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BackgroundRhodosporidium toruloides has emerged as a promising host for the production of bioproducts from lignocellulose, in part due to its ability to grow on lignocellulosic feedstocks, tolerate growth inhibitors, and co-utilize sugars and lignin-derived monomers. Ent-kaurene derivatives have a diverse range of potential applications from therapeutics to novel resin-based materials.ResultsThe Design, Build, Test, and Learn (DBTL) approach was employed to engineer production of the non-native diterpene ent-kaurene in R. toruloides. Following expression of kaurene synthase (KS) in R. toruloides in the first DBTL cycle, a key limitation appeared to be the availability of the diterpene precursor, geranylgeranyl diphosphate (GGPP). Further DBTL cycles were carried out to select an optimal GGPP synthase and to balance its expression with KS, requiring two of the strongest promoters in R. toruloides, ANT (adenine nucleotide translocase) and TEF1 (translational elongation factor 1) to drive expression of the KS from Gibberella fujikuroi and a mutant version of an FPP synthase from Gallus gallus that produces GGPP. Scale-up of cultivation in a 2 L bioreactor using a corn stover hydrolysate resulted in an ent-kaurene titer of 1.4 g/L.ConclusionThis study builds upon previous work demonstrating the potential of R. toruloides as a robust and versatile host for the production of both mono- and sesquiterpenes, and is the first demonstration of the production of a non-native diterpene in this organism.

BackgroundOur previous work has shown peroxiredoxin-1 (PRDX1), one of major antioxidant enzymes, to be a biomarker in human breast cancer. Hereby, we further investigate the role of PRDX1, compared to its close homolog PRDX2, in mammary malignant cells.MethodsCRISPR/Cas9- or RNAi-based methods were used for genetic targeting PRDX1/2. Cell growth was assessed by crystal violet, EdU incorporation or colony formation assays. In vivo growth was assessed by a xenotransplantation model. Adenanthin was used to inhibit the thioredoxin-dependent antioxidant defense system. The prooxidant agents used were hydrogen peroxide, glucose oxidase and sodium L-ascorbate. A PY1 probe or HyPer-3 biosensor were used to detect hydrogen peroxide content in samples.ResultsPRDX1 downregulation significantly impaired the growth rate of MCF-7 and ZR-75-1 breast cancer cells. Likewise, xenotransplanted PRDX1-deficient MCF-7 cells presented a retarded tumour growth. Furthermore, genetic targeting of PRDX1 or adenanthin, but not PRDX2, potently sensitised all six cancer cell lines studied, but not the non-cancerous cells, to glucose oxidase and ascorbate.ConclusionsOur study pinpoints the dominant role for PRDX1 in management of exogeneous oxidative stress by breast cancer cells and substantiates further exploration of PRDX1 as a target in this disease, especially when combined with prooxidant agents.

Covering: 2011-2017Radical cyclizations have a rich history in organic chemistry and have been particularly generous to the field of natural product synthesis. Owing to their ability to operate in highly congested molecular quarters, and with significant functional group compatibility, these transformations have enabled the synthesis of numerous polycyclic terpenoid natural products over the past several decades. Moreover, when programmed accordingly into a synthetic plan, radical cascade processes can be used to rapidly assemble molecular complexity, much in the same way nature rapidly constructs terpene frameworks through cationic cyclization pathways. This review highlights recent total syntheses of complex terpenoids (from 2011-2017) employing C-C bond-forming radical cascade sequences.

