Complex Natural Products Research Articles

De novo biosynthesis of the hops bioactive flavonoid xanthohumol in yeast

The flavonoid xanthohumol is an important flavor substance in the brewing industry that has a wide variety of bioactivities. However, its unstable structure results in its low content in beer. Microbial biosynthesis is considered a sustainable and economically viable alternative. Here, we harness the yeast Saccharomyces cerevisiae for the de novo biosynthesis of xanthohumol from glucose by balancing the three parallel biosynthetic pathways, prenyltransferase engineering, enhancing precursor supply, constructing enzyme fusion, and peroxisomal engineering. These strategies improve the production of the key xanthohumol precursor demethylxanthohumol (DMX) by 83-fold and achieve the de novo biosynthesis of xanthohumol in yeast. We also reveal that prenylation is the key limiting step in DMX biosynthesis and develop tailored metabolic regulation strategies to enhance the DMAPP availability and prenylation efficiency. Our work provides feasible approaches for systematically engineering yeast cell factories for the de novo biosynthesis of complex natural products.

Direct trifluoromethylselenolations of electron-rich (hetero)aromatic rings with N-trifluoromethylselenolating saccharin.

A novel, easily synthesizable, shelf-stable electrophilic trifluoromethylselenolating reagent, N-trifluoromethylselenosaccharin, has been developed. This reagent can be synthesized in good yield by a two-step one-pot reaction from BnSeCF3, SO2Cl2, and silver saccharin. N-Trifluoromethylselenosaccharin proves to be an efficient trifluoromethylselenolating reagent, enabling the direct trifluoromethylselenolation of various electron-rich aromatic and heteroaromatic rings under mild reaction conditions. It exhibits excellent chemoselectivity and excellent compatibility with various functional groups, making it suitable for late-stage trifluoromethylselenolation applications in complex natural product and drug synthesis.

Preparation, assay, and application of 4-fluorothreonine transaldolase from Streptomyces sp. MA37 for β-hydroxyl amino acid derivatives.

β-Hydroxy-α-amino acids (βHAAs) are an essential class of building blocks of therapeutically important compounds and complex natural products. They contain two chiral centers at Cα and Cβ positions, resulting in four possible diastereoisomers. Many innovative asymmetric syntheses have been developed to access structurally diverse βHAAs. The main challenge, however, is the control of the relative and absolute stereochemistry of the asymmetric carbons in a sustainable way. In this respect, there has been considerable attention focused on the chemoenzymatic synthesis of βHAAs via a one-step process. Nature has evolved different enzymatic routes to produce these valuable βHAAs. Among these naturally occurring transformations, L-threonine transaldolasespresent potential biocatalysts to generate βHAAs in situ. 4-Fluorothreonine transaldolase from Streptomyces sp. MA37 (FTaseMA) catalyzes the cross-over transaldolation reaction between L-Thr and fluoroacetaldehydeto give 4-fluorothreonineand acetaldehyde (Ad). It has been demonstrated that FTaseMA displays considerable substrate plasticity toward structurally diverse aldehyde acceptors, leading to the production of various βHAAs. In this chapter, we describe methods for the preparation of FTaseMA, and the chemoenzymatic synthesis of βHAAs from various aldehydes and L-Thr using FTaseMA.

Exploring the synthetic potential of epoxide ring opening reactions toward the synthesis of alkaloids and terpenoids: a review.

Epoxides are oxygen containing heterocycles which are significantly employed as crucial intermediates in various organic transformations. They are considered highly reactive three-membered heterocycles due to ring strain and they undergo epoxide ring opening reactions with diverse range of nucleophiles. Epoxide ring-opening reactions have gained prominence as flexible and effective means to obtain various functionalized molecules. These reactions have garnered substantial attention in organic synthesis, driven by the need to comprehend the synthesis of biologically and structurally important organic compounds. They have also found applications in the synthesis of complex natural products. In this review article, we have summarized the implementation of epoxide ring opening reactions in the synthesis of alkaloids and terpenoids based natural products reported within the last decade (2014-2023).

Photoinduced copper catalyzed nitrogen-to-alkyl radical relay Sonogashira-type coupling of o-alkylbenzamides with alkynes.

