3-Acetoxy-fatty acid isoprenyl esters from androconia of the ithomiine butterfly Ithomia salapia

Male ithomiine butterflies (Nymphalidae: Danainae) have hairpencils on the forewings (i.e., androconia) that disseminate semiochemicals during courtship. While most ithomiines are known to contain derivatives of pyrrolizidine alkaloids, dihydropyrrolizines, or γ-lactones in these androconia, here we report on a new class of fatty acid esters identified in two subspecies, Ithomia salapia aquinia and I. s. derasa. The major components were identified as isoprenyl (3-methyl-3-butenyl) (Z)-3-acetoxy-11-octadecenoate, isoprenyl (Z)-3-acetoxy-13-octadecenoate (12) and isoprenyl 3-acetoxyoctadecanoate (11) by GC/MS and GC/IR analyses, microderivatizations, and synthesis of representative compounds. The absolute configuration of 12 was determined to be R. The two subspecies differed not only in the composition of the ester bouquet, but also in the composition of more volatile androconial constituents. While some individuals of I. s. aquinia contained ithomiolide A (3), a pyrrolizidine alkaloid derived γ-lactone, I. s. derasa carried the sesquiterpene α-elemol (8) in the androconia. These differences might be important for the reproductive isolation of the two subspecies, in line with previously reported low gene exchange between the two species in regions where they co-occur. Furthermore, the occurrence of positional isomers of unsaturated fatty acid derivatives indicates activity of two different desaturases within these butterflies, Δ9 and Δ11, which has not been reported before in male Lepidoptera.


Samples
Male butterflies of Ithomia salapia aquinia and I. s. derasa were collected in northeastern Peru from 2011-2012 (sampling information in Table S2 below). The androconial hairpencils were dissected and extracted in ultrapure dichloromethane shortly after capture. Samples were kept at -20ºC until analysis.  Table S2).  Table S2). Natural extracts were analyzed using either an HP 7890A/MSD 5975 combination and an ALS 7683 Autosampler (Agilent) or a HP 7890B/MSD 5977 combination (Agilent) and an MPS auto-sampler (Gerstel) in EI mode (70 eV). For gas chromatographic separations a fused silica capillary column HP5-MS (Agilent, 30 m, 0.25 mm OD, 0.25 µm film thickness) was used. Helium was used as carrier gas. The samples were concentrated to approx. 20 µL in a nitrogen stream before injection. The starting temperature of the oven was 50 °C, which was maintained for 5 minutes. The oven was then heated to 320 °C at 5 °C/minute and the temperature was maintained for another 10 minutes. Determination of the enantiomeric compositions were performed on a chiral Hydrodex β-6TBDM type (Macherey & Nagel, 25 m, 0.25 mm OD, 0.25 µm film thickness) GC phase using the combination HP 7890B/MSD 5977 from Agilent and an MPS Autosampler from Gerstel. The mass spectrometer was operated in EI-mode at 70 eV. Helium was used as carrier gas. The starting temperature was 160 °C, which was maintained for 360 minutes. Subsequently, the temperature was raised to 220 °C at 25 °C/min. IR spectra were recorded with the Tensor 27 instrument from Bruker using the diamond ATR technique. GC/DD-IR analyses was performed using a Dani Instruments DiscovIR IR detector coupled to an Agilent Technologies 7890B gas chromatograph. Gas chromatographic separations were performed with a fused silica capillary column HP5-MS (Agilent, 30 m, 0.25 mm OD, 0.25 µm film thickness). Helium was used as carrier gas and residual water was removed using a conventional in-line water trap for gases.
The positions of the absorption bands are indicated as wave numbers in cm -1 . The intensities are marked with s (strong), m (medium) and w (weak), broadened bands are additionally marked with br (broad). UV/VIS-Spectra were measured with Cary 100 Bio spectrometer (Varian). The wavelength λ of the absorption maxima is listed in nm, the extinction ε in cm -2 •mmol -1 . Products were purified by column chromatography on silica gel (Fluka, silica gel 60, particle size 0.040-0.063 mm, mesh 230-440 ASTM). Various solvent mixtures of pentane and diethyl ether were used as indicated. Thin layer chromatography was performed with silica gel foil Polygram SIL G/UV254 (Macherey & Nagel). Detection was performed by UV (254 nm), potassium permanganate immersion, vanillin immersion, molybdatophosphoric acid immersion or by Vaughn's reagent. The rotation value of optically active compounds was determined with a Propol Digital Automatic Polarimeter from Dr. Kernchen, measured in a 1 cm cuvette at a wavelength of 589 nm.

