Plasma-purified human AAT (99% purity, Zemaira, CSL Behring, Kankakee, IL, USA) was used for experiments after buffer exchange to the sterile Hank’s Balanced Salt Solution (HBSS) (Merck Millipore, Darmstadt, Germany) using 10K centrifugal filter columns (Sartorius, Göttingen, Germany). The protein concentration was determined using the BCA Protein Assay Kit (Pierce™, Rockford, IL, USA) according to the instructions of the supplier. The oxAAT was prepared from AAT (Zemaira®) by adding N-chlorosuccinimide (Sigma-Aldrich, Merck, Darmstadt, Germany) at a molar ratio of 1:20 (AAT: N-chlorosuccinimide) for 20 min at room temperature. Afterward, to remove the N-chlorosuccinimide, AAT preparations were washed with phosphate-buffered saline (PBS; Sigma-Aldrich, St. Louis, MO, USA) using Vivaspin 20 centrifugal filter devices with a cutoff of 10K. The oxAAT did not form complexes with elastase and showed a retarded electrophoretic mobility relative to a native AAT. A highly purified (90%), glycosylated form of recombinant AAT protein produced in CHO cells was a gift from ExcellGene, Monthey, Switzerland.
N chlorosuccinimide
Manufactured by Merck Group
Sourced in Germany, United States
N-chlorosuccinimide is a chemical compound used as a halogenating agent in organic synthesis. It is a crystalline solid that serves as a source of positive chlorine in various chemical reactions.
Automatically generated - may contain errors
Lab products found in correlation
N bromosuccinimide, by Merck Group (3 mentions)
Methacryloyl chloride, by Merck Group (2 mentions)
Lysogeny broth agar medium, by Thermo Fisher Scientific (2 mentions)
Triethylamine, by Merck Group (2 mentions)
Trifluoroacetic acid, by Merck Group (2 mentions)
Dopamine hydrochloride, by Merck Group (2 mentions)
N chlorosuccinimide, by Sartorius (2 mentions)
Viva spin 20 centrifugal columns, by Sartorius (2 mentions)
Phosphate buffer saline (pbs), by Thermo Fisher Scientific (1 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (1 mentions)
Aapba, by Merck Group (1 mentions)
Mueller hinton broth medium, by Thermo Fisher Scientific (1 mentions)
Calcein am pi, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Azobisisobutyronitrile, by Merck Group (1 mentions)
N n methylenebisacrylamide, by Merck Group (1 mentions)
Tris hydroxymethyl aminomethane, by Merck Group (1 mentions)
N hydroxyethyl acrylamide, by Merck Group (1 mentions)
Zemaira, by CSL Behring (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
12 protocols using n chlorosuccinimide
1
Oxidation and Purification of Human AAT
2
Oxidized alpha-1 antitrypsin protocol
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Oxidized AAT (oxAAT) was prepared as previously described (14 (link)). Briefly, N-chlorosuccinimide (Sigma-Aldrich) was added to AAT protein at a molar ratio of 25:1, incubated for 30 min at room temperature, and excess of N-chlorosuccinimide was removed by ultrafiltration. The quality of oxidation was controlled by testing anti-elastase activity. Native or oxAAT was pre-incubated with NE (at a molar ratio of 1.5:1) for 30 min. Thereafter, samples were analyzed by SDS-PAGE in 7.5% polyacrylamide gels followed by staining with Brilliant Blue.
3
Antimicrobial Hydrogel Synthesis and Evaluation
4
Oxidation of Plasma-Purified AAT
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Oxidized AAT was prepared from native plasma-purified AAT (see above) by adding N-chlorosuccinimide (Sigma-Aldrich, Merck, Darmstadt, Germany) to AAT solution at a molar ratio of 20:1. The mixture was allowed to react for 20 min at room temperature, and N-chlorosuccinimide was then removed by washing with PBS 3 times using viva spin-20 centrifugal columns with a 10 k MWCO (Sartorius, Göttingen, Germany).
5
Synthesis of 2-ChloroHexadecynoic Acid
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The alkyne analog of 2-ClHDA (2-ClHDyA), was synthesized according to protocols described previously (Halland et al., 2004 (link); Nusshold et al., 2016 (link)). Sequentially, (1) hexadec-7-ynol (Alfa Aesar, cat. B22113) was converted to hexadec-15-ynol using sodium hydride (Sigma-Aldrich, cat. 452912) and diaminopropane (Sigma-Aldrich, cat. D23602) (Nusshold et al., 2016 (link)), hexadec-15-ynol was oxidized to HDyA in a solution of 2-iodoxybenzoic acid (Sigma-Aldrich, cat. 661384) in DMSO (Sigma-Aldrich, cat. D2650) (Nusshold et al., 2016 (link)), and HDyA was chlorinated using N-chlorosuccinimide (Sigma-Aldrich, cat. 109681) and proline (Sigma-Aldrich, cat. P0380) for 16 h (Halland et al., 2004 (link)). Products were purified by flash chromatography (30 g silica gel, high purity grade, pore size 60 Å, Sigma-Aldrich, cat. 227196) (Hartman et al., 2018 (link)). 2-ClHDyA was subsequently quantitated by GC-FID following conversion to its dimethyl acetal derivative using heptadecanoic acid and its methyl ester derivative as internal standard (Gross, 1985 (link); Albert et al., 2001 (link)).
