Materials and Methods
Substrate
(1S,5R)-(-)-alpha-pinene and (1R,5S)-(+)-alpha-pinene were purchased from Merck (Darmstadt, Germany). (1S,5S)-(-)-verbenone was purchased from Sigma-Aldrich Chemical Co. (St. Louis, USA).
Cell culture stocks
The callus tissues of P. brachyceras Müll Arg. (Rubiaceae) used in this investigation were induced from young stem segments (developed under indoor conditions) of cuttings cultured in nutrient solution, as described by Gregianini et al. (2003). After surface sterilization using standard procedures, stem segments were cultured under darkness in MS (Murashige and Skoog) medium containing 3% w/v of sucrose, 1% w/v soluble polyvinylpolypyrrolidone (PVP), 0.75% w/v microbiological grade agar, 10 mg/l of naphthaleneacetic acid (NAA, Sigma Chemical Co. St. Louis, USA) and 1 mg/l kinetin (KIN, Sigma Chemical Co. St. Louis, USA). Calli were developed and maintained in this medium with monthly subcultures at the Laboratory of Plant Physiology, UFRGS. A voucher of the plant, which was harvested at Morro Santana (campus of UFRGS, Porto Alegre, RS, Brazil), is deposited in the University Herbarium (ICN Sobral and Kerber 7899). Cell suspension cultures of R. sellowii Müll Arg. (Apocynaceae) were originally developed by Rech et al. (1998) and maintained on B5 (Gamborg) medium containing 1 mg/l of 2,4-dichlorophenoxyacetic acid (2,4-D, Sigma Chemical Co. St. Louis, USA) and 4% w/v sucrose. Suspension cultures were maintained in this medium, being transferred onto fresh medium fortnightly.
Biotransformations
Biotransformations of (1S,5R)-(-)-alpha-pinene and (1R,5S)-(+)-alpha-pinene were carried out by Psychotria brachyceras and Rauvolfia sellowii cell suspension cultures. Before each experiment, ca. 30 g of cells or callus tissue were transferred to a 250 ml conical flask containing 30 ml of freshly prepared SH medium (Schenk and Hildebrandt, 1972) containing 1 mg/l of 2,4-D and 3% w/v sucrose. Cells were then grown for 1 week at 25 ± 2ºC, under diffuse light (3 µmol. s-1.m2), on a rotary shaker (100 rpm). After this time, 1.0 ml of a methanolic solution (60 mg/ml) of substrate, without prior sterilization, was added to the cell suspensions, and the cultures were returned to the shaker for 15 days. Controls were prepared by the addition of 1 ml of a methanolic solution (60 mg/ml) of alpha-pinene to 30 ml of medium, and, in other flasks, ca. 30.0 g cells and 30 ml medium. Experiments (each with four replicates per sampling time) were independently repeated three times with similar results.
Extraction and analysis
For optimization of extraction procedure, portions of the incubation mixture were pipetted out and extracted with different polarity solvents, such as hexane, chloroform and ethyl acetate (EtOAc), in order to establish the best solvent to extraction procedure. A reminiscent strong emulsion could be observed by the use of EtOAc, even after treatment with sodium chloride-saturated aqueous solution and centrifugation steps, giving samples with a lower concentration of products than that obtained using the other solvents. When the chloroform and hexane were used as solvent, a good performance could be observed, resulting samples with qualitatively equivalent compounds. The exchange of the solvent chloroform to hexane did not change the quantity of obtained monoterpenes. In addition, the use of hexane to the time course analyses reduced another disadvantage associated with the use of chloroform, as the water and medium components solubility being lower in hexane than in chloroform; and the use of hexane extend the lifetime of the water-sensitive chiral gas chromatography (GC) columns that we employed for our analyses. Besides that, hexane afforded more environmentally benign conditions. Even so, for the time course analysis, at the desired time intervals, 10 ml portions of the incubation mixture were harvested from quadruplicate flasks, vigorous shaking, and extracted with 5 ml of hexane. The organic fraction was dried over sodium sulphate, filtered and evaporated under vacuum. The residue obtained was made up to 1 ml with hexane and 3 ml of the solution was subjected to gas chromatography-mass spectrometry (GC-MS) to the qualitative analysis and to GC with flame ionization detector (FID) detector to the quantitative analysis. To the confirmatory studies, the crude extract were saturated with sodium chloride-saturated aqueous solution, extracted with chloroform, evaporated to dryness under reduced pressure and purified by column chromatography on silica gel (70-230 mesh - Aldrich) using as mobile phase hexane:EtOAc (90:10 and 80:20), collecting 10 ml fractions, to give verbenones as yellow oils.
