Genome mining in Streptomyces coelicolor: molecular cloning and characterization of a new sesquiterpene synthase

The terpene synthase encoded by the SCO5222 (SC7E4.19) gene of Streptomyces coelicolor was cloned by PCR and expressed in Escherichia coli as an N-terminal-His6-tag protein. Incubation of the recombinant protein, SCO5222p, with farnesyl diphosphate (1, FPP) in the presence of Mg(II) gave a new sesquiterpene, (+)-epi-isozizaene (2), whose structure and stereochemistry were determined by a combination of 1H, 13C, COSY, HMQC, HMBC, and NOESY NMR. The steady-state kinetic parameters were kcat 0.049 +/- 0.001 s-1 and a Km (FPP) of 147 +/- 14 nM. Individual incubations of recombinant epi-isozizaene synthase with [1,1-2H2]FPP (1a), (1R)-[1-2H]-FPP (1b), and (1S)-[1-2H]-FPP (1c) and NMR analysis of the resulting deuterated epi-isozizaenes supported an isomerization-cyclization-rearrangement mechanism involving the intermediacy of (3R)-nerolidyl diphosphate (3).     

Three New Bisabolocurcumin Ethers from the Rhizomes of Curcuma longa L.

Three new bisabolocurcumin ethers, named demethoxybisabolocurcumin ether ( 1 ), bisabolocurcumin ether ( 2 ), and didemethoxybisabolocurcumin ether ( 3 ), along with two known compounds, 4 and 5 , were isolated from the AcOEt extract of the rhizomes of Curcuma longa L. Their structures were established by the analysis of NMR and MS data. The new compounds 1 – 3 , which possess a new 1,7‐diarylheptanoid skeleton linked with a bisabolone‐type sesquiterpene substructure by a C? O bond, were found for the first time in a natural source.     

Trichodiene Synthase: Synthesis and Inhibition Kinetics of 12‐Fluoro‐farnesylphosphonophosphate for Sesquiterpene Cyclases

trans, trans‐Farnesyl diphosphate (FPP) serves as a universal substrate for a large family of sesquiterpene cyclases that are responsible for biosynthesis of more than 300 structurally diverse sesquiterpenes in nature. A new FPP substrate analogue, 12‐fluoro‐farnesylphosphonophosphate (12‐F‐F‐CH₂PP), was synthesized in this paper for applications on kinetic and mechanistic studies of the enzyme family. Trichodiene synthase (TS), a sesquiterpene cyclase, catalyzes the conversion of trans, trans‐farnesyl diphosphate (FPP) to trichodiene. 12‐F‐F‐CH₂PP was tested as a potential inhibitor of TS. Inactivation and inhibition kinetic experiments showed that 12‐F‐F‐CH₂PP was not a mechanism‐based inactivator for TS; instead, a mixed‐type reversible inhibition was observed with inhibition constants K_i1 = 2.33 ± 0.50 μM and K_i2 = 25.80 ± 7.70 μM, values close to those previously determined for farnesylphosphonophosphate, K_i1 = 3.25 μM and K_i2 = 9.10 μM. Although 12‐F‐F‐CH₂PP did not irreversibly inactivate TS, this new analogue serves as a potential active‐site directed inactivator and mechanistic probe of other sesquiterpene cyclases and FPP‐utilizing enzymes, which utilize FPP as a common acyclic substrate.     

Biosynthesis of anthecotuloide,an irregular sesquiterpene lactone from Anthemis cotula L. (Asteraceae) via a non-farnesyl diphosphate route

Retrobiosynthetic analysis of the allergenic sesquiterpene lactone, anthecotuloide, suggested that this natural product could be formed either by head to head condensation of geranyl diphosphate with dimethylallyl diphosphate, or from farnesyl diphosphate (FPP), the accepted regular sesquiterpene precursor via the rearrangement of a germacranolide precursor. Isotopic labelling of anthecotuloide has now been achieved by feeding [1-(13)C]-glucose, [U-13C6]-glucose and [6,6-(2)H2]-glucose to aseptically grown plantlets of Anthemis cotula(family Asteraceae). Analysis of labelling patterns and absolute 13C abundances using quantitative 13C NMR spectroscopy showed that the isoprene building blocks of this sesquiterpene are formed exclusively via the MEP terpene biosynthetic pathway. This was supported by results from an experiment using [U-13C6]-glucose. A deuterium labelling experiment using [6,6-(2)H2]-glucose supported the original proposal and showed that anthecotuloide is formed from a non FPP precursor. Isotope ratio mass spectrometry suggested that there were two pathways for sesquiterpene biosynthesis in A. cotula.     

