Fragmentation pattern of permethyl 6-C-glycosylflavones in electron impact mass spectrometry

A mass spectral fragmentation pattern of permethyl 6-C-glycosylflavones is proposed from the MS data of permethyl derivatives mono-O-deuteriomethylated in the 2″-, 3″-, 4″- or 6″-positions. The synthesis of these compounds via O″-glycosyl-6-C-glucosylflavones is described.     

FABMS/derivatisation strategies for the analysis of heparin-derived oligosaccharides

Derivatisation/FABMS strategies applicable to the structure analysis of low microgramme quantities of heparin-derived oligosaccharides are described. Negative and positive FAB data from permethyl derivatives and positive FAB data from the products of subsequent methanolysis are reported for sulfated tetrasaccharides prepared by nitropus acid degradation of heparin. The preparation and FAB behaviour of acetylated derivatives of sulfated oligosaccharides are described for the first time, and the stability of the sulfate groups to base-catalysed acetylation is demonstrated. The acetylation/FABMS methodology, which yields high quality data, shows promise for the characterisation of a wide range of sulfated glycoconjugates.     

Trans-zeatin in culture filtrates of Agrobacterium tumefaciens

Cytokinin-active bases and nucleosides have been isolated from the culture filtrates of Agrobacterium tumefaciens by trace enrichment onto octadecyl-silica and have been identified by GLC-MS of their permethyl and trimethylsilyl derivatives. Besides the expected 6-(3-methylbut-2-enylamino) purine, the filtrate contained zeatin (85% trans, 15% cis), 2-methylthio-ribosylzeatin and smaller quantities of ribosylzeatin and other cytokinin-active nucleosides.     

2″-p-coumaroylvitexin 7-glucoside from Mollugo oppositifolia

Besides vitexin, two compounds have been isolated from Mollugo oppositifolia and identified as vitexin 7-glucoside and 2″-p-coumaroylvitexin 7-glucoside. The latter is a new natural compound. Some features common to the electron-impact mass spectra of permethyl vitexin 7-glucoside and permethyl isovitexin 7-glucoside are discussed.     

Structural studies on santalin permethyl ether

The chloroform extract of the heartwood of Pterocarpus santalinus yielded a mixture of red pigments which could be separated by polyamide column chromatography into two major compounds, santalin-A and santalin-B. Both gave the same permethyl ether, C₃₈H₃₆O₁₀ which had 8 methoxyls and formed a number of derivatives typical of anhydrobenzopyranols. IR and UV spectra confirmed the same. NMR and MS suggested the presence of homoveratrayl group supported by the formation of veratraldehyde in alkali degradation. Permanganate oxidation gave 2,4-dimethoxy benzoic acid, veratric acid and 3,4,6-trimethoxy phthalic acid. On a basic fluorone skeleton, the substituents in the A ring are indicated by 2,4-dihydroxy-5-methoxy benzaldehyde, an alkali fission product and, further, 2,4-dimethoxy phenyl and homoveratryl units are located in ring C based on NMR, MS and biogenetic considerations. The residues constitute another benzene ring fused to ring C leading to the complete structures of the permethyl ether as (VII) which explains all its degradations and which constitutes a highly condensed biflavonoid of a new type.     

Viscosol,a c-3′ prenylated flavonoid from dodonaea viscosa

The structure of viscosol, a new prenylated flavonoid isolated from the aerial parts of Dodonaea viscosa, was established on the basis of spectral studies as well as by the conversion of the flavonol penduletin into permethyl viscosol.     

Solid-phase synthesis of two glycopeptides containing the amino acid sequence 5 to 9 of somatostatin

2,3,4,6 Tetra-O-acetyl-1-N-[N-(tert-butyloxycarbonyl)-l-aspart-4-oyl]-d-glucopyranosylamine and 2-acetamido-3,4,6-tri-O-acetyl-1-N-[N-(tert-butyloxycarbonyl)-l-aspart-4-oyl]-2-deoxy-d-glucopyranosylamine were introduced, respectively, by the solid-phase procedure in the amino acid sequence 5 to 9 of somatostatin. The two resulting glycopeptides β-d-Glcp-(1→4)- and β-d-GlcpNAc-(1→4)-Asn-Phe-Phe-Trp-Lys-OH were homogeneous on examination by t.l.c. and l.c., and their structures were confirmed by m.s. of the N-acetyl, permethyl derivatives.     

Diosgenin saponins from Dioscorea floribunda

Five spirostanol glycosides and two furostanol glycosides were isolated from Dioscorea floribunda. In addition to the IR spectra of the free glycosides and the MS of the peracetates and permethyl ethers, the most effective method for structural determination proved to be the NMR spectra of the free saponins in pyridine-d₅.     

