Chemistry
General
Racemic mixture of cis/trans (35%:65%) isomers of phytol (1) (PYT) (97% purity), N-bromosuccinimide (NBS, 99% purity) and N-chlorosuccinimide (NCS, 98% purity) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), while trimethylortoacetate was purchased from Fluka. Analytical grade acetic acid, sodium hydrogen carbonate, acetone, hexane, diethyl ether, tetrahydrofuran (THF), anhydrous magnesium sulfate, sodium chloride were purchased from Chempur (Poland).
Analytical Thin Layer Chromatography (TLC) was carried out on silica gel coated aluminium plates (DC-Alufolien Kieselgel 60 F254, Merck, Darmstadt, Germany) with a mixture of hexane, acetone and diethyl ether in various ratios as the developing systems. Compounds were visualized by spraying the plates with solution of 1% Ce(SO4)2 and 2% H3[P(Mo3O10)4] (2 g) in 10% H2SO4, followed by heating to 120–200 °C.
The products of chemical synthesis were purified by column chromatography on silica gel (Kieselgel 60, 230–400 mesh ASTM, 40–63 μm, Merck) using a mixture of hexane, acetone, and diethyl ether (in various ratios) as eluents.
Gas chromatography (GC) analysis was carried out on an Agilent Technologies 6890 N Network GC instrument (Santa Clara, CA, USA) equipped with autosampler, split injection (20:1) and FID detector using a DB-5HT column (Agilent, Santa Clara, USA) (polyimide-coated fused silica tubing, 30 m × 0.25 mm × 0.1 µm) with hydrogen as the carrier gas. Products of the chemical reactions were analysed using the following temperature programme: injector 250 °C, detector (FID) 250 °C, initial column temperature: 100 °C, 100–300 °C (rate 30 °C/min), final column temperature 300 °C (hold 2 min).
Nuclear magnetic resonance spectra 1H NMR, 13C NMR, DEPT 135, HSQC, 1H–1H COSY and NOESY were recorded in CDCl3 solutions with signals of residual solvent (δH = 7.26 δC = 77) on a Brüker Avance II 600 MHz (Brüker, Rheinstetten, Germany) spectrometer.
High-resolution mass spectra (HRMS) were recorded using electron spray ionization (ESI) technique on spectrometer Waters ESI-Q-TOF Premier XE (Waters Corp., Milford, MA, USA).
General procedure for the synthesis of compounds (2–7)
The preparation of ester 2 and acid 3 has been illustrated in detail in our previous work39, and so the synthesis method would not be listed here.
To a solution of acid 3 (7.8 mmol) in THF (30 mL) the N-bromosuccinimide (7.8 mmol) or N-chlorosuccinimide (7.8 mmol) was added. The mixture was stirred at room temperature for 48–96 h. When the substrate reacted completely (TLC, GC) the mixture was diluted with diethyl ether and washed with saturated NaHCO3 solution and brine. Organic layer of ether extract was separated and dired over anhydrous magnesium sulfate and evaporated on a rotary evaporator. New δ-halogeno-γ-lactones (4–7) were separated by silica gel column using for elution hexane/diethyl eter in gradient system. Bromo- and chlorolactonization afforded products with the following physical and spectral data presented below:
trans-5-Bromomethyl-4-methyl-4-(4′,8′,12′-trimethyltridecyl)dihydrofuran-2-one ( 4 )
(25% yield); 1H NMR (600 MHz, CDCl3): δ 0.85 (four t, J = 6.4 Hz, 12H, CH3-4′, CH3-8′, (CH3)2–12′), 1.05–1.55 (m, 21H, CH2-1′, CH2-2′, CH2-3′, CH2-5′, CH2-6′, CH2-7′, CH2-9′, CH2-10′, CH2-11′, H-4′, H-8′, H-12′), 1.24 (s, 3H, CH3-4), 2.30 and 2.60 (two d, J = 17.2 Hz, 2H, CH2-3), 3.47 (dd, J = 11.3, 7.3 Hz, 1H, one of CH2-Br), 3.55 (dd, J = 11.3, 4.5 Hz, 1H, one of CH2-Br), 4.39 (dd, J = 7.2, 4.5 Hz, 1H, H-5); 13C NMR (150 MHz, CDCl3): δ 19.61, 19.69 (CH3)2–12′), 22.65, 22.75 (CH3-4′, CH3-8′), 24.50 (CH3-4), 29.21 (CH2-Br), 41.49 (CH2-3), 42.86 (C-4), 22.00, 24.47, 24.82, 34.08, 37.28, 37.41, 37.61, 37.70, 39.38 (CH2-1′, CH2-2′, CH2-3′, CH2-5′, CH2-6′, CH2-7′, CH2-9′, CH2-10′, CH2-11′), 28.00, 32.69, 32.80 (H-4′, H-8′, H-12′), 87.66 (H-5), 174.92 (C-2); HRMS (ESI): m/z calcd. for C22H41BrO2 [M + Na]+ 439.2188; found 439.2182.
