Metabolism of Ecdysteroids During the Embryogenesis of Manduca Sexta

Abstract

The combination of ion suppression, reverse phase high pressure liquid chromatography (IS-RPHPLC) and radioimmunoassay (RIA) employing two relatively non-specific, complimentary antisera was used to identify and quantify ecdysteroids of divergent polarities during embryogenesis of the tobacco hornworm, Manduca sexta. Newly layed eggs (0–1 hr) contained high levels (>30 μg/g) of a maternally derived, polar conjugate of 26-hydroxy-ecdysone (26E) but less than 0.6 and 0.2 μg/g of the polar conjugates of ecdysone (E) and 20-hydroxyecdysone (20E), respectively. The only free ecdysteroid detected was 26E (0.4 μg/g). Between 4 and 12 hr after oviposition, marked deconjugation activity occurred as titers of free 26E increased to 14 μg/g while levels of conjugated 26E fell to 19 μg/g. By this time there were only negligible levels of free E (less than 0.1 μg/g). After 12 hr, the deconjugation of the maternal polar 26E-26-phosphate conjugate slowed and levels of free 26E fell as it was metabolized. At 24 hr, low titers of free 20,26-dihydroxyecdysone (20,26E, 0.4 μg/g) and a β-D-glucose conjugate of 26E (0.8 μg/g) were detected, presumably formed by embryonic 20-monooxygenase and uridine diphospho-α-D-glucose (UDPG) glucosyltransferase activities, respectively. About the time of the deposition of the first larval cuticle (66–72 hr), these 26E metabolites reached high concentrations, (5 μg/g free 20,26E and 7 μg/g 26E-glucose conjugate) while free 26E fell to 2 μg/g. In newly hatched larvae, the 26E-glucose conjugate was the major detectable ecdysteroid. At no time during embryogenesis did the concentrations of free E and 20E exceed 0.5 μg/g and 0.3 μg/g, respectively.     

Profiling of ecdysteroids in complex biological samples using liquid chromatography/ion trap mass spectrometry

A sensitive method using high-performance liquid chromatography coupled to a mass spectrometer with electrospray ionization source (HPLC/ESI-MS) was developed for detection of ecdysteroids in biological samples. We report here for the first time that ecdysteroids can be classified into three groups based on ESI full-scan mass spectra: group 1 (ecdysone (E), 2-deoxyecdysone (2dE), 2,22-dideoxyecdysone (3beta5beta-KT), and 3alpha5alpha[H]-dihydroxycholest-7-en-6-one (3alpha5alpha-KD)), in which loss of one molecule of water from the protonated molecular ion ([M+H](+)) represents the dominant ion; group 2 (20-hydroxyecdysone (20E), makisterone A (MakA), 3beta5beta-KD, and 3beta5alpha-KD), in which [M+H](+) is a major ion but some water loss is observed; and group 3 (muristerone A (MurA) and ponasterone A (PonA)), in which [M+H](+) is the dominant ion with no water loss observed. Based on the analytical procedure in combination with structural information from the group classification and with the application of source-induced dissociation, we identified free ecdysteroids in biological samples: 20,26-dihydroxyecdysone and ecdysonic acid in the larval hemolymph, and the progressive metabolism of 26-hydroxyecdysone (26E) to 3alpha-26E from day-1 to day-3 embryos of the tobacco hornworm Manduca sexta.     

Identification and quantitative analysis of the phytoecdysteroids in Silene species (Caryophyllaceae) by high-performance liquid chromatography. Novel ecdysteroids from S. pseudotites.

