Use of heteronuclear multiple bond coherence NMR spectroscopy to monitor nitrogen metabolism in a transformed root culture of Datura stramonium

Abstract

Abstract In order to examine the factors that regulate the flux of primary metabolites into tropane alkaloids, Nuclear Magnetic Resonance spectroscopy (NMR) has been used to monitor nitrogen metabolism in transformed root cultures of Datura stramonium fed with (15NH4)2SO4 and K15NO3. This study employs the technique of Heteronuclear Multiple Bond Coherence (HMBC) NMR spectroscopy, which combines the advantages of both bidimensional resolution and 1H NMR sensitivity. Moreover, the HMBC sequence allows the 15N bound to labile protons to be observed via 2J and/or 3J couplings, which is very appropriate for the observation of tropane alkaloids. In transformed roots of Datura stramonium, in addition to the amino acids normally observed in monodimensional 15N NMR, other amino acids are resolved by HMBC. Labelled peaks due to N-acetyl compounds and to uridine are identified. While these primary metabolites were also seen in cell cultures of Nicotiana plumbaginifolia, cross peaks corresponding to secondary metabolites, such as tropine, were also observed. This is the first time that such secondary products have been found by this technique.