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Formation of gutingimycin: analytical investigation of trioxacarcin A-mediated alkylation of dsDNA.

Fitzner A, Frauendorf H, Laatsch H, Diederichsen U - Anal Bioanal Chem (2008)

Bottom Line: Formation and fragmentation of recognition complexes between trioxacarcin A and various DNA sequences were examined by temperature-dependent UV and CD spectroscopy, HPLC analysis, and ESI mass spectrometry with regard to reaction conditions, intermediates, products, mechanism, and sequence specificity.Cleavage of the trioxacarcin-DNA complexes provided the natural product gutingimycin by guanine abstraction.The resulting DNA with an abasic site was further cleaved into a DNA fragment with a furanyl unit at the 3'-end and an oligonucleotide with a phosphorylated 5'-end.

View Article: PubMed Central - PubMed

Affiliation: Institut für Organische und Biomolekulare Chemie, Georg-August-Universität Göttingen, Tammannstr. 2, 37077, Göttingen, Germany.

ABSTRACT
Formation and fragmentation of recognition complexes between trioxacarcin A and various DNA sequences were examined by temperature-dependent UV and CD spectroscopy, HPLC analysis, and ESI mass spectrometry with regard to reaction conditions, intermediates, products, mechanism, and sequence specificity. Cleavage of the trioxacarcin-DNA complexes provided the natural product gutingimycin by guanine abstraction. The resulting DNA with an abasic site was further cleaved into a DNA fragment with a furanyl unit at the 3'-end and an oligonucleotide with a phosphorylated 5'-end.

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HPLC chromatogram of a mixture of oligonucleotide 4a and trioxacarcin A (2): a at room temperature; b after heating to 90 °C; next to DNA 4a further oligonucleotides and oligonucleotide fragments are eluting between 11 and 17 min
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Fig5: HPLC chromatogram of a mixture of oligonucleotide 4a and trioxacarcin A (2): a at room temperature; b after heating to 90 °C; next to DNA 4a further oligonucleotides and oligonucleotide fragments are eluting between 11 and 17 min

Mentions: For a more detailed picture of trioxacarcin A (2) adduct formation with dsDNA, high-performance liquid chromatography was applied determining the components present after mixing at room temperature and after heating to 90 °C. The HPLC chromatogram of the mixture of oligonucleotide 4a with trioxacarcin A (2) before starting the heating cycle provided three peaks (Fig. 5a). Next to DNA (4a) and trioxacarcin A (2), an additional compound was indicated which was assigned to the stable covalent complex between the oligonucleotide and the natural product trioxacarcin A (4a + 2). The compound corresponding to peak (4a + 2) was isolated and characterized by high-resolution ESI MS as the DNA–trioxacarcin A adduct. After heating to 90 °C additional peaks appeared in the oligonucleotide area together with gutingimycin (1) and trioxacarcin B (3) generated by epoxide ring opening of trioxacarcin A (2) with water (Fig. 5b). The assignment of all HPLC peaks was confirmed by co-injection and mass spectrometry.Fig. 5


Formation of gutingimycin: analytical investigation of trioxacarcin A-mediated alkylation of dsDNA.

Fitzner A, Frauendorf H, Laatsch H, Diederichsen U - Anal Bioanal Chem (2008)

HPLC chromatogram of a mixture of oligonucleotide 4a and trioxacarcin A (2): a at room temperature; b after heating to 90 °C; next to DNA 4a further oligonucleotides and oligonucleotide fragments are eluting between 11 and 17 min
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2228378&req=5

Fig5: HPLC chromatogram of a mixture of oligonucleotide 4a and trioxacarcin A (2): a at room temperature; b after heating to 90 °C; next to DNA 4a further oligonucleotides and oligonucleotide fragments are eluting between 11 and 17 min
Mentions: For a more detailed picture of trioxacarcin A (2) adduct formation with dsDNA, high-performance liquid chromatography was applied determining the components present after mixing at room temperature and after heating to 90 °C. The HPLC chromatogram of the mixture of oligonucleotide 4a with trioxacarcin A (2) before starting the heating cycle provided three peaks (Fig. 5a). Next to DNA (4a) and trioxacarcin A (2), an additional compound was indicated which was assigned to the stable covalent complex between the oligonucleotide and the natural product trioxacarcin A (4a + 2). The compound corresponding to peak (4a + 2) was isolated and characterized by high-resolution ESI MS as the DNA–trioxacarcin A adduct. After heating to 90 °C additional peaks appeared in the oligonucleotide area together with gutingimycin (1) and trioxacarcin B (3) generated by epoxide ring opening of trioxacarcin A (2) with water (Fig. 5b). The assignment of all HPLC peaks was confirmed by co-injection and mass spectrometry.Fig. 5

Bottom Line: Formation and fragmentation of recognition complexes between trioxacarcin A and various DNA sequences were examined by temperature-dependent UV and CD spectroscopy, HPLC analysis, and ESI mass spectrometry with regard to reaction conditions, intermediates, products, mechanism, and sequence specificity.Cleavage of the trioxacarcin-DNA complexes provided the natural product gutingimycin by guanine abstraction.The resulting DNA with an abasic site was further cleaved into a DNA fragment with a furanyl unit at the 3'-end and an oligonucleotide with a phosphorylated 5'-end.

View Article: PubMed Central - PubMed

Affiliation: Institut für Organische und Biomolekulare Chemie, Georg-August-Universität Göttingen, Tammannstr. 2, 37077, Göttingen, Germany.

ABSTRACT
Formation and fragmentation of recognition complexes between trioxacarcin A and various DNA sequences were examined by temperature-dependent UV and CD spectroscopy, HPLC analysis, and ESI mass spectrometry with regard to reaction conditions, intermediates, products, mechanism, and sequence specificity. Cleavage of the trioxacarcin-DNA complexes provided the natural product gutingimycin by guanine abstraction. The resulting DNA with an abasic site was further cleaved into a DNA fragment with a furanyl unit at the 3'-end and an oligonucleotide with a phosphorylated 5'-end.

Show MeSH
Related in: MedlinePlus