Biosynthesis of kelsoene and prespatane in cultured cells of the liverwort Ptychanthus striatus

Abstract

Biosynthesis of the unusual sesquiterpenes, (+)-kelsoene and (−)-prespatane, that accumulate in the cultured cells of the liverwort Ptychanthus striatus was investigated. A mixture of (+)- and (−)-alloaromadendrene was isolated from a variety of unidentified sesquiterpenes from cultured cells. However, the (−)-isomer, which is thought to be an immediate precursor to kelsoene, was present only as a minor component [a ratio of (−)-isomer to (+)-isomer of 1∶25]. When the cells were incubated with a higher dosage (1.0 mmol) of mevalonate (MVA, 3,5-dihydroxy-3-methylvalerate) under heterotrophic conditions or with a normal dosage (0.5 mmol) under photoautotrophic conditions, the ²H- and ¹³C-labels from ²H- and ¹³C-MVA were incorporated into the sesquiterpenes at an extremely high level (30 to 40 and 46 atom% excess, respectively). Labelling patterns of the biosynthetically ²H- or ¹³C-labelled kelsoene and prespatane were determined by GLC-MS and ²H- and ¹³C-NMR analyses. The labelling pattern of the kelsoene demonstrates the randomization of the ²H- and ¹³C-labels between the isopropenylmethyl and the methylene groups, with loss of one H-1 proton of farnesyl diphosphate (FPP, farnesyl = 3,7,11-trimethyldodeca-2,6,10-trienyl), suggesting that kelsoene is biosynthesized from a germacradienyl cation with 7R configuration by means of a (−)-alloaromadendranyl cation. Randomization of the labels between the isopropenylmethyl and the methylene and loss of one H-1 proton of FPP were also observed in ²H- and ¹³C-labelled prespatane. Thus, prespatane is postulated to be biosynthesized from a (7S)-germacradienyl cation by means of a guaianyl cation. During the third cyclization of the guaianyl cation, one proton at the C-1 position of FPP was lost.