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

Introduction
N-15 NMR chemical shifts
Three dimensional structure using NOE and C-H coupling constants
Literature

Three dimensional structure using NOE and C-H coupling constants:

The ¹⁵N NMR data together with NOE’s uniquely define the overall structure of HO₂-Co(III)Ble A2. However there is considerable spectral overlap in the spectral region of the disaccharide protons reducing the numbers of distance restraints available for this part of the molecule. As the biological function of the disaccaride unit is still a matter of debate a good structural description of these residues was desirable.

Additional restraints were therefore obtained for CCCH and COCH dihedral angles by measuring ³J_CH coupling constants using the HSQMBC experiment [6] (Figure 6). While the correlation of ³J_CH coupling constants with dihedral angles is typically somewhat more scattered in carbohydrates, the information was sufficient to result in a considerable improvement of the conformation within and between the two sugar rings. The ensemble of the 18 final structures of lowest energy is shown in Figure 7.

Figure 6: (Top right) Pulse sequence of phase sensitive HSQMBC experiment [6 ]. φ1 = x,-x; φ2 = x,x,-x,-x; φ3 = x,x,x,x,-x,-x,-x,-x; ψ = x,-x,x,-x,-x,x,-x,x. The gradient ratios were 80:50:20 and the delay Δ = 31 ms. (left bottom) Selected expansion of the ¹H{¹³C} HSQMBC spectrum of HO₂-Co(III)-Blm showing the H-20/C24 and H20/C22 cross peaks. (left top) 1D slices at the f1 frequencies of C-22 and C-24 overlaid with the experimental line shapes obtained from a superposition of two HSQC slices through the corresponding carbon shifted by ³J_CH. (right bottom) Comparison of the experimental COCH coupling constants to the ones calculated from the final structure using the corresponding Karplus relation [7]

Figure 7: Overlay of the 18 lowest energy structures obtained for HO₂-Co(III)-Blm from ARIA/CNS using 243 distance restraints and 18 J coupling restraints (9 CH and 9 HH). The final RMSD is 0.35(13) for all non terminal heavy atoms and 0.61(0.11) including all non terminal atoms.

Converted to HTML by F. Holger Försterling March 2003

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

Three dimensional structure using NOE and C-H coupling constants:

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