Biomimetic Cationic Cyclopropanation Enables an Efficient Chemoenzymatic Synthesis of 6,8-Cycloeudesmanes

Cationic cyclopropanation involves the γ-elimination at carbocations to form a new σ-C–C bond through proton loss. While exceedingly rare in bulk solution, it is recognized as one of the main biosynthetic cyclopropanation pathways. Despite the rich history of bioinspired synthetic chemistry, cationic cyclopropanation has not been appropriated for the synthetic toolbox, likely due to the preference of carbocations to undergo competing E1 β-elimination pathways. Here, we present an in-depth synthetic and computational study of cationic cyclopropanation, focusing on the 6,8-cycloeudesmanes as a platform for this investigation. We were able to apply biomimetic cationic cyclopropanation to the synthesis of several 6,8-cycloeudesmanes and non-natural analogues—in doing so, we showcase the power of this transformation in the preparation of complex cyclopropanes.


General Information
Unless otherwise stated, all glassware was flame-dried before use and all reactions were performed under an atmosphere of argon. All solvents were distilled from appropriate drying agents prior to use or directly taken from commercial sealed bottles under an atmosphere of argon. All reagents and commercially available substrates were used as received from commercial suppliers unless otherwise stated. Experiments conducted without external heating or cooling are designated as at "room temperature" (rt), which ranged between 21 °C and 23 °C. Reaction progress was monitored by thin layer chromatography (TLC) performed on aluminium plates coated with silica gel F254 with 0.2 mm thickness. Chromatograms were visualised by fluorescence quenching with UV light at 254 nm or by staining using potassium permanganate. Flash column chromatography was performed using silica gel 60 (230-400 mesh, Merck and co.). Neat infrared spectra were recorded using a Bruker Vertex 70 FT-IR spectrometer. Wavenumbers are reported in cm -1 . Mass spectra were obtained using a Bruker maXis UHR-TOF spectrometer, using electrospray ionization (ESI) and by Agilent 7200B GC/Q-TOF spectrometer, using electron impact (EI). All 1 H NMR, 13 C NMR and 19 F NMR spectra were recorded using a Bruker AV III 400, AV NEO 500, AV III 600 or AV III HD 700 spectrometer in CDCl3 or DMSO-d6. Chemical shifts are given in parts per million (ppm, δ), referenced to the solvent residual peak of CDCl3 or DMSO-d6, defined at δ = 7.26 ppm ( 1 H NMR) and δ = 77.16 ( 13 C NMR) for CDCl3, and δ = 2.52 ppm ( 1 H NMR) and δ = 39.52 ( 13 C NMR) for DMSO-d6. Coupling constants are quoted in Hz (J). 1 H NMR splitting patterns were designated as singlet (s), doublet (d), triplet (t), quartet (q), pentet (p).
Splitting patterns that could not be interpreted or easily visualised were designated as multiplet (m), apparent (app) or broad (br). Enantiomeric excess was measured on a Shimadzu LC-8A preparative HPLC system using Lux Cellulose-1 or Lux Cellulose-3 chiral columns.

Additional synthetic schemes 2.1 Investigation of 21
The synthesis of acyclic diene 21 was achieved over three steps. Subjection of 21 to the brominative cationic cyclopropanation conditions resulted in complex mixtures from which no cyclopropanation products could be identified. Scheme S1. Synthesis and investigation of 21 2.2 Optimsation of cationic cyclopropanation to give 3  1 H-NMR by comparison to an internal standard (mesitylene); experiment procedure conducted analogously to preparative procedure (see pg S27).

Computational studies
The conformational space of all molecules has been initially searched using meta-dynamics simulations based on semiempirical tight-binding quantum chemical calculations as implemented in CREST. [1,2] Structures located with CREST have then been subjected to PBE0-D3BJ/def2-SVP [3][4][5][6][7][8][9]  The DFT calculations have been performed with the Gaussian 16 program package. [10] The polarizable continuum model (PCM) with SMD parameters [11,12] of dichloromethane was applied to consider solvent effects for both geometries and energies. The same method was applied for the computational model benchmarks with B3LYP [13] . The RI-MP2 [14,15] calculation was performed using Orca 5.0 program package [16] .
The chosen level of theory has been shown to be applicable to systems containing carbocations in previous benchmark studies [17,18] and recent works [19] .
Free energies in solution have been corrected to a reference state of 1 mol l -1 at 298. 15 K through the addition of RTln(24.46) = +7.925 kJ mol -1 to the gas phase (1 atm) free energies.

