Copper-catalysed dehydrogenation or lactonization of C(sp3)-H bonds.

Cytochrome P450 enzymes are known to catalyse bimodal oxidation of aliphatic acids via radical intermediates, which partition between pathways of hydroxylation and desaturation1,2. Developing analogous catalytic systems for remote C-H functionalization remains a significant challenge3-5. Here, we report the development of Cu(I)-catalysed bimodal dehydrogenation/lactonization reactions of synthetically common N-methoxyamides through radical abstractions of the γ-aliphatic C-H bonds. The feasibility of switching from dehydrogenation to lactonization is also demonstrated by altering reaction conditions. The use of a readily available amide as both radical precursor and internal oxidant allows for the development of redox-neutral C-H functionalization reactions with methanol as the sole side product. These C-H functionalization reactions using a Cu(I) catalyst with loading as low as 0.5 mol.% is applied to the diversification of a wide range of aliphatic acids including drug molecules and natural products. The exceptional compatibility of this catalytic system with a wide range of oxidatively sensitive functionality demonstrates the unique advantage of using a simple amide substrate as a mild internal oxidant.

Enzyme inhibitor discovery by activity-based protein profiling.

Eukaryotic and prokaryotic organisms possess huge numbers of uncharacterized enzymes. Selective inhibitors offer powerful probes for assigning functions to enzymes in native biological systems. Here, we discuss how the chemical proteomic platform activity-based protein profiling (ABPP) can be implemented to discover selective and in vivo-active inhibitors for enzymes. We further describe how these inhibitors have been used to delineate the biochemical and cellular functions of enzymes, leading to the discovery of metabolic and signaling pathways that make important contributions to human physiology and disease. These studies demonstrate the value of selective chemical probes as drivers of biological inquiry.

Insights into stereoselective ring formation in canonical strigolactone: Identification of a dirigent domain-containing enzyme catalyzing orobanchol synthesis.

Strigolactones (SLs) are plant apocarotenoids with diverse roles and structures. Canonical SLs, widespread and characterized by structural variations in their tricyclic lactone (ABC-ring), are classified into two types based on C-ring configurations. The steric C-ring configuration emerges during the BC-ring closure, downstream of the biosynthetic intermediate, carlactonoic acid (CLA). Most plants produce either type of canonical SLs stereoselectively, e.g., tomato (Solanum lycopersicum) yields orobanchol with an α-oriented C-ring. The mechanisms driving SL structural diversification are partially understood, with limited insight into functional implications. Furthermore, the exact molecular mechanism for the stereoselective BC-ring closure reaction is yet to be known. We identified an enzyme, the stereoselective BC-ring-forming factor (SRF), from the dirigent protein (DIR) family, specifically the DIR-f subfamily, whose biochemical function had not been characterized, making it a key enzyme in stereoselective canonical SL biosynthesis with the α-oriented C-ring. We first confirm the precise catalytic function of the tomato cytochrome P450 SlCYP722C, previously shown to be involved in orobanchol biosynthesis [T. Wakabayashi et al., Sci. Adv. 5, eaax9067 (2019)], to convert CLA to 18-oxocarlactonoic acid. We then show that SRF catalyzes the stereoselective BC-ring closure reaction of 18-oxocarlactonoic acid, forming orobanchol. Our methodology combines experimental and computational techniques, including SRF structure prediction and conducting molecular dynamics simulations, suggesting a catalytic mechanism based on the conrotatory 4π-electrocyclic reaction for the stereoselective BC-ring formation in orobanchol. This study sheds light on the molecular basis of how plants produce SLs with specific stereochemistry in a controlled manner.

Lactonization as a general route to ß-C(sp3)-H functionalization.

