Cyclic boronates in the mass spectrometry of ecdysteroids

Abstract

For the structural elucidation of ecdysteroids a reaction with phenylboronic acid has been employed. This reaction takes place exclusively on the C20,C22 diol moiety, thus facilitating fast and easy detection of this moiety in the molecule of ecdysteroid. This derivatization also redirects fragmentation to C17/C20 bond cleavage with charge retention on both fragments enabling assessment of the structure both of the steroid nucleus and of the side-chain. Possible reaction of phenylboronic acid with the diol moiety of the glycosyl group of the glycoside of ecdysteroid, producing di-adduct, is demonstrated; participation of the 5p-hydroxy group of 58,20-dihydroxyecdysone in the formation of such a di-adduct is discussed; and the role of the enol form of the 6-carbonyl group is proved.Ecdysteroids are polyhydroxylated steroid compounds, which take part in the regulation of developmental processes of invertebrates. Many compounds of this structural type have also been isolated from plants. ' Structure elucidation of these polyfunctional substances is usually accomplished by spectral methods, mainly nuclear magnetic resonance (NMR) and mass spectrometry because of the low amounts of the individual compounds that are available. Mass spectrometry is especially suitable because it requires only a very small amount of sample. Its combination with NMR and infra-red spectroscopy can give a large amount of information about the structure of the compound under investigation.Fast-atom bombardment (FAB) mass spectra of ecdysteroids give molecular weight information' and further information can be obtained from the spectra of daughter ions from collision-activated dissociation (CAD) in either the positive-or negative-ion m~d e .~.~ On the other hand, in an electron-impact (EI) mass spectrum the molecular ion is usually missing or is very weak, but from extensive fragmentation by sequential losses of molecules of water, cleavage of bonds C20/C22 for those compounds with a C20,C22 diol group or of bonds C17/C20 for compounds without this group as well as from steroid nucleus fragmentation, the main structural features can be Desorption chemical ionization (DCI) mass spectra can provide both molecular weight information and structural information from the extensive fragmentation that can be observed. However, in order to achieve both kinds of information it is necessary to set the experimental conditions precisely .'Another way of obtaining structural information is via the preparation of derivatives of ecdysteroids in order to change the mass spectrum in a defined way. For this purpose the derivatization reaction should fulfil the following criteria: it should be quantitative, it should produce only one product, and a mass spectrum should give information which cannot be obtained from the spectrum of an underivatized ecdysteroid. Trimethylsilylation has been used for increasing the volatility for GC/MS analysis,' while acetonides and Author to whom correspondence should be addressed. acetates have been prepared for elucidation of...

H/D isotopic exchange in the fast atom bombardment of ecdysteroids

“…As reported earlier, an enolization of the C6 carbonyl functionality of polypodine B (5-b-20-dihydroxyecdysone) 7 was determined by the formation of a bis-adduct from the reaction with phenylboronic acid, where the second adduct was found to be formed on the C5 OH and enolized C6 carbonyl. 20 In the H/D exchange experiment, polypodine B 7 exchanges additional hydrogen like other ecdysteroids to 0.6% in positive and 8.4% in negative mode. On the other hand, 22-oxo-20-hydroxyecdysone 3 shows much higher additional exchange (7.8% in positive and 23.6% in negative mode), pointing to a higher propensity of the alicyclic C22 carbonyl to enolization than the cyclic C6 enone (Scheme 2).…”

Positive-Ion Analysis of Boropeptides by Chemical Ionization and Liquid Secondary Ionization Mass Spectrometry

“…The derivatization technique was extended to negative-ion liquid secondary ion mass spectrometry (LSIMS) where it was presumed that mixtures of boronic acids and certain trifunctional nucleophilic compounds (e.g., glycerol) reacted in situ to produce negatively charged boronate complexes which were subsequently sputtered from the liquid surface and mass analyzed . This approach has been used to study a number of polyfunctional nucleophilic compounds as their corresponding boronate esters. − In situ derivatization was also used to characterize 20-hydroxyecdysteroids as their phenylboronate esters by positive-ion LSIMS, − which seems to belie the mechanism of direct boronate complex ion formation (“pre-ionization”) postulated by Rose et al Subsequently, there have been broader negative-ion LSIMS studies of boron-containing compounds. − The presumption that pre-ionization of a boronic acid to a boronate complex anion occurs in situ may account for the preferential use of negative LSIMS mode in conjunction with the derivatization technique. However, this assumption has apparently precluded the investigation of the positive-ion LSIMS of boronic acids and esters; no positive-ion LSIMS studies of boronic acids have been reported.…”