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Covering: 2015 to 2020Nitrogen heterocyclic natural products (NHNPs) are primary or secondary metabolites containing nitrogen heterocyclic (N-heterocyclic) skeletons. Due to the existence of the N-heterocyclic structure, NHNPs exhibit various bioactivities such as anticancer and antibacterial, which makes them widely used in medicines, pesticides, and food additives. However, the low content of these NHNPs in native organisms severely restricts their commercial application. Although a variety of NHNPs have been produced through extraction or chemical synthesis strategies, these methods suffer from several problems. The development of biotechnology provides new options for the production of NHNPs. This review introduces the recent progress of two strategies for the biosynthesis of NHNPs: enzymatic biosynthesis and microbial cell factory. In the enzymatic biosynthesis part, the recent progress in the mining of enzymes that synthesize N-heterocyclic skeletons (e.g., pyrrole, piperidine, diketopiperazine, and isoquinoline), the engineering of tailoring enzymes, and enzyme cascades constructed to synthesize NHNPs are discussed. In the microbial cell factory part, with tropane alkaloids (TAs) and tetrahydroisoquinoline (THIQ) alkaloids as the representative compounds, the strategies of unraveling unknown natural biosynthesis pathways of NHNPs in plants are summarized, and various metabolic engineering strategies to enhance their production in microbes are introduced. Ultimately, future perspectives for accelerating the biosynthesis of NHNPs are discussed.

Tropane alkaloids, including clinically important hyoscyamine and scopolamine, are produced in the roots of medicinal plant species, such as Atropa belladonna, from the Solanaceae family. Recent molecular and genomic approaches have advanced our understanding of the metabolic enzymes involved in tropane alkaloid biosynthesis. A noncanonical type III polyketide synthase, pyrrolidine ketide synthase (PYKS) catalyzes a two-step decarboxylative reaction, which involves imine–ketide condensation indispensable to tropane skeleton construction. In this study, we generated pyks mutant A. belladonna hairy roots via CRISPR/Cas9-mediated genome editing and analyzed the metabolic consequences of the loss of PYKS activity on tropane alkaloids, providing insights into a crucial role of the scaffold-forming reaction in the biosynthetic pathway.

Piper sarmentosum Roxb. (PS) and Piper cathayanum M. G. Gilbert & N. H. Xia (PC) are both listed as Traditional Chinese Medicine herbs. In this study, ultra-performance liquid chromatography mass spectrometry (UPLC-MS/MS) was used to analyze secondary metabolites and metabolically profile the leaves, stems and roots in PS and PC. A total of 110 and 92 differential metabolites were shared from various organs of PS and PC, respectively. Compared to the stems and roots of PS or PC, alkaloids and phenols in leaves of PS or PC accounted for 70% and 57% of the total alkaloids and phenols, respectively. An analysis of enriched metabolic pathways demonstrated that L-tyrosine and 4-hydroxycinnamic acid in PS were directly involved in five and three metabolic pathways, which are related to the biosynthesis of isoquinoline alkaloids and phenol backbones, respectively. The L-glutamic acid and oxoglutaric acid in PC were involved in seven and two metabolic pathways, respectively. L-glutamic acid synthesizes heme, tropane, piperidine and pyridine alkaloids, and the type Ⅱ polyketide backbone. Moreover, oxoglutaric acid generated via L-glutamic acid was mainly involved in the citrate cycle and played a fundamental role in determining these important metabolic processes.

Datura stramonium L. (Solanaceae) possesses a rich tropane alkaloids (TAs) spectrum. The plant contains, in particular, the allelopathic compounds scopolamine and atropine, which are poorly soluble in water, thus limiting their use in agrochemical formulations as biocidal and deterrent agents against herbivore insects. The efficacy of the hydrophobic TAs extracts could be increased with the improvement of their dissolution/leaching properties. This is important for improving screening and test performance and for elucidating the activity of environmentally friendly agricultural approaches, with new perspectives for the production and use of those biodegradable insecticidal products. The present study explores the aspects of atropine and scopolamine complexation with cyclodextrin (CDs) through FT-IR and UV–Vis spectroscopies. In addition, the structures of the inclusion complex of atropine, scopolamine and β-CD have been investigated by molecular modeling techniques. The results obtained indicate that β-CDs are a promising carriers for improving the properties of TAs, therefore increasing their application potential in agrochemical formulations. Graphic abstract

Pharmacologically important tropane alkaloids such as hyoscyamine, and scopolamine, are synthesized in the roots of several Solanaceae and also accumulate in their shoots. The tropane alkaloids content in roots of D. innoxia seedlings was almost 3-fold higher than in their shoots. Moreover, scopolamine accounted for 77 % of the total tropane alkaloid content in shoots as compared to only 20 % in roots. All candidate genes in the scopolamine biosynthetic pathway were significantly expressed in D. innoxia mature roots with limited expression in mature leaves. Using exogenous isotopically labeled 2H5 L-phenylalanine, we show here that root tissues, but not detached shoots, readily biosynthesize scopolamine. Tropane alkaloids newly synthesized by roots, and preferentially scopolamine, were further transported to shoots. Our evidence indicates that D. innoxia seedlings have the ability to synthesize tropane alkaloids in their roots and differentially transport them to the shoots.

Hyoscyamine, a member of the class of compounds known as tropane alkaloids (TAs), is clinically used as an anticholinergic drug. Previous research has predicted that hyoscyamine is produced via the...

A new tropane alkaloid 7β-acetoxy-6β-benzoyloxy-3α-hydroxytropane (1) was isolated from the twigs of Erythroxylum macrocalyx Mart. (Erythroxylaceae), along with the known substances: 6β,7β-dibenzoyloxy-3α-hydroxytropane (2), 6β,7β-dihydroxy-3α-(phenylacetoxy)tropane (3), 3α-benzoyloxy-6β,7β-dihydroxytropane (4), 6β-benzoyloxy-3α-(4-hydroxy-3,5-dimetoxybenzoyloxy)tropane (5), ombuin-3-rutinoside-5-glucoside (6), lupeol (7), taraxerol (8) and lupenone (9). Compounds 1 and 2 were also isolated from the leaves. The structures were established by analyses of 1D- and 2D-NMR and MS data, as well as by comparison with literature data for known compounds. The structure of 2 was also supported by X-ray crystallography analyses. The compounds were evaluated in vitro for their antibacterial and antiproliferative activities. Compound 5 showed high antiproliferative activity on liver hepatocellular carcinoma cells (HepG2) with IC50 value of 3.66 μg mL−1 (8.29 μmol L-1), but no cytotoxic effect (IC50 > 25 μg mL-1) on human lymphoblast cell line. This study reveals the potential use of 5 as prototype for the synthesis of new antiproliferative agents.

