IDENTIFICATION AND ELECTROPHYSIOLOGICAL STUDIES OF (4S,5S)-5-HYDROXY-4-METHYL-3-HEPTANONE AND 4-METHYL-3,5-HEPTANEDIONEIN MALE LUCERNE WEEVILS

C.R. Unelius,1,3* K.-C. Park,1 M. McNeill,2 S. L. Wee,1,4 B. Bohman,3,5& D.M. Suckling1

1The New Zealand Institute for Plant & Food Research Limited, PB 4704, Christchurch 8140, New Zealand

2AgResearch Limited, Lincoln, PB 4749, Christchurch 8140, New Zealand

3School of Natural Sciences, Linnaeus University, SE-391 82 Kalmar, Sweden

4Present address: School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Malaysia

5Present address: Research School of Chemistry and Research School of Biology, The Australian National University, Canberra, ACT 0200, Australia.

*To whom correspondence should be addressed. E-mail: .

Tel: +46-480-446271. Fax: +46-480-446262.

Supplementary material

Chemical analyses. For all synthesized compounds, 1H-NMR and 13C-NMR spectra of CDCl3 solutions were recorded at 500 MHz and 125 MHz, using a Varian Unity spectrometer. Chemical shifts were expressed in ppm in relation to tetramethylsilane, multiplicity (s, singlet; d, doublet; t, triplet; q, quintet and m, multiplet), coupling constants (Hz) and number of protons. The starting materials employed were obtained from commercial suppliers and used without further purification. GC-MS analyses were conducted using a Varian CP-3800 gas chromatograph equipped with a VF-5MS column (30 m × 0.25 mm i.d. × 0.25 µm fi lm, Varian) and connected to an ion trap Varian Saturn 2200 MS with an electron impact of 70 eV and source temperature of 250 oC. Injection volume was 1 µl in splitless mode. The carrier gas was helium and the oven temperature was programmed to increase from 40 oC (5 min hold) to 250 oC with 5 oC/min. Chemical identity of volatiles from all treatments was confirmed by comparing the retention times and mass fragment patterns with synthetic compounds. For enantioselective analyses, a CYCLOSILb column (30 m × 0.25 mm i.d. × 0.25 µm film, J & W Scientific) and an isothermal GC column temperature of 89 °C was used. All other conditions were identical.

Synthesis of 4-methyl-3,5-heptanedione. Synthesis of 4-methyl-3,5-heptanedione was based on a modification of a literature procedure (Kalaitzakis et al. 2006). A mixture of 3,5-heptanedione (6.0 g, 47 mmol), potassium carbonate (8.5 g, 62 mmol) and methyl iodide (6.65 g, 46 mmol) in acetone (15 ml) was heated to a gentle reflux until GCMS analysis indicated that the reaction was completed (2 h). The mixture was cooled to room temperature and acetone (200 ml) was added followed by petroleum ether (100 ml). The solids were removed by filtration and the solvents removed in vacuo. The crude product was purified by column chromatography using 2% EtOAc in petroleum ether as eluant. The product was obtained as colorless oil (4.2 g, 64%). The product consisted of a mixture of dione and enol forms, the dione tautomer dominating (>10 times as abundant). GC-MS and NMR data corresponded to literature data. GC-MS: 142(5), 114(5), 113(8), 86(45), 57(100) (Blight et al. 1984). 1HNMRδ: 3.68 (q, 1H, J=7.1 Hz), 2.49 (m, 4H), 1.31 (d, 3H, J=7.1 Hz), 1.04 (t, 6H, J=7.2 Hz), 13CNMR δ: 207.9, 60.5, 35.0, 13.1, 7.8 ppm (Kalaitzakis et al. 2006).

Synthesis of 5-hydroxy-4-methyl-3-heptanone. Diisopropylamine (42 ml, 0.30 mol) was dissolved in THF (100 ml). The stirred solution was cooled to 0°C and butyllithium (2.5 M in hexane, 80 ml, 0.20 mol) was added dropwise over 20 min. After 20 min of stirring at 0°C the temperature was lowered to -78°C and 3-pentanone (21.0 ml, 0.20 mol) was added dropwise over 20 min. The mixture was stirred at -(70-80)°C for 30 min before propanal (14.4 ml, 0.20 mmol) was added dropwise over 30 min and the reaction mixture was kept at the same temperature for 1 h. Then NH4Cl (400 ml, sat., aq.) was added to the reaction mixture, which was allowed to warm to RT. The aqueous phase was extracted 3 times with diethyl ether and the combined organic phases were washed twice with brine and dried over MgSO4. Concentration in vacuo gave a yellow oil of 95% purity (29.13 g, 96%). The product consisted of a mixture of syn and anti isomers in a 3:2 ratio, the syn isomer dominating.

NMR data corresponded with published data (Kalaitzakis et al. 2006; Heathcock et al. 1979; Bohman et al. 2009). 1H-NMR δ: 3.82 (m, 1H, syn), 3.62 (m, 1H, anti), 2.43 - 2.67 (m, 3H), 1.41 -1.57 (m, 2H), 1.13 (d, 3H, J=7.2 Hz), 1.06 (t, 3H, J=7.2 Hz), 0.98 (t, 3H, J=7.4 Hz). 13C-NMR syn δ: 217.2, 72.8, 49.5, 35.3, 27.1, 10.6, 10.1, 7.8 ppm; anti δ: 217.1, 75.2, 50.8, 36.3, 27.8, 14.5, 10.1, 7.8 ppm. GCMS: 126(15), 97(14), 86(37), 70(18), 69(11), 59(16), 57(100), 55(15).

LITERATURE CITED

Kalaitzakis D, Rozzell JD, et al. (2006). Synthesis of valuable chiral intermediates by isolated ketoreductases: application in the synthesis of a-alkyl-b-hydroxy ketones and 1,3-diols. Adv Synth Catal 348(14): 1958-1969

Heathcock CH, Pirrung MC, et al. (1979). Acyclic stereoselection. 4. Assignment of stereostructure to b-hydroxycarbonyl compounds by carbon-13 nuclear magnetic resonance. J Org Chem 44(24): 4294-4299

Bohman B, Cavonius LR, et al. (2009). Vegetables as biocatalysts in stereoselective hydrolysis of labile organic compounds. Green Chemistry 11(11): 1900-1905

Blight MM, Pickett JA, et al. (1984). An aggregation pheromone of Sitona lineatus: identification and initial field studies. Naturwissenschaften 71(9): 480-480

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