Chrysanthemyl diphosphate synthase. The relationship among chain elongation,branching, and cyclopropanation reactions in the isoprenoid biosynthetic pathway

The genes for chrysanthemyl diphosphate (CPP) synthase and farnesyl diphosphate (FPP) synthase from sagebrush, Artemisia tridentata spiciformis, were used to prepare a series of chimeric proteins to investigate the 1'-4 chain elongation, 1'-2 branching, and c1'-2-3 cyclopropanation reactions that join isoprenoid units to build more complex structures. The two genes were modified by site-directed mutagenesis to generate an identical set of six unique restriction sites at identical locations. The locations were selected to place a restriction site between each of the five conserved regions found in prenyltransferases that catalyze chain elongation. A series of chimeric proteins were generated by replacing amino acids in FPP synthase, beginning at the N-terminus of the enzyme, with increasing stretches of peptide from CPP synthase. An analysis of the products produced by the chimeras revealed a transition from 1'-4 chain elongation, to 1'-2 branching, and ultimately to c1'-2-3 cyclopropanation. These results demonstrate that the catalytic site for chain elongation, with minor modifications in its architecture, also catalyzes 1'-2 branching and c1'-2-3 cyclopropanation, and suggest that the branching and cyclopropanation reactions, in analogy to chain elongation, are electrophilic alkylations.     

Mechanism and stereochemistry of the germacradienol/germacrene D synthase of Streptomyces coelicolor A3(2)

Incubation of farnesyl diphosphate (1, FPP) with recombinant germacradienol synthase from Streptomyces coelicolor A3(2) gave, in addition to (4S,7R)-germacra-1(10)E,5E-diene-11-ol (2), 15% of (-)-germacrene D (5). Incubations of [1,1-2H2]FPP (1a), (1R)-[1-2H]FPP (1b), and (1S)-[1-2H]FPP (1c) with germacradienol/germacrene D synthase and analysis of the resulting samples of germacradienol (2) and germacrene D (5) by a combination of 1H, 2H, and 13C NMR and mass spectrometry established that it is H-1si of FPP that is lost in the formation of germacradienol (2) and that undergoes 1,3-hydride transfer in the formation of (-)-germacrene D (5). The proportion of the two products was also sensitive to isotopic labeling, with cyclization of (1S)-[1-2H]FPP (1c) giving an increased proportion (35%) of 5. These results could be explained by a mechanism involving partitioning of a common helminthogermacradienyl cation intermediate 7.     

Development of Petri Net-Based Dynamic Model for Improved Production of Farnesyl Pyrophosphate by Integrating Mevalonate and Methylerythritol Phosphate Pathways in Yeast

In this case study, we designed a farnesyl pyrophosphate (FPP) biosynthetic network using hybrid functional Petri net with extension (HFPNe) which is derived from traditional Petri net theory and allows easy modeling with graphical approach of various types of entities in the networks together. Our main objective is to improve the production of FPP in yeast, which is further converted to amorphadiene (AD), a precursor of artemisinin (antimalarial drug). Natively, mevalonate (MEV) pathway is present in yeast. Methyl erythritol phosphate pathways (MEP) are present only in higher plant plastids and eubacteria, but not present in yeast. IPP and DAMP are common isomeric intermediate in these two pathways, which immediately yields FPP. By integrating these two pathways in yeast, we augmented the FPP synthesis approximately two folds higher (431.16 U/pt) than in MEV pathway alone (259.91 U/pt) by using HFPNe technique. Further enhanced FPP levels converted to AD by amorphadiene synthase gene yielding 436.5 U/pt of AD which approximately two folds higher compared to the AD (258.5 U/pt) synthesized by MEV pathway exclusively. Simulation and validation processes performed using these models are reliable with identified biological information and data.     

An Unusual Skeletal Rearrangement in the Biosynthesis of the Sesquiterpene Trichobrasilenol from Trichoderma

The skeletons of some classes of terpenoids are unusual in that they contain a larger number of Me groups (or their biosynthetic equivalents such as olefinic methylene groups, hydroxymethyl groups, aldehydes, or carboxylic acids and their derivatives) than provided by their oligoprenyl diphosphate precursor. This is sometimes the result of an oxidative ring‐opening reaction at a terpene‐cyclase‐derived molecule containing the regular number of Me group equivalents, as observed for picrotoxan sesquiterpenes. In this study a sesquiterpene cyclase from Trichoderma spp. is described that can convert farnesyl diphosphate (FPP) directly via a remarkable skeletal rearrangement into trichobrasilenol, a new brasilane sesquiterpene with one additional Me group equivalent compared to FPP. A mechanistic hypothesis for the formation of the brasilane skeleton is supported by extensive isotopic labelling studies.     

