Biosynthesis of strychnine.

Strychnine is a natural product that, through isolation, structural elucidation and synthetic efforts, shaped the field of organic chemistry. Currently, strychnine is used as a pesticide to control rodents1 because of its potent neurotoxicity2,3. The polycyclic architecture of strychnine has inspired chemists to develop new synthetic transformations and strategies to access this molecular scaffold4, yet it is still unknown how plants create this complex structure. Here we report the biosynthetic pathway of strychnine, along with the related molecules brucine and diaboline. Moreover, we successfully recapitulate strychnine, brucine and diaboline biosynthesis in Nicotiana benthamiana from an upstream intermediate, thus demonstrating that this complex, pharmacologically active class of compounds can now be harnessed through metabolic engineering approaches.

Reinventing metabolic pathways: Independent evolution of benzoxazinoids in flowering plants.

Benzoxazinoids (BXDs) form a class of indole-derived specialized plant metabolites with broad antimicrobial and antifeedant properties. Unlike most specialized metabolites, which are typically lineage-specific, BXDs occur sporadically in a number of distantly related plant orders. This observation suggests that BXD biosynthesis arose independently numerous times in the plant kingdom. However, although decades of research in the grasses have led to the elucidation of the BXD pathway in the monocots, the biosynthesis of BXDs in eudicots is unknown. Here, we used a metabolomic and transcriptomic-guided approach, in combination with pathway reconstitution in Nicotiana benthamiana, to identify and characterize the BXD biosynthetic pathways from both Aphelandra squarrosa and Lamium galeobdolon, two phylogenetically distant eudicot species. We show that BXD biosynthesis in A. squarrosa and L. galeobdolon utilize a dual-function flavin-containing monooxygenase in place of two distinct cytochrome P450s, as is the case in the grasses. In addition, we identified evolutionarily unrelated cytochrome P450s, a 2-oxoglutarate-dependent dioxygenase, a UDP-glucosyltransferase, and a methyltransferase that were also recruited into these BXD biosynthetic pathways. Our findings constitute the discovery of BXD pathways in eudicots. Moreover, the biosynthetic enzymes of these pathways clearly demonstrate that BXDs independently arose in the plant kingdom at least three times. The heterogeneous pool of identified BXD enzymes represents a remarkable example of metabolic plasticity, in which BXDs are synthesized according to a similar chemical logic, but with an entirely different set of metabolic enzymes.

Enantioselective Divergent Syntheses of Diterpenoid Pyrones.

Capitalizing a synergy between late-stage C(sp3)-H alkynylation and a series of transition metal-catalyzed alkyne functionalization reactions, we reported herein enantioselective divergent synthesis of 10 diterpenoid pyrones within 14-16 steps starting from chiral pool enoxolone, including the first enantioselective synthesis of higginsianins A, B, D, E, and metarhizin C. Our synthesis also highlights an unprecedented biomimetic oxidative rearrangement of α-pyrone into 3(2H)-furanone, as well as applications of Echavarren C(sp3)-H alkynylation reaction and Toste chiral counterion-mediated Au-catalyzed intramolecular allene hydroalkoxylation in natural product synthesis.

Phylogeny-Aware Chemoinformatic Analysis of Chemical Diversity in Lamiaceae Enables Iridoid Pathway Assembly and Discovery of Aucubin Synthase.

Countless reports describe the isolation and structural characterization of natural products, yet this information remains disconnected and underutilized. Using a cheminformatics approach, we leverage the reported observations of iridoid glucosides with the known phylogeny of a large iridoid producing plant family (Lamiaceae) to generate a set of biosynthetic pathways that best explain the extant iridoid chemical diversity. We developed a pathway reconstruction algorithm that connects iridoid reports via reactions and prunes this solution space by considering phylogenetic relationships between genera. We formulate a model that emulates the evolution of iridoid glucosides to create a synthetic data set, used to select the parameters that would best reconstruct the pathways, and apply them to the iridoid data set to generate pathway hypotheses. These computationally generated pathways were then used as the basis by which to select and screen biosynthetic enzyme candidates. Our model was successfully applied to discover a cytochrome P450 enzyme from Callicarpa americana that catalyzes the oxidation of bartsioside to aucubin, predicted by our model despite neither molecule having been observed in the genus. We also demonstrate aucubin synthase activity in orthologues of Vitex agnus-castus, and the outgroup Paulownia tomentosa, further strengthening the hypothesis, enabled by our model, that the reaction was present in the ancestral biosynthetic pathway. This is the first systematic hypothesis on the epi-iridoid glucosides biosynthesis in 25 years and sets the stage for streamlined work on the iridoid pathway. This work highlights how curation and computational analysis of widely available structural data can facilitate hypothesis-based gene discovery.