Plant alkaloids, are one of the largest groups of pharmaceutical metabolites. Scopolamine and atropine are most common tropane alkaloids in Atropa belladonna L. with great application in pharmaceutical industry. Phytochemical biosynthetic pathways induction via various elicitors is an effective strategy for increasing the production of valuable substances. Therefore, in this research effect of yeast extract were studied on expression of h6h gene, antioxidant enzymes activity and production of tropane alkaloid in A. belladonna hairy roots. Hairy roots of A. belladonna were obtained from cotyledonary leaf explants by A7 strain of Agrobacterium rhizogenes. Polymerase chain reaction (PCR) analysis with specific primers confirmed the insertion of rolB in putative transgenic hairy roots genome. Different concentrations of yeast extract (YE) (0, 0.5, 1 and 1.5 mg/L) at different exposure time (24 and 48 h) were used as elicitor. Scopolamine and atropine content and activity of antioxidant enzymes such as guaiacol peroxidase, ascorbate peroxidase and catalase increased and expression of h6h (hyoscyamine 6 beta-hydroxylase) gene in elicited hairy roots were investigated. High concentration of YE decreased the growth of hairy roots but increased the atropine content compared to the control. According to the results, the highest content of scopolamine (9.21%) and atropine (43.39%) which were 9 and 5 fold than control, were obtained by 1 and 1.5 mg/L YE at 48 h exposure time, respectively. The results demonstrated that YE can be used as effective elicitor to increase tropane alkaloids production in hairy root cultures of A. belladonna.

Hyoscyamine, anisodamine and scopolamine are tropane alkaloids present in some Solanaceae species and used in modern medicine. L-Hyoscyamine is hydroxylated to 6β-hydroxyhyoscyamine (anisodamine) and then epoxidated to scopolamine by the dual action of hyoscyamine 6β-hydroxylase (H6H), a 2-oxoglutarate dependent dioxygenase. A natural mutation in the Gly-220 residue to Cys was previously shown to be associated with the loss of function of H6H in Mandragora officinarum, preventing the accumulation of anisodamine and scopolamine in these plants. We show here that a deliberate Gly220Cys mutation in the Datura innoxia DiH6H protein caused a loss of both its enzymatic abilities and rendered it unable to hydroxylate L-hyoscyamine into anisodamine and to epoxidate anisodamine into scopolamine. By using protein modeling based on an available crystal structure of H6H from Datura metel, we show how the Cys220 residue causes a steric interference in the active site cavity impairing the interaction of both substrates, hyoscyamine and anisodamine with the active site of the protein. We also address the enantiomeric preference of DiH6H based on molecular modeling.

The development of radiometal‐labelled pharmaceuticals for neuroimaging could offer great potential due to easier handling during labelling and availability through radionuclide generator systems. Nonetheless, to date, no such tracers are available for positron emission tomography, primarily owing to the challenge of crossing the blood–brain barrier (BBB) and loss of affinity through chelator attachment. We have prepared a variety of 68Ga‐labelled phenyltropanes showing that, through a simple hydrocarbon‐linker, it is possible to introduce a chelator onto the lead structure while maintaining its high affinity for hDAT (human dopamine transporter) and simultaneously achieving adequate lipophilicity. One of the candidates, [68Ga]Ga‐HBED‐hexadiyne‐tropane, showed an IC50 value of 66 nM, together with a log D 7.4 of 0.96. A μPET study in a hemi‐parkinsonian rat model showed a fast wash‐out of the tracer, and no specific uptake in the brain, thus implying an inability to penetrate the BBB., Tracer metal: Tropanes are commonly used in radiopharmacy to assess the status of the dopaminergic system. However, no suitable radiometal‐labelled tracers are available for PET, primarily owing to the challenge of crossing the blood–brain barrier and loss of affinity through chelator attachment. We have developed 68Ga‐labelled phenyltropanes showing promising affinity and lipophilicity, and conducted a first PET study.

Made available in DSpace on 2021-06-25T19:56:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-11-11 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) INCT BioNat Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Tropane alkaloids are specialized plant metabolites mostly found in the Erythroxylaceae and Solanaceae families. Although tropane alkaloids have a high degree of structural similarity because of the tropane ring, their pharmacological actions are quite distinct. Brazil is one of the main hotspots of Erythroxylum spp. diversity with 123 species (almost 66% of the species catalogued in tropical America). Erythroxylum pungens occurs in the Caatinga, a promising biome that provides bioactive compounds, including tropane alkaloids. As part of our efforts to investigate this species, 15 alkaloids in specimens harvested under different environmental conditions are presented herein. The occurrence of 3-(2-methylbutyryloxy)tropan-6,7-diol in the stem bark of plants growing in their natural habitat, greenhouse controlled conditions, and after a period of water restriction, suggests that it is a potential chemical marker for the species. This alkaloid was evaluated for several parameters in zebrafish ( Danio rerio ) as a model organism. Regarding toxicity, teratogenic effects were observed at 19.5 mu M and the lethal dose for embryos was 18.4 mu M. No mortality was observed in adults, but a behavioral screen showed psychostimulatory action at 116.7 mu M. Overall, the alkaloid was able to cause zebrafish behavioral changes, prompting further investigation of its potential as a new molecule in the treatment of depression-like symptoms. In silico , targets involved in antidepressant pathways were identified by docking. Univ Fed Rio Grande do Norte, Dept Pharm, Gal Gustavo Cordeiro Farias St S-N, BR-59012570 Natal, RN, Brazil Univ Fed Rio Grande do Norte, Dept Physiol & Behav, Natal, RN, Brazil Univ Fed Mato Grosso do Sul, Fac Pharmaceut Sci Food & Nutr, Campo Grande, MS, Brazil Univ Fed Rio Grande do Sul, Fac Pharm, Porto Alegre, RS, Brazil Univ Fed Rio Grande do Sul, Ctr Biotechnol & Dept Bot, Porto Alegre, RS, Brazil Sao Paulo State Univ, Chem Inst, Araraquara, SP, Brazil Sao Paulo State Univ, Chem Inst, Araraquara, SP, Brazil CNPq: 445149/2014-0 INCT BioNat: 465637/2014-0 CAPES: 001 FAPESP: 2014/50926-0

Setting of maximum limits for a number of plant alkaloids is under discussion in the EU. The novel method developed and optimized in this study enables simultaneous determination of 21 tropane alkaloids (TAs) and 33 pyrrolizidine (PAs) together with their N-oxides (PANOs). For analysis of aqueous-methanolic extract, reversed phase ultra-high-performance liquid chromatography and tandem mass spectrometry (RP-U-HPLC-MS/MS) was employed. The method was validated for frequently contaminated matrices (i) sorghum, (ii) oregano, and (iii) mixed herbal tea. The recoveries at two spiking levels were in the range of 82–115%, 80–106%, and 78–117%, respectively, and repeatabilities were less than 19% for all analyte/matrix combinations. As regards the achieved limits of quantification (LOQ), their values were in the range of 0.5–10 μg kg−1. The crucial problem encountered during method development, co-elution of multiple groups of isomeric alkaloids, was overcome by subsequent sample separation in the second chromatographic system, hydrophilic interaction liquid chromatography (HILIC), providing different separation selectivity. Lycopsamine, echinatine, and indicine (co-elution group 1) and N-oxides of indicine and intermedine (co-elution group 2), which could not be resolved on the commonly used RP column, were possible to separate fully by using the HILIC system.

