ABSTRACT
Chlororaphens A and B are structurally unique non-canonical C17 sesquiterpenoids from Pseudomonas chlororaphis that are made by two SAM-dependent methyltransferases and a type I terpene synthase. This study addresses the mechanism of their formation in isotopic labelling experiments and DFT calculations. The results demonstrate an astonishing complexity with distribution of labellings within a cyclopentane core that is reversely connected to two acyclic fragments in chlororaphen A and B. In addition, the uptake of up to 14 deuterium atoms from D2O was observed. These findings are explainable by a repeated late stage multistep rearrangement sequence. The absolute configurations of the chlororaphens and their biosynthetic intermediates were elucidated in stereoselective labelling experiments.
ABSTRACT
Two terpene synthases from the pleuromutilin producing fungus Clitopilus passeckerianus were functionally characterised. The first enzyme CpTS1 produces the new diterpene clitopilene with a novel 6-6-5-5 tetracyclic skeleton, while the second enzyme CpTS2 makes the new sesquiterpene isopentalenene. The CpTS1 reaction mechanism was studied in depth using experimental and theoretical approaches.
Subject(s)
Alkyl and Aryl Transferases , Diterpenes , Alkyl and Aryl Transferases/metabolism , Diterpenes/chemistry , Diterpenes/metabolism , Sesquiterpenes/chemistry , Sesquiterpenes/metabolism , PleuromutilinsABSTRACT
The biosynthesis of six recently reported non-canonical C16 sesquiterpenoids named after ancient Greek philosophers, archimedene, aristotelene, eratosthenene, pythagorene, α-democritene and anaximandrene, was investigated through density functional theory (DFT) calculations and isotopic labeling experiments. The results revealed for all compounds except archimedene a unique fragmentation-recombination mechanism as previously demonstrated for sodorifen biosynthesis, in addition to a remarkable "dancing" mechanism for anaximandrene biosynthesis.
ABSTRACT
Mining of two multiproduct sesterterpene synthases from Lentzea atacamensis resulted in the identification of the synthases for lentzeadiene (LaLDS) and atacamatriene (LaATS). The main product of LaLDS (lentzeadiene) is a new compound, while one of the side products (lentzeatetraene) is the enantiomer of brassitetraene B and the other side product (sestermobaraene F) is known from a surprisingly distantly related sesterterpene synthase. LaATS produces six new compounds, one of which is the enantiomer of the known sesterterpene Bm1. Notably, for both enzymes the products cannot all be explained from one and the same starting conformation of geranylfarnesyl diphosphate, demonstrating the requirement of conformational flexibility of the substrate in the enzymes' active sites. For lentzeadiene an intriguing thermal [1,5]-sigmatropic rearrangement was discovered, reminiscent of the biosynthesis of vitamin D3. All enzyme reactions and the [1,5]-sigmatropic rearrangement were investigated through isotopic labeling experiments and DFT calculations. The results also emphasize the importance of conformational changes during terpene cyclizations.
Subject(s)
Sesterterpenes , Terpenes , Terpenes/metabolism , Terpenes/chemistry , Sesterterpenes/chemistry , Sesterterpenes/metabolism , Molecular Conformation , Alkyl and Aryl Transferases/metabolism , Alkyl and Aryl Transferases/chemistry , StereoisomerismABSTRACT
The substrate analogue 19-nor-geranylgeranyl diphosphate (19-nor-GGPP) was synthesised and incubated with 20â diterpene synthases, resulting in the formation of diterpenoids in all cases. A total of 23â different compounds were isolated from these enzyme reactions and structurally characterised, if possible including the experimental determination of absolute configurations through a stereoselective deuteration approach. In several cases the missing 19-Me group in the substrate analogue resulted in opening of completely new reaction paths towards compounds with novel skeletons. DFT calculations were applied to gain a deeper understanding of these observed methyl group effects in diterpene biosynthesis.
