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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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Proposed cleavage reaction of DNA–trioxacarcin A adduct
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Fig10: Proposed cleavage reaction of DNA–trioxacarcin A adduct

Mentions: Further insight into the trioxacarcin A (2)-initiated substitution reaction and the DNA cleavage products were obtained by additional ESI MS experiments stepwise increasing the capillary-skimmer dissociation energy in the ion source [24–30]. Like increasing the temperature this also initiated fragmentation of alkylated DNA followed by strand cleavage. The adduct of trioxacarcin A (2) with oligomer 5d was unequivocally assigned by ESI MS with a low capillary exit voltage of −80 V providing exclusively ions of the DNA–trioxacarcin complex 5d + 2 (Fig. 9a): ([M − 3H]3−, [M − 4H]4−, [M − 5H]5−, and [M − 6H]6−). By applying capillary–skimmer dissociation by raising the capillary exit voltage to −140 V the signals for the DNA–trioxacarcin A adduct were also found (Fig. 9c). Nevertheless, the adduct ions were less intense. One of the peaks with the highest intensity appears at m/z 1026.37; this is assigned to the single negatively charged ion of gutingimycin (1) comparable to the thermal cleavage product observed already by HPLC. Depurination is known to result in oligonucleotide strand scission [19, 20, 27]. Furthermore, it has been reported that collision-activated dissociation of negatively charged oligonucleotide ions starts with cleavage of a base followed by cleavage of the 3′ C–O bond of the sugar unit from which the base was lost [31]. In accordance, two ESI MS peaks were correlated to the doubly charged w5-fragment ion at m/z 781.64 (calculated 781.63) and the doubly charged (a6-B)-type fragment ion at m/z 821.66 (calculated 821.64) of the oligonucleotide 5d. The accompanying smaller peaks to the right of w52− and [a6-GT]2− were assigned to the corresponding [M + Na − 3H]2− ions. These fragments might mechanistically be explained by the initiation of strand cleavage at C3′ leading to a furanyl unit at the 3′-end of d(AATTA) and a phosphoryl group at the 5′-end of the second fragment d(AAATT) [31]. An additional small peak m/z 801.90 [10 − 4H]4− can be assigned to the depurinated oligonucleotide 10 (Scheme 1, together with its sodium peak m/z 807.40 [10 + Na − 5H]4−). Nevertheless, oligonucleotide 10 with a double bond between C1′ and C2′ (Scheme 1) has a calculated mass [10 − 4H]4− of m/z 801.89 and [10 + Na − 5H]4− of m/z 807.38 corresponding to the mass of the intermediate fragment. This assignment is clearly supported by the corresponding deconvoluted mass spectra (Figs. 9b and d).Fig. 9


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

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

Proposed cleavage reaction of DNA–trioxacarcin A adduct
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2228378&req=5

Fig10: Proposed cleavage reaction of DNA–trioxacarcin A adduct
Mentions: Further insight into the trioxacarcin A (2)-initiated substitution reaction and the DNA cleavage products were obtained by additional ESI MS experiments stepwise increasing the capillary-skimmer dissociation energy in the ion source [24–30]. Like increasing the temperature this also initiated fragmentation of alkylated DNA followed by strand cleavage. The adduct of trioxacarcin A (2) with oligomer 5d was unequivocally assigned by ESI MS with a low capillary exit voltage of −80 V providing exclusively ions of the DNA–trioxacarcin complex 5d + 2 (Fig. 9a): ([M − 3H]3−, [M − 4H]4−, [M − 5H]5−, and [M − 6H]6−). By applying capillary–skimmer dissociation by raising the capillary exit voltage to −140 V the signals for the DNA–trioxacarcin A adduct were also found (Fig. 9c). Nevertheless, the adduct ions were less intense. One of the peaks with the highest intensity appears at m/z 1026.37; this is assigned to the single negatively charged ion of gutingimycin (1) comparable to the thermal cleavage product observed already by HPLC. Depurination is known to result in oligonucleotide strand scission [19, 20, 27]. Furthermore, it has been reported that collision-activated dissociation of negatively charged oligonucleotide ions starts with cleavage of a base followed by cleavage of the 3′ C–O bond of the sugar unit from which the base was lost [31]. In accordance, two ESI MS peaks were correlated to the doubly charged w5-fragment ion at m/z 781.64 (calculated 781.63) and the doubly charged (a6-B)-type fragment ion at m/z 821.66 (calculated 821.64) of the oligonucleotide 5d. The accompanying smaller peaks to the right of w52− and [a6-GT]2− were assigned to the corresponding [M + Na − 3H]2− ions. These fragments might mechanistically be explained by the initiation of strand cleavage at C3′ leading to a furanyl unit at the 3′-end of d(AATTA) and a phosphoryl group at the 5′-end of the second fragment d(AAATT) [31]. An additional small peak m/z 801.90 [10 − 4H]4− can be assigned to the depurinated oligonucleotide 10 (Scheme 1, together with its sodium peak m/z 807.40 [10 + Na − 5H]4−). Nevertheless, oligonucleotide 10 with a double bond between C1′ and C2′ (Scheme 1) has a calculated mass [10 − 4H]4− of m/z 801.89 and [10 + Na − 5H]4− of m/z 807.38 corresponding to the mass of the intermediate fragment. This assignment is clearly supported by the corresponding deconvoluted mass spectra (Figs. 9b and d).Fig. 9

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