A simple but effective method was developed for the ultra-trace determination of two tropane alkaloid residues, hyoscyamine and scopolamine, in honey. Honey samples were dissolved in a concentrated aqueous solution of sodium acetate from which the alkaloids were subsequently extracted using acetonitrile. The resulting extract was analyzed using HILIC–ESI-MS/MS without any additional extract cleanup or enrichment steps. Enhanced sensitivity associated with the combination of HILIC and ESI-MS/MS techniques permitted the quantitation of the two tropane alkaloids at concentrations as low as 0.01 µg kg−1. The use of isotopically labelled analogs permitted accuracies ranging from 86.9 to 106.1% for honey samples fortified at 0.02, 0.2 and 2 µg kg−1 with precisions ranging from 0.8 to 6.2%. The estimated method detection limits for hyoscyamine and scopolamine were 0.002 and 0.003 µg kg−1, respectively. Twenty-three samples of honey were analyzed and only two samples were found to contain residues of the tropane alkaloids at concentrations above the limit of quantitation of 0.01 µg kg−1. Hyoscyamine was detected at 0.012 µg kg−1 in two honey samples, one of which also contained 0.012 µg kg−1 of scopolamine.

Ornithine decarboxylase (ODC) plays an important role in various biological processes; however, its role in plant secondary metabolism, especially in the biosynthesis of tropane alkaloids (TAs) suc...

A straight synthetic approach to fused polycyclic tropane scaffold formation through an asymmetric dearomatization cycloaddition process of 2-nitrobenzofurans with cyclic azomethine ylides was successfully developed. In the presence of a chiral copper complex, derived from Cu(OAc)

Some medicinal plants of the Solanaceae produce pharmaceutical tropane alkaloids (TAs), such as hyoscyamine and scopolamine. Littorine is a key biosynthetic intermediate in the hyoscyamine and scopolamine biosynthetic pathways. However, the mechanism underlying littorine formation from the precursors phenyllactate and tropine is not completely understood. Here, we report the elucidation of littorine biosynthesis through a functional genomics approach and functional identification of two novel biosynthesis genes that encode phenyllactate UDP-glycosyltransferase (UGT1) and littorine synthase (LS). UGT1 and LS are highly and specifically expressed in Atropa belladonna secondary roots. Suppression of either UGT1 or LS disrupted the biosynthesis of littorine and its TA derivatives (hyoscyamine and scopolamine). Purified His-tagged UGT1 catalysed phenyllactate glycosylation to form phenyllactylglucose. UGT1 and LS co-expression in tobacco leaves led to littorine synthesis if tropine and phenyllactate were added. This identification of UGT1 and LS provides the missing link in littorine biosynthesis. The results pave the way for producing hyoscyamine and scopolamine for medical use by metabolic engineering or synthetic biology.

Przewalskia tangutica is a traditional medicinal plant from Tibet used for the analgesic effect from the tropane alkaloids (TAs) produced by the plant. Its roots have the highest yield of hyoscyamine in all plant species and so have been overharvested becoming an endangered medicinal plant species. Metabolic engineering is a good way to improve the yield of TAs in plants. In our study, two functionally distinct tropinone reductases genes, PtTRI and PtTRII, were cloned from P. tangutica and the functional divergence were characterized. The enzyme kinetics of PtTRI and PtTRII were investigated. The phylogenetic analysis classified them into different clades: PtTRI and PtTRII were in the clade of tropine-forming reductase and pseudotropine-forming reductase, respectively. We found PtTRI to be expressed in the roots but less in leaves, whereas PtTRII was expressed in the roots at higher levels than in the leaves. The kinetic parameters (Km , Vmax , and Kcat ) were analyzed using purified recombinant enzymes at their optimum pH. Enzymatic analysis results showed that tropinone is a better substrate for PtTRII compared with PtTRI, suggesting that PtTRII might be a potential gene target for TA biosynthesis engineering. Compared with the reported TRIs, PtTRI exhibited a higher affinity for tropinone.

Several lines of evidence suggest that selective sigma-2 (σ2) ligands might be useful for the treatment of solid tumors. However, very few selective σ2 ligands have been identified. This study was aimed at identifying new selective σ2 receptor ligands using a previously identified agent, SYA 013 as a lead. Four groups, homopiperazine, piperazine, tropane and selected oxime analogs of the homopiperazines were identified, synthesized and subsequently screened at the σ1 and σ2 receptors. The results demonstrate that these scaffolds can be modified to obtain selective σ2 receptor ligands. 1-(5-Chloropyridin-2-yl)-4-(3-((4-fluorophenyl)thio)propyl)-1,4-diazepane, 7 and 3-(4-chlorophenyl)-8-(3-((2-fluorophenyl)thio)propyl)-8-azabicyclo[3.2.1]octan-3-ol, 21 were identified as the highest binding affinity ligands (σ2Ki = 2.2 nM) and (4-(4-(5-chloropyridin-2-yl)-1,4-diazepan-1-yl)-1-(4-fluorophenyl)-butan-1-one oxime, 22 as a high affinity and the most selective ligand for the σ2 receptor (σ1Ki/σ2Ki = 41.8).

Datura, a genus of medicinal herb from the Solanaceae family, is credited with toxic as well as medicinal properties. The different plant parts of Datura sp., mainly D. stramonium L., commonly known as Datura or Jimson Weed, exhibit potent analgesic, antiviral, anti-diarrheal, and anti-inflammatory activities, owing to the wide range of bioactive constituents. With these pharmacological activities, D. stramonium is potentially used to treat numerous human diseases, including ulcers, inflammation, wounds, rheumatism, gout, bruises and swellings, sciatica, fever, toothache, asthma, and bronchitis. The primary phytochemicals investigation on plant extract of Datura showed alkaloids, carbohydrates, cardiac glycosides, tannins, flavonoids, amino acids, and phenolic compounds. It also contains toxic tropane alkaloids, including atropine, scopolamine, and hyoscamine. Although some studies on D. stramonium have reported potential pharmacological effects, information about the toxicity remains almost uncertain. Moreover, the frequent abuse of D. stramonium for recreational purposes has led to toxic syndromes. Therefore, it becomes necessary to be aware of the toxic aspects and the potential risks accompanying its use. The present review aims to summarize the phytochemical composition and pharmacological and toxicological aspects of the plant Datura.

