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Abstract. Ecdysteroids in diapause eggs of the silkworm, Bornbyx mori, were analyzed using high-performance liquid chromatography (HPLC) combined with radioimmunoassay (RIA). A relatively large amount of an unidentified free ecdysteroid and its phosphoric ester (conjugated form) were detected. These two compounds were isolated by a combination of column chromatography on silicic acid, thin-layer chromatography (TLC), and HPLC using a reverse-phase (RP) column. The purified compounds were identified as 3-epi-22-deoxy-20-hydroxyecdysone (22d20E') and 3-epi-22-deoxy-20-hydroxyecdysone 2-phosphate (22d20E'2P) by means of mass spectrometry and nuclear magnetic resonance spectroscopy. To our knowledge, this is the first report of 22d20E' and 22d20E'2P.Key words. Molting hormone; 3-epi-ecdysteroids; ecdysteroid conjugate; silkworm; Bombyx mori.The occurrence of large amounts of ecdysteroids in the ovaries and eggs of insects including the silkworm, Bombyx mori, has been welI documented ~-3, Ovaries and eggs thus serve as convenient materials for the isolation and identification of new species of ecdysteroids. In the ovaries of the Bombyx silkworm, the Ohnishi group identified six free ecdysteroids: 20-hydroxyecdysone (20E), ecdysone (E), 2-deoxy-20-hydroxyecdysone (2d20E), 2-deoxyecdysone (2dE), 2,22-dideoxy-20-hydroxyecdysone (2,22d20E) and bombycosterol (B), and their phosphoric ester (conjugated form), 20E 22-phosphate (20E22P), E22P, 2d20E22P, 22d20E3P, 2dE22P, 2,22d20E3P and B3P 4-7. We have recently isolated and identified six additional ecdysteroids in the ovaries and diapause eggs of the Bombyx silkworm, 22-deoxy-20-hydroxyecdysone (22d20E), 3-epi-ecdysone (E') and 3-epi-2-deoxyecdysone (2dE') and their phosphoric esters (22d20E3P, E'22P and 2dE'22P), and furthermore indicated the presence of several other unidentified ecdysteroids s,9. In the present communication we report the isolation and identification of two compounds detected in diapause eggs of the Bombyx silkworm, provisionally termed UKE (unknown ecdysteroids in the free eccysteroid fraction) and C-UKE (conjugated form of UKE). Materials and methodsInsects. Hybrid strains (Tokai-Fuyou, Tokai-Asahi, Kinshuu-Shouwa and Shunrei-Shougetu) of the silk-* To whom all correspondence should be addressed.worm, Bombyx mori, were used as a source of diapause eggs. Diapause eggs which were maintained for 10 days at 25 ~ after oviposition (embryos cease their development at the gastrula stage and enter diapause m) were used as the source of ecdysteroids for extraction. The eggs were stored at -80 ~ until extraction. Preparation of ecflysteroids for analytical high-performance liquid chromatography (HPLC). The procedure for extraction of ecdysteroids from diapause eggs was essentially the same as that for the preparation of ovarian ecdysteroids 8. Briefly, 10 g (wet wt) of diapause eggs were homogenized in 23.5 ml of 70% aqueous methanol and the homogenate was centrifuged. The supernatant was shaken with an equal volume of hexane to remove lipids. The methanol layer ...

Successful application of tandem mass spectrometry on a series of the ecdysteroid phosphates (eddysteroids conjugated with phosphoric acid) led to general information for their characterization. The 22-phosphates and the 2-(or 3-)phosphates can be definitely distinguished by their charge-remote fragmentation patterns. In the case of the 22-phosphates, information about the side-chain moiety is readily obtained. In the case of the 2-(or 3-)phosphates, information about the ring as well as the side-chain is readily available. Neither the positional isomers (2-and 3-phosphates) in the A-ring nor the configurational isomers (a and fi) can be distinguished.20-Hydroxyecdysone (2p, 3p, 14a, 20R, 22R, 25-hexahydroxy-5/3-cholest-7-en-6-one) (1) and its immediate precursor, ecdysone (2p, 3p, 14a, 22R, 25-pentahydroxy-5P-cholest-7-en-6-one) (2) are well known as moulting hormones of insects and crustaceans. All compounds structurally related to ecdysone (2) are generally called 'ecdysteroids'. Monophosphates of ecdysteroids at the 2, 3 and 22 positions, respectively, have been isolated, mostly from ovaries and eggs of the silkworm (Bombyx m~r i ) . ' -~ Free ecdysteroids resulting from the enzymatic hydrolysis of the phosphates are found to be linked to embryogenesis. Thus, the phosphates are proposed to be storage forms of the active hormone^.^In the course of our study on the hormonal control of crustacean moulting, ecdysteroid conjugates were also found from the American crayfish, Procambarus clarkii, when 3-dehydroecdysone was i n j e~t e d .~ Since hormonal ecdysteroids and their derivatives are generally found only in very small amounts, we need to establish a simple and effective method for their characterization.Fast-atom bombardment (FAB) mass spectrometry and nuclear magnetic resonance (NMR) are known to be effective methods for structure elucidation. Especially, tandem mass spectrometry (MS/MS) is suited to the study of compounds only available in limited amounts; the characteristic or supposed pseudomolecular ion isolated by the first mass analyzer undergoes collisionally activated dissociation in a collision cell to yield product ions to be characterized by a second mass analyzer. Thus, MUMS has come to the fore as a useful tool for determining the molecular weight of compounds that have only been roughly purified from natural sources. Indeed, Mauchamp et a1.6 and Lafont et a1.' succeeded in detecting the pseudomolecular ions and then identifying the free ecdysteroids by the aid of MS/MS. In addition, collisioninduced dissociation (CID) spectra are often found to give more informative structural data; the chargeremote fragmentation (CRF) patterns, which are proposed to occur, are clearly recorded with bile acids.'-") Naturally, in the negative-ion mode, the pseudomolecular ions [M-HI-, and the intense fragment ions at mlz 79 (PO;) and mlz 97 (H'PO;) are apparently observed when studying the phosphates.In the case of the monophosphates of ecdysteroids, in which either the 2-, 3-or 22-hydroxyl group is esterified...

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