Halide effect
To better understand the halide effect in the cyclization event, NBO analysis was performed on the transition state structures of the first step of the profiles for the reaction of 7 with NCS, NBS and NIS ( Figure S1). The obtained natural charges for carbon nuclei Ca and Cb, and the halogen (Cl, Br or I) show that for the case of the reaction of 7 with NCS and NBS, the halogen moiety presents neutral partial charge, and the carbon Cb is partially positive. As a result, the tertiary carbocation that would form would be too unstable, forcing a concerted mechanism for the formation of the two C-C bonds and the Cl-C or Br-C bond. However, the reaction of 7 with NIS presents an initial transition state in which the partial charge of the Iodine is 0.46 (partially positive), and consequently Cb presents a partial charge of 0.14 (significantly lower than 0.39 with NBS and 0.5 with NCS). This strongly suggests that the positive charge is shared between Cb and the halonium moiety, stabilizing an intermediate tertiary S7 carbocation species. Therefore, the reaction of 7 with NIS presents the obtained stepwise energy profile.

Thorpe-Ingold effect
To study the Thorpe-Ingold effect exerted by the isopropyl group, energy profiles leading to the

Substituent group replacement
The Gibbs free energies of the profiles obtained through substitutent group replacement are present in

Isolation of germacrene d
Gemacrene d was obtained from ylang ylang oil (Farfalla GmbH, 'Ylang Ylang Complete organic Grand Cru, essential oil', batch number #03.2026/032130) by the following protocol: 1.0 g of ylang ylang oil was purified by flash column chromatography over 5% AgNO3-impregnated silica

Expression and purification of germacradien-4-ol synthase
The DNA sequence of N-terminal His10-tagged Germacradien-4-ol synthase (GdolS, gene name: SC1) from Streptomyces citricolor was ordered in a pET-21 a (+) plasmid from BioCat GmbH (69120 Heidelberg, Germany) and transformed into E.coli strain Rosetta II using heat shock transformation.
Briefly, 5 μg of plasmid were dissolved in 25 μL of double-distilled water (ddH2O) and 5 μL of the plasmid solution was added to 100 μL of a solution of E.coli Rosetta II competent cells. After an incubation on ice for 10 minutes cells heat shocked at 42°C for 90 seconds before another incubation on ice for 5 minutes. After the addition of 1 mL of lysogeny broth (LB) medium the cells were allowed to recover at 37°C with gentle shaking (300 rpm) on a thermomixer before the solution was centrifuged for 2 minutes at 10 000 g. The cell pellet was resuspended in 100 μL of LB medium and the solution plated on a LB/agar plate treated with ampicillin (100 μg/mL) and chloramphenicol (30 μg/mL). The plate was incubated at 37°C overnight and a single colony was picked for the inoculation of 3 mL LB medium containing ampicillin (100 μg/mL) and chloramphenicol (30 μg/mL). After incubation overnight at 37°C and 170 rpm, the culture was treated with glycerol (final concentration 20%, v/v) and stored at -80°C as cell stock solution.
GdolS was expressed in ampicillin (100 μg/mL) and chloramphenicol (30 μg/mL) supplemented LB medium by inoculating the media to a starting OD of 0.2 using an overnight culture following an incubation at 37°C at 170 rpm. Induction was initiated at an OD of 0.7-0.8 using 0.5 mM isopropyl-β-Dthiogalactopyranosid (IPTG) following an incubation at 37°C, 170 rpm for 3h. Subsequently, cells were harvested at 10 000 g for 20 minutes at 4°C and cell pellets stored at -80°C before continuing with the cell workup. After thawing on ice, TBS buffer (50 mM Tris, 150 mM NaCl pH 7.5) was added to the cell S30 pellets (70 mL buffer for 1L LB media cell pellet) and the solution homogenized using an ultra turrax.
The cell solution was submitted for lysis using a cell disruptor (Constant Systems, Northants, United Kingdom) at 1.9 kbar for three cycles before separating the cell debris from the soluble protein fraction via centrifugation at 50 000 g for 45 min at 4°C. The supernatant was loaded on TBS equilibrated 5mL Ni-sepharose column (Cytiva) using an ÄKTAprime plus system. The column was washed with 20 mM imidazole in TBS buffer and the protein eluted using a linear gradient from 20 to 500 mM imidazole in 10 column volumes. Fractions containing the target protein were pooled and dialysed into TBS, pH 8 at 4°C over-night. The protein solution was concentrated using Amicon Ultra-15 centrifugal filters (10 kDa molecular weight cut-off) and stored at -80°C upon the addition of 10 % glycerol (v/v).
Protein concentration was measured using a NanoDrop 2000c and the yield determined as 45 mg / liter cell culture. Final analysis of the protein was performed by Electrospray ionization mass spectrometry (ESI-MS) ( Figure S4) and analytical reversed phase high pressure liquid chromatography (RP-HPLC) ( Figure S5).

