Taxane Skeleton Research Articles

Recent Research Progress in Taxol Biosynthetic Pathway and Acylation Reactions Mediated by Taxus Acyltransferases.

sTaxol is one of the most effective anticancer drugs in the world that is widely used in the treatments of breast, lung and ovarian cancer. The elucidation of the taxol biosynthetic pathway is the key to solve the problem of taxol supply. So far, the taxol biosynthetic pathway has been reported to require an estimated 20 steps of enzymatic reactions, and sixteen enzymes involved in the taxol pathway have been well characterized, including a novel taxane-10β-hydroxylase (T10βOH) and a newly putative β-phenylalanyl-CoA ligase (PCL). Moreover, the source and formation of the taxane core and the details of the downstream synthetic pathway have been basically depicted, while the modification of the core taxane skeleton has not been fully reported, mainly concerning the developments from diol intermediates to 2-debenzoyltaxane. The acylation reaction mediated by specialized Taxus BAHD family acyltransferases (ACTs) is recognized as one of the most important steps in the modification of core taxane skeleton that contribute to the increase of taxol yield. Recently, the influence of acylation on the functional and structural diversity of taxanes has also been continuously revealed. This review summarizes the latest research advances of the taxol biosynthetic pathway and systematically discusses the acylation reactions supported by Taxus ACTs. The underlying mechanism could improve the understanding of taxol biosynthesis, and provide a theoretical basis for the mass production of taxol.

Identification of potential genes that contributed to the variation in the taxoid contents between two Taxus species (Taxus media and Taxus mairei).

Taxol is currently a valuable anticancer drug; however, the accumulated mixture of taxoids can vary greatly among Taxus species. So far, there is very little genomic information for the genus Taxus, except for Taxus baccata. Transcriptome analysis is a powerful approach to explore the different regulatory mechanisms underlying the taxoid biosynthesis pathway in Taxus species. First, we quantified the variation in the taxoid contents between Taxus media and Taxus mairei. The contents of paclitaxel and 10-deacetylpaclitaxel in T. media are higher than that in T. mairei. Then, the transcriptome profiles of T. media and T. mairei were analyzed to investigate the altered expressions. A total of 20,704 significantly differentially expressed genes (DEGs), including 9865 unigenes predominantly expressed in T. media and 10,839 unigenes predominantly expressed in T. mairei, were identified. In total, 120 jasmonic acid-related DEGs were analyzed, suggesting variations in 'response to JA stimulus' and 'JA biosynthetic process' pathways between T. media and T. mairei. Furthermore, a number of genes related to the precursor supply, taxane skeleton formation and hydroxylation, and C13-side chain assembly were also identified. The differential expression of the candidate genes involved in taxoid biosynthetic pathways may explain the variation in the taxoid contents between T. media and T. mairei.

Crystal structures of (±)-(1SR,5SR,6SR,7SR,10SR,11SR,13RS,14SR)-13-hy-droxy-7-meth-oxy-meth-oxy-11,15,18,18-tetra-methyl-3-oxo-2,4-dioxa-tetra-cyclo-[12.3.1.0(1,5).0(6,11)]octa-dec-15-en-10-yl benzoate, its 13-epimer and 13-one derivative.

The title compounds, C29H38O8·0.25C5H12, (A), C29H38O8, (B), and C29H36O8, (C), are tetra-cyclic benzoates possessing a taxane skeleton with a fused dioxolane ring as the core structure. In the asymmetric unit of (A), there are two independent benzoate mol-ecules (A and A') and a half mol-ecule of solvent pentane disordered about an inversion center. The mol-ecular conformations of (A), (B) and (C) are similar except for the flexible meth-oxy-meth-oxy group. The cyclo-hexane, cyclo-hexene and central cyclo-octane rings adopt chair, half-chair and chair-chair (extended crown) forms, respectively. The dioxolane rings are essentially planar, while the dioxolane ring of A' is slightly twisted from the mean plane. In the crystal of (A), inter-molecular O-H⋯O, C-H⋯O and C-H⋯π inter-actions link the independent benzoates alternately, forming a chain structure. In the crystals of (B) and (C), mol-ecules are linked through O-H⋯O and C-H⋯π inter-actions, and C-H⋯O hydrogen bonds, respectively, into similar chains. Further, weak inter-molecular C-H⋯O inter-actions connect the chains into a three-dimensional network in (A) and a sheet in (B), whereas no other interactions are observed for (C).

