All chemical reagents and organic solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA), FUJIFILM Wako Pure Chem. (Osaka, Japan), or Nacalai Tesque (Kyoto, Japan) and used without further purification. The authentic compound 4 was synthesized according to the procedure reported previously.19 (link) The commercially-available compound 20 (MNI-137) was purchased from Nacalai Tesque. 11C was produced using a cyclotron (CYPRIS HM-18; Sumitomo Heavy Industries, Tokyo, Japan). 1H NMR (300 MHz) spectra were recorded on a JEOL-AL-300 spectrometer (JEOL, Tokyo, Japan) using tetramethylsilane (TMS) as an internal standard. All chemical shifts (δ) are reported as the ppm downfield relative to the TMS signal. Signals are quoted as s (singlet), d (doublet), t (triplet), br (broad), or m (multiplet). High-resolution fast atom bombardment mass spectra (HRMS) were acquired using a JEOL NMS-SX102 102A spectrometer. Silica gel column chromatography was performed using Wakosil C-200 (FUJIFILM Wako Pure Chem.). HPLC separation and analysis were performed using a JASCO HPLC system (JASCO, Tokyo, Japan). All semi-preparative HPLC separations were performed using CAPCELL PAK C18 columns (10 mm ID × 250 mm; Shiseido, Tokyo, Japan). All HPLC analyses were performed by using CAPCELL PAK C18 columns (4.6 mm ID × 250 mm; Shiseido) to determine the chemical purities (>98%) of 5a –5i .
Capcell pak c18 column
Manufactured by Shiseido
Sourced in Japan, United States, Germany
The Capcell Pak C18 column is a reversed-phase high-performance liquid chromatography (HPLC) column. It is designed for the separation and analysis of a variety of compounds, including small molecules, peptides, and proteins. The column features a silica-based packing material with chemically-bonded C18 alkyl chains, providing a hydrophobic stationary phase for the separation of analytes based on their polarity.
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Lab products found in correlation
Agilent 1260 infinity, by Agilent Technologies (3 mentions)
Sephadex lh 20, by GE Healthcare (3 mentions)
Smart 15, by Hanil (2 mentions)
Prominence hplc system, by Shimadzu (2 mentions)
Tcc 100, by Thermo Fisher Scientific (2 mentions)
Asi 100, by Thermo Fisher Scientific (2 mentions)
Agilent 1260 infinity lc system, by Agilent Technologies (2 mentions)
Gel ods c18 column, by YMC (2 mentions)
Vertex 70 spectrometer, by Bruker (2 mentions)
Agilent 1260 quat pump, by Shiseido (2 mentions)
Hplc system, by Jasco (1 mentions)
Pyruvate kinase from rabbit muscle, by Merck Group (1 mentions)
U 3000, by Hitachi (1 mentions)
Peptrex r48, by Peptron (1 mentions)
Lc ms, by Shimadzu (1 mentions)
Capcell pak adme column, by Shiseido (1 mentions)
Agilent 6120, by Agilent Technologies (1 mentions)
Jsm 5600, by JEOL (1 mentions)
3130 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
68 protocols using capcell pak c18 column
1
Synthesis and Characterization of Fluorine-18 Radiotracers
2
HPLC Analysis of Catecholamine Metabolites
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For analysis of β‐(carboxymethylthio)dopamine (CMT‐DA) and other catecholamine metabolites formed upon HCl hydrolysis, the mobile phase was 0.1 M sodium citrate buffer, pH 3.0, containing 1 mM sodium octanesulfonate and 0.1 mM EDTA.2Na (buffer A): methanol, 95 : 5 (v/v) (Wakamatsu et al. 2003 ). Analyses were performed at 35°C with an electrochemical detector set at +700 mV versus Ag/AgCl electrode. For the analysis of 5‐S‐Cys‐NE and 5‐S‐cysteinyl β‐(carboxymethylthio)dopamine (5‐S‐Cys‐CMT‐DA), we used a Capcell pak C18 column with 0.1 M potassium phosphate buffer, pH 2.1:methanol, 99 : 1 (v/v), at 25°C, with a UV‐VIS detector set at 290 nm. For the preparative separation of 5‐S‐Cys‐NE, we used a Capcell pak C18 column (type MG, 20 × 250 mm, 5 μm particle size; from Shiseido) attached with a Capcell pak C18 column (20 × 35 mm, 5 μm particle size; from Shiseido), with 0.1 M potassium phosphate buffer, pH 2.1:methanol, 99 : 1 (v/v), at 55°C, respectively, with a UV‐VIS detector set at 290 nm, at a flow rate of 7.0 mL/min. Determination of melanin markers was performed using HPLC as previously reported (Wakamatsu et al. 2002 , 2003 ; Ito et al. 2011 ).
