Gene discovery and virus-induced gene silencing reveal branched pathways to major classes of bioactive diterpenoids in .

Most macro- and polycyclic Euphorbiaceae diterpenoids derive from the common C20 precursor casbene. While the biosynthetic pathway from casbene to the lathyrane jolkinol C is characterized, pathways to other more complex classes of bioactive diterpenoids remain to be elucidated. A metabolomics-guided transcriptomic approach and a genomics approach that led to the discovery of two casbene-derived diterpenoid gene clusters yielded a total of 68 candidate genes that were transiently expressed in Nicotiana benthamiana for activity toward jolkinol C and other lathyranes.

Engineering Production of a Novel Diterpene Synthase Precursor in .

Diterpene biosynthesis commonly originates with the methylerythritol phosphate (MEP) pathway in chloroplasts, leading to the C substrate, geranylgeranyl pyrophosphate (GGPP). The previous work demonstrated that over-expression of genes responsible for the first and last steps in the MEP pathway in combination with () and () is optimal for increasing flux through to casbene in . When the gene responsible for the last step in the MEP pathway, (), is removed from this combination, casbene is still produced but at lower amounts.

Developing a Nicotiana benthamiana transgenic platform for high-value diterpene production and candidate gene evaluation.

To engineer Nicotiana benthamiana to produce novel diterpenoids, we first aimed to increase production of the diterpenoid precursor geranylgeranyl pyrophosphate (GGPP) by up-regulation of key genes of the non-mevalonate (MEP) pathway sourced from Arabidopsis thaliana. We used transient expression to evaluate combinations of the eight MEP pathway genes plus GGPP synthase and a Jatropha curcas casbene synthase (JcCAS) to identify an optimal combination for production of casbene from GGPP. AtDXS and AtHDR together with AtGGPPS and JcCAS gave a 410% increase in casbene production compared to transient expression of JcCAS alone.

A Solution NMR Approach To Determine the Chemical Structures of Carbohydrates Using the Hydroxyl Groups as Starting Points.

An efficient NMR approach is described for determining the chemical structures of the monosaccharide glucose and four disaccharides, namely, nigerose, gentiobiose, leucrose and isomaltulose. This approach uses the H resonances of the -OH groups, which are observable in the NMR spectrum of a supercooled aqueous solution, as the starting point for further analysis. The 2D-NMR technique, HSQC-TOCSY, is then applied to fully define the covalent structure (i.

Detailed Phytochemical Analysis of High- and Low Artemisinin-Producing Chemotypes of .

Chemical derivatives of artemisinin, a sesquiterpene lactone produced by , are the active ingredient in the most effective treatment for malaria. Comprehensive phytochemical analysis of two contrasting chemotypes of resulted in the characterization of over 80 natural products by NMR, more than 20 of which are novel and described here for the first time. Analysis of high- and low-artemisinin producing (HAP and LAP) chemotypes of confirmed the latter to have a low level of (artemisinic aldehyde Δ reductase) gene expression.

Artemisia annua mutant impaired in artemisinin synthesis demonstrates importance of nonenzymatic conversion in terpenoid metabolism.

Artemisinin, a sesquiterpene lactone produced by Artemisia annua glandular secretory trichomes, is the active ingredient in the most effective treatment for malaria currently available. We identified a mutation that disrupts the amorpha-4,11-diene C-12 oxidase (CYP71AV1) enzyme, responsible for a series of oxidation reactions in the artemisinin biosynthetic pathway. Detailed metabolic studies of cyp71av1-1 revealed that the consequence of blocking the artemisinin biosynthetic pathway is the redirection of sesquiterpene metabolism to a sesquiterpene epoxide, which we designate arteannuin X.

A Cytochrome P450-Mediated Intramolecular Carbon-Carbon Ring Closure in the Biosynthesis of Multidrug-Resistance-Reversing Lathyrane Diterpenoids.

The Euphorbiaceae produce a wide variety of bioactive diterpenoids. These include the lathyranes, which have received much interest due to their ability to inhibit the ABC transporters responsible for the loss of efficacy of many chemotherapy drugs. The lathyranes are also intermediates in the biosynthesis of range of other bioactive diterpenoids with potential applications in the treatment of pain, HIV and cancer.

Asymmetric cyclopropanation of conjugated cyanosulfones using a novel cupreine organocatalyst: rapid access to δ(3)-amino acids.

An organocatalytic asymmetric synthesis of a novel, highly functionalised cyclopropane system furnished with versatile substituents and containing a quaternary centre is described. The process utilises a new bifunctional catalyst based on the cinchona alkaloid framework and the products made using this catalyst were obtained as single diastereoisomers, with very high enantioselectivities (up to 96% ee). We have also demonstrated that these resulting cyclopropanes are very useful synthetic intermediates to interesting products, such as the difficult to access δ(3)-amino acids.

Proteomic analysis of Artemisia annua--towards elucidating the biosynthetic pathways of the antimalarial pro-drug artemisinin.

Background: MS-based proteomics was applied to the analysis of the medicinal plant Artemisia annua, exploiting a recently published contig sequence database (Graham et al. (2010) Science 327, 328-331) and other genomic and proteomic sequence databases for comparison. A.

Production of bioactive diterpenoids in the euphorbiaceae depends on evolutionarily conserved gene clusters.