Many species in the genus Silene (Caryophyllaceae) have previously been shown to contain ecdysteroids and this genus is recognised as a good source of novel ecdysteroid analogues. We have used ecdysteroid-specific radioimmunoassays and the microplate-based Drosophila melanogaster B(II) cell bioassay for ecdysteroid agonist and antagonist activities to identify further phytoecdysteroid-containing species in this genus. The main ecdysteroid components from 10 Silene species (S. antirrhina, S. chlorifolia, S. cretica, S. disticha, S. echinata, S. italica, S. portensis, S. pseudotites, S. radicosa, S. regia) were isolated and identified, mainly by normal-phase and reversed-phase high-performance liquid chromatography. The amount of each ecdysteroid was determined by comparing chromatogram peak areas with those for reference 20-hydroxyecdysone (20E) on reversed-phase HPLC. 20E is the most abundant ecdysteroid in each of the Silene extracts. Polypodine B, 2-deoxy-20-hydroxyecdysone and ecdysone are also common ecdysteroids in these Silene species, but the proportions of these ecdysteroids vary between the Silene species. HPLC proved to be a quick and effective way to screen Silene species, determine ecdysteroid profiles and, hence, identify extracts containing novel analogues. An extract of the aerial parts of S. pseudotites was found to contain several new ecdysteroids. These have been isolated and identified spectroscopically (by NMR and mass spectrometry) as 2-deoxyecdysone 22beta-D-glucoside, 2-deoxy-20,26-dihydroxyecdysone and 2-deoxypolypodine B 3beta-D-glucoside. Additionally, (5alpha-H)-2-deoxyintegristerone A (5alpha-2H 91%, 5alpha-1H 9%) was isolated as an artefact. This study contributes to the understanding of ecdysteroid distribution in Silene species and provides further information on the chemotaxonomic significance of ecdysteroids in Silene species.     

Isolation of a new class of ecdysteroid conjugates (glucosyl-ferulates) using a combination of liquid chromatographic methods

The Polynesian medicinal fern Microsorum membranifolium contains very large amounts of ecdysteroids, including ecdysone, 20-hydroxyecdysone, 2-deoxy-20-hydroxyecdysone, and 2-deoxyecdysone. It also contains large amounts of unusual ecdysteroids which have been unambiguously identified by mass spectrometry and nuclear magnetic resonance. A new class of ecdysteroid conjugates (3-glucosyl-ferulates of 2-deoxyecdysone and 2-deoxy-20-hydroxyecdysone) is isolated, together with a new glycoside (2-deoxyecdysone 25-rhamnoside). The simultaneous presence of a sugar and an aromatic moiety results in a very particular chromatographic behavior of these conjugates. They behave like flavonoids and polyphenols when using the classical purification on polyamide, aimed at removing the latter from crude plant extracts, and would therefore be lost. They elute as non-polar ecdysteroids on reversed-phase high-performance liquid chromatography (RP-HPLC), whereas their behavior on normal-phase (NP) HPLC is strongly dependent on the mobile phase composition. Our data highlight the importance of selectivity in the choice of HPLC methods used for ecdysteroid separations.     

Preparative-scale chromatography of ecdysteroids: a class of biologically active steroids

A simple separation procedure is developed for the isolation of the main phytoecdysteroid 20-hydroxyecdysone from the herb Silene viridiflora. The purification in four steps uses only a simple preparative-scale separation technique (i.e., liquid-liquid extraction, precipitation, solid-phase extraction on octadecyl silica, and crystallization). This procedure is extended using classical normal-phase liquid column chromatography, rotation planar chromatography, and preparative high-performance liquid chromatography for the isolation of the minor ecdysteroids: integristerone A, 26-hydroxypolypodine B, 2-deoxy-20,26-dihydroxyecdysone, and polypodine B. 2-Deoxy-20,26-dihydroxyecdysone is isolated from this species for the first time. The isolation of these ecdysteroids in adequate amounts makes them readily available for insect physiology experiments and for structure-activity relationship studies. The preparative-scale separation work also results in a minor, as yet unknown ecdysteroid.     

Extraction and monitoring of phytoecdysteroids through HPLC

The size of the phytoecdysteroids family is rapidly growing. Recent data shows over 250 ecdysteroid analogs have been identified so far in plants. It is theorized that there are over 1000 possible structures, which might occur in nature, but it is a fact that ecdysteroids usually occur in plants as a complex cocktail of structurally different compounds. Among these compounds, the major component is usually the common ecdysteroid-like 20-hydroxyecdysone. Ecdysteroids are polar steroids, almost sugar-like in their solubility properties. Extraction and purification of ecdysteroids (polyhydroxy steroids) is complicated by their polar nature and poor crystallizing properties. These properties make them difficult to separate from other polar plant constituents. Besides, this plant extract is very often processed by multistep procedures to isolate the major and minor ecdysteroids from the new or existing sources. A simplified scheme consisting of a few extraction steps for the purification of ecdysteroid from plants is in great demand. A quantitative approach through high-performance liquid chromatography has been initiated for developing an easy method for the extraction of ecdysteroids from Ipomoea hederacea (kaladana) seeds.     

