Synthesis of methylerythritol phosphate analogues and their evaluation as alternate substrates for IspDF and IspE from Agrobacterium tumefaciens.

The methylerythritol phosphate biosynthetic pathway, found in most Bacteria, some parasitic protists, and plant chloroplasts, converts D-glyceraldehyde phosphate and pyruvate to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where it intersects with the mevalonate pathway found in some Bacteria, Archaea, and Eukarya, including the cytosol of plants. D-3-Methylerythritol-4-phosphate (MEP), the first pathway-specific intermediate in the pathway, is converted to IPP and DMAPP by the consecutive action of the IspD-H proteins. We synthesized five D-MEP analogues-D-erythritol-4-phosphate (EP), D-3-methylthrietol-4-phosphate (MTP), D-3-ethylerythritol-4-phosphate (EEP), D-1-amino-3-methylerythritol-4-phosphate (NMEP), and D-3-methylerythritol-4-thiolophosphate (MESP)-and studied their ability to function as alternative substrates for the reactions catalyzed by the IspDF fusion and IspE proteins from Agrobacterium tumefaciens, which covert MEP to the corresponding eight-membered cyclic diphosphate.

Alternative synthesis of the Colorado potato beetle pheromone.

A concise preparation of the pheromone secreted by the male Colorado potato beetle [viz. (3S)-1,3-dihydroxy-3,7-dimethyl-6-octen-2-one] was accomplished in four steps starting from 2-fluoronerol or 2-fluorogeraniol. The key step in the synthesis involves a 6-endo epoxide ring-opening with ester participation that simultaneously inverts the 3R-configuration of the (3R)-2,3-epoxy-2-fluoroprenyl acetate intermediate and installs the ketone functionality of the semiochemical.

Formation of the unusual semivolatile diterpene rhizathalene by the Arabidopsis class I terpene synthase TPS08 in the root stele is involved in defense against belowground herbivory.

Secondary metabolites are major constituents of plant defense against herbivore attack. Relatively little is known about the cell type-specific formation and antiherbivore activities of secondary compounds in roots despite the substantial impact of root herbivory on plant performance and fitness. Here, we describe the constitutive formation of semivolatile diterpenes called rhizathalenes by the class I terpene synthase (TPS) 08 in roots of Arabidopsis thaliana.

Dissecting paclitaxel-microtubule association: quantitative assessment of the 2'-OH group.

Paclitaxel (PTX) is a microtubule-stabilizing agent that is widely used in cancer chemotherapy. This structurally complex natural product acts by binding to β-tubulin in assembled microtubules. The 2'-hydroxyl group in the flexible side chain of PTX is an absolute requirement for activity, but its precise role in the drug-receptor interaction has not been specifically investigated.

Functional identification of valerena-1,10-diene synthase, a terpene synthase catalyzing a unique chemical cascade in the biosynthesis of biologically active sesquiterpenes in Valeriana officinalis.

Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes, found in V. officinalis roots.

Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase.

The structure of ent-copalyl diphosphate synthase reveals three α-helical domains (α, β and γ), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the βγ domains in ent-copalyl diphosphate synthase but exclusively in the α domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.

2-azapinanes: aza analogues of the enantiomeric pinyl carbocation intermediates in pinene biosynthesis.

The enantiomeric 2-azapinanes, aza analogues of the pinyl carbocation intermediates in pinene biosynthesis, were synthesized from (-)- and (+)-cis-pinonic acids. The individual reactions in the 5-step sequence were Beckmann rearrangement of the pinonic acid oximes, cyclization to the N-acetyl lactams, hydrolysis to the NH-lactams, N-methylations, and LiAlH(4) reductions. The anti stereochemistry of the N-methyl groups in the salts with respect to the gem-dimethyl bridge was established by NOE measurements and by X-ray diffraction analysis.

Taxadiene synthase structure and evolution of modular architecture in terpene biosynthesis.

With more than 55,000 members identified so far in all forms of life, the family of terpene or terpenoid natural products represents the epitome of molecular biodiversity. A well-known and important member of this family is the polycyclic diterpenoid Taxol (paclitaxel), which promotes tubulin polymerization and shows remarkable efficacy in cancer chemotherapy. The first committed step of Taxol biosynthesis in the Pacific yew (Taxus brevifolia) is the cyclization of the linear isoprenoid substrate geranylgeranyl diphosphate (GGPP) to form taxa-4(5),11(12)diene, which is catalysed by taxadiene synthase.

