Lactones 41.† Synthesis and Microbial Hydroxylation of Unsaturated Terpenoid Lactones with p-Menthane Ring Systems

Racemic [(±)-4-isopropyl-1-methyl-7-oxa-cis-bicyclo[4.3.0]non-4-en-8-one] and optically active δ,ε-unsaturated lactones [(-)-(1R,6R)-4-isopropyl-1-methyl-7-oxabicyclo[4.3.0]non-4-en-8-one and (+)-(1S,6S)-4-isopropyl-1-methyl-7-oxabicyclo[4.3.0]non-4-en-8-one)] with the p-menthane system were obtained and their odoriferous properties were evaluated. Biotransformations of the racemic lactone with three fungal strains: Absidia cylindrospora AM336, Absidia glauca AM177 and Syncephalastrum racemosum AM105, were carried out. Microbial transformations afforded hydroxylactones with the hydroxy group in the allylic position.


OPEN ACCESS
As a continuation of our interest in the synthesis of odoriferous and biologically active lactones [10], here we present the synthesis of racemic and enantiomeric pairs of new ,-unsaturated lactones with the p-menthane system. The odour characteristics of the obtained lactones were also evaluated. The synthesis of enantiomeric lactones was carried out because it is commonly known, that the biological effect of bioactive compounds often depends on their chirality. The complicated stereochemistry-bioactivity relationships of insect pheromones, as an example, has been clearly presented by Kenji Mori [11].
The introduction of the hydroxy group into a molecule often leads to changes in its biological activity. Examples of such dependence are well-documented in the literature, especially among monoterpenes, e.g., limonene and carveol, sabinene and sabinol, α-pinene and verbenol [12][13][14][15]. This information inspired us to check the influence of the hydroxy group in the lactones obtained on their odoriferous properties.
Encouraged by the literature reports about regio-and stereoselective biohydroxylation of miscellaneous molecules, we applied some fungal strains to this purpose. Filamentous fungal strains Absidia cylindrospora [16], Absidia glauca [17,18] and Syncephalastrum racemosum [19,20] are known for their hydroxylation ability in relation to a broad variety of compounds. The aforementioned fungal strains were applied to transform the racemic ,-unsaturated lactone (±)-2.

Biotransformation of Racemic
Six fungi strains were tested for their ability to transform racemic lactone (±)-2. Three of them: Absidia cylindrospora AM336, Absidia glauca AM177 and Syncephalastrum racemosum AM105 transformed the substrate into the same products -hydroxylactones 3 and 4 (Scheme 2). No formation of any biotransformation products of (±)-2 in the cultures of another three microorganisms (Fusarium culmorum AM3/1, F. oxysporum AM145 and Penicillium vinaceum AM110), was observed after 10 days of incubation. The progress of biotransformation was monitored by chiral gas chromatography. Hydroxylactone 4 was the major product in all biotransformations carried out. The enantioselectivity of its formation depended on the fungi strains applied. The composition of the products mixture of biotransformation of (±)-2 by selected microorganisms are presented in Table 2. After the first day of incubation (±)-2 in the culture of A. cylindrospora AM336 the product mixture contained 66% of unreacted substrate (2), 14% of hydroxylactone 3 and 20% of product 4. In the next days the amount of 2 decreased and after four days the formation of 39% of 3 and 61% of 4 was observed. The enantiomeric excess of hydroxylactone (+)-3 obtained in this way was the highest one and reached a value of 39%.
The preparative biotransformation of unsaturated lactone (±)-2 (120 mg) in the shaken cultures of A. cylindrospora AM336 after four days afforded: 15 mg (12% isolated yield) of (+)-3-hydroxy-4isopropyl-1-methyl-7-oxa-cis-bicyclo . The structure of these products was confirmed by their spectral data. The presence of the hydroxy group in products 3 and 4 was confirmed by absorption bands in the IR spectra at 3438 and 3450 cm −1 , respectively. Furthermore, the absorption bands at 1760 and 1773 cm −1 , respectively, indicated that the γ-lactone ring has been retained. The final proof of the structure of hydroxylactones 3 and 4 was delivered by NMR spectroscopy.
Analysis of the 1 H-NMR spectrum of product 3 indicates that biohydroxylation had taken place at the C-3 position. The presence of multiplet at 4.37 ppm for proton H-3 and the change of multiplicity and chemical shift of protons CH 2 -2 (compared to the spectrum of the substrate) confirm this assignation. The signal from the pseudoequatorial H-2 proton (δ = 2.07) gave a doublet of doublets with a geminal coupling constant J H2a-H2e = 14.0 Hz and a small coupling constant J = 5.2 Hz with the pseudoequatorial H-3 proton. The pseudoaxial H-2 proton also gave a doublet of doublets (δ = 1.67) with the same geminal coupling constant (J H2a-H2e = 14.0 Hz) and with the vicinal coupling constant J = 7.1 Hz with the pseudoequatorial H-3 proton. On the basis of these data and according to model analysis (Dreiding models) a pseudoequatorial position was assigned to the H-3 proton. This analysis indicates also that the H-3 proton is situated close to the plane of the double bond, so it is deshielded and its signal is located at 4.37 ppm. Additionally, a significant increase in the chemical shift difference of the CH 2 -9 protons (0.22 ppm) in comparison to difference in the spectrum of (±)-2 (0.08 ppm) indicates the nearness of the hydroxy group to one of these protons and the cis-orientation of the OH group to the γ-lactone ring in product 3. The fungal strain A. cylindrospora AM336 is known for its allylic position hydroxylation ability. Our previous biotransformation of unsaturated lactones with A. cylindrospora AM336 afforded hydroxylactones with pseudoaxially oriented hydroxy groups in the allylic position [22,23].
The next product 4 of the biotransformation of (±)-2 was also identified as a hydroxylactone with the hydroxy group in the allylic position. Spectral data provided evidence that the hydroxy group was introduced in the C-11 position. The methyl groups at C-11 gave two singlets (δ = 1.34 and 1.35) whereas in the spectrum of substrate (±)-2 two doublets (J = 6.8 Hz) at 1.01 and 1.02 ppm were observed. Disappearance of the signal from H-11 and the lack of new signals between 3-4 ppm confirm the structure of lactone 4. Additionally, in the 13 C-NMR spectrum the signal from the carbon atom directly connected to the hydroxy group was situated at 72.61 ppm. Moreover, the absence of this signal in the DEPT135 spectrum undoubtedly confirms of the position of hydroxy group in product 4 at C-11.
The retention time of product 3 obtained from the conversion of (-)-(1R,6R)-2 was tR = 115.45 min and the retention times of the two enantiomers of 3 formed during the biotransformation of (±)-2 were tR = 114.30 and 115.45 min, respectively. These results indicate that stereoisomer with tR = 115.45 min has R configuration at C-1 and C-6. Knowing that the hydroxy group at C-3 is cis-oriented with respect to the γ-lactone ring it was possible to ascribe the absolute configuration at this chirality centre. On the basis of these data 1S,3S,6S configuration of (+)-isomer of 3 with tR = 114.30 min and 1R,3R,6R configuration of the (-)-enantiomer of 3 with tR = 115.45 min were assigned.
Unfortunately, it was impossible to evaluate the enantiomeric excess of hydroxylactone 4 by chromatographic methods using the chiral columns at our disposal. However, the analysis of the composition of the products mixture obtained from transformation of lactone (±)-2 ( Table 2) indicates that 1R,6R isomer of 2 was transformed faster than the 1S,6S one, so it could be suggested that the (+)-isomer of 4 has a 1R,6R configuration.

