Evaluation of alpha-cyano ethers as fluorescent substrates for assay of cytochrome P450 enzyme activity.
We have previously reported the synthesis of four alpha-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 alpha-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 alpha-cyano ether compounds suggest that they are possibly good general P450 substrates.
Kang
Kyung-Don
KD
Department of Entomology and Cancer Research Center, University of California, Davis, CA 95616, USA.
Jones
Paul D
PD
Huang
Huazhang
H
Zhang
Rong
R
Mostovich
Lyudmila A
LA
Wheelock
Craig E
CE
Watanabe
Takaho
T
Gulyaeva
Lyudmila F
LF
Hammock
Bruce D
BD
D000447:Aldehydes / Q000138:chemical synthesis / Q000737:chemistry*; D000818:Animals; D003486:Cyanides / Q000737:chemistry*; D019363:Cytochrome P-450 CYP1A1 / Q000032:analysis; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000096:biosynthesis; D003640:Dealkylation; D004790:Enzyme Induction; D004987:Ethers / Q000737:chemistry*; D005456:Fluorescent Dyes; D006801:Humans; D051379:Mice; D008862:Microsomes, Liver / Q000378:metabolism; D009249:NADP / Q000737:chemistry; D009281:Naphthalenes / Q000138:chemical synthesis / Q000737:chemistry*; D051381:Rats
C506914:6-dimethylamino-2-naphthaldehyde; D000447:Aldehydes; D003486:Cyanides; D004987:Ethers; D005456:Fluorescent Dyes; D009281:Naphthalenes; D009249:NADP; C018686:2-naphthaldehyde; D003577:Cytochrome P-450 Enzyme System; D019363:Cytochrome P-450 CYP1A1