3
Kinetic Characterization of Nucleoside Kinases
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The kcat and KM values of ttCK and hsUCK2 variants for cytidine and uridine were determined by using an NADH-dependent enzyme-coupled assay [14] (link) with a UV spectrophotometer, U-3000 (Hitachi). Measurements were performed at 25 °C in solutions containing 20 mM HEPES (pH 7.2), 100 mM KCl, 2 mM MgCl2, 50–2000 μM cytidine, 100–200 μM ATP, 15 μM NADH, 15 μM phosphoenolpyruvate, 0.5 mU of lactate dehydrogenase from pig heart (Toyobo), 0.5 mU of pyruvate kinase from rabbit muscle (Sigma) and 0.15 μM ttCK. The concentration of ATP was the concentration when ttCK had the highest activity towards cytidine. The kinetic constants, kcat and KM, were determined by fitting the data to Michaelis-Menten equation with IGOR Pro ver. 4.0.3.0 (WaveMetrics).
Reversed-phase HPLC was used to identify reaction products. The reaction mixture contained 20 mM Tris-HCl, 100 mM KCl, 10 mM MgCl2, 50 μM each nucleoside (adenosine, guanosine, uridine, cytidine, deoxyadenosine, deoxyguanosine, thymidine, and deoxycytidine), 500 μM ATP, and 50 nM ttCK at pH 8.0. The reaction mixtures were incubated at 25 °C or 37 °C for 1 h. The reaction products were separated by a CAPCELL PAK C18 column (Shiseido).
Reversed-phase HPLC was used to identify reaction products. The reaction mixture contained 20 mM Tris-HCl, 100 mM KCl, 10 mM MgCl2, 50 μM each nucleoside (adenosine, guanosine, uridine, cytidine, deoxyadenosine, deoxyguanosine, thymidine, and deoxycytidine), 500 μM ATP, and 50 nM ttCK at pH 8.0. The reaction mixtures were incubated at 25 °C or 37 °C for 1 h. The reaction products were separated by a CAPCELL PAK C18 column (Shiseido).
4
Synthetic Peptide Purification and Characterization
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The peptide was chemically synthesized at the peptide synthesizer facility of PepTron Inc. (Daejeon, Korea). The peptides were synthesized using the Fmoc solid-phase method with a peptide synthesizer (PeptrEX-R48; Peptron, Inc., Deajeon, Korea). The synthetic peptides were purified using HPCL (Shimadzu, Kyoto, Japan) on a Capcell Pak C18 column (4.6 × 50 mm, 5 μm, Shiseido, Kyoto, Japan). The column was developed at a flow rate of 1.0 mL/min by a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid. The identity of synthetic peptides was confirmed by liquid chromatography-mass spectroscopy (LC-MS) (Shimadzu, Japan), and the purity of the synthetic peptide was confirmed to be over 95%.
5
Quantification of Edaravone, PHZ, GSH, and GSSG
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Edaravone was quantified by high-performance liquid chromatography (HPLC) separation on the CAPCELL PAK ADME column (5 µm, 4.6 × 250 mm, Shiseido, Tokyo, Japan) using methanol/40 mM aqueous sodium phosphate (60/40 by volume) as the mobile phase (0.5 ml/min) with detection at 295 nm. PHZ was measured at the same HPLC conditions, with the exception of detection at 280 nm. GSH and its oxidized form (GSSG) were quantified by HPLC separation on the CAPCELL PAK C18 column (5 µm, 4.6 × 250 mm, Shiseido) using 3% methanol aqueous solution containing 0.05% trifluoroacetic acid as the mobile phase (1.0 ml/min) with detection at 210 nm.
