The chemical and enzymatic oxidation of hematohemin IX. Identification of hematobiliverdin IX alpha as the sole product of the enzymatic oxidation.

Oxidative cleavage of hematohemin IX in pyridine solution in the presence of ascorbic acid (coupled oxidation), followed by esterification of the products with boron trifluoride/methanol produced the four possible hematobiliverdin dimethyl esters in 11.1% overall yield. Transetherifications took place simultaneously with the esterification reaction and resulted in the formation of the dimethyl ester of hematobiliverdin IX gamma 8a,13a-dimethyl ether (1.8%), the dimethyl ester of hematobiliverdin IX beta 13a,18a-dimethyl ether (1.9%), the dimethyl ester of hematobiliverdin IX delta 8a-monomethyl ether (1.4%), and the dimethyl ester of hematobiliverdin IX alpha 18a-monomethyl ether (0.4%). The latter was the sole product obtained after the enzymatic oxidation of hematohemin with heme oxygenase, after esterification of the reaction product with boron trifluoride/methanol. When the esterification step was omitted hematobiliverdin IX alpha was obtained from the enzymatic oxidation. The structures of the hematobiliverdin derivatives were secured by their NMR and mass spectra data. Saponification of the dimethyl esters afforded the hematobiliverdin methyl ethers, which were excellent substrates of biliverdin reductase and were readily reduced to the corresponding bilirubins. Hematobiliverdin IX alpha was also a good substrate of biliverdin reductase. It is concluded that the enzymatic oxidation of hematohemin IX by heme oxygenase is alpha-selective, while biliverdin reductase shows no selectivity in the reduction of the four hematobiliverdin isomers.