A Novel Class of Defensive Compounds in Harvestmen: Hydroxy-γ-Lactones from the Phalangiid Egaenus convexus

When threatened, the harvestman Egaenus convexus (Opiliones: Phalangiidae) ejects a secretion against offenders. The secretion originates from large prosomal scent glands and is mainly composed of two isomers of 4-hydroxy-5-octyl-4,5-dihydro-3H-furan-2-one (1), a β-hydroxy-γ-lactone. The compounds were characterized by GC-MS of their microreaction derivatives, HRMS, and NMR. After the synthesis of all four possible stereoisomers of 1, followed by their separation by chiral-phase GC, the absolute configurations of the lactones in the Egaenus secretion was found to be (4S,5R)-1 (90%) and (4S,5S)-1 (10%). Hydroxy-γ-lactones represent a new class of exocrine defense compounds in harvestmen.

Immediately after reaching this temperature, vigorous gas evolution started. After 5 h, the reaction mixture was cooled to ambient temperature. The reaction mixture was concentrated under reduced pressure. Purification of the residue by flash chromatography (cyclohexane:Et2O = 19:1 → 14:1) yielded the product as pale yellow liquid (1.92 g, 42%). The physical properties of the product were in good accordance with the reported values 13 .
The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. Purification of the residue by flash chromatography (cyclohexane:EtOAc = 4:5) yielded the product as colorless wax (150 mg, 70%).
After 3 h, the starting material was completely consumed. The reaction mixture was poured onto a saturated ammonium chloride solution (15 mL). The layers were separated and the aqueous layer was extracted with CH2Cl2 (2x20 mL). The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. Purification of the residue (cyclohexane: tertbutylmethylether = 98:2 → 97:3) yielded the product as colorless oil (813 mg 89%).
The hydrogen atmosphere was replaced by nitrogen and a second batch of palladium on carbon (10%, 105 mg) was added. The nitrogen was again replaced by hydrogen atmosphere and the reaction mixture was further stirred at ambient temperature. After 2 d, the starting material was almost completely consumed. The reaction mixture was concentrated under reduced pressure.
After 1.5 h, the starting material was completely consumed (TLC cyclohexane:EtOAc = 7:3, PMA stain) a much more product, which was by TLC identical with the racemate ANF-096, was formed.
The reaction mixture was concentrated under reduced pressure. Purification of the residue by flash chromatography (cyclohexane:EtOAc = 6:4) yielded the product as colorless film/wax (3 mg, 70%).
Two-dimensional NMR spectra of the authentic purified secretion, containing natural compound (4S,5R)-1). A) COSY spectrum with the resonances labeled in 1 and 2 (label rotated by 90°). B) TOCSY spectrum recorded with a mixing time of 120 ms. C) Key correlations observed in the COSY spectrum are indicated as red arrows on the structure of 1. The cross-peak corresponding to the arrow with the dotted line was too close to the diagonal and could not be observed (chemical shifts of H-4 and H-5 are too similar). Both spectra were recorded at 293 K, using 4 scans and 1024x256 complex points.

4) Supporting figures (Comparison of sampling techniques)
Total ion chromatogram of whole body-extracts of Egaenus convexus, showing gland-derived lactones as well as cuticle-derived hydrocarbons (above). Total ion chromatogram of directly sampled secretion (i.e. secretion dabbed from ozopores on filter paper) showing lactones only, and lacking hydrocarbons (below).