UWM Chemistry NMR facility
bar.gif

UWM Logo

The Internal Axial Ligand of Hydroperoxy-Co(III)-Bleomycin: 1H-15N HSQC NMR investigation of Bleomycin, Deglycobleomycin, and their Hydroperoxy-Co(III) Complexes.

Chuanwu Xia#; Chunching Zhao; F. Holger Försterling, and David Petering
Department of Chemistry and UWM-NIEHS Marine and Freshwater Biomedical Core Center, University of Wisconsin-Milwaukee, Milwaukee WI 53211


# Current address: Dept. of Biochemistry, Medical College of Wisconsin, Milwaukee, WI

bar.gif
bar.gif

Introduction

Bleomycin (Blm) is one of only few metallo-natural products proven to be an anti-cancer drug. In its metal bound form HO2-Fe(III)Blm associates with double stranded DNA and causes single- and double strand cleavage. Structural studies performed with HO2-Co(III)Blm bound to DNA oligomers reveal that the metal domain and linker are folded into a unit interacting with the minor groove while the drug's bithiazole is intercalated between adjacent base pairs [1].(Figure 1)

bar.gif
Figure 1: HO2-Co(III)-Blm A2 binds to major and minor groove of d(GGAAGCTTCC)2 [1]
bar.gif
While NMR studies support the assumption that HO2-Co(III)Blm is a a valid structural model for the biologically active Fe(III) complex there is still uncertainty about some details of its structure. In particular there is still controversy concerning the nature of the fifth internal axial ligand opposite of the hydroperoxide ligand. It has been assumed that the primary amine (N-2) serves as that residue already provides another ligand to the metal (N-3) and would thus act as a chelating ligand (Figure 2) [2].

bar.gif
Figure 2: Structure of BleomycinA2 (Blm) with the nitrogen atoms involved or considered to be involved in binding to the metal ion highlighted (O)
bar.gif
However this assumption was challenged by the recent publication of the structure of HO2-Co(III)Pep-Blm that differs from Blm-A2 only in the nature of its R group (Figure 2). The authors places the mannose carbamoyl group (N-26) in the axial position and state that upon removal of the disaccharide in HOi2-Co(III)Pep-dBlm the primary amine moves into that position [3 ].

We thus engaged into a closer examination of the structure of HO2-Co(III)Blm. NOE based structure calculations of HO2-Co(III)Blm result in acceptable structures for both coordination modes as the two residues are located in close spatial proximity to each other (Figure 3). We therefore turned to probing for N-15 NMR chemical shift changes upon coordination to metal using natural abundance 15N{1H} HSQC and HMBC spectroscopy of apo-Blm, HO2-Co(III)Blm and their deglyco counterparts dgBlm and HO2-Co(III)dgBlm to unambiguously determine the nature of the remaining ligand [4]
bar.gif
> Figure: Two possible coordination modes of HO2-Co(III)Blm. (a) the NH2 of the β-aminoanalin NH2 (N-2 ) as terminal ligand (b) Mannose-carbamoyl NH2 (N-26) as terminal ligand. Both structures are consistent with all experimental NOE's even though structure (b) is slightly higher in energy than (a).
bar.gif
bar.gif
To Top of This Page NMR Home back to NMR lab on line posters To Poster-On-Line
Converted to HTML by F. Holger Försterling March 2003