Elizabeth S. Sattely

Highly efficient molybdenum-based catalysts for enantioselective alkene metathesis

Discovery of efficient catalysts is one of the most compelling objectives of modern chemistry. Chiral catalysts are in particularly high demand, as they facilitate synthesis of enantiomerically enriched small molecules that are critical to developments in medicine, biology and materials science. Especially noteworthy are catalysts that promote—with otherwise inaccessible efficiency and selectivity levels—reactions demonstrated to be of great utility in chemical synthesis. Here we report a class of chiral catalysts that initiate alkene metathesis with very high efficiency and enantioselectivity. Such attributes arise from structural fluxionality of the chiral catalysts and the central role that enhanced electronic factors have in the catalytic cycle. The new catalysts have a stereogenic metal centre and carry only monodentate ligands; the molybdenum-based complexes are prepared stereoselectively by a ligand exchange process involving an enantiomerically pure aryloxide, a class of ligands scarcely used in enantioselective catalysis. We demonstrate the application of the new catalysts in an enantioselective synthesis of the Aspidosperma alkaloid, quebrachamine, through an alkene metathesis reaction that cannot be promoted by any of the previously reported chiral catalysts.

Design and Stereoselective Preparation of a New Class of Chiral Olefin Metathesis Catalysts and Application to Enantioselective Synthesis of Quebrachamine: Catalyst Development Inspired by Natural Product Synthesis

A total synthesis of the Aspidosperma alkaloid quebrachamine in racemic form is first described. A key catalytic ring-closing metathesis of an achiral triene is used to establish the all-carbon quaternary stereogenic center and the tetracyclic structure of the natural product; the catalytic transformation proceeds with reasonable efficiency through the use of existing achiral Ru or Mo catalysts. Ru- or Mo-based chiral olefin metathesis catalysts have proven to be inefficient and entirely nonselective in cases where the desired product is observed. In the present study, the synthesis route thus serves as a platform for the discovery of new olefin metathesis catalysts that allow for efficient completion of an enantioselective synthesis of quebrachamine. Accordingly, on the basis of mechanistic principles, stereogenic-at-Mo complexes bearing only monodentate ligands have been designed. The new catalysts provide significantly higher levels of activity than observed with the previously reported Ru- or Mo-based complexes. Enantiomerically enriched chiral alkylidenes are generated through diastereoselective reactions involving achiral Mo-based bispyrrolides and enantiomerically pure silyl-protected binaphthols. Such chiral catalysts initiate the key enantioselective ring-closing metathesis step in the total synthesis of quebrachamine efficiently (1 mol % loading, 22 degrees C, 1 h, >98% conversion, 84% yield) and with high selectivity (98:2 er, 96% ee).

A new cyanogenic metabolite in Arabidopsis required for inducible pathogen defence

Thousands of putative biosynthetic genes in Arabidopsis thaliana have no known function, which suggests that there are numerous molecules contributing to plant fitness that have not yet been discovered. Prime among these uncharacterized genes are cytochromes P450 upregulated in response to pathogens. Here we start with a single pathogen-induced P450 (ref. 5), CYP82C2, and use a combination of untargeted metabolomics and coexpression analysis to uncover the complete biosynthetic pathway to 4-hydroxyindole-3-carbonyl nitrile (4-OH-ICN), a previously unknown Arabidopsis metabolite. This metabolite harbours cyanogenic functionality that is unprecedented in plants and exceedingly rare in nature; furthermore, the aryl cyanohydrin intermediate in the 4-OH-ICN pathway reveals a latent capacity for cyanogenic glucoside biosynthesis in Arabidopsis. By expressing 4-OH-ICN biosynthetic enzymes in Saccharomyces cerevisiae and Nicotiana benthamiana, we reconstitute the complete pathway in vitro and in vivo and validate the functions of its enzymes. Arabidopsis 4-OH-ICN pathway mutants show increased susceptibility to the bacterial pathogen Pseudomonas syringae, consistent with a role in inducible pathogen defence. Arabidopsis has been the pre-eminent model system for studying the role of small molecules in plant innate immunity; our results uncover a new branch of indole metabolism distinct from the canonical camalexin pathway, and support a role for this pathway in the Arabidopsis defence response. These results establish a more complete framework for understanding how the model plant Arabidopsis uses small molecules in pathogen defence.

