SYNTHESES OF LABELED HEMES FOR PROTEIN NMR STUDIES

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Hemes regioselectively labeled in predetermined positions with deuterium, carbon-13, and fluorine will be used as NMR probes to gain an understanding of the relationship between hyperfine shifts and structure/function correlations in biologically important heme proteins such as myoglobins, hemoglobins, cytochromes,and peroxidases; the proposed work will facilitate understanding of heme protein function, and of structural and electronic factors with cause several debilitating diseases such as anemias. The synthelic labeled materials will also be used for making definitive band assignments in resonance Raman spectra, and also in studies of electron paramagnetic characteristics. After either chemical or biosynthetic reconstitution into appropriate apoproteins, the synthetic compounds will be used to enable assignment of heme associated resonances by difference spectroscopy for protons, and directly in the deuterium, carbon-13, and fluorine spectra. Two fundamental approaches will be followed in synthesis of the labeled hemes; carbon-13 and some deuterium labeled derivatives of protoporphyrin-IX will be obtained through total synthesis from acyclic precursors using our recently developed tripyrrene approaches, and cupric-catalyzed cyclization of a,c-biiadienes (the mechanism of which will also be studied). A mono-meso-deuterated, and carbon-13 labeled derivative of protoporphyrin-IX will also be synthesized to test an NMR model for heme catabolism to bilirubin (which is associated with hepatic problems such as jaundice and hyperbilirubinemia), and in this connection, NMR spectra of oxophlorin/protein aggregates will also be investigated. The second route to labeled hemes will utilize deuterium exchange on protoporphyrin-IX, to give directly the required labeled hemes and an attempt will be made to rationalize the exchange processes on the basis of electronic structure within the porphyrin nucleus. Fluorine and some carbon-13 labeled hemes will be prepared by modification of protoporphyrin-IX derivatives. Certain novel, unlabeled porphyrins will also be synthesized, either by total synthesis or by modification of existing commercially available samples; they will be used in NMR studies, and also in proposed resonance Raman, X-ray and electron paramagnetic resonance investigations.