Optical rotation measurements were carried out on a PerkinElmer model 341 polarimeter with a 10 cm length cell at room temperature. IR and UV spectra were recorded on a Bruker tensor 27 FTIR spectrometer and a Hitachi U-3010 spectrophotometer, respectively. ECD spectra were obtained from a Jasco J-715 spectropolarimeter. 1H, 13C, DEPT135, COSY, HSQC, HMBC, and ROESY NMR spectra were collected on an Agilent 600 MHz NMR apparatus. Chemical shifts (δ) are referenced to tetramethylsilane (TMS) at 0.00 ppm and the residual solvent peak of DMSO-d6 at 39.52 ppm, for proton and carbon, respectively. HRESIMS and HRESIMS/MS spectra were determined on a Waters Xevo G2-XS QTOF spectrometer. HPLC-MS spectra were collected using a Waters HPLC system equipped with a Waters Acquity QDa spectrometer and a Waters XBridge C18 column (4.6 × 250 mm, 5 μm). Analytical thin-layer chromatography (TLC) systems were performed on silica gel 60 F254 plates. Column chromatography was performed using silica gel 60 (200–300 mesh; Yantai). Preparative MPLC was carried out on an Interchim Puriflash 450 apparatus. Semipreparative reversed-phased HPLC (RP-HPLC) was carried out using a Waters 1525 pump equipped with a 2998 photodiode array detector and a Waters XBridge C18 column (10 × 250 mm, 5 μm).
Xbridge c18 column
Manufactured by Waters Corporation
Sourced in United States, Ireland, Japan, Germany, Belgium, United Kingdom, France
The XBridge C18 column is a reversed-phase high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a 3.5 μm particle size and a C18 stationary phase, providing efficient chromatographic separations.
Automatically generated - may contain errors
Lab products found in correlation
Ultimate 3000 system, by Thermo Fisher Scientific (20 mentions)
Htc pal autosampler, by CTC Analytics (8 mentions)
Ultimate 3000, by Thermo Fisher Scientific (7 mentions)
Ultimate 3000 lc system, by Thermo Fisher Scientific (6 mentions)
1290 series liquid chromatography system, by Agilent Technologies (6 mentions)
Xevo g2 xs qtof mass spectrometer, by Waters Corporation (6 mentions)
Tsq vantage am, by Thermo Fisher Scientific (5 mentions)
Ultimate 3000, by Waters Corporation (5 mentions)
Hplc system, by Shimadzu (5 mentions)
High performance liquid chromatography (hplc), by Waters Corporation (5 mentions)
Labsolutions software, by Shimadzu (4 mentions)
Winnonlin version 3, by Pharsight (4 mentions)
Agilent 1260 infinity bio inert hplc, by Agilent Technologies (4 mentions)
Aquity uplc system, by Waters Corporation (4 mentions)
Analyst software version 1, by Thermo Fisher Scientific (4 mentions)
Orbitrap fusion, by Thermo Fisher Scientific (4 mentions)
Hplc system, by Waters Corporation (4 mentions)
Oasis hlb column, by Waters Corporation (4 mentions)
1100 series hplc system, by Agilent Technologies (4 mentions)
Tmt10plex isobaric label reagent set, by Thermo Fisher Scientific (4 mentions)
428 protocols using xbridge c18 column
1
Structural Characterization of Natural Products
2
Tritium Labeling of Levocetirizine
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Precursor (S)-28 (0.8 mg, 1.55 μmol), Pd (10% on carbon, 0.5
mg, 0.47 μmol), and Et3N (5 μL, 0.04 mmol)
were mixed in EtOH (200 μL). The flask was fitted to the tritium
manifold. The mixture was freeze–pump–thaw degassed
and was then stirred under tritium gas (63.5 GBq) at 162 mbar for
2.5 h. The reaction mixture was filtered through a poly(tetrafluoroethylene)
filter (Whatman 0.45 μm) and washed thoroughly with more EtOH
(5 mL). The solution was lyophilized to remove labile tritium, more
EtOH (5 mL) was addeAmyloid Inhbitorsd, and the mixture was again
lyophilized. Purification by preparative HPLC (Waters XBridge C18
column, 5 μm, 4.6 × 150 mm2) using decreasingly
polar mixtures of water (containing 0.1% TFA) and MeCN as eluents
followed by further preparative HPLC (Waters XBridge C18 column, 5
μm, 4.6 × 150 mm2), using decreasingly polar
mixtures of water (containing 0.1% ammonia) and MeCN as eluents, afforded
[3H]levocetirizine (728 MBq), which was dissolved in EtOH
(10 mL) for storage as a colorless solution.
