Chemoenzymatic preparation of germacrene analoguesw

Terpenoids represent a valuable class of bioactive fine chemicals and are therefore attractive targets for synthetic modification; modulation of their natural properties may lead to new medicinal and agrochemical compounds with improved properties. However, the complexity of the hydrocarbon skeletons and the often significant chemical instability of many terpenoids can present a formidable challenge to the synthetic chemist. Synthetic biology approaches have focused on the preparation of natural terpenoids in living organisms, but they operate with whole biochemical pathways using fundamental biosynthetic building blocks (e.g. isopentenyl diphosphate) and can therefore not easily be applied to generate modified terpenes. One attractive synthetic approach that complements current terpene synthetic biology and circumvents the difficult task of engineering full metabolic pathways to generate alternative substrates in vivo, could rely on the chemical preparation of FDP analogues as substrates of recombinant terpene synthases to create modified terpenoids. Modified FDPs have been used extensively to study the mechanisms of the cationic reactions mediated by (sesqui)terpene synthases. However, despite the fact that several unnatural FDPs are indeed turned over by these enzymes, only a few reports have explored the synthetic utility of terpene synthases toward the production of valuable novel terpenoids. Germacrene A and germacrene D synthases (GAS and GDS) are two plant sesquiterpene synthases that catalyze the Mg-dependent conversion of FDP (1a) to germacrene A (3a) and germacrene D (5a), respectively (Fig. 1). These two macrocyclic sesquiterpenes have been shown to act as semiochemicals affecting the olfactory response of insects. While a synthesis of the rather unstable germacrene D (5a) has been reported, the extreme thermal and photochemical instability of the acid labile germacrene A (3a) has so far hampered the development of a satisfactory chemical synthesis. Nevertheless, fluorinated germacrene A analogues with improved stabilities have previously been produced enzymatically from fluorinated FDP analogues. Thus, based on the biological and potential economic significance of compounds 3a and 5a, germacrene A and D synthases from Solidago canadensis were selected to investigate their capability to produce non-natural germacrenes from modified FDPs. To this end, recombinant GAS and GDS were overproduced in E. coli and purified as previously described. Several fluorine and methyl modified FDPs were screened by GC-MS on an analytical scale for substrate activity. Germacrene A analogues were readily identified through their ability to undergo thermal Cope rearrangements to the corresponding b-elemene analogues under GC-MS conditions; germacrene D analogues were identified from their mass spectra since the presence of the more stable (i.e. more abundant, 100%) [M 43] fragment in the EI-MS is diagnostic of the parent 5a. Only modified FDP analogues (Fig. 1, framed) that gave a relatively strong ion count in the total ion chromatogram (GC-MS) as compared with the natural substrate 1a were considered suitable for this study (vide infra). Interestingly, in contrast to what has been observed with other sesquiterpene synthases, 2-fluoro-FDPwas not turned over significantly by GAS or GDS. In addition, the H/F and in particular the H/CH3 substitution at the C15 position of FDP Fig. 1 Proposed biosynthesis of germacrenes A (3a) and D (5a). Modified substrate analogues of GAS and GDS (framed).