¹³C-²H correlation NMR spectroscopy studies of the in vivo transformations of natural products from Artemisia annua

Abstract

¹³C-²H correlation NMR spectroscopy (¹³C-²H COSY) permits the identification of ¹³C and ²H nuclei which are connected to one another by a single chemical bond via the sizeable ¹J_CD coupling constant. The practical development of this technique is described using a ¹³C-²H COSY pulse sequence which is derived from the classical ¹³C-¹H correlation experiment. An example is given of the application of ¹³C-²H COSY to the study of the biogenesis of natural products from the anti-malarial plant Artemisia annua, using a doubly-labelled precursor molecule. Although the biogenesis of artemisinin, the anti-malarial principle from this species, has been extensively studied over the past twenty years there is still no consensus as to the true biosynthetic route to this important natural product – indeed, some published experimental results are directly contradictory. One possible reason for this confusion may be the ease with which some of the metabolites from A. annua undergo spontaneous autoxidation, as exemplified by our recent in vitro studies of the spontaneous autoxidation of dihydroartemisinic acid, and the application of ¹³C-²H COSY to this biosynthetic problem has been important in helping to mitigate against such processes. In this in vivo application of ¹³C-²H COSY, [15-¹³C²H₃]-dihydroartemisinic acid (the doubly-labelled analogue of the natural product from this species which was obtained through synthesis) was fed to A. annua plants and was shown to be converted into several natural products which have been described previously, including artemisinin. It is proposed that all of these transformations occurred via a tertiary hydroperoxide intermediate, which is derived from dihyroartemisinic acid. This intermediate was observed directly in this feeding experiment by the ¹³C-²H COSY technique; its observation by more traditional procedures (e.g., chromatographic separation, followed by spectroscopic analysis of the purified product) would have been difficult owing to the instability of the hydroperoxide group (as had been established previously by our in vitro studies of the spontaneous autoxidation of dihydroartemisinic acid). This same hydroperoxide has been reported as the initial product of the spontaneous autoxidation of dihydroartemisinic acid in our previous in vitro studies. Its observation in this feeding experiment by the ¹³C-²H COSY technique, a procedure which requires the minimum of sample manipulation in order to achieve a reliable identification of metabolites (based on both ¹³C and ²H chemical shifts at the 15-position), provides the best possible evidence for its status as a genuine biosynthetic intermediate, rather than merely as an artifact of the experimental procedure.