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The BI-3802 was designed by Boehringer Ingelheim and could be obtained free of charge through the Boehringer Ingelheim open innovation portal opnMe.com, associated with its negative control.
ADHP is a fluorescent peroxidase substrate (λex=530 nm, λem=590 nm).
Synonyms: 10-Acetyl-3,7-dihydroxyphenoxazine; A 6550; Amplex red reagent
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Liu, Huan ; Tonoyan, Lusine ; Reiz, Béla ; Siraki, Arno G ;
Abstract: Amplex Red (AR) is commonly used to detect extracellular hydrogen peroxide (H2O2) and is considered a cellimpermeable compound. However, it would appear capable of entering cells based on its phenoxazine substructure and the report of its mitochondrial membrane permeability. Additionally, myeloperoxidase (MPO) oxidation of AR produces a fluorescent compound, resorufin, which has been reported, though the mechanism is not well-studied. EPR spin trapping using glutathione (GSH) revealed that AR metabolism produced AR radicals and glutathionyl radicals (GS•). An intermediate metabolite, 3,7-dihydroxyphenoxazine, was observed by liquid chromatography-mass spectrometry (LC-MS), which supported AR radical disproportionation first and subsequently N-oxidation. Besides, in the presence of GSH, the formation of resorufin decreased significantly evidencing the reactivity of radical intermediates. Three types of AR-GS adduct were found using LC-MS and the resorufin GS-adduct was the dominant one. Regarding intracellular findings in HL-60 cells (that highly express MPO), LC-MS and fluorescence analysis showed AR penetrated the cell membrane and was oxidized by cellular MPO. Interestingly, we demonstrated that the oxidation of AR in HL-60 cells showed a significant time dependence; PF-1355, an MPO inhibitor, inhibited the oxidation of AR by MPO. Cell viability (ATP) revealed that 200 μM AR significantly decreased viability in HL-60 cells in 6 h. We also found that AR-mediated decreased total GSH and increased protein-radical formation. These findings revealed that AR is cell-permeable, and AR radicals induce cellular oxidative distress and lead to the formation of protein radicals, which correlate with the MPO-mediated mechanism of cytotoxicity.
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Keywords: Amplex Red ; Myeloperoxidase ; EPR ; Free radicals ; Oxidative stress ; Protein radicals
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| CAS No. : | 119171-73-2 |
| Formula : | C14H11NO4 |
| M.W : | 257.24 |
| SMILES Code : | CC(N1C2=C(C=C(O)C=C2)OC3=CC(O)=CC=C13)=O |
| Synonyms : |
10-Acetyl-3,7-dihydroxyphenoxazine; A 6550; Amplex red reagent
|
| English Name : | 1-(3,7-Dihydroxy-10H-phenoxazin-10-yl)ethanone |
| MDL No. : | MFCD00467861 |
* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.

| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 39% | With dihydrogen peroxide In aq. phosphate buffer for 0.25h; Irradiation; | |
| With dihydrogen peroxide | ||
| With horseradish peroxidase-biotin immobilized on microbeads; dihydrogen peroxide In dimethyl sulfoxide |
| With dihydrogen peroxide; horseradish peroxidase In phosphate buffer at 30℃; for 0.5h; Enzymatic reaction; | ||
| With Tris-HCl buffer; dihydrogen peroxide In dimethyl sulfoxide | ||
| With dihydrogen peroxide In phosphate buffer; dimethyl sulfoxide | ||
| With dihydrogen peroxide; horseradish peroxidase In phosphate buffer at 24℃; | ||
| With dihydrogen peroxide; horseradish peroxidase at 20℃; aq. phosphate buffer; Enzymatic reaction; | ||