Total Synthesis of Diverse Tetramic Acid Bearing cis-Decalin Natural Products.

We report herein the first total syntheses of four natural antibiotics, vermisporin, PF1052/AB4015-A, AB4015-L, AB4015-B, and one hydrogenated natural product derivative, AB4015-A2, that all feature a tetramic acid bearing cis-decalin ring. The construction of the functionalized cis-decalin ring was achieved by a diastereoselective intramolecular Diels-Alder (IMDA) reaction, which proceeded via a rare endo-boat transition state. Through an intramolecular neighboring-group-oriented strategy, the sterically hindered epoxy group in vermisporin, PF1052/AB4015-A and AB4015-L was installed efficiently. A one-pot aminolysis/Dieckmann condensation cascade using l-amino acid derivatives afforded the desired tetramic acid structure. The total synthesis led to the unambiguous verification of the absolute configuration of these natural products.

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

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.

Enantioselective Total Synthesis of Hyperforin and Pyrohyperforin.

Capitalizing on the late-stage diversification of an essential 1,3-diene intermediate, we describe herein a 9-step enantioselective total synthesis of (+)-hyperforin and (+)-pyrohyperforin, starting from commercially available allylacetone. Our convergent synthesis features a series of critical reactions: 1) an enantioselective deconjugative α-alkylation of α,ß-unsaturated acid using chiral lithium amides as noncovalent stereodirecting auxiliaries; 2) a HfCl4 -mediated carbonyl α-tert-alkylation to forge the intricate bicyclo[3.3.1]nonane framework; 3) an abiotic cascade pyran formation; and 4) a selective 1,4-semihydrogenation of polyenes. During the course of our synthesis, we also identified a 1,2-cyclopropanediol overbred intermediate which was responsible for the 1,3-diene precursor formation through a controlled fragmentation.

Expansion of the Catalytic Repertoire of Alcohol Dehydrogenases in Plant Metabolism.

Medium-chain alcohol dehydrogenases (ADHs) comprise a highly conserved enzyme family that catalyse the reversible reduction of aldehydes. However, recent discoveries in plant natural product biosynthesis suggest that the catalytic repertoire of ADHs has been expanded. Here we report the crystal structure of dihydroprecondylocarpine acetate synthase (DPAS), an ADH that catalyses the non-canonical 1,4-reduction of an α,ß-unsaturated iminium moiety. Comparison with structures of plant-derived ADHs suggest the 1,4-iminium reduction does not require a proton relay or the presence of a catalytic zinc ion in contrast to canonical 1,2-aldehyde reducing ADHs that require the catalytic zinc and a proton relay. Furthermore, ADHs that catalysed 1,2-iminium reduction required the presence of the catalytic zinc and the loss of the proton relay. This suggests how the ADH active site can be modified to perform atypical carbonyl reductions, providing insight into how chemical reactions are diversified in plant metabolism.

Two bi-functional cytochrome P450 CYP72 enzymes from olive (Olea europaea) catalyze the oxidative C-C bond cleavage in the biosynthesis of secoxy-iridoids - flavor and quality determinants in olive oil.

Olive (Olea europaea) is an important crop in Europe, with high cultural, economic and nutritional significance. Olive oil flavor and quality depend on phenolic secoiridoids, but the biosynthetic pathway of these iridoids remains largely uncharacterized. We discovered two bifunctional cytochrome P450 enzymes, catalyzing the rare oxidative C-C bond cleavage of 7-epi-loganin to produce oleoside methyl ester (OeOMES) and secoxyloganin (OeSXS), both through a ketologanin intermediary. Although these enzymes are homologous to the previously reported Catharanthus roseus secologanin synthase (CrSLS), the substrate and product profiles differ. Biochemical assays provided mechanistic insights into the two-step OeOMES and CrSLS reactions. Model-guided mutations of OeOMES changed the product profile in a predictable manner, revealing insights into the molecular basis for this change in product specificity. Our results suggest that, in contrast to published hypotheses, in planta production of secoxy-iridoids is secologanin-independent. Notably, sequence data of cultivated and wild olives point to a relation between domestication and OeOMES expression. Thus, the discovery of this key biosynthetic gene suggests a link between domestication and secondary metabolism, and could potentially be used as a genetic marker to guide next-generation breeding programs.

New Strategies in the Efficient Total Syntheses of Polycyclic Natural Products.