Application of combined chromatographic techniques in the screening and purification of ecdysteroids

Summary Plants were found to contain ecdysteroids in concentrations in the order of 0.1% which are higher than those of the insects, and are adequate for the preparative production of ecdysteroids. A TLC screening method is elaborated for the qualitative and semiquantitative analysis of the ecdysteroid content of plants. The combined TLC method differentiates several characteristic ecdysteroids ranging from the apolar 2-deoxyecdysone to the polar 20,26-dihydroxyecdysone. A preparative purification method was developed consisting of solvent extraction, precipitation, crystallization and column liquid chromatography; these procedures permit the isolation of the main ecdysteroids in gram quantities. In some cases, droplet counter-current chromatography was also applied. Altogether, the isolation of thirteen ecdysteroids from five different plant species was accomplished by using a fast, simple and low-cost procedure, resulting in pure substances.     

Preparative scale purification of shidasterone, 2-deoxy-polypodine b and 9a,20-dihydroxyecdysone from Silene italica ssp. nemoralis

A suitable combination of preparative scale separation methods results in effective clean-up of the ecdysteroids of Silene italica ssp. nemoralis (Waldst. and Kit.) Nyman. The isolation of minor ecdysteroids from the partially purified extract is based on the use of both droplet counter-current chromatography and low-pressure reversed-phase liquid chromatography. The purification is completed by preparative thin-layer chromatography and preparative high-performance liquid chromatography to obtain the minor ecdysteroids, such as 2-deoxy-20-hydroxyecdysone, shidasterone, 2-deoxy-polypodine B, makisterone C, and 9alpha,20-dihydroxyecdysone.     

Metastable ions arising from pseudomolecular [M-H]- ions produced by fast-atom bombardment negative-ion mass spectrometry of ecdysteroids

Metastable ions arising from pseudomolecular [M-H]- ions produced by fast-atom bombardment negative-ion mass spectrometry of a range of free ecdysteroids, ecdysteroid conjugates and polar metabolites were investigated by means of linked scanning at constant B/E. Free ecdysteroids displayed daughter-ion spectra which allow 20-hydroxyecdysteroids and ecdysteroids lacking C-20 hydroxylation to be readily distinguished. The ejection of acetic acid from acetylated ecdysteroids was also readily detectable. Characteristic metastable-ion decomposition of ecdysteroid acids was not observed, presumably as a result of charge localization. High-mass daughter ions were also lacking in the case of phosphate conjugates.     

Isolation of a new member of the ecdysteroid glycoside family: 2-deoxy-20-hydroxyecdysone 22-O-beta-D-glucopyranoside

A new ecdysteroid glycoside, 2-deoxy-20-hydroxyecdysone 22-O-beta-D-glucopyranoside, is isolated from the herb Silene italica ssp. nemoralis (Waldst. and Kit.) Nyman. The compound is purified with multistep chromatography, such as classical column chromatography on alumina and droplet countercurrent distribution. Also, it is expanded using twice low-pressure reversed-phase liquid column chromatography. Chromatography in four steps results in the purified 2-deoxy-20-hydroxyecdysone 22-O-beta-D-glucopyranoside. Two other ecdysteroids have also been separated, including the formerly identified integristerone A and 24(28)-dehydromakisterone A.     

Concerning carbon-13 n.m.r. spectroscopy of 5β-hydroxylated phytoecdysones

The ¹³C n.m.r. spectra of the 5β-hydroxylated phytoecdysones polypodine B, muristerone A and kaladasterone are presented and briefly discussed together with the spectrum of makisterone A. Comparisons with previously reported spectra of ecdysone, ecdysterone and poststerone are made and allow a correction for the C-20/C-24 assignment for ecdysone.     

