Exploiting Nuclear Spin Polarization to Investigate Enzyme-Mediated Free Radical Reactions via in situ NMR

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Radicals occuring in enzyme-mediated reactions will be studied via NMR spectroscopy using the CIDNP method. A threefold strategy will be followed: 1. Initially simple model radicals for the postulated intermediates will be generated either photochemically using an UV eximer laser or by mixing appropriate components in a flow system. Photoinitiated electron transfer between a donor (triethylamine) and as acceptor a halogen-containing precursor yields the desired neutral free radicals after eliminating a halide from the halogen-containing radical anion. The radical cation expels a proton, yielding a neutral free radical likewise. The resulting radical pairs cause CIDNP, which should allow to differentiate between the two alternative reaction pathways, i.e. fragmentation followed by recombination versus addition followed by elimination. 2. Analogously, the enzyme-produced radicals NO., HO., O2-. and their follow-up species will be generated via a flow system. 3. The associated phenomena and polarization patterns of the products will be studied initially using a conventional NMR probe, but eventually via a cylindrical torus probe, which insures higher sensitivity. This same setup has been used successfully to study homogeneous hydrogenation of unsaturated substrates using only one spin isomers of H2, typically of parahydrogen. This phenomenon, termed Parahydrogen Induced Polarization (PHIP), though different in detail, has features similar to CIDNP.