Synthesis Of Artemisinin Research Articles

Synthesis of Artemisinin G from Artemisinin via Photocatalysis

AbstractPhotoredox catalysis has become a mainstay in synthetic methodology over the past decade. However, its application in the functionalization of natural products remains to be further explored. Herein, we report an efficient approach to synthesize artemisinin G from artemisinin via photocatalysis in high yield.

Comparison of phytochemical properties and expressional profiling of artemisinin synthesis-related genes in various Artemisia species

The Artemisia genus belongs to the Asteraceae family and is used in the treatment of many different diseases such as hepatitis and cancer. So far, around 500 species of Artemisia have been found in different regions of the world. Artemisinin is one of the medicinal compounds found in Artemisia species. Hence, this medical feature encourages researchers to pay attention to various species of this genus to discover more genetic and phytochemical information. In the present study, five species of Artemisia including A. fragrans, A. annua, A. biennis, A. scoparia, and A. absinthium were compared to each other in terms of the artemisinin content and other phytochemical components. Moreover, the relative expression profiles of eight genes related to the accumulation and synthesis of artemisinin [including 4FPSF, DBR2, HMGR1, HMGR2, WIRKY, ADS, DXS, and SQS] were determined in investigated species. The result of high-performance liquid chromatography (HPLC) analysis showed that the content of artemisinin in various species was in the order of A. fragrans > A. annua > A. biennis > A. scoparia > A. absinthium. Based on the gas chromatography–mass spectrometry (GC-MS) analysis, 34, 26, 26, 24, and 20 phytochemical compounds were identified for A. scoparia, A. biennis, A. fragrans, A. absinthum, and A. annua species, respectively. Moreover, camphor (38.86%), β-thujone (68.42%), spathulenol (48.33%), β-farnesene (48.16%), and camphor (29.04%) were identified as the considerable compounds A. fragrans, A. absinthium, A. scoparia, A. biennis, and A. annua species, respectively. Considering the relative expression of the targeted genes, A. scoparia revealed higher expression for the 4FPSF gene. The highest relative expression of the DBR2, WIRKY, and SQS genes was found in A. absinthium species. Moreover, A. annua showed the highest expression of the ADS and DXS genes than the other species. In conclusion, our findings revealed that various species of Artemisia have interesting breeding potential for further investigation of different aspects such as medicinal properties and molecular studies.

Synthesis of Novel Artemisinin, Ciprofloxacin, and Norfloxacin Hybrids with Potent Antiplasmodial Activity.

The synthesis and antiplasmodial evaluation of new hybrids combining the pharmacophore structures of artemisinin, ciprofloxacin or norfloxacin, and 7-chloroquinoline are reported in this study. The first step for all of the syntheses is the obtainment of key piperazine esters intermediates bearing the drugs ciprofloxacin and norfloxacin. Using these platforms, 18 final compounds were synthesized through a multistep procedure with overall yields ranging between 8 and 20%. All compounds were screened for their antiplasmodial activity against the chloroquine-resistant Plasmodium falciparum FcB1 strain. Compounds 20, 21, 22, and 28, bearing an artesunate fragment with ciprofloxacin, exhibited IC50 values in the range of 3.5-5.4 nM and excellent selectivity indices. Among the compounds bearing the artesunate moiety on the norfloxacin, two of them, 23 and 24, afforded IC50 values of 1.5 nM and 1.9 nM, respectively. They also showed excellent selectivity indices. The most potent compounds were also evaluated against the CQ-resistant Dd2 strain of Plasmodium falciparum, demonstrating that those compounds incorporating the artesunate fragment were the most potent. Finally, the combination of artesunate with either ciprofloxacin or norfloxacin moieties in a single molecular entity proved to substantially enhance the activity and selectivity when compared to the administration of the unconjugated counterparts artesunate/ciprofloxacin and artesunate/norfloxacin.

Foliar treatment with MSB (menadione sodium bisulphite) to increase artemisinin content in Artemisia annua plants

