Enantioselective synthesis of tri-deuterated ( – )-geosmin to be used as internal standard in quantitation assays

For the accurate and sensitive quantitation of the off-flavor compound geosmin, particularly in complex matrices, a stable isotopologue as internal standard is highly advantageous. In this work, we present a versatile synthetic strategy leading from (4a R )-1,4a-dimethyl-4,4a,5,6,7,8-hexahydronaphthalen-2(3 H )-one to tri-deuterated ( – )-geosmin ((4 S ,4a S ,8a R )-4,8a-dimethyl(3,3,4- 2 H 3 ) octahydronaphthalen-4a(2 H )-ol). The starting material was readily accessible from inexpensive 2-methylcyclohexan-1-one using previously published procedures.


| INTRODUCTION
(-)-Geosmin ( Figure 1) is a highly odorous molecule with a characteristic musty and earthy smell and a low odor detection threshold value in the range of 1-20 ng/kg. 1,2 Its name is derived from the ancient Greek words "geo" meaning earth and "osme" meaning odor.
In nature, geosmin is produced as secondary metabolite by several types of microorganisms, including actinomycetes, cyanobacteria, myxobacteria, and fungi. [3][4][5] The biosynthesis involves a Mg 2+ -dependent sesquiterpene synthase, which converts farnesyl diphosphate (FPP) to a mixture of sesquiterpenoids including geosmin. 3 The compound can cause a musty and earthy off-flavor in foods and beverages such as drinking water, wine, fish, and cereals. [6][7][8] In the worst case, the off-flavor may lead to consumers' rejection and significant economic loss. Recently, we identified geosmin in fermented cocoa and demonstrated that it may be transferred in odor-active amounts to chocolate. To avoid this, an accurate and sensitive method for its detection and quantitation in fermented cocoa is essential. In gas chromatography-mass spectrometry (GC-MS) and in liquid chromatographymass spectrometry (LC-MS), the use of a stable isotopically substituted analog of the target compound as internal standard is currently considered the best approach. 9,10 Although racemic deuterated geosmin is available from chemical companies, it is highly expensive (20 000 € for 150 mg). Therefore, we attempted to find a convenient synthetic route to deuterated geosmin as an alternative to the commercial product.

Enantiopure
(4aR)-1,4a-dimethyl-4,4a,5,6,7,8-hexahydronaphthalen-2(3H)-one 1 was converted to trideuterated geosmin by a sequence of four synthetic steps as depicted in Scheme 1. The first step was the epoxidation of the double bond in 1. Based on the work of Gosselin et al., 13 mCPBA was chosen as oxidizing agent. For the synthesis of isotopically unmodified geosmin, they compared the suitability of mchloroperbenzoic acid and hydrogen peroxide for the epoxidation of 1. The use of the peroxy acid afforded a 96:4 mixture of the αand β-epoxyketones and an overall yield of 80% of the α-epimer, whereas such a high stereoselectivity could not be achieved with hydrogen peroxide. Gosselin et al. 13 concluded that the steric hindrance induced by the angular methyl group over the β-face in 1 accounts for the preferential attack of the bulky aromatic peroxy acid molecule on the α-face. We adopted the approach of Gosselin et al. for the epoxidation of 1 but applied NaHCO 3 as an additional base because preliminary experiments had revealed that this slightly increased the yield (data not shown). This was to be expected as the acidity of m-chloroperbenzoic acid can lead to side products. 18 Protonation of the double bond in the educt could lead to the formation of an alcohol whereas protonation of the epoxide would lead to the formation of a diol. The epoxidation step proceeded with a yield of 78%. The epoxide was then subjected to reduction with LiAlD 4 , which led to the incorporation of two deuterium atoms and finally resulted in diol 3.
By selective tosylation, the secondary hydroxy group of 3 was converted into a good leaving group, affording 4. Without isolation of 4, a second reduction step with LiAlD 4 replaced the tosyl group by deuterium, finally leading to the trideuterated target molecule (4S,4aS,8aR)-4,8a-dimethyl(3,3,4-2 H 3 )octahydronaphthalen-4a(2H)-ol 5, that is, ( 2 H 3 )geosmin. The compound was purified by flash chromatography. The overall yield from 1 was 24%. The enantiomeric distribution of ( 2 H 3 )geosmin was determined by GC-MS using a β-cyclodextrin-based chiral column. The elution order was taken from a previous report on the enantioseparation of geosmin in wine. 19 Results indicated an enantiomeric purity of 91%, which confirmed the proposed enantioselectivity of the synthetic approach.
The incorporation of three deuterium atoms was confirmed by GC-MS. The EI mass spectrum of ( 2 H 3 ) geosmin ( Figure 2A) showed a molecular ion of m/z 185, whereas the spectrum of the isotopically unmodified geosmin showed a molecular ion of m/z 182 ( Figure 2B). No signals of m/z 182, 183, and 184 were present in the spectrum of the synthesized molecule, showing that no undeuterated, monodeuterated, and dideuterated geosmin isotopologues were present. Thus, the approach resulted in a uniformly trideuterated product. Further evidence was achieved by NMR. 1 H and 13 C NMR spectra allowed to unambiguously assign the positions of the three deuterium atoms. The singlet obtained in the 1 H NMR spectrum confirmed the presence of the deuterium atom at C4. Moreover, the multiplicity of the signals obtained in the 13 C NMR spectrum for carbons C3 and C4 indicated the coupling with two and one deuterium atoms, respectively.

