Before complex formation, TCEP was removed from each protein preparation using Superdex 75 10/300 GL column. TCEP-free Syb (100 to 200 μM) was mixed with 400 to 500 μM 2,2-dithiodipyridine (Sigma-Aldrich). The reaction was monitored at 343 nm using an Agilent 8453 ultraviolet-visible spectrometer. Upon completion, the excess of 2,2-dithiodipyridine was removed using a PD MiniTrap G-25 Sephadex column with 100 mM phosphate at pH 7.0, 100 mM NaCl, 6 M urea, and 0.2 mM EDTA as the elution buffer. An excess of activated Syb was then mixed with TCEP-free SyxLE and left incubated at room temperature overnight. The efficiency of disulfide bond formation between Syb and SyxLE was estimated on the basis of the absorbance at 343 nm. The SyxLE/Syb conjugate was purified by size exclusion chromatography using Superdex 75 10/300 GL and 20 mM Hepes (pH 7.4), 150 mM KCl as the running buffer. TCEP-free Munc18-1 D326K was then mixed with purified SyxLE/Syb and left rotated overnight at room temperature. The template complex was finally purified by size exclusion chromatography using a Superdex 75 10/300 GL column in 20 mM Hepes (pH 7.4) with 150 mM KCl.
2 2 dithiodipyridine
Manufactured by Merck Group
Sourced in United States
2,2′-dithiodipyridine is a laboratory reagent used in various analytical and synthetic applications. It is a colorless crystalline solid with a molecular formula of C10H8N2S2. The product's core function is to serve as a sulfhydryl-reactive agent, facilitating the detection and quantification of thiol-containing compounds.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 8453, by Agilent Technologies (1 mentions)
Fluozin 3, by Thermo Fisher Scientific (1 mentions)
Alpha lipoic acid, by Merck Group (1 mentions)
Cystamine dihydrochloride, by Merck Group (1 mentions)
Enspire multilabel plate reader, by PerkinElmer (1 mentions)
Phenyl disulfide, by Merck Group (1 mentions)
4 dimethylaminopyridine dmap, by Merck Group (1 mentions)
Hydroxybenzotriazole, by Merck Group (1 mentions)
Glutaric anhydride, by Merck Group (1 mentions)
Triphosgene, by Merck Group (1 mentions)
1 3 diisopropylcarbodiimide, by Merck Group (1 mentions)
Benzotriazol 1 yl oxytripyrrolidinophosphonium hexafluorophosphate, by Merck Group (1 mentions)
4 amino tempo, by Merck Group (1 mentions)
O phthalaldehyde, by Merck Group (1 mentions)
Xylenol orange, by Merck Group (1 mentions)
2 4 dinitrophenylhydrazine, by Merck Group (1 mentions)
Acetylthiocholine iodide, by Merck Group (1 mentions)
2 4 6 tripyridyl s triazine, by Merck Group (1 mentions)
Isopropyl alcohol, by Merck Group (1 mentions)
Dichloromethane, by Merck Group (1 mentions)
7 protocols using 2 2 dithiodipyridine
1
Formation of SNARE Complex Template
2
Zn2+ Ejection from SARS-CoV-2 Proteins
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Zn2+-ejecting
agents were purchased from Sigma-Aldrich (USA), including 2,2′-dithiobis(benzothiazole),
cystamine dihydrochloride, 5,5′-dithiobis(2-nitrobenzoic acid)
(DTNB), phenyl disulfide, 3-nitrobenzamide, tetraethylthiuram disulfide
(disulfiram), 2,2′-dithiodipyridine, (±)alpha-lipoic acid,
ebselen, and cisplatin. The Zn2+-specific fluorophore FluoZinTM-3
(Invitrogen/Life Technologies) was used to monitor the release of
Zn2+ ions from SARS-CoV-2 nsp13 and nsp14. The Zn2+-ejecting agents were dissolved in DMSO to a stock solution of 100
μM. SARS-CoV-2 His-tagged nsp13 (5 μM) and nsp14 (5 μM)
were respectively mixed with each Zn2+-ejecting agent (5
μM) and FluoZin-3 (1 μM) in a total reaction volume of
200 μL at room temperature. Fluorescence emission was then measured
by EnSpire Multilabel Plate Reader (PerkinElmer, USA) at an excitation
wavelength of 494 nm and emission wavelength of 516 nm.
agents were purchased from Sigma-Aldrich (USA), including 2,2′-dithiobis(benzothiazole),
cystamine dihydrochloride, 5,5′-dithiobis(2-nitrobenzoic acid)
(DTNB), phenyl disulfide, 3-nitrobenzamide, tetraethylthiuram disulfide
(disulfiram), 2,2′-dithiodipyridine, (±)alpha-lipoic acid,
ebselen, and cisplatin. The Zn2+-specific fluorophore FluoZinTM-3
(Invitrogen/Life Technologies) was used to monitor the release of
Zn2+ ions from SARS-CoV-2 nsp13 and nsp14. The Zn2+-ejecting agents were dissolved in DMSO to a stock solution of 100
μM. SARS-CoV-2 His-tagged nsp13 (5 μM) and nsp14 (5 μM)
were respectively mixed with each Zn2+-ejecting agent (5
μM) and FluoZin-3 (1 μM) in a total reaction volume of
200 μL at room temperature. Fluorescence emission was then measured
by EnSpire Multilabel Plate Reader (PerkinElmer, USA) at an excitation
wavelength of 494 nm and emission wavelength of 516 nm.
