Plant carbonic anhydrase-like enzymes in neuroactive alkaloid biosynthesis.

Plants synthesize numerous alkaloids that mimic animal neurotransmitters1. The diversity of alkaloid structures is achieved through the generation and tailoring of unique carbon scaffolds2,3, yet many neuroactive alkaloids belong to a scaffold class for which no biosynthetic route or enzyme catalyst is known. By studying highly coordinated, tissue-specific gene expression in plants that produce neuroactive Lycopodium alkaloids4, we identified an unexpected enzyme class for alkaloid biosynthesis: neofunctionalized α-carbonic anhydrases (CAHs). We show that three CAH-like (CAL) proteins are required in the biosynthetic route to a key precursor of the Lycopodium alkaloids by catalysing a stereospecific Mannich-like condensation and subsequent bicyclic scaffold generation. Also, we describe a series of scaffold tailoring steps that generate the optimized acetylcholinesterase inhibition activity of huperzine A5. Our findings suggest a broader involvement of CAH-like enzymes in specialized metabolism and demonstrate how successive scaffold tailoring can drive potency against a neurological protein target.

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.

Sesquiterpene Cyclase BcBOT2 Promotes the Unprecedented Wagner-Meerwein Rearrangement of the Methoxy Group.

Presilphiperfolan-8ß-ol synthase (BcBOT2), a substrate-promiscuous sesquiterpene cyclase (STC) of fungal origin, is capable of converting two new farnesyl pyrophosphate (FPP) derivatives modified at C7 of farnesyl pyrophosphate (FPP) bearing either a hydroxymethyl group or a methoxymethyl group. These substrates were chosen based on a computationally generated model. Biotransformations yielded five new oxygenated terpenoids. Remarkably, the formation of one of these tricyclic products can only be explained by a cationically induced migration of the methoxy group, presumably via a Meerwein-salt intermediate, unprecedented in synthetic chemistry and biosynthesis. The results show the great principle and general potential of terpene cyclases for mechanistic studies of unusual cation chemistry and for the creation of new terpene skeletons.

Chemistry and bioactivity of lindenane sesquiterpenoids and their oligomers.

Covering: 1925 to July 2023Among the sesquiterpenoids with rich structural diversity and potential bioactivities, lindenane sesquiterpenoids (LSs) possess a characteristic cis, trans-3,5,6-carbocyclic skeleton and mainly exist as monomers and diverse oligomers in plants from the Lindera genus and Chloranthaceae family. Since the first identification of lindeneol from Lindera strychnifolia in 1925, 354 natural LSs and their oligomers with anti-inflammatory, antitumor, and anti-infective activities have been discovered. Structurally, two-thirds of LSs exist as oligomers with interesting skeletons through diverse polymeric patterns, especially Diels-Alder [4 + 2] cycloaddition. Fascinated by their diverse bioactivities and intriguing polycyclic architectures, synthetic chemists have engaged in the total synthesis of natural LSs in recent decades. In this review, the research achievements related to LSs from 1925 to July of 2023 are systematically and comprehensively summarized, focusing on the classification of their structures, chemical synthesis, and bioactivities, which will be helpful for further research on LSs and their oligomers.

Huperzine alkaloids: forty years of total syntheses.

Covering: up to 2023Huperzine alkaloids are a group of natural products belonging to the Lycopodium alkaloids family. The representative member huperzine A has a unique structure and exhibits potent inhibitory activity against acetylcholine esterase (AChE). This subfamily of alkaloids provides a great opportunity for developing synthetic methodologies and asymmetric synthesis. The efforts towards the synthesis of huperzine A have cultivated dozens of total syntheses and a rich body of new chemistry. Impressive progress has also been made in the synthesis of other huperzine alkaloids. The total syntheses of huperzines B, U, O, Q and R, structure reassignment and total syntheses of huperzines K, M and N have been reported in the past decade. This review focuses on the synthetic organic chemistry and the biosynthesis and medicinal chemistry of huperzines are also covered briefly.

Chemoenzymatic Synthesis of a New Germacrene Derivative Named Germacrene F.

