Chemoenzymatic Synthesis of a New Germacrene Derivative Named Germacrene F

The new farnesyl pyrophosphate (FPP) derivative with a shifted olefinic double bond from C6‐C7 to C7‐C8 is accepted and converted by the sesquiterpene cyclases protoilludene synthase (Omp7) as well as viridiflorene synthase (Tps32). In both cases, a so far unknown germacrene derivative was found to be formed, which we name “germacrene F”. Both cases are examples in which a modification around the central olefinic double bond in FPP leads to a change in the mode of initial cyclization (from 1→11 to 1→10). For Omp7 a rationale for this behaviour was found by carrying out molecular docking studies. Temperature‐dependent NMR experiments, accompanied by NOE studies, show that germacrene F adopts a preferred mirror‐symmetric conformation with both methyl groups oriented in the same directions in the cyclodecane ring.


Introduction
Germacrenes AÀ E 2-6 belong to the large natural product class of sesquiterpenes (Figure 1), whose biosynthetic precursor is farnesylpyrophosphate (FPP, 1) and their formation is catalyzed by sesquiterpene cyclases (STCs).Germacrenes are characterised by a cyclodecane nucleus, which can contain two to three alkene moieties and which bear three substituents.(À )-Germacrene A 2 was first isolated from the Gorgonian Eunicea mammosa. [1]It was also described as the alarm pheromone of the aphid Terioaphis maculata. [2]ermacrene B 3 was first obtained from Humulus lupulus [3] and from Citrus junos. [4]Later, this derivative was also isolated from Stenocalyx michelii, [5] and Citrus aurantifolia. [6]In general, it is an important aroma component of lime peel oil which also contains germacrene C 4. [7] Germacrene D 5 has been found in the essential oils of the red deadnettle and hedgenettles and is a major component of patchouli oil. [8]Finally, germacrene E 6 was first isolated from the Indo-Pacific soft coral Sinularia erecta. [9]n recent years, the substrate specificity of STCs has been studied in more detail. [10]Allemann and coworkers intensively used mono-fluorinated FPP-derivatives as substrates for germacrene A synthase (GAS) and (S)-germacrene D synthase (GDS), respectively, and found a remarkable promiscuity of the STCs for these modified substrates commonly yielding new fluorinated germacrene A and D derivatives. [11]he fungal STC presilphiperfolan-8β-ol synthase (BcBOT2) is responsible for the formation of the tricyclic sesquiterpene presilphiperfolan-8β-ol (7) from farnesylpyrophosphate (FPP, 1) (Scheme 1, A) and the initial step after diphosphate activation is a 1!11 ringclosure. [12]BcBOT2 is remarkable, as it shows a broad substrate promiscuity towards modified farnesyl pyrophosphates [13] and selected such FPP derivatives 8-11 are listed in Scheme 1B along with their initially formed ring sizes.The cyclizations generally lead to 11-membered rings in the first step.In only one case, however, we encountered ring closure to a 10-membered macrocycle and formation of a germacrene derivative 13 (Scheme 1C). [14]A subtle change around the central trisubstituted olefinic double has led to an altered mode of cyclization.

