Asymmetric Synthesis of Oxygenated Monoterpenoids of Importance for Bark Beetle Ecology

Herein we report the asymmetric syntheses of a number of oxygenated terpenoids that are of importance in the chemical ecology of bark beetles. These are pinocamphones, isopinocamphones, pinocarvones, and 4-thujanols (= sabinene hydrates). The camphones were synthesized from isopinocampheol, the pinocarvones from β-pinene, and the thujanols from sabinene. The NMR spectroscopic data, specific rotations, and elution orders of their stereoisomers on a chiral GC-phase (β-cyclodextrin) are also reported. This enables facile synthesis of pure compounds for biological activity studies and identification of stereoisomers in mixed natural samples.

T he colonization of trees by bark beetles is generally influenced by an intricate release of chemical signals in strict chronological order. The signals recruit conspecifics to a suitable host tree, and later in the colonization, other compounds are produced to convey to conspecifics that this tree is becoming overexploited. 1 Thereby competition for food and larvae is avoided. These attractant chemical signals can originate from metabolized monoterpenoids, like for the noxious larger spruce bark beetle, Ips typographus, where cisverbenol is converted to verbenone, but the pheromones can also be synthesized de novo. 2 In recent work with semiochemicals for tree-killing bark beetles we have encountered a number of oxygenated monoterpenoids that are physiologically active (antiattractive, i.e., reduce the effect of aggregation pheromone) in these beetles ( Figure 1). 3,4 We recently published that the production of oxygenated monoterpenoids is related to tree stress and that it might be a signal for a suitable or nonsuitable host for bark beetles. 3 Investigations by gas chromatographic electroantennographic detection (GC-EAD) of monoterpenones by us 3 and Kalinova et al. 5 revealed that both isopinocamphones and pinocamphones elicit antennal responses in I. typographus. There are relatively few syntheses of pinocamphone and isopinocamphone reported. In one report in Chinese, Wang et al. reacted α-pinene with borane to obtain diisopinocampheylborane, which was oxidized to isopinocampheol by sodium perborate to afford isopinocampheol. 6 The isopinocampheol was finally oxidized by H 2 O 2 with vanadium phosphorus oxide as catalyst to yield isopinocamphone. Pitínova-Sťekrováand co-workers utilized different titanosilicate catalysts to convert α-pinene to obtain campholenic aldehyde. 7 Some of these catalysts produced pinocamphone as side-product. In another report, thermolysis of α-pinene epoxide in supercritical anhydrous isopropanol afforded up to ∼25% pinocamphone, but in an inseparable mixture of oxygenated monoterpenoids. 8 These syntheses do not yield pure stereoisomers or are tedious and have low yields. For short and convenient synthesis without heating, we developed a simple method using pure isopinocampheol stereoisomers that are commercially available. Pinocarvone is reported as a pheromone for the southern pine beetle (Dendroctonus f rontalis) 9 and has been found in the hindguts of male white pine cone beetles, Conophthorus coniperda. 10 In the GC-EAD analysis of I. typographus, pinocarvone gave strong antennal responses, indicating biological activity. 3 Pinocarvone has been isolated from Eucalyptus oil and has been produced by oxidation of βpinene with SeO 2 . In this reaction, myrtenal is formed by a rearrangement, and this byproduct was either overlooked or lost during spinning band distillation. 11,12 In previous reports, we reported that one of the two trans-4thujanol stereoisomers showed strong GC-EAD activity for bark beetles 3 and that this (+)-trans-thujanol is a field-active semiochemical for the bark beetle, I. typographus. 13 Blazyte-Cereskienėet al. reported that young spruce trees release more 4-thujanol than older trees and that 4-thujanol plays an important role in both host defense and tree choice by bark beetles. 14 Thus, 4-thujanol is seemingly an indicator of healthy strong trees which should be avoided and could be of interest in forest protection. Several publications on the synthesis of 4thujanol have been published, including the biotransformation of α-pinene to cis-4-thujanol using the microorganism Fusarium saloni, 15 Baeckstrom's synthesis of trans-4-thujanol from 3-thujol, 16 Galopin's synthesis of the trans isomers from methyl vinyl ketone, 17 Cheng's syntheses of cis-thujanol, 18,19 and Fanta's synthesis of trans-thujanol. 20 However, all these synthetic procedures involve many steps and/or expensive starting materials, and there are no effective synthetic routes for all possible stereoisomers. In order to develop a short synthesis of all stereoisomers of 4-thujanol for the investigation of GC-EAD activity, herein these were synthesized from commercial sabinene. The absolute configuration of each stereoisomer was unambiguously assigned by the deduction from the original sabinene in combination with NMR spectroscopy and chiral-phase GC-MS analysis.
These compounds are obviously important in bark beetle ecology, and some of them act as indicators of tree health; they are also of interest for managing bark beetle populations. It is well known that the stereochemistry of pheromones and other semiochemicals is often extremely important. 21−24 Thus, there is a need to develop analytical procedures to be able to use enantioselective gas chromatography to differentiate between the stereoisomers of these semiochemicals in a biological sample, as well as their facile synthesis. We herein report the syntheses, specific rotations, and elution orders on a chiral GC phase (β-cyclodextrin) of pinocamphones, isopinocamphones, pinocarvones, and 4-thujanols (sabinene hydrate).