Radiochemical purity
>98% by HPLC. Chiral purity 93% enantiomeric excess by HPLC (obtained
on ethyl ester derivative by standing in ethanol with TFA for 3 days).
LC–MS (ESI) m/z 391 [M +
H]+. 1H NMR (640 MHz, DMSO-d6) 7.20 (t, J = 7.8). Specific activity
by mass spectrometry: 956 GBq mmol–1.
mg, 0.47 μmol), and Et3N (5 μL, 0.04 mmol)
were mixed in EtOH (200 μL). The flask was fitted to the tritium
manifold. The mixture was freeze–pump–thaw degassed
and was then stirred under tritium gas (63.5 GBq) at 162 mbar for
2.5 h. The reaction mixture was filtered through a poly(tetrafluoroethylene)
filter (Whatman 0.45 μm) and washed thoroughly with more EtOH
(5 mL). The solution was lyophilized to remove labile tritium, more
EtOH (5 mL) was addeAmyloid Inhbitorsd, and the mixture was again
lyophilized. Purification by preparative HPLC (Waters XBridge C18
column, 5 μm, 4.6 × 150 mm2) using decreasingly
polar mixtures of water (containing 0.1% TFA) and MeCN as eluents
followed by further preparative HPLC (Waters XBridge C18 column, 5
μm, 4.6 × 150 mm2), using decreasingly polar
mixtures of water (containing 0.1% ammonia) and MeCN as eluents, afforded
[3H]levocetirizine (728 MBq), which was dissolved in EtOH
(10 mL) for storage as a colorless solution.
Radiochemical purity
>98% by HPLC. Chiral purity 93% enantiomeric excess by HPLC (obtained
on ethyl ester derivative by standing in ethanol with TFA for 3 days).
LC–MS (ESI) m/z 391 [M +
H]+. 1H NMR (640 MHz, DMSO-d6) 7.20 (t, J = 7.8). Specific activity
by mass spectrometry: 956 GBq mmol–1.
3
Organic Compound Purification and Characterization
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All solvents and reagents were purchased
from commercial vendors and were used without further purification
unless otherwise mentioned. Column chromatography was conducted using
prepacked silica gel cartridges (Biotage) on a Biotage Isolera Prime
system. LC–MS was conducted on an Agilent 1100 series HPLC
apparatus and a 6120 Quadrupole LC–MS apparatus in API-ES mode
using a Waters Xbridge C18 column (4.6 mm × 50 mm, 3.5 μm).
Mobile phase A was 0.1% trifluoroacetic acid in water, and mobile
phase B was 0.1% trifluoroacetic acid in acetonitrile. 1H NMR spectra were obtained using a Varian Mercury 300 MHz spectrometer.
The final products were purified by preparative HPLC on a Waters XBridge
C18 column (19 mm × 100 mm, 3.5 μm). The purity of all
tested compounds was ≥95%, as determined by HPLC with UV detection
at 220 nm and 1H NMR analyses.
All animal experiments
performed in the manuscript were conducted in compliance with institutional
guidelines, as defined by the Institutional Animal Care and Use Committee
(IACUC).
from commercial vendors and were used without further purification
unless otherwise mentioned. Column chromatography was conducted using
prepacked silica gel cartridges (Biotage) on a Biotage Isolera Prime
system. LC–MS was conducted on an Agilent 1100 series HPLC
apparatus and a 6120 Quadrupole LC–MS apparatus in API-ES mode
using a Waters Xbridge C18 column (4.6 mm × 50 mm, 3.5 μm).
Mobile phase A was 0.1% trifluoroacetic acid in water, and mobile
phase B was 0.1% trifluoroacetic acid in acetonitrile. 1H NMR spectra were obtained using a Varian Mercury 300 MHz spectrometer.
The final products were purified by preparative HPLC on a Waters XBridge
C18 column (19 mm × 100 mm, 3.5 μm). The purity of all
tested compounds was ≥95%, as determined by HPLC with UV detection
at 220 nm and 1H NMR analyses.