Results and Discussion
In view of this finding, we investigated, whether the shift of the central olefinic double bond (C6-C7) by one position to C7-C8 of FPP would provide a so far unknown FPP derivative 14 that could exert a similar effect on different STCs with respect to the mode of cyclization.
The synthesis of double bond shifted FPP derivative 14 commenced from 3-butyn-1-ol 15 and proceeded over seven steps (Scheme 2).Isogeraniol 16 was obtained via the established Zr-catalyzed carboalumination followed by an Pdcatalyzed cross-coupling with prenylbromide. [15,16]The homoallylic alcohol was converted to the corresponding sulfone 17, [17,18] which was employed in the alkylation towards 19 by coupling with bromide 18. [19] A reductive desulfonylation step and the removal of the silyl protection yielded the desired allylic alcohol, [13a,20] which was converted to the diphosphate 14 by an established procedure. [21]mong nine STCs tested [22] two provided a new transformation product with FPP derivative 14.These are a) protoilludene synthase (Omp7) which has been found in various basidiomycetes including Omphalotus olearius and that produces the tricyclic sesquiterpene Δ 6 -protoilludene (20) [23] and b) viridiflorene synthase (Tps32) found in Solanum lycopersicum [24] that forms viridiflorene 22 from 1 (Scheme 2) All enzymes including Omp7 and Tsp32 were cloned and expressed in E. coli as detailed in the SI.To determine enzyme activity and substrate tolerance, in vitro enzyme assays were conducted with both FPP 1 and derivative 14 (500 μL scale, 0.1 g/L Omp7, 37 °C, 70 min) which yielded one major product as judged by GC-MS (R I = 1464, M = 204 g/mol).For upscaling Omp7 was chosen as judged from the semiquantitative analysis of the assay experiments.It provided a sufficient amount of material (6.5 mg crude product) to characterize the structure of the main product.This turned out to be germacrene derivative 23, a 1! 10 cyclization product, which we name "germacrene F" which is another example that marginal structural differences around the central olefinic double bond in 1, can lead to a new mode of initial cyclization (1!10 instead of 1!11 cyclization) (Scheme 3).
Based on GC-HRMS measurements we determined the purity of germacrene F (23) to be 93.1 % (see SI; Figure S19).In order to shed light on this unexpected behaviour, we carried out molecular docking studies.The substrate, the product, and the expected reaction intermediates formed from the cyclization of 1!10 and 1!11 pathways were docked into the active site of the structural model of Omp7.The reaction intermediates resulting from the 1!10 transformation yield a tertiary carbocation, a structural motif exhibiting superior stability compared to the secondary carbocation arising from the 1!11 conversion.Notably, in agreement with the investigations by Christianson [25] carbocation intermediates derived from FPP and its derivatives [26] experience stabilization through π-system interactions mainly through phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp) amino acid residues.Our molecular docking analysis (Figure 2) revealed that the reaction intermediate stemming from the cyclization event 1!10 predominantly engages in arene-H interactions with the neighbouring Phe81 residue, with occasional occurrences involving Trp303.Moreover, a noteworthy observation was the favourable H-bond interaction between the intermediate and Met77, which serves to expedite dehydrogenation and double bond formation, thereby facilitating product synthesis.In contrast, molecular docking of the intermediate formed via 1! 11 did not manifest comparable interactions.This difference potentially underpins the molecular basis for the preference of a 1!10 cyclization pathway by the Omp7 in this scenario.
The fact that the NMR spectra along with the GC-HRMS data indicate mirror symmetry necessitates a more general and comparative analysis of the preferred conformation of this germacrene.The conformational dynamics around the cyclodecandiene core of the (E,E)-germacrenes [27] have been widely studied in solution.It became apparent from NMR studies that the iso-propenyl group (in 2) is usually equatorially oriented.As a result, four distinct conformations, commonly labelled DD, UU, UD, and DU (D = down, U = up with respect to orientation of the methyl groups) can be adopted (Figure 3).
NMR spectroscopic analysis of germacrene A (2) at room temperature revealed a mixture of three conformational isomers with the most stable conformation being the DD form (52 %), while the other conformers found in lower proportions have UD (29 %) and DU (19 %) orientations.Here, the ring methyl groups are on opposite sides of the cyclodecadiene. [27]imilar studies were performed for germacrene B (3), however, these were based on molecular mechanics calculations.They revealed that germacrene B (3) has two comparably stable conformations.The lowest barrier to ring inversion of the most stable conformer to its mirror image is estimated to be 23 kcal/ mol but no further structural details were provided. [28]he reduced set of signals in the 1 H and 13 C NMR spectra of germacrene F (23) indicate the mirror symmetry present under routine NMR experimental conditions at 298 K. Thus, a conformation with a UU (or the identical DD) orientation with respect to the methyl groups can be concluded.For the olefinic protons H6 and H6' a syn-orientation can be proposed from the strong splitting into a doublet signal with a J-coupling constant of 3 J HH = 11.1 Hz (DD or UU). [29]his can be understood from the Karplus relation of J-Figure 2. A. Binding orientation of the intermediates formed by cyclization of 1!10 (red) and 1!11 (green) at the active site of Omp7.A: Holo model of Omp7 predicted by Alpha Fold2 [31] (see SI). B. A closeup of the active site of Omp7 with the top scoring binding poses from the molecular docking of the two reaction intermediates formed from the two cyclization modes: the active site pocket residues along with the diphosphate group (magenta) coordinating with the triad of Mg 2 + shown in stick representation.C. A 2D overlay of the ligand interaction diagram of the two intermediates and their interactions with the neighbouring residues; Diagrams: legend for the ligand interaction diagram shown.coupling constants in six-membered rings.Furthermore, the conformation was investigated by 1 H-1 H NOESY NMR experiments.Here, the cross-correlation signals between H6/H6' and H4 indicate the orientation of the same ring side.Additionally, no strong correlation signals between the olefinic protons and the methyl groups were identified indicating the proposed UU conformation.
Interestingly, chemical exchange signals with a minority compound were also identified for the olefinic proton in the 1 H-1 H NOESY NMR spectrum at 298 K, indicating the presence of a second conformer associated with an interconversion at 298 K.The chemical exchange signals were also identified for the olefinic proton.However, when the temperature was lowered to 265 K, the chemical exchange signals were no longer present in the 1 H-1 H-NOESY NMR spectrum.Thus, the conversion of 23 is frozen in deuterated toluene below ambient temperature.
Importantly, the new product represents a germacrene derivative that e. g. unlike germacrene A (2) cannot undergo a facile Cope rearrangement to an elemene derivative under thermal conditions a process that has been encountered during isolation of 2. [28,13b]