cis-5-Bromomethyl-4-methyl-4-(4′,8′,12′-trimethyltridecyl)dihydrofuran-2-one ( 5 )
(46% yield); 1H NMR (600 MHz, CDCl3): δ 0.85 (four t, J = 6.4 Hz, 12H, CH3-4′, CH3-8′, (CH3)2–12′), 1.04–1.55 (m, 21H, CH2-1′, CH2-2′, CH2-3′, CH2-5′, CH2-6′, CH2-7′, CH2-9′, CH2-10′, CH2-11′, H-4′, H-8′, H-12′), 1.08 (s, 3H, CH3-4), 2.38 and 2.49 (two d, J = 17.2 Hz, 2H, CH2-3), 3.48 (m, 2H, one CH2-Br), 4.41 (dd, J = 7.5, 4.5 Hz, 1H, H-5); 13C NMR (150 MHz, CDCl3): δ 18.96 (CH3-4), 19.69, 19.76 (CH3)2–12′), 22.65, 22.75 (CH3-4′, CH3-8′), 29.17 (CH2-Br), 42.70 (CH2-3), 43.09 (C-4), 22.20, 24.46, 24.82, 37.28, 37.39, 37.41, 37.49, 39.38, 39.96 (CH2-1′, CH2-2′, CH2-3′, CH2-5′, CH2-6′, CH2-7′, CH2-9′, CH2-10′, CH2-11′), 28.00, 30.95, 32.72 (H-4′, H-8′, H-12′), 86.46 (H-5), 174.76 (C-2); HRMS (ESI): m/z calcd. for C22H41BrO2 [M + Na]+ 439.2188; found 439.2183.
trans-5-Chloromethyl-4-methyl-4-(4′,8′,12′-trimethyltridecyl)dihydrofuran-2-one ( 6 )
(21% yield); 1H NMR (600 MHz, CDCl3): δ 0.84 (four t, J = 6.4 Hz, 12H, CH3-4′, CH3-8′, (CH3)2–12′), 1.03–1.54 (m, 21H, CH2-1′, CH2-2′, CH2-3′, CH2-5′, CH2-6′, CH2-7′, CH2-9′, CH2-10′, CH2-11′, H-4′, H-8′, H-12′), 1.23 (s, 3H, CH3-4), 2.23 and 2.60 (two d, J = 17.2 Hz, 2H, CH2-3), 3.67 (dd, J = 12.1, 6.1 Hz, 1H, one of CH2-Cl), 3.73 (dd, J = 12.1, 4.6 Hz, 1H, one of CH2-Cl), 4.33 (dd, J = 7.2, 4.6 Hz, 1H, H-5); 13C NMR (150 MHz, CDCl3): δ 19.61, 19.67 (CH3)2–12′), 22.65, 22.75 (CH3-4′, CH3-8′), 24.67 (CH3-4), 42.30 (CH2-Cl), 41.48 (CH2-3), 42.38 (C-4), 22.09, 24.46, 24.82, 34.21, 37.29, 37.39, 37.61, 37.70, 39.38 (CH2-1′, CH2-2′, CH2-3′, CH2-5′, CH2-6′, CH2-7′, CH2-9′, CH2-10′, CH2-11′), 28.00, 32.70, 32.80 (H-4′, H-8′, H-12′), 87.45 (H-5), 175.21 (C-2); HRMS (ESI): m/z calcd. for C22H41ClO2 [M + Na]+ 395.2693; found 395.2698.