The new farnesyl pyrophosphate (FPP) derivative with a shifted olefinic double bond from C6-C7 to C7-C8 is accepted and converted by the sesquiterpene cyclases protoilludene synthase (Omp7) as well as viridiflorene synthase (Tps32). In both cases, a so far unknown germacrene derivative was found to be formed, which we name "germacrene F". Both cases are examples in which a modification around the central olefinic double bond in FPP leads to a change in the mode of initial cyclization (from 1→11 to 1→10). For Omp7 a rationale for this behaviour was found by carrying out molecular docking studies. Temperature-dependent NMR experiments, accompanied by NOE studies, show that germacrene F adopts a preferred mirror-symmetric conformation with both methyl groups oriented in the same directions in the cyclodecane ring.

Functional and Mechanistic Characterization of the 4,5-diepi-Isoishwarane Synthase from the Liverwort Radula lindenbergiana.

The microbial type sesquiterpene synthase RlMTPSL4 from the liverwort Radula lindenbergiana was investigated for its products, showing the formation of several sesquiterpene hydrocarbons. The main product was structurally characterized as the new compound 4,5-diepi-isoishwarane, while the side products included the known hydrocarbons germacrene A, α-selinene, eremophilene and 4,5-diepi-aristolochene. The cyclization mechanism towards 4,5-diepi-isoishwarane catalyzed by RlMTPSL4 was investigated through isotopic labeling experiments, revealing the stereochemical course for the deprotonation step to the neutral intermediate germacrene A, a reprotonation for its further cyclization, and a 1,2-hydride shift along the cascade. The absolute configuration of 4,5-diepi-isoishwarane was determined using a stereoselective deuteration approach, revealing an absolute configuration typically observed for a microbial type sesquiterpene.

Dipeniroqueforins A-B and Peniroqueforin D: Eremophilane-Type Sesquiterpenoid Derivatives with Cytotoxic Activity from Penicillium roqueforti.

Guided by the Global Natural Products Social (GNPS) molecular networking strategy, five undescribed eremophilane-type sesquiterpenoid derivatives (1-5) were isolated and identified from fungus Penicillium roqueforti, which was separated from the root soil of plant Hypericum beanii collected in Shennongjia Forestry District, Hubei Province. Dipeniroqueforins A-B (1-2), representing a lactam-type sesquiterpenoid skeleton with a highly symmetrical and homodimeric 5/6/6-6/6/5 hexacyclic system, are reported within the eremophilane-type family for the first time. Peniroqueforin D (5) represents the first example of a 1,2-seco eremophilane-type sesquiterpenoid derivative featuring an undescribed 7/6-fused ring system. The structures of these compounds were elucidated by various spectroscopic analyses, DP4+ probability analyses, ECD calculations, and single-crystal X-ray diffraction experiments. Furthermore, these isolates were evaluated for cytotoxicity, and the result uncovered that compound 1 displayed broad-spectrum activity. Further mechanistic study revealed that compound 1 could significantly upregulate the mRNA expression of genes related to the oxidative induction, leading to the abnormal ROS levels in tumor cells and ultimately causing tumor cell apoptosis.

Inubritantrimers A-D: Trimerized Sesquiterpenoid [4 + 2] Adducts Featuring a Distinctive Spiro-Polycyclic Scaffold from Inula britannica.

Inubritantrimer A (1), a trace trimerized sesquiterpenoid [4 + 2] adduct featuring an unusual exo-exo type spiro-polycyclic scaffold, together with three new endo-exo [4 + 2] adducts, inubritantrimers B-D (2-4), were discovered from the flowers of Inula britannica. Their structures were elucidated using 1D/2D NMR, X-ray diffraction, and ECD approaches. 1 is characterized as a novel exo-exo trimer, synthesized biogenetically from three sesquiterpenoid monomers, featuring a unique linkage of C-11/C-1', C-13/C-3' and C-13'/C-3″, C-11'/C-1″ through a two-step exo [4 + 2] cycloaddition process. Compounds 1-4 exhibited modest cytotoxicity against breast cancer cells with IC50 values in the range of 5.84-12.01 µM.

Zerumbone exhibits anti-inflammatory effects by suppressing eicosanoid signaling: Evidence from LPS-induced peripheral blood leukocytes.