Conclusions
In summary, we report the Omp7-promoted synthesis of a new germacrene derivative, which we name germacrene F. An unnatural FPP derivative in which the central (E)-alkene is shifted by one position.Within the germacrene family, this derivative is the first symmetric member and it adopts conformations in which the two methyl groups point in the same direction, as judged by NMR analysis.This was first reported for the STC BcBOT2 (12!13), and in the presented work we provide with Omp7, another example where the initial cyclization can switch from 1!11 to 1!10 by choosing other FPP derivatives.In addition, we disclose a plausible mechanistic rationale using molecular modeling and docking studies which showed intermediates formed via 1!11 did not manifest comparable interactions with the residues in the active site of Omp7 especially the critically important arene-H interactions with neighbouring Phe, Tyr, and Trp residues that were seen in 1!10 cyclizations.

Heterologous protein expression and cell lysis via ultrasound:
In order to cultivate the E. coli BL21 (DE3) cells, carrying the required plasmids, a seed culture (50 μL) was incubated with Kanamycin (50 mg/mL, 3 μL) in LB-media (3 mL) for 4.5 h at 37 °C and 200 rpm.From this pre-culture (1 mL) a main culture was created by incubation with Kanamycin (50 mg/mL) in 2-TY media (50 mL) at 37 °C and 200 rpm until the culture reached an OD 600 value of a 0.4 to 0.8.To initiate the protein overexpression IPTG (1 M, 25 μL) was added to the culture that was stirred at 16 °C and 180 rpm for approximately 22 h.After centrifugation, the cell pellets were stored at À 20 °C or used immediately for cell lysis.Cells were resuspended in lysis buffer (20 mL) at 0 °C and lysed by ultrasonication (10 min, 45 % amplitude, 4 s ultrasound to 6 s pause).The resulting solution was centrifuged (4 °C, 20 min, 10000 g) to give the crude enzyme solution.
Immobilized metal-affinity chromatography: For conditioning the column was rinsed with water (10× the column volume) and lysis buffer (5× column volume).The lysate was loaded onto the column (2×) and eluted with Ni-NTA buffers (5 mL each) with increasing imidazol concentrations (25 mM, 50 mM, 100 mM, 250 mM, 500 mM).During this time the solutions were cooled at 0 °C.The fractions were analysed using a Bradford assay and those fractions containing protein were united and concentrated by centrifugation (20 min, 4 °C, 4500 rpm).

Buffer exchange
To perform the buffer exchange the column was rinsed with water (10× column volume) and HEPES buffer (5× column volume).The protein solutions were loaded onto the column and eluted with HEPES buffer (5 mL).After centrifugation (4 °C, 4500 rpm) the solutions can be used or stored as a mixture of water and glycerol (1/1) between À 70 °C and À 80 °C.