Characterization of the kaurene oxidase CYP701A3, a multifunctional cytochrome P450 from gibberellin biosynthesis.

KO (kaurene oxidase) is a multifunctional cytochrome P450 catalysing three sequential oxidations in gibberellin phytohormone biosynthesis. These serve to transform the C4α methyl of the ent-kaurene olefin intermediate into the carboxylic acid moiety of ent-kauren-19-oic acid. To investigate the unknown catalytic mechanism and properties of KO, we have engineered the corresponding CYP701A3 from Arabidopsis thaliana (AtKO) for functional recombinant expression in Escherichia coli, involving use of a fully codon-optimized construct, along with additional N-terminal deletion and modification.

Enantioselective inhibition of squalene synthase by aziridine analogues of presqualene diphosphate.

Squalene synthase catalyzes the conversion of two molecules of (E,E)-farnesyl diphosphate to squalene via the cyclopropylcarbinyl intermediate, presqualene diphosphate (PSPP). Since this novel reaction constitutes the first committed step in sterol biosynthesis, there has been considerable interest and research on the stereochemistry and mechanism of the process and in the design of selective inhibitors of the enzyme. This paper reports the synthesis and characterization of five racemic and two enantiopure aziridine analogues of PSPP and the evaluation of their potencies as inhibitors of recombinant yeast squalene synthase.

A single residue switch for Mg(2+)-dependent inhibition characterizes plant class II diterpene cyclases from primary and secondary metabolism.

Class II diterpene cyclases mediate the acid-initiated cycloisomerization reaction that serves as the committed step in biosynthesis of the large class of labdane-related diterpenoid natural products, which includes the important gibberellin plant hormones. Intriguingly, these enzymes are differentially susceptible to inhibition by their Mg(2+) cofactor, with those involved in gibberellin biosynthesis being more sensitive to such inhibition than those devoted to secondary metabolism, which presumably limits flux toward the potent gibberellin phytohormones. Such inhibition has been suggested to arise from intrasteric Mg(2+) binding to the DXDD motif that cooperatively acts as the catalytic acid, whose affinity must then be modulated in some fashion.

Bisabolyl-derived sesquiterpenes from tobacco 5-epi-aristolochene synthase-catalyzed cyclization of (2Z,6E)-farnesyl diphosphate.

We report the structures and stereochemistry of seven bisabolyl-derived sesquiterpenes arising from an unprecedented 1,6-cyclization (cisoid pathway) efficiently catalyzed by tobacco 5-epi-aristolochene synthase (TEAS). The use of (2Z,6E)-farnesyl diphosphate as an alternate substrate for recombinant TEAS resulted in a robust enzymatic cyclization to an array of products derived exclusively (>/=99.5%) from the cisoid pathway, whereas these same products account for ca.

Structural elucidation of cisoid and transoid cyclization pathways of a sesquiterpene synthase using 2-fluorofarnesyl diphosphates.

Sesquiterpene skeletal complexity in nature originates from the enzyme-catalyzed ionization of (trans,trans)-farnesyl diphosphate (FPP) (1a) and subsequent cyclization along either 2,3-transoid or 2,3-cisoid farnesyl cation pathways. Tobacco 5-epi-aristolochene synthase (TEAS), a transoid synthase, produces cisoid products as a component of its minor product spectrum. To investigate the cryptic cisoid cyclization pathway in TEAS, we employed (cis,trans)-FPP (1b) as an alternative substrate.

Doubly deuterium-labeled patchouli alcohol from cyclization of singly labeled [2-(2)H(1)]farnesyl diphosphate catalyzed by recombinant patchoulol synthase.

Incubations of isotopically pure [2-(2)H(1)](E,E)-farnesyl diphosphate with recombinant patchoulol synthase (PTS) from Pogostemon cablin afforded a 65:35 mixture of monodeuterated and dideuterated patchoulols as well as numerous sesquiterpene hydrocarbons. Extensive NMR analyses ((1)H and (13)C NMR, (1)H homodecoupling NMR, HMQC, and (2)H NMR) of the labeled patchoulol mixture and comparisons of the spectra with those of unlabeled alcohol led to the conclusion that the deuterium label was located at positions (patchoulol numbering system) C5 (both isotopomers, ca. 100%) and C12 (minor isotopomer, 30-35%), that is, an approximately 2:1 mixture of [5-(2)H(1)]- and [5,12-(2)H(2)]-patchoulols.