Plants produce an immense diversity of natural products (i.e. secondary or specialized metabolites) that offer a rich source of known and potentially new pharmaceuticals and other desirable bioproducts. The Traditional Chinese Medicinal plant Isodon rubescens (Lamiaceae) contains an array of bioactive labdane-related diterpenoid natural products. Of these, the ent-kauranoid oridonin is the most prominent specialized metabolite that has been extensively studied for its potent antimicrobial and anticancer efficacy. Mining of a previously established transcriptome of I. rubescens leaf tissue identified seven diterpene synthase (diTPSs) candidates. Here we report the functional characterization of four I. rubescens diTPSs. IrTPS5 and IrTPS3 were identified as an ent-copalyl diphosphate (CPP) synthase and a (+)-CPP synthase, respectively. Distinct transcript abundance of IrTPS5 and the predicted ent-CPP synthase IrTPS1 suggested a role of IrTPS5 in specialized ent-kaurene metabolism possibly en route to oridonin. Nicotiana benthamiana co-expression assays demonstrated that IrTPS4 functions sequentially with IrTPS3 to form miltiradiene. In addition, IrTPS2 converted the IrTPS3 product (+)-CPP into the hydroxylated tricyclic diterpene nezukol not previously identified in I. rubescens. Metabolite profiling verified the presence of nezukol in I. rubescens leaf tissue. The proposed IrTPS2-catalyzed reaction mechanism proceeds via the common ionization of the diphosphate group of (+)-CPP, followed by formation of an intermediary pimar-15-en-8-yl+ carbocation and neutralization of the carbocation by water capture at C-8 to yield nezukol, as confirmed by nuclear magnetic resonance (NMR) analysis. Oxygenation activity is rare for the family of class I diTPSs and offers new catalysts for developing metabolic engineering platforms to produce a broader spectrum of bioactive diterpenoid natural products.

Ent-kaurane diterpene compounds have attracted considerable attention in recent years due to its antitumor, antibacterial, and antiviral activities. However, the clinical development of natural kaurane diterpenes, for example, oridonin for cancer therapy has been hampered by its relatively moderate potency, limited bioavailability. Herein, we report a newly synthetic analog of natural ent-kaurane diterpene, DS2, which exhibits significantly improved activity of antiproliferation against various cancer cell lines relative to oridonin. DS2 treatment triggers the mitochondria-mediated apoptosis and cell cycle arrest in human esophageal cancer cell lines (EC9706, EC109). Interestingly, normal human esophageal epithelial cells (HEECs) and normal human liver cells (HL-7702) are both significantly more resistant to the growth inhibition by DS2 compared with esophageal cancer cells. The DS2-induced apoptosis in EC9706 cells correlated with the drop of mitochondrial membrane potential (MMP), release of cytochrome c into the cytosol and activation of caspase-9 and -3. The induction of proapoptotic proteins p21 and Bax were also observed in DS2-treated cells. The DS2-induced apoptosis was significantly attenuated by knockdown of Bax proteins. Meanwhile, the DS2 treatment caused generation of reactive oxygen species (ROS) in human esophageal cancer cells, but not in HEECs, which was attenuated by pretreatment with ROS scavenger N-acetylcysteine (NAC). More interestingly, the antioxidants pretreatment completely attenuated DS2 mediated loss of the MMP and apoptosis, as well as Bax expression and growth inhibition. In conclusion, the present study reveals that the mitochondria-mediated cell death by DS2 is associated with Bax regulation and ROS generation, and understanding the function and mechanism of DS2 will help us to design better anti-cancer drugs.

• Kaurenoic or beyerenoic acid as substrates to explore stereoselective Prins reaction. • Quirality from natural diterpenes directly influences the Prins-cyclization process. • Highly stereoselective Prins-cyclization in diterpene skeletons favors fused oxane ring. • The mechanism of Prins-cyclization is proposed and supported by computational means. • Cytotoxic potential of kauroxane and beyeroxane is explored in vitro assays. A semisynthetic strategy to access novel kauroxane or beyeroxane compounds from ent ‑kauranal or ent ‑beyerenal, respectively, is described. A Prins cyclization was used as a key step reaction to synthesize diterpene-tetrahydropyran hybrid compounds with high stereoselectivity by using 3-buten-1-ol as oxane precursor and indium trichloride as the Lewis acid catalyst. The absolute configurations of new compounds were determined by mechanistic means and supported with DFT calculations, as well as considering the stereoselectivity behavior associated with the chiral purity of diterpenes and their conformational restrictions. The structures were elucidated by their physical and spectroscopic data. Finally, the antiproliferative activity of the target molecules was done, being moderately bioactive. [Display omitted] [ABSTRACT FROM AUTHOR]