Quantification of taxanes in a leaf and twig extract from Taxus baccata L. using 13C NMR spectroscopy

In the course of our ongoing work on the chemical characterization of Taxus baccata L. growing wild in Corsica, we have developed and validated a method for direct quantification of taxane derivatives by (13)C NMR using 10-deacetylbaccatin III as reference compound and 1,6-hexanediol as internal standard. We have observed good accuracy (relative errors between 0.3% and 3.5%), linearity (R(2) = 0.999) and precision (reproducibility 8.5 mg ± 1.1%) of the measurements. The experimental procedure was applied to the quantification of six identified taxanes in a fraction of chromatography of a methanol extract of T. baccata leaves. This method can be applied to other compounds bearing the taxane skeleton.

TAXOIDES: METABOLITOS SECUNDARIOS DEL ÁRBOL DEL TEJO (Taxus spp.)

El árbol del tejo (Taxus spp.) es una gimnosperma dicotiledónea que pertenece a la familia Taxaceae; se distribuye en America, Europa y Asia. Taxus spp. contiene en la corteza y en las hojas, alcaloides diterpénicos conocidos como taxoides. Éstos se caracterizan químicamente por la presencia de un esqueleto de taxano y un anillo de oxetano, tal como el taxol, que es utilizado en el tratamiento del cáncer de ovario, seno, próstata y pulmón; enfermedad que actualmente es una de las principales causas de muerte a nivel mundial. El contenido de taxol es bajo en los árboles, por tanto, es necesario el uso de alternativas biotecnológicas como lo es el cultivo in vitro de tejidos vegetales para incrementar la cantidad de taxoides y preservar el recurso natural. En esta revisión científica se reportan las características químicas de los taxoides, el mecanismo de acción biológico y semisíntesis orgánica del taxol, se detalla también la ruta biosintética de los principales taxoides, así como las principales enzimas involucradas para la obtención de éstos a nivel bioquímico y, finalmente, se indica de manera general la producción de taxoides en cultivos in vitro.

Relationship between the Structures of Taxane Derivatives and Their Microtubule Polymerization Activity

Paclitaxel (Taxol), one of the most potent anticancer drugs, is a microtubule-stabilizing compound that inhibits microtubule depolymerization within the cell. The structure of paclitaxel is composed of two key elements, a taxane ring and an N-benzoylphenylisoserine side chain at C-13. A number of natural and artificial compounds with taxane skeletons have been isolated, but almost none of their bioactivities have been evaluated. In this study, we focused on compounds having a taxane skeleton structure and examined their effects on tubulin dynamics. Although none of these compounds had an N-benzoylphenylisoserine side chain, three were found to promote tubulin assembly. On the other hand, one compound inhibited tubluin assembly in a way similar to nocodazole. These compounds exhibited novel structure-activity relationships of taxane compounds.

Isolation of taxol, an anticancer drug produced by the endophytic fungus, Phoma betae

Phoma betae, an endophytic fungus, was isolated from the healthy leaves of Ginkgo biloba . The fungus was screened for the production of taxol on a modified liquid medium for the first time. The fungal species were identified by their characteristic culture morphology and molecular analysis. The presence of taxol was confirmed by spectroscopic: ultraviolet (UV), infrared (IR), nuclear magnetic resonance (NMR), liquid chromatography–mass spectrometry (LC-MS); and chromatographic: thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) methods of analysis. The taxol production was quantified by HPLC analysis. The maximum amount of taxol production was recorded as 795 μg/L. The production rate was 15,900-fold more than that found in the culture broth of earlier reported fungus, Taxomyces andreanae . The extracted fungal taxol demonstrate a strong cytotoxic activity in the in vitro culture of tested human cancer cells by apoptotic assay. This indicates that the increase in taxol concentration induced an increased cell death. A polymerase chain reaction (PCR) based screening for taxadiene synthase (ts), a unique gene in the formation of the taxane skeleton was confirmed as a molecular blueprint for taxol biosynthesis. These results designate that the fungus, P. betae is an excellent candidate for taxol supply and can serve as a potential species for genetic engineering to enhance the production of taxol to a higher level. Key words : Taxol production, Phoma betae, analytical methods, cytotoxicity assay, taxadiene synthase.

