A Family of Related Fungal and Bacterial Di‐ and Sesterterpenes: Studies on Fusaterpenol and Variediene

Abstract The absolute configuration of fusaterpenol (GJ1012E) has been revised by an enantioselective deuteration strategy. A bifunctional enzyme with a terpene synthase and a prenyltransferase domain from Aspergillus brasiliensis was characterised as variediene synthase, and the absolute configuration of its product was elucidated. The uniform absolute configurations of these and structurally related di‐ and sesterterpenes together with a common stereochemical course for the geminal methyl groups of GGPP unravel a similar conformational fold of the substrate in the active sites of the terpene synthases. For variediene, a thermal reaction observed during GC/MS analysis was studied in detail for which a surprising mechanism was uncovered.

. Structure elucidation of fusaterpenol (ent-8) including methylene group assignment. H,H-COSY correlations are shown in bold grey, single headed arrows represent HMBC correlations and NOESY correlations are shown by double headed arrows.  Figure S1. [b] Chemical shifts  in ppm, multiplicity: s = singlet, d = doublet, m = multiplet, coupling constants J are given in Hertz.
(Madison, USA). The correct incorporation of the target genes was verified by analytic digest with PvuII and XhoI, as well as gene sequencing to yield plasmid pYE-OJJ72250 (AbVS).  Figure S9. Amino acid sequence of AbVS (OOJ72250).

Gene expression and protein purification
Expression of AbVS from an E. coli BL21 (DE3) culture carrying the plasmid pYE-OJJ72250 was performed according to the same procedure as described above for FgGS. The obtained fraction was checked by SDS-PAGE ( Figure S10) and directly used for incubation experiments. Protein concentrations were determined by Bradford assay (calibrated with bovine serum albumin). [13] Typical concentrations were 1.48 mg mL -1 for AbVS.

APCI-MS measurements
High resolution mass spectra using APCI were recorded on an Orbitrap XL instrument (Thermo Fisher Scientific, Waltham, MA, USA).

Identification of AbVS as variediene synthase and product isolation
Test incubations to identify the substrate scope of recombinant AbVS were performed with GPP, FPP, GGPP, GFPP or DMAPP and IPP (1 mg) dissolved in substrate buffer (1 mL) and diluted with binding buffer (2.5 mL) and incubation buffer (4 mL). Protein preparations (0.5 mL) obtained from 100 mL expression culture were added, followed by incubation with shaking at 28 °C for 4 h. The products were extracted with hexane (100 μL), the extracts were dried with MgSO4 and analysed by GC/MS. Only the incubations with GGPP and DMAPP with IPP showed the production of variediene (3). For preparative isolation of 3, large scale incubations were done by dissolving GGPP (trisammonium salt, 80 mg) in substrate buffer (20 mL). This solution was diluted by binding buffer (80 mL) and incubation buffer (200 mL). To start the conversion, AbVS elution fraction (100 mL; from 8 L expression culture) was added and the reaction mixtures were incubated for 3 h at 28 °C and were extracted with pentane (2x 200 mL), the extracts were dried with MgSO4 and concentrated in vacuo. Column chromatography on silica gel with pentane yielded the pure diterpene (1.82 mg) as a colorless oil. GC/MS and NMR data measured in C6D6 (Table  S3 and Figures S11-S18) were comparable to published data in CDCl3 [14] and identified the diterpene as variediene (3) [14] and independently by isotopic labelling experiments (Figures S19-S20).   Figure S11. [b] Chemical shifts  in ppm, multiplicity: s = singlet, d = doublet, m = multiplet, coupling constants J are given in Hertz.
[c] 13 C data in CDCl3 from reference [14] for comparison.         Figure S11), C8 cannot be used to correlate the absolute configuration. Instead, its orientation can be determined from these experiments. Black dots represent 13 C-labelled atoms. . Stereochemical assignment of the geminal methyl groups of 3. Partial 13 C-NMR spectra of A) unlabelled 3, B) a C6D6 extract from the incubation of (12-13 C)FPP with IPP, GGPPS and AbVS and C) a C6D6 extract from the incubation of (9-13 C)GPP with IPP, GGPPS and AbVS. The minor peak visible in B) for C17 arises from partial labelling of C13 in synthetic (12-13 C)FPP. Black dots represent 13 C-labelled atoms.

Thermal isomerisation of isotopically labelled samples of variediene (3)
The four isotopically labelled variediene samples, prepared as described above, were concentrated in vacuo and dissolved in nitrobenzene (0.2 mL). After thermal treatment at 210 °C in a 1 mL pressure tube for 1 h, the samples were passed through a glass pipette charged with SiO2 and a wool-filter by pentane after cooling. The flowthrough was collected shortly before elution of nitrobenzene was observed, the solutions were concentrated under reduced pressure and dissolved in C6D6 for NMR measurement.  Figure S23). Black dots represent 13 C-labelled carbon atoms. Figure S32. Thermal isomerisation of isotopically labelled variediene (3) samples at positions C1, C5, C9 and C13 to 17. Partial HSQC of unlabelled 17 (top) compared to deuterated HR (middle) and deuterated HS (bottom). The observed incorporation for C1 is in line with the methylene configuration observed by NOESY ( Figure S23). For C5, a partial epimerisation can be observed after isomerisation, pointing to a role of the corresponding hydrogens in the isomerisation mechanism. Black dots represent 13 C-labelled carbon atoms. Figure S32 (continued). Enlarged representation of HSQC spectra from thermally isomerised labelled variediene (3) samples for C9 and C13. Whereas a selective incorporation is observed for the hydrogen atoms of C13, a conclusion for deuterium scrambling for C9 is not possible because of overlaying signals.