■ RESULTS AND DISCUSSION
Synthesis of Isopinocamphones and Pinocamphones. Scheme 1 summarizes the syntheses of the four stereoisomers of pinocamphone. The pure enantiomers of isopinocampheol (1 and 2) were separately oxidized with pyridinium dichromate (PDC) to obtain both enantiomers of isopinocamphone in >98% optical purity. The oxidation was improved by adding silica gel to the reaction mixture, which prevents the formation of lumps and tar and in turn leads to higher yields and easier filtration at workup.
To produce pinocamphones, NaOEt was used to epimerize C-2 of isopinocamphone. The thermodynamic equilibrium seems to be 4:1 in favor of pinocamphone, and thus, 20% of isopinocamphone had to be removed by chromatography to obtain pure pinocamphone (Scheme 1).
Specific Rotation of Isopinocamphones and Pinocamphones. The sign of the specific rotation changed when going from isopinocampheols (1 and 2) to isopinocamphones (3 and 4), but not during epimerization from isopinocamphones to pinocamphones (5 and 6). The specific rotations are listed in Table 1.
As the ratio of stereoisomers in the sabinene (10)  The NMR spectrum and specific rotation proved that the isomer purchased from Sigma-Aldrich was the (+)-transisomer, and the major product could be assigned as (−)-cis-4thujanol (13), based on the retention of ring configuration in the synthesis sequence, as well as regioselective considerations and reported NMR spectroscopic data. 30,31 GC Elution Order of 4-Thujanol Stereoisomers. On the HP-5MS GC column, the trans diastereomers eluted first. On the β-cyclodextrin column the first peak of four synthetic isomers coeluted with the commercial (+)-trans-4-thujanol stereoisomer (15) purchased from Sigma-Aldrich, and the last peak coeluted with the isolated (−)-cis-thujanol (13). The elution order of all isomers was (+)-trans, (−)-trans, (+)-cis, (−)-cis ( Figure 5). The elution order is in accordance with those reported by Larkov et al. 31 and Marriott et al. 32 Specific Rotation of Sabina Ketone and 4-Thujanol Stereoisomers. It should be noted that (−)-sabinene (10) yields (+)-sabina ketone (12), which is subsequently transformed to thujanols with (−)-cis-thujanol (13) as the major isomer (Scheme 3). The commercial sabinene (apparently from a natural source) has a specific rotation of −73 (c 1.0, EtOH) and −81 (c 1.0, DCM). Moreover, the chemical purity of the commercial sabinene (10) was only 75%, with 25% βpinene as an impurity and with an ee of 86%. Sabina ketone (12) was obtained in 86% ee and with specific rotations of (1) (Sigma-Aldrich, Schnelldorf, Germany) (12.32 g, 80.0 mmol, chemical purity 98% and optical purity 95% ee) was dissolved in CH 2 Cl 2 (150 mL). 32 Silica gel (18 g) and PDC (60 g, 160 mmol) were added, and the mixture was stirred for 3.5 h at RT before leaving it in a fridge overnight. The slurry was diluted with cyclohexane and filtered. The solid material was washed twice with 1:1 cyclohexane/ CH 2 Cl 2 (50 mL). The filtrate was concentrated and subjected to MPLC, yielding 87% (10.6 g, 69.7 mmol) of (1S,2S,5R)-2,6,6trimethylbicyclo   (8), and (C) mix of pinocarvone enantiomers. The temperature program: Initial oven temperature was 40°C (held for 5 min) increased to 150°C at 3°C/min and finally increased to 220°C at 10°C/min (held for 5 min at the final temperature). Synthesis of the Pinocarvone Stereoisomers (Scheme 2). To a solution of (−)-β-pinene (7) (0.25 g, 1.8 mmol) in DCM (3 mL) was added SeO 2 (0.20 g, 1.8 mmol), and the mixture was refluxed for 2.5 h until GC-MS showed complete transformation. The solution was filtered through silica gel (in a Pasteur pipet), and the product washed out of the silica gel with additional aliqouts of DCM. The product was concentrated under reduced pressure at 30°C to obtain a mixture of pinocarvone and myrtenal. These two compounds were close on TLC and difficult to purify by column chromatography. To the concentrate, MeCN (2 mL), NaH 2 PO 4 (70 mg) in Milli-Q-water (1 mL), and 35% H 2 O 2 (0.2 mL) were added. The solution was stirred for approximately 1 h until the solution became clear. On an ice bath, NaClO 2 (0.32 g) in MQ-water (3 mL) was added dropwise, and the mixture was stirred overnight. One spatula of anhydrous Na 2 SO 3 was added, and the mixture was extracted with DCM (3 × 5 mL). After removal of the solvents, the concentrate was purified on silica gel. The combined fractions were concentrated by rotatory lowvacuum evaporation to afford (+)-pinocarvone (8) (yield 60%, 165 mg, 1.1 mmol). The same experimental procedure was followed to produce (−)-pinocarvone (9) from (+)-β-pinene. The chemical purity of (+)-pinocarvone was 95%, and the optical purity was 97% ee.