Direct observation of substrate-enzyme complexation by surface forces measurement

The substrate-enzyme complexation of heptaprenyl diphosphate synthase was directly investigated using colloidal probe atomic force microscopy (AFM) and a quartz crystal microbalance (QCM) in order to obtain new insights into the molecular mechanism of the enzyme reaction. This enzyme is composed of two dissociable subunits that exhibit a catalytic activity only when they are associated together in the presence of a cofactor, Mg2+, and a substrate, farnesyl diphosphate (FPP). The QCM measurement revealed that FPP was preferentially bound to subunit II in the presence of Mg2+, while the AFM measurement showed that the adhesive force between the subunits was observed only in the presence of both Mg2+ and FPP. This is the first direct demonstration of the specific interaction involved in the enzyme reaction. The dependence of the Mg2+ concentration on the specific interaction between subunits I and II well agreed with that on the enzyme activity of heptaprenyl diphosphate synthase. This indicated that the observed adhesive forces were indeed involved in the catalytic reaction of this enzyme. On the basis of these results, we discussed the processes involved in the substrate-enzyme complexation. The first, the substrate FPP bound to subunit II using Mg2+, followed by the formation of the subunit I-FPP-Mg2+-subunit II complex. Our study showed a very useful methodology for examining the elemental processes of biological reactions such as an enzyme reaction.     

Dicobalt octacarbonyl-catalyzed cationic polymerization of allylic and enolic ethers

In the presence of silanes bearing Si H groups, dicobalt octacarbonyl [Co₂(CO)₈] efficiently catalyzes the cationic polymerization of a wide variety of enol ether and other related monomers including vinyl ethers, 1-propenyl ethers, 1-butenyl ethers, 2,3-dihydrofuran, 3,4-dihydro-2H-pyran, ketene acetals, and allene ethers. In addition, this catalyst system is also effective for the polymerization of complimentary allylic and propargylic ethers by a process involving tandem isomerization and cationic polymerization. This latter process occurs by a stepwise mechanism in which the allylic or propargylic ether is first isomerized, respectively, to the corresponding enol ether or allenic ether and then this latter compound is rapidly cationically polymerized in the presence of the catalyst. In accord with this mechanism, it has been shown that the structure of the polymers prepared from related enol and allyl ethers using the above catalyst system are identical. © 1997 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 35: 1579–1591, 1997     

Farnesyl diphosphate synthase: the art of compromise between substrate selectivity and stereoselectivity

Farnesyl diphosphate (FPP) synthase catalyzes the consecutive head-to-tail condensations of isopentenyl diphosphate (IPP, C5) with dimethylallyl diphosphate (DMAPP, C5) and geranyl diphosphate (GPP, C10) to give (E,E)-FPP (C15). The enzyme belongs to a genetically distinct family of chain elongation enzymes that install E-double bonds during each addition of a five-carbon isoprene unit. Analysis of the C10 and C15 products from incubations with avian FPP synthase reveals that small amounts of neryl diphosphate (Z-C10) and (Z,E)-FPP are formed along with the E-isomers during the C5 --> C10 and C10 --> C15 reactions. Similar results were obtained for FPP synthase from Escherichia coli, Artemisia tridentata (sage brush), Pyrococcus furiosus, and Methanobacter thermautotrophicus and for GPP and FPP synthesized in vivo by E. coli FPP synthase. When (R)-[2-2H]IPP was a substrate for chain elongation, no deuterium was found in the chain elongation products. In contrast, the deuterium in (S)-[2-2H]IPP was incorporated into all of the products. Thus, the pro-R hydrogen at C2 of IPP is lost when the E- and Z-double bond isomers are formed. The synthesis of Z-double bond isomers by FPP synthase during chain elongation is unexpected for a highly evolved enzyme and probably reflects a compromise between optimizing double bond stereoselectivity and the need to exclude DMAPP from the IPP binding site.     

