Aspergillus unguis 158SC-067 was isolated from a seawater sample, as previously described [9 (link)]. The mycelium extract was separated into 10 fractions (1–10 m), as described earlier [9 (link)]. Fraction 8 m was subjected to an analytical HPLC (YMC-Pack ODS-A, 250 × 4.6 mm i.d., 5 µm, flow rate 0.9 mL/min) with an isocratic elution of 70% MeOH in H2O to obtain compounds 11 (3.0 mg, tR = 10 min) and 15 (3.0 mg, tR = 24 min). Compounds 12 (1.3 mg, tR = 25 min) and 13 (3.0 mg tR = 29 min) were purified from fraction 9 m by a semipreparative HPLC (YMC-Pack ODS-A, 250 × 10 mm i.d., 5 µm, flow rate 2.0 mL/min) with an isocratic elution of 80% MeOH in H2O.
Pack ods a
Manufactured by YMC
Sourced in Japan, United States
YMC-Pack ODS-A is a reversed-phase chromatography column. It is designed for the separation and purification of a wide range of chemical compounds.
Automatically generated - may contain errors
Lab products found in correlation
Chirascan circular dichroism spectrometer, by Applied Photophysics (11 mentions)
Sephadex lh 20, by Cytiva (8 mentions)
Mpc 500, by PerkinElmer (7 mentions)
Mixis tof qii mass spectrometer, by Bruker (6 mentions)
Avance spectrometer, by Bruker (6 mentions)
Silica gel, by Qingdao Haiyang Chemical (6 mentions)
Ir affinity 1 spectrometer, by Shimadzu (5 mentions)
Agilent 500 mhz dd2 spectrometer, by Agilent Technologies (5 mentions)
Uv 2600 pc spectrometer, by Shimadzu (4 mentions)
Tensor 27, by Bruker (3 mentions)
Gf254, by Qingdao Marine Chemical (3 mentions)
P 1020, by Jasco (3 mentions)
Model 1525 pump, by YMC (2 mentions)
Model 2489, by YMC (2 mentions)
Hplc system, by Waters Corporation (2 mentions)
Ac 500, by Bruker (2 mentions)
Avance 3 hd 700 nmr spectrometer, by Bruker (2 mentions)
Microtof q 2 mass spectrometer, by Bruker (2 mentions)
Uv 2401 pc spectrophotometer, by Shimadzu (2 mentions)
Sephadex lh 20, by GE Healthcare (2 mentions)
52 protocols using pack ods a
1
Isolation and Purification of Fungal Compounds
2
HPLC Analysis of Mutant Compound Differences
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A 10 µL extract was injected into the HPLC system (Waters Inc., Milford, MA, USA), which contained a model 1525 pump, an ODS column (Pack ODS-A, 250 × 4.6 mm, 5 µm, YMC Co., Ltd., Japan), and a model 2489 UV detector. The gradient increased from 20% to 100% MeOH over 30 min and then was retained at 100% MeOH for 10 min. The fold differences of these compounds between the mutant and WT were calculated by HPLC peak area according to the following formula: [Area (Sample)—Area (Control)]/[Area (WT)–Area (Control)].
3
Isolation and Characterization of Microbial Metabolites
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A two-liter-scale culture and extract preparation of the ΔhdaA strain were performed using the method mentioned above. The obtained extract (1.3 g) was gained and separated by silica gel vacuum liquid chromatography using MeOH-H2O to give four fractions (Fractions 1–4). Fraction 1 was further separated by Sephadex LH-20 eluted with MeOH and then on a semipreparative HPLC column (Pack ODS-A, 250 × 10 mm, 5 µm, YMC Co., Ltd., Japan) eluted with MeOH-H2O (50:50, 3 mL/min) to provide compound 1 (2.6 mg, tR 8.5 min). Fraction 2 was separated by semipreparative HPLC eluted with MeOH-H2O (60:40, 3 mL/min) to obtain compound 2 (5.7 mg, tR 10.5 min). Fraction 4 was separated on a Sephadex LH-20 column with CH2Cl2-MeOH (1:1) and then on a semipreparative HPLC eluted with MeOH-H2O (75:25, 3 mL/min) to obtain compound 3 (3.5 mg, tR 11.5 min) and compound 4 (3.6 mg, tR 12.5 min). The structures of the compounds were identified using MS and NMR data. MS spectra were recorded on a Q-TOF Ultima Global GAA076 LC mass spectrometer (Wasters Inc., Milford, MA, USA). NMR spectra were collected on a Varian 500 spectrometer (Varian Medical Systems Inc., Palo Alto, CA, USA).