Subject(s)
Alkyl and Aryl Transferases , Diterpenes , Diterpenes/chemistryABSTRACT
The diterpene synthase AlTS was identified from Aspergillus luchuensis. AlTS catalyses the formation of the diterpene hydrocarbon spiroluchuene A, which exhibits a novel skeleton characterised by a spirocyclic ring system. The cyclisation mechanism towards this compound was elucidated through isotopic labelling experiments in conjunction with DFT calculations and metadynamic simulations. The biosynthetic intermediate luchudiene, besides the derivative spiroluchuene B, was captured from an enzyme variant obtained through site-directed mutagenesis. With its 10-membered ring luchudiene is structurally related to germacrenes and can undergo a Cope rearrangement to luchuelemene.
Subject(s)
Diterpenes , Aspergillus/genetics , CyclizationABSTRACT
In the past decade, there has been an increased interest in applying supramolecular capsule and cage catalysis to the current challenges in synthetic organic chemistry. In this context, we recently reported the resorcin[4]arene capsule-catalyzed conversion of α-glycosyl halides into ß-glycosides with high selectivity. Interestingly, this methodology enabled the formation of a wide range of ß-pyranosides as well as ß-furanosides, although these two donor classes exhibit different reactivities and usually require different reaction conditions and catalysts. Evidence was provided that a proton wire plays a key role in this reaction by enabling dual activation of the glycosyl donor and acceptor. Here, we describe a detailed investigation of several aspects of this reactivity. Besides a mechanistic study, we elucidated the size limitation, the origin of catalytic turnover, and the electrophile scope of nonglycosylic halides. Moreover, a screening of the sensitivity to changes in the reaction conditions provides guidelines to facilitate reproducibility. Furthermore, we demonstrate the compatibility with environmentally benign solvent alternatives, including the renewable solvent limonene.
ABSTRACT
Mining of a terpene synthase from Streptomyces subrutilus resulted in the identification of the hexacyclic sesterterpene subrutilane, besides eight pentacyclic side products. Subrutilane represents the first case of a saturated sesterterpene hydrocarbon. Its structure, including the absolute configuration, was unambiguously determined through X-ray crystallographic analysis and stereoselective deuteration. The cyclisation mechanism to subrutilane and its side products was investigated in all detail by isotopic labelling experiments and DFT calculations. The subrutilane synthase (SrS) also converted (2Z)-GFPP into one major product. Additional compounds were obtained from the substrate analogues (7R)-6,7-dihydro-GFPP and (2Z,7R)-6,7-dihydro-GFPP with blocked reactivity at the C6-C7 bond. Interestingly, the early steps of the cyclisation cascade with (2Z)-GFPP and the saturated substrate analogues were analogous to those of GFPP, but then deviations from the natural cyclisation mode occur.
Subject(s)
Alkyl and Aryl Transferases , Streptomyces , Humans , Sesterterpenes/chemistry , Terpenes/chemistryABSTRACT
A gene coding for a terpene synthase homolog from Kitasatospora viridis was cloned and expressed in Escherichia coli. The purified recombinant protein possessed sesterterpene synthase activity and efficiently converted geranylfarnesyl diphosphate (GFPP) with 19 % yield into the sesterterpene hydrocarbon sestervirideneâ A. Large scale enzymatic conversions also allowed for the isolation of two side products that are generated with very low yields of ca. 0.1 %. Several derivatives of sestervirideneâ A were obtained by chemical transformations, securing the NMR-based structural assignments. The absolute configuration of sestervirideneâ A was determined by chemical correlation using stereoselectively deuterated precursors and by anomalous dispersion X-ray crystallography. The cyclisation mechanism from GFPP to sestervirideneâ A was extensively studied through isotopic labelling experiments and DFT calculations.