Bromide 6
To a solution of germacrene d (42.0 mg, 77% purity, 0.158 mmol) in DCM (6 mL) at -78 °C was added NBS (31.0 mg, 1.1 eq, 0.174 mmol) as a solution in DCM (0.5 mL). The resulting solution was stirred for 1 h at -78 °C, then allowed to warm to rt. The reaction mixture was then extracted with sat.aq. Na2S2O3 (10 mL), and the organic phase was collected. The aqueous phase was extracted with DCM (2 × 5 mL), and the combined organic phases were dried over Na2SO4 and concentrated in vacuo. 1 H-NMR analysis of the crude oil showed  1,9 -6 and  1,2 -6 were formed in 22% and 8% yield, respectively (mesitylene internal standard). The crude oil was then purified by flash chromatography (pentane) to give  1,9 -6 and  1,2 -6 (3 Signals arising exclusively from the minor isomer are indicated by an * and those arising solely from the major isomer are indicated by a † ; undesignated signals arise from a mixture of both.
This procotol was adapted from procedure of Polter et al. [20] Germacradien-4-ol (7) Based on the yield and loading of GdolS, the total number of turnovers under these conditions is 360 (assuming quantitative yield of the preceding diphosphorylation step).
The spectra matched those reported in the literature. [21] Germacradien-4-methyloxy (17) To NaH (25.2 mg, 60% dispersion in mineral oil, 0.63 mmol, 4.0 eq) in THF (1 mL) at rt was added germacradien-4-ol (7)  was then extracted with sat. aq. Na2S2O3 (10 mL), and the organic phase was collected. The aqueous phase was extracted with DCM (2 × 10 mL), and the combined organic phases were dried over Na2SO4 and concentrated in vacuo. The crude mixture was dissolved again in DCM (3 mL) and cooled to 0 °C. NaHCO3 (37.8 mg, 0.45 mmol, 2.0 eq) and mCPBA (60.5 mg, 77% purity, 0.27 mmol, 1.2 eq) were then added, and the resulting mixture was stirred at 0 °C for 1 h. The mixture was then warmed to rt and sat.
aq. Na2S2O3 (10 mL) was added. The biphasic mixture was separated, and the aqueous phase was extracted with DCM (2 × 10 mL). The combined organic phases were washed with brine (20 mL), and dried over Na2SO4, then concentrated in vacuo. The resultant crude oil was purified by flash chromatography (15 -40% Et2O in heptane) to give 3 (23.7 mg, 35%) as a clear oil.
The absolute stereochemistry of 3 was not assigned in the isolation report, however, the optical rotation  To a solution of NCS (3.2 mg, 0.024 mmol, 1.1 eq) in DCM (0.6 mL) at -78 °C was added germacradien-4-ol (5.0 mg, 0.023 mmol) as a solution in DCM (0.3 mL). The resulting solution was stirred for 1 h at -78 °C, then allowed to warm to rt. The reaction mixture was then extracted with sat. aq. Na2S2O3 (10 mL), and the organic phase was collected. The aqueous phase was extracted with DCM (2 × 5 mL), and the combined organic phases were dried over Na2SO4 and concentrated in vacuo. 1 H-NMR analysis of the crude oil showed that 9 was formed in 33% (mesitylene internal standard). The crude mixture was dissolved again in DCM (1 mL) and cooled to 0 °C. NaHCO3 (3.8 mg, 0.045 mmol, 2 eq) and mCPBA (6.1 mg, 77% purity, 0.027 mmol, 1.2 eq) were then added, and the resulting mixture was stirred at 0 °C for 1 h. The mixture was then warmed to rt and sat. aq. Na2S2O3 (3 mL) was added. The biphasic mixture was separated, and the aqueous phase was extracted with DCM (2 × 5 mL). The combined organic phases were washed with brine (5 mL), and dried over Na2SO4, then concentrated in vacuo. The resultant crude oil was purified by flash chromatography (15 -40% Et2O in heptane) to give 9 (2.1 mg, 36%) as a clear oil.

