The reagents, solvents, and restriction enzymes were purchased from standard commercial sources and used directly. PCR amplifications were carried out on Bio-Rad T100 thermal cycler using Phanta® Super Fidelity DNA Polymerase (P505-d3, Vazyme, China). 3-oxo-glutaric acid (OGA, 7 , 165115-25g) and malonyl-CoA (M4263-5mg) were purchased from sigma-aldrich Co. (USA). Triacetic acid lactone (TAL, 8 ) was purchased from Macklin (H811426-25g). Primer synthesis and DNA sequencing were performed by TsingKe Co. (China). LC-MS analysis was conducted on AGILENT 1290/6530 system and analyzed by Agilent MassHunter. HPLC analysis was conducted on a HITACHI Chromaster system equipped with a DAD detector, a YMC-Triart C18 column (I.D. 4.6 mm × 250 mm, Japan), and a flow rate of 1.0 mL/min at a column temperature of 25 °C. ChemBioDraw Ultra 14.0 was used for drawing chemical structures. OriginPro 9.0 was used for LC-MS and HPLC data visualization.
Chromaster system
Manufactured by YMC
Sourced in Japan
The Chromaster system is a high-performance liquid chromatography (HPLC) instrument designed for analytical and preparative applications. It provides reliable and accurate separation and detection of a wide range of chemical compounds. The system includes a solvent delivery unit, an autosampler, a column oven, and a variety of detectors to suit different analytical needs.
Automatically generated - may contain errors
Lab products found in correlation
Phanta super fidelity dna polymerase, by Vazyme (1 mentions)
Chembiodraw ultra 14, by Revvity Signals Software (1 mentions)
Avance 3 600 spectrometer, by Bruker (1 mentions)
Silica gel, by Qingdao Marine Chemical (1 mentions)
Xevo tq s mass spectrometer, by Waters Corporation (1 mentions)
C18 column, by YMC (1 mentions)
1290 uplc 6540 q tof mass instrument, by Agilent Technologies (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Fv1000, by Olympus (1 mentions)
Uplansapo 100, by Olympus (1 mentions)
αplan apochromat 100, by Zeiss (1 mentions)
Mercury 400 spectrometer, by Agilent Technologies (1 mentions)
C8 column, by YMC (1 mentions)
4 protocols using chromaster system
1
Biosynthesis of Triacetic Acid Lactone
2
Spectroscopic Analysis of Natural Compounds
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NMR spectra were recorded in CD3OD using a Bruker AVANCE III-600 spectrometer, and TMS was used as internal standard. ESIMS spectra were recorded using a Waters Xevo TQS mass spectrometer. HRESIMS data were obtained using an Agilent 1290 UPLC/6540 Q-TOF mass instrument. Column chromatography (CC) was performed using silica gel (200–300 mesh, Qingdao Marine Chemical Inc., China) and MCI gel CHP20P (75–150 µm, Mitsubishi Corp., Japan). Semipreparative HPLC was conducted on a HITACHI Chromaster system equipped with a DAD detector, a YMC-Triart C18 column (250 × 10 mm i.d., 5 μm).
3
Detailed NMR and Mass Spectrometry Analysis
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Unless stated otherwise, all commercial reagents were used as received. Thin-layer chromatography (TLC) was performed on silica gel 60F254 (Merck). 1H NMR spectra were obtained on a Varian Mercury 400 spectrometer with residual non-deuterated solvents (CD3OD: 3.31 ppm for 1H) as the internal reference. ESI mass spectra were recorded using an Exactive (Thermo Scientific). Reversed-phase HPLC (RP-HPLC) was carried out on a Hitachi Chromaster system equipped with a diode array and YMC-Pack Triart C18 or ODS-A columns. All runs used linear gradients of acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA) and 0.1% aqueous TFA. The images of confocal laser scanning microscopy (CLSM) were acquired by a FV1000 (Olympus) and a LSM800 (Carl Zeiss Microscopy). UPlanSApo 100× (1.40 numerical aperture, oil immersion, Olympus), UPlanSApo 4× (0.16 numerical aperture, Olympus), Plan-Apochromat 20× (0.8 numerical aperture, Carl Zeiss), and αPlan-Apochromat 100× (1.46 numerical aperture, Carl Zeiss) were used. The fluorescent intensity of the CLSM images was calculated by Fiji49 (link).
4
Budesonide Prodrug Synthesis and Characterization
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The prodrug was synthesized via esterification in the presence of 1,3-diisopropylcarbodiimide (DISC) and 4-dimethylaminopyridine (DMAP). DISC (7.3 mg, 0.06 mmol) and DMAP (7.5 mg, 0.06 mmol) were added to a solution of budesonide (21.5 mg, 0.05 mmol) and LA (14.0 mg, 0.05 mmol) in 4 mL anhydrous dichloromethane (DCM) (Marquez Ruiz et al., 2013 (link)). The reaction mixture was refluxed overnight. After confirming the completion of the reaction, the crude product was washed with 5% citric acid, saturated NaHCO3, and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated under vacuum. The residue was further purified using flash column chromatography on silica gel (DCM:MeOH = 30:1) to yield the budesonide prodrug (23.0 mg, 71.4%). The chemical structure of final product was confirmed by 1H nuclear magnetic resonance (NMR) spectroscopy, and the purity was validated by high-performance liquid chromatography (HPLC). HPLC was carried out on a Hitachi Chromaster system, and a YMC-Pack C8 column (5 μm, 250 × 4.6 mm) at a flow rate of 1.0 mL/min. UV detection was performed at 220 nm. All runs used a linear gradient of 50–100% acetonitrile/water containing 0.1% TFA within 30 min.
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