This report describes a copper-catalyzed, photoinduced N-to-alkyl radical relay Sonogashira-type reactions at benzylic sites in o-alkylbenzamides with alkynes. The process employs an N-to-alkyl radical mechanism, initiated through the copper-catalyzed reductive generation of nitrogen radicals. Radical translocation is facilitated by a 1,5-hydrogen atom transfer (1,5-HAT), leading to the formation of translocated carbon radicals. These radicals are then subjected to copper-catalyzed alkynylation. The methodology exhibits broad sub-strate scope and applicability to the synthesis of complex natural products.

Concise synthesis of 3-C-glycosyl isocoumarins and 2-glycosyl-4H-chromen-4-ones.

A new Ru-catalyzed C-H activation/cyclization reaction for the synthesis of 3-C-glycosyl isocoumarins and 2-glycosyl-4H-chromen-4-ones with carbonyl sulfoxonium ylide glycogen are reported. In this catalytic system, benzoic acid and its derivatives react with carbonyl sulfoxonium ylide glycogen to yield isocoumarin C-glycosides, while 2-hydroxybenzaldehyde substrates react to produce chromone C-glycosides. These reactions were characterized by mild reaction conditions, broad substrate scope, high functional-group compatibility, and high stereoselectivity to yield several high-value isocoumarins and chromone skeleton-containing C-glycosides. The methods were successfully implemented in the context of large-scale reactions and the late-stage modification of complex natural products.

Purification and characterization of a Rieske oxygenase and its NADH-regenerating partner proteins.

The Rieske non-heme iron oxygenases (Rieske oxygenases) comprise a class of metalloenzymes that are involved in the biosynthesis of complex natural products and the biodegradation of aromatic pollutants. Despite this desirable catalytic repertoire, industrial implementation of Rieske oxygenases has been hindered by the multicomponent nature of these enzymes and their requirement for expensive reducing equivalents in the form of a reduced nicotinamide adenine dinucleotide cosubstrate (NAD(P)H). Fortunately, however, some Rieske oxygenases co-occur with accessory proteins, that through a downstream reaction, recycle the needed NAD(P)H for catalysis. As these pathways and accessory proteins are attractive for bioremediation applications and enzyme engineering campaigns, herein, we describe methods for assembling Rieske oxygenase pathways in vitro. Further, using the TsaMBCD pathway as a model system, in this chapter, we provide enzymatic, spectroscopic, and crystallographic methods that can be adapted to explore both Rieske oxygenases and their co-occurring accessory proteins.

The Intermolecular Pauson‐Khand Reaction: Applications, Challenges, and Opportunities

AbstractThe rapid assembly of complex organic molecules from simple and structurally diverse building blocks is a prevalent challenge in organic synthesis. The Pauson‐Khand reaction (PKR) is a method of choice for the construction of five membered rings that has historically been popularized as a late‐stage intramolecular cyclization method. The intermolecular version of the PKR, on the other hand, constitutes a powerful approach for the rapid assembly of densely functionalized cyclopentanic cores at early stages of a synthetic sequence. Despite its potential, the intermolecular PKR is much less prevalent in the organic synthesis literature due to several historical limitations, most importantly a reduced scope with respect to the alkene component of the reaction. The last decade has witnessed important developments in the area including a) experimental and theoretical studies that provide a good mechanistic understanding of the reaction and its selectivity, b) methodological developments that have broadened the scope of potential alkene partners, and c) the development of catalytic enantioselective versions that provide useful levels of enantioselectivity. In parallel, remarkable synthetic applications of the intermolecular PKR have emerged, including (enantioselective) total syntheses of complex natural products (polycyclic terpenes, alkaloids, prostanes) as well as examples of industrial relevance. A fundamental limitation of the PKR that needs to be addressed in the future is the current lack of a ligand‐accelerated version of the reaction, which would be a promising advance towards developing more efficient and general catalytic (enantioselective) reactions.

Using Mechanochemistry to Activate Commodity Plastics as Initiators for Radical Chain Reactions of Small Organic Molecules.