Plant alkaloids represent a diverse group of nitrogen-containing natural products. These compounds are considered valuable in drug discovery and development. High-throughput identification of such plant secondary metabolites in complex plant extracts is essential for drug discovery, lead optimization, and understanding the biological pathway. The present study aims to rapidly identify different classes of alkaloids in plant extracts through the liquid chromatography with electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) approach using 161 isolated and purified alkaloids. These are biologically important unique alkaloids belonging to different sub-classes such as isoquinoline, quinoline, indole, tropane, pyridine, piperidine, quinolizidine, aporphine, steroidal, and terpenoid. The majority of these are not available commercially and are known to manifest valuable biological activities. Four pools of a maximum of 50 phytostandards each were prepared, based on their log P value to minimize co-elution for rapid and cost-effective analyses. MS/MS spectra were acquired in the positive ionization mode by using their [M + H]+ and/or [M + Na]+ with both the average collisional energy (25.5-62 eV) and individual collisional energies (10, 20, 30, and 40 eV). Accurate mass, high-resolution mass spectrometry (HR-MS) data, MS/MS data, and retention times were curated for each compound. The developed LC-MS/MS method was successfully used to interrogate and fast dereplicate alkaloids in 13 medicinal plant extracts and a herbal formulation. A total of 56 alkaloids were identified based on the reference standard retention times (RTs), HR-MS spectra, and/or MS/MS spectra. The MS data have been submitted to the MetaboLights online database (MTBLS2914). The mass spectrometric and chromatographic data will be useful for the discovery of new congeners and the study of biological pathways of alkaloids in the plant kingdom.

Microbial biosynthesis of plant natural products (PNPs) can facilitate access to valuable medicinal compounds and derivatives. Such efforts are challenged by metabolite transport limitations, which arise when complex plant pathways distributed across organelles and tissues are reconstructed in unicellular hosts without concomitant transport machinery. We recently reported an engineered yeast platform for production of the tropane alkaloid (TA) drugs hyoscyamine and scopolamine, in which product accumulation is limited by vacuolar transport. Here, we demonstrate that alleviation of transport limitations at multiple steps in an engineered pathway enables increased production of TAs and screening of useful derivatives. We first show that supervised classifier models trained on a tissue-delineated transcriptome from the TA-producing plant Atropa belladonna can predict TA transporters with greater efficacy than conventional regression- and clustering-based approaches. We demonstrate that two of the identified transporters, AbPUP1 and AbLP1, increase TA production in engineered yeast by facilitating vacuolar export and cellular reuptake of littorine and hyoscyamine. We incorporate four different plant transporters, cofactor regeneration mechanisms, and optimized growth conditions into our yeast platform to achieve improvements in de novo hyoscyamine and scopolamine production of over 100-fold (480 μg/L) and 7-fold (172 μg/L). Finally, we leverage computational tools for biosynthetic pathway prediction to produce two different classes of TA derivatives, nortropane alkaloids and tropane N-oxides, from simple precursors. Our work highlights the importance of cellular transport optimization in recapitulating complex PNP biosyntheses in microbial hosts and illustrates the utility of computational methods for gene discovery and expansion of heterologous biosynthetic diversity.

The 8-azabicyclo[3.2.1]octane scaffold is the central core of the family of tropane alkaloids, which display a wide array of interesting biological activities. As a consequence, research directed towards the preparation of this basic structure in a stereoselective manner has attracted attention from many research groups worldwide across the years. Despite this, most of the approaches rely on the enantioselective construction of an acyclic starting material that contains all the required stereochemical information to allow the stereocontrolled formation of the bicyclic scaffold. As an alternative, there are a number of important methodologies reported in which the stereochemical control is achieved directly in the same transformation that generates the 8-azabicyclo[3.2.1]octane architecture or in a desymmetrization process starting from achiral tropinone derivatives. This review compiles the most relevant achievements in these areas.

The toxic alkaloids in forage grass present a serious health hazard to humans and livestock, especially ergot alkaloids (EAs), pyrrolizidine alkaloids (PAs) and tropane alkaloids (TAs). Hence, there is a need for a simultaneous method that allows these dangerous plant toxins to be determined. A simple and effective method was developed to determine fifteen toxic alkaloids (EAs, PAs and TAs) in forage grass using the QuEChERS method and liquid chromatography tandem mass spectrometry (LC-MS/MS). The developed method was validated with average recoveries ranging from 63.10 to 102.10%, and relative standard deviations lower than or equal to 6.39% were obtained. Good linearity over the concentration range of 10-600 µg/kg dry matter (DM) was observed for the target alkaloids. The determination coefficients R2 calculated for each of the matrix calibration curves were greater than 0.99. The limits of detection and quantification were 5 µg/kg DM and 10 µg/kg DM, respectively. The reproducibility of the method was verified in three laboratories: all of the mean recoveries of 15 alkaloids were higher than 60%, and the relative standard deviations in alkaloids between laboratories were all less than 14.24%. The proposed method was applied to analyse 134 forage grass samples from the meadow steppe of Inner Mongolia to monitor toxic alkaloids. A significant difference in the frequency of contamination was found between different herbage species and different regions.

We recently reported a convergent strategy to access the polycyclic guanidinium alkaloid (+)-batzelladine B via an aldol addition–retro-aldol–aza-Michael addition cascade. Here we describe the application of this approach toward the total syntheses of (+)-batzelladine E, (−)-dehydrobatzelladine C, and (+)-batzelladine K. The identification of suitable methods to functionalize a common tropane core by electrophilic alkynylation and nucleophilic 1,2-addition were essential to generalizing this approach. We provide evidence for the intermediacy of an acylallene species in the cascade reaction.

Covering: 1917 to 2020Tropane alkaloids (TAs) are a remarkable class of plant secondary metabolites, which are characterized by an 8-azabicyclo[3.2.1]octane (nortropane) ring. Members of this class, such as hyoscyamine, scopolamine, and cocaine, are well known for their long history as poisons, hallucinogens, and anaesthetic agents. Since the structure of the tropane ring system was first elucidated in 1901, organic chemists and biochemists have been interested in how these mysterious tropane alkaloids are assembled in vitro and in vivo. However, it was only in 2020 that the complete biosynthetic route of hyoscyamine and scopolamine was clarified, and their de novo production in yeast was also achieved. The aim of this review is to present the innovative ideas and results in exploring the story of tropane alkaloid biosynthesis in plants from 1917 to 2020. This review also highlights that Robinson's classic synthesis of tropinone, which is one hundred years old, is biomimetic, and underscores the importance of total synthesis in the study of natural product biosynthesis.

The secondary metabolites of nightshade and coca plant families contain important compounds known as tropane alkaloids (TAs). TAs possess pharmaceutical properties and are used in the treatment of several neurological disorders such as cardiac arrhythmia, Parkinson’s disease, etc. These compounds are also used as bronchodilators and anesthetics, although their prolonged use has many side effects. Comparing the global consumption rate of the TAs to the production by the original plant source, their yield is too low. Thus, it is important to increase their production. Several TAs such as scopolamine, spermidine, and hyoscyamine have been subjected to the de novo manufacturing processes for their large-scale production. This chapter summarizes the scientific efforts used to scale up the production of TAs for medicinal purposes. Several strategies such as tissue culture, root culture, hairy root culture, and genetically modified microbe (bacteria and yeast)-mediated large-scale production have been described. However, the best strategies are expected to come in near future, which could fulfill the gap of requirement and the production rate of TAs along with their preserved bioactivity.

