M. Przybylski

Isolation and structural characterization of polypeptide antibiotics of the peptaibol class by high-performance liquid chromatography with field desorption and fast atom bombardment mass spectrometry

Abstract A number of polypeptide antibiotics of the peptaibol class, i.e., trichotoxin, alamethicin, suzukacillin, hypelcin and paracelsin, have been separated into components and isolated by high-performance liquid chromatography on spherical, porous octadecylsilyl bonded phases. All peptaibols were found to reveal a strong microheterogeneity due to single or multiple amino acid exchange. Most of the closely related and partially isobaric sequence analogue could be resolved using mixed alcohol—water eluents. As demonstrated by the structure analysis of the paracelsins and the main component of trichotoxin A-50, high-performance liquid chromatography with field desorption and fast atom bombardment mass spectrometry have been found to be a powerful and universally applicable method for the direct and unequivocal sequence determination of components of this class of polypeptides.

Methods for the rapid detection, isolation and sequence determination of “peptaibols” and other aib-containing peptides of fungal origin. I. Gliodeliquescin a fromGliocladium deliquescens

SummaryA rapid and simple detection procedure of Aib-containing “peptaibol” polypeptide antibiotics (mycotoxins) in fungal culture broths is described which employs alkylsilica (Sep-Pak®) cartridges for a selective enrichment, and the utilization of the unusualα-aminoisobutyric acid (Aib) and amino alcohols as specific marker constituents, which are easily detectable in total hydrolysates. Preparative isolations from fermentation broths is facilitated by adsorber resin (XAD) column chromatography, and purification of crude isolates is achieved by silica gel- and sephadex LH-20 chromatography. Preparative HPLC using spherical, totally porous ODS-bonded phases enables the isolation of uniform peptides, completely free of sequence analogs, from microheterogeneous peptaibol mixtures, and analytical HPLC of highest resolution allows a “fingerprint” assignment of microheterogeneous peptaibols of different fungal origin. Fast atom bombardment mass spectrometry is demonstrated to be a rapid and precise method for the complete sequence determination of peptaibols when employing sophisticated liquid matrix systems including both, lipophilic tetraethylenglycol and hydrophilic glycerol combined with a selective, trifluoroacetolytic cleavage step carried out directly “on target” (in situ). The versatility of the methods described is demonstrated with the detection and sequence determination of the new membrane-altering eicosapeptide named gliodeliquescin A from the soil fungusGliocladium deliquescens NRRL 3091 establishing the occurence of peptaibols in that fungal genus for the first time.

Chromatographic and mass spectrometric characterization of the structures of the polypeptide antibiotics samarosporin and stilbellin and identity with emerimicin

SummaryThe structural identity of the polypeptide antibiotics, samarosporin I(II) and stilbellin I(II) with emerimicin IV(III) has been established by thin-layer chromatography, quantitative amino acid analysis by ion-exchange chromatography, gas-liquid chromatography of the N-pentafluoropropionyl amino acid n-propyl esters and N,O-bis-pentafluoropropionyl phenylalaninol with quartz capillaries coated with the chiral stationary phase N-propionyl-L-valine-tert-butylamide, and determination of the relative molecular masses and sequence-specific fragments by field desorption fast atom bombardment mass spectrometry. The separation of closely related sequence analogues of the above polypeptides could be achieved by reversed-phase high-performance liquid chromatography with spherical, fully porous octadecylsilyl bonded phases, and 86% aqueous methanol as eluent. The application of both chromatographic and mass spectrometric methods is demonstrated to be most valuable for the characterization of antibiotics with the unusual constituents α-aminoisobutyric acid, isovaline and phenylalaninol. The methology employed is regarded to be applicable to all polypeptide antibiotics of the peptaibol class.

Thermal degradation of bridged polyaromatic sulphides investigated by direct pyrolysis in the mass spectrometer

Abstract The thermal degradation of three bridged polyaromatic sulphides (I–III) was investigated in the ion source of a mass spectrometer. The reactions were followed by detecting the thermal and electron impact induced fragments. The electron impact induced fragmentation reactions of the pyrolytic fragments are influenced by ortho-methyl substitution, and the mechanisms of these processes are discussed. The results indicate also that the disulphide bridge content in the methyl substituted polymers considerably lowers the thermal stability of these materials with respect to poly(phenylene sulphide).