6
Isolation and Characterization of Copaiba Diterpenes
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(−)-Polyalthic acid (1 ) and kaurenoic acid (2 ) were isolated from the oleoresin of Copaifera reticulata Ducke, Fabaceae, as described in our previous studies (Çiçek et al. 2018 (link); Pfeifer Barbosa et al. 2019 (link)). N-Chlorosuccinimide (98%) and triphenylphosphine (ReagentPlus, 99%), sulphuric acid (puriss., analytical grade) and LC-MS grade formic acid were purchased from Sigma-Aldrich Co., St. Louis, MO, USA. Hydrochloric acid (25%, analytical grade) was obtained from Honeywell, Seelze, Germany, while sodium hydroxide solution (2N) was purchased from Carl Roth GmbH, Karlsruhe, Germany. Diethylamine (for synthesis), acetone (analytical grade), methanol (gradient grade or LC-MS grade) and water (LC-MS grade) as well as other analytical grade solvents used for purification were obtained from VWR International GmbH, Darmstadt, Germany. Solid phase extraction (SPE) columns (Chromabond SB 3 ml/500 mg) were obtained from Macherey-Nagel GmbH & Co. KG, Düren, Germany. Deuterated methanol (Lot P3021, 99.80%), deuterated DMSO (Lot S1051, 99.80%) and deuterated chloroform (Lot Q1981, 99.80%) for NMR spectroscopy were obtained from Eurisotop GmbH, Saarbrücken, Germany. Conventional 5-mm sample tubes were purchased from Rototec-Spintec GmbH, Griesheim, Germany.
7
Synthetic Peptide Synthesis Protocol
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Fmoc-protected amino acids (except of listed below), HATU, HOAt were purchased from GL Biochem (Shanghai) Ltd., Shanghai, China. Fmoc-Cys(Acm)-OH and Fmoc-Cys(Mob)-OH were from Bachem, Bubendorf, Germany. Fmoc-Lys(Boc)-Wang resin, TFA and PyOxim were from Merck, Darmstadt, Germany. Fmoc-Lys(Boc)-HMPA ChemMatrix resin was from PCAS BioMatrix, Inc., St-Jean-sur-Richelieu, QC, Canada. Fmoc-Ser(tBu)-Thr(ψMe,Mepro)-OH, Fmoc-Leu-Ser(ψMe,Mepro)-OH, Fmoc-Asn(Trt)-Thr(ψMe,Mepro)-OH, Cys(Mmt)-OH and Fmoc-Sec(Mob)-OH from Novabiochem, Läufelfingen, Switzerland. Fmoc-Lys(Boc)-PHB resin was purchased from Rapp Polymere, Tübingen, Germany. N-chlorosuccinimide was from Sigma-Aldrich, Darmstadt, Germany. Commercial, native HBD-3 was purchased from Peptide Institute, Inc., Osaka, Japan.
8
Synthesis and Evaluation of Functional Molecules
9
Borohydride Reduction Reactions Optimization
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Unless otherwise noted, all manipulations
were carried out under open air conditions. 11B,19F,13C, and 1H NMR spectra were recorded at
room temperature, on a Varian INOVA 300 MHz NMR spectrophotometer.
Chemical shifts (δ values) are reported in parts per million
relative to BF3·Et2O for 11B
NMR. Data are reported as: δ value, multiplicity (s, singlet;
d, doublet; t, triplet; q, quartet; p, pentet; h, hextet; hept, heptet;
m, multiplet; br, broad) and integration. All solvents for routine
isolation of products were reagent-grade. Sodium borohydride (powder,
purity >99% by hydride estimation 1) was purchased from Oakwood
Chemical.
Tetrahydrofuran (THF, ACS reagent >99.0% containing 0.004% water
and
0.025% BHT), toluene (anhydrous, ≥99.8%), iodine (ACS reagent,
≥99.8%), bromine (reagent grade), N-chlorosuccinimide
(ReagentPlus, 99%), and N-bromosuccinimide (ReagentPlus,
99%) were purchased from Sigma-Aldrich. All amines, aryl halides,
and pinacol were purchased from commercial sources and used without
further purification. Flash chromatography was performed using silica
gel 40–63 um, 60 Å with diethyl ether as the eluent.
were carried out under open air conditions. 11B,19F,13C, and 1H NMR spectra were recorded at
room temperature, on a Varian INOVA 300 MHz NMR spectrophotometer.
Chemical shifts (δ values) are reported in parts per million
relative to BF3·Et2O for 11B
NMR. Data are reported as: δ value, multiplicity (s, singlet;
d, doublet; t, triplet; q, quartet; p, pentet; h, hextet; hept, heptet;
m, multiplet; br, broad) and integration. All solvents for routine
isolation of products were reagent-grade. Sodium borohydride (powder,
purity >99% by hydride estimation 1) was purchased from Oakwood
Chemical.
Tetrahydrofuran (THF, ACS reagent >99.0% containing 0.004% water
and
0.025% BHT), toluene (anhydrous, ≥99.8%), iodine (ACS reagent,
≥99.8%), bromine (reagent grade), N-chlorosuccinimide
(ReagentPlus, 99%), and N-bromosuccinimide (ReagentPlus,
99%) were purchased from Sigma-Aldrich. All amines, aryl halides,
and pinacol were purchased from commercial sources and used without
further purification. Flash chromatography was performed using silica
gel 40–63 um, 60 Å with diethyl ether as the eluent.
10
Synthesizing 1-Acetyl-1-Cyclohexene
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1-Acetyl-1-cyclohexene (≥98% purity), propionic acid (≥99.5% purity), N-bromosuccinimide (NBS), N-Chlorosuccinimide (NCS) were purchased from Sigma–Aldrich. Triethylorthoacetate (≥98% purity) were purchased from Fluka. Iodine, potassium iodide, hydrochloric acid, sodium bicarbonate, sodium hydroxide and sodium thiosulphate were purchased from POCh (Gliwice, Poland). All the solvents used in column chromatography, purchased from Chempur, were of analytical grade.
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