A known kaurenoic acid (1), together with a new kaurane derivative named 3α-isobutyryloxykaurenoic acid (2), was isolated from the roots of Lasianthaea aurea, a plant endemic to Mexico. The compounds were characterized using their physical and spectroscopic data. In order to verify the structure and absolute configuration of 2, the derived methyl ester 3, obtained after diazomethane treatment, was subjected to single-crystal X-ray diffraction analysis, whose Flack and Hooft parameter calculations evidenced that the new natural product is an ent-kaurane derivative. [ABSTRACT FROM AUTHOR]

A phytochemical investigation on the flesh fruits of atemoya led to the isolation of seven new kaurane type diterpenoids, (4S*,5S*,8S*,9R*,10S*,13R*,16R*)-16-hydro-18-nor-kauran-4,17-diol (1), (4S*,5S*,8S*,9R*,10S*,13R*,16S*)-18-nor-kauran-4,16,17-triol (2), (4S*,5S*,8S*,9R*,10S*,13R*,16S*)-17-acetoxy-18-nor-kauran-4,16-diol (3), (4S*,5S*,8S*,9R*,10S*,13R*,16R*)-18-nor-kauran-4,16,17-triol (4), (4S*,5S*,8S*,9R*,10S*,13R*,16S*)-17-acetoxy-16-hydro-18-nor-kauran-4-ol (5), (4R*,5S*,8S*,9R*,10S*,13R*,16S*)-16,17-dihydroxy-19-nor-kauran-4-hydroperoxide (6), and (4R*,5S*,8S*,9R*,10S*,13R*,16S*)-kauran-16,19-diol (7) along with 26 known ent-kaurane compounds. Their structures are determined on the basis of spectroscopic data and optical rotation. Compounds 1-5 were new 18-nor-kauran-4-ol type diterpenoids, which are very rarely obtained from natural sources.

Abstract The surface extract of an accession of Psiadia punctulata (DC.) Vatke (Asteraceae) growing in Saudi Arabia was investigated for its phytochemical composition. A bio-guided investigation of the extract led to the isolation of thirteen ent -kaurane and trachylobane diterpenes and seventeen compounds previously described, including nine flavonoids and eight diterpenes. Three flavonoids and one ent -kaurane diterpene showed antimicrobial activity with MIC100 values ranging from 25 to 150 μg/ml. The extract showed antibacterial activity against Staphylococcus aureus (MIC 100 = 180 μg/ml) and antifungal activity against Candida albicans (MIC 0 = 130 μg/ml). The isolated 3′,4′,5,7-tetramethoxyflavone, at a concentration of 40 μg/ml, displayed the ability to reduce biofilm formation of S. aureus and C. albicans by 50% and 90% respectively. Graphical abstract Image 1 Highlights • The surface extract of Psiadia punctulata from Saudi Arabia was investigated. • Thirteen previously undescribed ent -kaurane and trachylobane diterpenes were isolated. • Nine known flavonoids and eight known diterpenes were also isolated. • The antimicrobial activity of the extracts, fractions and isolates was evaluated. [ABSTRACT FROM AUTHOR]

The fruits of Zanthoxylum leprieurii Guill. & Perr. (Rutaceae) are traditionally used in Africa, particularly in Cameroon, as a spice and in the treatment of sickle cell anaemia. Phytochemical investigation on the fruits of this plant afforded five kaurane diterpenes, i.e., kaurenoic acid (1), xylopic acid (2), ent-kauran-16β-ol (3), ent-16β-hydroxykauran-19-al (4) and ent-16β-hydroxykauran-19-oic acid (5). The structures of these diterpenes were determined comprehensively by spectroscopic means (1D and 2D NMR spectroscopy and MS analyses) and also by comparison with respective literature data. Among the isolated compounds, only kaurenoic acid (1) exhibited cytotoxicity against the PC3 cell line with an IC50 value of 33.28 ± 9.14 μg/mL. To the best of our knowledge, this is the first report on the isolation of these kaurane diterpenes (1-5) from the genus Zanthoxylum. [ABSTRACT FROM AUTHOR]

An efficient retro-biomimetic procedure for the synthesis of two natural products having atisanic and beyeranic structure is reported. The readily available ent-kaurenoic acid is used as the substrate, which provides under superacidic treatment a mixture of natural ent-beyer-15-en-19-oic and ent-atis-16-en-19-oic acids. These compounds have been reported as components of medicinal plants and possess relevant biological activity. Their structure was confirmed on the basis of chemical transformations and spectral data.