Enzymatic Tailoring of Ornithine in the Biosynthesis of the Rhizobium Cyclic Trihydroxamate Siderophore Vicibactin

To acquire iron, the N(2)-fixing, symbiotic bacterium Rhizobium sp. produce the cyclic trihydroxamate siderophore vicibactin, containing a 30-membered trilactone scaffold. Herein we report the overproduction and purification of the six proteins VbsACGOLS in the bacterial host Escherichia coli and the reconstitution of the biosynthesis of vicibactin from primary metabolites. The flavoprotein VbsO acts as a pathway-initiating l-ornithine N(5)-hydroxylase, followed by VbsA, which transfers (R)-3-hydroxybutyryl- from the CoA thioester to N(5)-hydroxyornithine to yield N(5)-((R)-3-hydroxybutyryl)-N(5)-hydroxy-l-ornithine. VbsL is a PLP-dependent epimerase acting at C(2) of the 10 atom monomer unit. VbsS, a nonribosomal peptide synthetase free-standing module, then activates N(5)-((R)-3-hydroxybutyryl)-N(5)-hydroxy-d-ornithine as the AMP anhydride on the way to cyclotrimerization to the vicibactin scaffold. The last step, tris-acetylation of the C(2) amino group of desacetyl-d-vicibactin to the mature siderophore vicibactin, is catalyzed distributively by VbsC, using three molecules of acetyl-CoA.

Three siderophores from one bacterial enzymatic assembly line.

Siderophores play a vital role in the survival of bacteria, as they facilitate the transport of iron in low-concentration environments. Nature employs a variety of coordinating functional groups in siderophore scaffolds as a way of creating structural diversity. We have successfully shown that the pseudomonine synthetase can produce three distinct siderophore natural products and five siderophore-like compounds. The in vitro enzymatic production of acinetobactin has prompted a revision of the reported structure from an oxazoline to an isoxazolidinone. Our results reveal the inherent flexibility of the pseudomonine synthetase and thus provide insight into the evolution of siderophore biosynthetic gene clusters in bacteria.

A Latent Oxazoline Electrophile for N-O-C Bond Formation in Pseudomonine Biosynthesis

Nitrogen-heteroatom bonds figure prominently in the structural, chemical, and functional diversity of natural products. In the case of Pseudomonas siderophore pseudomonine, an N-O hydroxamate linkage is found uncommonly configured in an isoxazolidinone ring. In an effort to understand the biogenesis of this heterocycle, we have characterized the pseudomonine synthetase in vitro and reconstituted the complete biosynthetic pathway. Our results indicate that the isoxazolidinone of pseudomonine arises from spontaneous rearrangement of an oxazoline precursor. To the best of our knowledge, this is a previously uncharacterized mode of post-assembly line heterocyclization. Our results establish the oxygen of the ubiquitous siderophore hydroxamate functionality as a nucleophile and may be indicative of general strategy for N-O-C bond formation in nature.

Minimum Set of Cytochromes P450 for Reconstituting the Biosynthesis of Camalexin, a Major Arabidopsis Antibiotic

Bringing it all together: The missing key step in the biosynthesis of camalexin was uncovered by in vitro biochemical characterization. The coupling of Trp- and Cys-derived fragments through CS bond formation is promoted by an unusual cytochrome P450 CYP71A13. The in vitro reconstitution of the camalexin biosynthesis (left) from Trp and Cys was achieved using just three cytochromes P450. IAN=indole-3-acetonitrile.