| Chemicals and materials
The chemicals used were obtained from commercial sources: m-chloroperbenzoic acid (77%), ptoluenesulfonyl chloride, pyridine, sodium sulfate, and lithium aluminum deuteride were purchased from Merck (Darmstadt, Germany); sodium bicarbonate from Alfa Aesar (Karlsruhe, Germany); tetrahydrofuran from Santa Cruz Biotechnology (Heidelberg, Germany). Diethyl ether and dichloromethane were purchased in technical grade from Fisher Scientific (Loughborough, UK) and VWR (Darmstadt, Germany), respectively, and they were freshly distilled before use. Hexane, tetrahydrofuran, and chloroform were purchased in technical grade and stored over molecular sieves (4 Å). Chloroform was filtered through alumina before use to eliminate traces of ethanol present as stabilizer. Silica gel 60 (particle size: 0.035-0.070 mm) and LiChroprep ® DIOL (particle size: 0.040-0.063 mm) used for purification as well as precoated silica gel thin-layer chromatography (TLC) plates (layer thickness 750 μm, no fluorescence indicator) used for reaction monitoring were purchased from Merck. Hexane and diethyl ether mixtures in different proportions were used as mobile phase. Cerium ammonium molybdate or potassium permanganate solutions were employed in TLC as stains for substance detection, followed by heat treatment (200 C).