Radical initiators such as azo compounds and organic peroxides have been widely used to facilitate numerous transformations of free radicals, which enable the efficient synthesis of structurally complex molecules, natural products, polymers, and functional materials. However, these high-energy reagents are potentially explosive and thus often require special precautions or delicate operating conditions. We postulated that a more convenient and safer alternative for radical chain initiation could be developed by mechanical activation of thermodynamically stable covalent bonds. Here, we show that commodity plastics such as polyethylene and poly(vinyl acetate) are capable of acting as efficient initiators for radical chain reactions under solvent-free mechanochemical conditions. In this approach, polymeric mechanoradicals, which are generated by homolytic cleavage of the polymer chains in response to the applied mechanical energy provided by ball milling, react with tris(trimethylsilyl)silane to initiate radical chain dehalogenation of organic halides. Preliminary calculations support our proposed force-induced radical chain mechanism.

Metal Ion-Induced Large Fragment Deactivation: A Different Strategy for Site-Selectivity in a Complex Molecule.

Complex natural product functionalizations generally involve the use of highly engineered reagents, catalysts, or enzymes to react exclusively at a desired site through lowering of a select transition state energy. In this communication, we report a new, complementary strategy in which all transition states representing undesirable sites in a complex ionophore substrate are simultaneously energetically increased through the chelation of a metal ion to the large fragment we wish to neutralize. In the case of an electrophilic, radical based fluorination reaction, charge repulsion (electric field effects), induced steric effects, and electron withdrawal provide the necessary deactivation and proof of principle to afford a highly desirable natural product derivative. We envisage that many other electrophilic or charge based synthetic methods may be amenable to this approach as well.

Metal Ion‐Induced Large Fragment Deactivation: A Different Strategy for Site‐Selectivity in a Complex Molecule

AbstractComplex natural product functionalizations generally involve the use of highly engineered reagents, catalysts, or enzymes to react exclusively at a desired site through lowering of a select transition state energy. In this communication, we report a new, complementary strategy in which all transition states representing undesirable sites in a complex ionophore substrate are simultaneously energetically increased through the chelation of a metal ion to the large fragment we wish to neutralize. In the case of an electrophilic, radical based fluorination reaction, charge repulsion (electric field effects), induced steric effects, and electron withdrawal provide the necessary deactivation and proof of principle to afford a highly desirable natural product derivative. We envisage that many other electrophilic or charge based synthetic methods may be amenable to this approach as well.

A novel off-line multi-dimensional high-speed countercurrent chromatography strategy for preparative separation of bioactive neolignan isomers from Piper betle. L

Separation and purification of naturally occurring isomers from herbs are still challenging. High-speed counter-current chromatography (HSCCC) has been applied to isolate natural products. In this study, an off-line multi-dimensional high-speed counter-current chromatography (multi-D HSCCC) strategy was developed utilizing the in situ concentration technique with online storage recycling elution to rapidly separate bioactive isomeric neolignans from chloroform-partitioned samples of the plant Piper betle L. In the procedure, the crude sample (105 mg) was implemented using the online storage recycling technique in a two-phase solvent system composed of petroleum ether-ethyl acetate–methanol-water (7: 5: 12: 3), which first simply afforded a neolignane kadsurenone (1, 5.3 mg) and its epimer (-)-denudatin B (2, 6.4 mg). Then, the remains fr a was subjected to the second-dimensional HSCCC elution using the in situ concentration technique with online storage recycling technique in another solvent system of petroleum ether-ethyl acetate–methanol–water (5: 5: 11, 15). As a result, kadsurenin I (3, 0.6 mg) and its regioisomer pibeneolignan C (4, 5.0 mg), together with the fractional remaining fr b and fr c, were obtained. Thirdly, the fr c was reloaded to allow the HSCCC for recycling elution with the former solvent system employing the in situ concentration strategy and yielded a pair of epimers, (7R,8S,1′S)-1′-allyl-5-methoxy-8-methyl-7-piperonyl-7,8,3,6-tetrahydro-2-oxobenzofuran (5, 10.2 mg), and 3-epi-(-)-burchullin (6, 2.6 mg). Finally, the three pairs of less amount and the structurally similar isomers 1–6 were isolated from the crude fraction of P. betle with a high HPLC purity of over 95.0 % for compound 2, 4-6 and 92.5 % for compound 1, 91.0 % for 3, while the purity of 1 and 3 in 1H NMR were 89.9 % and 91.1 %, respectively. The whole isolation process was quick and efficient. Compounds 1, 2, 4 and 5 showed significantly synergistic activities combining several antibiotics against five drug-resistant Staphylococcus aureus with FICIs from 0.156 to 0.375. This novel off-line multi-dimensional HSCCC strategy could be broadened to application for the rapid separation of complex natural products.

