UV was acquired on a UV/EV300 spectrometer (Thermo Scientific, Waltham, MA, USA), and IR spectra were obtained on a Nicolet iS20 instrument (Thermo Fisher Scientific, MA, USA). Optical rotation data were performed by a Jasco P-2000 polarimeter (Jasco, Hachiojishi, Tokyo, Japan). 1H and 13C NMR spectra were acquired on an Agilent 600 MHz spectrometer (Agilent Technologies, Santa Clara, CA, USA), with CDCl3 (δH 7.26 and δC 77.16) as the solvent and internal standard. HRESIMS data were collected with a Bruker micrOTOF-Q II mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany). A Shimadzu LC-16 series instrument (Shimadzu, Kyoto, Japan) was equipped with C-18 column (5 µm, 10 mm × 250 mm, YMC, Kyoto, Japan) and an SPD-M20A diode array detector (Shimadzu, Kyoto, Japan) for high-performance liquid chromatography (HPLC) analysis. For column chromatography, Silica gel 60 (200–300 mesh; Yantai, China) and octadecylsilyl (ODS) (50 µm, YMC, Kyoto, Japan) were used. A silica gel 60 F254 plate (Merck, Darmstadt, Germany) was used for analytical thin-layer chromatography.
Agilent 600 mhz spectrometer
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 600 MHz spectrometer is a high-performance nuclear magnetic resonance (NMR) instrument designed for analytical and research applications. It operates at a frequency of 600 MHz, providing precise and sensitive measurements of molecular structures and compositions.
Automatically generated - may contain errors
Lab products found in correlation
Nicolet is20 instrument, by Thermo Fisher Scientific (2 mentions)
Lc 16 series instrument, by Shimadzu (2 mentions)
Octadecylsilyl ods, by YMC (2 mentions)
Spd m20a diode array detector, by Shimadzu (2 mentions)
Microtof qii mass spectrometer, by Bruker (1 mentions)
Silica gel 60 f254 plate, by Merck Group (1 mentions)
C18 column, by YMC (1 mentions)
P 2000 polarimeter, by Jasco (1 mentions)
Microtof q 2 mass spectrometer, by Bruker (1 mentions)
4 mm ghx nanoprobe, by Agilent Technologies (1 mentions)
Simca p 12, by Sartorius (1 mentions)
600 mhz library database, by Chenomx (1 mentions)
Q exactive hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Onenmr probe, by Agilent Technologies (1 mentions)
Dionex ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Synapt esi quadrupole tof mass spectrometry system, by Waters Corporation (1 mentions)
Acquity ultra performance liquid chromatography system, by Waters Corporation (1 mentions)
Hitrap columns for ni nta affinity chromatography, by GE Healthcare (1 mentions)
Ultraflextreme maldi tof tof mass spectrometer, by Bruker (1 mentions)
One probe, by Agilent Technologies (1 mentions)
8 protocols using agilent 600 mhz spectrometer
1
Spectroscopic Analysis of Organic Compounds
2
NMR Characterization of Peptide-Zn(II) Complex
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All spectra were measured with a DDR2 Agilent 600 MHz spectrometer equipped with a TRIAX probe. Assignment of 1H and 13C signals (Table S2, Supporting Information) was based on the previously described assignment of a peptide complexed with ZnII.33 2D homonuclear TOCSY88 (mixing time 65 ms), NOESY89 (mixing time 150 ms), and heteronuclear 1H‐13C HSQC90 spectra recorded at 25 °C were used to confirm the assignment (relevant parameters of the NMR spectra are given in Table S3, Supporting Information). All chemical shifts in the 1H NMR spectra are reported with respect to external sodium trimethylsilylpropanesulfonate ([D4]DSS). Chemical shifts of the 13C spectra were referenced indirectly by using the 0.251449530 frequency ratios for 13C/1H.91 Processed spectra were analyzed with SPARKY software.92
3
Spectroscopic Characterization of Organic Compounds
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The UV spectrum was obtained using SPD-M20A diode array detector (Shimadzu, Kyoto, Japan). Optical rotation data were tested on an Autopol IV polarimeter (Rudolph, Hackettstown, NJ, USA). The FT-IR spectra were recorded using a Nicolet iS20 instrument (Thermo Fisher Scientific, Waltham, MA, USA), with a resolution of 4 cm−1 and 32 scans in % transmittance mode. A KBr-disk was used for preparing the samples. 1H and 13C NMR spectra were recorded in CDCl3 containing Me4Si as internal standard on Agilent 600 MHz spectrometer (Agilent Technologies, Santa Clara, CA, USA). HRESIMS was performed with a Bruker micrOTOF-Q II mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany). HPLC was carried out on a Shimadzu LC-16 series instrument (Shimadzu, Kyoto, Japan), equipped with C-18 column (5 µm, 10 mm × 250 mm, SilGreen, Beijing, China) and an SPD-M20A diode array detector (Shimadzu, Kyoto, Japan). As for column chromatography, silica gel 60 (200–300 mesh; Yantai Jiangyou Silica Gel Development Co. LTD, Yantai, China) and octadecylsilyl (ODS) (50 μm, YMC, Kyoto, Japan) were used.
