Monoterpene Indole Alkaloids Research Articles

Alscholarines A and B, two rearranged monoterpene indole alkaloids from Alstonia scholaris.

Alscholarines A and B (1 and 2), two unprecedented rearranged monoterpene indole alkaloids, were isolated from Alstonia scholaris. Alscholarine A (1) features an imidazole ring fused with a rearranged vallesamine-type alkaloid possessing an unparalleled 6/5/6/6 tetracyclic skeleton through an unprecedented C7-C-19 connectivity. Alscholarine B (2), incorporating an unusual 7-oxa-1-azabicyclo[3.2.1]octane moiety, represents a unique rearranged vallesamine-type alkaloid with a 6/5/6/6/5 ring system via an unprecedented C-6-C-20 connectivity. Their structures were established by spectroscopic analysis, X-ray crystallography, and quantum-chemical calculations. Their plausible biosynthetic pathways were proposed. The vasorelaxant and anti-inflammatory activities of them were also evaluated. Compounds 1-3 showed moderate vasorelaxant activities.

An updated version of the Madagascar periwinkle genome.

The Madagascar periwinkle, Catharanthus roseus, belongs to the Apocynaceae family. This medicinal plant, endemic to Madagascar, produces many important drugs including the monoterpene indole alkaloids (MIA) vincristine and vinblastine used to treat cancer worldwide. Here, we provide a new version of the C. roseus genome sequence obtained through the combination of Oxford Nanopore Technologies long-reads and Illumina short-reads. This more contiguous assembly consists of 173 scaffolds with a total length of 581.128 Mb and an N50 of 12.241 Mb. Using publicly available RNAseq data, 21,061 protein coding genes were predicted and functionally annotated. A total of 42.87% of the genome was annotated as transposable elements, most of them being long-terminal repeats. Together with the increasing access to MIA-producing plant genomes, this updated version should ease evolutionary studies leading to a better understanding of MIA biosynthetic pathway evolution.

Transcriptome Analysis in Response to Infection of Xanthomonas oryzae pv. oryzicola Strains with Different Pathogenicity

Bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most important quarantine diseases in the world. Breeding disease-resistant varieties can solve the problem of prevention and treatment of BLS from the source. The discovery of the molecular mechanism of resistance is an important driving force for breeding resistant varieties. In this study, a BLS-resistant near isogenic line NIL-bls2 was used as the material. Guangxi Xoc strain gx01 (abbreviated as WT) and its mutant strain (abbreviated as MT) with a knockout type III effectors (T3Es) gene were used to infect rice material NIL-bls2. The molecular interaction mechanism of rice resist near isogenic lines in response to infection by different pathogenic strains was analyzed by transcriptome sequencing. The results showed that there were 415, 134 and 150 differentially expressed genes (DEGs) between the WT group and the MT group at 12, 24 and 48 h of post inoculation (hpi). Through GO and KEGG enrichment analysis, it was found that, compared with non-pathogenic strains, the T3Es secreted by pathogenic strains inhibited the signal transduction pathway mediated by ethylene (ET), jasmonic acid (JA) and salicylic acid (SA), and the MAPKK (MAPK kinase) and MAPKKK (MAPK kinase kinase) in the MAPK (mitogen-activated protein kinase) cascade reaction, which prevented plants from sensing extracellular stimuli in time and starting the intracellular immune defense mechanism; and inhibited the synthesis of lignin and diterpenoid phytochemicals to prevent plants from establishing their own physical barriers to resist the invasion of pathogenic bacteria. The inhibitory effect was the strongest at 12 h, and gradually weakened at 24 h and 48 h. To cope with the invasion of pathogenic bacteria, rice NIL-bls2 material can promote wound healing by promoting the synthesis of traumatic acid at 12 h; at 24 h, hydrogen peroxide was degraded by dioxygenase, which reduced and eliminated the attack of reactive oxygen species on plant membrane lipids; and at 48 h, rice NIL-bls2 material can resist the invasion of pathogenic bacteria by promoting the synthesis of lignin, disease-resistant proteins, monoterpene antibacterial substances, indole alkaloids and other substances. Through transcriptome sequencing analysis, the molecular interaction mechanism of rice resistance near isogenic lines in response to infection by different pathogenic strains was expounded, and 5 genes, Os01g0719300, Os02g0513100, Os03g0122300, Os04g0301500, and Os10g0575100 closely related to BLS, were screened. Our work provides new data resources and a theoretical basis for exploring the infection mechanism of Xoc strain gx01 and the resistance mechanism of resistance gene bls2.