Concentration measurement
Concentrations were determined by measuring the absorption of the purified protein solutions, using the extinction coefficient for reduced cysteine side chains.

In-vitro biotransformation in an analytical scale
In order to find STCs that accept FPP-derivative 18 as a substrate we tested nine different enzymes, namely BcBot2, PenA, Tri5, Cyc1, Hvs1, GCoA, Tps32, Omp7, Cop4.Since Omp7 turned out to be most susceptible to 14, the protocol for this enzyme will be described in detail here.Screening for new biotransformation products was performed in a reaction scale of 500 μL containing the corresponding enzyme (0.05 mg), the FPP-derivative 14 (1.5 μL, 50 mM) and a MgCl 2 solution (1.25 μL, 2 M).In parallel with the enzyme-derivative combinations performed, we also performed negative (without derivative or without enzyme) and positive controls (reaction of FPP 14 with different enzymes) under analogous conditions.All reactions were carried out in HEPES buffer.The reactions were incubated for 70 min at 37 °C and 100 rpm (30 °C, 0 rpm for FPP).

Biotransformation for product isolation:
In order to obtain sufficient amounts of the biotransformation product detected in the analytical studies for the purpose of structural elucidation, the reaction was repeated on a 50 mL scale and the total volume was divided into four parallel reactions.These were composed of the following components: Tween® 20 (2.5 μL) and diphosphatase (0.25 μL) in HEPES buffer (12.5 mL).The Omp7 solution (15.5 μL, 40.21 g/mL), derivative solution (in 0.05 M NH 4 HCO 3 , 18.75 mM, 125 μL) and finally MgCl 2 (2 M, 62.5 μL) were added and this mixture was shaken at 37 °C and 100 rpm.Every 30 minutes solutions of derivative 14 (total of 32.5 mg, 18.75 mM, 125 μL) were added up to a total volume of 1 mL each.After 2 h another batch of an Omp7 solution (15.5 μL each, 40.21 g/mL) was added.One hour after the last batch of derivative 14 had been added, the four batches were combined and n-pentane (25 mL) was added.The phases were separated, the aqueous phase was extracted with n-pentane (3×) and after short ultrasonic treatment the combined organic phases were washed with brine, dried over MgSO 4 • H 2 O, filtered and the solvent was removed in vacuo.The crude product was purified by column chromatography (n-pentane 100 %!n-pentane : Et 2 O 1 : 1).Germacrene F 23 (6.5 mg) was obtained still containing some n-pentane (~10 : 1 as judged by 1 H-NMR).To remove npentane residues the NMR sample was exposed for 30 minutes to a stream of N 2 with a cold trap (À 78 °C) located downstream.R f = 0.46 (n-pentane 100 %); 1  Protein modelling: There is no available protein structure for Omp7.Thus, an Apo model for Omp7 was generated with Alpha Fold2 [30] and evaluated according to structural, geometrical, and statistical metrics [31][32][33][34][35][36][37][38] The Holo form with the Mg 2 + triad was generated using the templates from the PDB structure with the closest sequence identity (PDB ID : 8H72) and the BcBOT crystal from Lou et al. [39] Molecular docking of substrate, intermediates, and product: A ligand database containing the substrate, intermediates, and products was prepared in ChemDraw. [40,41]The molecular docking of the substrate was used to validate the model and then the model was prepared for the molecular docking of the intermediates and product.All the molecular docking was performed in MOE [42] and 300 poses were taken into consideration according to London dG Score criteria of which the top 10 poses were selected for visualization based on GBVI/WSA dG Score criteria.

Figure 3 .
Figure 3. Left: Preferred conformation of germacrene F (23) based on the analysis of the 1 H-1 H NOESY spectra (cf. Figure S16-S18).Blue arrows refer to selected NOESY correlation signals; right: Comparison of conformational features and populations (%) of germacrenes A (2) and the new germacrene F (23) at room temperature:[a]  by NMR studies,[b]  DD and UU cannot be distinguished because of the mirror symmetry of 23, therefore both conformers of 23 are treated as one by referring to 100 %.