All animal experiments
performed in the manuscript were conducted in compliance with institutional
guidelines, as defined by the Institutional Animal Care and Use Committee
(IACUC).
4
Glycan, Glycopeptide, and Payload Derivative Analysis
5
Purification of Bioactive Compounds
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6 compounds were purified based on 2 injections of 40 mg: 70 (0.5 mg, tR 22.5 min), 65 (1.6 mg, tR 35.8 min), 66 (0.5 mg, tR 41.6 min), 67 (5.4 mg, tR 42.5 min), 68 (0.4 mg, tR 54.8 min), 69 (0.7 mg, tR 70.6 min). The fractions collected from 17.5 to 19.0 min (3.6 mg) were further purified on a smaller XBridge C18 column (250 × 10 mm i.d., 5 μm; Waters) by using isocratic conditions at 30% MeOH to give compound 71 (0.7 mg, tR 50 min). The fractions collected from 33.5 to 35.0 min (2.3 mg) were further purified on a smaller XBridge C18 column (250 × 10 mm i.d., 5 μm; Waters) by using isocratic conditions at 42% MeOH to give compound 72 (1.1 mg,, tR 68 min) and 73 (0.2 mg, tR 92.5 min).
6
Medicinal Chemistry Characterization Protocol
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All solvents and reagents were purchased
from commercial vendors and were used without further purification
unless otherwise mentioned. Column chromatography was conducted using
prepacked silica gel cartridges (Biotage) on a Biotage Isolera Prime
system. LC–MS was conducted on an Agilent 1100 series HPLC
and 6120 Quadrupole LC/MS in the API-ES mode using a Waters Xbridge
C18 column (4.6 mm × 50 mm, 3.5 μm). Mobile phase A was
0.01% trifluoroacetic acid in water, and mobile phase B was 0.01%
trifluoroacetic acid in acetonitrile. 1H NMR spectra were
recorded using a Varian Mercury 300 MHz spectrometer. The final products
were purified by preparative HPLC on a Waters XBridge C18 column (19
mm × 100 mm, 5 μm). The purity of all tested compounds
was ≥95%, as determined by HPLC with UV detection at 220 nm
and 1H NMR analyses.
Calculations of clog P and tPSA were performed using ChemDraw (Pro, Version 19.0).
For clog P, ChemDraw uses the calculator from Biobyte
Corp, Claremont, CA (www.biobyte.com ).45 (link) For tPSA, calculations were based
on fragment contributions using the SMILES Daylight Toolkit module.46 (link)All animal experiments performed in the
article were conducted
in compliance with institutional guidelines as defined by the Institutional
Animal Care and Use Committee (IACUC).
from commercial vendors and were used without further purification
unless otherwise mentioned. Column chromatography was conducted using
prepacked silica gel cartridges (Biotage) on a Biotage Isolera Prime
system. LC–MS was conducted on an Agilent 1100 series HPLC
and 6120 Quadrupole LC/MS in the API-ES mode using a Waters Xbridge
C18 column (4.6 mm × 50 mm, 3.5 μm). Mobile phase A was
0.01% trifluoroacetic acid in water, and mobile phase B was 0.01%
trifluoroacetic acid in acetonitrile. 1H NMR spectra were
recorded using a Varian Mercury 300 MHz spectrometer. The final products
were purified by preparative HPLC on a Waters XBridge C18 column (19
mm × 100 mm, 5 μm). The purity of all tested compounds
was ≥95%, as determined by HPLC with UV detection at 220 nm
and 1H NMR analyses.
Calculations of clog P and tPSA were performed using ChemDraw (Pro, Version 19.0).
For clog P, ChemDraw uses the calculator from Biobyte
Corp, Claremont, CA (
on fragment contributions using the SMILES Daylight Toolkit module.46 (link)All animal experiments performed in the
article were conducted
in compliance with institutional guidelines as defined by the Institutional
Animal Care and Use Committee (IACUC).