Functionalization of the ß-C-H bonds of aliphatic acids is emerging as a valuable synthetic disconnection that complements a wide range of conjugate addition reactions1-5. Despite efforts for ß-C-H functionalization in carbon-carbon and carbon-heteroatom bond-forming reactions, these have numerous crucial limitations, especially for industrial-scale applications, including lack of mono-selectivity, use of expensive oxidants and limited scope6-13. Notably, the majority of these reactions are incompatible with free aliphatic acids without exogenous directing groups. Considering the challenge of developing C-H activation reactions, it is not surprising that achieving different transformations requires independent catalyst design and directing group optimizations in each case. Here we report a Pd-catalysed ß-C(sp3)-H lactonization of aliphatic acids enabled by a mono-N-protected ß-amino acid ligand. The highly strained and reactive ß-lactone products are versatile linchpins for the mono-selective installation of diverse alkyl, alkenyl, aryl, alkynyl, fluoro, hydroxyl and amino groups at the ß position of the parent acid, thus providing a route to many carboxylic acids. The use of inexpensive tert-butyl hydrogen peroxide as the oxidant to promote the desired selective reductive elimination from the Pd(IV) centre, as well as the ease of product purification without column chromatography, render this reaction amenable to tonne-scale manufacturing.

Computational planning of the synthesis of complex natural products.

Training algorithms to computationally plan multistep organic syntheses has been a challenge for more than 50 years1-7. However, the field has progressed greatly since the development of early programs such as LHASA1,7, for which reaction choices at each step were made by human operators. Multiple software platforms6,8-14 are now capable of completely autonomous planning. But these programs 'think' only one step at a time and have so far been limited to relatively simple targets, the syntheses of which could arguably be designed by human chemists within minutes, without the help of a computer. Furthermore, no algorithm has yet been able to design plausible routes to complex natural products, for which much more far-sighted, multistep planning is necessary15,16 and closely related literature precedents cannot be relied on. Here we demonstrate that such computational synthesis planning is possible, provided that the program's knowledge of organic chemistry and data-based artificial intelligence routines are augmented with causal relationships17,18, allowing it to 'strategize' over multiple synthetic steps. Using a Turing-like test administered to synthesis experts, we show that the routes designed by such a program are largely indistinguishable from those designed by humans. We also successfully validated three computer-designed syntheses of natural products in the laboratory. Taken together, these results indicate that expert-level automated synthetic planning is feasible, pending continued improvements to the reaction knowledge base and further code optimization.

Enzyme-catalysed [6+4] cycloadditions in the biosynthesis of natural products.

Pericyclic reactions are powerful transformations for the construction of carbon-carbon and carbon-heteroatom bonds in organic synthesis. Their role in biosynthesis is increasingly apparent, and mechanisms by which pericyclases can catalyse reactions are of major interest1. [4+2] cycloadditions (Diels-Alder reactions) have been widely used in organic synthesis2 for the formation of six-membered rings and are now well-established in biosynthesis3-6. [6+4] and other 'higher-order' cycloadditions were predicted7 in 1965, and are now increasingly common in the laboratory despite challenges arising from the generation of a highly strained ten-membered ring system8,9. However, although enzyme-catalysed [6+4] cycloadditions have been proposed10-12, they have not been proven to occur. Here we demonstrate a group of enzymes that catalyse a pericyclic [6+4] cycloaddition, which is a crucial step in the biosynthesis of streptoseomycin-type natural products. This type of pericyclase catalyses [6+4] and [4+2] cycloadditions through a single ambimodal transition state, which is consistent with previous proposals11,12. The [6+4] product is transformed to a less stable [4+2] adduct via a facile Cope rearrangement, and the [4+2] adduct is converted into the natural product enzymatically. Crystal structures of three pericyclases, computational simulations of potential energies and molecular dynamics, and site-directed mutagenesis establish the mechanism of this transformation. This work shows how enzymes are able to catalyse concerted pericyclic reactions involving ambimodal transition states.

A broad specificity ß-propeller enzyme from Rhodopseudomonas palustris that hydrolyzes many lactones including γ-valerolactone.