Chimgin from Ferula haussknechtii as AChE inhibitor and confirmation of the absolute configuration

Alzheimer’s disease (AD) is the most common cause of dementia worldwide and is classified as a neurodegenerative disorder. From a drug design perspective, natural products (NPs) are more drug-like and are highly compatible with biological systems compared to most synthetic libraries. NPs provide a more efficient and cost-effective approach to new drug discovery. However, the complexity of NPs makes their identification a challenging task. Chimgin, a bicyclic monoterpene with three chiral centers, exhibits a wide range of biological activity. Despite this, the exact structure of chimgin has remained unclear until now. In this study, we quantified the amount of chimgin in Ferula haussknechtii using analytical Reversed-phase high-pressure liquid chromatography equipped with photodiode array detector (RP-HPLC-PDA). Furthermore, we determined the absolute configuration of chimgin through electronic circular dichroism (ECD) spectroscopy and time-dependent density functional theory (TDDFT) calculations. Finally, we evaluated its inhibitory effect on AChE through in vitro and in silico studies. The extraction process yielded an output of 2.82 ± 0.10% with an exact amount of 0.62 ± 0.04 mg of chimgin per 100 g of plant. Based on the results of ECD and TDDFT calculation, the absolute configuration of chimgin was determined to be 1S, 2S, 4S. Chimgin exhibited an inhibitory effect on AChE with an IC50 of 37.43 µM and its mechanism of action was found to be competitive. Highlights Chimgin was isolated from the roots of Ferula haussknechtii. The amount of chimgin in the plant was determined by RP-HPLC-PDA. Its absolute configuration of chimgin was determined using ECD. In vitro acetylcholinesterase activity of the chimgin was evaluated. The docking and molecular dynamic simulation of chimgin was done. Communicated by Ramaswamy H. Sarma

Efficient screening of pancreatic lipase inhibitors from Rheum palmatum by affinity ultrafiltration-high-performance liquid chromatography combined with high-resolution inhibition profiling.

Pancreatic lipase is one of the most important key targets in the treatment of obesity. Inhibition of pancreatic lipase can effectively reduce lipid absorption and treat obesity and other related metabolic disorders. The goal of this study is the efficient screening of pancreatic lipase inhibitors in the root and rhizome of Rheum palmatum using affinity ultrafiltration-high-performance liquid chromatography (AUF-HPLC) combined with high-resolution inhibition profiling (HRIP). Potential pancreatic lipase ligands and pancreatic lipase inhibitors in ethyl acetate fraction of R. palmatum were screened using AUF-HPLC and HRIP, respectively. All screened compounds were identified by HPLC- quadrupole time-of-flight (Q-TOF)/MS. Eight compounds were screened out by both AUF-HPLC and HRIP, and six compounds were screened out by either AUF-HPLC or HRIP alone. The pancreatic lipase inhibitory activities of all screened compounds were verified by enzyme inhibition assay and molecular docking. Five new potent pancreatic lipase inhibitors were discovered, namely procyanidin B5 3,3'-di-O-gallate (IC50 = 0.06 ± 0.01 μM), 1,6-di-O-galloyl-2-O-cinnamoyl-β-D-glucoside (IC50 = 12.83 ± 0.67 μM), 1-O-(1,3,5-trihydroxy)phenyl-2-O-galloyl-6-O-cinnamoyl-β-D-glucoside (IC50 = 17.84 ± 1.33 μM), 1,2-di-O-galloyl-6-O-cinnamoyl-β-D-glucoside (IC50 = 18.39 ± 1.52 μM), and 4-(4'-hydroxyphenyl)-2-butanone-4'-O-β-D-(2"-O-galloyl-6"-O-cinnamoyl)-glucoside (IC50 = 2.91 ± 0.40 μM). It was found that procyanidin B5 3,3'-di-O-gallate showed higher pancreatic lipase inhibitory activity than the positive control orlistat (IC50 = 0.12 ± 0.02 μM). The combination of affinity ultrafiltration-high-performance liquid chromatography (AUF-HPLC) and high-resolution inhibition profiling (HRIP) could reduce the risk of false-negative screening and missed screening and could achieve more efficient screening of bioactive compounds in complex natural products.