4
High-Resolution NMR Metabolic Profiling
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The high-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) spectra were recorded using an Agilent 600 MHz spectrometer that was equipped with a 4 mm gHX NanoProbe (Agilent Technologies, Santa Clara, CA, USA). All spectra were acquired at 600.167 MHz. The acquisition time was 1.703 s, relaxation delay was 1 s, and a total of 128 scans was obtained. The Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence was used for the suppression of water and compounds with high molecular mass. For data processing, Chenomx NMR Suit 7.1 professional with the Chenomx 600 MHz library database were used (Chenomx Inc., Edmonton, AB, Canada). The bin size for spectra was 0.001 ppm. The binning data were normalized to the total area. PCA, partial least-squares discriminant analysis (PLS-DA), and orthogonal partial least-squares discriminant analysis (OPLS-DA) were performed using SIMCA-P+ 12.0 (Umetrics, Malmö, Sweden). For visualization of VIP scores of metabolites and Metabolic Set Enrichment Analysis, web-based software MetaboAnalyst 3.0 (http://www.metaboanalyst.ca ) was used.
5
NMR and Mass Spectrometry Analysis of O-PnAS
6
Characterization of Natural Compounds
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UV measurements were obtained by the extraction of the Diode Array Detector (DAD) signal of the Ultra-High Pressure Liquid Chromatography (UHPLC) Dionex Ultimate 3000 (Thermo Scientific, Waltham, MA, USA). Optical rotation measurements were obtained at the sodium D line (589.3 nm) with a 10-cm cell at 20 °C on a UniPol L1000 polarimeter (Schmidt + Haensch, Berlin, Germany). NMR spectra were acquired on an Agilent 600 MHz spectrometer equipped with a cryoprobe with pulse field gradient, and signals were referenced in ppm to the residual solvent signals (CD3OD, at δH 3.31 and δC 49.0). HRESIMS data were obtained with a UHR-qTOF Agilent 6540 mass spectrometer. Purification was carried out on a JASCO HPLC equipped with a PU4087 pump and a UV4070 UV/Vis detector.
7
Heterologous Protein Production and Purification
8
NMR Analysis of Bean Cotyledon
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For NMR analysis, the bean coat was manually separated from the cotyledon. The cotyledon were powdered, and approximately 30.0 mg were soaked in a mixture of 600 μL of D 2 O and 1 mM of TMSP-d 4 (sodium-3-trimethylsilylpropionate-2,2,3,3-d 4 ). The solutions were mixed for 2 min at room temperature and centrifuged at 804.6 g for 2 min. The supernatants were transferred to 5 mm NMR tubes. The NMR experiments were performed on an Agilent 600-MHz spectrometer equipped with a 5 mm (H-F/ 15 N-31 P) inverse detection One Probe™ with actively shielded Z-gradient. The 1 H NMR spectra were acquired with 32 free induction decays (FID), 48 k of time domain points for a spectral window of 16 ppm, under quantitative parameters at 298 K: acquisition time of 5.0 s and a recycling delay of 25.0 s (determined by the inversion-recovery pulse sequence). The spectra were processed by applying exponential line broadening of 0.3 Hz, zero filling of 32 k points, and referenced to the TMSP-d 4 resonance at δ 0.0. The identification of the constituents was performed through 1 H-1 H COSY, 1 H-13 C HSQC, and 1 H-13 C HMBC experiments, supplementary open access database (http://www.hmdb.ca) and literature reports (Alves Filho, Silva, Teofilo, Larsen, & de Brito, 2017; (link)Choze et al., 2013; (link)Lião et al., 2011) . Complete signaling is provided in Supporting Information (SI).
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