Lactones are prevalent in biological and industrial settings, yet there is a lack of information regarding enzymes used to metabolize these compounds. One compound, γ-valerolactone (GVL), is used as a solvent to dissolve plant cell walls into sugars and aromatic molecules for subsequent microbial conversion to fuels and chemicals. Despite the promise of GVL as a renewable solvent for biomass deconstruction, residual GVL can be toxic to microbial fermentation. Here, we identified a Ca2+-dependent enzyme from Rhodopseudomonas palustris (Rpa3624) and showed that it can hydrolyze aliphatic and aromatic lactones and esters, including GVL. Maximum-likelihood phylogenetic analysis of other related lactonases with experimentally determined substrate preferences shows that Rpa3624 separates by sequence motifs into a subclade with preference for hydrophobic substrates. Additionally, we solved crystal structures of this ß-propeller enzyme separately with either phosphate, an inhibitor, or a mixture of GVL and products to define an active site where calcium-bound water and calcium-bound aspartic and glutamic acid residues make close contact with substrate and product. Our kinetic characterization of WT and mutant enzymes combined with structural insights inform a reaction mechanism that centers around activation of a calcium-bound water molecule promoted by general base catalysis and close contacts with substrate and a potential intermediate. Similarity of Rpa3624 with other ß-propeller lactonases suggests this mechanism may be relevant for other members of this emerging class of versatile catalysts.

Enzymatic Assembly of Diverse Lactone Structures: An Intramolecular C-H Functionalization Strategy.

Lactones are cyclic esters with extensive applications in materials science, medicinal chemistry, and the food and perfume industries. Nature's strategy for the synthesis of many lactones found in natural products always relies on a single type of retrosynthetic strategy, a C-O bond disconnection. Here, we describe a set of laboratory-engineered enzymes that use a new-to-nature C-C bond-forming strategy to assemble diverse lactone structures. These engineered "carbene transferases" catalyze intramolecular carbene insertions into benzylic or allylic C-H bonds, which allow for the synthesis of lactones with different ring sizes and ring scaffolds from simple starting materials. Starting from a serine-ligated cytochrome P450 variant previously engineered for other carbene-transfer activities, directed evolution generated a variant P411-LAS-5247, which exhibits a high activity for constructing a five-membered ε-lactone, lactam, and cyclic ketone products (up to 5600 total turnovers (TTN) and >99% enantiomeric excess (ee)). Further engineering led to variants P411-LAS-5249 and P411-LAS-5264, which deliver six-membered δ-lactones and seven-membered ε-lactones, respectively, overcoming the thermodynamically unfavorable ring strain associated with these products compared to the γ-lactones. This new carbene-transfer activity was further extended to the synthesis of complex lactone scaffolds based on fused, bridged, and spiro rings. The enzymatic platform developed here complements natural biosynthetic strategies for lactone assembly and expands the structural diversity of lactones accessible through C-H functionalization.

Natural ten-membered lactones: sources, structural diversity, biological activity, and intriguing future.

Covering: 2012 to 2022Ten-membered lactones (TMLs) are an interesting and diverse group of natural polyketides that are abundant in fungi and, to a lesser extent, in bacteria, marine organisms, and insects. TMLs are known for their ability to exhibit a wide spectrum of biological activity, including phytotoxic, cytotoxic, antifungal, antibacterial, and others. However, the random discovery of these compounds by scientific groups with various interests worldwide has resulted in patchy information about their distribution among different organisms and their biological activity. Therefore, despite more than 60 years of research history, there is still no common understanding of the natural sources of TMLs, their structural type classification, and most characteristic biological activities. The controversial nomenclature, incorrect or erroneous structure elucidation, poor identification of producing organisms, and scattered information on the biological activity of compounds - all these factors have led to the problems with dereplication and the directed search for TMLs. This review consists of two parts: the first part (Section 2) covers 104 natural TMLs, published between 2012 and 2022 (after the publishing of the previous review), and the second part (Section 3) summarizes information about 214 TMLs described during 1964-2022 and as a result highlights the main problems and trends in the study of these intriguing natural products.

