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A divergent synthesis of (2E,4E,8E,10E)- and (2E,4E,8E,10Z)-N-isobutyldodeca-2,4,8,10-tetraenamides from pent-4-yn-1-ol allowed identification of the (2E,4E,8E,10Z)-isomer for the first time in Echinacea species. A short, stereoselective synthesis of the (2E,4E,8E,10Z)-isomer is also described which allowed further biological evaluation of this material, and the demonstration that this isomer does not occur in Spilanthes mauritiana as previously reported.

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ABSTRACT:A common feature of many CYP2C9 ligands is their weak acidity. As revealed by crystallography, the structural basis for this behavior involves a charge-pairing interaction between an anionic moiety on the substrate and an active site R108 residue. In the present study we attempted to re-engineer CYP2C9 to better accept basic ligands by charge reversal at this key residue. We expressed and purified the R108E and R108E/D293N mutants and compared their ability with that of native CYP2C9 to interact with (S)-warfarin, diclofenac, pyrene, propranolol, and ibuprofen amine. As expected, the R108E mutant maintained all the native enzyme's pyrene 1-hydroxylation activity, but catalytic activity toward diclofenac and (S)-warfarin was abrogated. In contrast, the double mutant displayed much less selectivity in its behavior toward these control ligands. Neither of the mutants displayed significant enhancement of propranolol metabolism, and all three preparations exhibited a type II (inhibitor) rather than type I (substrate) spectrum with ibuprofen amine, although binding became progressively weaker with the single and double mutants. Collectively, these data underscore the importance of the amino acid at position 108 in the acid substrate selectivity of CYP2C9, highlight the accommodating nature of the CYP2C9 active site, and provide a cautionary note regarding facile re-engineering of these complex cytochrome P450 active sites.CYP2C9 is a major form of human liver cytochrome P450 (P450) involved in the metabolism of ϳ15% of all the therapeutic agents that are cleared by oxidative metabolic processes (Williams et al., 2004). Many of these drugs, including (S)-warfarin, phenytoin, and a wide variety of nonsteroidal anti-inflammatory drugs (NSAIDs), are hydrophobic and weakly acidic (Miners and Birkett, 1998;Rettie and Jones, 2005). Early site-directed mutagenesis studies suggested that CYP2C9 residues F114, V113, and F476 were involved in hydrophobic interactions with the substrates (S)-warfarin, diclofenac, and the competitive inhibitor sulfaphenazole (Haining et al., 1999;Melet et al., 2003). In addition, the I helix residues S286 and N289 have been implicated as important determinants of CYP2C9-dependent NSAID metabolism and high affinity sulfaphenazole binding (Jung et al., 1998;Klose et al., 1998). Mutagenesis of the charged residues at R97, R108, D293, and D360 was also found to alter CYP2C9 substrate binding and metabolism (Ridderström et al., 2000;Flanagan et al., 2003;Dickmann et al., 2004). In particular, the R108A mutant displayed a 100-fold lower activity toward diclofenac compared with wild-type (Ridderström et al., 2000), and previous studies from this laboratory showed that the R108F mutant exhibited diminished catalytic activity toward diclofenac and (S)-warfarin while maintaining most of the wild-type activity toward the uncharged molecule pyrene (Dickmann et al., 2004). This latter finding of ligand-dependent activity changes showed that activity alterations were not a global phenomenon and indicat...