cis-5-chloromethyl-4-methyl-4-(4′,8′,12′-trimethyltridecyl)dihydrofuran-2-one ( 7 )
(39% yield); 1H NMR (600 MHz, CDCl3): δ 0.85 (four t, J = 6.6 Hz, 12H, CH3-4′, CH3-8′, (CH3)2–12′), 1.04–1.56 (m, 21H, CH2-1′, CH2-2′, CH2-3′, CH2-5′, CH2-6′, CH2-7′, CH2-9′, CH2-10′, CH2-11′, H-4′, H-8′, H-12′), 1.06 (s, 3H, CH3-4), 2.38 and 2.47 (two d, J = 17.2 Hz, 2H, CH2-3), 3.67 (m, 2H, one CH2-Cl), 4.35 (dd, J = 6.4, 4.9 Hz, 1H, H-5); 13C NMR (150 MHz, CDCl3): δ 19.06 (CH3-4), 19.69, 19.76 (CH3)2–12′), 22.65, 22.74 (CH3-4′, CH3-8′), 42.52 (CH2-Cl), 42.39 (CH2-3), 42.54 (C-4), 22.14, 24.46, 24.83, 37.28, 37.37, 37.41, 37.49, 39.38, 40.16 (CH2-1′, CH2-2′, CH2-3′, CH2-5′, CH2-6′, CH2-7′, CH2-9′, CH2-10′, CH2-11′), 28.00, 32.64, 32.80 (H-4′, H-8′, H-12′), 86.24 (H-5), 175.04 (C-2); HRMS (ESI): m/z calcd. for C22H41ClO2 [M + Na]+ 395.2693; found 395.2697.
Deterrent activity of phytol and its derivatives
Aphids, plants and compound application
The peach potato aphids Myzus persicae (Sulzer) and the Chinese cabbage Brassica rapa subsp. pekinensis (Lour.) Hanelt were reared in laboratory at 20 °C, 65% r.h., and L16:8D photoperiod. One to seven days old apterous females of M. persicae and 3-week old plants with 4–5 fully developed leaves were used for experiments. M. persicae were obtained from the laboratory culture maintained at the Department of Botany and Ecology for many generations since 2000. All experiments were carried out under the same conditions of temperature, relative humidity, and photoperiod. The bioassays were started at 10–11.a.m. Each compound was dissolved in 70% ethanol to obtain the recommended 0.1% solution40. All compounds were applied on the adaxial and abaxial leaf surfaces by immersing a leaf in the ethanolic solution of a given compound for 30 s.20. Control leaves of similar size were immersed in 70% ethanol that was used as a solvent for the studied compounds. Experiments were performed 1 h after the compounds application to allow the evaporation of the solvent. Every plant and aphid were used only once.
Aphid settling (choice test)
This bioassay allows the study of aphid host preferences under semi-natural conditions41. In the present study, aphids were given free choice between control and treated excised leaves that were placed in a Petri dish. Aphids were placed in the dish equidistance from treated and untreated leaves, so that aphids could choose between treated (on one half of a Petri dish) and control leaves (on the other half of the dish). Aphids that settled, i.e. they did not move, and the position of their antennae indicated feeding, on each leaf were counted at 1 h, 2 h, and 24 h intervals after access to the leaf. Each experiment was replicated 8 times (n = 8 replicates, 20 viviparous apterous females/replicate). Aphids that were moving or not on any of the leaves were not counted.
