Sequenzierung von Peptaibol-Antibiotika mittels Flüssigkeitschromatographie-Elektrosprayionisierungs-Massenspektrometrie

Abstract

The topic of this work was the development of fast and reliable methods to permit the sequencing of microheterogeneous linear peptides. Owing to the presence of a-aminoisobutyric acid (Aib) residues and a C-terminal amino-alcohol, these peptides belong to the family of peptaibol antibiotics. The sequence determination was carried out using high-performance liquid chromatography (HPLC) coupled to electrospray ionization mass-spectrometry (ESI-MS) and MSⁿ (multiple MS) of characteristic fragments in the positive- and negative-ionization mode. This method made possible the sequence elucidation of components without previous isolation and allowed, in certain cases, the sequence determination of peptides which were incompletely or not resolved by HPLC. Application of the negative ionization mode completed and ensured the sequence determination in the positive ion mode. A proposed mechanism for the formation of fragments in the negative ionization mode was formulated.

The use of a fluorocarbon-coated silica stationary phase made possible the sequencing of peptides which were not resolved on common used octadecylsilyl- (ODS) stationary phases. The GC-MS (gas chromatography-mass-spectrometry) analysis of generated trifluoroacetyl-dipeptide-methylesters allowed the determination of positions of isobaric amino acids (AA) Val and Iva, which could not be distinguished by ESI-MS. Reference dipeptides not available, were generated by methanolysis from peptaibols of known sequences and identified by their mass spectrum via GC-MS.

The determination of chirality and stoichiometry of contained AA was carried out using perfluoroacyl-AA-alkylester derivatives determined by GC-MS applying chiral and non-chiral stationary phases.

By application of these methods the sequences of twelve peptaibols (Trichovirin II 1a - 6b) isolated from Trichoderma viride strain NRRL 5243 were elucidated and were correlated to the HPLC elution profile (fingerprint). Bactericidal and hemolytic activities of the microheterogeneous mixture of peptides were determined.

Peptaibol trichotoxins A-40 (TT A-40) were reinvestigated. Elution of TT A-40 peptides from the employed ODS stationary phase required the use of an acidic mobile phase. By the use of HPLC-ESI-MSⁿ the structures of two major and one minor component could be confirmed and hitherto not known sequences of a further major and two minor peptides could be determined and correlated to the HPLC elution profile.

Mixtures of the microheterogeneous 16-mer peptaibol antibiotic antiamoebin (AAM) were isolated from the culture broths of the filamentous fungi Stilbella erythrocephala ATCC 28144, Stilbella fimetaria CBS 548.84 and Gliocladium catenulatum CBS 511.66. Further, HPLC elution profiles and sequences from a sample originally used for sequencing AAM (from Hindustan Antibiotics), and a sample of AAM commercially available (from Sigma Chemicals) were assigned. Sequences of AAM previously isolated from Emericellopsis synnematicola CBS 176.60 and Emericellopsis salmosynnemata CBS 382.62 were also determined. The peptide designated AAM I was the most abundant in all isolates and its structure could be confirmed. AAM II was detectable as a minor component (1.9 %) only in the original sample of AAM, but not in the other isolates. The structures of AAM III, IV and V, which had previously been assigned partly, were definitely established, and the new sequences AAM VI - XVI were elucidated. AAM showing Phe¹/Leu¹ or Phe¹/Val¹ exchange, respectively, are produced in amounts only by Stilbella erythrocephala. Sequences, HPLC elution profiles, and relative amounts of peptides of all isolates were correlated.

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