| With superoxide dismutase; horseradish peroxidase aq. buffer; | ||
| With pyruvate oxidase; pyridoxal 5'-phosphate; 2-oxo-propionic acid; horseradish peroxidase aq. phosphate buffer; Enzymatic reaction; | ||
| With cytochrome c; cardiolipin | ||
| With CYP175A1 L80Q mutant; dihydrogen peroxide at 70℃; for 0.166667h; aq. phosphate buffer; Enzymatic reaction; | ||
| With tyrosamine; recombinant human monoamine oxidase B; horseradish peroxidase In aq. phosphate buffer at 37℃; for 0.25h; Darkness; Enzymatic reaction; | ||
| With C44H44Fe2N8O14(2+)*2Cl(1-); dihydrogen peroxide In aq. buffer for 48h; | ||
| With [Fe(2-[N-methyl-N-pyridin-2-ylmethyl(-H)]-amino-N′-quinolin-8-yl-acetamidate)(H2O)2](ClO4)2; dihydrogen peroxide In water at 25℃; | ||
| With dihydrogen peroxide; myoglobin In aq. phosphate buffer | ||
| With horseradish peroxidase (HRP)-APSN-APTES; dihydrogen peroxide In aq. phosphate buffer; dimethyl sulfoxide at 20 - 25℃; Flow reactor; Enzymatic reaction; | ||
| With dihydrogen peroxide; horseradish peroxidase In aq. phosphate buffer for 0.5h; Enzymatic reaction; | ||
| With dihydrogen peroxide In aq. buffer for 2h; | ||
| With dihydrogen peroxide; horseradish peroxidase In aq. phosphate buffer Electrochemical reaction; Enzymatic reaction; | ||
| With dihydrogen peroxide; horseradish peroxidase In aq. buffer Enzymatic reaction; | ||
| With dihydrogen peroxide | ||
| With silver orthophosphate Photolysis; | ||
| With RKRLQVQLSIRT In aq. acetate buffer | ||
| With nickel(II) oxide In aq. buffer at 20℃; for 0.5h; Darkness; | ||
| With potassium chloride; dihydrogen peroxide; horseradish peroxidase In aq. phosphate buffer at 24.84℃; | Measurement of UV-Vis Absorption and Amplex RedOxidation Kinetics. General procedure: UV-Vis absorption spectra were recordedat 298K with a Beckman DU 640 spectrophotometer using 50mM potassium phosphate buffer, pH 6.80, containing 300mMKCl as the solvent. Horseradish peroxidase (HRP) waspurchased as a lyophilized powder from Sigma-Aldrich Co.and used without further purification. The chromogenic substrate,Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine),was purchased from Sigma-Aldrich, and the reaction was monitored by following the appearance of the oxidized product,7-hydroxyphenoxazin-3-one (Resorufin) (Scheme 2), whichabsorbs light at ~570 nm.32Kinetic studies were performed on a Beckman DU640spectrometer. (d(TTAGGG))4 and (d(TTAGGGA))4 in 50mMpotassium phosphate buffer, pH 6.80, were heat-denatured at363K for 5min., followed by cooling to 298 K. Amplex Redwas dissolved in dimethylformamide to prepare a 10mM stocksolution. Heme(Fe3+) and then Amplex Red were added to 0.1 μM and 50 μM, respectively, to 10 M DNA in 50mM potassiumphosphate buffer, pH 6.80. To initiate the oxidation reactions,5200mMhydrogen peroxide (H2O2) were added to thesolution mixture. For the kinetic measurements of the HRPsystem, 1-100 μM H2O2 was added to the solution mixture of0.01 μM protein and 50 μM Amplex Red in 50mM potassiumphosphate buffer, pH 6.80, to initiate the reactions. The initialslope (R0) of the time evolution of 570-nm absorbance due toResorufin was used as an index for the peroxidase activities ofthe complexes and HRP. | |
| With gold; dihydrogen peroxide Darkness; | ||
| With dihydrogen peroxide; horseradish peroxidase In aq. phosphate buffer at 20℃; UV-irradiation; Enzymatic reaction; | ||
| With dihydrogen peroxide In aq. buffer Flow reactor; | ||