Gas Chromatography analyses were performed using a Shimadzu GC-17A chromatograph equipped with a fused silica capillary column (30 m x 0.25 mm x 0.25 mm, coated with DB-5). Injector and detector temperatures were set at 220ºC and 250ºC, respectively; the oven temperature was programmed from 60-230ºC at 3ºC/min. All the samples were analyzed by GC-MS in the same apparatus and chromatographic conditions as described above, using a quadrupole MS system (QP 5000) operating at 70 eV. The percentage composition of unreacted substrate and the amount of products were obtained from electronic integration measurements using flame ionization detection, without taking into account relative response factors. Compounds identification was based on a comparison of retention indexes (determined relatively to the retention times of a series of n-alkanes) and mass spectra with those of authentic standard purchased from Sigma-Aldrich and literature data (van Dyk et al. 1998; Adams, 2001). The retention indexes obtained were 945 to alpha-pinene, 1132 to trans-pinocarveol, 1140 to trans-verbenol, 1189 to myrtenol, 1199 to unidentified, and 1202 to verbenone. Chiral Gas Chromatography analyses were carried out using the same GC-MS system, equipped with a chiral beta-cyclodextrin (30 m x 0.25 mm x 0.25 µm, coated with B-CDEX 120) fused silica capillary column. The oven temperature was programmed from 60 - 220ºC at 3ºC/min. Injector and detector temperatures were set at 200ºC and 230ºC, respectively. Helium was employed as carrier gas (1 ml/min). Under these conditions, the retention times obtained were 10.071 min to (-)-alpha-pinene, 10.338 min to (+)-alpha-pinene, 27.858 min to (-)-verbenone, and 28.033 min to (+)-verbenone. In the confirmatory studies, the purified verbenones were identified by physical data comparison with authentic samples purchased from Sigma-Aldrich based on a comparison of [α]D values and Nuclear Magnetic Resonance Spectroscopy (NMR) chemical shifts (reported in ppm) and compared with previously reported data (van Dyk et al. 1998; Lajunen et al. 2000).The optical rotation values of purified verbenones ([α]D20 = -258º to (-)-verbenone and +258º to (+)-verbenone, c 1.0, CHCl3), were measured with a Perkin Elmer 341 Polarimeter. The NMR spectra were obtained on a Varian VXR200 apparatus, using CDCl3 as an internal standard. 1H NMR. (200 MHz, CDCl3) δ:1.02 (s, 3H, H-9); 1.47 (s, 3H, H-10); 2.00 (d, 3H, J = 1,5 Hz, H-8); 2.10 - 2.08 (m, 1H, H-6); 2.42 (m, 1H, H-5); 2.70 - 2.62 (m, 1H, H-1); 2.91 - 2.80 (m, 1H, H-6); 5.71 (m, 1H, H-3). 13C RMN. (50.0 MHz, CDCl3)δ; 22.0 (C-9); 23.3 (C-8); 26.7 (C-10); 40.8 (C-7); 49.7 (C-5); 53.8, (C-6); 57.6 (C-1); 121.4 (C-3); 170.0 (C-4); 203.9 (C-2).
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