Artemisinin is a sesquiterpene lactone endoperoxide, recognized for its application as a treatment for malaria. Besides, it has activity against several types of cancers, as well as anti-viral and anti-inflammatory properties. The chemical synthesis of artemisinin is quite complex and economically unfeasible. Hence, artemisinin is typically obtained from Artemisia annua plants in low quantities in relation to the biomass. A strategy to increase the biosynthesis of secondary metabolites would be the application of elicitors. In this work, the effect of treating A. annua plants with menadione sodium bisulphite (MSB) is studied. An initial dose-optimization was conducted by treating the plants with different MBS concentrations (0, 0.2, 0.8, 1, 2, 3 and 4 mM) and artemisinin was quantified 48 h after treatment. The highest artemisinin content (3.71 mg artemisinin g−1 DW) was obtained with 1 mM of MSB. Then, harvesting time was optimised (24, 48 and 72 h after treatment application) with 1 mM of MSB. The largest increase in artemisinin content was observed at 48 h with an increment of 62.37 % over the control. The treatment increased H2O2 content, as well as the activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX). Besides, MSB up-regulated HMGR (the gene for 3-hydroxy-3-methylglutaryl-CoA reductase) and DXS (the gene for 1-deoxy-D-xylulose-5-phosphate synthase) 24 h after treatment. Both genes are involved in the MVA (mevalonate) and MEP (2C-methylerythritol 4-phosphate) pathways, respectively. These pathways represent the initiation of artemisinin biosynthesis. Thus, MSB application and the consequent up-regulation of early biosynthetic pathway genes along with the triggered oxidative stress may have been contributed to the observed increase in artemisinin production.

Heterologous Production of Artemisinin in Physcomitrium patens by Direct in vivo Assembly of Multiple DNA Fragments.

The sesquiterpene lactone compound artemisinin is a natural medicinal product of commercial importance. This Artemisia annua-derived secondary metabolite is well known for its antimalarial activity and has been studied in several other biological assays. However, the major shortcoming in its production and commercialization is its low accumulation in the native plant. Moreover, the chemical synthesis of artemisinin is difficult and expensive due to its complex structure. Hence, an alternative and sustainable production system of artemisinin in a heterologous host is required. Previously, heterologous production of artemisinin was achieved by Agrobacterium-mediated transformation. However, this requires extensive bioengineering of modified Nicotiana plants. Recently, a technique involving direct in vivo assembly of multiple DNA fragments in the moss, P. patens, has been successfully established. We utilized this technique to engineer artemisinin biosynthetic pathway genes into the moss, and artemisinin was obtained without further modifications with high initial production. Here, we provide protocols for establishing moss culture accumulating artemisinin, including culture preparation, transformation method, and compound detection via HS-SPME, UPLC-MRM-MS, and LC-QTOF-MS. The bioengineering of moss opens up a more sustainable, cost effective, and scalable platform not only in artemisinin production but also other high-value specialized metabolites in the future.

From Plant to Yeast-Advances in Biosynthesis of Artemisinin.

Malaria is a life-threatening disease. Artemisinin-based combination therapy (ACT) is the preferred choice for malaria treatment recommended by the World Health Organization. At present, the main source of artemisinin is extracted from Artemisia annua; however, the artemisinin content in A. annua is only 0.1–1%, which cannot meet global demand. Meanwhile, the chemical synthesis of artemisinin has disadvantages such as complicated steps, high cost and low yield. Therefore, the application of the synthetic biology approach to produce artemisinin in vivo has magnificent prospects. In this review, the biosynthesis pathway of artemisinin was summarized. Then we discussed the advances in the heterologous biosynthesis of artemisinin using microorganisms (Escherichia coli and Saccharomyces cerevisiae) as chassis cells. With yeast as the cell factory, the production of artemisinin was transferred from plant to yeast. Through the optimization of the fermentation process, the yield of artemisinic acid reached 25 g/L, thereby producing the semi-synthesis of artemisinin. Moreover, we reviewed the genetic engineering in A. annua to improve the artemisinin content, which included overexpressing artemisinin biosynthesis pathway genes, blocking key genes in competitive pathways, and regulating the expression of transcription factors related to artemisinin biosynthesis. Finally, the research progress of artemisinin production in other plants (Nicotiana, Physcomitrella, etc.) was discussed. The current advances in artemisinin biosynthesis may help lay the foundation for the remarkable up-regulation of artemisinin production in A. annua through gene editing or molecular design breeding in the future.

ARTEMISININA E DERIVADOS: DESCOBERTA, ESTRATÉGIAS SINTÉTICAS E OBTENÇÃO INDUSTRIAL

ARTEMISININ AND DERIVATIVES: DISCOVERY, SYNTHETIC STRATEGIES, AND INDUSTRIAL PRODUCTION. Artemisinin, a natural sesquiterpene isolated from the Artemisia annua L. plant, represents a remarkable achievement in the treatment against malaria. Since its discovery in the 1970’s, researchers readily started synthesizing this complex natural product containing an unusual endoperoxide fragment. Despite various total syntheses reported, only in the last decade the production jumped from extraction/bench scale to industrial manufacturing. In this sense, the objective of this contribution is to highlight the discovery, the most representative works towards the synthesis of artemisinin and its main derivatives used worldwide against malaria, and the path to the industrial production of these compounds.