Rapid Phytotransformation of Benzotriazole Generates Synthetic Tryptophan and Auxin Analogs in Arabidopsis

Benzotriazoles (BTs) are xenobiotic contaminants widely distributed in aquatic environments and of emerging concern due to their polarity, recalcitrance, and common use. During some water reclamation activities, such as stormwater bioretention or crop irrigation with recycled water, BTs come in contact with vegetation, presenting a potential exposure route to consumers. We discovered that BT in hydroponic systems was rapidly (approximately 1-log per day) assimilated by Arabidopsis plants and metabolized to novel BT metabolites structurally resembling tryptophan and auxin plant hormones; <1% remained as parent compound. Using LC-QTOF-MS untargeted metabolomics, we identified two major types of BT transformation products: glycosylation and incorporation into the tryptophan biosynthetic pathway. BT amino acid metabolites are structurally analogous to tryptophan and the storage forms of auxin plant hormones. Critical intermediates were synthesized (authenticated by (1)H/(13)C NMR) for product verification. In a multiple-exposure temporal mass balance, three major metabolites accounted for >60% of BT. Glycosylated BT was excreted by the plants into the hydroponic medium, a phenomenon not observed previously. The observed amino acid metabolites are likely formed when tryptophan biosynthetic enzymes substitute synthetic BT for native indolic molecules, generating potential phytohormone mimics. These results suggest that BT metabolism by plants could mask the presence of BT contamination in the environment. Furthermore, BT-derived metabolites are structurally related to plant auxin hormones and should be evaluated for undesirable biological effects.

Key applications of plant metabolic engineering.

Elizabeth Sattely, Anne Osbourn, and colleagues discuss in this Essay four long-standing challenges in plant metabolic engineering: to create plants that provide their own nitrogen, have improved nutrient content, function better as biofuels, and have increased photosynthetic efficiency.

Biosynthesis of cabbage phytoalexins from indole glucosinolate

Significance Cruciferous vegetables—such as broccoli, mustard greens, and turnip—produce a variety of indole- and sulfur-containing chemicals when attacked by pathogens. These compounds, termed phytoalexins, are thought to serve as an important defense mechanism for the plant. Several phytoalexins have been shown to also have anticancer properties, raising the possibility that dietary exposure may be important to human health. Here, we report a minimal set of enzymes required to make brassinin, the parent cruciferous phytoalexin, from the well-studied glucosinolates. These genes enabled production of brassinin and related phytoalexins in tobacco leaves at levels comparable to those measured in cruciferous vegetables. The complete biosynthetic pathway to brassinin is critical for using metabolic engineering to characterize the biological role of indole–sulfur phytoalexins. Brassica crop species are prolific producers of indole–sulfur phytoalexins that are thought to have an important role in plant disease resistance. These molecules are conspicuously absent in the model plant Arabidopsis thaliana, and little is known about the enzymatic steps that assemble the key precursor brassinin. Here, we report the minimum set of biosynthetic genes required to generate cruciferous phytoalexins starting from the well-studied glucosinolate pathway. In vitro biochemical characterization revealed an additional role for the previously described carbon–sulfur lyase SUR1 in processing cysteine–isothiocyanate conjugates, as well as the S-methyltransferase DTCMT that methylates the resulting dithiocarbamate, together completing a pathway to brassinin. Additionally, the β-glucosidase BABG that is present in Brassica rapa but absent in Arabidopsis was shown to act as a myrosinase and may be a determinant of plants that synthesize phytoalexins from indole glucosinolate. Transient expression of the entire pathway in Nicotiana benthamiana yields brassinin, demonstrating that the biosynthesis of indole–sulfur phytoalexins can be engineered into noncruciferous plants. The identification of these biosynthetic enzymes and the heterologous reconstitution of the indole–sulfur phytoalexin pathway sheds light on an important pathway in an edible plant and opens the door to using metabolic engineering to systematically quantify the impact of cruciferous phytoalexins on plant disease resistance and human health.