Assessing Gibberellins Oxidase Activity by Anion Exchange/Hydrophobic Polymer Monolithic Capillary Liquid Chromatography-Mass Spectrometry

Bioactive gibberellins (GAs) play a key regulatory role in plant growth and development. In the biosynthesis of GAs, GA3-oxidase catalyzes the final step to produce bioactive GAs. Thus, the evaluation of GA3-oxidase activity is critical for elucidating the regulation mechanism of plant growth controlled by GAs. However, assessing catalytic activity of endogenous GA3-oxidase remains challenging. In the current study, we developed a capillary liquid chromatography – mass spectrometry (cLC-MS) method for the sensitive assay of in-vitro recombinant or endogenous GA3-oxidase by analyzing the catalytic substrates and products of GA3-oxidase (GA₁, GA₄, GA₉, GA₂₀). An anion exchange/hydrophobic poly([2-(methacryloyloxy)ethyl]trimethylammonium-co-divinylbenzene-co-ethylene glycol dimethacrylate)(META-co-DVB-co-EDMA) monolithic column was successfully prepared for the separation of all target GAs. The limits of detection (LODs, Signal/Noise = 3) of GAs were in the range of 0.62–0.90 fmol. We determined the kinetic parameters (K _m) of recombinant GA3-oxidase in Escherichia coli (E. coli) cell lysates, which is consistent with previous reports. Furthermore, by using isotope labeled substrates, we successfully evaluated the activity of endogenous GA3-oxidase that converts GA₉ to GA₄ in four types of plant samples, which is, to the best of our knowledge, the first report for the quantification of the activity of endogenous GA3-oxidase in plant. Taken together, the method developed here provides a good solution for the evaluation of endogenous GA3-oxidase activity in plant, which may promote the in-depth study of the growth regulation mechanism governed by GAs in plant physiology.     

Multidrug resistance reversal activity of permethyl ningalin B amide derivatives

A series of amide derivatives of permethyl ningalin B were prepared and examined as multidrug resistance (MDR) reversal agents illustrating that the C5 carboxylate is widely tolerant of such derivatization.     

Determination of O-glycosidic bond position in 6-C-glycosylglucosylflavones by mass spectrometry

2″,3″,4″ and 6″-O-glycosides of 6-C-glucosylflavones can be differentiated by the MS of the hydrolysis products of their permethyl ethers.     

Structure of the antibiotic virenomycin

To identify the structure of virenomycin, a new antitumor antibiotic consisting of components V and M, its acetyl and permethyl derivatives, as well as products of acid methanolysis and their derivatives were obtained. The IR-, NMR- and mass-spectra of the above compounds are presented. Based on an analysis of the spectral data the structure of virenomycin is suggested.     

Qualitative and semi-quantitative analysis of gibberellins

A method for the qualitative and semi-quantitative analysis of gibberellins (GAs) was examined, and a systematic method consisting of six steps was established. By this method endogenous GAs in some organs of Pharbitis nil were quantitatively analysed.     

Fractionation of gibberellins in plant extracts by reverse phase high performance liquid chromatography

In studies on endogenous plant gibberellins (GAs), reverse phase (Bondapak C₁₈) high performance liquid chromatography (HPLC) has proved to be a useful method for the fractionation of plant extracts. The behavior of 18 authentic GAs in such a chromatographic system is described. The main factors determining chromatographic behavior are the degree and the position of hydroxylation of the GA. Generally, dihydroxylated GAs elute before monohydroxylated GAs, whereas 13-hydroxylated GAs elute before 3-hydroxylated GAs. The number of carboxyl groups and the degree of saturation of the A-ring have little effect. For 20-carbon GAs, the oxidation state at C-20 is only relevant insofar as GAs having a methyl group at this position elute later than those with other groups (lactone, aldehyde, or carboxyl).     

Enhanced recovery of four antitumor ganoderic acids from Ganoderma lucidum mycelia by a novel process of simultaneous extraction and hydrolysis

Ganoderic acids (GAs) Mk, T, S and R exhibit promising anti-tumor effect, but they are difficult to purify from Ganoderma lucidum mycelia due to the presence of numerous analogs. In this work, a novel and efficient extraction/hydrolysis method was developed for the recovery of these four GAs from the mycelia of G. lucidum. By using a 50% aqueous ethanol solution containing 50 mmol/l HCl as extractant, extraction of GAs from mycelia and conversion of analogs impurities into the products of interest could be achieved in one step. This one-pot extraction/hydrolysis process increased the yield of GA-Mk, -T, -S and -R to 242%, 389%, 189% and 420%, respectively, compared to a raw sample without hydrolysis. Simultaneous purification of these four GAs was readily achieved in a single RP-HPLC run due to the conversion of analog impurities into corresponding desired GAs, and the purity and recovery of these four GAs were over 97% and 90%, respectively. The results demonstrated that the simultaneous extraction and hydrolysis process is simple and efficient and thus can act as a useful approach for enhanced recovery of those four GAs from G. lucidum mycelia.     