Polycyclic natural products are an inexhaustible source of medicinal agents, and their complex molecular architecture renders challenging synthetic targets where innovative and effective approaches for their rapid construction are urgently required. The total synthesis of polycyclic natural products has witnessed exponential progression along with the emergence of new synthetic strategies and concepts, such as sequential C-H functionalizations, radical-based transformations, and functional group pairing strategies. Our group exerts continued interest in the construction of bioactive and structurally complex natural products as well as evaluation of the mode of action of these molecules. In this Account, we will showcase how these new synthetic strategies are employed and guide our total synthesis endeavors.During the last two decades, a series of remarkable advances in C-H functionalization have led to the emergence of many new approaches to directly functionalize C-H bonds into useful functional groups. These selective transformations have provided a great opportunity for the step- and atom-economical construction of key fragments in complex molecule synthesis. We recently furnished the total syntheses for polycyclic natural products: incarviatone A, chrysomycin A, polycarcin V, and gilvocarcin V by employing a multiple C-H bond functionalization strategy. The polysubstituted benzene or naphthalene skeleton was constructed through sequential and site-selective C-H functionalizations from readily available simple starting materials, which reduced the number of steps and streamlined synthesis.Recently, we have also completed the total syntheses for a number of skeletally diverse tetracyclic Isodon diterpenoids inspired by their biogenesis and radical-based retrosynthetic disconnections. Radical transformations are strategically and tactically utilized in our syntheses, and radical-based reactions, including organo-SOMO catalysis, Birch reduction, regioselective 1,6-dienyne reductive cyclization, visible-light-mediated Schenck ene reaction, and photoradical-mediated late-stage skeletal rearrangement, play significant roles in our synthetic endeavors. Protecting-group-free and scalable syntheses are also built into our work to achieve the "ideal" synthesis. Furthermore, our synthetic work reveals that late-stage skeletal rearrangement through a photo radical process is possible in a biological setting in complement with nature's carbocation chemistry in complex natural product biosynthesis.Lycopodium alkaloids are a large family of structurally unique polycyclic natural products with impressive biological activities. Owing to their fascinating polycyclic architectures and diverse biological activities, these alkaloids have continued to serve as targets as well as inspirations for the synthetic community for decades. To access these bioactive natural products or natural product-like molecules for biological exploration and drug discovery, we applied a novel functional group pairing strategy to furnish the total syntheses for several Lycopodium alkaloids and obtained numerous skeletally diverse compounds with structural complexity comparable to natural products.

Enantioselective Total Synthesis of Berkeleyone†A and Preaustinoids.

Herein we report the first enantioselective total synthesis of 3,5-dimethylorsellinic acid-derived meroterpenoids (-)-berkeleyone A and its five congeners ((-)-preaustinoids A, A1, B, B1, and B2) in 12-15 steps, starting from commercially available 2,4,6-trihydroxybenzoic acid hydrate. Based upon the recognition of latent symmetry within D-ring, our convergent synthesis features two critical reactions: 1) a symmetry-breaking, diastereoselective dearomative alkylation to assemble the entire carbon core, and 2) a Sc(OTf)3 -mediated sequential Krapcho dealkoxycarbonylation/carbonyl α-tert-alkylation to forge the intricate bicyclo[3.3.1]nonane framework. We also conducted our preliminary biomimetic investigations and uncovered a series of rearrangements (α-ketol, α-hydroxyl-ß-diketone, etc.) responsible for the biomimetic diversification of (-)-berkeleyone A into its five preaustinoid congeners.

Protecting-Group-Free Syntheses of ent-Kaurane Diterpenoids: [3+2+1] Cycloaddition/Cycloalkenylation Approach.

The Yu's Rh-catalyzed [3+2+1] cycloaddition followed by a Pd-mediated 5-endo cycloalkenylation is shown to be a general and powerful approach for efficient construction of the tetracyclic core structure of ent-kaurane diterpenoids. The utility of this strategy was further demonstrated by concise and protecting-group-free total syntheses of ent-1α-hydroxykauran-12-one, 12-oxo-9,11-dehydrokaurene, and 12α-hydroxy-9,11-dehydrokaurene.

Total synthesis of (±)-antroquinonol.

We report the total synthesis of (±)-antroquinonol based on a concise and efficient route. Key features include two different strategies to install the required three contiguous stereogenic centers, which not only furnish the desired natural product but also provide natural product congeners for further biological studies.

Enantioselective Total Synthesis of (+)-Jungermatrobrunin†A.

A concise and enantioselective total synthesis of (+)-jungermatrobrunin A (1), which features a unique bicyclo[3.2.1]octene ring skeleton with an unprecedented peroxide bridge, was accomplished in 13 steps by making use of a late-stage visible-light-mediated Schenck ene reaction of (-)-1α,6α-diacetoxyjungermannenone C (2). Along the way, a UV-light-induced bicyclo[3.2.1]octene ring rearrangement afforded (+)-12-hydroxy-1α,6α-diacetoxy-ent-kaura-9(11),16-dien-15-one (4). These divergent photo-induced skeletal rearrangements support a possible biogenetic relationship between (+)-1, (-)-2, and (+)-4.