Zerumbone, a sesquiterpene isolated from Zingiber zerumbet, has many bioactivities, exhibiting anti-inflammatory properties. However, the effect of zerumbone on the eicosanoid signaling pathway has yet to be examined. Here, we deciphered the anti-eicosanoid properties of zerumbone isolated from ginger. The molecular interaction between zerumbone and eicosanoid metabolizing enzymes (COX-2, 5-LOX, FLAP, and LTA4-hydrolase) and receptors (EP-4, BLT-1, and ICAM-1) along with NOS-2 were assessed using Auto-Dock 4.2 and visualized by chimera and Liggplot+ software. Further, the leukocytes were treated with zerumbone (1-20 µM) and activated using bacterial lipopolysaccharide (LPS-10 nM). The oxidative stress (OS) markers, antioxidant enzymes, and the eicosanoid pathway mediators such as COX-2, 5-LOX, BLT-1, and EP-4 were assessed. The molecular interaction of zerumbone with eicosanoids showed a higher binding affinity with mPGES-1, followed by NOS-2, FLAP, COX-2, LTA-4-hydrolase, and BLT-1. The concentration of 5 µM zerumbone effectively prevented the generation of reactive oxygen species (ROS) and nitric oxide (NO). Likewise, zerumbone significantly (p<0.05) inhibited COX-2, 5-LOX, NOS-2, EP-4, BLT-1, and ICAM-1 expression in LPS-induced peripheral blood leukocytes from rats. Further, the zerumbone treatment on the human PBMCs activated with LPS showed significant inhibition in the expression of ICAM1, COX-2, 5-LOX, and the generation of inflammatory cytokines compared to the control. Overall, the data presented infers that zerumbone positively modulates critical enzymes and receptors of eicosanoids in leukocytes activated with lipopolysaccharides. Thus, zerumbone can be a potential anti-eicosanoid drug in managing inflammation.

Antimicrobial sesterterpenoids with a unique 5/8/6/5 tetracyclic carbon-ring-system and diepoxide polyketides from a deep sea-sediment-sourced fungus Chaetomium globosum SD-347.

Two new sesterterpenoids, sesterchaetins A and B (1 and 2), and two new diepoxide polyketides, chaetoketoics A and B (3 and 4), were characterized from the culture extract of Chaetomium globosum SD-347, a fungal strain derived from deep sea-sediment. Their structures and absolute configurations were unambiguously determined by detailed NMR, mass spectra, and X-ray crystallographic analysis. Compounds 1 and 2 contained a distinctive 5/8/6/5 tetracyclic carbon-ring-system, which represented a rarely occurring natural product framework. The new isolates 1-4 exhibited selective antimicrobial activities against human and aquatic pathogenic bacteria and plant-pathogenic fungi.

Missing Measurements of Sesquiterpene Ozonolysis Rates and Composition Limit Understanding of Atmospheric Reactivity.

Emissions of biogenic reactive carbon significantly influence atmospheric chemistry, contributing to the formation and destruction of secondary pollutants, such as secondary organic aerosol and ozone. While isoprene and monoterpenes are a major fraction of emissions and have been extensively studied, substantially less is known about the atmospheric impacts of higher-molecular-weight terpenes such as sesquiterpenes. In particular, sesquiterpenes have been proposed to play a significant role in ozone chemical loss due to the very high ozone reaction rates of certain isomers. However, relatively little data are available on the isomer-resolved composition of this compound class or its role in ozone chemistry. This study examines the chemical diversity of sesquiterpenes and availability of ozone reaction rate constants to evaluate the current understanding of their ozone reactivity. Sesquiterpenes are found to be highly diverse, with 72 different isomers reported and relatively few isomers that contribute a large mass fraction across all studies. For the small number of isomers with known ozone reaction rates, estimated rates may be 25 times higher or lower than measurements, indicating that estimated reaction rates are highly uncertain. Isomers with known ozone reaction rates make up approximately half of the mass of sesquiterpenes in concentration and emission measurements. Consequently, the current state of the knowledge suggests that the total ozone reactivity of sesquiterpenes cannot be quantified without very high uncertainty, even if isomer-resolved composition is known. These results are in contrast to monoterpenes, which are less diverse and for which ozone reaction rates are well-known, and in contrast to hydroxyl reactivity of monoterpenes and sesquiterpenes, for which reaction rates can be reasonably well estimated. Improved measurements of a relatively small number of sesquiterpene isomers would reduce uncertainties and improve our understanding of their role in regional and global ozone chemistry.

Unusual Sesquiterpenes from Streptomyces olindensis DAUFPE 5622.