The Euphorbiaceae produce a diverse range of diterpenoids, many of which have pharmacological activities. These diterpenoids include ingenol mebutate, which is licensed for the treatment of a precancerous skin condition (actinic keratosis), and phorbol derivatives such as resiniferatoxin and prostratin, which are undergoing investigation for the treatment of severe pain and HIV, respectively. Despite the interest in these diterpenoids, their biosynthesis is poorly understood at present, with the only characterized step being the conversion of geranylgeranyl pyrophosphate into casbene.

I-motif formation in gene promoters: unusually stable formation in sequences complementary to known G-quadruplexes.

I-motif formation has been confirmed in a number of gene promoter sequences known to form G-quadruplex structures. I-motif formation can occur close to physiological temperature and pH for h-tert and PDGF-A. The i-motif structure formed by a HIF-1α promoter sequence shows unexpected stability near neutral pH.

Impact of gut passage and mucus secretion by the earthworm Lumbricus terrestris on mobility and speciation of arsenic in contaminated soil.

Earthworms inhabiting arsenic contaminated soils may accelerate the leaching of As into surface and ground waters. We carried out three experiments to determine the impact of passage of As contaminated soil (1150 mg As kg(-1)) through the gut of the earthworm Lumbricus terrestris on the mobility and speciation of As and the effects of earthworm mucus on As mobility. The concentration of water soluble As in soil increased (from 1.

The biosynthesis of artemisinin (Qinghaosu) and the phytochemistry of Artemisia annua L. (Qinghao).

The Chinese medicinal plant Artemisia annua L. (Qinghao) is the only known source of the sesquiterpene artemisinin (Qinghaosu), which is used in the treatment of malaria. Artemisinin is a highly oxygenated sesquiterpene, containing a unique 1,2,4-trioxane ring structure, which is responsible for the antimalarial activity of this natural product.

Terpenoids from the seeds of Artemisia annua.

Fourteen sesquiterpenes, three monoterpenes and one diterpene natural product have been isolated from the seeds of Artemisia annua. The possible biogenesis of some of these natural products are discussed by reference to recently reported experimental results for the autoxidation of dihydroartemisinic acid and other terpenoids from Artemisia annua.

Maculalactone M from the marine cyanobacterium Kyrtuthrix maculans.

Maculalactone M, a seco-dibenzyldiphenyl-4,5,6,7-tetrahydrobenzofuranone, was isolated from the epilithic encrusting cyanobacterium Kyrtuthrix maculans. A possible biogenetic route to this compound is proposed involving cleavage of an allylic hydroperoxide.

Dimeric guaianolides and a fulvenoguaianolide from Artemisia myriantha.

The aerial parts of Artemisia myriantha have afforded one new fulvenoguaianolide and four dimeric guaianolides in addition to seven known guaianolides. The structures of all the compounds were elucidated by 2D NMR. It is speculated that the dimeric guianolides are formed via Diels-Alder type reactions of fulvenoguaianolide derivatives.

Germacranolides from Artemisia myriantha and their conformation.

The CH(2)Cl(2) extract of the aerial parts of Artemisia myriantha afforded three germacranolides derived from 13-acetoxy-3beta-hydroxy-germacra-1(10)E,4E,7(11)-trien-12,6alpha-olide, whose structures were elucidated by 2D-NMR spectroscopic analyses. Some conclusions are drawn about the possible conformations of the ten-membered germacranolide ring system from the exchange peaks seen in the NOESY spectra, and an estimate is made of the energy barrier to ring-flipping from variable-temperature NOESY experiments. The conclusions reached were supported by molecular modeling studies and an NMR spectroscopic investigation of the commercially available germacranolide, parthenolide.

beta-Methoxy-gamma-methylene-alpha,beta-unsaturated-gamma-butyrolactones from Artabotrys hexapetalus.

The dichloromethane extract of the aerial parts of Artabotrys hexapetalus afforded three beta-methoxy-gamma-methylene-alpha,beta-unsaturated-gamma-butyrolactones, which are proposed to be derived from a C(18) unsaturated fatty acid by a biosynthetic route similar to that proposed for the Annonaceous acetogenins. The structure of the unique beta-methoxy-gamma-methylene-substituted, alpha,beta-unsaturated-gamma-butyrolactone ring of artapetalins A-C (1-3) was determined by 2D-NMR spectroscopic analyses. Two unusual simple butyrolactones, (+)-tulipalin B and (2R,3R)- 3-hydroxy-2-methylbutyrolactone were also isolated from this species.

Determination of the absolute stereochemistry of the epoxide group in alpinia epoxide by NMR.

The absolute stereochemistry of the epoxide group in alpinia epoxide [14,15-epoxylabda-8(17),12-dien-16-al (E)] has been determined by simultaneous reduction of the aldehyde and epoxide functional groups in this molecule to primary and secondary alcohols, followed by selective protection of the primary alcohol and derivitization of the secondary alcohol with S(+) and R(-) MTPCl as Mosher esters. Changes in (1)HNMR chemical shifts for all positions in these two esters were determined by 2D-NMR and used to infer the absolute stereochemistry of the epoxide group in the natural product alpinia epoxide.

A prezizaane sesquiterpene from Illicium angustisepalum.

The prezizaane sesquiterpene angustisepalin has been isolated from the aerial parts of Illicium angustisepalum, and its structure determined by 2D-NMR spectroscopy. Angustisepalin is formally the 10-benzoyl ester of neomajucin, previously reported from Illicium majus.