Biotransformation of (1S)-2-Carene and (1S)-3-Carene by Picea abies Suspension Culture

Biotransformation of (1S)-2-carene and (1S)-3-carene by Picea abies suspension culture led to the formation of oxygenated products. (1S)-2-Carene was transformed slowly and the final product was identified as (1S)-2-caren-4-one. On the other hand, the transformation of (1S)-3-carene was rapid and finally led to the formation of (1S)-3-caren-5-one and (1S)-2-caren-4-one as equally abundant major products. The time-course of the reaction indicates that some products abundant at the beginning of the reaction (e.g. (1S,3S,4R)-3,4-epoxycarane and (1R)-p-mentha-1(7),2-dien-8-ol) were consumed by a subsequent transformations. Thus, a precise selection of the biotransformation time may be used for a production of specific compounds.


Introduction
Biotransformations are environmentally friendly methods to obtain valuable chemicals which are often used as flavours, fragrances and pharmaceuticals. There has been an extensive interest in biotransformations for they are chemo-, regio-and stereospecific and are a precious source of "natural products", chemical compounds synthesized enzymatically without the use of toxic organic reagents and solvents [1].
Their significance is also inflicted in their variability. Biotransformations may be carried out by a vast range of organisms and on an ample variety of compounds, even exogenous ones [2,3]. Microbial systems may seem advantageous over the others because their biomass doubling times are short and methods for their genetic manipulation are well established [2]. On the other hand, plants possess unique enzymes which enhance their potential. For example, plants are involved in the biosynthesis of some very complicated pharmaceuticals such as paclitaxel or artemisinin [4,5].
(1S)-2-Carene (1) and (1S)-3-carene (2) are monoterpenes produced by conifers as components of their resin, which is engaged in plant defence against herbivores. On the other hand, oxygenated monoterpenes are the main source of aromas in spices and herbs, and they are in great demand for their antibacterial, antifungal and anticancer effects. The biotransformation of carenes by plant cell cultures may lead to oxygenated products which may themselves possess such properties or may be employed as the structural scaffolds in the synthesis of active compounds (for example: sesquiterpenoids, diterpenoids, β-lactam antibiotics) [6].
Since there exist only two reports on carenes transformation by plants [7,8], we have utilized Picea abies suspension culture to explore this area. The pine tree, P. abies, is the natural source of carenes and thus its enzymatic system is accustomed to their bioconversion and its cells are resistant to their toxicity to a great extent. Similar approach was taken by Miyazawa and Kano [9], who studied the biotransformation of (1S)-3-carene by larvae of Spodoptera litura which feeds on plants producing terpenes. Our study focuses on the identification of the biotransformation products and the determination of the dependence of the relative quantitative product yields on incubation time. The absolute configuration of the biotransformation products was assigned according to the synthesized reference compounds. For those compounds not synthesized, the configuration was assigned only when indisputable. The quantitative yields of (1S)-3-carene (2) biotransformation products are also given.
The quantitative product yields in μg/L are entered in Table 1. The maximum yield was obtained for (1R)-p-mentha-1(7),2-dien-8-ol (4) after one day (58.3 μg/L) and for m-cymen-8-ol (13) after four days (32.3 μg/L). The total yield of biotransformation products decreased continually with time due to their volatility. The overall yield (3.5%) was the highest the first day and decreased further on, ending with 1% yield after eleven days. The compound recovery from the Sep-Pak cartridges, which was 60%, also affected the yield.
Importantly, in our case, biotransformation of carenes led to the formation of narrow range of highly abundant compounds which is fundamental for their possible isolation and further utilization. Moreover, the precise selection of biotransformation time-course could maximize the production of the desired compounds. For example, biotransformation of (1S)-2-carene (1) afforded (1R,4R)-p-menth-2en-1,8-diol (6) and (1S)-2-caren-4-one (5) as the most abundant products after five and twelve days, respectively, based to their relative abundance. On the other hand, in the biotransformation of (1S)-3carene (2), (1R)-p-mentha-1(7),2-dien-8-ol (4) was the most abundant product after 24 h and m-cymen-8-ol (13) after four and eight days. Unfortunately, the biotransformation has still its main drawback in low overall conversion to products, which in our case was at the most 3.5%.