In nature, the vast majority of sesquiterpenes are produced by type I mechanisms, and glycosylated sesquiterpenes are rare in actinobacteria. Streptomyces olindensis DAUFPE 5622 produces the sesquiterpenes olindenones A-G, a new class of rearranged drimane sesquiterpenes. Olindenones B-D are oxygenated derivatives of olindenone A, while olindenones E-G are analogs glycosylated with dideoxysugars. 13C-isotope labeling studies demonstrated olindenone A biosynthesis occurs via the methylerythritol phosphate (MEP) pathway and suggested the rearrangement is only partially concerted. Based on the structures, one potential mechanism of olindenone A formation proceeds by cyclization of the linear terpenoid precursor, likely occurring via a terpene cyclase-mediated type II mechanism whereby the terminal alkene of the precursor is protonated, triggering carbocation-driven cyclization followed by rearrangement. Diphosphate hydrolysis may occur either before or after cyclization. Although a biosynthetic route is proposed, the terpene cyclase gene responsible for producing olindenones currently remains unidentified.

Heterodimers of Aromadendrane Sesquiterpenoid with Benzoquinone from the Chinese Liverwort Mylia nuda.

Mylnudones A-G (1-7), unprecedented 1,10-seco-aromadendrane-benzoquinone-type heterodimers, and a highly rearranged aromadendrane-type sesquiterpenoid (8), along with four known analogs (9-12), were isolated from the liverwort Mylia nuda. Compounds 1-6 and 7, bearing tricyclo[6.2.1.02,7] undecane and tricyclo[5.3.1.02,6] undecane backbones, likely formed via a Diels-Alder reaction and radical cyclization, respectively. Their structures were determined by spectroscopic analysis, computational calculation, and single-crystal X-ray diffraction analysis. Dimeric compounds displayed cytoprotective effects against glutamic acid-induced neurological deficits.

Discovery of Diverse Sesquiterpenoids from Crossiella cryophila through Genome Mining and NMR Tracking.

Terpenoids, the largest and most structurally diverse natural product family, are predominantly found in fungi and plants, with bacterial terpenoids forming a minor fraction. Here, we established an efficient platform that integrates genome mining and NMR-tracking for prioritizing strains and tracking bacterial terpenoids. By employing this platform, we selected Crossiella cryophila for a comprehensive investigation of its capacity for terpenoid production, resulting in the characterization of 15 sesquiterpenoids. These compounds comprise nine new sesquiterpenoids (1-9), along with six known analogs (10-15), which are categorized into five distinctive carbon skeletons: bicyclogermacrane, maaliane, cadinane, eudesmane, and nor-eudesmane. Their chemical structures were determined through a combination of spectroscopic analysis, single-crystal X-ray diffraction, and quantum chemical calculations. Notably, the absolute configurations of compounds 1, 2, 5-7, 9, and 13-15 were determined via single-crystal X-ray diffraction analyses. The selected compounds were evaluated for their anticancer, antimicrobial, and anti-inflammatory bioactivities; however, none of these compounds displayed any significant bioactivity. This study enriches the repertoire of bacterial terpenoids, offers a practical process for prioritizing strains for bacterial terpenoids discovery, and establishes a foundation for exploring terpenoid biosynthesis.

Protoilludane-Type and Related Nor-Sesquiterpenes from Phellinus hartigii and Their Anti-Hypertrophic Activities in Rat Cardiomyocytes.

Three nor-sesquiterpenes, phellinharts A-C (1-3), isolated from Phellinus hartigii, exhibited unprecedented protoilludane and cerapicane-type structures. The structures of compounds 1-3 were elucidated via spectroscopic analysis, quantum chemical calculations, and X-ray diffraction. Potential biogenic pathways involving demethylation, ring cleavage, and rearrangement were proposed. Compounds 1-3 displayed potent anti-hypertrophic activities with low cytotoxicity (CC50 > 50 µM) in rat cardiomyocytes, underscoring their therapeutic potential.

Arenarialins A-F, Anti-inflammatory Meroterpenoids with Rearranged Skeletons from the Marine Sponge Dysidea arenaria.

