Analytical RP-HPLC was carried out on a Thermo Scientific instrument with a YMC C18 column (5 μm, 4.6 × 250 mm). The column was eluted with a linear gradient of 10–50 % acetonitrile with 0.1 % formic acid (v/v) for 30 min at a flow rate of 1 ml/min under UV 266 nm. The retention time of sulfated N-glycans (2 , 3 , 5 ) were 23.5 min, 22.6 min, and 22.2 min, respectively. Reverse phase preparative HPLC was executed on a Waters 600 HPLC instrument with a SymmetryPrep C18 column (7 μm, 19 × 300 mm). The column was eluted with a linear gradient of 20–60 % acetonitrile with 0.1 % formic acid (v/v) for 30 min at a flow rate of 10 ml/min under UV 266 nm.
Symmetryprep c18 column
Manufactured by Waters Corporation
Sourced in United States, Germany
The SymmetryPrep C18 column is a reverse-phase high-performance liquid chromatography (HPLC) column designed for the separation and purification of a wide range of organic compounds. It features a silica-based sorbent with a chemically bonded C18 alkyl chain, which provides a hydrophobic stationary phase for the retention and separation of analytes.
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Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
P 1020 polarimeter, by Jasco (2 mentions)
600 hplc instrument, by Waters Corporation (1 mentions)
1100 series system, by Agilent Technologies (1 mentions)
Cortecs c18 column, by Waters Corporation (1 mentions)
Infinity 2 lc system, by Agilent Technologies (1 mentions)
Xevo g2 xs qtof, by Waters Corporation (1 mentions)
Symmetry c18 column, by Waters Corporation (1 mentions)
Nanodrop instrument, by Thermo Fisher Scientific (1 mentions)
Avance 600 spectrometer, by Bruker (1 mentions)
Avance 500 spectrometer, by Bruker (1 mentions)
6545 q tof mass spectrometer, by Agilent Technologies (1 mentions)
High performance liquid chromatography (hplc), by Merck Group (1 mentions)
Agilent 1100 hplc, by Agilent Technologies (1 mentions)
Avance 500, by Bruker (1 mentions)
Uhplc system, by Waters Corporation (1 mentions)
Dpx 400, by Bruker (1 mentions)
Uv 1800 spectrophotometer, by Jasco (1 mentions)
Ft ir 4600 typea spectrometer, by Jasco (1 mentions)
Gcms qp2010 se, by Shimadzu (1 mentions)
11 protocols using symmetryprep c18 column
1
Analytical and Preparative RP-HPLC of Sulfated N-Glycans
2
Analytical Characterization of Chemical Compounds
3
NMR and Mass Spectrometry Analysis
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NMR spectra were measured on Bruker Avance 500 or 600 MHz spectrometers equipped with [2H, 15N, 13C, 1H], z-gradient cryoprobes. Chemical shifts (δ) were reported in parts per million (ppm) relative to the chemical shift of the solvent. LR-ESI-MS was carried out on an Agilent 1100 HPLC instrument and an Agilent 6310 Quadrupole mass spectrometer (Santa Clara, CA, USA). HR-ESI-MS was carried out on a Waters Time-of-Flight (TOF, Milford, MA, USA) mass spectrometer (Milford MA, USA) model LCT Premier or Agilent 6545 Q-TOF mass spectrometer. Semi-preparative HPLC was performed on an Agilent 1100 HPLC equipped with a SymmetryPrep C18 column (Waters, 7 μm, 7.8 × 300 mm) or Phenomenex kintex biphenyl column (5 uM, 100 Å, 10 × 250 mm). All solvents used for HPLC were of HPLC grade (Sigma Aldrich, St. Louis, MO, USA).