Biomimetic tail-to-head terpene cyclizations using the resorcin[4]arene capsule catalyst.

The tail-to-head terpene (THT) cyclization is a biochemical process that gives rise to many terpene natural product skeletons encountered in nature. Historically, it has been difficult to achieve THT synthetically without using an enzyme. In this protocol, a hexameric resorcin[4]arene capsule acts as an artificial enzyme mimic to carry out biomimetic THT cyclizations and related carbocationic rearrangements. The precursor molecule bears a leaving group (usually an alcohol or acetate group) and undergoes the THT reaction in the presence of the capsule catalyst and HCl as a cocatalyst. Careful control of several parameters (including water content, amount of HCl cocatalyst, temperature and solvent) is crucial to successfully carrying out the reaction. To facilitate the application of this unique capsule-catalysis methodology, we therefore developed a very detailed procedure that includes the preparation and analysis of all reaction components. In this protocol, we describe how to prepare two different terpenes: isolongifolene and presilphiperfolan-1β-ol. The two procedures differ in the water content required for efficient product formation, and thus exemplify the two common use cases of this methodology. The influence of other crucial reaction parameters and means of precisely controlling them are described. A commercially available substrate, nerol, can be used as simple test substrate to validate the reaction setup. Each synthetic procedure requires 5-7 d, including 1-5 h of hands-on time. The protocol applies to the synthesis of many complex terpene natural products that would otherwise be difficult to access in synthetically useful yields.

Chiral Hydroxamic Acid Ligands in the Asymmetric Synthesis of Natural Products

Chiral hydroxamic acid (HA) and bis-hydroxamic acid (BHA) ligands have made significant contributions to the field of asymmetric synthesis, particularly in the synthesis of natural products. These ligands possess unique molecular structures that allow for exceptional stereochemical control, leading to their widespread use in catalytic systems. This review highlights the advancements made in asymmetric synthesis using chiral hydroxamic acid and bis-hydroxamic acid ligands and their impact on the synthesis of complex natural products. This discussion encompasses their role in enantioselective C–C bond formation, the functionalization of C–H bonds, the asymmetric transformations involving heteroatoms, and their application in the total synthesis of natural products. The versatility and efficiency of chiral hydroxamic acid ligands and bis-hydroxamic acid ligands make them invaluable tools for synthetic chemists working towards the efficient and selective synthesis of natural products. This review provides a comprehensive overview of their contributions, showcasing their potential to expand the boundaries of chemical synthesis and access the diverse array of natural product scaffolds.

How Acyl Carrier Proteins (ACPs) Direct Fatty Acid and Polyketide Biosynthesis.

Megasynthases, such as type I fatty acid and polyketide synthases (FASs and PKSs), are multienzyme complexes responsible for producing primary metabolites and complex natural products. Fatty acids (FAs) and polyketides (PKs) are built by assembling and modifying small acyl moieties in a stepwise manner. A central aspect of FA and PK biosynthesis involves the shuttling of substrates between the domains of the multienzyme complex. This essential process is mediated by small acyl carrier proteins (ACPs). The ACPs must navigate to the different catalytic domains within the multienzyme complex in a particular order to guarantee the fidelity of the biosynthesis pathway. However, the precise mechanisms underlying ACP-mediated substrate shuttling, particularly the factors contributing to the programming of the ACP movement, still need to be fully understood. This Review illustrates the current understanding of substrate shuttling, including concepts of conformational and specificity control, and proposes a confined ACP movement within type I megasynthases.

