17 research outputs found

    Recycling Upstream Redox Enzymes Expands the Regioselectivity of Cycloaddition in Pseudo-Aspidosperma Alkaloid Biosynthesis

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    Nature uses cycloaddition reactions to generate complex natural product scaffolds. Dehydrosecodine is a highly reactive biosynthetic intermediate that undergoes cycloaddition to generate several alkaloid scaffolds that are the precursors to pharmacologically important compounds such as vinblastine and ibogaine. Here we report how dehydrosecodine can be subjected to redox chemistry, which in turn allows cycloaddition reactions with alternative regioselectivity. By incubating dehydrosecodine with reductase and oxidase biosynthetic enzymes that act upstream in the pathway, we can access the rare pseudoaspidosperma alkaloids pseudo-tabersonine and pseudo-vincadifformine, both in vitro and by reconstitution in the plant Nicotiana benthamiana from an upstream intermediate. We propose a stepwise mechanism to explain the formation of the pseudo-tabersonine scaffold by structurally characterizing enzyme intermediates and by monitoring the incorporation of deuterium labels. This discovery highlights how plants use redox enzymes to enantioselectively generate new scaffolds from common precursors

    Moose, caribou and fire: have we got it right yet?

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    Natural disturbance plays a key role in shaping community dynamics. Within Canadian boreal forests, the dominant form of natural disturbance is fire, and its effects are thought to influence the dynamics between moose (Alces alces (Linnaeus, 1758)) and the boreal ecotype of woodland caribou (Rangifer tarandus caribou (Gmelin, 1788)). Boreal caribou are considered “threatened” and population declines are attributed, at least in part, to disturbance-mediated apparent competition (DMAC) with moose. Here, we tested a primary prediction of the DMAC hypothesis: that moose respond positively to burns within and adjacent to the caribou range. We assessed moose selection for ≀25-year-old burns (when selection is predicted to be strongest) at multiple spatial scales and evaluated whether moose density was correlated with the extent of ≀40-year-old burns (a time frame predicted to negatively affect caribou). Against expectation, moose showed avoidance and low use of ≀25-year-old burns at all scales, regardless of burn age, season, and type of land cover burned. These findings mirrored the demographic response, as we found no correlation between ≀40-year-old burns and moose density. By contradicting the prevailing hypothesis linking fires to caribou population declines, our results highlight the need to understand regional variation in disturbance impacts on caribou populations.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

    Controlled Oxidation of Remote sp<sup>3</sup> C–H Bonds in Artemisinin via P450 Catalysts with Fine-Tuned Regio- and Stereoselectivity

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    The selective oxyfunctionalization of isolated sp<sup>3</sup> C–H bonds in complex molecules represents a formidable challenge in organic chemistry. Here, we describe a rational, systematic strategy to expedite the development of P450 oxidation catalysts with refined regio- and stereoselectivity for the hydroxylation of remote, unactivated C–H sites in a complex scaffold. Using artemisinin as model substrate, we demonstrate how a three-tier strategy involving first-sphere active site mutagenesis, high-throughput P450 fingerprinting, and fingerprint-driven P450 reactivity predictions enabled the rapid evolution of three efficient biocatalysts for the selective hydroxylation of a primary and a secondary C–H site (with both <i>S</i> and <i>R</i> stereoselectivity) in a relevant yet previously inaccessible region of this complex natural product. The evolved P450 variants could be applied to provide direct access to the desired hydroxylated derivatives at preparative scales (0.4 g) and in high isolated yields (>90%), thereby enabling further elaboration of this molecule. As an example, enantiopure C7-fluorinated derivatives of the clinical antimalarial drugs artesunate and artemether, in which a major metabolically sensitive site is protected by means of a C–H to C–F substitution, were afforded via P450-mediated chemoenzymatic synthesis

    Biochemical and Structural Characterization of MycCI, a Versatile P450 Biocatalyst from the Mycinamicin Biosynthetic Pathway

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    Cytochrome P450 monooxygenases (P450s) are some of nature’s most ubiquitous and versatile enzymes for performing oxidative metabolic transformations. Their unmatched ability to selectively functionalize inert C–H bonds has led to their increasing employment in academic and industrial settings for the production of fine and commodity chemicals. Many of the most interesting and potentially biocatalytically useful P450s come from microorganisms, where they catalyze key tailoring reactions in natural product biosynthetic pathways. While most of these enzymes act on structurally complex pathway intermediates with high selectivity, they often exhibit narrow substrate scope, thus limiting their broader application. In the present study, we investigated the reactivity of the P450 MycCI from the mycinamicin biosynthetic pathway toward a variety of macrocyclic compounds and discovered that the enzyme exhibits appreciable activity on several 16-membered ring macrolactones independent of their glycosylation state. These results were corroborated by performing equilibrium substrate binding experiments, steady-state kinetics studies, and X-ray crystallographic analysis of MycCI bound to its native substrate mycinamicin VIII. We also characterized TylHI, a homologous P450 from the tylosin pathway, and showed that its substrate scope is severely restricted compared to MycCI. Thus, the ability of the latter to hydroxylate both macrocyclic aglycones and macrolides sets it apart from related biosynthetic P450s and highlights its potential for developing novel P450 biocatalysts with broad substrate scope and high regioselectivity

    Regiodivergent Glycosylations of 6‑Deoxy-erythronolide B and Oleandomycin-Derived Macrolactones Enabled by Chiral Acid Catalysis

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    This work describes the first example of using chiral catalysts to control site-selectivity for the glycosyl­ations of complex polyols such as 6-deoxy­erythro­nolide B and oleando­mycin-derived macrolactones. The regio­divergent introduction of sugars at the C3, C5, and C11 positions of macrolactones was achieved by selecting appropriate chiral acids as catalysts or through introduction of stoichio­metric boronic acid-based additives. BINOL-based chiral phosphoric acids (CPAs) were used to catalyze highly selective glycosyl­ations at the C5 positions of macrolactones (up to 99:1 rr), whereas the use of SPINOL-based CPAs resulted in selectivity switch and glycosyl­ation of the C3 alcohol (up to 91:9 rr). Additionally, the C11 position of macrolactones was selectively function­alized through traceless protection of the C3/C5 diol with boronic acids prior to glycosyl­ation. Investigation of the reaction mechanism for the CPA-controlled glycosyl­ations revealed the involvement of covalently linked anomeric phosphates rather than oxo­carbenium ion pairs as the reactive intermediates
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