Total Synthesis and Structural Reassignment of Aspergillomarasmine†A.

The increase and spread of Gram-negative bacteria that resistant are to almost all currently available ß-lactam antibiotics is a major global health problem. The primary cause for drug resistance is the acquisition of metallo-ß-lactamases such as metallo-ß-lactamase-1 (NDM-1). The fungal natural product aspergillomarasmine A (AMA), a fungal natural product, is an inhibitor of NDM-1 and has shown promising in vivo therapeutic potential in a mouse model infected with NDM-1-expressing Gram-negative bacteria. The first total synthesis and stereochemical configuration reassignment of aspergillomarasmine A is reported. The synthesis highlights a flexible route and an effective strategy to achieve the required oxidation state at a late stage. This modular route is amenable to the efficient preparation of analogues for the development of metallo-ß-lactamase inhibitors to potentiate ß-lactam antibiotics.

Enantioselective Total Synthesis of (-)-Incarviatone A.

We report herein the first total synthesis of (-)-incarviatone A (1) in 14 steps starting from commercially available inexpensive phenylacetic acid (9). Our early stage synthesis relies on the scalable and sequential C-H functionalization to rapidly assemble the indanyl dialdehyde framework. Further biomimetic cascade strategy allows us to obtain the natural product in a one-pot operation. We also conduct detailed mechanistic studies and disclose all the possible intermediates and isomers formed during the biomimetic cascade process.

Total syntheses of (-)-huperzine Q and (+)-lycopladines B and C.

Utilizing a late-stage enamine bromofunctionalization strategy, the twelve-step total synthesis of (-)-huperzine Q was accomplished. Furthermore, the first total syntheses of (+)-lycopladines B and C are described. An unprecedented X-ray crystal structure of an unusual epoxyamine intermediate is also reported, and the synthetic application of this intermediate in natural product synthesis is demonstrated.

Collective synthesis of Lycopodium alkaloids and tautomer locking strategy for the total synthesis of (-)-lycojapodine A.

The collective total synthesis of Lycopodium alkaloids (+)-fawcettimine (1), (+)-fawcettidine (2), (+)-alopecuridine (4), (-)-lycojapodine A (6), and (-)-8-deoxyserratinine (7) has been accomplished from a common precursor (15) based on a highly concise route inspired by the proposed biosynthesis of the fawcettimine- and serratinine-type alkaloids. An intramolecular C-alkylation enabled efficient installation of the challenging spiro quaternary carbon center and the aza-cyclononane ring. The preparation of the tricyclic skeleton as well as the establishment of the correct relative stereochemistry of the oxa-quaternary center were achieved by hydroxyl-directed SmI(2)-mediated pinacol couplings. An unprecedented tandem transannular N-alkylation and removal of a Boc group was discovered to realize a biosynthesis-inspired process to furnish the desired tetracyclic skeleton. Of particular note is the unique and crucial tautomer locking strategy employed to complete the enantioselective total synthesis of (-)-lycojapodine A (6). The central step in this synthesis is the late-stage hypervalent iodine oxidant (IBX or Dess-Martin periodinane)/TFA-mediated tandem process, which constructed the previously unknown carbinolamine lactone motif and enabled a biomimetic transformation to generate (-)-lycojapodine A (6) in a single operation.

Engineering the Biosynthesis of Late-Stage Vinblastine Precursors Precondylocarpine Acetate, Catharanthine, Tabersonine in Nicotiana benthamiana.

Vinblastine is a chemotherapy agent produced by the plant Catharanthus roseus in small quantities. Currently, vinblastine is sourced by isolation or semisynthesis. Nicotiana benthamiana is a plant heterologous host that can be used for reconstitution of biosynthetic pathways as an alternative natural product sourcing strategy. Recently, the biosynthesis of the late-stage vinblastine precursors precondylocarpine acetate, catharanthine, and tabersonine have been fully elucidated. However, the large number of enzymes involved in the pathway and the unstable nature of intermediates make the reconstitution of late-stage vinblastine precursor biosynthesis challenging. We used the N. benthamiana chassis and a state-of-art modular vector assembly to optimize the six biosynthetic steps leading to production of precondylocarpine acetate from the central intermediate strictosidine (∼2.7 mg per 1 g frozen tissue). After selecting the optimal regulatory element combination, we constructed four transcriptional unit assemblies and tested their efficiency. Finally, we successfully reconstituted the biosynthetic steps leading to production of catharanthine and tabersonine.