7
Fmoc-Based Solid-Phase Peptide Synthesis
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The peptides were prepared via standard Fmoc-based solid-phase synthesis on Rink amide 4-methylbenzhydrylamine resin. Diisopropylcarbodiimide and 1-hydroxybenzotriazole were used as coupling reagents, and a threefold excess of the Fmoc-amino acid was added during every coupling cycle. After cleavage and deprotection using a mixture of TFA/triisopropylsilane/H2O (95:2.5:2.5, v/v/v) for 3 h at room temperature, the crude peptide was repeatedly extracted with diethyl ether, dried under vacuum, and purified by RP-HPLC on a preparative Waters XBridge C18 column (length: 150 mm, internal diameter: 19 mm, pore size: 100 Å, particle size: 5 mm) using an appropriate 5–80% water/acetonitrile gradient in the presence of 0.1% TFA. The appropriate fractions were combined and lyophilized, and the final purity of the obtained peptides (> 95%) was determined by RP-HPLC on an analytical Waters XBridge C18 column (length: 250 mm, internal diameter: 4.6 mm, pore size: 100 Å, particle size: 5 mm). ESI-MS was used to determine the molecular weight.
8
High-pH Peptide Fractionation Protocol
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The mixed peptides were re-dissolved in buffer A (buffer A: 20 mM ammonium formate in water, pH 10.0, adjusted with ammonium hydroxide) and then loaded onto a reverse phase column (XBridge C18 column, 4.6 mm × 250 mm, 5 μm, Waters Corporation, MA) using Ultimate 3000 HPLC system (Thermo Fisher scientific, MA). High pH separation was achieved in 40 minute with a linear gradient starting from 5% B to 45% B (B: 20 mM ammonium formate in 80% ACN, pH 10.0, adjusted with ammonium hydroxide). The column was re-equilibrated at 30°C for 15 minute (flow rate: 1 mL/min). A total of 10 fractions were obtained and dried by vacuum concentrator for future use.
9
Spectroscopic Characterization of Compounds
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The NMR spectra were recorded on a Bruker AVIII-500 NMR spectrometer (Bruker Corporation, Karlsruhe, Germany) and Bruker DRX-600 spectrometer (Bruker Biospin AG, Fällanden, Germany). The chemical shifts of 1H NMR (500 MHz, 600 MHz) and 13C NMR (125 MHz, 150 MHz,) data were shown in δ (ppm) and referenced against the solvent signal (DMSO-d6, δH 2.50 and δC 39.52; acetone-d6, δH 2.05 and δC 29.84). HRESIMS data were measured on an API QSTAR Pulsar mass spectrometer (Bruker, Bremen, Germany). UV and IR data were tested on a UV-2550 spectrometer (Shimadzu, Kyoto, Japan) and Nicolet 380 Infrared Spectrometer (Thermo Electron Corporation, Madison, WI, USA), respectively. The semi-preparative HPLC was conducted on a Waters 1525 HPLC equipped with an XBridge C18 column (5 μm, 250.0 mm × 10.0 mm; Waters Corporation, Milford, MA, USA). Thin-layer chromatography (TLC) was conducted on pre-coated glass plates (silica gel GF254, Qingdao Marine Chemical Inc., Qingdao, China). Column chromatography (CC) was conducted on silica gel (45–75 µm; Qingdao Marine Chemical Inc., Qingdao, China).
10
Peptide Fractionation and Mass Spectrometry
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The peptides were vacuum dried and fractionated
using the basic
pH/reverse-phase liquid chromatography (bRPLC) method, and separated
using a Hitachi LaChrom Elite HPLC System (Hitachi High-Technologies
Corporation, Tokyo, Japan) equipped with an XBridge C18 column (130
Å, 5 μm, 250 mm × 4.6 mm; Waters Corporation, Milford,
MA) with a linear increase in the gradient from 5 to 100% of 10 mM
TEABC with 90% acetonitrile over 120 min. Initially, 96 fractions
were obtained, which were then concatenated to six fractions and dried
before being desalted with C18 cartridges. The desalted peptides were
then vacuum dried and stored at −80 °C until LC-MS/MS
analysis.
using the basic
pH/reverse-phase liquid chromatography (bRPLC) method, and separated
using a Hitachi LaChrom Elite HPLC System (Hitachi High-Technologies
Corporation, Tokyo, Japan) equipped with an XBridge C18 column (130
Å, 5 μm, 250 mm × 4.6 mm; Waters Corporation, Milford,
MA) with a linear increase in the gradient from 5 to 100% of 10 mM
TEABC with 90% acetonitrile over 120 min. Initially, 96 fractions
were obtained, which were then concatenated to six fractions and dried
before being desalted with C18 cartridges. The desalted peptides were
then vacuum dried and stored at −80 °C until LC-MS/MS
analysis.
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