6,8-cycloeudesmane 14
To a solution of germacradien-4-ol 7 (30.0 mg, 0.135 mmol, 1 eq) and NaHCO3 (17.0 mg, 0.202 mmol, 1.5 eq) in DCM (2 mL) at 0 °C was added mCPBA (33.3 mg, 0.148 mmol, 1.1 eq). The resulting suspension was stirred at 0 °C for 20 min, then warmed to rt, and filtered through a syringe filter, washing with DCM until the filtrate diluted to 4 mL total volume. At this point, pivalic acid (41.3 mg, 0.405 mmol, 3 eq) was added. The resulting solution was stirred for rt for 48 h, then concentrated in vacuo. 1 H-NMR analysis of the crude oil showed that 14 was formed in 13% (mesitylene internal standard). [23] An analytical sample was purified by flash column chromatography.
The resulting solution was stirred for 1 h at -78 °C, then allowed to warm to rt. The reaction mixture was then extracted with sat. aq. Na2S2O3 (5 mL), and the organic phase was collected.
The aqueous phase was extracted with DCM (2 × 5 mL), and the combined organic phases were dried over Na2SO4 and concentrated in vacuo. The crude mixture was dissolved again in DCM (1 mL) and cooled to 0 °C. NaHCO3 (14.2 mg, 0.17 mmol, 2 eq) and mCPBA (20.9 mg, 77% purity, 0.093 mmol, 1.2 eq) were then added, and the resulting mixture was stirred at 0 °C for 1 h. The mixture was then warmed to rt and sat. aq. Na2S2O3 (10 mL) was added. The biphasic mixture was separated, and the aqueous phase was extracted with DCM (2 × 10 mL). The combined organic phases were washed with brine (20 mL), and dried over Na2SO4, then concentrated in vacuo. The resultant crude oil was purified by flash chromatography (0 -5% Et2O in heptane) to give 19 (9.1 mg, 34%) as a clear oil. for C15H24Br + .