In vitro screening of taxol, an anticancer drug produced by the fungus, Colletotrichum capsici

AbstractThe fungus Colletotrichum capsici was isolated from the diseased fruits of Chilli plant, Capsicum annuum. The isolated test fungus was identified by its morphological and molecular characteristic features. For the first time, the fungus was screened for the production of taxol on modified liquid medium. The presence of taxol was confirmed by the spectroscopic and chromatographic methods of analyses. The amount of taxol produced by this fungus was quantified by HPLC. The maximum amount of fungal taxol production was recorded as 687 μg/L. The production rate was 13 740‐fold higher than that, previously reported for the fungus Taxomyces andreanae. The extracted fungal taxol showed a strong cytotoxic activity in an in vitro culture of human cancer cells indicating that the increase in taxol concentration induces increased cell death. A PCR‐based screening for taxadiene synthase (ts), a unique gene in the formation of the taxane skeleton, confirmed the molecular blueprint for taxol biosynthesis. The results show that the fungus C. capsici is an excellent candidate for an alternate source of taxol supply and can serve as a potential species for genetic engineering to enhance the production of taxol to a higher level.

Screening the endophytic flora of Wollemia nobilis for alternative paclitaxel sources

The endophytic flora of Wollemia nobilis was investigated in search for alternative paclitaxel producers. On one hand, metabolic profiling of the obtained specimens using an immunoenzymatic technique was carried out. On the other, we aimed at revealing the genetic background of presumed paclitaxel biosynthesis in the isolates. We found an indication of endophytic taxane production in the extracts of two strains, Phomopsis sp. and Cladosporium langeronii. A PCR-based screening for taxadiene synthase, a gene unique to the formation of the taxane skeleton, confirmed the molecular blueprint for paclitaxel biosynthesis to be an inherent characteristic of the latter. Although this result makes C. langeronii an interesting candidate for further study, we postulate that proclaiming it ‘a fungus factory for paclitaxel’, as has been done for several other endophytes in the past, might still be premature.

Taxol promising fungal endophyte, Pestalotiopsis species isolated from Taxus cuspidata

The endophytic fungi, Pestalotiopsis versicolor and Pestalotiopsis neglecta, were isolated from the healthy leaves and bark of the Japanese Yew tree, Taxus cuspidata. The fungal species were identified by their characteristic culture morphology and molecular analysis. For the first time, the test fungi were screened for the production of taxol in modified liquid medium. The presence of taxol was confirmed by HPLC, (1)H NMR, and LC-MS methods of analysis. The maximum amount of taxol production in P. versicolor was recorded as 478 μg/l. The production rate was increased to 9560-fold than that found in the culture broth of earlier reported fungus, Taxomyces andreanae. The extracted fungal taxol showed a strong cytotoxic activity in the in vitro culture of tested human cancer cells by apoptotic assay indicating that the increase in taxol concentration induces increased cell death. A PCR-based screening for ts, a unique gene in the formation of the taxane skeleton, confirmed the molecular blueprint for taxol biosynthesis. The results designate that the fungal endophyte, P. versicolor, is an excellent candidate for an alternate source of taxol supply and can also serve as a potential species for genetic engineering to enhance the production of taxol to a higher level.

Synthetic studies on the taxane skeleton: effective construction of eight-membered carbocyclic ring by palladium-catalyzed intramolecular α-alkenylation of a methyl ketone

A new method for the construction of the eight-membered carbocyclic ring in the taxane skeleton is described. The palladium-catalyzed intramolecular α-alkenylation of a methyl ketone effectively constructed the eight-membered carbocyclic ring in the taxol model compound. To the best of our knowledge, this reaction is the first example of forming an eight-membered carbocyclic ring by the palladium-catalyzed intramolecular α-alkenylation of a ketone.