3
Synthesis of Peptide-Drug Conjugates
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All required organic solvents were purchased from Wilkem Scientific (Pawtucket, RI, USA). The Fmoc-protected building block of amino acids, including Fmoc-Trp(Boc)-OH, Fmoc-Arg(Pbf)-OH, Dde-K(Boc)-OH, Boc-βAla-OH, 3-(tritylthio)propionic acid, and H-Trp(Boc)-2-chlorotrityl resin, were purchased from Chem-Impex International Inc. (Wood Dale, IL, USA). Hydroxy-7-azabenzotriazole (HOAT), 1,3-diisopropylcarbodiimide (DIC), O-(benzotriazole-1-yl)-N,N,N’,N’-tetramethyluroniumhexafluorophosphate (HBTU), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), and hydroxybenzotriazole (HOBt), glutaric anhydride (95%), triphosgene (99%), 4-dimethylaminopyridine (DMAP, 99%), N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), β-mercaptoethylamine hydrochloride (cystamine hydrochloride), and 2,2′-dithiodipyridine were purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI, USA). Camptothecin (CPT) was purchased from Santa Cruz Biotech, Inc. (Dallas, TX, USA) and Paclitaxel (PTX) was purchased from Euroasias Group Inc. (Woodbridge, NJ, USA).
4
Spin Label and Redox Assay Reagents
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The following chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA): 4-iodoacetamide-TEMPO (iodoacetamide spin label; ISL), 4-Amino-TEMPO (tempamine), 2, 4, 6-tripyridyl-s-triazine (TPTZ), o-phthalaldehyde (OPA), xylenol orange, 5-dithiobis-2-nitrobenzoic acid (DTNB), acetylthiocholine iodide, 2, 4-dinitrophenylhydrazine (DNPH), and 2, 2′-dithiodipyridine. Bis-(2, 2, 5, 5-tetramethyl-3-imidazoline-1-oxyl-4-yl) disulfide biradical (•RSSR•) was obtained from Enzo Life Sciences, Inc. (New York, USA). All other reagents of analytical purity were obtained from POCH S.A. (Gliwice, Poland).
5
Silica-Filled Styrene-Butadiene Rubber Compounding
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(3-mercaptopropyl)methyldimethoxysilane (MPDMS, ≥95%), 2,2’-dithiodipyridine, dichloromethane (anhydrous, 99.8%), petroleum ether, and isopropyl alcohol (IPA, 99.7%) were purchased from Sigma-Aldrich, St. Louis, MO, USA. SBR 1723 (styrene content: 23.5%, oil type: TDEA 37.5 phr, mooney viscosity (ML1 + 4): 49, coagulant: acid salt) was obtained from Kumho Petrochemical, Seoul, Korea. F-175 silica cake (pH: 7.0, loss on drying/105 °C × 2H: 6.0%, ignition loss at 1000 °C: 13.0%, oil absorption: 250 mL/100 g, Brunaeur–Emmett–Teller (BET): 175 m2/g) was purchased from Namhae Chemical, Yeosu, Korea. Bis[3-(triethoxysilyl)propyl] disulfide (TESPD (Si-75)) was purchased from Evonik Industries, Essen, Germany. Sodium hydroxide (NaOH, ≥96%) was obtained Samchun, Seoul, Korea. Various compounding processing additives such as zinc oxide (ZnO), stearic acid (S/A), and N-(1,3-dimethyl-butyl)-N’-phenyl-p-phenylenediamine (6PPD) were purchased from Sigma-Aldrich, USA. In the final compounding step, sulfur (Samchun, Seoul, Korea) was used as a crosslinking agent. N-cyclohexyl-2-benzothiazole sulfonamide (CBS) (Tokyo Chemical Industry, Tokyo, Japan) and diphenyl guanidine (DPG, Sigma-Aldrich, St. Louis, MO, USA) were used as crosslinking accelerators.
6
Synthesis and Purification of Thiol-Modified Oligonucleotides
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1.0 mg of an oligonucleotide (Biomers) composed of 30 cytosines with a 5′-thiol (hexamethylene linker) and a 3′-biotin (triethylene glycol spacer) was dissolved in 1 mL of 10 mM Tris·HCl, 1 mM EDTA, 5 mM TCEP, pH 7.5. After heating at 65 °C for 1.5 h, TCEP was removed with an Amicon Ultra column (0.5 mL, 3 kDa). The reduced oligonucleotide was then treated with the maleimide-CBT crosslinker (15 equivalents dissolved in a minimal volume of dimethyl sulfoxide) at room temperature for 2 h. The modified oligonucleotide (5′-CBT-oligo(dC)30-biotin-3′) was purified by ion-exchange chromatography on a monoQ FF Sepharose column (GE Healthcare) with an AKTApurifier FPLC (GE Healthcare) eluted with 0–1 M KCl in TE buffer (10 mM Tris·HCl, 1 mM EDTA, pH 8.0). The identity of the product was confirmed by MS (Supplementary Fig. 1c–f ).
5′-S-thiopyridyl-oligo(dA)30-biotin-3′ was synthesized by incubating 1.0 mg of a reduced oligonucleotide composed of 30 adenines with a 5′-thiol (hexamethylene linker) and a 3′-biotin (triethylene glycol spacer) with 20 mM 2,2′-dithiodipyridine (Sigma) in acetonitrile for 2 h at room temperature (Supplementary Fig.3b ). The modified oligonucleotide was purified by ion-exchange chromatography on the monoQ FF Sepharose column.
5′-S-thiopyridyl-oligo(dA)30-biotin-3′ was synthesized by incubating 1.0 mg of a reduced oligonucleotide composed of 30 adenines with a 5′-thiol (hexamethylene linker) and a 3′-biotin (triethylene glycol spacer) with 20 mM 2,2′-dithiodipyridine (Sigma) in acetonitrile for 2 h at room temperature (Supplementary Fig.
7
Synthesis of Multifunctional Polymer Therapeutics
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