On the Early Steps of Cineol Biosynthesis in Eucalyptus globulus

Samples of [4-2H1]-1-deoxyxylulose (17a) and [2-13C, 4-2H1]-1-deoxyxylulose (17b), have been prepared by modification of known procedures and fed in aqueous solution to twiglets of Eucalyptus globulus. The probes of cineol (6) isolated from these experiments were analyzed by GC/MS, 2H- and 13C-NMR techniques. In the experiments with 17b, the formation of five isotopomers of 6 could be detected. Their structure and relative abundance demonstrate that the 13C-label is incorporated to the same extent into the two C5-units of 6, and that the 2H label is retained to an extent of 57% in the starter dimethylallyl-diphosphate unit (DMAPP; 12), but completely or almost completely lost in the unit derived from isopentenyl diphosphate (IPP; 11), in the elongation step which leads to geranyl diphosphate (GPP; 1). These results confirm that the recently discovered mevalonate-independent pathway to IPP and DMAPP is operative in the biosynthesis of cineol, and indicate, together with previous finding, that, within this pathway, formation of IPP and DMAPP occurs in independent rather than in sequential steps. In addition, the demonstration of different metabolic origins for the olefinic H-atoms of GPP (1), the aliphatic C10-precursor of 6, paves the way for a realistic interpretation of the strikingly consistent but hitherto unexplained anomalies detected in the natural-abundance 2H-NMR spectra of (+)- and (−)-α-pinene and of (+)-limonene.