Six new sesquiterpene quinone/hydroquinone meroterpenoids, arenarialins A-F (1-6), were isolated from the marine sponge Dysidea arenaria collected from the South China Sea. Their chemical structures and absolute configurations were determined by HRMS and NMR data analyses coupled with DP4+ and ECD calculations. Arenarialin A (1) features an unprecedented tetracyclic 6/6/5/6 carbon skeleton, whereas arenarialins B-D (2-4) possess two rare secomeroterpene scaffolds. Arenarialins A-F showed inhibitory activity on the production of inflammatory cytokines TNF-α and IL-6 in LPS-induced RAW264.7 macrophages with arenarialin D regulating the NF-κB/MAPK signaling pathway.

Genome Mining Leads to Diverse Sesquiterpenes with Anti-inflammatory Activity from an Arctic-Derived Fungus.

With the advancement of bioinformatics, the integration of genome mining with efficient separation technology enables the discovery of a greater number of novel bioactive compounds. The deletion of the key gene responsible for triterpene cyclase biosynthesis in the polar strain Eutypella sp. D-1 instigated metabolic shunting, resulting in the activation of dormant genes and the subsequent production of detectable, new compounds. Fifteen sesquiterpenes were isolated from the mutant strain, with eight being new compounds. The structural elucidation of these compounds was obtained through a combination of HRESIMS, NMR spectroscopy, and ECD calculations, revealing six distinct skeleton types. Compound 7 possessed a unique skeleton of 5/10 macrocyclic ether structure. Based on the gene functions and newly acquired secondary metabolites, the metabolic shunting pathway in the mutant strain was inferred. Compounds 6, 8, 11, 14, and 15 exhibited anti-inflammatory effects without cytotoxicity through the release of nitric oxide from lipopolysaccharide-stimulated RAW264.7 cells. Notably, acorane-type sesquiterpene 8 inhibited nitric oxide production and modulated the MAPK and NLRP3/caspase-1 signaling pathways. Compound 8 also alleviated the CuSO4-induced systemic neurological inflammation symptoms in a transgenic fluorescent zebrafish model.

Discovery of P450-Modified Sesquiterpenoids Levinoids A-D through Global Genome Mining.

Cytochrome P450-modified bacterial terpenoids remain in a vast chemical space to be explored. In the present study, we conducted global genome mining of 223,829 bacterial genomes and identified 2892 bacterial terpenoid biosynthetic gene clusters (BGCs) with cytochrome P450 genes. Among these, we selected 562 with multiple P450 enzymes, which were further clustered as 355 gene cluster families by sequence similarity analysis. We then chose lev, a BGC from Streptomyces levis MCCC1A01616, for heterologous expression and discovered four new α-amorphene-type sesquiterpenoids, levinoids A-D (1-4). The structures and absolute configurations of these four new compounds were determined by employing extensive NMR analysis, NMR chemical shift calculations with DP4+, and ECD calculations. Furthermore, levinoid C (3) exhibited a moderate level of neuroprotective activity (EC50 = 21 µM) in the glutamate-induced excitotoxicity cell model. Our findings highlight the untapped chemical diversity of P450-modified bacterial terpenoids, opening new avenues for further exploration and discovery.

Divergent Biosynthesis of Bridged Polycyclic Sesquiterpenoids by a Minimal Fungal Biosynthetic Gene Cluster.

The bridged polycyclic sesquiterpenoids derived from sativene, isosativene, and longifolene have unique structures, and many chemical synthesis approaches with at least 10 steps have been reported. However, their biosynthetic pathway remains undescribed. A minimal biosynthetic gene cluster (BGC), named bip, encoding a sesquiterpene cyclase (BipA) and a cytochrome P450 (BipB) is characterized to produce such complex sesquiterpenoids with multiple carbon skeletons based on enzymatic assays, heterologous expression, and precursor experiments. BipA is demonstrated as a versatile cyclase with (-)-sativene as the dominant product and (-)-isosativene and (-)-longifolene as minor ones. BipB is capable of hydroxylating different enantiomeric sesquiterpenes, such as (-)-longifolene and (+)-longifolene, at C-15 and C-14 in turn. The C-15- or both C-15- and C-14-hydroxylated products are then further oxidized by unclustered oxidases, resulting in a structurally diverse array of sesquiterpenoids. Bioinformatic analysis reveals the BipB homologues as a discrete clade of fungal sesquiterpene P450s. These findings elucidate the concise and divergent biosynthesis of such intricate bridged polycyclic sesquiterpenoids, offer valuable biocatalysts for biotransformation, and highlight the distinct biosynthetic strategy employed by nature compared to chemical synthesis.