TFMU-ADPr triethylamine 

TFMU-ADPr triethylamine is a selective reporter substrate of SARS-CoV-2 Macro1 (IC₅₀=0.59 μM), with an excitation wavelength (λ_Ex) of 385 nm, and an emission wavelength (λ_Em) of 502 nm (or 495 nm). TFMU-ADPr triethylamine can also undergo enzymatic hydrolysis with Poly(ADP-ribose) Glycohydrolase (PARG) sourced from human, Tetrahymena thermophila and ADP-ribosylhydrolase 3 from human to release fluorophores, thereby directly reporting total poly (ADP-ribose) hydrolase activity. TFMU-ADPr triethylamine binds to the ADPr-binding site of SARS-CoV-2 Macro1, and its TFMU moiety inserts into the narrow hydrophobic groove of this protein. TFMU-ADPr triethylamine can thus be used to evaluate small-molecule inhibitors targeting PAR hydrolases under in vitro conditions, to investigate the regulatory mechanisms of ADP-ribosyl catabolic enzymes, or to detect PAR hydrolase activity in whole-cell lysate assays. TFMU-ADPr triethylamine is also applicable to COVID-19-related research^[1][2][3].

TFMU-ADPr triethylamine (0-500 μM, 0-120 μM; 5 minutes) serves as a continuous fluorescent substrate for hPARG (full-length PARG from Homo sapiens), ttPARG (PARG from Tetrahymena thermophila) and hARH3 (full-length ARH3 from H. sapiens), enabling robust enzyme kinetic assays. The corresponding K_M values for the three enzymes are 66.2 μM, 210 μM and 6.3 μM, respectively^[1].
TFMU-ADPr triethylamine (200 μM) can report the combined hydrolase activity of PARG and ARH3 in U2OS cell lysates, and this activity is detected in both wild-type cells and ARH3 knockout cells (the latter represents PARG-only activity)^[1].
TFMU-ADPr triethylamine (0-50 μM) serves as an effective reporter substrate for detecting the inhibitory activity of hARH3, which is confirmed by the potent inhibitory effect of the endogenous metabolite ADPr-Arg (K_i=18 nM)^[1].
TFMU-ADPr triethylamine (in a dose-response manner; incubation duration 30 min) potently binds SARS-CoV-2 Macro1 (IC₅₀=0.59 μM), human MacroD1, human MacroD2, VEEV Macro, and human PARP9 Macro2; compared with ADPr, it shows significantly improved binding potency for all tested macrodomains except CHIKV Macro^[2].
TFMU-ADPr triethylamine (2 mM; 5 d) binds to the conserved ADPr-binding pocket of SARS-CoV-2 Macro1, and its TFMU group forms favorable interactions with hydrophobic residues in the adjacent hydrophobic groove, thereby contributing to high binding affinity^[2].
TFMU-ADPr triethylamine acts as an inhibitor of the ADPr hydrolase activity of SARS-CoV-2 Macro1 and human MacroD1 in cell lysates^[2].
Experimental procedure for in vitro detection using TFMU-ADPr triethylamine mainly focuses on enzyme kinetics and inhibition assays, as specified below^[3]:
1. Experimental Preparation:
Establish a reaction system in a 384-well plate. The system contains 50 mM Na₂HPO₄ (pH 7.4), 10 mM MgCl₂, 5 mM DTT, TFMU-ADPr triethylamine at a specified concentration as the substrate, and the enzyme to be tested (such as hARH1, hARH3, etc.). For inhibitor studies, pre-incubate the enzyme with the inhibitor at room temperature for 5 minutes first.
2. Reaction Initiation and Signal Detection:
After shaking the reaction system for 5 seconds, record the fluorescence signal using a Molecular Devices SpectraMax M3 microplate reader. Set the excitation wavelength (λ_Ex) to 385 nm and the emission wavelength (λ_Em) to 502 nm (or 495 nm). Read each well 6 times in low-gain mode, and record the fluorescence value every 5 seconds for a continuous detection duration of 15 min.
3. Data Processing:
Determine the initial reaction rate by fitting the linear segment of the reaction progress curve. Fit the initial rate to the substrate concentration, use the nonlinear curve fitting algorithm in GraphPad Prism 6, and analyze enzyme kinetic parameters based on the Michaelis-Menton equation. For inhibition assays, use enzyme-free reactions and inhibitor-free reactions as positive and negative controls, respectively, calculate the inhibition percentage, and obtain relevant inhibition parameters through dose-response curve fitting.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

1024.42

C₂₅H₂₆F₃N₅O₁₆P₂.2.5C₆H₁₅N

Viscous Liquid

Colorless to light yellow

O[C@H]1[C@](O[C@@H]([C@H]1O)COP(OP(OC[C@H]2O[C@@H]([C@@H]([C@@H]2O)O)OC3=CC=C(C(C(F)(F)F)=CC(O4)=O)C4=C3)(O)=O)(O)=O)([H])N5C6=NC=NC(N)=C6N=C5.CCN(CC)CC.[2.5]

Room temperature in continental US; may vary elsewhere.

-20°C, sealed storage, away from moisture

*In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)

• [1]. Drown BS, et al. Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate. Cell Chem Biol. 2018;25(12):1562-1570.e19.  [Content Brief]

  [2]. Anmangandla A, et al. A Fluorescence Polarization Assay for Macrodomains Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2 Macrodomain. ACS Chem Biol. 2023;18(5):1200-1207.  [Content Brief]

  [3]. Rack JGM, et al. (ADP-ribosyl)hydrolases: Structural Basis for Differential Substrate Recognition and Inhibition. Cell Chem Biol. 2018 Dec 20;25(12):1533-1546.e12.  [Content Brief]