Mycotoxins and pesticides regularly co-occur in agricultural products worldwide. Thus, humans can be exposed to both toxic contaminants and pesticides simultaneously, and multi-methods assessing the occurrence of various food contaminants and residues in a single method are necessary. A two-dimensional high performance liquid chromatography tandem mass spectrometry method for the analysis of 40 (modified) mycotoxins, two plant growth regulators, two tropane alkaloids, and 334 pesticides in cereals was developed. After an acetonitrile/water/formic acid (79:20:1, v/v/v) multi-analyte extraction procedure, extracts were injected into the two-dimensional setup, and an online clean-up was performed. The method was validated according to Commission Decision (EC) no. 657/2002 and document N° SANTE/12682/2019. Good linearity (R2 > 0.96), recovery data between 70-120%, repeatability and reproducibility values < 20%, and expanded measurement uncertainties < 50% were obtained for a wide range of analytes, including very polar substances like deoxynivalenol-3-glucoside and methamidophos. However, results for fumonisins, zearalenone-14,16-disulfate, acid-labile pesticides, and carbamates were unsatisfying. Limits of quantification meeting maximum (residue) limits were achieved for most analytes. Matrix effects varied highly (−85 to +1574%) and were mainly observed for analytes eluting in the first dimension and early-eluting analytes in the second dimension. The application of the method demonstrated the co-occurrence of different types of cereals with 28 toxins and pesticides. Overall, 86% of the samples showed positive findings with at least one mycotoxin, plant growth regulator, or pesticide. Graphical abstract

Graphical abstract, Highlights • Antivirals against SARS-CoV-2 are needed. • The papain-like protease represents an important target for antivirals. • We investigated tropane alkaloids from Schizanthus porrigens. • By molecular docking and MS simulations we identified two leads. • Shizanthine Z has favorable ADME properties and can be considered a lead., This paper presents identification of potential inhibitors of SARS-CoV-2 papain-like protease from tropane alkaloids from Schizanthus porrigens, using molecular docking method. Binding affinities were compared with those obtained with Lopinavir as a SARS-CoV-2 papain-like protease inhibitor. Overall, our findings indicate that Schizanthine Z binds to the SARS-CoV-2 papain-like protease with relatively high affinity and favorable ADME properties. Therefore, Schizanthine Z may represent an appropriate compound for further evaluation in antiviral assays.

The tropane alkaloids (TAs) hyoscyamine and scopolamine function as acetylcholine receptor antagonists and are used clinically as parasympatholytics to treat neuromuscular disorders in humans. While TAs are synthesized in a small subset of plant families, these specialized metabolites are only accumulated in limited quantities in plant organs. The complex chemical structures of these compounds make their industrial production by chemical synthesis very challenging, Therefore, the supply of these TAs still relies on intensive farming of Duboisia shrubs in tropical countries. Many adverse factors such as climate fluctuations and pandemics can thus influence annual world production. Based on the landmark microbial production of the antimalarial semi-synthetic artemisinin, the Smolke group recently developed a yeast cell factory capable of de novo synthesizing hyoscyamine and scopolamine, thus paving the way for an alternative production of these compounds.

Here we demonstrate that the antigen binding function of a humanized anti-cocaine mAb (h2E2) can be directly and easily determined using simple and inexpensive absorption spectroscopy and dyes commonly used for differential scanning fluorimetry, such as DASPMI and SYPRO Orange. Therapeutic monoclonal antibodies are commonly formulated in buffers which can interfere with necessary functional assays, containing additives and excipients such as mild detergents. Using the undiluted therapeutic product h2E2 mAb in its formulation buffer containing 0.01% polysorbate 80, the number of antigen/cocaine binding sites can be determined by the increase in absorbance (for DASPMI dye) or by the decrease in absorbance maximum wavelength (for SYPRO Orange dye), confirming proper function of the therapeutic mAb product. This ligand-induced visible dye absorption change can also be used to qualitatively evaluate the relative affinities of various metabolites of cocaine. These results are confirmed and extended by binding data obtained in the same formulation buffer using intrinsic tyrosine and tryptophan fluorescence quenching by cocaine, as well as by differential scanning fluorimetry. Interestingly, the binding of the cocaine metabolite norcocaine was demonstrated to be differentially affected by the pH 6 formulation buffer used for this mAb, presumably due to the differential ionizability of the demethylated norcocaine tropane ring nitrogen. This simple, direct, and inexpensive technique should prove useful for evaluation of other small molecule binding mAbs directly in their formulation buffers containing detergent, allowing rapid functional assessment of the produced therapeutic proteins.

Tropane alkaloids from nightshade plants are neurotransmitter inhibitors that are used for treating neuromuscular disorders and are classified as essential medicines by the World Health Organization1,2. Challenges in global supplies have resulted in frequent shortages of these drugs3,4. Further vulnerabilities in supply chains have been revealed by events such as the Australian wildfires5 and the COVID-19 pandemic6. Rapidly deployable production strategies that are robust to environmental and socioeconomic upheaval7,8 are needed. Here we engineered baker's yeast to produce the medicinal alkaloids hyoscyamine and scopolamine, starting from simple sugars and amino acids. We combined functional genomics to identify a missing pathway enzyme, protein engineering to enable the functional expression of an acyltransferase via trafficking to the vacuole, heterologous transporters to facilitate intracellular routing, and strain optimization to improve titres. Our integrated system positions more than twenty proteins adapted from yeast, bacteria, plants and animals across six sub-cellular locations to recapitulate the spatial organization of tropane alkaloid biosynthesis in plants. Microbial biosynthesis platforms can facilitate the discovery of tropane alkaloid derivatives as new therapeutic agents for neurological disease and, once scaled, enable robust and agile supply of these essential medicines.

A unified synthetic approach was developed that enabled the synthesis of diverse tropane-related scaffolds. The key intermediates that were exploited were cycloadducts formed by reaction between 3-hydroxy-pyridinium salts and vinyl sulfones or sulfonamides. The diverse tropane-related scaffolds were formed by addition of substituents to, cyclisation reactions of, and fusion of additional ring(s) to the key bicyclic intermediates. A set of 53 screening compounds was designed, synthesised and evaluated in order to determine the biological relevance of the scaffolds accessible using the synthetic approach. Two inhibitors of Hedgehog signalling, and four compounds with weak activity against the parasite P. falciparum, were discovered. Three of the active compounds may be considered to be indotropane or pyrrotropane pseudo natural products in which a tropane is fused with a fragment from another natural product class. It was concluded that the unified synthetic approach had yielded diverse scaffolds suitable for the design of performance-diverse screening libraries.