Organocatalytic Enantioselective Pictet–Spengler Reaction of α‐Ketoesters: Development and Application to the Total Synthesis of (+)‐Alstratine A

AbstractWe report herein an asymmetric Pictet–Spengler reaction of α‐ketoesters. In the presence of a catalytic amount of simple alanine‐derived squaramide and p‐nitrobenzoic acid, reaction of tryptamines with methyl 2‐oxoalkanoates afforded the corresponding 1‐alkyl‐1‐methoxycarbonyl tetrahydro‐β‐carbolines (THBCs) in high yields and ee values. A primary kinetic isotope effect (KIE=4.5) using C2‐deteurium‐labelled tryptamine indicates that rearomatization through deprotonation of the pentahydro‐β‐carbolinium ion could be the rate‐ and enantioselectivity‐determining step. A concise enantioselective total synthesis of (+)‐alstratine A, a hexacyclic cagelike monoterpene indole alkaloid, featuring this reaction as a key step, was subsequently accomplished. Remeasurement of the [a]D value of the natural product indicates that natural alstratine A is dextrorotatory rather than levorotatory as it was initially reported in the isolation paper.

Organocatalytic Enantioselective Pictet-Spengler Reaction of α-Ketoesters: Development and Application to the Total Synthesis of (+)-Alstratine A.

We report herein an asymmetric Pictet-Spengler reaction of α-ketoesters. In the presence of a catalytic amount of simple alanine-derived squaramide and p-nitrobenzoic acid, reaction of tryptamines with methyl 2-oxoalkanoates afforded the corresponding 1-alkyl-1-methoxycarbonyl tetrahydro-β-carbolines (THBCs) in high yields and ee values. A primary kinetic isotope effect (KIE=4.5) using C2-deteurium-labelled tryptamine indicates that rearomatization through deprotonation of the pentahydro-β-carbolinium ion could be the rate- and enantioselectivity-determining step. A concise enantioselective total synthesis of (+)-alstratine A, a hexacyclic cagelike monoterpene indole alkaloid, featuring this reaction as a key step, was subsequently accomplished. Remeasurement of the [a]D value of the natural product indicates that natural alstratine A is dextrorotatory rather than levorotatory as it was initially reported in the isolation paper.

Gene coexpression networks allow the discovery of two strictosidine synthases underlying monoterpene indole alkaloid biosynthesis in Uncaria rhynchophylla

Plant-derived monoterpene indole alkaloids (MIAs) from Uncaria rhynchophylla (UR) have huge medicinal properties in treating Alzheimer's disease, Parkinson's disease, and depression. Although many bioactive UR-MIA products have been isolated as drugs, their biosynthetic pathway remains largely unexplored. In this study, untargeted metabolome identified 79 MIA features in UR tissues (leaf, branch stem, hook stem, and stem), of which 30 MIAs were differentially accumulated among different tissues. Short time series expression analysis captured 58 pathway genes and 12 hub regulators responsible for UR-MIA biosynthesis and regulation, which were strong links with main UR-MIA features. Coexpression networks further pointed to two strictosidine synthases (UrSTR1/5) that were coregulated with multiple MIA-related genes and highly correlated with UR-MIA features (r > 0.7, P < 0.005). Both UrSTR1/5 catalyzed the formation of strictosidine with tryptamine and secologanin as substrates, highlighting the importance of key residues (UrSTR1: Glu309, Tyr155; UrSTR5: Glu295, Tyr141). Further, overexpression of UrSTR1/5 in UR hairy roots constitutively increased the biosynthesis of bioactive UR-MIAs (rhynchophylline, isorhynchophylline, corynoxeine, etc), whereas RNAi of UrSTR1/5 significantly decreased UR-MIA biosynthesis. Collectively, our work not only provides candidates for reconstituting the biosynthesis of bioactive UR-MIAs in heterologous hosts but also highlights a powerful strategy for mining natural product biosynthesis in medicinal plants.

