Enantioselective Total Synthesis of (R,R)-Blumenol B and d9-(R,R)-Blumenol B

C13-norisoprenoids are of particular importance to grapes and wines, as these molecules influence wine aroma and have been shown to significantly contribute to the distinct character of various wine varieties. Blumenol B is a putative precursor to a number of important wine aroma compounds, including the well-known compounds theaspirone and vitispirane. The enantioselective synthesis of (R,R)-blumenol B from commercially available 4-oxoisophorone was achieved using a short and easily scaleable route, which was then successfully applied to the synthesis of poly-deuterated d9-blumenol B.


Introduction
Norisoprenoids are molecules found in grapes and wines that result from the direct degradation of carotenoids either photochemically, chemically or via oxidase-coupled mechanisms [1,2]. Those with 13 carbon atoms, i.e., C 13 -norisoprenoids, are of particular importance as these molecules, even at very low concentrations, are key contributors to the aroma profile of wine. C 13 -norisoprenoids are known to influence the distinctive sensory character of many wine varieties including Cabernet Sauvignon, Chenin blanc, Sauvignon blanc, Syrah and Riesling [3][4][5].
To date, blumenol B 1 has only been synthesized via routes that were either no stereospecific, lengthy (17-steps) or involved a semi-synthetic route from complex isolat natural products, such as the case of (S,R)-and (R,S)-blumenol B [17,25,29]. We wished develop an efficient stereospecific synthesis of blumenol B from commercially availab materials that would also be suitable for the preparation of isotopically labelled standard Such standards could be employed for the identification and quantification of aglycon that are enzymatically and/or biochemically released from glycosidic precursors found wine.

Results
A previous synthesis of structurally similar C13-norisoprenoids such as (±)-theasp rone and (±)-(Z)-vomifoliol utilized the coupling of a organometallic reagent with a cyc ketone to form the key carbon-carbon bond at the tertiary alcohol center [17,25]. The previously endeavors were only able to form ring-opened derivatives such as 1 and 4 an acidic ring cleavage of spiro-like molecules similar to 2. This not only resulted in rin opened compounds but numerous rearranged and dehydrated species. We aimed to em ploy a similar organometallic strategy of addition to a ketone when attempting the rac mic synthesis of blumenol B 1, but would avoid the formation of spiro-compoun Scheme 1. Blumenol B (1) is a precursor in the formation of theaspirone (2) and other important C 13 -norisoprenoids including vitispirane (3), megastigm-4-ene-3,6,9-triol (4), TDN (5) and Riesling acetal (6) in grapes and wines.
To date, blumenol B 1 has only been synthesized via routes that were either nonstereospecific, lengthy (17-steps) or involved a semi-synthetic route from complex isolated natural products, such as the case of (S,R)-and (R,S)-blumenol B [17,25,29]. We wished to develop an efficient stereospecific synthesis of blumenol B from commercially available materials that would also be suitable for the preparation of isotopically labelled standards. Such standards could be employed for the identification and quantification of aglycones that are enzymatically and/or biochemically released from glycosidic precursors found in wine.