Wie Acyl‐Carrier‐Proteine (ACPs) die Biosynthese von Fettsäuren und Polyketiden steuern

AbstractMegasynthasen wie die Typ I Fettsäuresynthasen (engl. fatty acid synthases, FASs) und Polyketidsynthasen (PKSs) sind Multienzymkomplexe, die für die Produktion von primären Metaboliten und komplexen Naturstoffen verantwortlich sind. Fettsäuren (engl. fatty acids, FAs) und Polyketide (PKs) werden durch das schrittweise Zusammenfügen und Modifizieren kleiner Acyleinheiten aufgebaut. Ein zentraler Aspekt der FA‐ und PK‐Biosynthese besteht in dem Transport der Substrate zwischen den Multienzymkomplex‐bildenden Domänen. Dieser essenzielle Prozess wird von kleinen Acyl‐Carrier‐Proteinen (ACPs) übernommen. Die ACPs müssen die katalytischen Domänen in einer bestimmten Sequenz ansteuern, um zu gewährleisten, dass die Biosyntheseschritte stets in derselben Reihenfolge erfolgen. Die genauen Mechanismen, die dem ACP‐vermittelten Substrattransport zugrunde liegen, insbesondere die Faktoren, die zur Programmierung der ACP‐Bewegung beitragen, müssen jedoch noch vollständig entschlüsselt werden. Dieser Übersichtsartikel veranschaulicht das aktuelle Verständnis des ACP‐vermittelten Substrattransports und bietet Konzepte zur Kontrolle der ACP‐Bewegung auf Basis der konformationellen Variabilität des Multienzymkomplexes und der Spezifitäten der involvierten Domänen und schlägt als Resultat eine eingeschränkte Bewegung der ACP‐Domänen innerhalb von Typ I Megasynthasen vor.

Establishing Modular Cell‐Free Expression System for the Biosynthesis of Bicyclomycin from a Chemically Synthesized Cyclodipeptide†

Comprehensive SummaryCell‐free expression systems have emerged as a versatile and powerful platform for metabolic engineering, biosynthesis and synthetic biology studies. Nevertheless, successful examples of the synthesis of complex natural products using this system are still limited. Bicyclomycin, a structurally unique and complex diketopiperazine alkaloid, is a clinically promising antibiotic that selectively inhibits the transcription termination factor Rho. Here, we established a modular cell‐free expression system with cascade catalysis for the biosynthesis of bicyclomycin from a chemically synthesized cyclodipeptide. The six cell‐free expressed biosynthetic enzymes, including five iron‐ and α‐ketoglutarate‐dependent dioxygenases and one cytochrome P450 monooxygenase, were active in converting their substrates to the corresponding products. The co‐expressed enzymes in the cell‐free module were able to complete the related partial pathway. In vitro biosynthesis of bicyclomycin was also achieved by reconstituting the entire biosynthetic pathways (i.e., six enzymes) using the modular cell‐free expression system. This study demonstrates that the modular cell‐free expression system can be used as a robust and promising platform for the biosynthesis of complex antibiotics.

Total Synthesis of Taxol Enabled by Intermolecular Radical Coupling and Pd-Catalyzed Cyclization.

Taxol (1) is a clinically used antineoplastic diterpenoid. The tetracyclic ring system comprises a 6/8/6-membered carbocycle (ABC-ring) and a fused oxetane ring (D-ring) embedded with a bridgehead double bond and decorated with multiple oxygen functionalities. Here, we report a convergent total synthesis of this exceedingly complex natural product. The C-ring fragment was designed to possess a bromocyclohexenone and an extra tetrahydrofuran ring to control the reactivity and selectivity, as well as to minimize functional group manipulations en route to 1. The α-alkoxyacyl telluride of the A-ring served as a radical precursor, and intermolecular radical coupling with the C-ring realized the installation of the C2- and C3-stereocenters and reductive removal of the bromide. After the C8-quaternary stereocenter was constructed by exploiting the three-dimensional shape of the intermediate, the C11-vinyl triflate of A-ring and the C8-methyl ketone of C-ring were utilized for Pd(0)-catalyzed cyclization of the central eight-membered B-ring with the bridgehead olefin. Adjustment of the oxidation level and attachment of the oxetane D-ring completed the total synthesis of 1 (28 steps, as the longest linear sequence). The fragment design principle and implementation of the powerful radical coupling reaction described in the present synthesis provide valuable information for planning and executing syntheses of diverse densely oxygenated terpenoids.