SYNTHESIS OF ALKYL AND ARYL 1-PROPENYL ETHER MONOMERS: A NEW APPROACH

A new, convenient synthesis of alkyl and aryl 1-propenyl ether monomers in good to excellent yields has been developed. Alkyl and aryl allyl ethers can be smoothly isomerized to the desired 1-propenyl ethers by refluxing in a basic ethanolic solution containing pentacarbonyliron as a catalyst. A simplified two-step, one-pot procedure has also been developed which consists of combining an alcohol with allyl bromide in the presence of base and then adding pentacarbonyliron to isomerize the in-situ generated allyl ether to directly give the 1-propenyl ether. Good yields of alkyl 1-propenyl ethers were obtained using this process. Factors affecting the isomerization reaction were investigated and a mechanism was proposed.     

Recombinant squalene synthase. Synthesis of non-head-to-tail isoprenoids in the absence of NADPH

Squalene synthase (SQase) catalyzes two consecutive reactions in sterol biosynthesis. The first is the condensation of two molecules of farnesyl diphosphate (FPP) to form a cyclopropylcarbinyl intermediate, presqualene diphosphate (PSPP). The subsequent conversion of PSPP to squalene (SQ) involves an extensive rearrangement of the carbon skeleton and a NADPH-dependent reduction. Incubation of a truncated soluble form of recombinant yeast SQase with FPP in buffer lacking NADPH gave (1R,2R,3R)-PSPP. As the incubation continued, SQase catalyzed the subsequent conversion of PSPP to a mixture of triterpenes. Two of the major products, (Z)-dehydrosqualene (DSQ) and (R)-12-hydroxysqualene (HSQ), have the same 1'-1 linkage between the farnesyl units from FPP that is found in squalene. The other major product, (10S,13S)-10-hydroxybotryococcene (HBO), has a 1'-3 linkage between the farnesyl units. Small quantities of (S)-HSQ and (10R,13S)-HBO were also formed. Three additional triterpenes, the allylic isomers of HSQ and HBO, and an unidentified alcohol were produced in minor amounts. A methyl ether corresponding to HSQ was detected when methanol was present in the incubation buffer. These compounds are the expected "solvolysis" products from PSPP. They provide strong support for mechanisms that propose cyclopropylcarbinyl cations as intermediates in the SQase-catalyzed rearrangement of PSPP to SQ and unambiguously demonstrate that the catalytic machinery of SQase is capable of synthesizing a variety of irregular isoprenoids.     

Synthesis of Aryl Ethers via a Sulfonyl Transfer Reaction

A general synthesis of aryl ethers from primary and secondary alcohols and aryl mesylates is presented. The reaction proceeds via a sulfonyl-transfer mechanism. In this paper, we compare the sulfonyl transfer reaction to Mitsunobu ether formation. The reaction can be employed in a multistep synthesis where the aryl mesylate is used as a phenol protecting group and then as an activating group for ether formation. This protecting/activating group strategy is demonstrated using raloxifene as the target.     

γ-Ray-induced copolymerization of carbon monoxide with cyclic ethers

The γ-ray copolymerization of carbon monoxide with cyclic ethers, such as ethylene oxide, phenyl glycidyl ether, 1,3-dioxolane, 2-vinyl-1,3-dioxolane, terahydrofuran, 1,4-dioxane, and acetaldehyde was studied. A yellowish or brownish powdery copolymer was obtained in most of the cases examined. The infrared spectra showed that copolymers containing the ester structural unit were produced in the copolymerization with cyclic ethers which have no vinyl groups, and that a copolymer containing a ketone structure was produced from cyclic ether having vinyl group. It was found that the copolymer with ethylene oxide also had a β-propiolactone ring structure at the chain end or the side chain. The copolymers were confirmed to be partially crystalline from the x-ray diffraction diagrams. Further, a ring-opening polymerizability of the cyclic ether by γ-radiation was discussed. And it was found that as the bond dissociation energy between the carbon–oxygen linkage of the cyclic ether is small, the polymer yield both in the homopolymerization and copolymerization with carbon monoxide is high. A mechanism for the copolymerization is proposed on the basis of the results.     