Results
A previous synthesis of structurally similar C 13 -norisoprenoids such as (±)-theaspirone and (±)-(Z)-vomifoliol utilized the coupling of a organometallic reagent with a cyclic ketone to form the key carbon-carbon bond at the tertiary alcohol center [17,25]. These previously endeavors were only able to form ring-opened derivatives such as 1 and 4 by an acidic ring cleavage of spiro-like molecules similar to 2. This not only resulted in ring-opened compounds but numerous rearranged and dehydrated species. We aimed to employ a similar organometallic strategy of addition to a ketone when attempting the racemic synthesis of blumenol B 1, but would avoid the formation of spiro-compounds enroute to 1. We began using the commercially available 4-oxoisophorone 7 as the substrate for coupling with a lithiated acetylide. The first step in this pathway was protection of the less hindered carbonyl in 4-oxoisophorone 7 to afford the known ketal 8, which was reacted with lithiated TMS-acetylene giving tertiary alcohol 9 in 65% yield across the two steps (Scheme 2) [30,31]. Removal of the TMS-group formed the free acetylide 10, which was reacted with acetaldehyde to afford diol 11 as an inseparable mixture of diastere-omers. Acetylene 11 then underwent exhaustive hydrogenation of the exocyclic alkyne, followed by deprotection of the ketone acetal to give (±)-blumenol B 1 as an inseparable 2:1 diastereomeric mixture in 53% yield over two steps. It was found that hydrogenation for 20 h reaction time resulted in an exclusive reactive of the endocyclic alkene [32,33]. However, longer reaction times were found to result in unwanted over-reduction. The spectroscopic data of the mixture of isomers of (±)-1 were in agreement with those reported by Matsunami et al. [29] (Tables S1 and S2). enroute to 1. We began using the commercially available 4-oxoisophorone 7 as the substrate for coupling with a lithiated acetylide. The first step in this pathway was protection of the less hindered carbonyl in 4-oxoisophorone 7 to afford the known ketal 8, which was reacted with lithiated TMS-acetylene giving tertiary alcohol 9 in 65% yield across the two steps (Scheme 2) [30,31]. Removal of the TMS-group formed the free acetylide 10, which was reacted with acetaldehyde to afford diol 11 as an inseparable mixture of diastereomers. Acetylene 11 then underwent exhaustive hydrogenation of the exocyclic alkyne, followed by deprotection of the ketone acetal to give (±)-blumenol B 1 as an inseparable 2:1 diastereomeric mixture in 53% yield over two steps. It was found that hydrogenation for 20 h reaction time resulted in an exclusive reactive of the endocyclic alkene [32,33]. However, longer reaction times were found to result in unwanted over-reduction. The spectroscopic data of the mixture of isomers of (±)-1 were in agreement with those reported by Matsunami et al. [29] (Tables S1 and S2). Following the development of this short racemic synthesis of 1, the next goal was the stereoselective synthesis of (R,R)-blumenol B using enantiopure reagents to direct the stereochemistry of the two chiral centers (Scheme 3). The first enantiopure reagent employed was commercially available (R)-(+)-3-butyn-2-ol 12, which was protected with a TBDPS group giving 13 in quantitative yield [34]. The second enantiopure reagent used was 2R,3R-(-)-2,3-butanediol, which was used to protect the less-hindered carbonyl on 4-oxoisophorone 7, forming the known chiral ketal 14 [35]. Alkynylation of ketal 14 using lithiated 13 afforded a 1.2:1 diastereomeric mixture of the R-and S-configuration at the newly formed tertiary alcohol stereocenter. Subsequent recrystallization using petroleum ether gave the major R-isomer 15 in 47% yield as a white solid and minor S-isomer 15a in 12% yield as a colourless oil, allowing for 15 to be easily separated. Similar facial selectivity, and solid versus oil for the two diastereoisomers, has been observed for the additional of alternative acetylenes with chiral ketone 14, which allowed for the determination of absolute stereochemistry in 15 [35]. This was further confirmed when crystals of d9-16 were formed (see below) and X-ray unequivocally determined the absolute stereochemistry. With 15 formed, both the acetal group and TBDPS-protecting group were removed to form propargyl alcohol 17 in 91% yield, across the two steps. The hydrogenation of propargyl alcohol 17 in the presence of Lindlar catalyst afforded (R,R)-blumenol B 1 in 57% yield, which exhibited identical 1 H and 13 C NMR data to reported values for (S,S)-blumenol B (Table S3) [29]. Following the development of this short racemic synthesis of 1, the next goal was the stereoselective synthesis of (R,R)-blumenol B using enantiopure reagents to direct the stereochemistry of the two chiral centers (Scheme 3). The first enantiopure reagent employed was commercially available (R)-(+)-3-butyn-2-ol 12, which was protected with a TBDPS group giving 13 in quantitative yield [34]. The second enantiopure reagent used was 2R,3R-(-)-2,3-butanediol, which was used to protect the less-hindered carbonyl on 4-oxoisophorone 7, forming the known chiral ketal 14 [35]. Alkynylation of ketal 14 using lithiated 13 afforded a 1.2:1 diastereomeric mixture of the Rand Sconfiguration at the newly formed tertiary alcohol stereocenter. Subsequent recrystallization using petroleum ether gave the major R-isomer 15 in 47% yield as a white solid and minor S-isomer 15a in 12% yield as a colourless oil, allowing for 15 to be easily separated. Similar facial selectivity, and solid versus oil for the two diastereoisomers, has been observed for the additional of alternative acetylenes with chiral ketone 14, which allowed for the determination of absolute stereochemistry in 15 [35]. This was further confirmed when crystals of d 9 -16 were formed (see below) and X-ray unequivocally determined the absolute stereochemistry. With 15 formed, both the acetal group and TBDPS-protecting group were removed to form propargyl alcohol 17 in 91% yield, across the two steps. The hydrogenation of propargyl alcohol 17 in the presence of Lindlar catalyst afforded (R,R)-blumenol B 1 in 57% yield, which exhibited identical 1 H and 13 C NMR data to reported values for (S,S)-blumenol B (Table S3) [29].
