Boltorn H40 (Sigma-Aldrich) was purified by precipitation from acetone and diethyl ether and dried under a vacuum at room temperature before use. Amino acids, coupling agents and the Wang resin (0.87 mmol/g) were obtained from EMD Millipore USA and used as received. The Rink amide resin (Rink amide chemMatrix, 0.49 mmol/g) was purchased from Biotage. Fmoc–N–amido–dPEG4–acid (Quantabiode-sign), mannosamine hydrochloride (Aldrich), triethylamine (TEA, Aldrich), dimethylamino pyridine (Aldrich), azido-dPEG12–NHS ester (Quanta Biodesign), pHRodo red succinimidyl ester (Thermofisher), O-(2-azidoethyl)-O′-methyl-triethylene glycol (Aldrich), 1,2-ethanedithiol (EDT, Al-drich), phenol (Acros Organics), diisopropylethylamine (DIPEA, Aldrich), trifluoroacetic acid (TFA, Alfa Aesar), thioanisole (Aldrich), triisopropylsilane (TIPS, Aldrich), succinic anhydride (Acros Organics), N-(3-(dimethylamino)-propyl)-N′-ethylcarbodiimide hydrochloride, EDC·HCl (Al-drich), N-hydroxybenzotriazole (HOBT, Aldrich) and dibenzylcylooctyne (Click Chemistry Tools) were used as received. Methanol (Fisher) and dimethylformamide (Fisher) were dried over molecular sieves (Aldrich) of 3 and 4 Å, respectively. Azidovaleric acid was synthesized in accordance with a previously published protocol.70 All other solvents were purchased from Fisher at the highest purity available and used as received.
1 2 ethanedithiol edt
Manufactured by Thermo Fisher Scientific
Sourced in Belgium, United States
1,2-Ethanedithiol (EDT) is a colorless liquid chemical compound with the molecular formula C2H6S2. It is a dithiol, meaning it contains two sulfhydryl (-SH) functional groups. EDT is commonly used as a chemical reagent and an intermediate in the synthesis of various organic compounds.
Automatically generated - may contain errors
Lab products found in correlation
Dimethylaminopyridine, by Merck Group (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Phenol, by Thermo Fisher Scientific (1 mentions)
Molecular sieves, by Merck Group (1 mentions)
Dimethylformamide, by Thermo Fisher Scientific (1 mentions)
Succinic anhydride, by Thermo Fisher Scientific (1 mentions)
Trifluoroacetic acid tfa, by Thermo Fisher Scientific (1 mentions)
N 3 dimethylaminopropyl n ethylcarbodiimide hydrochloride, by Merck Group (1 mentions)
Triethylamine tea, by Merck Group (1 mentions)
Phrodo red succinimidyl ester, by Thermo Fisher Scientific (1 mentions)
Wang resin, by Merck Group (1 mentions)
Thioanisole, by Thermo Fisher Scientific (1 mentions)
Acetic anhydride ac2o, by Merck Group (1 mentions)
N methylmorpholine nmm, by Merck Group (1 mentions)
Fmoc d phe oh, by Iris Biotech (1 mentions)
Acetonitrile, by Scharlab (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Flash edt2, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Collagenase type 4, by Thermo Fisher Scientific (1 mentions)
5 protocols using 1 2 ethanedithiol edt
1
Synthesis and Characterization of Functionalized Boltorn H40
2
Synthesis of Triazolyl-Containing MT-II Analogues
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The ω-alkynyl-alcohols
were purchased from Alfa Aesar; Fmoc-l -Trp-(Boc)-OH, Fmoc-d -Phe-OH, and Fmoc-l -Pra-OH amino acids, Fmoc-Rink
amide resin, and HOBt were purchased from Iris Biotech GmbH (Marktredwitz,
Germany); Fmoc-l -Arg(Pbf)-OH and Fmoc-l -His(Trt)-OH
amino acids were purchased from CBL (Patras, Greece); Fmoc-l -Nle was purchased from NeoMPS (Strasbourg, France); TBTU from Advanced
Biotech Italy (Milan, Italy). Peptide-synthesis grade N,N-dimethylformamide (DMF) was purchased from Scharlau
(Barcelona, Spain); acetonitrile from Carlo Erba (Milan, Italy); dichloromethane
(DCM), trifluoroacetic acid (TFA), piperidine, acetic anhydride (Ac2O), and N-methyl morpholine (NMM) were purchased
from Sigma-Aldrich (Milan, Italy). The scavengers for cleavage of
peptides from resin, 1,2-ethanedithiol (EDT), thioanisole, and phenol
(PhOH), were purchased from Acros Organics (Geel, Belgium), Jansenn
Chimica (Beerse, Belgium), and Carlo Erba (Milan, Italy).