Kinetic analysis of the partial synthesis of artemisinin: Photooxygenation to the intermediate hydroperoxide

The price of the currently best available antimalarial treatment is driven in large part by the limited availability of its base drug compound artemisinin. One approach to reduce the artemisinin cost is to efficiently integrate the partial synthesis of artemisinin starting from its biological precursor dihydroartemisinic acid (DHAA) into the production process. The optimal design of such an integrated process is a complex task that is easier to solve through simulations studies and process modelling. In this article, we present a quantitative kinetic model for the photooxygenation of DHAA to an hydroperoxide, the essential initial step of the partial synthesis to artemisinin. The photooxygenation reactions were studied in a two-phase photo-flow reactor utilizing Taylor flow for enhanced mixing and fast gas-liquid mass transfer. A good agreement of the model and the experimental data was achieved for all combinations of photosensitizer concentration, photon flux, fluid velocity and both liquid and gas phase compositions. Deviations between simulated predictions and measurements for the amount of hydroperoxide formed are 7.1 % on average. Consequently, the identified and parameterized kinetic model is exploited to investigate different behaviors of the reactor under study. In a final step, the kinetic model is utilized to suggest attractive operating windows for future applications of the photooxygenation of DHAA exploiting reaction rates that are not affected by mass transfer limitations.

Photochemical Hydrothiolation of Amorphadiene and Formal Synthesis of Artemisinin via a Pummerer Rearrangement.

A new access to artemisinin is reported based on a selective photochemical hydrothiolation of amorphadiene, a waste product of the industrial semisynthetic route. This study highlights the discovery of two distinctive activation pathways under solvent-free conditions or using a photocatalyst promoting H-abstraction. Subsequently, a chemoselective oxidation of the resulting photochemically generated thioether, followed by a Pummerer rearrangement, affords dihydroartemisinic aldehyde, a key intermediate in the synthesis of artemisinin.

Chemo- and Diastereoselective Hydrosilylation of Amorphadiene toward the Synthesis of Artemisinin.

A formal synthesis of artemisinin starting from amorphadiene is described. This new route relies on the development of a catalytic chemo- and diastereoselective hydrosilylation. The practicability of this method is demonstrated by converting amorphadiene to dihydroartemisinic aldehyde using a one-pot hydrosilylation/oxidation sequence, minimizing the number of purifications and maximizing the productivity through a practical one-pot procedure. In addition, this approach can be coupled with a crystallization-induced diastereoselective transformation (CIDT) to enhance the optical purity of the key target intermediate, dihydroartemisinic aldehyde.

Kinetics study on reaction between dihydroartemisinic acid and singlet oxygen: An essential step to photochemical synthesis of artemisinin

Artemisinin is an excellent antimalarial drug widely used in clinical medicine. However, due to the limitation of natural source of artemisinin, the chemical synthesis of artemisinin has achieved substantial attention. Dihydroartemisinic acid is a key precursor for the synthesis of artemisinin. The reaction of dihydroartemisinic acid with singlet oxygen to form peroxide is a pivotal step in the photochemical preparation of artemisinin. Nevertheless, the reaction kinetics of dihydroartemisinic acid with singlet oxygen has not been investigated previously. Herein, we report the rate constants of the reaction between dihydroartemisinic acid and singlet oxygen. By directly detecting the luminescence decay kinetics of singlet oxygen at 1270 nm at room temperature, the reaction rate constants of singlet oxygen and dihydroartemisinic acid in different solvents are obtained to be 1.81×105 (mol/L)−1·s−1 in CCl4, 5.69×105 (mol/L)−1·s−1 in CH3CN, and 3.27×106 (mol/L)−1·s−1 in DMSO, respectively. It is found that the reaction rate constants of dihydroartemisinic acid with singlet oxygen increase as polarity of the solvent increases among the three solvents. These results provide fundamental knowledge to optimize experiment conditions of photochemical synthesis of artemisinin for improving the yields of artemisinin.

Wanbin Zhang.

"My greatest achievement has been the synthesis of artemisinin. My biggest motivation was to make the ambitions of my youth become reality today …" Find out more about Wanbin Zhang in his Author Profile.