Following the successful synthesis of (R,R)-blumenol B 1, the next goal was to apply this methodology to synthesize isotopically labelled (R,R)-1, for possible use as an analytical standard for quantifying (R,R)-1. This would require the synthesis of an isotopically labelled precursor and it was decided to use the previously reported d 9 -8 in this study [36]. The synthesis began with treating 1,4-cyclohexanedione monoethylene acetal 18 with four equivalents of CD 3 I, which afforded the target tri-substituted d 9 -19 in 77% (Scheme 4). Next, the α,β-unsaturated ketone was installed in two steps by converting d 9 -19 to the TMSprotected enol d 9 -20, which was then subjected to one-pot halogenation and elimination, using Br 2 followed by DBU to give the target enone d 9 -8 in 70% yield over the two steps. Subsequent removal of the acetal group using 2M HCl afforded d 9 -7, which could then be utilized for synthesis of d 9 -(R,R) blumenol B 1 via the aforementioned methods. In brief, d 9 -7 was protected using 2R,3R-(-)-2,3-butanediol to give chiral ketal d 9 -14, which was then reacted with acetylide 13 affording d 9-15 in 44% yield over the two steps. Chiral acetal d 9 -15 was removed using 2M HCl and subsequent TBDPS deprotection with TBAF gave d 9 -17 in 94% yield. X-ray diffraction studies on TBDPS-protected d 9-16 intermediate confirmed the desired R,R diastereomer had formed ( Figure S1). Lastly, the alkyne motif was fully hydrogenated using H 2 and Lindlar catalyst, forming d 9 -(R,R) blumenol B 1 in 44% yield. Following the successful synthesis of (R,R)-blumenol B 1, the next goal was to apply this methodology to synthesize isotopically labelled (R,R)-1, for possible use as an analytical standard for quantifying (R,R)-1. This would require the synthesis of an isotopically labelled precursor and it was decided to use the previously reported d9-8 in this study [36]. The synthesis began with treating 1,4-cyclohexanedione monoethylene acetal 18 with four equivalents of CD3I, which afforded the target tri-substituted d9-19 in 77% (Scheme 4). Next, the α,β-unsaturated ketone was installed in two steps by converting d9-19 to the TMS-protected enol d9-20, which was then subjected to one-pot halogenation and elimination, using Br2 followed by DBU to give the target enone d9-8 in 70% yield over the two steps. Subsequent removal of the acetal group using 2M HCl afforded d9-7, which could then be utilized for synthesis of d9-(R,R) blumenol B 1 via the aforementioned methods. In brief, d9-7 was protected using 2R,3R-(-)-2,3-butanediol to give chiral ketal d9-14, which was then reacted with acetylide 13 affording d9-15 in 44% yield over the two steps. Chiral acetal d9-15 was removed using 2M HCl and subsequent TBDPS deprotection with TBAF gave d9-17 in 94% yield. X-ray diffraction studies on TBDPS-protected d9-16 intermediate confirmed the desired R,R diastereomer had formed ( Figure S1). Lastly, the alkyne motif was fully hydrogenated using H2 and Lindlar catalyst, forming d9-(R,R) blumenol B 1 in 44% yield.