Scope and mechanism of intramolecular aziridination of cyclopent-3-enyl-methylamines to 1-azatricyclo[2.2.1.0(2,6)]heptanes with lead tetraacetate.

A series of seven cyclopent-3-en-1-ylmethylamines bearing one, two, or three methyl substituents at the C2, C3, C4, or C(alpha) positions, including the unsubstituted parent, was accessed by ring-closing metatheses of alpha,alpha-diallylacetonitrile (or methallyl variants) and alpha,alpha-diallylacetone followed by hydride reductions or reductive amination, or by Curtius degradations of alpha,alpha-dimethyl- and 2,2,3-trimethylcyclopent-3-enylacetic acids. Oxidation of the primary amines with Pb(OAc)(4) in CH(2)Cl(2), CHCl(3) or benzene in the presence of K(2)CO(3) effected efficient intramolecular aziridinations, in all cases except the alpha-methyl analogue (16), to form the corresponding 1-azatricyclo[2.2.

Characteristic flavonoids from Acacia burkittii and A. acuminata heartwoods and their differential cytotoxicity to normal and leukemia cells.

Bioassay-guided fractionation of extracts from Acacia burkittii and A. acuminata heartwoods against an L1210 (mouse lymphoma) cell line led to the isolation of two flavan-3,4-diols, melacacidin (1) and isomelacacidin (2), and three flavonoids, 3,7,8,3',4'-pentahydroxyflavone (3), 7,8,3',4'-tetrahydroxyflavanone (4) and 3,7,8,3',4'-pentahydroxyflavanone (5). HPLC analyses (280 nm) of the 70% acetone extracts of the two plants showed different profiles in terms of the relative concentration of the five identified compounds.

Specificity of the N-benzoyl transferase responsible for the last step of Taxol biosynthesis.

The last few steps in the biosynthesis of the anticancer drug Taxol in yew (Taxus) species are thought to involve the attachment of beta-phenylalanine to the C13-O-position of the advanced taxane diterpenoid intermediate baccatin III to yield N-debenzoyl-2'-deoxytaxol, followed by hydroxylation on the side chain at the C2'-position to afford N-debenzoyltaxol, and finally N-benzoylation to complete the pathway. A cDNA encoding the N-benzoyl transferase that catalyzes the terminal step of the reaction sequence was previously isolated from a family of transferase clones (derived from an induced Taxus cell cDNA library) by functional characterization of the corresponding recombinant enzyme using the available surrogate substrate N-debenzoyl-2'-deoxytaxol [K. Walker, R.

X-ray crystallographic studies of substrate binding to aristolochene synthase suggest a metal ion binding sequence for catalysis.

The universal sesquiterpene precursor, farnesyl diphosphate (FPP), is cyclized in an Mg(2+)-dependent reaction catalyzed by the tetrameric aristolochene synthase from Aspergillus terreus to form the bicyclic hydrocarbon aristolochene and a pyrophosphate anion (PP(i)) coproduct. The 2.1-A resolution crystal structure determined from crystals soaked with FPP reveals the binding of intact FPP to monomers A-C, and the binding of PP(i) and Mg(2+)(B) to monomer D.

The dioxanone approach to (2S,3R)-2-C-methylerythritol 4-phosphate and 2,4-cyclodiphosphate, and various MEP analogues.

Efficient syntheses of the non-mevalonate pathway intermediates 2-C-methylerythritol 4-phosphate (MEP) and 2-C-methylerythritol 2,4-cyclodiphosphate (ME-2,4-cycloPP), as well as the parent tetrol 2-C-methylerythritol, in enantiopure form from (2S,4R)-cis-2-phenyl-4-tert-butyldimethylsilyloxy-1,3-dioxan-5-one are reported. The 2S configuration of the C-methyl group was installed by highly axial-face selective addition of CH3MgBr (20:1) to the chiral dioxanone carbonyl group. Primary selective mono-phosphorylation and 2,4-bis-phosphorylation, followed by desilation and hydrogenolysis to the free mono- and diphosphates, and, in the latter case, cyclization to form the eight-membered phosphoryl anhydride, afforded MEP and ME-2,4-cycloPP in good yields.

