Diverged Plant Terpene Synthases Reroute the Carbocation Cyclization Path towards the Formation of Unprecedented 6/11/5 and 6/6/7/5 Sesterterpene Scaffolds

Abstract Sesterterpenoids are a relatively rare class of plant terpenes. Sesterterpene synthase (STS)‐mediated cyclization of the linear C25 isoprenoid precursor geranylfarnesyl diphosphate (GFPP) defines sesterterpene scaffolds. So far only a very limited number of STSs have been characterized. The discovery of three new plant STSs is reported that produce a suite of sesterterpenes with unprecedented 6/11/5 and 6/6/7/5 fused ring systems when transiently co‐expressed with a GFPP synthase in Nicotiana benthamiana. Structural elucidation, feeding experiments, and quantum chemical calculations suggest that these STSs catalyze an unusual cyclization path involving reprotonation, intramolecular 1,6 proton transfer, and concerted but asynchronous bicyclization events. The cyclization is diverted from those catalyzed by the characterized plant STSs by forming unified 15/5 bicyclic sesterterpene intermediates. Mutagenesis further revealed a conserved amino acid residue implicated in reprotonation.


Isolation of sesterterpenes following large-scale vacuum-infiltration of N. benthamiana leaves.
To direct the metabolic flux towards sesterterpene production, we also co-infiltrated an A. tumefaciens strain harboring an expression construct for 1-deoxy-D-xylulose 5-phosphate synthase (DXS) (At4g15560), [3] an enzyme from the non-mevalonate pathway, with AtGFPPS1 and STS genes in our scale-up experiments. Seed cultures of A. tumefaciens strains harboring expression constructs for the relevant AtGFPPS1 and STSs were prepared as described above and 100 ml inoculated into 1 l LB broth containing 50 g/ml rifampicin, 50 g/ml kanamycin and 100 g/ml streptomycin. Cultures were incubated in a shaker (28 o C and 220 rpm) for about 15 h until the OD600 reached ca. 2.0. A. tumefaciens cells were pelleted by centrifuging at 6500 g for 25 min. Supernatants were discarded.
The pellets were then resuspended in MMA buffer (200 ml) and kept in the dark for 1 h before performing vacuum infiltration. Batches of four N. benthamiana plants were simultaneously infiltrated using a custom-built vacuum chamber with an oil pump, as described in the literature [4] . Briefly, the A. tumefaciens suspensions containing the different expression constructs were mixed and topped up to ca. 10 l with MMA buffer in a 10 l basin. Plants were secured in a rack and inverted to submerge the leaves into the bacterial suspension in the basin. The basin was placed into a vacuum chamber and the plants vacuum-infiltrated by imposing a vacuum to ca. 0.1 bar followed by venting. The plants were then maintained in a containment growth room (16h light, 8h dark) for five days before the leaves were harvested and lyophilized. Dried leaf material was ground to a powder using a mortar and pestle and extracted with hexanes at room temperature with agitation for three days. Extracts were filtered each day and the leaf material re extracted with fresh solvent.
To quench the reaction, hexanes (1 mL) was added to the reaction mixture. The suspension was filtered through a silica plug which was further rinsed with 10% ethyl acetate (in hexanes, 3 mL) three times. Combined filtrates were dried under N2 and dry reaction products purified by SCC to furnish (-)-brarapone B (9, 22.3 mg) in ~60% yield as a colorless liquid.  Table S12.

Structural characterization of sesterterpenes by NMR and other spectroscopic techniques.
Standard 1D and 2D NMR spectra including 1 H, 13 C, DEPT135, COSY, HSQC, HMBC and NOESY were acquired on a Bruker 400 MHz Topspin NMR spectrometer. All signals were acquired at 298 K. Samples were dissolved in CDCl3 or C6D6 for data acquisition and calibrated by referencing to either residual solvent 1 H and 13 C signal or TMS. Detailed structural assignments and tabulated NMR data for compounds 2-9 were presented in the Tables  Single crystal X-ray diffraction analysis.
Single crystal X-ray analysis was carried out for compound 3b on a Bruker D8-QUEST instrument equipped with a PHOTON-100 area detector and Incoatec IS Cu microsource (wavelength = 1.5418 Å, beam diameter at the crystal ca 100 m). Crystals were mounted on an X-ray transparent loop (Mitegen) using an inert oil and cooled to 180(2) K using an open-flow N2 cryostat. Data were collected and processed using the APEX3 software package (Bruker). Structures were solved and refined using SHELXT and SHELXL (Bruker).
In the absence of any significant anomalous scatterers, reliable indications of the absolute structure could not be obtained. The crystallographic data have been deposited at the Cambridge Crystallographic Data Centre: CCDC1583364

Time course and feeding experiments
Three four week old N. benthamiana plants were each infiltrated with combinations of A.tumefacien strains carrying different expression constructs: AtGFPPS1+Cr089, AtGFPPS1+Br580, and AtGFPPS1+AtTPS17, respectively, using a needless syringe as outlined above.
Five leaves of each plant were infiltrated. The infiltrated plants were maintained in a growth chamber with 16 h light/ 8 h dark cycle at 22 o C. One leaf from each plant was harvested on the thrid day, before watering the plants with D2O (~5 ml/day) until the tenth day.
Infiltrated leaves expressing different combinations of genes were harvested on the fourth, fifth, eight and tenth days, respectively.
Harvested leaves were lyophilized and analyzed by GC-MS as described above.
Site-directed mutagenesis was performed by PCR amplification using the entry vector pDONR207 harbouring the wild type genes as templates and the mutated complementary sequences as primers (listed in Table S2). DMSO (5%) was added to the PCR reaction to alleviate formation of primer dimers. PCR-amplified products were purified with a PCR quick purification kit and eluted with 15 l elution Quantum chemical calculations. All quantum calculations were performed with the GAUSSIAN09 software suite. [7] Geometries were optimized using the B3LYP density functional theory (DFT) method and the 6-31+G(d,p) basis set. [8] All stationary points were characterized as minima or transition state structures using frequency calculations. All reported energies include zero-point energy corrections (unscaled) from these frequency calculations. mPW1PW91 single point energies were calculated for all structures [9] . These methods are well established for examining carbocation rearrangement reactions. [10] Structures of intermediate carbocations and ransition state structures are presented in Figure S73-74.