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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

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N-15 NMR chemical shifts

Careful adjustment of the pH allowed to observe and assign all NH and NH2 groups in HO2-Co(III)-Blm and HO2-Co(III)-deglyco-Blm at pH 6.7 using 2D 1H{15N}-HSQC and 1H/1 H COSY spectroscopy. In particular the &beta7-amino-alanine NH2-2 protons and nitrogens which were very elusive in previous studies could be uniquely identified (Figure 4). In addition 1H{15N}-HMBC allowed to identify amide N-12 which is non protonated due to coordination to the metal In addition the imidazole nitrogens N28 and N29 were observed. In a similar manner all NH groups except NH2-2 could be detected in the apo-bleomycins.The primary amine of the pyridilpropionamide (NH2-8) was only observed with the pH lowered to pH 6.4.

Large negative N-15 chemical shift changes up to 45 ppm are observed for N-2, N-3, N-12 and N-29 when comparing apo-Blm with the HO2-Co(III)-Blm (Figure 5). Such shifts are consistent with coordination to a metal ion [5]. No significant shift is observed for the disaccharide carbamoyl nitrogen N-26 indicating that this group is not involved in metal binding.

In addition the nitrogen shifts in HO2-Co(III)-deglyco-Blm are basically identical to the ones in HO2-Co(III)-Blm (Figure 5) suggesting that the metal coordination is identical in both cases and does not change upon removal of the disaccharide ligand as suggested. bar.gif
Figure 4: (a) Amide-region of natural abundance 1H{15N}-HSQC spectrum of 10 mM (pH 6.7) HO2-Co(III)-Blm (b) The amine region depicting N-3 and N-2 is shown in with the assigngment through 1H/1H COSY indicated in (c). Natural abundance 1H{15N}HSQC experiments were performed with gradient selection and sensitivity enhancement employing water flip back pulses to minimize saturation of exchanging protons.
A typical experiment comprised 144 t1 increments with 1024 transients of 2048 points each, covering a spectral width of 15205 and 8012 Hz in f1and f2, respectively. 1H{15N}HMBC experiments were carried out using a standard gradient selected sequence with additional presaturation of the water resonance for improved water suppression. A total of 132 and 4096 data points were acquired in t1 and t2, respectively, with 1600 scans accumulated for each t1 data point with a spectral width of 18245 and 8012 Hz in f1 and f2.
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Figure 5: (a) 15N chemical shift difference between apo-Blm and HO2-Co(III)-Blm. Large negative shift changes are observed for N2, N3, N12 and N29, but no significant shift is observed for N-26. The positive shift of N-8 arises from the coordination of the neighbored N-10 (not observed). (b) 15N chemical shift difference betweenHO2-Co(III)deglycoBlm and HO2-Co(III)-Blm. No large change is observed for N-2 between the two compounds indicating that the coordination is the same in both cases.
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