Solanaceous medicinal plants produce tropane alkaloids (TAs). We discovered a novel gene from Atropa belladonna, AbPPAR, which encodes a phenylpyruvic acid reductase required for TA biosynthesis. AbPPAR was specifically expressed in root pericycles and endodermis. AbPPAR was shown to catalyze reduction of phenylpyruvic acid to phenyllactic acid, a precursor of TAs. Suppression of AbPPAR disrupted TA biosynthesis through reduction of phenyllactic acid levels. In summary, we identified a novel enzyme involved in TA biosynthesis.

Under hypoxic conditions, the expression of Vitreoscilla hemoglobin (VHb) in plants is proposed to increase the productivity of certain oxygen-requiring metabolic pathways by promoting the delivery of oxygen. Tropane alkaloids (TAs) are a class of important plant secondary metabolites with significant medicinal value; the final step in their biosynthesis requires oxygen. Whether heterologous expression of VHb, especially in different subcellular compartments, can accelerate the accumulation of TAs is not known. Herein, the effect of heterologous expression of VHb in different subcellular locations on the TA profile of H. niger hairy roots was investigated. The targeted expression of VHb in the plastids (using pVHb-RecA construct), led to the accumulation of 197.68 μg/g hyoscyamine in the transgenic H. niger hairy roots, which was 1.25-fold of the content present in the lines in which VHb expression was not targeted, and 3.66-fold of that present in the wild type (WT) lines. The content of scopolamine was increased by 2.20- and 4.70-fold in the pVHb-RecA transgenic lines compared to that in the VHb transgenic and WT lines. Our results demonstrate that VHb could stimulate the accumulation of TAs in the transgenic H. niger hairy roots. Quantitative RT-PCR analysis revealed that the expression of key genes involved in TA biosynthesis increased significantly in the VHb transgenic lines. We present the first description of a highly efficient strategy to increase TA content in H. niger. Moreover, our results also shed light on how the production of desired metabolites can be efficiently enhanced by using more accurate and appropriate genetic engineering strategies.

Stem bark, root bark, and leaf extracts of Erythroxylum pungens were subjected to phytochemical analysis. N,N-dimethyltryptamine (DMT) was isolated and characterized from E. pungens roots. This unprecedented result is remarkable since no indole alkaloid has been previously reported from Erythroxylaceae so far. Eleven known tropane alkaloids were identified by their mass spectra and 3-(2-methylbutyryloxy)tropan-6,7-diol as well as 3-(2-methylbutyryloxy)nortropan-6,7-diol were isolated and characterized based on mass spectrometry, 1H, 13C, COSY, and NOESY NMR analysis. The complete NMR data are reported for the first time. Inverse Structure-based and Ligand-Based virtual screening were carried out to identify possible targets for 3-(2-methylbutyryloxy)tropan-6,7-diol. The level of cytotoxicity of this tropane alkaloid aliphatic ester was discrete with potencies on the order of 0.3–1.0 mg/mL and better results against HeLa (50% cell viability reduction). Otherwise, atropine (0.3 mg/mL), a Solanaceae tropane alkaloid, and DMT (0.5 mg/mL) from E. pungens roots impaired at 50% the cell viability against HeLa, SiHa, PC3, and 786-0. This study stimulates scientific investigation of the impact of edaphoclimatic features in a semi-arid environment on tropane alkaloid biosynthesis.

N-methylputrescine is the precursor of nicotine and pharmaceutical tropane alkaloids such as hyoscyamine. Putrescine N-methyltransferase (PMT) catalyzes the N-methylation of putrescine to form N-methylputrescine. While the role of PMT in nicotine biosynthesis is clear, knowledge of PMT in the biosynthesis of tropane alkaloids (TAs) and the regulation of polyamines remains limited. We characterized a PMT gene from Hyoscyamus niger, designated HnPMT that was specifically expressed in roots, especially in the secondary roots and dramatically induced by methyl jasmonate (MeJA). The GUS gene was specifically expressed in Arabidopsis roots or in the vascular tissues, including pericycles and endodermis, of the H. niger hairy root cultures, when it was driven by the 5'-flanking promoter region of HnPMT. The recombinant HnPMT was purified for enzymatic assays. HnPMT converted putrescine to form N-methylputrescine, as confirmed by LC-MS. The kinetics analysis revealed that HnPMT had high affinity with putrescine but low catalytic activity, suggesting that it was a rate-limiting enzyme. When HnPMT was suppressed in the H. niger plants by using the VIGS approach, the contents of N-methylputrescine and hyoscyamine were markedly decreased, but the contents of putrescine, spermidine and a mixture of spermine and thermospermine were significantly increased; this suggested that HnPMT was involved in the biosynthesis of tropane alkaloids and played a competent role in regulating the biosynthesis of polyamines. Functional identification of HnPMT facilitated the understanding of TA biosynthesis and thus implied that the HnPMT-catalyzed step might be a target for metabolic engineering of the TA production in H. niger.

This Article describes the development of a second-generation catalyst system for the transannular C–H functionalization of alicyclic amines. Pyridine- and quinoline-carboxylate ligands are shown to be highly effective for increasing the reaction rate, yield, and scope of Pd-catalyzed transannular C–H arylation reactions of azabicyclo[3.1.0]hexane, azabicyclo[3.1.1]heptane, azabicyclo[3.2.1]octane, and piperidine derivatives. Mechanistic studies reveal that the pyridine/quinoline-carboxylates play a role in impeding both reversible and irreversible catalyst decomposition pathways. These ligands enable the first reported examples of the transannular C–H arylation of the ubiquitous tropane, 7-azanorbornane, and homotropane cores. Finally, the pyridine/quinoline-carboxylates are shown to promote both transannular C–H arylation and transannular C–H dehydrogenation on a homotropane substrate.

Scopolia lurida, a medicinal plant native to the Tibetan Plateau, is among the most effective producers of pharmaceutical tropane alkaloids (TAs). The hyoscyamine 6β-hydroxylase genes of Hyoscyamus niger (HnH6H) and S. lurida (SlH6H) were cloned and respectively overexpressed in hairy root cultures of S. lurida, to compare their effects on promoting the production of TAs, especially the high-value scopolamine. Root cultures with SlH6H/HnH6H overexpression were confirmed by PCR and real-time quantitative PCR, suggesting that the enzymatic steps defined by H6H were strongly elevated at the transcriptional level. Tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were analyzed by HPLC. Scopolamine and anisodamine contents were remarkably elevated in the root cultures overexpressing SlH6H/HnH6H, whereas that of hyoscyamine was more or less reduced, when compared with those of the control. These results also indicated that SlH6H and HnH6H promoted anisodamine production at similar levels in S. lurida root cultures. More importantly, HnH6H-overexpressing root cultures had more scopolamine in them that did SlH6H-overexpressing root cultures. This study not only provides a feasible way of overexpressing H6H to produce high-value scopolamine in engineered root cultures of S. lurida but also found that HnH6H was better than SlH6H for engineering scopolamine production.