4
Synthesis and Characterization of 19-Eicosynoic Acid
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All chemical reagents and anhydrous solvents for synthesis were purchased from commercial suppliers (Sigma-Aldrich, Fluka, Acros) and were used without further purification or distillation. 19-eicosynoic acid was custom synthesized by Spirochrome AG. The composition of mixed solvents is given by the volume ratio (v/v). 1H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DPX 400 (400 MHz for 1H) with chemical shifts (δ) reported in ppm relative to the solvent residual signals (7.26 ppm for of CDCl3; 3.31 ppm for MeOD). Coupling constants are reported in Hz. LC-MS was performed on a Shimadzu MS2020 connected to a Nexerra UHPLC system equipped with a Waters ACQUITY UPLC BEH Phenyl 1.7 µm 2.1 x 50mm column. Buffer A: 0.05% HCOOH in H2O Buffer B: 0.05% HCOOH in acetonitrile. LC gradient: 10% to 90% B within 6.0 min with 0.5 ml/min flow. Unless otherwise stated, preparative HPLC was performed on a Dionex system equipped with an UltiMate 3000 diode array detector for product visualization on a Waters SymmetryPrep C18 column (7 µm, 7.8 × 300 mm). Buffer A: 0.1% v/v TFA in H2O; Buffer B: acetonitrile. Gradient was from 25% to 90% B within 30 min with 3 ml/min flow.
5
Analytical Techniques for Chemical Analysis
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UV/Vis spectra were measured with a Shimadzu UV-1800 spectrophotometer, IR spectra with a Jasco FT-IR-4600 type A spectrometer, and optical rotations with a Jasco P-1020 polarimeter. ECD spectra were obtained on a J-715 spectropolarimeter (Jasco) at room temperature, using a standard cell (0.02 cm) and spectrophotometric-grade methanol, and are reported in Δε values (cm2 mol−1) at the given wavelength λ (nm). GC-MSD analyses were performed on a GCMS-QP 2010SE (Shimadzu). 1D and 2D NMR spectra were recorded on Bruker Avance III HD 400 (400 MHz) and 600 (600 MHz) instruments in deuterated methanol. Chemical shifts (δ) are reported in parts per million (ppm) with the 1H and 13C signals of the solvent (1H, δ = 3.31 ppm; 13C, δ = 49.15 ppm) as the internal reference. HRESIMS spectra were obtained on a microTOF-focus and micrOTOF-Q III mass spectrometers (Bruker). Preparative HPLC separation was performed on a Jasco HPLC system (PU-2087, UV-2077, LC-NetII/ADC), using a SymmetryPrep C18 column (Waters, 19 × 300 mm, 7 μm) with the UV absorption wavelengths set at 232, 254, and 310 nm. Organic solvents were analytical grade or distilled prior to use.
6
Peptide Synthesis and Purification
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PG-1 was kindly provided by Prof. R. Lehrer (University of California, Los Angeles, CA, USA); it was synthesized by SynPep Corporation (Dublin, CA, USA) with assessed purity of 99%. ChBac3.4 and RFR-ChBac3.4(1–14) were produced as previously described [47 (link)] according to a standard solid-phase peptide synthesis protocol utilizing Fmoc/tBu protecting groups scheme on a Symphony X peptide synthesizer (Protein Technologies, Tucson, AZ, USA). Trifluoroacetic acid (TFA) cleavage cocktail (TFA/triisopropylsilane/water/ethandithiol = 94/1/2.5/2.5) was used for the final deprotection and cleavage of the assembled linear peptides from the 2-chlorotrityl chloride resin. Samples were purified via semi-preparative RP-HPLC using a Waters SymmetryPrep C18 column, 9 × 300 mm, 100Å, 7µm, and then verified for a purity of no less than 96% by analytical RP-HPLC using Luna C18 column, 4.6 × 250 mm, 100 Å, 5 µm on a Gilson chromatograph. The molecular weight of the peptides was checked to be as expected by MALDI-TOF mass spectrometry with alpha-cyano-4-hydroxycinnamic acid as a matrix.
