Halimane Diterpenes in the Alpine DaisyCelmisia viscosa: Absolute Configuration, 2,6-Dideoxyhexopyran-3-ulosides, Conformational Flexibility, and Intraspecific Variation

Abstract

The methyl-migrated bicyclic skeleton of the halimane diterpenes has been found in a wide range of organisms, including flowering plants, liverworts, marine animals, and bacteria. The discovery of halima-1(10),14-dien-13-ol (3) from the Aotearoa New Zealand endemic alpine daisy Celmisia viscosa is now reported. The full configuration was assigned for the first time by X-ray crystallography, enantiomeric to that of a liverwort isolate. The absolute configuration at C-5 of the halimane is opposite to that at C-5 of the labdane epimanool (1) found in some C. viscosa specimens. Two new 2,6-dideoxyhexopyran-3-uloside halimane derivatives (4 and 5) were also found, and the absolute configuration of 5 was determined by 1H NMR analysis of the Mosher esters. Line broadening in the 13C NMR spectra of these halim-1(10)-enes was due to conformational exchange in the decalin ring A, as shown by molecular modeling and DFT calculations. 1H NMR and GC analyses of leaf extracts of individual plants from across the full geographic range of C. viscosa revealed intraspecific variation of diterpenes: 37 samples had halimadienol as the main diterpene in large amounts and 2 specimens had predominantly epimanool, again in large amounts. Three other viscid (sticky leaved) Celmisia species also contained diterpenes, but none was detectable in four nonviscid Celmisia species.

O-Acylated Flavones in the Alpine Daisy Celmisia viscosa: Intraspecific Variation

“…These results are similar to the discovery of two diterpene chemotypes in C. viscosa using the same sample set. 11 However, the two stand-out labdane diterpene-dominated individual plants were not the same as the two stand-out dimethoxyflavone-rich individuals. These flavone results again emphasize the importance of examining multiple collections of any taxon before drawing conclusions about characteristic secondary metabolite profiles.…”

“…8 Elsholtzia blanda (Lamiaceae) gave a 7-(2methylbutanoate)flavone with C-methylation and a prenyl group (not shown). 7 ■ RESULTS AND DISCUSSION Possible flavones in C. viscosa were noted during studies of the major diterpenes in resinous leaf coatings (see accompanying paper 11 ). 1 H NMR analyses of crude leaf extracts showed vinyl and other signals due to halimane and/or labdane diterpenes, 11 but also all showed coupled doublet signals at 7. bulk C. viscosa leaf extract (Figure S1, Supporting Information) gave several fractions showing these 1 H NMR signals but all containing different compounds.…”

“…These previously unreported 8-O-acyl flavones 5−7 and 10−14 co-occur at high concentrations with diterpenes in C. viscosa leaf extracts, and these diterpenes show intraspecific variation. 11 Intraspecific flavonoid variation is well known, 15 so it seemed possible that the flavones might also vary between individual C. viscosa plants. The 1 H NMR signals of the various flavone derivatives identified above were too similar to detect such variation (Tables 1−3), and these compounds could not be analyzed by GC.…”

“…It seems that the flavonoid contents of C. viscosa leaves are more heterogeneous than the diterpene contents. 11 Expressing the flavone class peaks as percent of total flavone peak areas showed consistent results for the composite sample (Figure S3, Supporting Information). Within a single plant, absolute flavone concentrations were highest in leaves (new or old) compared to roots and other parts.…”

“…Therefore, single plants per site were used in a study of leaf flavone variation across the full geographic range of C. viscosa, i.e., the mountains of the South Island of New Zealand. 2 The 39 leaf extracts described in the accompanying paper 11 were analyzed by RPLC for intraspecific variation of acylated flavones. These were CDCl 3 extracts, so the more polar flavones were probably not representatively extracted, and any flavonoid glycosides would not have been extracted.…”