Plant cytochrome P450s directing monoterpene indole alkaloid (MIA) and benzylisoquinoline alkaloid (BIA) biosynthesis

Plant cytochrome P450s directing monoterpene indole alkaloid (MIA) and benzylisoquinoline alkaloid (BIA) biosynthesis

Uncariphyllin A-J, indole alkaloids from Uncaria rhynchophylla as antagonists of dopamine D2 and Mu opioid receptors

Ten new indole alkaloids (1–10) as well as eleven known analogs (11–21) were isolated from the stems and hooks of Uncaria rhynchophylla. Their structure elucidation was based on extensive NMR studies, MS and ECD data, with the essential aid of DFT prediction of ECD spectra. Compound 1 was determined as a 17,19-seco-cadambine-type alkaloid, and compound 3 was confirmed to be a 3,4-seco-tricyclic monoterpene indole alkaloid, which are the first seco-alkaloids possessing such cleavage positions from U. rhynchophylla. All the isolated compounds were evaluated for their bioactivities on dopamine D2 and Mu opioid receptors for discovering natural therapeutic drugs targeting central nervous system (CNS) diseases. Compounds 1, 2, 4, 5, 20 and 21 showed antagonistic bioactivities on the D2 receptor (IC50 0.678–15.200 μM), and compounds 1, 3, 6, 9, 10, 13, 18, 19 and 21 exhibited antagonistic effects on the Mu receptor (IC50 2.243–32.200 μM). Among them, compounds 1 and 21 displayed dual-target activities. Compound 1 showed conspicuous antagonistic activity on D2 and Mu receptors with the IC50 values of 0.678 ± 0.182 μM and 13.520 ± 2.480 μM, respectively. Compound 21 displayed moderate antagonistic activity on the two receptors with the IC50 values at 15.200 ± 1.764 μM and 32.200 ± 5.695 μM, respectively.

Genome Assembly of the Medicinal Plant Voacanga thouarsii.

The Apocynaceae tree Voacanga thouarsii, native to southern Africa and Madagascar, produces monoterpene indole alkaloids (MIA), which are specialized metabolites with a wide range of bioactive properties. Voacanga species mainly accumulates tabersonine in seeds making these species valuable medicinal plants currently used for industrial MIA production. Despite their importance, the MIA biosynthesis in Voacanga species remains poorly studied. Here, we report the first genome assembly and annotation of a Voacanga species. The combined assembly of Oxford Nanopore Technologies long-reads and Illumina short-reads resulted in 3,406 scaffolds with a total length of 1,354.26 Mb and an N50 of 3.04 Mb. A total of 33,300 protein-coding genes were predicted and functionally annotated. These genes were then used to establish gene families and to investigate gene family expansion and contraction across the phylogenetic tree. A transposable element (TE) analysis showed the highest proportion of TE in Voacanga thouarsii compared with all other MIA-producing plants. In a nutshell, this first reference genome of V. thouarsii will thus contribute to strengthen future comparative and evolutionary studies in MIA-producing plants leading to a better understanding of MIA pathway evolution. This will also allow the potential identification of new MIA biosynthetic genes for metabolic engineering purposes.

