During the current grant period have discovered that the photochemistry of simple aromatic aides are remarkably sensitive to temperature. Photolysis of phenyl azide at room temperature produces dehydroazepine which is relatively unreactive and prone to polymerization to form tar. However, photolysis of phenyl azide in a rigid matrix (RH) at 77K produces triplet phenyl nitrene which at this temperature is indefinitely stable in the dark. However, photolysis of triplet nitrenes results in hydrogen atom abstraction from the matrix (RH) to form radical pair, which when warmed to 100K followed by radical pair collapses to form coupling product. The sequences of photochemical steps at 77K efficiently leads to coupling products such as in high yields. We have exploited this by studying the photochemistry and low temperature EPR spectroscopy of modified chymotrypsins at 77K. In this proposal experiments are outlined which extend the low temperature method to other enzymes and which introduce new non covalently bound photoprobe reagents which are expected to be particularly eficient at 77K.
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