Cultivation of in Vitro Cultures
Embryogenic culture of P. abies was induced from immature zygotic embryos and maintained on sterile medium, solidified with 0.75% (w/v) agar. The medium was prepared according to Gupta and Durzan [21] and supplemented with 5 μM 2,4-D, 2 μM kinetin, 2 μM BAP and 30 g/L sucrose. Its pH was adjusted to 5.80 ± 0.05 before autoclaving. The cultures were subcultivated every 7 days. The suspension culture was initiated from the embryogenic culture. The same supplemented maintenance medium (excluding agar) was used as the nutrient medium. The suspension cultures were kept on rotary shakers at 100 rpm in 250 mL Erlenmeyer flasks at 24 °C in darkness. The flasks were sealed with aluminium foil.

Autooxidation of Carenes
(1S)-2-Carene (1) and (1S)-3-carene (2) (20 μL, 23 mg), respectively, were added to a 100 mL medium without cells in a 250 mL Erlenmeyer flask. The flasks, sealed with aluminium foil, were kept on a rotary shaker at 100 rpm at 24 °C in darkness for one day in case of (1S)-3-carene (2) and two days for (1S)-2-carene (1). The experiments were performed in duplicate. After incubation, the medium (50 mL) was applied to a Sep-Pak C-18 syringe cartridge (Waters, Milford). The sugar and salts originating from the nutrient medium were washed out from the cartridge with 10 mL distilled water, whereupon the products were eluted with 1.5 mL of TBME. The resulting samples were analyzed by GC-MS and compared.

Biotransformation of Carenes
(1S)-2-Carene (1) and (1S)-3-carene (2) (20 μL, 23 mg), respectively, were added to a 100 mL suspension culture in a 250 mL Erlenmeyer flask, which was sealed with aluminium foil and kept on a rotary shaker at 100 rpm at 24 °C in darkness for up to 12 days. The experiments were performed in duplicate. After incubation, the medium was filtered through a filter paper (No. 388, filtrate volume 50 mL) and then it was applied to a Sep-Pak C-18 syringe cartridge (Waters, Milford). The samples were processed and analysed the same way as autooxidation samples.

Quantification of Product Formation
In case of (1S)-3-carene (2), the amount of products formed throughout the biotransformation course was quantified. To an eluate from the Sep-Pak cartridge was added 100 μL of 1-adamantol solution of 3 mg/mL concentration as an internal standard. One microlitre of the final solution was subjected to GC-MS.

GC-MS Analyses
The biotransformation products were identified by gas chromatography -mass spectrometry by the use of the MS library Wiley 275 and NIST or by comparisons of their retention times and mass spectra with those of the synthesized reference substances [24][25][26][27][28][29][30]. The relative quantitative yield of each product (in %) was determined as the GC-MS integration area of that product divided by the GC-MS integration area of all products.

Synthesis of Reference Compounds
These were prepared as shown in Scheme 4. (1S)-2-Caren-4-one (5) [24]. The mixture of (1S,4R)-2-caren-4-ol and (1S,3S)-4-caren-3-ol (0.5 g, 3.3 mmol) was added to a stirred solution of pyridiniumchlorochromate (1.42 g, 6.6 mmol) and sodium acetate (92 mg, 1.1 mmol) in dry dichloromethane. The resulting solution was stirred at RT for 4 h. Then, the solution was poured into diethyl ether (50 mL). The solids formed were filtered off through celite and the filtrate was evaporated under reduced pressure. The residue (0.4 g, 80%) was a mixture of two compounds, (1S)-2-caren-4-one (5) and m-cymen-8-ol (13)   and oxygenated compounds are formed. By the proper choice of biotransformation duration, the required compounds may be generated selectively which may simplify their isolation, although the overall yield of the biotransformations is rather low yet comparable to that seen in similar biotransformations.