Inhibition of monoterpene cyclases by inert analogues of geranyl diphosphate and linalyl diphosphate.

The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which then cyclizes to the various monoterpene skeletons. X-ray crystal structures of these enzymes complexed with suitable analogues of the substrate and intermediate could provide a clearer view of this universal, but cryptic, step of monoterpenoid cyclase catalysis. Toward this end, the functionally inert analogues 2-fluorogeranyl diphosphate, (+/-)-2-fluorolinalyl diphosphate, and (3R)- and (3S)-homolinalyl diphosphates (2,6-dimethyl-2-vinyl-5-heptenyl diphosphates) were prepared, and compared to the previously described substrate analogue 3-azageranyl diphosphate (3-aza-2,3-dihydrogeranyl diphosphate) as inhibitors and potential crystallization aids with two representative monoterpenoid cyclases, (-)-limonene synthase and (+)-bornyl diphosphate synthase.

16-Aza-ent-beyerane and 16-Aza-ent-trachylobane: potent mechanism-based inhibitors of recombinant ent-kaurene synthase from Arabidopsis thaliana.

The secondary ent-beyeran-16-yl carbocation (7) is a key branch point intermediate in mechanistic schemes to rationalize the cyclic structures of many tetra- and pentacyclic diterpenes, including ent-beyerene, ent-kaurene, ent-trachylobane, and ent-atiserene, presumed precursors to >1000 known diterpenes. To evaluate these mechanistic hypotheses, we synthesized the heterocyclic analogues 16-aza-ent-beyerane (12) and 16-aza-ent-trachylobane (13) by means of Hg(II)- and Pb(IV)-induced cyclizations onto the Delta12 double bonds of tricyclic intermediates bearing carbamoylmethyl and aminomethyl groups at C-8. The 13,16-seco-16-norcarbamate (20a) was obtained from ent-beyeran-16-one oxime (17) by Beckmann fragmentation, hydrolysis, and Curtius rearrangement.

Interception of the enzymatic conversion of farnesyl diphosphate to 5-epi-aristolochene by using a fluoro substrate analogue: 1-fluorogermacrene A from (2E,6Z)-6-fluorofarnesyl diphosphate.

Tobacco 5-epi-aristolochene synthase (TEAS) catalyzes the Mg(II)-dependent cyclizations and rearrangements of (E,E)-farnesyl diphosphate (PP) to the bicyclic sesquiterpene hydrocarbon via a tightly bound (+)-germacrene A as a deprotonated intermediate. With the native enzyme, only a few percent of the putative germacrene A intermediate is released from the active site during the catalytic cycle. 6-Fluorofarnesyl PP was designed and synthesized with the aim of arresting the cyclization-rearrangement mechanism en route to 5-epi-aristolochene.

Conformational Analysis of (+)-Germacrene A by Variable Temperature NMR and NOE Spectroscopy.

(+)-Germacrene A, an important intermediate in sesquiterpene biosynthesis, was isolated in pure form from a genetically engineered yeast and was characterized by chromatographic properties (TLC, GC), MS, optical rotation, UV, IR, (1)H NMR and (13)C NMR data. Variable-temperature 500 MHz (1)H NMR spectra in CDCl(3) showed that this flexible cyclodecadiene ring exists as three NMR-distinguishable conformational isomers in a ratio of about 5:3:2 at or below ordinary probe temperature (25° C). The conformer structures were assigned by (1)H NMR data comparisons, NOE experiments, and vicinal couplings as follows: 1a (52%, UU), 1b (29% UD), and 1c (19%, DU).

Functional characterization of premnaspirodiene oxygenase, a cytochrome P450 catalyzing regio- and stereo-specific hydroxylations of diverse sesquiterpene substrates.

Solavetivone, a potent antifungal phytoalexin, is derived from a vetispirane-type sesquiterpene, premnaspirodiene, by a putative regio- and stereo-specific hydroxylation, followed by a second oxidation to yield the alpha,beta-unsaturated ketone. Mechanistically, these reactions could occur via a single, multifunctional cytochrome P450 or some combination of cytochrome P450s and a dehydrogenase. We report here the characterization of a single cytochrome P450 enzyme, Hyoscyamus muticus premnaspirodiene oxygenase (HPO), that catalyzes these successive reactions at carbon 2 (C-2) of the spirane substrate.