Kinetic isotope effects (KIEs) provide a powerful tool to interrogate transition states of both enzymic and non-enzymic reactions, [1][2][3][4] provided that one can measure the intrinsic KIE on the chemical step of interest, that is, the isotope effect undiminished by other isotope-insensitive steps that may contribute to the overall rate of reaction. For small molecules reacting in solution the KIE measured usually represents the intrinsic value; however, for enzymes this is seldom the case. Here we report the first measurement of the intrinsic KIE in an adenosylcobalamin enzyme (AdoCbl, coenzyme B 12 ) for hydrogen atom transfer from substrate to coenzyme, which is a key step in the mechanism of this class of enzymes. For the B 12 enzyme glutamate mutase the intrinsic deuterium KIE for hydrogen transfer from the substrate, (2S,3S)-3-methylaspartate, to 5'-deoxyadensosine is 4.1. This value is well within the semiclassical limit for a deuterium isotope effect and is much smaller than the anomalously large KIEs previously measured in other B 12 enzymes and non-enzymatic model reactions, which were attributed to extensive hydrogen tunneling.Glutamate mutase one is of a group of AdoCbl-dependent enzymes that catalyze unusual isomerization reactions that formally involve a 1,2 hydrogen atom migration and proceed through a mechanism involving carbon-based free radical intermediates (Scheme 1). [5][6][7][8][9][10] Radicals are generated by homolysis of the reactive cobalt-carbon bond of the coenzyme to form cob(II)alamin, a cobalt(II) intermediate, and the 5'-deoxyadenosyl radical. The adenosyl radical then abstracts the migrating hydrogen from the substrate to form 5'-deoxyadenosine and the substrate radical. The substrate radical next undergoes rearrangement to give the product radical, which is then quenched by hydrogen transfer from 5'-deoxyadenosine to give the product and regenerate the 5'-deoxyadenosyl radical. Finally, recombination of the adenosyl radical and cob(II)alamin to reform the coenzyme completes the catalytic cycle.Our interest in the mechanisms by which enzymes generate free radicals, as exemplified by dependent glutamate mutase, [10] led us to undertake an extensive set of KIE measurements to examine how hydrogen abstraction from the substrate and coenzyme homolysis are coupled together. [11][12][13][14][15] KIE measurements using deuterium-and tritium-labeled substrates and coenzyme have proved especially informative probes of the key steps of Co À C bond homolysis and hydrogen atom abstraction from substrate. Pre-steady-state measurements on a number of enzymes have shown that hydrogen abstraction is kinetically coupled to CoÀC bond homolysis, [11,[16][17][18] as evidenced by the appearance of a kinetic isotope effect on cobalt-carbon bond homolysis when the enzymes are reacted with deuterated substrates.This observation implies that the 5'-dA radical is a highenergy intermediate that only has a fleeting existence. Furthermore, the KIEs reported for several AdoCbl enzymes are extremely large (r...

Photochemical reactions taking place between 1,2-diketones and silyl ketene acetals and their excited state reaction mechanisms have been explored. Irradiation of benzene, acetone, or acetonitrile solutions containing 1,2-diketones and silyl ketene acetals is observed to promote formation of 1,4-dioxenes, resulting from [4 + 2]-cycloaddition, oxetanes, arising by Paterno-Buchi processes, and beta-hydroxy-gamma-ketoesters, generated by SET-promoted Claisen-type condensation. These competitive pathways leading from the excited states of the 1,2-diketones to these products are influenced by solvent polarity and the nature of the silyl ketene acetal and 1,2-diketone. The Claisen-type condensation process, following an SET desilylation pathway and predominating when the photoreactions are carried out in the polar solvent acetonitrile, represents an efficient method to prepare a variety of diversely substituted beta-hydoxy-gamma-ketoesters.

Biosynthetic pathways can be hijacked to yield novel compounds by introduction of novel starting materials. Here we have altered tryptamine, which serves as the starting substrate for a variety of alkaloid biosynthetic pathways, by replacing the indole with one of four aza-indole isomers. We show that two aza-tryptamine substrates can be successfully incorporated into the products of the monoterpene indole alkaloid pathway in Catharanthus roseus. Use of unnatural heterocycles in precursor directed biosynthesis, in both microbial and plant natural product pathways, has not been widely demonstrated, and successful incorporation of starting substrate analogs containing the aza-indole functionality has not been previously reported. This work serves as a starting point to explore fermentation of aza-alkaloids from other tryptophan and tryptamine derived natural product pathways.