Isolation of 24-Methylene-cholesterol from Honey Bees (Apis mellifica L)

Abstract

IN connexion with an attempt to isolate pheromone¹ from the queens of the honey bee, Apis mellifica L., an examination was carried out on the neutal portion of the extract obtained by perfusion of the powdered whole-bodies of the queens with ethanol and tert-butanol. By chromatographing the neutral portion on alumina, or silica gel, a crystalline substance, m.p. 138–145° C. [α]_D ²⁵ = −31.6 ± 6° (c = 0.39 in chloroform), was obtained, which resembled cholesterol in behaviour and chemical properties. The Liebermann-Burchard reaction gave the same coloration as that obtained with cholesterol and the mixed melting point was the same. However, there were two bands (6.08µ and 11.33µ) in the infra-red spectrum of the substance which are not found in the spectrum of cholesterol and its derivatives. The same substance was isolated by similar means from worker bees. The constitution 1 for the sterol was elucidated from the following: An Oppenauer oxidation using cyclohexanone and aluminium isopropylate in toluene produced a conjugated unsaturated ketone; m.p. 77–84° C., λ_max = 242 mµ, log ɛ = 4.0 calculated for C₂₈H₄₄O, 396.6. Besides, the double bond of the αβ-unsaturated ketone function, a second double bond was present as shown by titration with bromine³ (1.155 mgm. of substance used, in 4 hr. 0.934 mgm. bromine which corresponds to 2.01 moles C₂₈H₄₄O by calculation. From the absorption bands at 6.08µ and 11.33µ (pressed in potassium bromide) it was inferred that one of the double bonds was present⁴ as a methylene group (C = CH₂). Similarly 26 per cent of the theoretical yield of formaldehyde was formed on reaction with 10 moles of ozone. Based on these properties we have compared the free sterol and the O-acetyl-compound with 24-methylene-cholesterol and its O-acetyl-derivative⁵ (Table 1).