7
Radiolabeling of Tributylstannane Precursor
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Synthesis of tributylstannane precursor ICF15060 is detailed in supplementary data. To a solution of ICF15060 (50 μl, 1 mg/ml in EtOH) were successively added, in a sealed vial, [131I]NaI (1517 MBq) or [125I]NaI (304 MBq), an aqueous HCl solution (50 μl, 1 N for 131I-radiolabeling or 0.5 N for 125I-radiolabeling), and an aqueous solution of Chloramine-T monohydrate (20 μl, 1 mg/ml). The reaction mixture was left at room temperature for 1 hour (vortexed every 20 minutes). The resulting solution was quenched by addition of 1 N aqueous NaOH solution (100 μl for 131I-radiolabeling or 25 μl for 125I-radiolabeling) and aqueous sodium metabisulfite solution (10 μl, 20 mg/ml). The mixture was purified by semipreparative HPLC [Waters Symmetry Prep C18 column (300 × 7.8 mm, porosity 7 μm); MeOH/H2O (0.2% NH4OH) 70:30 to 100:0 v/v linear gradient in 15 minutes then 70:30 v/v isocratic during 15 minutes] at a flow rate of 1.2 ml/min. The product peak was collected (retention time 20.04 minutes) and evaporated to dryness. The formulation was achieved in EtOH/saline 05:95 v/v.
8
Isolation and Purification of Bioactive Compounds
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The ferment culture was extracted with EtOAc (three times, each 6 l) and vacuum-dried to afford the crude extract (~10 g). The extract was fractionated by silica gel vacuum liquid chromatography (VLC) in petroleum ether-Acetone-MeOH gradient elution (Supplementary Figure S1). The fraction (2 g) eluted with 10% MeOH was then loaded on a silica gel column (2.5 × 45 cm) eluted with CH 2 Cl 2 -MeOH. The fraction (538 mg) eluted from 20:1 CH 2 Cl 2 :MeOH was isolated by Sephadex LH-20 column chromatography then eluted with MeOH, the subfractions were selected and purified by RP HPLC (Waters Symmetry Prep C 18 column; 7 μm; 7.8 × 300 mm; 60-70% MeOH in H 2 O with 0.1% HCOOH for 45 min; 2.2 ml min -1 ) to afford 1 (10.0 mg, t R 19 min) and 2 (6.0 mg, t R 17.00 min) (Supplementary Figure S2). The fraction (368 mg) eluted from 10:1 CH 2 Cl 2 :MeOH was isolated by Sephadex LH-20 column chromatography eluted in MeOH. The subfractions were purified by RP HPLC (Waters Symmetry Prep C 18 column; 7 μm; 7.8 × 300 mm; 40-60% MeOH in H 2 O with 0.1% HCOOH for 50 min; 2.2 ml min -1 ) to afford 3 (10.0 mg, t R 27.20 min), 4 (8.0 mg, t R 20.50 min) and 5 (6.0 mg, t R 23.00 min) (Supplementary Figure S2).
9
Antioxidant and ACE-Inhibitory Fractions
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Fractions obtained from SEC of AHP and RJ were re-dissolved in 130 μL and 100 μL bidistilled water, respectively, and 20 μL was injected into an Agilent 1100 HPLC system (Agilent Technologies) using a Symmetry Prep™ C18 column (7.8 × 300 mm, 7μm) from Waters (Milford, MA). Solvent A was 0.1% TFA in bidistilled water and solvent B consisted of acetonitrile/ bidistilled water (60:40, v/v) containing 0.085% of TFA. The mobile phases were filtered through a 0.45μm filter and degassed. The elution was 100% solvent A for 2 min, followed by a linear gradient from 0% to 50% of solvent B in 50 min at a flow rate of 3.5 mL/min. Collected fractions (1 mL) were monitored at 214 nm and assayed for antioxidant and ACE-inhibitory activities for AHP. Fractions showing remarkable activities were freeze dried and further analysed.
10
Purification of ACE-Inhibitory Peptides
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The active fractions separated by the above method were further purified by a Symmetry PrepTM C18 column (7 μm, 7.8 mm × 300 mm, Waters corporation, Milford, MA, USA) equipped with an Agilent series 1100 HPLC instrument (Agilent, California, CA, USA,) with flow rate of 2.0 mL·min−1. Peptides were eluted and monitored at 220 nm using the mobile phase composed of solution A (water containing 0.1‰, TFA, v/v) and solution B (acetonitrile) with a linear gradient of B from 5% to 15% for 30 min, and then the gradient was from 15% to 30% B within 25 min. Major peaks were collected, lyophilized and tested for ACE-inhibitory activities.
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