Isolation and structural identification of a direct-acting mutagen derived from N-nitroso-N-methylpentylamine and Fenton's reagent with copper ion

Motofumi Miura, Keiko Inami, Masafumi Yoshida, Kentaro Yamaguchi, Tadahiko Mashino, Masataka Mochizuki

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

N-Nitrosodialkylamines show their mutagenicity by forming α-hydroxynitrosamines in the presence of rat S9 mix in the Ames assay. The hydroxyl radical derived from Fe 2+-H 2O 2 (Fenton's reagent) with Cu 2+ activates N-nitrosamines, with an alkyl chain longer than a propyl constituent, to a direct-acting mutagen. The reactivity of Fe 2+-Cu 2+-H 2O 2 on nitrosamines in relation to their metabolic activation is not fully characterized. Here, we report the identification of the direct-acting mutagen derived from N-nitroso-N-methylpentylamine (NMPe) in the presence of Fe 2+, Cu 2+, H 2O 2 and nitric oxide (NO), which is a product of nitrosamine metabolism. A dichloromethane extract of the NMPe reaction mixtures was fractionated by silica gel column chromatography several times and by a preparative high performance liquid chromatography (HPLC); we obtained white crystals as a product. The direct-acting mutagen that was isolated was provisionally identified as 5-ethyl-5-nitro-1-pyrazoline 1-oxide by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, infrared (IR) spectroscopy and X-ray crystallography. To confirm the structure of the mutagen, the authentic compound was synthesized from 2-nitrobutene and diazomethane, followed by N-oxidation with m-chloroperoxybenzoic acid. The 1H NMR spectral data from the direct-acting mutagen that was synthesized was identical to the data from the isolated mutagen. Furthermore, the authentic 5-ethyl-5-nitro-1-pyrazoline 1-oxide was mutagenic in Salmonella typhimurium TA1535. The results showed that 5-ethyl-5-nitro-1-pyrazoline 1-oxide was a direct-acting mutagen derived from the reaction of NMPe and Fe 2+-Cu 2+-H 2O 2-NO.

Original languageEnglish
Pages (from-to)5693-5697
Number of pages5
JournalBioorganic and Medicinal Chemistry
Volume19
Issue number18
DOIs
Publication statusPublished - 2011 Sep 15

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Mutagens
Copper
Ions
Nitrosamines
Oxides
Nitric Oxide
Magnetic Resonance Spectroscopy
Diazomethane
Column chromatography
Salmonella
Methylene Chloride
X ray crystallography
X Ray Crystallography
Silica Gel
High performance liquid chromatography
Salmonella typhimurium
Fenton's reagent
Metabolism
Hydroxyl Radical
Nuclear magnetic resonance spectroscopy

Keywords

  • Fenton's reagent
  • Hydroxyl radical
  • Metabolic activation
  • N-Nitrosodialkylamine
  • Nitric oxide
  • Pyrazoline

ASJC Scopus subject areas

  • Pharmaceutical Science
  • Drug Discovery
  • Organic Chemistry
  • Molecular Medicine
  • Molecular Biology
  • Clinical Biochemistry
  • Biochemistry

Cite this

Isolation and structural identification of a direct-acting mutagen derived from N-nitroso-N-methylpentylamine and Fenton's reagent with copper ion. / Miura, Motofumi; Inami, Keiko; Yoshida, Masafumi; Yamaguchi, Kentaro; Mashino, Tadahiko; Mochizuki, Masataka.

In: Bioorganic and Medicinal Chemistry, Vol. 19, No. 18, 15.09.2011, p. 5693-5697.

Research output: Contribution to journalArticle

Miura, Motofumi ; Inami, Keiko ; Yoshida, Masafumi ; Yamaguchi, Kentaro ; Mashino, Tadahiko ; Mochizuki, Masataka. / Isolation and structural identification of a direct-acting mutagen derived from N-nitroso-N-methylpentylamine and Fenton's reagent with copper ion. In: Bioorganic and Medicinal Chemistry. 2011 ; Vol. 19, No. 18. pp. 5693-5697.
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AU - Yamaguchi, Kentaro

AU - Mashino, Tadahiko

AU - Mochizuki, Masataka

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AB - N-Nitrosodialkylamines show their mutagenicity by forming α-hydroxynitrosamines in the presence of rat S9 mix in the Ames assay. The hydroxyl radical derived from Fe 2+-H 2O 2 (Fenton's reagent) with Cu 2+ activates N-nitrosamines, with an alkyl chain longer than a propyl constituent, to a direct-acting mutagen. The reactivity of Fe 2+-Cu 2+-H 2O 2 on nitrosamines in relation to their metabolic activation is not fully characterized. Here, we report the identification of the direct-acting mutagen derived from N-nitroso-N-methylpentylamine (NMPe) in the presence of Fe 2+, Cu 2+, H 2O 2 and nitric oxide (NO), which is a product of nitrosamine metabolism. A dichloromethane extract of the NMPe reaction mixtures was fractionated by silica gel column chromatography several times and by a preparative high performance liquid chromatography (HPLC); we obtained white crystals as a product. The direct-acting mutagen that was isolated was provisionally identified as 5-ethyl-5-nitro-1-pyrazoline 1-oxide by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, infrared (IR) spectroscopy and X-ray crystallography. To confirm the structure of the mutagen, the authentic compound was synthesized from 2-nitrobutene and diazomethane, followed by N-oxidation with m-chloroperoxybenzoic acid. The 1H NMR spectral data from the direct-acting mutagen that was synthesized was identical to the data from the isolated mutagen. Furthermore, the authentic 5-ethyl-5-nitro-1-pyrazoline 1-oxide was mutagenic in Salmonella typhimurium TA1535. The results showed that 5-ethyl-5-nitro-1-pyrazoline 1-oxide was a direct-acting mutagen derived from the reaction of NMPe and Fe 2+-Cu 2+-H 2O 2-NO.

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