Abstract: The main diterpenoids in the roots of Arctopus (Apiaceae, tribe Saniculeae) have been identified for the first time. The major compounds are manool, ent-trachyloban-19-oic acid and a kauren-19-oic acid, while methyl-16β-hydroxy-ent-kaur-11-en-19-oate is a minor diterpene. Comparative studies of non-polar extracts by means of LC–MS showed that the isolated main compounds are present in all three species of Arctopus. The study also revealed that the diterpenoid pattern in Arctopus is very similar to that of the genus Alepidea. The tuberous roots of Arctopus and the rhizomes and roots of Alepidea are important traditional medicines in South Africa, used mainly for infections and respiratory ailments. Their efficacy is ascribed to antimicrobial and anti-inflammatory activity. The known biological activities of the isolated diterpenes provide a scientific rationale for the traditional uses. The chemical similarity in diterpenoids also supports the idea that the two genera are closely related, despite their conspicuous morphological differences. [Copyright &y& Elsevier]

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In this study, two Sideritis species, endemic to Turkey, S. niveotomentosa Huber -- Morathii, S. brevidens P.H. Davis have been studied for their diterpenic compounds and the antioxidant properties. Eight known diterpenoids, which have ent-kaurene skeleton, were isolated from acetone and methanol extracts of these species. The structures of the isolated diterpenes were determined by using the NMR (¹H-NMR, 13C-NMR, COSY, HMQC, and HMBC) spectroscopy. The analysis of the phenolic compounds of the extracts was performed by high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Also the antioxidant capacity of the extracts was investigated namely by two methods; free radical scavenging and β-carotene bleaching activities. [ABSTRACT FROM AUTHOR]

Abstract: A new ent-kaurane diterpenoid, together with eight known ent-kauranes, were isolated from the petroleum ether and acetone extracts of the whole plant of Sideritis congesta P.H. Davis & Hub.-Mor. and their structures were elucidated as the new compound ent-7α-acetoxy-16β,18-dihydroxy-kaurane (7-acetyldistanol) (1) and the known compounds ent-3β,7α-dihydroxy,18-acetoxy-15β,16β-epoxykaurane (epoxyisolinearol) (2), sideroxol (3), sideridiol (4), siderol (5), 7-epicandicandiol (6), foliol (7), linearol (8) and sidol (9). Characterization of compounds 1–9 was based on spectral analyses and comparison with reported data, particularly the new compound 1 was identified by 1D- and 2D-NMR and mass spectroscopic analyses. The antioxidant potential of the extracts, and also of the ent-kauranes except for 7, was investigated by three methods including β-carotene bleaching method, free radical scavenging activity and superoxide anion scavenging activity. The anticholinesterase activity was also evaluated for the ent-kauranes except for 7, and most of the diterpenes exhibited weak acetylcholinesterase inhibitory activity. However, almost all diterpenes exhibited some inhibitory activity against butyrylcholinesterase; particularly, compounds 3 and 6 exhibited better BChE inhibitory activity than the standard compound galanthamine. [Copyright &y& Elsevier]

Abstract: The paper presents the influence of mechanochemical activation of the Slovak brown coal to make the organic structure of coal accessible for the extraction agent. It was proved that mechanical activation of optimally ground coal sample (20min) produced ultra-fine and nano-particles with specific surface of 3.79m2/g. The distribution density curves show two maxima at 6.5μm and 42nm, respectively. The identification of 16β(H)-kaurane in the coal extract was performed after the low-temperature supercritical fluid extraction of the coal powders with and without modifier. GC–MS chromatograms present the favourable effect of the modifier. It is well known, that the kaurane derivatives display biological activity. [Copyright &y& Elsevier]

