HR-MS Analysis of the Covalent Binding of Edaravone to 5-Formylpyrimidine Bases and a DNA Oligonucleotide Containing a 5-Formylcytidine Residue

Abstract

Rationale: Edaravone (EDA) is a radical scavenger and an antioxidant drug approved to treat amyotrophic lateral sclerosis and used as a research tool to explore treatment of neurodegenerative diseases and cancers. It is also a reactive agent, known as PMP (1-phenyl-3-methyl-5-pyrazolone), used for the analysis of polysaccharides composition. EDA can react with sugars and aromatic aldehydes. In this context, we have investigated the reactivity of EDA toward the biologically relevant formylated nucleobases, nucleosides, and an oligonucleotide containing a formylated residue.

Methods: The formation of both mono- and bis-adducts between EDA and the formylated nucleobases (5-formyluracil (5fU) and 5-formylcytosine (5fC)) or the corresponding nucleosides 5-fdU and 5-fdC was characterized using high-resolution mass spectrometry (HR-MS). Similarly, the covalent binding of EDA to an 8-mer palindromic oligonucleotide d (TATG[*C]ATA) containing a single 5-fdC residue [*C] under physiological conditions was investigated using mass spectrometry.

Results: For the first time, EDA is shown to react with formylated pyrimidines. Covalent and stable adducts were identified. EDA was found to react efficiently with the formylated oligonucleotide to generate mono- and bis-adducts. The rate of formation of the mono-adduct was five times higher than that of the bis-adduct. The reaction of EDA with aldehydic DNA modifications such as 5fU/5fC may have important consequences in terms of gene expression.

Conclusions: These observations raise implications for an epigenetic contribution to the mechanism of action of EDA. The biological implications of our in vitro results are discussed, notably in the frame of neurodegenerative diseases and cancers.

Keywords: Edaravone; covalent adducts; epigenetic; nucleosides; oligonucleotide.

FIGURE 1

Structures of edaravone, the formylated pyrimidines bases or nucleosides, and the oligonucleotide d (TATG*CATA) incorporating a 5‐fdC residue (*C).

FIGURE 2

Reaction of edaravone with the formylated bases and nucleosides. (a) Mass spectra obtained upon reaction of EDA (0.5 mM) with the nucleobases 5‐formyluracil (5fU) or 5‐formylcytosine (5fC) (0.5 mM), for 24 h at 37°C in PBS buffer, diluted to 10 μM in methanol and recorded in the ESI negative‐ion mode. (b) Mass spectra obtained upon reaction of EDA (0.5 mM) with the nucleosides 2′‐deoxy‐5‐formyluracil (5fdU) or 2′‐deoxy‐5‐formyl cytidine (5fdC) (0.5 mM), for 24 h at 37°C in PBS buffer, diluted to 10 μM in methanol and recorded in the ESI negative‐ion mode.

FIGURE 3

Reaction scheme of EDA with 5‐formylcytosine (5fC) leading to the formation of the mono‐ and bis‐adducts. Molecular models of the two adducts are presented.

FIGURE 4

Compared reactivity of edaravone toward the formylated bases and nucleosides. The histograms refer to the log (intensity) for each adducts observed at 5 or 24 h of reaction at 37°C in PBS buffer. The level of mono‐ and bis‐adducts were displayed in black and grey bars, respectively.

FIGURE 5

Reaction of edaravone with the oligonucleotide. (a) Mass spectrum of the formylated oligonucleotide d (TATG*CATA) (5 μM) in a PBS medium. (b) Mass spectrum obtain upon reaction of EDA (2000 μM) and the formylated oligonucleotide d (TATG*CATA) (5 μM) for 24 h at 37°C in PBS buffer in the presence of triethanolamine (TEA, 2.23 mM) and 2% DMSO, prior to direct infusion of the mixture in the ESI negative‐ion mode.

FIGURE 6

Deconvoluted mass spectrum obtained upon reaction of the formylated oligonucleotide d (TATG*CATA) with edaravone for 24 h at 37°C. Experimental conditions as for Figure 5.

FIGURE 7

Kinetic analysis of the formation of the mono‐adduct (♦) and bis‐adduct (●) upon reaction of edaravone with the formylated oligonucleotide d (TATG*CATA). Edaravone (2000 μM) was incubated with the modified oligonucleotide (5 μM) at 37°C for up to 25 h min in PBS buffer, pH 7.4. The reaction products were analyzed in the ESI negative‐ion mode. Intensity ratio [M1‐3H]³⁻/[M0‐3H]³⁻ and intensity ratio [M2‐3H]³⁻/[M0‐3H]³⁻ versus time are presented with solid and dotted lines, respectively.

FIGURE 8

Mechanism of the expected chemistry between EDA and the oligonucleotide d (TATG*CATA) containing a 2′‐deoxy‐5‐formyl cytidine (*C) residue. The top and bottom parts show the potential reaction mechanism leading to the formation of the mono‐ and bis‐adducts, respectively. The keto‐enol tautomerism of EDA is represented.