Isotopologue Profiling of Triterpene Formation under Physiological Conditions. Biosynthesis of Lupeol-3-(3′‑<i>R</i>‑hydroxy)-stearate in <i>Pentalinon andrieuxii</i>

The biosynthesis of lupeol-3-(3′<i>R</i>-hydroxy)-stearate (procrim b, <b>1</b>) was investigated in the Mexican medicinal plant <i>Pentalinon andrieuxii</i> by ¹³CO₂ pulse-chase experiments. NMR analyses revealed positional enrichments of ¹³C₂-isotopologues in both the triterpenoid and the hydroxystearate moieties of <b>1</b>. Five of the six isoprene units reflected a pattern with [1,2-¹³C₂]- and [3,5-¹³C₂]-isotopologues from the respective C₅-precursors, IPP and DMAPP, whereas one isoprene unit in the ring E of <b>1</b> showed only the [3,5-¹³C₂]-connectivity of the original C₅-precursor, due to rearrangement of the dammarenyl cation intermediate during the cyclization process. The presence of ¹³C₂-isotopologues was indicative of [¹³C₂]­acetyl-CoA being the precursor units in the formation of the fatty acid moiety and of the triterpene via the mevalonate route. The observed labeling pattern was in agreement with a chair-chair-chair-boat conformation of the (<i>S</i>)-2,3-oxidosqualene precursor during the cyclization process, suggesting that the lupeol synthase from <i>P. andrieuxii</i> is of the same type as that from <i>Olea europea</i> and <i>Taraxacum officinale</i>, but different from that of <i>Arabidopsis thaliana</i>. The study shows that ¹³CO₂ pulse-chase experiments are powerful in elucidating, under <i>in vivo</i> conditions and in a single experiment, the biosynthesis of complex plant products including higher terpenes