4
Comprehensive Spectroscopic Characterization
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The 1D (1H and 13C) and 2D (COSY, ROESY, HSQC, and HMBC) NMR spectra were acquired on a Bruker 600 MHz spectrometer. UV spectra were obtained on a Shimadzu UV-1650PC spectrophotometer. IR spectra were recorded on a JASCO FT/IR-4100 spectrophotometer. Optical rotations were measured on a Rudolph Research Analytical (Autopol III) polarimeter. HRESIMS spectra were recorded on a hybrid ion-trap time-of-flight mass spectrometer (Shimadzu LC/MS-IT-TOF). HPLC was performed on a PrimeLine Binary pump with RI-101 (Shodex). Analytical HPLC was conducted on an ODS column (YMC-Pack-ODS-A, 250 × 4.6 mm i.d, 5 µm).
5
Isolation and Purification of Bioactive Compounds
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Large-scale culture (10 L) and extract preparation of the strain Lcu-Fe1712 were performed in PDB liquid medium using the method mentioned above. 2.7 g of the EtOAc extract was gained and separated by silica gel vacuum liquid chromatography using CH 2 Cl 2 -MeOH (20:1) to give five fractions (Fractions 1 to 5). Fraction 2 was further separated by Sephadex LH-20 chromatograph eluted with CH 2 Cl 2 -MeOH (1:1) and then on a semi-preparative HPLC column (Pack ODS-A, 250 × 10 mm, 5 μm, YMC Co., Ltd.) eluted with MeOH-H 2 O (80:20, 3 ml/min) to provide compound 4 (5.6 mg, t R 10.5 min) and compound 5 (20.2 mg, t R 12.0 min). Fraction 3 was further separated on a Sephadex LH-20 column with MeOH to provide three subfractions (fractions 3-1 to 3-3). Fraction 3-2 was separated by semi-preparative HPLC eluted with MeOH-H 2 O (56:36, 3 ml/min), to obtain compound 1 (2.7 mg, t R 12.5 min), compound 2 (3.2 mg, t R 15.5 min) and compound 3 (6.2 mg, t R 23.5 min).
The structures of the compounds were elucidated from extensive MS and NMR. High-resolution electrospray ionization MS (HRESI-MS) spectra were measured on a Micromass EI-4000 Autospec-Ultima-TOF (Micromass communication Inc., UK). NMR spectra were recorded on a Varian 500 spectrometer (Varian Medical Systems Inc., USA) using tetramethylsilane as an internal standard, and chemical shifts were recorded as δ values.
The structures of the compounds were elucidated from extensive MS and NMR. High-resolution electrospray ionization MS (HRESI-MS) spectra were measured on a Micromass EI-4000 Autospec-Ultima-TOF (Micromass communication Inc., UK). NMR spectra were recorded on a Varian 500 spectrometer (Varian Medical Systems Inc., USA) using tetramethylsilane as an internal standard, and chemical shifts were recorded as δ values.
6
HPLC Analysis of Organic Compounds
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The extracts were analyzed in a HPLC system (Waters Inc., USA), which contained a model 1525 pump, a model 2489 UV detector, and a HPLC column (Pack ODS-A, 250 × 4.6 mm, 5 μm, YMC Co., Ltd., Japan). The gradient increased from 10 to 100% MeOH over 30 min and was retained at 100% for 10 min.
7
Spectroscopic Characterization of Natural Products
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Optical rotations were measured on a Perkin-Elmer 343 digital polarimeter (Perkin Elmer, Waltham, MA, USA). The 1H and 13C NMR spectra were recorded in CDCl3 using Bruker Avance III 500 (Bruker BioSpin GmbH, Rheinstetten, Germany) (500.13/125.77 MHz) or Avance III 700 Bruker FT-NMR (Bruker BioSpin GmbH, Rheinstetten, Germany) (700.13/176.04 MHz) spectrometers. HRESI and ESI mass spectra were recorded on an Agilent 6510 Q-TOF LC/MS mass spectrometer (Agilent Technologies, Santa Clara, CA, USA), and samples were dissolved in methanol (c 0.01 mg/mL). TLC was carried out on silica gel plates (CTX-1A, 5-17 µm, Sorbfil, Russia) and spots were visualized by spraying with aqueous 10% H2SO4 followed by heating. Column chromatography (CC) was performed on silica gel (KSK, 50−160 mesh, Sorbfil, Russia) and YMC ODS-A (12 nm, S-75 um, YMC Co., Ishikawa, Japan). HPLC was performed using an Agilent 1100 Series chromatograph with a differential refractometer (Agilent Technologies, Santa Clara, CA, USA). The reversed-phase columns (YMC-Pack ODS-A, YMC Co., Ishikawa, Japan, 10 mm × 250 mm, 5 µm and 4.6 mm × 250 mm, 5 µm) and normal-phase column (Ultrashere-Si, Beckman Instruments, Inc., Berkeley, CA, USA, 10 mm × 250 mm, 5 µm) were used for HPLC. Yields are based on dry weight of the sponge.