Chemistry of Strigolactones, Key Players in Plant Communication.

Today, the use of artificial pesticides is questionable and the adaptation to global warming is a necessity. The promotion of favorable natural interactions in the rhizosphere offers interesting perspectives for changing the type of agriculture. Strigolactones (SLs), the latest class of phytohormones to be discovered, are also chemical mediators in the rhizosphere. We present in this review the diversity of natural SLs, their analogs, mimics, and probes essential for the biological studies of this class of compounds. Their biosynthesis and access by organic synthesis are highlighted especially concerning noncanonical SLs, the more recently discovered natural SLs. Organic synthesis of analogs, stable isotope-labeled standards, mimics, and probes are also reviewed here. In the last part, the knowledge about the SL perception is described as well as the different inhibitors of SL receptors that have been developed.

Cracking the enigma: understanding strigolactone signalling in the rhizosphere.

The rhizosphere is a complex physical and chemical interface between plants and their underground environment, both biotic and abiotic. Plants exude a large number of chemicals into the rhizosphere in order to manipulate these biotic and abiotic components. Among such chemicals are strigolactones, ancient signalling molecules that in flowering plants act as both internal hormones and external rhizosphere signals. Plants exude strigolactones to communicate with their preferred symbiotic partners and neighbouring plants, but at least some classes of parasitic organisms are able to 'crack' these private messages and eavesdrop on the signals. In this review, we examine the intentional consequences of strigolactone exudation, and also the unintentional consequences caused by eavesdroppers. We examine the molecular mechanisms by which strigolactones act within the rhizosphere, and attempt to understand the enigma of the strigolactone molecular diversity synthesized and exuded into the rhizosphere by plants. We conclude by looking at the prospects of using improved understanding of strigolactones in agricultural contexts.

Enabling High Activation of Glucose-6-Phosphate Dehydrogenase Activity Through Liquid Condensate Formation and Compression.

Droplet formation via liquid-liquid phase separation is thought to be involved in the regulation of various biological processes, including enzymatic reactions. We investigated a glycolytic enzymatic reaction, the conversion of glucose-6-phosphate to 6-phospho-D-glucono-1,5-lactone with concomitant reduction of NADP+ to NADPH both in the absence and presence of dynamically controlled liquid droplet formation. Here, the nucleotide serves as substrate as well as the scaffold required for the formation of liquid droplets. To further expand the process parameter space, temperature and pressure dependent measurements were performed. Incorporation of the reactants in the liquid droplet phase led to a boost in enzymatic activity, which was most pronounced at medium-high pressures. The crowded environment of the droplet phase induced a marked increase of the affinity of the enzyme and substrate. An increase in turnover number in the droplet phase at high pressure contributed to a further strong increase in catalytic efficiency. Enzyme systems that are dynamically coupled to liquid condensate formation may be the key to deciphering many biochemical reactions. Expanding the process parameter space by adjusting temperature and pressure conditions can be a means to further increase the efficiency of industrial enzyme utilization and help uncover regulatory mechanisms adopted by extremophiles.

From the Total Synthesis of Semi-Viriditoxin, Semi-Viriditoxic Acid and Dimeric Naphthopyranones to their Biological Activities in Burkitt B Cell Lymphoma.

Dimeric naphthopyranones are known to be biologically active, however, for the corresponding monomeric naphthopyranones this information is still elusive. Here the first enantioselective total synthesis of semi-viriditoxic acid as well as the synthesis of semi-viriditoxin and derivatives is reported. The key intermediate in the synthesis of naphthopyranones is an α,ß-unsaturated δ-lactone, which we synthesized in two different ways (Ghosez-cyclization and Grubbs ring-closing metathesis), while the domino-Michael-Dieckmann reaction of the α,ß-unsaturated δ-lactone with an orsellinic acid derivative is the key reaction. A structure-activity relationship study was performed measuring the cytotoxicity in Burkitt B lymphoma cells (Ramos). The dimeric structure was found to be crucial for biological activity: Only the dimeric naphthopyranones showed cytotoxic and apoptotic activity, whereas the monomers did not display any activity at all.