Behavioral responses of aphids Myzus persicae during probing and feeding (no-choice test)
Aphid probing and the phloem sap uptake by M. persicae was monitored using the technique of electronic registration of aphid probing in plant tissues, known as EPG (= Electrical Penetration Graph), that is frequently employed in insect–plant relationship studies considering insects with sucking-piercing mouthparts42,43,44. In this experimental set-up, aphid and plant are connected to electrodes and thus made parts of an electric circuit, which is completed when the aphid inserts its stylets into the plant. Weak voltage is supplied in the circuit, and all changing electric properties are recorded as EPG waveforms that can be correlated with aphid activities and stylet position in plant tissues45,46. The parameters describing aphid behaviour during probing and feeding, such as total time of probing, proportion of phloem patterns E1 and E2, number of probes, etc., are good indicators of plant suitability or interference of probing by chemical or physical factors in individual plant tissues44,45,46. In the present study, aphids were attached to a golden wire electrode with conductive silver paint (epgsystems. eu) and starved for 1 h prior to the experiment. Probing behaviour of 12 apterous females/studied compound and control was monitored for 8 h continuously with Giga-4 and Giga-8 DC EPG with 1 GΩ of input resistance recording equipment (EPG Systems, Wageningen, The Netherlands). Each aphid was given access to a freshly prepared plant and each aphid/plant combination was used only once. Various behavioural phases were labelled manually using the Stylet + software (www.epgsystems.eu). The following aphid behaviours were distinguished: no penetration (waveform ‘np’ – aphid stylets outside the plant), pathway phase—penetration of non-phloem tissues (waveforms ‘ABC’), phloem phase (salivation into sieve elements, waveform ‘E1’ and ingestion of phloem sap, waveform ‘E2’), and xylem phase (ingestion of xylem sap, waveform ‘G’). Waveform ‘G’ occurred rarely irrespective of the treatment. Therefore, in all calculations the xylem phase was added to the pathway phase and termed as probing in non-phloem tissues. The E1/E2 transition patterns were included in E2. Waveform patterns that were not terminated before the end of the experimental period (8 h) were not excluded from the calculations. The parameters derived from EPG recordings were analyzed according to their frequency and duration in configuration related to activities in peripheral and vascular tissues.
Statistical analysis
The data of the choice-test were analyzed using Student’s t-test (STATISTICA 13.1. package). If aphids showed clear preference for the leaf treated with the tested compound (p < 0.05), the compound was described as having attractant properties. If aphids settled mainly on the control leaf (p < 0.05), the compound tested in the respective choice-test was stated a deterrent. From the data thus obtained the relative index of deterrence (DI) was calculated: DI = (C − T/C + T) where C was the number of aphids settled on control leaf, T was the number of aphids settled on the leaf treated with the tested compound. The value of DI ranged between 1 (ideal deterrent) and − 1 (ideal attractant)41.
All statistical calculations related to data of the no-choice test were performed using StatSoft, Inc. (2014) STATISTICA (data analysis software system), version 12, www.statsoft.com. EPG parameters describing aphid probing behaviour (Table 2) were calculated manually and individually for every aphid and the mean and standard errors were subsequently calculated using the EPG analysis Excel worksheet created for this study. The parameters derived from EPGs were analyzed according to their frequency and duration in configuration related to activities in peripheral and vascular tissues. The results were statistically analyzed using Mann–Whitney U-test where the values of EPG parameters recorded from aphids on treated plants were compared to control41.
Antiproliferative activity of phytol and its derivatives
Cell lines
Human biphenotypic B myelomonocytic leukemia MV4-11, human colon cancer LoVo cell line and normal mouse fibroblast BALB/3T3 cells were obtained from American Type Culture Collection (Rockville, Maryland, USA), human lung carcinoma A549 cells were obtained from European Collection of Authenticated Cell Cultures (UK). All the cell lines are being maintained at the Hirszfeld Institute of Immunology and Experimental Therapy, PAS, Wroclaw, Poland and were cultured according the procedure described before47.
Cell lines were cultured according to the protocol described before48. MV4-11 cells were cultured in RPMI 1640 medium (Gibco, UK) with 1.0 mM sodium pyruvate, and 10% fetal bovine serum (FBS) (all from Sigma-Aldrich, Germany). A549, LoVo and LoVo/DX cells were cultured in RPMI 1640 + Opti-MEM (1:1) (both from Gibco, UK) supplemented with 5% fetal bovine serum, 1.0 mM sodium pyruvate (LoVo and LoVo/DX cells) (all from Sigma Aldrich Germany) and 0.1 μg/mL doxorubicin chloride (Accord) (only LoVo/DX cells). BALB/3T3 cells in Dulbecco medium (IIET, Poland) supplemented with 10% fetal bovine serum (Sigma-Aldrich, Germany). All culture media were supplemented with 2 mM l-glutamine (Sigma-Aldrich, Germany), 100 units/mL penicillin, and 100 µg/mL streptomycin (both from Polfa Tarchomin S.A., Poland). All cell lines were grown at 37 °C with 5% CO2 humidified atmosphere.