Ecdysteroid glycosides: identification, chromatographic properties, and biological significance

Ecdysteroid glycosides are found in both animals and plants. The chromatographic behavior of these molecules is characteristic, as they appear much more polar than their corresponding free aglycones when analyzed by normal-phase high-performance liquid chromatography (HPLC), whereas the presence of glycosidic moieties has a very limited (if any) impact on polarity when using reversed-phase HPLC. Biological activity is greatly reduced because the presence of this bulky substituent probably impairs the interaction with ecdysteroid receptor(s). 2-Deoxy-20-hydroxyecdysone 22-O-beta-D-glucopyranoside, which has been isolated from the dried aerial parts of Silene nutans (Caryophyllaceae), is used as a model compound to describe the rationale of ecdysteroid glycoside purification and identification.     

Stereoselective synthesis and moulting activity of 2,3-diepi-20-hydroxyecdysone and 2,3-diepi-5α-20-hydroxyecdysone

The ecdysteroid analogues 2,3-diepi-20-hydroxyecdysone and 2,3-diepi-5α-20-hydroxyecdysone have been synthesized from the readily available ecdysteroid, 20-hydroxyecdysone, and moulting activity has been determined using the Musca bioassay. As expected, the 2,3-diepi-analogue was less active than the parent ecdysteroid, 20-hydroxyecdysone. However, the 2,3-diepi-5α-analogue, which was expected to be inactive in the assay, exhibited moulting activity though it was approximately 1.5-fold less active than its 5β-analogue. The activity of the 5α-analogue could possibly result from the ability of this compound to bind to the ecdysteroid receptor. Alternatively, a possible in vivo C-5 epimerization of the 2,3-diepi-5α-analogue to the corresponding 5β-analogue could account for its activity.     

An extensive ecdysteroid CoMFA

The ecdysteroid agonist activity of 71 HPLC-purified ecdysteroids was measured in the Drosophila melanogaster BII tumorous blood cell line assay. The resultant log(ED50) values, spanning almost 6 orders of magnitude, were used to construct a comparative molecular field analysis (CoMFA) model in which conformations were selected by homology to the crystal structure of ecdysone. Model A was constructed by utilization of the region- focused electrostatic indicator field (q2=0.631, r2=0.903, 5 components, 4 outliers). Model B made use of region-focused electrostatic and steric indicator fields along with MlogP (q2=0.694, r2=0.892, 5 components, 4 outliers). The model and its underlying bioassay data support a pharmacophore hypothesis in which ecdysteroid binding is understood to be due principally to the summation of localized interactions from approximately six specific loci. This is in contrast to previous structure-activity relationship hypotheses which are formulated in terms of the presence or absence of essential functional groups, without which ecdysteroid receptor affinity would be completely absent. The present CoMFA model is utilized to predict the activities of heretofore unknown ecdysteroids.     

Synthesis of the 3-0, 4-0 and 6-0 Sulfates of Methyl 2-amino-2-deoxy-α-D-Glucopyranoside

Abstract

Starting with methyl 2-(benzyloxycarbonyl)amino-2-deoxy-α-D-glucopyranoside (1), the isomeric methyl 2-amino-2-deoxy-α-D-glucopyranoside 3-, 4-, and 6-sulfates have each been prepared by sulfation of suitably blocked intermediates. Tritylation and acetylation of 1 followed by detritylation gave methyl 3,4-di-0-acetyl-2-(benzyloxycarbonyl)amino-2-deoxy-α-D-glucopyranoside (3), having a free 6-hydroxyl group. Base catalyzed 0–4→0–6 acetyl migration provided the corresponding 3,6 di-O-acetyl derivative (4) posessing a free 4-hydroxyl group. Preparation of methyl 4,6-0-benzylidene-2-(benzyloxycarbonyl)amino-2-deoxy-α-D-glucopyranoside (9) provided the intermediate bearing a free 3-hydroxyl group. 0-sulfation of 3, 4, and 9 was effected with the pyridine sulfur trioxide complex in dry pyridine.     

