ¹³C(¹H) dual cryoprobe at 298 K and data analyzed using Bruker Topspin 3.2. All spectra were processed with a Lorentzian line broadening of 0.3 Hz. The solutions were buffered in 50 mm Tris‐D₁₁⋅HCl pH 7.5, in 90:10 H₂O/D₂O. Bruker MATCH 3 mm diameter and 5 mm NMR tubes, with total sample volumes of 160 μL and 500 μL, respectively, were used. For the psBBOX‐catalyzed substrate‐turnover experiments, the assay mixture was incubated in an Eppendorf tube and whenever necessary the reaction was quenched (stopped) with the addition of 1 m HCl (5 μL) and the spectrum was recorded for analysis. To measure the ligand binding constant by psBBOX titration, separate samples were prepared. The PROJECT‐CPMG pulse sequence (90°_x–[τ–180°_y–τ–90°_y–τ–180°_y–τ] n–acquisition), as described by Aguilar et al.,27 was used to remove the broad resonances of the protein. The relaxation edited (CPMG) ¹H NMR experiments were recorded with a total filter time of 32 ms. Protein titration data were fitted using OriginPro 9.0 (Origin lab, Northampton, MA, USA) to calculate the ligand binding constant (K_D). Water suppression was achieved by presaturation.

¹H NMR spectra were recorded in methanol-d₄ on a spectrometer (Bruker Avance II 500 MHz spectrometer, Karlsruhe, Germany) using tetramethylsilane as an internal standard (IS), and the chemical shifts are given in δ (ppm). Additionally, the ¹H NMR and ¹³C NMR spectra were recorded in dimethyl sulfoxide (DMSO)-d₆ on a spectrometer (Bruker Avance III HD 400 MHz spectrometer, Karlsruhe, Germany) (Tables S1 and S2, Figures S1 and S2). The thermal transition properties were investigated using a differential scanning calorimeter (TA DSC Q20, New Castle, DE, USA). Each sample was transferred to a sealed aluminum pan and heated at a rate of 10 °C/min from 40 °C to 250 °C under nitrogen purging. The chemical structures were also evaluated using Fourier transform infrared spectroscopy (Nicolet iS10 FT-IR Spectrometer, Madison, WI, USA). Each spectrum was obtained over the wavelength range of 4000–450 cm⁻¹ with 32 scans at a resolution of 4 cm⁻¹.