Sideroxol ( 1), a kaurane diterpene which has the ent-7α,18-dihydroxy-15β,16β-epoxykaurane structure (MW = 320.47, C20H32O3) was obtained from the acetone extract of Sideritis leptoclada plant as well as from some other Sideritis species. It crystallizes in the orthorhombic space group P21, 21, 21 with a = 10.967(3), b = 24.555(5), c = 6.372(4) Å, Dc = 1.240 g cm−3, Z = 4, and refines to R = 0.065 for 721 independent reflections. The skeleton consists of three fused six-membered rings and a five-membered ring with fused epoxide. The six membered rings exhibited slightly distorted chair conformation. In addition to sideroxol, two kaurane and five kaurene diterpenes were isolated from the hexane and acetone extracts of the studied plant. [ABSTRACT FROM AUTHOR]

Some kaurane diterpenoids were isolated from 3 species of the genus Sideritis (Lamiaceae) growing in the Eastern Mediterranean region. Sideritis libanotica subsp. libanotica contained siderol 2 and sideridiol 3. Sideritis erythrantha var. erythrantha yielded sideridiol 3. Sideritis perfoliata gave siderol 2, sideridiol 3 and sideritriol 4. The products are known as they occur in another species of Sideritis growing in Italy and in other species growing also in Turkey. The products are isolated for the first time from these 3 species. The taxonomic significance of these results is discussed. [ABSTRACT FROM AUTHOR]

Acetone and ethanol extracts of the tubercula and several compounds isolated from Aristolochia pubescens (Wild) were bioassayed on velvetbean caterpillars, Anticarsia gemmatalis (Hübner), for evaluation of the insecticidal activities. Of the extracts subjected to bioassay, the acetone extract showed the highest activity. (-)-Cubebin did not show activity against soybean caterpillars, whereas aristolochic acid and ent-kaur-l5-en-17-o1 increased the larval period. These compounds, and (+)-eudesmin and (+)-sesamin, reduced the viability of this period, giving rise to malformed adults. These extracts and compounds are therefore potential botanical insecticide agents for the control of velvetbean caterpillars in soybean crops. [ABSTRACT FROM AUTHOR]

The Nsp9 replicase is a conserved coronaviral protein that acts as an essential accessory component of the multi-subunit viral replication/transcription complex. Nsp9 is the predominant substrate for the essential nucleotidylation activity of Nsp12. Compounds specifically interfering with this viral activity would facilitate its study. Using a native mass-spectrometry-based approach to screen a natural product library for Nsp9 binders, we identified an ent-kaurane natural product, oridonin, capable of binding to purified SARS-CoV-2 Nsp9 with micromolar affinities. By determining the crystal structure of the Nsp9-oridonin complex, we showed that oridonin binds through a conserved site near Nsp9's C-terminal GxxxG-helix. In enzymatic assays, oridonin's binding to Nsp9 reduces its potential to act as substrate for Nsp12's Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. We also showed using in vitro cellular assays oridonin, while cytotoxic at higher doses has broad antiviral activity, reducing viral titer following infection with either SARS-CoV-2 or, to a lesser extent, MERS-CoV. Accordingly, these preliminary findings suggest that the oridonin molecular scaffold may have the potential to be developed into an antiviral compound to inhibit the function of Nsp9 during coronaviral replication., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The article discusses research which examines the neuroprotective effects of kaurane diterpene isolated from the bulbs of Fritillaria ebeiensis against MPP+-induced neural cell death in human dopaminergic neuroblastomoa SH-SY5Y cells. The study showed that the structure of kaurane diterpenes were elucited on the basis of extensive spectroscopic analyses. It indicated the importance of diterpenoids that possessed biological activity for treating neurological disorders.

The current work presents the first results on the application of the radical addition methodology for the simultaneous attachment of a C-2 synthon and a functional group to the entkaurenoic acid methyl ester and its C-15 hydroxylated derivative at the C-17 carbon atom. [ABSTRACT FROM AUTHOR]