Competitive inhibition of aristolochene synthase by phenyl-substituted farnesyl diphosphates: evidence of active site plasticity

Analogues of farnesyl diphosphate (FPP, ) containing phenyl substituents in place of methyl groups have been prepared in syntheses that feature use of a Suzuki-Miyaura reaction as a key step. These analogues were found not to act as substrates of the sesquiterpene cyclase aristolochene synthase from Penicillium roqueforti (AS). However, they were potent competitive inhibitors of AS with K(I)-values ranging from 0.8 to 1.2 microM. These results indicate that the diphosphate group contributes the largest part to the binding of the substrate to AS and that the active sites of terpene synthases are sufficiently flexible to accommodate even substrate analogues with large substituents suggesting a potential way for the generation of non-natural terpenoids. Molecular mechanics simulations of the enzyme bound inhibitors suggested that small changes in orientations of active site residues and subtle alterations of the conformation of the backbones of the inhibitors are sufficient to accommodate the phenyl-farnesyl-diphosphates.     

Two Reaction Mechanisms via Iminium Ion Intermediates: The Different Reactivities of Diphenylprolinol Silyl Ether and Trifluoromethyl‐Substituted Diarylprolinol Silyl Ether

The reactions of α,β‐unsaturated aldehydes with cyclopentadiene in the presence of diarylprolinol silyl ethers as catalyst proceed via iminium cations as intermediates, and can be divided into two types; one involving a Michael‐type reaction (type A) and one involving a cycloaddition (type B). Diphenylprolinol silyl ethers and trifluoromethyl‐substituted diarylprolinol silyl ethers, which are widely used proline‐type organocatalysts, have been investigated in this study. As the LUMO of the iminium ion derived from trifluoromethyl‐substituted diarylprolinol silyl ether is lower in energy than that derived from diphenylprolinol silyl ether, as supported by ab initio calculations, the trifluoromethyl‐substituted catalyst is more reactive in a type B reaction. The iminium ion from an α,β‐unsaturated aldehyde is generated more quickly with diphenylprolinol silyl ether than with the trifluoromethyl‐substituted diarylprolinol silyl ether. When the generation of the iminium ion is the rate‐determining step, the diphenylprolinol silyl ether catalyst is the more reactive. Because acid accelerates the generation of iminium ions and reduces the generation of anionic nucleophiles in the Michael‐type reaction (type A), it is necessary to select the appropriate acid for specific reactions. In general, diphenylprolinol silyl ether is a superior catalyst for type A reactions, whereas the trifluoromethyl‐substituted diarylprolinol silyl ether catalyst is preferred for type B reactions.     

Ionic polymerization of p-vinylbenzyl methyl ether

Ionic polymerizations of vinylbenzyl methyl ether initiated by either carbanions or Lewis acids has been found to lead to crosslinked polymers. By comparative studies of strong carbanionic bases and Lewis acids with benzyl ethers, it has been possible to define details of mechanisms which in conjunction with cationic or anionic propagation lead to crosslinks. The α-hydrogens of benzyl ethers have been found to be sufficiently acidic to terminate anionic polymerization of styrene and displacement of alkoxide anion from the benzyl ether linkage by nucelophilic polymer anions is proposed as a mechanism leading to branching and eventual crosslinking in anionic polymerization of vinylbenzyl methyl ether. Cationic polymerization of vinylbenzyl methyl ether is quite complex. In addition to propagation, chain transfer, and spontaneous termination of cation chain carriers, there is evidence for complex formation between Lewis acid initiator and the benzyl ether substituent. A slow decomposition of ether–Lewis acid complexes produces benzylcarbonium ions which alkylate aromatic rings of polymer and thereby crosslink the polymer. Benzyl ether has been found to be an effective chain terminator for cationic styrene polymerization.     

Synthesis of Glycol Ethers and Their Use for Intensification of Oil Recovery

Monoalkyl ethers of ethylene and triethylene glycols were prepared and tested for intensification of oil recovery. The features of oil displacement with aqueous solutions of glycol ethers from bulk models of strata and the effect of glycol ethers on acid treatment of oil-saturated samples were examined. A correlation between the structure of ether and its performance was revealed. The interphase tension at the boundary between the aqueous solution of the glycol ether and kerosene was determined.     

Cross-dehydrogenative coupling of secondary benzylic ethers with indoles and pyrroles

Current studies on cross-dehydrogenative coupling of benzylic ethers for new C–C bond construction predominantly focus on primary ether moieties. Oxidative cross-coupling of secondary benzylic ethers remains elusive. Herein, we describe the first cross-dehydrogenative coupling of secondary benzylic ethers with indoles and pyrroles for tertiary ether construction. A broad range of α-aryl substituted isochromans react with a variety of electronically varied indoles and pyrroles smoothly under mild metal-free conditions in high efficiency. In addition, the catalytic asymmetric variant was preliminarily explored, and corresponding tertiary ether was obtained in 69% ee.