6
Fenofibrate Nanospherule Solubility Analysis
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An ample quantity of each nanospherule formulation (~10 mg) was added to 500 μL of distilled water in a 2 mL microtube and vortex-mixed (~1 minute). Subsequently, the tubes were agitated at 100 rpm for 7 days in a water bath at 25°C. Then, after vortex-mixing again, centrifugation was performed at 10,000× g for 10 minutes (Smart 15; Hanil Science Industrial Co, Gangneung, South Korea). A 300 μL aliquot of supernatant was diluted with 300 μL of acetonitrile in another clean microtube. The diluent was filtered through a syringe filter (0.45 μm, number 6789–1304; Whatmann Co, Shrewsbury, MA, USA) and the concentration of fenofibrate in the filtrate (50 μL) was quantified by high-performance liquid chromatography (HPLC) (Agilent 1260 Infinity; Agilent Technologies, Santa Clara, CA, USA). The HPLC system was equipped with a Capcell Pak C18 column (4.6 mm inner diameter ×250 mm, 5 μm particle size; Shiseido, Tokyo, Japan). The column temperature was set at 30°C. The mobile phase, consisting of acetonitrile and 0.1% (v/v) aqueous phosphoric acid at a volume ratio of 75:25, was eluted at a flow rate of 2 mL/minutes. The eluent was monitored at 286 nm for the quantification of fenofibrate.26
7
Synthesis and Purification of CopA3 Peptide
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CopA3 (LLCIALRKK-NH2) was synthesized by AnyGen (Gwangju, Korea). The peptide was
purified via high performance liquid chromatography (HPLC) using a Capcell Pak
C18 column (Shiseido, Ginza, Japan). The peptide was then dissolved in distilled
water and stored at −20°C until use.
purified via high performance liquid chromatography (HPLC) using a Capcell Pak
C18 column (Shiseido, Ginza, Japan). The peptide was then dissolved in distilled
water and stored at −20°C until use.
8
HPLC Analysis of Chemical Compounds
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Analyses were performed using a reversed-phase high-performance liquid chromatography (HPLC) system (Agilent model 1260 series, Santa Clara, CA, USA) with a Capcell pak C18 column (5 μm × 4.6 mm × 250 mm; Shiseido, Japan) and Agilent 6120 (Santa Clara, CA, USA) in the single-quadrupole positive ion mode. Chromatography was performed at room temperature at a flow rate of 1 mL/min, and 10 μL was analyzed for 50 min. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) in a ratio specified by the following binary gradient with linear interpolation: 0 min 5% B, 40 min 80% B and 50 min 5% B.
9
Taxonomic Characterization of Strain K13-0306
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Taxonomic studies of strain K13-0306 were carried out as described previously. 4 The International Streptomyces Project media recommended by Shirling and Gottlieb 20 and by Waksman 21 were used to investigate cultural characteristics. Cultures were observed after incubation for 2 weeks at 27 °C. The morphological characteristics were observed by scanning electron microscope (JSM-5600; JEOL) after incubation on inorganic salts-starch agar for 3 weeks at 27 °C and fixation with 4% osmium tetroxide vapor. Isomers of diaminopimelic acid in whole-cell hydrolysates were determined by TLC, following the standard methods of Becker et al. 22 and Hasegawa et al. 23 Menaquinones were extracted and purified by the method of Collins et al. 24 and then analyzed by LC/MS (JSM-T 100LP; JEOL) with a CAPCELL PAK C18 column (Shiseido, Tokyo, Japan) eluted with methanol/2-propanol (7:3). 16S rRNA gene sequence was amplified by PCR and sequenced on a 3130 Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA) using a BigDye Terminator v.3.1 cycle sequencing kit (Applied Biosystems) according to the manufacturer's instructions.
10
LC-MS/MS Plasma Sample Preparation
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To prepare the plasma sample for LC-MS/MS analysis (liquid chromatography coupled with tandem mass spectrometry), 150 μl internal standard solution (5 μg/mL, acetonitrile) was added to 50 μl rat plasma samples to precipitate proteins. After centrifugation for 30 min (13000 rpm, 4 °C), 20 μl of the supernatant was introduced into the LC-MS/MS system. Detection was carried out using a API 3000 mass spectrometer (Applied Biosystems, Foster City, CA) with TurboIonSpray source interface. The processed samples were injected on a CAPCELL PAK C18 column (2.0 mm × 50 mm, 5 μm; Shiseido, Japan). The system was run in isocratic mode with mobile phase consisting of methanol and water in the ratio of 90:10 (v/v) at a flow rate of 0.3 mL/min. An electrospray ion source in the negative-ion multiple reaction monitoring (MRM) mode was used for detection. The MRM transition channel was m/z 517.3 to m/z 233.2 for S116836 and m/z 260.0 to m/z 183.0 for internal standard (Propranolol, Sigma). Ionization temperature was set as 400 °C. Data acquisition and quantitation were performed using Analyst 1.4 (Applied Biosystems, MDS Sciex Toronto, Canada).
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