General
Unless otherwise stated, all chemicals were purchased as the highest purity commercially available and were used without further purification. DBU

Screening-Scale Biotransformations
Screening scale biotransformations of lactone (±)-2 by six microorganisms were carried out in 300 mL Erlenmeyer flasks. The corresponding microorganisms were cultivated at room temperature in Erlenmeyer flasks containing 100 mL medium, which had the following composition (per 1 L of distilled water): peptone (10 g) and glucose (30 g). Cultures were incubated in a rotary shaker at 150 rpm at 25 °C. After 3-5 days, 10 mg of substrate (±)-2 dissolved in 1 mL of acetone was added to the shaken cultures. The samples of biotransformation mixture were extracted with chloroform after 1, 2, 4 and 6 days. They were dried over anhydrous MgSO 4 , concentrated in vacuo and analyzed by TLC and GC. The same procedure was applied during the transformation of (-)-2 by Absidia cylindrospora AM336.
Additionally, two control flask were used for each biotransformations. A culture control contained sterile culture medium with microorganism inoculum. This experiment used to define and exclude the secondary metabolites generated by fungi. The second control flask contained substrate and sterile growth medium incubated without fungi. The substrates used for the biotransformation were stable in these conditions. It can be concluded that all products isolated from the cultures were bioconversion products.

Preparative-Scale Biotransformations
Preparative scale biotranasformations were carried out in 2 L flat bottomed flasks containing 400 mL of medium (the same as in the screening scale). Lactone (±)-2 (120 mg in 1 mL acetone) was added to the grown cultures of corresponding microorganisms (Absidia cylindrospora AM336, Absidia glauca AM177, Syncephalastrum racemosum AM105) prepared as described in the screening procedure. After 4 or 6 days the products were extracted with chloroform (3 × 150 mL). The organic solutions were dried over anhydrous MgSO4 and concentrated in vacuo. Mixtures of products as well as metabolites produced by the fungi were separated by column chromatography (hexane-diethyl ether, 9:1 → 4:1). The spectral data of biotransformation products are given below: (3). Colourless oily liquid.

Conclusions
Racemic (±)-2 and an enantiomeric pair of unsaturated γ-lactones with the p-menthane system (-)-2 and (+)-2 were synthesized and their odoriferous properties were evaluated. Comparison of the odour of racemic (±)-2 and optically active compounds (-)-2 and (+)-2 confirmed that their fragrance depends on the configuration of their chiral centers. The results obtained in this work indicate that hydroxylation of (±)-2 by all fungi strains studied has taken place in the allylic positions C-3 and C-11. Similar to the literature data [24] the product with the hydroxy group at C-11 was formed preferentially. Probably it is the result of easier oxidation of tertiary C-H bond than the primary or secondary ones. Unfortunately, in this case, the introduction of the hydroxy group into the substrate molecule resulted in a loss of odoriferous properties.