Field Desorption Mass Spectra of Gastrine Peptides and Glutathione Derivatives

Oligopeptides comprising the sequence of the C-terminal tetrapeptide of gastrine, Trp-Met-Asp-Phe-NH2, and several derivatives of glutathione, γ-Glu-Cys(SR)-Gly, were characterized by field desorption mass spectrometry. The field desorption mass spectra obtained at various field ion emitter temperatures reveal abundant molecular ions and fragmentation reactions that yield partial sequence information. In the series of glutathione derivatives investigated, characteristic ions formed by cleavage of the γ-Glu-Cys peptide bond determine the substituent at the Cys residue and can therefore be used to identify corresponding conjugation products of drug metabolites with glutathione.

Characterisation of disaccharide fragments from the enzymatic digestion of heparin by liquid secondary ion mass spectrometry

SummaryDisaccharide fragments resulting from the enzymatic digestion of heparins with heparinase have been purified by gel filtration chromatography and directly analyzed by positive and negative ion liquid secondary ion mass spectrometry (LSIMS). Following the chromatographic purification from excess sodium salt, the mass spectra of di- and tetrasaccharide fragment mixtures enabled the identification of up to three covalently bound sulfate moieties per glycosaminoglycan-disaccharide unit, by means of their molecular ions, containing the corresponding alkali-counterions.

High performance liquid chromatography and field desorption mass spectrometry of dimethylaminoazobenzenthiocarbamoyl-(DABTC)-peptides

Abstract Fractions obtained by gel-filtration of urine, containing low molecular weight peptides, amino acids and amino acids derivatives were reacted with N,N-dimethylaminoazobenzeneisothiocyanate (DABITC) to enhance sensitivity and selectivity of separation on the reversed phase h.p.l.c. column. These compounds are well suited for structure identification by field desorption mass spectrometry.

Reduction of azo-group-containing peptides in fast atom bombardment mass spectrometry

Azo-group-containing peptides in glycerol are reduced on bombardment with a xenon beam generated in a discharge source but not on bombardment with thermionically produced Cs+ ions; it is suggested that the reduction is due to short-wavelength radiation from the discharge source.

Flavonoide in Heilpflanzen - Vorkommen, Biosynthese und neue Methoden zur Strukturaufklärung

Es wird eine Ubersicht uber Flavonoide und deren Biosynthese in verschiedenen Heilpflanzen gegeben. Es sind hauptsachlich die physikalisch-chemischen Methoden der Analytik, die zur raschen Strukturaufklarung der Flavone beitragen. Dies gilt insbesondere fur die13C- und 1H-NMR-Spektroskopie. Verfeinerte Techniken, wie J-modulierte Spin-Echo-Experimente mit 400-MHz-Hochfeldgeraten erlauben wesentlich raschere und eindeutigere NMR-Spektreninterpretationen. FD-Massenspektren des Rutins und seiner Hydroxyethylderivate beweisen die Flavon-Glycosid-Bindung und zeigen eine Ionenbindung mit Natriumkationen im Rutinoseteil. Aufnahmen von CD-Spektren der Flavonoide sind direkt im Humanserum oder Plasma moglich. Sie erlauben, geringe Konzentrationsanderungen der Wirkstoffe zu erfassen.

Untersuchung von isomeren Tripeptiden und Derivaten des Thyrotropin-,Releasing" Hormons durch Elektronenstoß- und Felddesorptions-Massenspektrometrie / Investigation of Isomeric Tripeptides and Thyrotropin-Releasing Hormone Derivatives by Electron Impact and Field Desorption Mass Spectrometry

Abstract The complementary informations from field desorption and electron impact mass spectra of free tripeptides and thyrotropine releasing hormone derivatives can be used to determine primary structure and molecular weight. Characteristic and diagnostic fragmentation pathways, the formation of diketopiperazines and the temperature-dependent tendency to form amine fragments and diketopiperazines will be discussed.