Evaluation of α-cyano ethers as fluorescent substrates for assay of cytochrome P450 enzyme activity

Kyung Don Kang, Paul D. Jones, Huazhang Huang, Rong Zhang, Lyudmila A. Mostovich, Craig E. Wheelock, Takaho Watanabe, Lyudmila F. Gulyaeva, Bruce D. Hammock

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

We have previously reported the synthesis of four α-cyano-containing ethers based on 2-naphthaldehyde (2-NA) as cytochrome P450 (P450) fluorescent substrates. Activity detection was based on the formation of fluorescent 2-NA following substrate hydrolysis. A major limitation of these substrates was the need to remove NADPH, a required cofactor for P450 oxidation, before measuring 2-NA fluorescence. In this article, we report the synthesis of a new series of novel P450 substrates using 6-dimethylamino-2-naphthaldehyde (6-DMANA), which has a green fluorescent emission that is well separated from the NADPH spectrum. A major advantage of the 6-DMANA substrates is that NADPH removal is not required before fluorescence detection. We used eight α-cyano ether-based substrates to determine the O-dealkylation activity of human, mouse, and rat liver microsomes. In addition, substrate activities were compared with the commercial substrate 7-ethoxyresorufin (7-ER). The catalytic turnover rates of both the 6-DMANA- and 2-NA-based substrates were in some cases threefold faster than the catalytic turnover rate of 7-ER. The 2-NA-based substrates had greater turnover than did the 6-DMANA-based substrates. Murine and rat liver microsomes prepared from animals that had been treated with various P450 inducers were used to examine for isozyme-selective turnover of the substrates. The vastly improved optical properties and synthetic flexibility of the α-cyano ether compounds suggest that they are possibly good general P450 substrates.

Original languageEnglish (US)
Pages (from-to)183-192
Number of pages10
JournalAnalytical Biochemistry
Volume344
Issue number2
DOIs
StatePublished - Sep 15 2005

Keywords

  • α-Cyanohydrin ether
  • Cytochrome P450
  • Fluorescent substrate
  • Liver microsomes

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Fingerprint Dive into the research topics of 'Evaluation of α-cyano ethers as fluorescent substrates for assay of cytochrome P450 enzyme activity'. Together they form a unique fingerprint.

  • Cite this

    Kang, K. D., Jones, P. D., Huang, H., Zhang, R., Mostovich, L. A., Wheelock, C. E., Watanabe, T., Gulyaeva, L. F., & Hammock, B. D. (2005). Evaluation of α-cyano ethers as fluorescent substrates for assay of cytochrome P450 enzyme activity. Analytical Biochemistry, 344(2), 183-192. https://doi.org/10.1016/j.ab.2005.06.032