Enzymatic assembly of the salinosporamide γ-lactam-ß-lactone anticancer warhead.

The marine microbial natural product salinosporamide A (marizomib) is a potent proteasome inhibitor currently in clinical trials for the treatment of brain cancer. Salinosporamide A is characterized by a complex and densely functionalized γ-lactam-ß-lactone bicyclic warhead, the assembly of which has long remained a biosynthetic mystery. Here, we report an enzymatic route to the salinosporamide core catalyzed by a standalone ketosynthase (KS), SalC. Chemoenzymatic synthesis of carrier protein-tethered substrates, as well as intact proteomics, allowed us to probe the reactivity of SalC and understand its role as an intramolecular aldolase/ß-lactone synthase with roles in both transacylation and bond-forming reactions. Additionally, we present the 2.85-Å SalC crystal structure that, combined with site-directed mutagenesis, allowed us to propose a bicyclization reaction mechanism. This work challenges our current understanding of the role of KS enzymes and establishes a basis for future efforts toward streamlined production of a clinically relevant chemotherapeutic.

Engineering Biodegradable Polyurethanes with Precisely Controlled Hierarchical Structures to Access Shape Memory Effect and Enhanced Bioactivities.

Biodegradable polymers with shape memory effects (SMEs) offer promising solutions for short-term medical interventions, facilitating minimally invasive procedures and subsequent degradation without requiring secondary surgeries. However, achieving a good balance among desirable SMEs, mechanical performance, degradation rate, and bioactivities remains a significant challenge. To address this issue, we established a strategy to develop a versatile biodegradable polyurethane (PPDO-PLC) with tunable hierarchical structures via precise chain segment control. Initial copolymerization of l-lactide and ε-caprolactone sets a tunable Tg close to body temperature, followed by block copolymerization with poly(p-dioxanone) to form a hard domain. This yields a uniform microphase-separation morphology, ensuring robust SME and facilitating the development of roughly porous surface structures in alkaline environments. Cell experiments indicate that these rough surfaces significantly enhance cellular activities, such as adhesion, proliferation, and osteogenic differentiation. Our approach provides a methodology for balancing biodegradability, SMEs, three-dimensional (3D) printability, and bioactivity in materials through hierarchical structure regulation.

Absolute Configuration of 4-Chloroisoleucine in Cyclolithistide A: An Antifungal Peptide Lactone Discovered in Marine Lithistid Sponges.

Cyclolithistide A is a peptide lactone isolated from marine lithistid sponges. Its entire structure, including absolute configurations, has been reported except the relative and absolute configurations of its characteristic residue, 4-chloroisoleucine (4-CIle). We synthesized four isomers of 4-CIle from furfural-derived N-Boc imine and propionaldehyde. Analysis of the acid hydrolysate of cyclolithistide A and the synthetic samples of 4-CIle after derivatization with l- and d-FDAA permitted us to propose the absolute configuration of the 4-chloroisoleucine residue in cyclolithistide A as 2S,3R,4R.

Visible Light-Regulated Ring-Opening Polymerization of Lactones by Employing Indigo as a Photoacid Catalyst.

The development of visible light-regulated polymerizations for precision synthesis of polymers has drawn considerable attention in the past years. In this study, an ancient dye, indigo, is successfully identified as a new and efficient photoacid catalyst, which can readily promote the ring-opening polymerization of lactones under visible light irradiation in a well-controlled manner, affording the desired polyester products with predictable molecular weights and narrow dispersity. The enhanced acidity of indigos by excitation is crucial to the H-bonding activation of the lactone monomers. Chain extension and block copolymer synthesis are also demonstrated with this method.

Structure-Activity Relationships of Natural C-9-Methyl-Substituted 10-Membered Lactones and Their Semisynthetic Derivatives.