| With potassium phosphate; ethylenediaminetetraacetic acid; dihydrogen peroxide; cytochrome P450 In aq. phosphate buffer at 20℃; | Amplex Red peroxidation activity assayin the rapid screening Amplex Red peroxidation activity assay has a total of100 μL reaction volume that consist of 50 μL of enzyme extracted with Solu Lyse reagent and 50 μL of reaction mixture 2 (1 mm H2O2,10 μm Amplex Red, and 50 mm potassium phosphate buffer, pH 7.4). 1:1 v/v SoluLyse reagent and potassium phosphate buffer were used as blank, 1:1 v/v potassium phosphate buffer, and reaction mixture 2, and proteins extracted from E. coli BL21 (DE3) cells that contain pET20b mixed with reaction mixture 2 were used as negative controls. The positive control was 1 μU of HRP in1:1 v/v potassium phosphate buffer and reaction mixture 2. Samples were incubated at 25 °C for 10 min. Fluorescence emission by resorufin was measured at 595 nm with FLUO star Omega (BMG LABTECH). | |
| With dihydrogen peroxide; horseradish peroxidase In aq. phosphate buffer at 37℃; for 0.333333h; Irradiation; Enzymatic reaction; | ||
| With dihydrogen peroxide; catalase In aq. phosphate buffer; dimethyl sulfoxide Enzymatic reaction; | ||
| With 3-chloro-benzenecarboperoxoic acid In acetonitrile | ||
| With D-glu; glucose oxidase loaded polymersome vesicle; horse radish peroxidase In aq. phosphate buffer; dimethyl sulfoxide Enzymatic reaction; | ||
| With hemin; 2,3,4,5,6-pentahydroxy-hexanal; oxygen | ||
| With hemin; anhydrous sodium perchlorate; dihydrogen peroxide; 5'-6-FAM-TG GGT TAG GGA ATT CGG GTT AGG G-Iowa Black FQ-3'; 5'-CCC TAA CCC GAA TTC CCT AAC CCA-3' In aq. phosphate buffer; dimethyl sulfoxide | ||
| With horseradish peroxidase In aq. phosphate buffer at 58℃; for 0.166667h; Irradiation; Enzymatic reaction; | ||
| With dihydrogen peroxide; horseradish peroxidase Enzymatic reaction; | ||
| With dihydrogen peroxide In dimethyl sulfoxide | ||
| With dihydrogen peroxide; C10H16CuN2O9; horseradish peroxidase In aq. phosphate buffer; dimethyl sulfoxide Electrolysis; | ||
| With [Cu(2,5,8-trimethyl-1,9-bis(2-pyridyl)-2,5,8-triazanonane)](BF4)2; horseradish peroxidase In aq. phosphate buffer; dimethyl sulfoxide Enzymatic reaction; | ||
| With dihydrogen peroxide In aq. phosphate buffer | ||
| With dihydrogen peroxide; horseradish peroxidase | ||
| With H+ adsorbed BiOCl Irradiation; | ||
| With dihydrogen peroxide; C30H33ClCoN6NiO9 | ||
| With graphitic carbon nitride nanosheet In water | ||
| With dihydrogen peroxide | ||
| With dihydrogen peroxide; horseradish peroxidase In water for 0.5h; Irradiation; Enzymatic reaction; | ||
| With APEX2-displaying bacillus subtilis spore biocomposite assembled with benzalcyanoacetate-functionalized polymer In acetone | ||
| With dihydrogen peroxide; sodium 4-dodecylbenzenesulfonate; N-[4-ethoxy-4'-butoxyazobenzene]-N-octyl-N,N-dimethylammonium bromide; horseradish peroxidase In water Enzymatic reaction; | 2.7 Enzymatic reactions using Amplex red and HRP AzoN/SDBS dispersions (total concentrations: 1.0 mM) were prepared using thin-film hydration methods where distilled water (10 mL), Amplex red aqueous dispersions (5.0 mg/mL, 2.0 mL), and horseradish peroxidase (HRP) aqueous solutions (1.0 mg/mL, 10 mL) were used to hydrate the formed thin films. Then H2O2 aqueous solutions (35 wt%, 1.0 mL) were added to the AzoN/SDBS dispersions, and the samples were measured usingfluorescence spectroscopy (RF-6000, SHIMADZU, Kyoto, Japan) for 10 min. λex = 561 nm; λem = 570 to 800 nm. Regarding UV-irradiated samples, the dispersions were irradiatedwith UV for 10 min before the addition of H2O2 aqueous solutions and subsequent fluorescence measurement. |

| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With dihydrogen peroxide; native horseradish peroxidase In various solvent(s) at 25℃; for 0.333333h; | ||
| With dihydrogen peroxide at 25℃; | ||
| With myoglobin T39I, R45D, F46L, I107F, H93Nδ-methyl histidine, I28T,D45G, K63E, V21A, T95A, T67A, K140T mutant; dihydrogen peroxide In aq. phosphate buffer at 25℃; Enzymatic reaction; |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 1.b (b) (b) N-Acetyl-3,7-dihydroxyphenoxazine 2.39 g. (7 mmole) Triacetyl-3,7-dihydroxyphenoxazine and 1.9 g. (15 mmole) sodium sulphite were dissolved in 70 ml. of a dioxan-water mixture (1:1 v/v) which had been gassed with nitrogen and heated under reflux for 20 minutes. The reaction mixture was then cooled in an ice bath, acidified with 1.14 ml. (20 mmole) acetic acid, filtered off over a wadding from a small amount of insoluble material and concentrated to about 20 ml., a viscous oil hereby separating out. About 150 ml. water, which had been gassed with nitrogen, were added thereto and the mixture left to stand at 0° C. The precipitated crystals were filtered off with suction, washed with ice water and dried in a vacuum at 0° C. over anhydrous calcium chloride. Yield 1.5 g. (97% of theory) of a beige coloured product; m.p. >250° C. (decomp.). Rf value: 0.19 (TLC on silica gel; chloroform/ethyl acetate 2:1 v/v). |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With hemin; 5'-d(TTTGGGTAGGGCGGGTTGGG)-3'; ammonium acetate; oxygen at 25℃; for 0.5h; aq. buffer; |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| With dihydrogen peroxide; myoglobin In water |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| 92% | With pyridine In dichloromethane at 0 - 20℃; for 3h; | 61.1 [00325 Step 1: Aniplex Red (449 tug, 1.75 mmol) was taken up in CK2CI2 (45 mL) and cooled toO 7C. Pyridine (1.14 mE, 14.0 mmol, 8.0 eg) and trifluoromethanesulfonic anhydride (1.17 mE, 6.9 mmol,4.0 ec) were added, arid the ice bath was removed. ‘[he reaction was stirred at room temperature fhr 3 h. itwas subsequently diluted with water arid extracted with Cl-i 2C12 (2x). The combined organic extracts were washed with brine, dried (MgSO:), filtered, and concentrated in vuenu. Flash chromatography on silica gel (G---35% EtOAc/hexanes, linear gradient) affbrded 836mg (92%) of 10-acei-10J1-phenoxazine-3,7’diy1 bis(trifluoromethanesulfonate) as an offa-vhite solid. ‘11 NMR (CDC13, 400 MHz) 6 7.58 T54 (m, 21]). 7.14-”- 7.09 (in, 411), 2.35 (s, 311); ‘9FNMR(CDC13, 376 MHz) 64315 (s): tNMR(CDCh, 101 MHz)6 1 689 (C), 151 0 (C), 147.4 (C), 12c. I (C), 126.3 (Cli), 118.8 (q. Jcj- 320.9 Hz. CFO. 1 17.2 (CII), 111,1 (C K), 23.0 (CH); HRMS (ESI) caled for C16H1F6NOS2 [MH] 521.9747, found 521.9748. |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| Multi-step reaction with 3 steps 1: pyridine / dichloromethane / 3 h / 0 - 20 °C 2: tris-(dibenzylideneacetone)dipalladium(0); XPhos; caesium carbonate / 1,4-dioxane / 4 h / 80 °C / Sealed tube; Inert atmosphere 3: 2,3-dicyano-5,6-dichloro-p-benzoquinone / water; dichloromethane / 3 h / 0 - 20 °C |
| Yield | Reaction Conditions | Operation in experiment |
|---|---|---|
| Multi-step reaction with 2 steps 1: pyridine / dichloromethane / 3 h / 0 - 20 °C 2: tris-(dibenzylideneacetone)dipalladium(0); XPhos; caesium carbonate / 1,4-dioxane / 4 h / 80 °C / Sealed tube; Inert atmosphere |