Gibberellin conjugates: an overview

This article surveys the currently isolated and identified GA conjugates, their synthesis and evaluates modern methods for analysing GA glucose conjugates. The metabolism of applied GAs in higher plant systems leading, in most cases, to GA conjugates is also considered. The enzymology of the formation and hydrolysis of GA glucose conjugates is discussed in connection with their possible physiological function.Abbreviations API = atmospheric pressure ionization - FAB = fast atom bombardment - GA-GE = gibberellin glucosyl ester - GA-O-G = gibberellin-O-glucoside - GC = gas chromatography - GC-MS = combined gas chromatography-mass spectrometry - HPLC = high performance liquid chromatography - LC-MS = combined high performance liquid chromatography-mass spectrometry - MS = mass spectrometry - NMR = nuclear magnetic resonance - PME = permethyl - SIM = selected ion monitoring - TMS = trimethylsilyl     

Microbiological conversion of 12-oxygenated and other derivatives of ent-kaur-16-en-19-oic acid by Gibberella Fujikuroi, mutant B1-41a

The metabolism of several ring C and D-functionalized ent-kaur-16-en-19-oic acids by cultures of Gibberella fujikuroi, mutant B1-41a, to the corresponding derivatives of the normal fungal gibberellins (GAs) and ent-kaurenoids is described. A range of 12α- and 12β-hydroxyGAs and ent-kaurenoids are characterized by their mass spectra and GC Kovats retention indices. The mass spectral and GC data are used to identify the 12α-hydroxy derivatives of GA₁₂, GA₁₄, GA₃₇ and GA₄ (GA₅₈), and of the 12β-hydroxy derivatives of ent-7α-hydroxy- and ent-6α, 7α-dihydroxykaurenoic acids, in seeds of Cucurbita maxima. Similarly the metabolites of GA₉, formed in seeds of Pisum sativum and cultures of G.fujikuroi, mutant B1-41a, are identified as 12α-hydroxyGA₉. ent-11β-Hydroxy- and ent-11-oxo-kaurenoic acids are metabolized by the fungus to the corresponding 11-oxygenated derivatives of the normal fungal ent-kaurenoids and some C₂₀-GAs; no 11-oxygenated C₁₉-GAs are formed. Grandiflorenic acid, 11β-hydroxygrandiflorenic acid, attractyligen and ent-15β-hydroxykaurenoic acid are metabolized to unidentified products.     

Mass-spectrometric identification and sequence location of the ten residues of the new amino acid (gamma-Carboxyglutamic acid) in the N-terminal region of prothrombin.

The detailed mass-spectrometric evidence for our original findings [Magnusson et al. (1974) FEBS Lett. 44, 189-193] of ten gamma-carboxyglutamic acid residues in the N-terminal calcium-binding polypeptide of prothrombin is presented. The identification and sequence location of gamma-carboxyglutamic acid was made by electron-impact and field-desorption studies on acetyl permethyl peptide derivatives, and on the free amino acid. Details of the derivatives formed, and how this new amino acid may be easily recognized and sequenced from the mass spectrum, are given as a basis for future work.     

The identification of 3,3′,5,5′-tetraiodothyroformic acid within the rat liver

Homogenized rat livers were extracted with organic solvents and the extracts converted into more readily volatile derivatives (trimethylsilylmethyl esters and permethyl esters). Combined g.l.c.-mass-spectrometric analysis identified the presence of tetraiodothyroformate. It is postulated that mitochondrial beta-oxidation of tetraiodothyropyruvate results in the formation of this apparently undescribed metabolite.     

Endogenous Gibberellins of Pine Pollen: II. Changes during Germination of Pinus attenuata, P. coulteri, and P. ponderosa Pollen

The endogenous gibberellins (GAs) of pollen of Pinus attenuata, P. coulteri, and P. ponderosa were bioassayed at hour 0, 3, 15, 24, 48 and 72 of germination. Dormant pollen showed relatively high GA activity throughout the elution spectrum (i.e. ranging from relatively nonpolar to highly polar). The maximum GA activity was obtained at hour 15 in more polar regions and especially in the zone corresponding to GA₃ (for P. attenuata estimated as 250 micrograms of GA₃/kilogram pollen). It is probable that the “nonpolar” GAs present in high quantities in dormant pollen and in early stages of germination were converted to “more polar” GAs as germination progressed. The amount of all GAs decreased after hour 15 of germination and by hour 72 no GAs could be detected. Among the species tested P. attenuata showed the highest over-all GA activity.