Optimisation of 1D and 2D in vivo 1H NMR to study tropane alkaloid metabolism in Pseudomonas
Résumé
1H NMR has been used for the quantitative in vivo analysis of tropinone metabolism in Pseudomonas AT3 cultures. Quantification of the metabolites solely by 1D NMR proved difficult as a result of large overlaps in the 1H spectrum due to similar metabolite structures. We show, however, that good resolution of all target metabolites is obtained by using 2D TOCSY NMR. Several mixing times were tested in order to obtain spectra with high cross-peak intensities for accurate quantification. The metabolism of tropinone in Pseudomonas AT3 to tropine and pseudotropine was successfully quantified directly by NMR. Quantification was carried out with the aid of calibration curves obtained with standard mixtures of tropinone, tropine and pseudotropine at various concentrations.
L’analyse quantitative des me´tabolites de la tropinone dans des cultures de Pseudomonas AT3 a pu eˆtre re´alise´e in vivo par RMN 1
H. La quantification de ces me´tabolites par RMN monodimensionnelle est difficile, en raison de recouvrements spectraux provenant de me´tabolites de structures similaires. En revanche, il a e´te´ possible d’obtenir une se´paration suffisante en utilisant une se´-
quence bidimensionnelle TOCSY. Plusieurs temps de me´lange ont e´te´ teste´s afin d’obtenir des taches de corre´lation d’intensite´s
compatibles avec une analyse quantitative. Ainsi, le me´tabolisme de la tropanone en tropanol et en pseudotropanol a pu eˆtre directement quantifie´ par RMN a` l’aide de courbes de calibration.