Abstract
In cultured cells of liverworts which generally accumulate sesqui- and di-terpenes at significantly high levels, added precursors such as ^<13>C (or ^2H) labelled acetate and mevalonate (MVA) have been incorporated into sesquiterpenes at levels comparable to those normally associated with fungal cultures. Thus the cell cultures of the liverworts must be of increasing value for examining in detail the biosynthesis of these terpenes by NMR analyses. The cultured cells and gametophytes of the liverwort, Heteroscyphus planus produced sesquiterpenes of cadinane-type such as (1S,4R)-7-hydroxycalamenene (HC) and (1S)-7-methoxy-1,2-dihydrocadalene (MDC) together with diterpenoic acids of clerodane-type including heteroscyphic acid A (HAA). The biosynthetic sequence of these cadinanes has been examined by incorporation of differently ^2H and ^<13>C labelled mevalonate (MVA). GC-MS, and ^2H and ^<13>C NMR analyses of the isotopically labelled products provided evidences for 1,2- and 1,3-migration of hydrogens, and the hydroxylation of calamenene (CA) at C-7 without NIH shift to form HC and MDC. A cell-free extract from cultured cells indicated that (+)-cubenene (CU) and (+)-epicubenol (EP) was specifically formed from 2E, 6E-farnesyl diphosphate (FPP). ^2H-NMR analyses of (+)-CU and (+)-EP generated from 2E, 6E-[1,1-^2H_2]- and [6-_2H]-FPP confirmed the presence of 1,2- and 1,3-hydride shifts in their formation. These results together with the common absolute configurations suggest that MDC and HC are formed via CU, and that HC may be formed via the direct oxygenation of CU without the aromatization to CA. Experiments by incorporation of [2,2-^2H_2]-, [2-^<13>C] and [4,5-^<13>C_2]-MVAs, and [α-^<13>C]-glycine unexpectedly showed the preferential labelling in the FPP-derived portion in the biosynthesis of HAA and the phytyl moiety in chlorophyll a, while a-carbon in glycine was universally incorporated in the phytyl moiety. This may suggest that FPP is directly incorporated into chloroplasts. A 1,2-migration of the methyl (C19) originating at C-6 of MVA was also observed in the biosynthesis of HAA.