Fungal 10-membered lactones (TMLs), such as stagonolide A, herbarumin I, pinolidoxin, and putaminoxin, are promising candidates for the development of nature-derived herbicides. The aim of this study was to analyze the structure-activity relationships (SAR) of C-9-methyl-substituted TMLs with a multitarget bioassay approach to reveal compounds with useful (phytotoxic, entomotoxic, antimicrobial) or undesirable (cytotoxic) bioactivities. A new TML, stagonolide L (1), along with five known compounds (stagonolides D (2) and E (3), curvulides A (4) and B1/B2 (5a,b), and pyrenolide C (6)), were purified from cultures of the phytopathogenic fungus Stagonospora cirsii, and five semisynthetic derivatives of 3 and 4 (7-11) were obtained. The absolute configuration of 4 was revised to 2Z, 4S, 5S, 6R, and 9R. The identity of 5a,b and stagonolide H is discussed. The phytotoxicity of compound 4, the entomotoxicity of 5a,b, and nonselective toxicity of compound 6 are demonstrated. The latter confirms the hypothesis that the α,ß-unsaturated carbonyl group is associated with the high general toxicity of TML, regardless of its position in the ring and other substituents. The epoxide in compound 4 is important for phytotoxicity. The revealed SAR patterns will be useful for further rational design of TML-based herbicides including curvulide A analogs with a 4,5-epoxy group.

Isolation of Paraphaeoketones: A Plausible Biosynthetic Explanation for Paraphaeolactones via a Benzilic Acid Rearrangement Rather than a Favorskii Rearrangement.

Paraphaeoketones A-C (1-3) were isolated from the culture broth of Paraphaeosphaeria sp. KT4192. Their structures and relative configurations were determined using spectroscopic analysis and verified through density functional theory (DFT)-based chemical shift calculations. The absolute configurations of these compounds were determined by comparing the experimental electronic circular dichroism (ECD) spectra with those based on DFT calculations. We also propose a plausible biosynthetic route to 1-3. While our prior studies on the isolation and structural elucidation of paraphaeolactones (e.g., 4) led us to suggest a Favorskii rearrangement for their biosynthesis, the isolation of 2 prompted the proposal of an alternative biosynthesis for 4, featuring a benzilic acid rearrangement of 2. Moreover, an in vitro conversion of 2 into 4 was achieved successfully, suggesting that a biosynthetic pathway for paraphaeolactones involving a benzilic acid rearrangement is more plausible than the previously presumed Favorskii rearrangement pathway. Arguments based on DFT calculations for these pathways are also described.

(R)-(+)-γ-Decalactone is Conserved in North America as a Pheromone Component of Osmoderma eremicola (Coleoptera: Scarabaeidae) and a Kairomone of Elater abruptus (Coleoptera: Elateridae).

The scarab genus Osmoderma (Coleoptera: Scarabaeidae) includes several large species called hermit beetles that develop within dead and decaying hardwood trees. Males of at least three Palearctic species produce the aggregation-sex pheromone (R)-(+)-γ-decalactone, including the endangered O. eremita (Scopoli). However, hermit beetles have received less attention in the western hemisphere, resulting in a large gap in our knowledge of the chemical ecology of Nearctic species. Here, we identify (R)-( +)-γ-decalactone as the primary component of the aggregation-sex pheromone of the North American species Osmoderma eremicola (Knoch). Field trials at sites in Wisconsin and Illinois revealed that both sexes were attracted to lures containing (R)-(+)-γ-decalactone or the racemate, but only males of O. eremicola produced the pheromone in laboratory bioassays, alongside an occasional trace of the chain-length analog γ-dodecalactone. Females of the congener O. scabra (Palisot de Beauvois) were also significantly attracted by γ-decalactone, suggesting further conservation of the pheromone, as were females of the click beetle Elater abruptus Say (Coleoptera: Elateridae), suggesting that this compound may have widespread kairomonal activity. Further research is needed to explore the behavioral roles of both lactones in mediating behavioral and ecological interactions among these beetle species.