Functional group-mediated biotransformation by Curvularia lunata NRRL 2178: synthesis of 3-dehydro-2-deoxy-ecdysteroids from the 3-hydroxy-2-mesyloxy analogues

Microbial transformation of ecdysteroids with 3-hydroxy-2-mesyloxy functional group by the fungus Curvularia lunata NRRL 2178 furnished 3-dehydro-2-deoxy analogues. The metabolites included 3-dehydro-2-deoxy analogues of 20-hydroxyecdysone, pterosterone, ponasterone A, 20-hydroxyecdysone 20,22-acetonide, shidasterone, and poststerone. The mild biotransformation conditions prevented the metabolites from C-5 epimerization.     

Ecdysteroids from Silene claviformis

The ecdysteroid content ofSilene claviformishas been analyzed and the four major compounds have been identified as 20-hydroxyecdysone, 2-deoxy-20-hydroxyecdysone, 2-deoxyecdysone, and integristerone A. Two new minor compounds have also been identified An erratum to this article can be found at     

Synthesis of the Oligosaccharide Moieties of Musettamycin,Marcellomycin and Aclacinomycin A,Antitumor Antibiotics

Abstract

Condensation of benzyl 2,3,6-trideoxy-3-trifluoroacetamido-α-L-lyxo-hexopyranoside (5) with 4-O-acetyl-3-O-benzyl-2,6-dideoxy-α-L-lyxo-hexopyranosyl bromide (10) carried out under Koenigs-Knorr conditions gave 12. Total deprotection of 12 and N-dimethylation at C-3 led to 17 while selective removal of the 4-O-acetyl group led to 13, a synthetic intermediate for preparing 24 and 33. Condensation of 13 with di-O-acetyl-L-fucal (18) or 4-O-acetyl-L-amicetal (25) in the presence of N-iodosuccinimide followed by hydrogenolysis of the C-2-I bond gave 20 and 27 respectively. The trisaccharide 24 then was obtained from 20 by the same sequence of reactions used to convert 12 into 17. After deacetylation and oxidation, this set of reactions also transformed 27 into 33.     

On the Stereoselectivity of Fluorine and Acetylhypofluorite Additions to Glycals: The Synthesis of 2-Deoxy-2-Fldorohexoses

Electrophilic syn additions of fluorine and acetylhypofluorite across double bonds in 3,4,6-tri-0-acetyl-D-glucal (1a) and D-glucal (1b) followed by acid hydrolysis gave mixtures of 2-deoxy-2-fluoro-D-glucose (8) and 2-deoxy-2-fluoro-D-mannose (9). These addition reactions were conducted in various solvents with a view to investigating the reaction mechanism based on the product distribution analysis by ¹⁹F NMR. Tight ion pair intermediates (4 and 5) have been invoked to explain the stereospecific characteristics of the addition of fluorine or acetylhypofluorite to glycals. The relative stabilities of these intermediates control the product distributions and are governed by a) the anomeric effect (axial vs equatorial preference of C(1) electronegative substituents in pyranose rings), b) dipole-dipole interactions of the lone pairs of electrons on the ring oxygen and the electronegative substituents on C(2), and c) the gauche relationship that exists between the C(2) fluorine and polar groups in the molecule. The overall contribution of these three factors largely depends upon the polarity of the solvent. A rationale for the ¹⁹F NMR chemical shifts as well as the anomeric distributions of the α and β anomers of 2-FDG (8) and 2-FDM (9) has been proposed.     

High performance liquid chromatography coupled to nuclear magnetic resonance spectroscopy and mass spectrometry applied to plant products: Identification of ecdysteroids fromSilene otites

Summary HPLC coupled in parallel to nuclear magnetic resonance (NMR) and mass spectrometry (MS) has been used to obtain¹H NMR and mass spectra of a number of ecdysteroids present in an extract of the plantSilene otites. Reversed phase gradient chromatography was performed using a D₂0-acetonitrile-based solvent system. NMR and mass spectra were obtained for integristerone A, 20-hydroxyecdysone, 2-deoxy-20-hydroxyecdysone and 2-deoxyecdysone to provide structural confirmation using continuous and stopped flow HPLC-NMR. The combined HPLC-NMR-MS system described here provided a more comprehensive analysis of the ecdysteroids present in the extract than HPLC-NMR alone.