| Gas chromatography-mass spectrometry
EI mass spectra were recorded using a GC-MS system consisting of a Trace GC Ultra gas chromatograph coupled to a single quadrupole ISQ mass spectrometer (Thermo Fisher Scientific, Dreieich, Germany). Compounds were dissolved in dichloromethane at a concentration of 20 μg/mL. An aliquot (1 μL) was introduced by an autosampler GC PAL, PAL Firmware 2.5.2 (Chromtech, Bad Camberg, Germany), into a PTV injector (Thermo Fisher Scientific) at 40 C. The injector temperature was raised at 12 C/s to 60 C (held for 0.5 min) and then by 10 C/s to 240 C (held for 1 min). The carrier gas was helium at a flow rate of 2 mL/min. The splitflow was 24 mL/min. The column was a DB-1701 coated fused silica capillary, 30 m × 0.25 mm i.d., 0.25-μm film thickness (Agilent, Waldbronn, Germany). The initial oven temperature was 40 C. After 2 min, it was raised at 6 C/min to 230 C (held for 5 min). Mass spectra were acquired at an ionization energy of 70 eV and a scan range of 40-300 m/z. The mass spectra were evaluated using Xcalibur 2.0 software (Thermo Fisher Scientific). Chemical ionization (CI) mass spectra were recorded using an enantioGC-MS system consisting of a Trace 1310 gas chromatograph coupled to a Q Exactive mass spectrometer (Thermo Fisher Scientific). Compounds were dissolved in dichloromethane at a concentration of 10 μg/mL. An aliquot (1 μL) was introduced by a TRI Plus RSH autosampler (Thermo Fisher Scientific) into a PTV injector (Thermo Fisher Scientific) used in oncolumn mode. The carrier gas was helium at a flow rate of 1 mL/min. The column was a BGB-176 coated fused silica capillary, 30 m × 0.25 mm i.d., 0.25-μm film thickness (BGB Analytik, Rheinfelden, Germany). The initial oven temperature was 40 C. After 2 min, it was raised at 2 C/min to 200 C, and the final temperature was held for 5 min. Mass spectra were acquired with targeted SIM mode at an ionization energy of 70 eV and a scan range of 50-500 m/z. The reagent gas was isobutane. The mass spectra were evaluated using Xcalibur 2.0 software (Thermo Fisher Scientific).
Compound 1 (400 mg, 2.25 mmol) was dissolved in dichloromethane (30 mL), and sodium hydrogen carbonate (378 mg, 4.50 mmol) was added. The mixture was cooled on ice, and m-chloroperbenzoic acid (581 mg, 3.37 mmol) was added under argon over a period of 10 min. Quickly, a white precipitate was formed. After 2 h, the suspension was brought to room temperature and left under magnetic stirring for additional 24 h. The suspension was washed with a mixture of a saturated aqueous sodium thiosulfate solution and a saturated aqueous sodium carbonate solution (1 + 1, v + v; 2 × 50 mL), followed by brine (50 mL) and finally dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure, and the residue was purified by flash chromatography on silica gel to afford 342 mg of 2 (78% yield). TLC: R f 0.48 (hexane/diethyl ether, 4 + 1, v + v).
Under an argon atmosphere, lithium aluminum deuteride (185 mg, 4.41 mmol) was suspended in dry THF (15 mL), and the flask was heated to gentle reflux. Epoxide 2 (342 mg, 1.76 mmol) was dissolved in dry THF (5 mL) and added dropwise. After 2 h, the flask was cooled on ice, and a saturated aqueous solution of sodium sulfate (5 mL) was slowly added. Hydrochloric acid (1%; 1 mL) was added, and the mixture was stirred. The aqueous layer was separated and extracted with diethyl ether (2 × 20 mL). The organic phase and the diethyl ether extracts were combined, washed with brine (2 × 20 mL), and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica gel to afford 198 mg of 3 (56% yield). TLC: R f 0.28 (hexane/diethyl ether, 2 + 3, v + v). 4.6 | (4S,4aS,8aR)-4,8a-dimethyl(3,3,4-2 H 3 ) octahydronaphthalen-4a(2H)-ol (5) Diol 3 (198 mg, 0.99 mmol) was dissolved in chloroform (40 mL). Under an argon atmosphere, pyridine (800 μl, 9.90 mmol) and subsequently p-toluenesulfonyl chloride (1.91 g, 10.0 mmol) were added slowly under stirring. The mixture was kept for 72 h at 10 C. A suspension of lithium aluminum deuteride (83.4 mg, 1.99 mmol) in dry THF (10 mL) was added dropwise, and the reaction mixture was heated at reflux. After 4 h, the mixture was cooled down to room temperature. Diethyl ether (20 mL) was added, followed by water (5 mL), and subsequently aqueous hydrochloric acid (1%; 20 mL). The organic phase was separated and washed with an aqueous sodium hydrogen carbonate solution (5%; 20 mL) and brine (20 mL). After drying over anhydrous sodium sulfate and filtration, the solvents were removed under reduced pressure, and the crude product was purified by flash chromatography on a diol phase to give 98.5 mg of 5 (55% yield) with an enantiomeric purity of 91%