Determination of antiproliferative activity
The solutions of the compounds (50 mM) were prepared by dissolving the substances in DMSO (Sigma Aldrich, Germany). Then the tested compounds were diluted in culture medium to reach the final concentrations of 625, 125, 25 and 5 μM. Before adding of the tested compounds (24 h prior), the cells were plated in 96-well plates (Sarstedt, Germany) at a density of 1 × 104 or 0.5 × 104 (A549) cells per well. The assay was performed after 72 h of exposure to 625, 125, 25 and 5 μM of the tested agents. The in vitro cytotoxic effect of all agents was examined using the MTT (MV4-11) or SRB assay, described previously47. The results were calculated as an IC50 (inhibitory concentration 50%) the concentration of tested agent, which is cytotoxic for 50% of the cancer cells. IC values were calculated for each experiment separately and mean values ± SD are presented in Tables 3. Each compound in each concentration was tested in triplicate in a single experiment, which was repeated 3–5 times.
Cell cycle analysis
Cell cycle analysis was carried out according to the procedure previously described48. The MV4-11 cells were seeded at the density of 1 × 105 cells/well of culture medium on 24-well plates (Sarstedt, Germany) to the final volume of 2 mL and were exposed to the test compounds at concentrations 75 μM for 72 h. After incubation, the cells were collected and 1 × 106 of cells were washed twice in cold PBS and fixed for 24 h in 70% ethanol at − 20 °C. Then the cells were washed twice in PBS and incubated with RNAse (8 μg/mL, Fermentas, Germany) at 37 °C for 1 h. The cells were stained for 30 min. with propidium iodide (50 μg/mL, Sigma Aldrich, Germany) at 4 °C and the cellular DNA content was analyzed by flow cytometry using BD LSRFortessa cytometer (BD Bioscience, San Jose, USA). Compounds at each concentration were tested at least three times independently. Obtained results were analyzed using Flowing software 2 (Cell Imaging Core, Turku Centre for Biotechnology, University of Turku Åbo Akademi University).
Caspase-3/7 activity determination
Caspase-3/7 activity determination was performed according to the protocol previously described48. The MV4-11 cells were seeded at the density of 1 × 105 cells/mL of culture medium on 24-well plates (Sarstedt, Germany) to the final volume of 2 mL. The cells were exposed to the test compounds at concentrations 75 μM or campthothecin (0.05 μg/mL) as a positive control, for 72 h. After 72 h of the incubation, the cells were collected and centrifuged (5 min., 4 °C, 250×g). Cells were suspended in 50 µL of ice-cold lysis buffer (50 mM HEPES, 10% (w/v) sucrose, 150 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-100, pH 7.3, IIET, Poland) and incubated 30 min. at 4 °C. After the incubation, 40 µL of each sample was transferred to a white, 96-well plate (Corning, USA) containing 160 µL of the reaction buffer (20 mM HEPES, 10% sucrose, 100 mM NaCl, 1 mM EDTA, 10 mM DTT, 0.02% Trition X-100, pH 7.3) (IIET, Wroclaw, Poland) with 9 µM Ac-DEVD-ACC fluorogenic substrate (λex = 360 nm, λem = 460 nm). The fluorescence increase correlated with the caspase-3/7 level was continuously recorded at 37 °C for 120 min using a Biotek Synergy H4 (Biokom, Warsaw, Poland). Compounds were tested in duplicates in single experiment and each experiment was repeated at least three times independently. Results were normalized to the number of cells in each well and are reported as mean relative caspase-3/7 activity compared to untreated control sample ± SD.
Statistical analysis
Statistical analysis was performed in Statsoft Statistica 10. All datasets were analyzed using t-test. p Values lower than 0.05 were considered as statistically significant.
Source: Ecology - nature.com
