1H NMR spectral studies at 360 MHz have been conducted on bleomycin, a modified bleomycin, and the zinc-bleomycin complex. In these studies, the revised structure of bleomycin has been independently verified by (1) the pD dependence of chemical shifts, (2) observation of the exchangeable resonances with (CD3)2SO as solvent, and (3) acylation of the α-amine of the β-aminoalanine moiety. The 1H NMR spectra of the zinc-bleomycin complex (D2O as solvent) have been obtained and analyzed. Chemical shift changes larger than 0.1 ppm are observed upon metal binding for the following groups: (1) β-aminoalanine, (2) propion-amide, (3) pyrimidinyl methyl, (4) β-hydroxyhistidine, (5) mannose 3′, and (6) methylvalerate. Coordination to zinc also causes large changes in vicinal coupling constants for the hydroxyhistidine α- and β-CH protons and for the vicinal coupling in the β-aminoalanine moiety, thus indicating the predominance of specific rotamers around these bonds. Titration studies of the zinc-bleomycin complex establish the pAKa of the imidazole coordinated to zinc as 11.0. The de-protonation of the zinc-imidazole also causes a further 0.4-ppm shielding of the methylvalerate α-CH resonance. This shift is in addition to the 0.6-ppm upfield shift observed upon the initial formation of the zinc-bleomycin complex. These results establish that coordination to zinc confers a conformational rigidity on bleomycin and demonstrate the existence of a heretofore unsuspected association between the imidazole and methylvalerate groups.
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