Construction of the taxane skeleton via the stereoselective conjugate addition of cyanide and the intramolecular B-alkyl Suzuki–Miyaura coupling reaction

Construction of the taxane skeleton via the stereoselective conjugate addition of cyanide and the intramolecular B-alkyl Suzuki–Miyaura coupling reaction is described. A conjugate addition of cyanide to enone 17 proceeded diastereoselectively to provide the desired 18 incorporating the correct C3 stereogenic center in the taxol C-ring. The intramolecular B-alkyl Suzuki–Miyaura coupling reaction of 22, which was derived from 18, successfully furnished the taxol B-ring in 81% yield.

Models for Taxol Biosynthesis: SeO2 Oxidation of Taxadiene

Together with the expected mono‐oxidized taxa‐4(20),11(12)‐diene‐5‐ol (1) and taxa‐4(5),11,20‐diene‐20‐ol (2), a new taxane skeleton, 3,12‐cyclotaxane (3), was obtained from chemical oxidation of taxadiene by selenium dioxide/t‐butylhydroperoxide.

Taxol Biosynthesis and Molecular Genetics

Biosynthesis of the anticancer drug Taxol in Taxus (yew) species involves 19 steps from the universal diterpenoid progenitor geranylgeranyl diphosphate derived by the plastidial methyl erythritol phosphate pathway for isoprenoid precursor supply. Following the committed cyclization to the taxane skeleton, eight cytochrome P450-mediated oxygenations, three CoA-dependent acyl/aroyl transfers, an oxidation at C9, and oxetane (D-ring) formation yield the intermediate baccatin III, to which the functionally important C13-side chain is appended in five additional steps. To gain further insight about Taxol biosynthesis relevant to the improved production of this drug, and to draw inferences about the organization, regulation, and origins of this complex natural product pathway, Taxus suspension cells (induced for taxoid biosynthesis by methyl jasmonate) were used for feeding studies, as the foundation for cell-free enzymology and as the source of transcripts for cDNA library construction and a variety of cloning strategies. This approach has led to the elucidation of early and late pathway segments, the isolation and characterization of over half of the pathway enzymes and their corresponding genes, and the identification of candidate cDNAs for the remaining pathway steps, and it has provided many promising targets for genetically engineering more efficient biosynthetic production of Taxol and its precursors.

Verticillane Derivatives from Bursera suntui and Bursera kerberi

The stems of Bursera suntui afforded two new verticillane derivatives, (1S,3Z,7E,11S,12S)-(+)-verticilla-3,7-dien-12,20-diol (1) and (1S,3Z,7E,11S,12S)-(+)-verticilla-3,7-dien-12,20-diol 20-acetate (2), together with (1S,3E,7E,11R)-(+)-verticilla-3,7,12(18)-triene (3), (1R,3E,7E,11R,12Z)-(+)-verticilla-3,7,12-triene (4), (1R,7E,11Z)-(-)-verticilla-4(20),7,11-triene (5), and (1S,3E,7E,11S,12S)-(+)-verticilla-3,7-dien-12-ol (6). Compounds 3 and 4 are new enantiomerically pure natural products whose racemic mixtures, derived from synthetic approaches toward the taxane skeleton, were obtained previously. The stems of Bursera kerberi afforded the new (1S,3E,7E,11S,12R)-(+)-verticilla-3,7-dien-12-ol (7) together with 3-5. This is the first time that verticillane derivatives have been isolated from the genus Bursera. Their structures and stereochemistry were elucidated by 1D and 2D NMR data, including COSY, NOESY, HSQC, and HMBC experiments, while the absolute configuration was determined by comparison of the optical rotatory dispersion data with that of recently revised (1S,3E,7E,11S,12S)-(+)-verticilla-3,7-dien-12-ol (6), obtained from Sciadopitys verticillata, and those of (1R,3E,7E,11R,12R)-(-)-verticilla-3,7-dien-12-ol (8) and (1R,3E,7E,11R,12S)-(-)-verticilla-3,7-dien-12-ol (9), isolated from the liverwort Jackiella javanica. The conformational preferences of 1-7 were studied by molecular mechanics modeling employing the Monte Carlo protocol.

