Modular Chemoenzymatic Synthesis of Terpenes and their Analogues

Abstract

Non‐natural terpenoids offer potential as pharmaceuticals and agrochemicals. However, their chemical syntheses are often long, complex, and not easily amenable to large‐scale production. Herein, we report a modular chemoenzymatic approach to synthesize terpene analogues from diphosphorylated precursors produced in quantitative yields. Through the addition of prenyl transferases, farnesyl diphosphates, (2E,6E)‐FDP and (2Z,6Z)‐FDP, were isolated in greater than 80 % yields. The synthesis of 14,15‐dimethyl‐FDP, 12‐methyl‐FDP, 12‐hydroxy‐FDP, homo‐FDP, and 15‐methyl‐FDP was also achieved. These modified diphosphates were used with terpene synthases to produce the unnatural sesquiterpenoid semiochemicals (S)‐14,15‐dimethylgermacrene D and (S)‐12‐methylgermacrene D as well as dihydroartemisinic aldehyde. This approach is applicable to the synthesis of many non‐natural terpenoids, offering a scalable route free from repeated chain extensions and capricious chemical phosphorylation reactions.

In vitro Applications of the Terpene Mini‐Path 2.0

“…A 2.5 mM concentration of DMAOH ( vs 5 mM), a 15 mM concentration of Mg 2+ ( vs 10 mM), a pH of 7.5 ( vs 8) and the absence of Mn 2+ and NH 4 + were the most beneficial conditions among the tested ones. By combining all these values in a single experiment, we ended‐up with a 54 % yield of isolated FPP as its tris‐ammonium form, something comparable with a previous report, [7] but at a larger concentration. The use of the TMP 2.0 coupled to an efficient FPPS is thus a real alternative to the chemical synthesis of FPP, avoiding the cumbersome generation of activated farnesol as halide derivatives and the need of tris(tetrabutylammonium) hydrogen diphosphate as diphosphorylating agent [9] …”

“…To this end, we first incorporated to the TMP 2.0, the farnesyl diphosphate synthase from Geobacillus stearothermophilus (FPPS Gs ), a known stable and highly active FPPS, [8] in order to access FPP in a one pot – three steps cascade (Scheme 3). For note, a similar reaction was conducted by the group of Allemann, using two steps (firstly, synthesis of DMAPP and IPP then synthesis of FPP), instead of the one pot cascade presented here [7] …”

“…described the promiscuous kinase activity of the hydroxyethylthiazole kinase from E. coli (ThiM Ec kinase) on DMAOH [6] . This finding and the fact that IOH was also a substrate of this enzyme, stimulated the use of ThiM Ec as a catalyst for the mono‐phosphorylation of DMAOH and IOH in the context of the TMP, either in vivo or in vitro , to generate various terpenoids [4,7] . Within this contribution we wish to report i) the in vitro improvement of the TMP using the ThiM Ec kinase instead of a phosphatase in the first phosphorylation step, ii) the extension of the available terpene chemical space thanks to the simplicity of the TMP allowing the use of a structural equivalent of DMAOH as a starter unit and iii) the relevance of the TMP to identify the product specificity of a yet uncharacterized sesquiterpene synthase without the need to chemically synthesize farnesyl diphosphate (FPP).…”

Ternary complexes of isopentenyl phosphate kinase from Thermococcus paralvinellae reveal molecular determinants of non‐natural substrate specificity

“…This enzymatic reaction plays a crucial role in the alternate mevalonate pathway found in Archaea and plant cytoplasm 1 . Beyond their natural function, IPKs are currently receiving attention for their potential applications in various engineered pathways aimed at producing isoprenoid natural products (NPs) 2–11 . These pathways (hereafter called “isoprenol pathways”) seek to simplify the natural syntheses of the isoprenoid precursors (IPP and its structural isomer dimethylallyl diphosphate, DMAPP) by diphosphorylating their readily available alcohol analogs (IOH and DMAOH, respectively; Figure 1B).…”