Dopamine transporter (DAT) blockers like cocaine and many other abused and therapeutic drugs bind and stabilize an inactive form of the transporter inhibiting reuptake of extracellular dopamine (DA). The resulting increases in DA lead to the ability of these drugs to induce psychomotor alterations and addiction, but paradoxical findings in animal models indicate that not all DAT antagonists induce cocaine-like behavioral outcomes. How this occurs is not known, but one possibility is that uptake inhibitors may bind at multiple locations or in different poses to stabilize distinct conformational transporter states associated with differential neurochemical endpoints. Understanding the molecular mechanisms governing the pharmacological inhibition of DAT is therefore key for understanding the requisite interactions for behavioral modulation and addiction. Previously, we leveraged complementary computational docking, mutagenesis, peptide mapping, and substituted cysteine accessibility strategies to identify the specific adduction site and binding pose for the crosslinkable, photoactive cocaine analog, RTI 82, which contains a photoactive azide attached at the 2β position of the tropane pharmacophore. Here, we utilize similar methodology with a different cocaine analog N-[4-(4-azido-3-I-iodophenyl)- butyl]-2-carbomethoxy-3- (4-chlorophenyl) tropane, MFZ 2–24, where the photoactive azide is attached to the tropane nitrogen. In contrast to RTI 82, which crosslinked into residue Phe319 of transmembrane domain (TM) 6, our findings show that MFZ 2–24 adducts to Leu80 in TM1 with modeling and biochemical data indicating that MFZ 2–24, like RTI 82, occupies the central S1 binding pocket with the (+)-charged tropane ring nitrogen coordinating with the (−)-charged carboxyl side chain of Asp79. The superimposition of the tropane ring in the three-dimensional binding poses of these two distinct ligands provides strong experimental evidence for cocaine binding to DAT in the S1 site and the importance of the tropane moiety in competitive mechanisms of DA uptake inhibition. These findings set a structure-function baseline for comparison of typical and atypical DAT inhibitors and how their interactions with DAT could lead to the loss of cocaine-like behaviors.

Tropane alkaloids are a wide group of substances that comprises more than 200 compounds occurring especially in the Solanaceae family. The main aim of this study is the development of a method for the analysis of the principal tropane alkaloids as atropine, scopolamine, anisodamine, tropane, tropine, littorine, homatropine, apoatropine, aposcopolamine, scopoline, tropinone, physoperuvine, pseudotropine and cuscohygrine in cereals and related matrices. For that, a simple solid-liquid extraction was optimized and a liquid chromatographic method coupled to a single stage Exactive-Orbitrap was developed. The method was validated obtaining recoveries in the range of 60-109% (except for some compounds in soy), precision values (expressed as relative standard deviation) lower than 20% and detection and quantification limits equal to or lower than 2 and 3μg/kg respectively. Finally, the method was applied to the analysis of different types of samples as buckwheat, linseed, soy and millet, obtaining positives for anisodamine, scopolamine, atropine, littorine and tropinone in a millet flour sample above the quantification limits, whereas atropine and scopolamine were detected in a buckwheat sample, below the quantification limit. Contaminated samples with Solanaceaes seeds (Datura Stramonium and Brugmansia Arborea) were also analysed, detecting concentrations up to 693μg/kg (scopolamine) for contaminated samples with Brugmansia seeds and 1847μg/kg (atropine) when samples were contaminated with Stramonium seeds.

Tetraploidy improves overexpression of h6h and scopolamine production of H. muticus, while in H. senecionis, pmt overexpression and elicitation can be used as effective methods for increasing tropane alkaloids. The effects of metabolic engineering in a polyploid context were studied by overexpression of h6h in the tetraploid hairy root cultures of H. muticus. Flow cytometry analysis indicated genetic stability in the majority of the clones, while only a few clones showed genetic instability. Among all the diploid and tetraploid clones, the highest level of h6h transgene expression and scopolamine accumulation was interestingly observed in the tetraploid clones of H. muticus. Therefore, metabolic engineering of the tropane biosynthetic pathway in polyploids is suggested as a potential system for increasing the production of tropane alkaloids. Transgenic hairy root cultures of Hyoscyamus senecionis were also established. While overexpression of pmt in H. senecionis was correlated with a sharp increase in hyoscyamine production, the h6h-overexpressing clones were not able to accumulate higher levels of scopolamine than the leaves of intact plants. Applying methyl jasmonate was followed by a sharp increase in the expression of pmt and a drop in the expression of tropinone reductase II (trII) which consequently resulted in the higher biosynthesis of hyoscyamine and total alkaloids in H. senecionis.

Aromatic amino acid aminotransferases (AAA-ATs) catalyze the reversible transamination reactions of proteinogenic and non-proteinogenic aromatic amino acids to corresponding keto acids and vice versa. The products of plant AAA-ATs serve as key precursors of many primary and secondary metabolites that are crucial for both plant and human metabolism and physiology. In most microbes, l-tyrosine (Tyr) and l-phenylalanine (Phe) aminotransferases (Tyr and Phe-ATs) catalyze the final steps of Phe and Tyr biosynthesis. On the other hand, plants use different pathways to synthesize Tyr and Phe via arogenate, in which prephenate-specific aminotransferases (PPA-ATs) catalyze the committed step in the plastids. Plant Tyr and Phe-ATs, unlike microbial counterparts, often prefer the reverse reactions and metabolize Tyr and Phe to their respective aromatic keto acids, which serve as precursors of various plant natural products (e.g. benzenoid volatiles, tocochromanols, plastoquinone, and tropane and benzylisoquinoline alkaloids). Unlike plastidic PPA-ATs, plant Tyr/Phe-ATs are localized outside of the plastids, have broad substrate specificity, and interlink Tyr and Phe metabolism. l-Tryptophan (Trp) aminotransferases (Trp-ATs) are involved in biosynthesis of the plant hormone auxin. Although significant advancement has been made on biochemical, molecular, and genetic characterizations of plant AAA-ATs, there are still many critical knowledge gaps, which are highlighted in the current review.

The objective of this study was to investigate the effects of 2,3,4-trimethoxy-N'-(8-metyl-8-azabicyclo[3.2.1.] octan-3-ylidene) benzohydrazide hydrochloride (LK-933), a compound of the tropane group with anxiolytic and antimigraine activity, on the levels of monoamines and their metabolites in different brain structures of outbred mice using HPLC–ED. Administration of LK-933 caused an increase in the composite characteristics that describe the rate of dopamine transformation into its metabolites dioxyphenylacetic acid and homovanillic acid in the frontal cortex by 114 and 181%, respectively. The levels of homovanillic acid in the same structure increased by 49%. These data implicate the dopaminergic pathway in the mechanism of LK-933 action.