8
Detailed Analytical Techniques for Research
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Optical rotations were measured on a PerkinElmer MPC 500 (PerkinElmer, Waltham, MA, USA) polarimeter. The UV spectra were obtained with a Shimadzu UV-2600 PC spectrometer (Shimadzu, Kyoto, Japan). IR spectra were determined with an IR Affinity-1 spectrometer (Shimadzu, Kyoto, Japan). NMR spectra were recorded on a Bruker Avance spectrometer (Bruker, Billerica, MA, USA) operating at 500 MHz and 700 MHz for 1H NMR and 125 MHz and 175 MHz for 13C NMR used tetramethylsilane as an internal standard. HRESIMS spectra were acquired on a Bruker miXis TOF-QII mass spectrometer (Bruker, Billerica, MA, USA). TLC and column chromatography (CC) were performed on plates precoated with silica gel GF254 (10−40 μm) and over silica gel (200–300 mesh) (Qingdao Marine Chemical Factory, Qingdao, China), respectively. Spots were detected on TLC (Qingdao Marine Chemical Factory, Qingdao, China) under 254 nm UV light. All solvents employed were analytical grade (Tianjin Fuyu Chemical and Industry Factory, Tianjin, China). Semipreparative HPLC was performed using an ODS column (YMC-pack ODS-A, YMC Co., Ltd., 10 × 250 mm, 5 μm, Kyoto, Japan). Artificial sea salt was a commercial product (Guangzhou Haili Aquarium Technology Company, Guangzhou, China).
9
Spectroscopic Characterization of Organic Compounds
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1D and 2D NMR spectra were recorded on an AVANCE III HD 700 (Temperature 298.0 K, Bruker, Billerica, MA, USA). Optical rotations were measured with an MCP 500 automatic polarimeter (Anton Paar, Graz, Austria) with CH3OH as the solvent. IR spectra were measured on an IR Affinity-1 spectrometer (Shimadzu, Kyoto, Japan). UV spectra were recorded on a UV-2600 spectrometer (Shimadzu, Tokyo, Japan). Circular dichroism spectra were measured by Chirascan circular dichroism spectrometer with the same concentration of UV measurement (Pathlength 10 mm, Applied Photophysics, Surrey, UK). HRESIMS spectra data were recorded on a MaXis quadrupole-time-of-flight mass spectrometer. Thin layer chromatography (TLC) was performed on plates precoated with silica gel GF254 (10–40 μm). Column chromatography (CC) was performed over silica gel (100–200 mesh and 200–300 mesh) (Qingdao Marine Chemical Factory, Qingdao, China) and ODS (50 μm, YMC, Kyoto, Japan). High-performance liquid chromatography was performed on an Agilent 1260 HPLC equipped with a DAD detector using an ODS column (YMC-pack ODS-A, 250 × 10 mm, 5 μm, 3 mL/min). All solvents used in CC and HPLC were of analytical grade (Tianjin Damao Chemical Plant, Tianjin, China) and chromatographic grade (Oceanpak, Goteborg, Sweden), respectively.
10
Synthesis and Purification of 18F-FBPA
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FBPA was prepared as described previously [4 (link)], although with several modifications, using an F-1 synthesizer (Sumitomo Heavy Industries, Tokyo, Japan). In brief, 18 F-acetylhypofluorite was bubbled at a flow rate of 600 mL/min at room temperature into 5 mL of trifluoroacetic acid containing 30 mg of 4-borono-l -phenylalanine. Next, trifluoroacetic acid was removed by passing N2 under reduced pressure at a flow rate of 200 mL/min. The residue was dissolved in 3 mL of 0.1% acetic acid, and the solution was applied to YMC-Pack ODS-A, a high-performance liquid chromatography column (20 mm in inner diameter × 150 mm in length; YMC, Kyoto, Japan), under the following conditions: mobile phase, 0.1% acetic acid; flow rate, 10 mL/min; ultraviolet detector at 280 nm; and radioactivity detector. The FBPA fraction (retention time = 19 to 21 min) was collected. After drying of the FBPA fraction, the residue was dissolved in saline. The radiochemical purity of FBPA was >98%, and the specific activity at the end of the synthesis was 49.7 ± 17.3 GBq/mmol as determined by HPLC.
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