CARBON‐13 N.M.R. STUDY OF BLEOMYCIN‐A2 PROTONATION

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Abstract
The acid-base titration of bleomycin A2 in D2O solution at 35 .+-. 5.degree. C was monitored by 13C NMR spectroscopy at 67.89 MHz. The following .**GRAPHIC**. values were obtained: 3.68 .+-. 0.05 (secondary amine), 5.29 .+-. 0.03 (imidazole), and 8.23 .+-. 0.19 (primary amine), where .**GRAPHIC**. is the dissociation constant in D2O solution. The equilibrium isotope effects .**GRAPHIC**. - pKa in H2O) are: 0.70 .+-. 0.06 (secondary amine), 0.28 .+-. 0.04 (imidazole), and 0.85 .+-. 0.19 (primary amine). Titration of the imidazole group of Bleo-A2 occurs at N.pi., i.e., only N.tau. is protonated in basic solution. Significant protonation shifts are almost completely limited to carbons of the N-terminal tetrapeptide, suggesting that the C-terminal tripeptide extends into the solvent and interacts to a minimal extent with the rest of the molecule. Long range protonation shifts associated with titration of the imidazole and secondary amine groups indicate that protonation of one or both of these sites is probably accompanied by significant conformational changes. The observed protonation shifts generally fail to correlate with Zn(II) complexation shifts reported by Dabrowiak et al., indicating that ligation sites cannot unambiguously be determined from these complexation shifts. The complexation shifts previously attributed to coordination of the imidazole and carbamoyl groups probably result from conformational changes.