General Experimental Details
All reactions in non-aqueous solvents were carried out under an inert atmosphere using anhydrous AR grade solvents. Solvents used for reaction work up and purification were used as purchased, without further purification. Thin-layer chromatography (TLC) was performed using Merck silica gel F354 aluminium plates pre-coated with silica. Flash chromatography was carried out using Silica Gel 60 (40-63 μm, 230-430 mesh ASTM) uti-

General Experimental Details
All reactions in non-aqueous solvents were carried out under an inert atmosphere using anhydrous AR grade solvents. Solvents used for reaction work up and purification were used as purchased, without further purification. Thin-layer chromatography (TLC) was performed using Merck silica gel F354 aluminium plates pre-coated with silica. Flash chromatography was carried out using Silica Gel 60 (40-63 µm, 230-430 mesh ASTM) utilising solvent systems defined in the experimental procedure for each synthesized molecule. Infra-red (IR) spectra were obtained using a Perkin-Elmer Spectrum 1000 series Fourier Transform Infra-Red ATR spectrometer. Melting points were measured using a Reicher-Kofler block and are uncorrected. NMR spectra were obtained using a Bruker Avance DRX 400 MHz spectrometer at ambient temperature. Chemical shifts are reported relative to the residual solvent peak of either CDCl 3 (δ 7.26 for 1 H and δ 77.16 for 13 C) or CD 3 OD (δ 3.31 for 1 H and δ 49.00 for 13 C). 1 H NMR data are reported in the following sequence: position (δ), relative integral, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; m, multiplet; br s, broad singlet; br d, broad doublet), coupling constant (J, Hz), and proton assignment. 13 C NMR data were reported in the following sequence: position (δ), multiplicity (d, doublet; q, quartet), coupling constant (J, Hz), and the carbon assignment. NMR assignments were made using a combination of 1 H NMR, 13 C NMR, HSQC and HMBC experiments. High-resolution mass spectroscopy (HRMS) was carried out using electrospray ionisation (ESI) on a MicroTOF-Q mass spectrometer. in THF (6 mL) was added dropwise to the previously prepared reaction mixture. The resultant mixture was then stirred under a nitrogen atmosphere for 1 h, and the reaction was quenched with sat. aq. NH 4 Cl (5 mL), and was extracted with diethyl ether (3 × 5 mL), before the combined organic extracts were washed with water (5 mL), brine (5 mL) and dried over anhydrous MgSO 4 and the solvent was removed in vacuo. The crude product was purified by flash chromatography (14:1 hexanes, ethyl acetate) to yield the title compound (±)-9 (0.22 g, 75%) as a yellow oil. R F (14:1, 9:1 hexanes, ethyl acetate) = 0.39. 4,4-(Ethylenedioxy)-2,6,6-trimethyl-1-ethynylcyclohex-2-en-1-ol (±)-10: To a stirred solution of (±)-9 (1.80 g, 6.12 mmol) in methanol (50 mL), potassium carbonate (2.54 g, 18.8 mmol) was added, and the reaction mixture was stirred at room temperature for 1 h. The precipitate was filtered, the solvent was removed in vacuo, and was extracted with diethyl ether (3 × 50 mL); then, the combined organic extracts were washed with water, dried over anhydrous MgSO 4  (±)-Blumenol B (±)-1: To a stirred solution of (±)-11 (300 mg, 1.13 mmol) in MeOH (30 mL), Pa/BaSO 4 (120 mg, 40% w/w) was added, and was stirred under a hydrogen atmosphere at room temperature for 18-20 h. The mixture was filtered through Celite ® , washed with MeOH and the solvent was removed in vacuo to yield the alkane (160 mg, 53%) as a colorless oil, which was immediately dissolved in THF (8 mL). The solution was placed under a nitrogen atmosphere, 2 M HCl (0.8 mL) was added, and the resultant mixture was stirred at room temperature overnight. The solvent was removed in vacuo and extracted with ethyl acetate (3 × 5 mL), and the combined organic extracts were washed with brine (5 mL), and dried over anhydrous MgSO 4 , and the solvent was removed in vacuo. The  [25,29].