6,8-cycloeudesmane 20
To a solution NCS (4.4 mg, 1.1 eq, 0.033 mmol) in DCM (1.2 mL) at -78 °C was added germacradien-4-OMe 17 (7.0 mg, 0.030 mmol) as a solution of in DCM (0.3 mL). The resulting solution was stirred for 1 h at -78 °C, then allowed to warm to rt. The reaction mixture was then extracted with sat. aq. Na2S2O3 (2 mL), and the organic phase was collected. The aqueous phase was extracted with DCM (2 × 5 mL), and the combined organic phases were dried over Na2SO4 and concentrated in vacuo. The crude mixture was dissolved again in DCM (1 mL) and cooled to 0 °C. NaHCO3 (5.0 mg, 0.059 mmol, 2 eq) and mCPBA (7.3 mg, 77% purity, 0.033 mmol, 1.2 eq) were then added, and the resulting mixture was stirred at 0 °C for 1 h. The mixture was then warmed to rt and sat. aq. Na2S2O3 (5 mL) was added. The biphasic mixture was separated, and the aqueous phase was extracted with DCM (2 × 5 mL). The combined organic phases were washed with brine (5 mL), and dried over Na2SO4, then concentrated in vacuo. The resultant crude oil was purified by flash chromatography (0 -5% Et2O in heptane) to give 20 (2.6 mg, 32%) as a clear oil.
HCl (2 × 10 mL), then brine (10 mL). The organic phase was then dried over Na2SO4 and concentrated in vacuo. Traces of pyridine were removed from the crude oil by concentration from toluene (3 × 5 mL), to give the crude mesylate used without further purification.
The crude mesylate was dissolved in THF (20 mL), cooled to 0 °C, then BH3•THF (30.2 mL, 30.2 mmol, 6 eq) was added dropwise. After 5 min, the solution was then warmed to rt and stirred for 2 h. MeOH (1.2 mL) then NaOMe (4 mL, 2 M in MeOH, freshly prepared) were then added dropwise, and once the evolution of gas had subsided, the solution was brought to reflux for 30 min. The mixture was allowed to cool to rt, then sat. aq. NH4Cl (50 mL) was then added carefully. The biphasic mixture was extracted with pentane (3 × 150 mL), then the organic phases were dried over Na2SO4 and concentrated in vacuo.
The resultant crude oil was purified by flash chromatography (pentane) to afford cyclodecadiene 23 (486 mg, 50% yield) as a clear oil.
The resulting solution was stirred for 1 h at -78 °C, then allowed to warm to rt. The reaction mixture was then extracted with sat. aq. Na2S2O3 (5 mL), and the organic phase was collected.
The aqueous phase was extracted with DCM (2 × 5 mL), and the combined organic phases were dried over Na2SO4 and concentrated in vacuo. 1 H-NMR analysis of the crude oil showed that 28 was formed in 36% (mesitylene internal standard). The crude mixture was dissolved again in DCM (2 mL) and cooled to 0 °C. NaHCO3 (39.8 mg, 0.474 mmol, 3 eq) and mCPBA (39.0 mg, 77% purity, 0.174 mmol, 1.1 eq) were then added, and the resulting mixture was stirred at 0 °C for 1 h. The mixture was then warmed to rt and sat. aq. Na2S2O3 (10 mL) was added. The biphasic mixture was separated, and the aqueous phase was extracted with DCM (2 × 10 mL). The combined organic phases were washed with brine (5 mL), and dried over Na2SO4, then concentrated in vacuo. The resultant crude oil was purified by flash chromatography (pentane) to give 28 (10.8 mg, 25% yield) as a clear oil.
The mixture was then warmed to rt and filtered through a syringe filter. The filtrate was diluted to by addition of DCM (4.5 mL), then pivalic acid (48.4 mg, 0.474 mmol, 3 eq) was added. 1 H-NMR analysis of the crude oil showed that 29 was formed in 13% (mesitylene internal standard). The crude oil was then purified by flash chromatography (10 -25% acetone in heptane) to give 29 (2.5 mg, 6%) as a clear oil.
The resulting solution was stirred for 1 h at -78 °C, then allowed to warm to rt. The reaction mixture was then extracted with sat. aq. Na2S2O3 (5 mL), and the organic phase was collected.
The aqueous phase was extracted with DCM (2 × 5 mL), and the combined organic phases were dried over Na2SO4 and concentrated in vacuo. 1 H-NMR analysis of the crude oil showed that 27 was formed in 36% (mesitylene internal standard).The crude mixture was dissolved again in DCM (2 mL) and cooled to 0 °C. NaHCO3 (39.8 mg, 0.474 mmol, 3 eq) and mCPBA (39.0 mg, 77% purity, 0.174 mmol, 1.1 eq) were then added, and the resulting mixture was stirred at 0 °C for 1 h. The mixture was then warmed to rt and sat. aq. Na2S2O3 (10 mL) was added. The biphasic mixture was separated, and the aqueous phase was extracted with DCM (2 × 10 mL). The combined organic phases were washed with brine (5 mL), and dried over Na2SO4, then concentrated in vacuo. The resultant crude oil was purified by flash chromatography (pentane) to give 27 (11.1 mg, 31%) as a clear oil.