Cocaine, a widely abused psychostimulant, inhibits the dopamine transporter (DAT) by trapping the protein in an outward-open conformation, whereas atypical DAT inhibitors such as benztropine have low abuse liability and prefer less outward-open conformations. Here, we use a spectrum of computational modeling and simulation approaches to obtain the underlying molecular mechanism in atomistic detail. Interestingly, our quantum mechanical calculations and molecular dynamics (MD) simulations suggest that a benztropine derivative JHW007 prefers a different stereoisomeric conformation of tropane in binding to DAT compared to that of a cocaine derivative, CFT. To further investigate the different inhibition mechanisms of DAT, we carried out MD simulations in combination with Markov state modeling analysis of wild-type and Y156F DAT in the absence of any ligand or the presence of CFT or JHW007. Our results indicate that the Y156F mutation and CFT shift the conformational equilibrium toward an outward-open conformation, whereas JHW007 prefers an inward-occluded conformation. Our findings reveal the mechanistic details of DAT inhibition by JHW007 at the atomistic level, which provide clues for rational design of atypical inhibitors.

Yeast has been engineered to convert simple sugars and amino acids into drugs that inhibit a neurotransmitter molecule. The work marks a step towards making the production of these drugs more reliable and sustainable. An engineered strain of yeast produces tropane alkaloids.

Higher plants produce divergent classes of specialized (so called secondary) metabolites, such as alkaloids, phenolics, terpenoids, and so on. Among them, alkaloids, which contain nitrogen atom often in heterocyclic ring, are most biologically active and used as poisons and/or also pharmaceuticals. Whereas natural resources are commonly used for the preparation for these chemicals, shortage of natural resources in quantity and quality need more advanced technology for the production. Recent progresses in biochemistry, molecular biology and recombinant technology provide new way for the production of specialized metabolites without chemical synthesis. Metabolic engineering in target plants and reconstruction of biosynthetic pathway in heterologous hosts, especially microbes using synthetic biology, offers great potentials to improve the productivity and quality of these metabolites. In this report, recent progresses in the isolation and identification of molecular tools for metabolic engineering and synthetic biology are critically summarized and also some achievement of alkaloid engineering in benzylisoquinoline alkaloids (BIAs) such as berberine, morphine, noscapine and sanguinarine, monoterpenoid indole alkaloids (MIAs) such as vinca alkaloids, nicotine and tropane alkaloids, steroidal glycoalkaloids such as solanine and so on are summerized.

The hairy roots developed after Agrobacterium rhizogenes transformation can be used for the production of secondary metabolites which show medicinal properties. Plant secondary metabolites include various classes of chemical compounds, such as the tropane alkaloids found in some members of the Solanaceae family, for example, Atropa belladonna, Datura stramonium, D. innoxia, Hyoscyamus muticus, and H. niger. This chapter presents the strategy for producing tropane alkaloids such as scopolamine and hyoscyamine by the hairy roots of these species. High biomass production of D. stramonium and D. innoxia hairy roots and the associated bioactive compounds can be enabled by optimization of the medium. In addition, scopolamine and hyoscyamine production can be enhanced by optimizing various culture conditions, such as the type of the medium or sucrose concentration, and the ploidy level of H. muticus hairy roots. Their production by A. belladonna hairy roots can also be influenced by the ammonium or nitrate concentration of the medium. Tropane alkaloid production by hairy roots can be significantly increased by the use of elicitor treatments such as chitosan, acetic acid, citric acid, jasmonic acid, acetylsalicylic acid, or aluminum chloride: for example, treatment with Tween 20, combined with L-phenylalanine or DL-s-phenyllactic acid alone or with tropinone feeding, increased the level of hyoscyamine released into the medium by D. innoxia hairy roots. Secondary metabolite production can also be improved by transgenic manipulation: tropane alkaloid biosynthesis can be raised by overexpression of the genes encoding the enzymes involved in the tropane alkaloid biosynthesis pathway such as hyoscyamine-6s-hydroxylase (H6H), putrescine N-methyltransferase (PMT), or tropinone reductase I and II (TR I and TR II). Alkaloid production can be influenced by overexpression of genes encoding enzymes involved in the tropane alkaloid biosynthesis pathway, as well as by genetic manipulation based on gene silencing. The cultivation of D. innoxia and H. niger hairy roots in bubbling bioreactors may also serve as an efficient culture for large-scale tropane alkaloid production.

Six previously undescribed tropane alkaloids, designated as erythrobezerrines A-F, were isolated from the EtOH extract from the stem bark of Erythroxylum bezerrae Plowman. Their structures were elucidated based on the interpretation of the NMR and MS data and in some instances, confirmed by X-ray diffraction analysis. The cytotoxicity of the isolated compounds was evaluated against the cancer cell lines L929, PC-3, HCT-116, SNB-19 and NCI-H460, but only erythrobezerrine C showed moderate activity with IC50 values of 3.38 and 5.43 μM for HCT-116 and NCI-H460, respectively.

The application of ionic liquids (ILs) for the separation of bioactive compounds from various sample matrices is a burgeoning area. Here, a class of porous materials called metal–organic frameworks (MOFs) were combined with tropine-based ILs having structural similarity with natural tropane alkaloids for the quantitative analysis of Huashansheng tablets. Three MOFs (MIL-101, HKUST-1 and ZIF-8) were used to form the composite with the a tropine-based IL (N-propyl-substituted tropine hexafluorophosphate, [C3tr][PF6]), using the ship-in-a-bottle (SIB) synthesis approach. The performance of the hybrid IL/MOF composite (IL@MOF) was evaluated as a potential sorbent for application in the dispersive solid-phase-extraction-based enrichment of tropane alkaloids, followed by chromatographic analysis. It was found that [C3tr][PF6]@MIL-101 exhibited excellent adsorption capacity, and the extraction results showed satisfactory recoveries in the range of 91.5–104.7% (RSD < 5%). Further, the limit of detection went down to 10–20 μg L−1 with a linear range from 100–500 μg L−1, which proved that an adsorbent with the same nucleus as the target objects would be more suitable and efficient for their later extraction.

Tropane alkaloids (TAs) are a class of phytochemicals produced by plants of the nightshade family used for treating diverse neurological disorders. Here, we demonstrate de novo production of tropine, a key intermediate in the biosynthetic pathway of medicinal TAs such as scopolamine, from simple carbon and nitrogen sources in yeast (Saccharomyces cerevisiae). Our engineered strain incorporates 15 additional genes, including 11 derived from diverse plants and bacteria, and 7 disruptions to yeast regulatory or biosynthetic proteins to produce tropine at titers of 6 mg/L. We also demonstrate the utility of our engineered yeast platform for the discovery of TA derivatives by combining biosynthetic modules from distant plant lineages to achieve de novo production of cinnamoyltropine, a non-canonical TA. Our engineered strain constitutes a starting point for future optimization efforts towards realizing industrial fermentation of medicinal TAs and a platform for the synthesis of TA derivatives with enhanced bioactivities., Tropane alkaloids (TAs) are a group of phytochemicals that are used to treat neurological disorders. Here, the authors engineer baker’s yeast to produce tropine, a key intermediate in the biosynthetic pathway of TAs, and cinnamoyltropine, a non-canonical TA, from simple carbon and nitrogen sources.