Advances in synthesis of artemisinin based on plant genetic engineering

Artemisinin is a kind of sesquiterpene lactone containing endoperoxide bridge,which is the most effective anti-malarial drug at present. However,low content of artemisinin in Artemisia annua,ranging from 0. 1%-1. 0% of dry weight,as well as the complicated extraction process have resulted in low yield and high cost of artemisinin,making it difficult to meet market demand.Based on the development of high-throughput sequencing and molecular biology,the related enzyme genes and transcription factors involved in the artemisinin metabolic pathway were cloned and identified. Metabolic engineering and synthetic biology methods to modify the original metabolic pathway of A. annua and genetic engineering in heterologous host cells have become one of the hotspots in this field. Therefore,the molecular mechanism of artemisin biosynthesis,different strategies of genetic modifications of A. annua,and the research status and application prospect of artemisinin synthesis in heterologous host cells( Nicotiana benthamiana,Physcomitrella patens) were summarized in our review,hoping to provide molecular basis and theoretical basis for breeding new varieties of A. annua with high artemisinin output.

Synthesis of artemisinin derived glycoconjugates inspired by click chemistry

Cu(i)-catalysed click reactions between β-propargylated dihydroartemisinin and azido sugars were carried out to furnish artemisinin based glycoconjugates.

The Study of Artemisinin Content in Callus Artemisia annua L. Cultures Elicited with Endophytic Fungi Aspergillus sp.

Artemisinin, is a sesquiterpene lactone which is originally extracted from the medicinal plant Artemisia annua L. Artemisinin is a very effective antimalarial agent, however it is produced in very low amounts in plants. Chemical synthesis of artemisinin is complicated and not economically feasible. Researchers often use elicitation methods to increase artemisinin content in a laboratory scale with biotic or abiotic elicitor. This study examined the effect of elicitation using endophytic fungi Aspergillus sp on artemisinin content of callus A. annua cultures. Observations were made on its growth, texture and color, chlorophyll content and dry weight. Fungi were isolated from the stem tissue of A. annua and callus were grown on Murashige and Skoog medium. After 3 weeks of incubation, callus treated with four different elicitors concentration (0, 2, 4, 6 mg/mL). There were significant alterations on callus culture in terms of growth, texture, color and chlorophyll content after elicitation. The results clearly demonstrated that the endophytic fungal elicitor Aspergillus sp could increase artemisinin content but did not affect the callus dry weight of A. annua. The treatment of 4 mg/mL elicitor produced the highest artemisinin content, that increased 7.6 times compared to the control, from 0.0036% to 0.0275%.

Mechanism of high temperature promoting artemisinin biosynthesis in Artemisia annua

Artemisia annua also known as Qinghao, is a kind of traditional Chinese medicine. Its active ingredient is artemisinin, a sesquiterpene lactone compound with a peroxy bridging group structure. A. annua is an effective antimalarial drug. Artemisinin, a secondary metabolite in A. annua, can be induced by many physical and chemical factors, such as salinity, moisture, light, and plant hormones. Temperature, as an important growth factor, also has a great influence on the synthesis of artemisinin. This article aims to study the effect of high temperature on inducing artemisinin biosynthesis in A. annua. The A. annua seedlings were placed at 25, 40 °C, and the samples were taken after 0, 3, 12 and 36 h. The content of artemisinin in each sample was determined by liquid chromatography-mass spectrometry. Total RNA was extracted from the samples, and then transcriptome sequencing and real-time fluorescence quantitative PCR were used to quantitatively analyze the expression of the key enzyme genes in artemisinin synthesis pathway and competition pathway. The results showed that artemisinin content was increased by 20%, 42% and 68% after 3, 12, 36 h of treatment at 40 °C. The expression levels of FDS, ALDH1, CYP71AV1 and ADS were up-regulated by 4.3, 3.3, 2.5, 1.9 times, and the expression levels of SQS and BPS were down-regulated by 37% and 90% respectively. In summary, high temperature can promote the biosynthesis of artemisinin by promoting the expression of synthetase genes in artemisinin synthesis pathway and inhibiting the expression of synthetase genes in artemisinin-competition pathway.

Silencing amorpha-4,11-diene synthase Genes in Artemisia annua Leads to FPP Accumulation.