Secondary metabolites of the leaves of Tricalysia atherura N. Hallé (Rubiaceae) and their potential antiplasmodial activity

One monoterpene indole alkaloid, atheruramine (1) bearing an ether bridge linking, one hydrobenzoin derivative, tricalydioloside (2) and two ursane-type triterpenes, atherurosides (A and B) (3 and 4) were isolated from the leaves of Tricalysia atherura, together with eight known compounds. The structures of these new compounds were elucidated on the basis of the results of spectroscopic analysis, and the relative configurations of compounds 1–3 were established by NOE difference. Four of the metabolites were screened in vitro against both chloroquine (CQ)-sensitive (3D7) and -resistant (Dd2) strains of Plasmodium falciparum; they were found to exhibit moderate activity against chloroquine-resistant (Dd2) (IC50 64.99–92.29 μg/mL). Meanwhile, crude extract possesses high antiplasmodial activity against both 3D7 and Dd2 strains of P. falciparum (IC50 4.39–7.54 μg/mL) and high selectivity indices values (SI > 10) and was found to be safe.

The Vinca minor genome highlights conserved evolutionary traits in monoterpene indole alkaloid synthesis.

Vinca minor, also known as the lesser periwinkle, is a well-known species from the Apocynaceae, native to central and southern Europe. This plant synthesizes monoterpene indole alkaloids, which are a class of specialized metabolites displaying a wide range of bioactive- and pharmacologically important properties. Within the almost 50 monoterpene indole alkaloids it produces, V. minor mainly accumulates vincamine, which is commercially used as a nootropic. Using a combination of Oxford Nanopore Technologies long read- and Illumina short-read sequencing, a 679,098 Mb V. minor genome was assembled into 296 scaffolds with an N50 scaffold length of 6 Mb, and encoding 29,624 genes. These genes were functionally annotated and used in a comparative genomic analysis to establish gene families and to investigate gene family expansion and contraction across the phylogenetic tree. Furthermore, homology-based monoterpene indole alkaloid gene predictions together with a metabolic analysis across 4 different V. minor tissue types guided the identification of candidate monoterpene indole alkaloid genes. These candidates were finally used to identify monoterpene indole alkaloid gene clusters, which combined with synteny analysis allowed for the discovery of a functionally validated vincadifformine-16-hydroxylase, reinforcing the potential of this dataset for monoterpene indole alkaloids gene discovery. It is expected that access to these resources will facilitate the elucidation of unknown monoterpene indole alkaloid biosynthetic routes with the potential of transferring these pathways to heterologous expression systems for large-scale monoterpene indole alkaloid production.

Bioproduction of monoterpene indole alkaloids in a single cell factory

The de novo biosynthesis of vindoline and catharanthine, the direct precursors used for industrial production of the anti-cancer drug vinblastine, has been achieved in the yeast cell factory. To date, this is the longest natural product biosynthesis pathway that has been successfully transferred from plants to microorganisms, indicating the possibility of producing more than 3,000 other monoterpene indole alkaloids in yeast by synthetic genome engineering.

Interference between ER stress-related bZIP-type and jasmonate-inducible bHLH-type transcription factors in the regulation of triterpene saponin biosynthesis in Medicago truncatula.

Triterpene saponins (TS) are a structurally diverse group of metabolites that are widely distributed in plants. They primarily serve as defense compounds and their production is often triggered by biotic stresses through signaling cascades that are modulated by phytohormones such as the jasmonates (JA). Two JA-modulated basic helix-loop-helix (bHLH) transcription factors (TFs), triterpene saponin biosynthesis activating regulator 1 (TSAR1) and TSAR2, have previously been identified as direct activators of TS biosynthesis in the model legume Medicago truncatula. Here, we report on the involvement of the core endoplasmic reticulum (ER) stress-related basic leucine zipper (bZIP) TFs bZIP17 and bZIP60 in the regulation of TS biosynthesis. Expression and processing of M. truncatula bZIP17 and bZIP60 proteins were altered in roots with perturbed TS biosynthesis or treated with JA. Accordingly, such roots displayed an altered ER network structure. M. truncatula bZIP17 and bZIP60 proteins were shown to localize in the nucleus and appeared to be capable of interfering with the TSAR-mediated transactivation of TS biosynthesis genes. Furthermore, interference between ER stress-related bZIP and JA-modulated bHLH TFs in the regulation of JA-dependent terpene biosynthetic pathways may be widespread in the plant kingdom, as we demonstrate that it also occurs in the regulation of monoterpene indole alkaloid biosynthesis in the medicinal plant Catharanthus roseus.