The
purity of the final linear precursors (I′–X′)
and the [1,2,3]triazolyl-containing MT-II analogues (I–X) was
established by analytical RP-HPLC and exceeded 95%, their structural
integrity was established by ESI-MS.
were purchased from Alfa Aesar; Fmoc-
amide resin, and HOBt were purchased from Iris Biotech GmbH (Marktredwitz,
Germany); Fmoc-
amino acids were purchased from CBL (Patras, Greece); Fmoc-
Biotech Italy (Milan, Italy). Peptide-synthesis grade N,N-dimethylformamide (DMF) was purchased from Scharlau
(Barcelona, Spain); acetonitrile from Carlo Erba (Milan, Italy); dichloromethane
(DCM), trifluoroacetic acid (TFA), piperidine, acetic anhydride (Ac2O), and N-methyl morpholine (NMM) were purchased
from Sigma-Aldrich (Milan, Italy). The scavengers for cleavage of
peptides from resin, 1,2-ethanedithiol (EDT), thioanisole, and phenol
(PhOH), were purchased from Acros Organics (Geel, Belgium), Jansenn
Chimica (Beerse, Belgium), and Carlo Erba (Milan, Italy).
The
purity of the final linear precursors (I′–X′)
and the [1,2,3]triazolyl-containing MT-II analogues (I–X) was
established by analytical RP-HPLC and exceeded 95%, their structural
integrity was established by ESI-MS.
3
Optimized Labeling of β-Tubulin in Cells
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The protocol for the association of FlAsH-EDT2 with β-tubulin-TC in cell was adapted from the previous study (Hoffmann et al., 2010 (link)) so as to maximize the labeling fraction while maintaining cell viability. The engineered U2OS cells expressing β-tubulin-TC were grown to 80~90% confluency in a 30 mm cell culture dish, and then were gently washed with Opti-MEM (Thermo Fisher) twice, and then stained in 2 ml Opti-MEM with 1 μM FlAsH-EDT2 (Thermo Fisher) for 2 hr. To reduce the non-specific binding of FlAsH, the stained cells were subsequently incubated in Opti-MEM containing 250 μM 1,2-Ethanedithiol (EDT, Alfa Aesar) for 10 min, followed by a gentle wash with Opti-MEM. The cells were incubated in DMEM with 10% FBS for 6~10 hr before imaging, because they were found to be interphase-arrested for the first ~5 hr after the incubation with 250 μM EDT. Every buffers and media above were pre-warmed at 37°C before use. All incubation steps were performed at 37°C in a humidified atmosphere with 5% CO2.
4
Peptide Synthesis Using Rink Amide Resin
5
Peptide Synthesis and Cell Viability Assay
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TentaGel S Resin was purchased from Rapp Polymere (Germany, loading 0.31 mmol/g). 9-Fluorenylmethoxycarbonyl (Fmoc)-protected amino acids, 2-(1H-benzotriazole-1-yl)-1,1,3,3- tetramethyluronium hexafluoro-phosphate (HBTU), and Wang resin were obtained from GL Biochem (China). Trifluoroacetic acid (TFA), fluorescein isothiocyanate (FITC), N-methyl morpholine (NMM), piperidine, and N,N′-dimethylformamide (DMF) were all from Beijing Chemical Plant (China). 1,2-Ethanedithiol (EDT) was purchased from Alfa Aesar (USA). Dimethyl sulfoxide (DMSO) was purchased from Aldrich Chemical Co. and used without further purification. Cyanogen bromide (CNBr) was from J&K Chemical (China). The HeLa cell lines were received from the Cell Culture Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). Cell culture medium and fetal bovine serum were from WisentInc (Multicell, WisentInc, St. Bruno. The cell counting kit-8 assay (CCK-8) (Beyotime Institute of Biotechnology, China) was used. Hela cells were maintained Dulbecco’s modified eagle’s medium (DMEM) with 10% fetal bovine serum and 1% penicillin. All cells were cultured in a humidified atmosphere containing 5% CO2 at 37 °C.
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