Hydroxylation of sesquiterpenes by enzymes from chicory (Cichorium intybus L.) roots
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Introduction
Recent investigations at our laboratories have identified a biochemical pathway in chicory (Cichorium intybus L.) roots that leads to the formation of (+)-costunolide from farnesyl diphosphate (FPP) (Scheme 1).1., 2., 3. (+)-Costunolide is the most elementary structure of a germacrane sesquiterpene lactone and the precursor of the germacrane, eudesmane and guaiane lactones present in chicory. Moreover, it is the postulated key intermediate in the formation of the majority of sesquiterpene lactones found in plants.4., 5. Biosynthesis of sesquiterpene lactones in chicory roots involves various types of enzymes including microsomal (i.e. membrane bound) cytochrome P450 enzymes. These enzymes activate molecular oxygen and regioselectively insert one oxygen atom into the substrate at an allylic position.2., 3. Such a cytochrome P450 enzyme is the (+)-germacrene A hydroxylase (Scheme 1, step II) that hydroxylates (+)-germacrene A (5) to germacra-1(10),4,11(13)-trien-12-ol (17).2 Interestingly, the (+)-germacrene A hydroxylase of chicory has been demonstrated to hydroxylate both enantiomers of β-elemene (4, structure in Table 1) as well,2 in contrast to the common idea that cytochrome P450 hydroxylases of plant terpenoid biosynthesis have a narrow substrate specificity.6., 7.
Regio and stereoselective introduction of a hydroxyl group into an unactivated organic compound or at an allylic position such as catalysed by the (+)-germacrene A hydroxylase is still a challenge in synthetic organic chemistry.8 On the whole, the lack of specificity and the occurrence of undesired side reactions are a major drawback in the use of organic chemical methods for hydroxylation. However, hydroxylation of terpenes is of importance to the flavour and fragrance industry in the search for new production methods and new compounds.8., 9. Hence, it was investigated whether the microsomal fraction of a chicory root extract, containing the (+)-germacrene A hydroxylase, is capable of converting other sesquiterpene olefins in addition to the reported hydroxylation of β-elemene (4).2
Section snippets
Conversion of terpenes
Sixteen different terpenes were tested as possible substrates for the microsomal cytochrome P450 hydroxylase(s) present in chicory roots. GC–MS analysis showed that most of the tested sesquiterpenes were hydroxylated and that their molecular mass was correspondingly raised by 16 amu. Without the addition of NADPH the substrates were not hydroxylated, or only in negligible amounts. The accepted substrates (1–11) and their products (12–25) are depicted in Table 1. (−)-α-Cubebene and
Discussion
In the presence of NADPH, a microsomal enzyme preparation from chicory roots is able to catalyse the hydroxylation of a range of sesquiterpene olefins that are exogenous to the plant (Table 1). Most of these hydroxylations take place at an isopropenyl or isopropylidene group, yielding in some cases sesquiterpene alcohols that have not previously been described, e.g. alloisolongifolene alcohol (12) and amorpha-4,11-dien-12-ol (13). The novelty of the formed sesquiterpene alcohols in some cases
Substrates
Alloisolongifolene (1), (−)-α-gurjunene, (+)-γ-gurjunene (7), (+)-ledene (8) and (+)-valencene (11) were purchased from Fluka. ICN Biomedicals furnished (−)-α-cubebene. (−)-Limonene and (+)-limonene were purchased from Merck and Janssen, respectively. Amorpha-4,11-diene (2) was synthesised by Dr B. J. M. Jansen.24 Germacrone was isolated from the natural oil of Geranium macrorrhizum by Dr D. P. Piet, who also synthesised germacrene B (6) from this compound.25 (−)-α-trans-Bergamotene (3) and
Acknowledgements
The authors like to thank J. de Mik for the gift of the chicory roots, A. van Veldhuizen for collecting NMR data, Dr M. A. Posthumus for performing the HRMS measurements and F. W. A. Verstappen for technical assistance.
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Present address: Max-Planck Institute of Chemical Ecology, Beutenberg Campus, Winzerlaer Straße 10, 07745 Jena, Germany.