Microderivatizations Transesterification
Sodium methanolate in abs. methanol (0.2 mL, 0.5M) was added to about 20 µL of a natural extract or 0.5 mg synthetic material in 0.1 mL pentane. The sodium methanolate solution was prepared by adding freshly cut sodium pieces to abs. methanol (caution: H2 gas evolution). The mixture was heated to 60°C for 10 minutes in a closed cup vial and then mixed with glacial acetic acid (10 µL) and water (0.5 ml). The aqueous phase was extracted twice with pentane (0.5 mL) and the organic phase way dried with a small amount of MgSO4. The sample was concentrated in a stream of nitrogen.

DMDS adducts
To about 20 µL of a natural extract in 20 µL mL pentane was added dimethyl disulfide (50 µL) and a solution of iodine in diethyl ether (5 µL, 0.24 M). The mixture was heated to 40 °C for 15 h in a closed vial. Pentane (200 µL) was added, and the mixture was washed with 5% Na2S2O3-solution. The organic phase was separated, dried with MgSO4 and concentrated in a stream of nitrogen.

Hexadecanal (14)
According to the procedure of More and Finney [S2], Hexadecanol (13, 5.00 g, 20.62 mmol, 1 eq) was dissolved in ethyl acetate (150 ml) and IBX (16.8 g, 60 mmol) was added. The solution was heated to reflux until the alcohol was completely converted (2.5 to 3.25 hours). After cooling, the solid was filtered through a frit and washed three times with ethyl acetate. The solvent was removed under vacuum and the crude S14 S15 product purified by column chromatography with 20:1 pentane/tert-butyl methyl ether (TBME). A colorless waxy solid was obtained (4.9 g, 20.4 mmol, 99%

Ethyl 3-oxooctadecanoat (16)
According to the general procedure for the synthesis of -keto esters by Holmquist and Roskamp [S3], ethyl diazoacetate (15, 2.2 mL, 20.96 mmol, 1.05 eq) was dissolved in dry dichloromethane (40 mL) under inert gas and tin(II) chloride (0.379 g, 1.99 mmol, 0.1 eq) was added. A few drops of a solution of hexadecanal (4.9 g, 20.37 mmol, 1 eq.) in dry dichloromethane (10 mL) were slowly added. After the start of the reaction (gas formation) the remaining hexadecanal solution was slowly added over 10 minutes. The solution was stirred for 3 h at room temperature. Then the mixture was poured into 100 mL sat. NaCl solution. The resulting emulsion was destroyed with KOH. The phases were separated and the aqueous phase was extracted three times with dichloromethane. The combined organic phases were dried and concentrated over Na2SO4. The raw product was purified by column chromatography with a 20:1 to 10:1 pentane/TBME gradient. Yield: 4.67 g (14.3 mmol, 70%) of a white, waxy solid DC (20:1 pentane/TBME): Rf = 0.34.

Methyl 3-hydroxyoctadecanoate (17)
Ketoester 16 (750 mg, 2.3 mmol, 1 eq) was dissolved in methanol (2 mL) and NaBH4 (44 mg, 1.15 mmol, 0.5 eq) was slowly added while stirring. The reaction mixture was stirred for 12 h and then concentrated in vacuo. The residue was mixed with water and extracted three times with TBME. The combined organic layers were washed with brine and dried with MgSO4. The solvent was removed under vacuum and the product 17 was obtained in quantitative yield (723 mg, 2.3 mmol) as a yellowish, waxy solid. During the reaction transesterification to the methyl ester was observed.

3-Methyl-3-butenyl 3-acetoxyoctadecanoate (11)
Acetylation was performed using a modified general procedure [S5]. Ester 18 (426 mg, 1.16 mmol, 1 eq) was dissolved in dry dichloromethane (4.5 ml) under nitrogen gas atmosphere. The mixture was cooled to 0°C and mixed with pyridine (138 mg, 1.74 mmol) and DMAP (14 mg, 0.12 mmol). Then acetic anhydride (142 mg, 1.4 mmol) was slowly added. The reaction mixture was warmed to room temperature and stirred until complete conversion was achieved. Brine was added, and the phases were separated. The aqueous phase was extracted three times with dichloromethane and the combined organic phases dried with Na2SO4. The solvent was removed under vacuum and the raw product was purified by column chromatography with a 5:1 pentane/diethyl ether solvent mixture. Yield: 19 mg (0.78 mmol, 67%) of a white, waxy solid.