Synthesis and evaluation of 3D templates based on a taxane skeleton to circumvent P-glycoprotein-associated multidrug resistance of cancer

We have developed a practical synthetic route to construct C-aromatic taxane derivatives as three-dimension templates by way of intramolecular alkylation. The usefulness of synthesized compounds as the core template was evaluated by using a newly developed screening system for P-glycoprotein.

Synthetic Studies on the Taxane Skeleton: Construction of Eight‐Membered Carbocyclic Rings by the Intramolecular B‐Alkyl Suzuki—Miyaura Cross‐Coupling Reaction.

Construction of eight-membered carbocyclic rings via the intramolecular B-alkyl Suzuki−Miyaura cross-coupling reaction has been studied. This protocol proved its potency through the formation of the eight-membered ring possessing a quaternary carbon on its ring in high yield, affording promise of a new access to the eight-membered ring of Taxol.

Paclitaxel production by plant-cell-culture technology.

Plant cells catalyze multiple-step reactions of secondary metabolite biosynthesis, and selectively synthesize chiral compounds with polycyclic structures. Taking advantage of this characteristic, we studied the production of the anticancer drug paclitaxel, which is currently produced in limited supply. Callus culture investigations indicate that woody plant medium supplemented with 10(-5) mol L(-1) 1-naphthylacetic acid and without the NH4+ -type ion is the best condition for growth of the callus. The accumulation of paclitaxel and related taxanes in Taxus plants is thought to be a biological response to specific external stimuli. Several signal transducers were screened; taxane biosynthesis was strongly promoted by methyl jasmonate (MeJA) and silver thiosulfate (STS) as an anti-ethylene compound. Of ten taxane-type diterpenoids isolated from T. baccata suspension-cultured cells treated with MeJA, five have a phenylisoserine side-chain at the C-13 position of the taxane skeleton. Time-course analysis revealed two regulatory steps in taxane biosynthesis: the taxane-ring formation step and the acylation step of the C-13 position. Methyl jasmonate promoted the formation of the taxane-ring. The production of paclitaxel reached a maximum level of 295 mg L(-1) in a large-scale culture of T. x media cells using a two-stage process.

Semisynthesis of 7‐Deoxypaclitaxel Derivatives Devoid of an Oxetane D‐Ring, Starting from Taxine B

AbstractSix 7‐deoxypaclitaxel derivatives have been prepared from taxine B; they contribute to understanding of the effect of the oxetane D‐ring in paclitaxel on the cytotoxic activity of paclitaxel. Three of these analogues each contain a double bond instead of a D‐ring at the C‐4 position, while two others each contain a three‐membered ring in place of the oxetane ring. Both the C‐4 double bond and the small D‐ring in these paclitaxel derivatives were intended to act as substitutes for the paclitaxel oxetane D‐ring and were expected to impose on the taxane skeleton and the pendant groups a kind of rigidity and conformation similar to that produced by the oxetane ring in paclitaxel. All the derivatives demonstrated considerably reduced in vitro cytotoxic activity − relative to paclitaxel − against a panel of seven well‐characterized human tumor cell lines. The general picture coming out of the structure‐activity relationships of the paclitaxel derivatives reported here is that the presence of an oxygen atom at a spatial position resembling that of the oxygen atom in the oxetane ring of paclitaxel is important if a paclitaxel derivative is to retain considerable cytotoxic activity. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

Three new C-14 oxygenated taxanes from the wood of Taxus yunnanensis.

Three new C-14 oxygenated taxane-type diterpenes, hongdoushans A-C (1-3), were isolated from the wood of Taxus yunnanensis together with four known diterpenes and two lignans. The absolute stereochemistry of the 2-methylbutyryloxy group attached at C-14 of the taxane skeleton was determined to be S by GC analysis of the methyl ester of 2-methylbutyric acid obtained after alkaline hydrolysis of 1 and 4 followed by treatment with CH(2)N(2). The complete stereostructure of the known compound 2alpha,5alpha,10beta-triacetoxy-14beta-[(S)-2-methylbutyryloxy]-4(20),11-taxadiene (4) was established for the first time. The isolates obtained were evaluated for their antiproliferative activity toward murine colon 26-L5 carcinoma and human HT-1080 fibrosarcoma cell lines.