Artemisia annua is established as an efficient crop for the production of the anti-malarial compound artemisinin, a sesquiterpene lactone synthesized and stored in Glandular Secretory Trichomes (GSTs) located on the leaves and inflorescences. Amorpha-4,11-diene synthase (AMS) catalyzes the conversion of farnesyl pyrophosphate (FPP) to amorpha-4,11-diene and diphosphate, which is the first committed step in the synthesis of artemisinin. FPP is the precursor for sesquiterpene and sterol biosynthesis in the plant. This work aimed to investigate the effect of blocking the synthesis of artemisinin in the GSTs of a high artemisinin yielding line, Artemis, by down regulating AMS. We determined that there are up to 12 AMS gene copies in Artemis, all expressed in GSTs. We used sequence homology to design an RNAi construct under the control of a GST specific promoter that was predicted to be effective against all 12 of these genes. Stable transformation of Artemis with this construct resulted in over 95% reduction in the content of artemisinin and related products, and a significant increase in the FPP pool. The Artemis AMS silenced lines showed no morphological alterations, and metabolomic and gene expression analysis did not detect any changes in the levels of other major sesquiterpene compounds or sesquiterpene synthase genes in leaf material. FPP also acts as a precursor for squalene and sterol biosynthesis but levels of these compounds were also not altered in the AMS silenced lines. Four unknown oxygenated sesquiterpenes were produced in these lines, but at extremely low levels compared to Artemis non-transformed controls (NTC). This study finds that engineering A. annua GSTs in an Artemis background results in endogenous terpenes related to artemisinin being depleted with the precursor FPP actually accumulating rather than being utilized by other endogenous enzymes. The challenge now is to establish if this precursor pool can act as substrate for production of alternative sesquiterpenes in A. annua.

Literally Green Chemical Synthesis of Artemisinin from Plant Extracts.

Active pharmaceutical ingredients are either extracted from biological sources-where they are synthesized in complex, dynamic environments-or prepared in stepwise chemical syntheses by reacting pure reagents and catalysts under controlled conditions. A combination of these two approaches, where plant extracts containing reagents and catalysts are utilized in intensified chemical syntheses, creates expedient and sustainable processes. We illustrate this principle by reacting crude plant extract, oxygen, acid, and light to produce artemisinin, a key active pharmaceutical ingredient of the most powerful antimalarial drugs. The traditionally discarded extract of Artemisia annua plants contains dihydroartemisinic acid-the final biosynthetic precursor-as well as chlorophyll, which acts as a photosensitizer. Efficient irradiation with visible light in a continuous-flow setup produces artemisinin in high yield, and the artificial biosynthetic process outperforms syntheses with pure reagents.

Synthesis of novel artemisinin–triazole hybrids

AbstractSeveral substituted triazole–artemisinin hybrids were synthesized based on ‘click’ chemistry between propargyl‐substituted derivatives and artemisinin containing a 2‐hydroxypropane unit. The structures of all obtained compounds were confirmed by means of IR, NMR, and MS spectroscopy. Four new hybrids were evaluated as cytotoxic agents, revealing an interesting anticancer activity of 1‐(4‐(3‐Aminophenyl)‐1H‐1,2,3‐triazol‐1‐yl)‐3‐(((3R,6R,8aS,9R,10S,12R,12aR)‐3,6,9‐trimethyl‐decahydro‐12H‐3,12‐epoxy[1,2]dioxepino[4,3‐i]isochromen‐10‐yl)oxy)propan‐2‐ol compound in KB and HepG2 cancer cell lines.

Continuous Photo-Oxidation in a Vortex Reactor: Efficient Operations Using Air Drawn from the Laboratory.

We report the construction and use of a vortex reactor which uses a rapidly rotating cylinder to generate Taylor vortices for continuous flow thermal and photochemical reactions. The reactor is designed to operate under conditions required for vortex generation. The flow pattern of the vortices has been represented using computational fluid dynamics, and the presence of the vortices can be easily visualized by observing streams of bubbles within the reactor. This approach presents certain advantages for reactions with added gases. For reactions with oxygen, the reactor offers an alternative to traditional setups as it efficiently draws in air from the lab without the need specifically to pressurize with oxygen. The rapid mixing generated by the vortices enables rapid mass transfer between the gas and the liquid phases allowing for a high efficiency dissolution of gases. The reactor has been applied to several photochemical reactions involving singlet oxygen (1O2) including the photo-oxidations of α-terpinene and furfuryl alcohol and the photodeborylation of phenyl boronic acid. The rotation speed of the cylinder proved to be key for reaction efficiency, and in the operation we found that the uptake of air was highest at 4000 rpm. The reactor has also been successfully applied to the synthesis of artemisinin, a potent antimalarial compound; and this three-step synthesis involving a Schenk-ene reaction with 1O2, Hock cleavage with H+, and an oxidative cyclization cascade with triplet oxygen (3O2), from dihydroartemisinic acid was carried out as a single process in the vortex reactor.