Microscopic Studies and Preliminary Pharmacognostical Evaluation of Leaves and Roots of Ophiorrhiza eriantha Wight

Death and suffering caused by cancer have been a major concern throughout the history of mankind. Extensive research has been undertaken in the plant kingdom to find out an effective drug for the treatment of various cancer diseases. Drugs derived from plants and microorganism plays a major role in the chemotherapy of cancer. Monoterpene indole alkaloid camptothecin (CPT) is a potent anticancer agent first isolated and reported from extracts of the Chinese tree Camptotheca acuminata (Nyssaceae). Thereafter camptothecin was isolated from plants of different families mainly Ophiorrhiza mungos and Nothapodytes nimmoniana. The genus Ophiorrhiza is a potent natural source of camptothecin. The presence of fortyseven species and nine varieties of Ophiorrhiza was reported in the Indian subcontinent. Ophiorrhiza eriantha Wight is an erect subshrub species, its anticancer potential and detailed phytochemical analysis are yet to be studied. Our aim is to evaluate the pharmacognostic parameters of leaves and roots of Ophiorrhiza eriantha Wight. Our objective is to identify characteristic microscopic features of leaves and roots of the plant and to study the quantitative microscopy, ash value, loss on drying, phytochemical screening, fluorescence analysis, and powder analysis of leaf and roots of Ophiorrhiza eriantha Wight. The thickness of the midrib, lamina, leaf margin, and epidermal trichome is measured. It showed the presence of paracytic stomata. Non-glandular epidermal trichomes are common in leaf powder. Ophiorrhiza eriantha Wight is a promising anticancer plant; this is a pioneer work to explore the anatomical features of Ophiorrhiza eriantha Wight. Keywords: ,, , , And

Enantioselective Total Synthesis of (-)-Limaspermidine and (-)-Kopsinine by a Nitroaryl Transfer Cascade Strategy.

We report an intramolecular conjugate addition/Truce-Smiles/E1cb cascade of 2-nitrobenzenesulfonamide-functionalized cyclohexenones as a new entry to the core scaffold of monoterpene indole alkaloids. The method was applied to the asymmetric total synthesis of (-)-limaspermidine, (-)-kopsinilam, and (-)-kopsinine, as well as the framework of the kopsifoline alkaloids, thus highlighting its complementarity to existing approaches involving the use of indole-based starting materials or the interrupted Fischer indole synthesis. Furthermore, we show that the cascade tolerates various substituents on the nitroarene, opening the way to other natural products as well as non-natural analogues.

Enantioselective Total Synthesis of (−)‐Limaspermidine and (−)‐Kopsinine by a Nitroaryl Transfer Cascade Strategy

We report an intramolecular conjugate addition/Truce-Smiles/E1cb cascade of 2-nitrobenzenesulfonamide-functionalized cyclohexenones as a new entry to the core scaffold of monoterpene indole alkaloids. The method was applied to the asymmetric total synthesis of (−)-limaspermidine, (−)-kopsinilam, and (−)-kopsinine, as well as the framework of the kopsifoline alkaloids, thus highlighting its complementarity to existing approaches involving the use of indole-based starting materials or the interrupted Fischer indole synthesis. Furthermore, we show that the cascade tolerates various substituents on the nitroarene, opening the way to other natural products as well as non-natural analogues.

Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana

Monoterpene indole alkaloids (MIAs) are a diverse class of plant natural products that include a number of medicinally important compounds. We set out to reconstitute the pathway for strictosidine, a key intermediate of all MIAs, from central metabolism in Nicotiana benthamiana. A disadvantage of this host is that its rich background metabolism results in the derivatization of some heterologously produced molecules. Here we use transcriptomic analysis to identify glycosyltransferases that are upregulated in response to biosynthetic intermediates and produce plant lines with targeted mutations in the genes encoding them. Expression of the early MIA pathway in these lines produces a more favorable product profile. Strictosidine biosynthesis was successfully reconstituted, with the best yields obtained by the co-expression of 14 enzymes, of which a major latex protein-like enzyme (MLPL) from Nepeta (catmint) is critical for improving flux through the iridoid pathway. The removal of endogenous glycosyltransferases does not impact the yields of strictosidine, highlighting that the metabolic flux of the pathway enzymes to a stable biosynthetic intermediate minimizes the need to engineer the endogenous metabolism of the host. The production of strictosidine in planta expands the range of MIA products amenable to biological synthesis.

Analgesic monoterpene indole alkaloids from Gelsemium elegans stems

ObjectiveThe aim of this study is to search for potent analgesics from the stems of Gelsemium elegans. MethodsAlkaloids were isolated from the samples of G. elegans and purified using column chromatograph and High-Performance Liquid Chromatography. The chemical structures of the isolated alkaloids were determined using extensive high resolution electrospray ionization mass spectroscopy (HRESIMS) and nuclear magnetic resonance (NMR) spectroscopic data analyses, 13C NMR DP4+ analysis and electronic circular dichroism (ECD) calculations, and Rh2(OCOCF3)4-induced ECD data analysis. The analgesic activities of all the isolates were analyzed using an acetic acid-induced writhing test in mice. ResultsTwo new monoterpene indole alkaloids, elegansine A (1) and 14-hydroxysempervirine (2), and seven known monoterpene indole alkaloids were isolated from the stems of G. elegans. Elegansine A (1) represents the first example of sarpagine-type alkaloids with a Δ15(20) double bond. All the alkaloids (1–9) showed potential analgesic activities. Three alkaloids, namely 14-hydroxysempervirine (2), 14β,20α-dihydroxydihydrorankinidine (4), and 14-hydroxygelsenicine (6), exhibited significant analgesic activities in the acetic acid-induced writhing test in mice at a dose of 5.0 mg/kg with the writhing inhibition rates of 69.5%, 69.2%, and 72.7%, respectively. ConclusionThese results enlarged the diversity of monoterpene indole alkaloids and also provided a new basis to develop novel potent analgesics.

A microbial supply chain for production of the anti-cancer drug vinblastine

Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine1. As MIAs are difficult to chemically synthesize, the world’s supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.

First genomic insights into the Mandevilla genus

Mandevilla (Apocynaceae) is a greatly appreciated genus in the world ornamental market. In this study, we attempted to address the poor genetic knowledge and the huge taxonomic gaps existing in this genus by analyzing a collection of 55 accessions. After cytometrically determining the triploid genome size (1,512.64 Mb) of a reference sample (variety “Mandevilla 2001”), the plastidial genome (cpDNA, 0.18 Mb) and a draft of the nuclear genome (nuDNA, 207 Mb) were assembled. While cpDNA was effective in reconstructing the phylogenesis of the Apocynaceae family based on a DNA superbarcoding approach, the nuDNA assembly length was found to be only 41% of the haploid genome size (506 Mb, predicted based on the K-mer frequency distribution). Its annotation enabled the prediction of 37,811 amino acid sequences, of which 10,562 resulted full length proteins. Among them, we identified nine proteins whose orthologs (in Catharanthus roseus) are involved in the biosynthesis of monoterpene indole alkaloids (MIAs), including catharanthine, tabersonine, and vincadifformine. The nuclear genome draft was also useful to develop a highly informative (average polymorphism information content, PIC = 0.62) set of 23 simple sequence repeat (SSR) markers that was validated on the Mandevilla collection. These results were integrated with cytometric measurements, nuclear ITS1 haplotyping and chloroplast DNA barcoding analyses to assess the origin, divergence and relationships existing among the 55 accessions object of the study. As expected, based on the scarce information available in the literature, the scenario was extremely intricate. A reasonable hypothesis is that most of the accessions represent interspecific hybrids sharing the same species as maternal parent (i.e., Mandevilla sanderi).