Subject(s)
Alkyl and Aryl Transferases , Streptomycetaceae , Sesterterpenes/chemistry , Streptomycetaceae/metabolism , Recombinant ProteinsABSTRACT
Terpenes constitute the largest class of natural products. Their skeletons are formed by terpene cyclases (TCs) from acyclic oligoprenyl diphosphates through sophisticated enzymatic conversions. These enzyme reactions start with substrate ionization through diphosphate abstraction, followed by a cascade reaction via cationic intermediates. Based on isotopic-labelling experiments in combination with a computational study, the cyclization mechanism for sodorifen, a highly methylated sesquiterpene from the soil bacterium Serratia plymuthica, was resolved. A peculiar problem in its biosynthesis lies in the formation of several methyl groups from chain methylene carbons. The underlying mechanism involves a methyltransferase-mediated cyclization and unprecedented ring contraction with carbon extrusion from the chain to form a methyl group. A terpene cyclase subsequently catalyses a fragmentation into two reactive intermediates, followed by hydrogen transfers between them and recombination of the fragments by [4 + 3] cycloaddition. This study solves the intricate mechanistic problem of extra methyl group formation in sodorifen biosynthesis.
Subject(s)
Sesquiterpenes , Terpenes , Cycloaddition Reaction , Bridged Bicyclo Compounds , CyclizationABSTRACT
A sesquiterpene synthase from Kitasatospora viridis was discovered and shown to produce kitaviridene, a sesquiterpene hydrocarbon with an additional methyl group equivalent in comparison to a regular sesquiterpene. Isotopic labeling experiments together with DFT calculations gave detailed insights into the cyclization cascade toward kitaviridene and explained the formation of the additional methyl group equivalent.
ABSTRACT
The sesterviolene synthase from Streptomyces violens was identified and represents the second known sesterterpene synthase from bacteria. Isotopic labelling experiments in conjunction with DFT calculations were performed that provided detailed insight into its complex cyclisation mechanism. Enzyme engineering through site-directed mutagenesis gave access to a high-yielding enzyme variant that provided six additional minor products and the main product in sufficient quantities to study its chemistry.
Subject(s)
Streptomyces , Mutagenesis, Site-Directed , Streptomyces/genetics , CyclizationABSTRACT
Different mechanisms for the cyclisation of farnesyl pyrophosphate to patchoulol by the patchoulol synthase are discussed in the literature. They are based on isotopic labelling experiments, but the results from these experiments are contradictory. The present work reports on a reinvestigation of patchoulol biosynthesis by isotopic labelling experiments and computational chemistry. The results are in favour of a pathway through the neutral intermediates germacrene A and α-bulnesene that are both reactivated by protonation for further cyclisation steps, while previously discussed intra- and intermolecular hydrogen transfers are not supported. Furthermore, the isolation of the new natural product (2S,3S,7S,10R)-guaia-1,11-dien-10-ol from patchouli oil is reported.
ABSTRACT
The multiproduct chimeric sesterterpene synthase AcAS from Aspergillus calidoustus yielded spirocyclic calidoustene, which exhibits a novel skeleton, besides five known sesterterpenes. The complex cyclisation mechanism to all six compounds was investigated by isotopic labelling experiments in combination with DFT calculations. Chemically synthesised 8-hydroxyfarnesyl diphosphate was converted with isopentenyl diphosphate and AcAS into four oxygenated sesterterpenoids that structurally resemble cytochromeâ P450 oxidation products of the sesterterpene hydrocarbons. Protein engineering of AcAS broadened the substrate scope and gave significantly improved enzyme yields.
Subject(s)
Alkyl and Aryl Transferases , Sesterterpenes , Alkyl and Aryl Transferases/metabolism , Aspergillus , Cyclization , Sesterterpenes/chemistryABSTRACT
A reinvestigation of the linalool synthase from Chryseobacterium polytrichastri uncovered its diterpene synthase activity, yielding polytrichastreneâ A and polytrichastrolâ A with new skeletons, besides known wanju-2,5-diene and thunbergol. The enzyme mechanism was investigated by isotopic labeling experiments and DFT calculations to explain an unusual ethyl group formation. Rationally designed exchanges of active site residues showed major functional switches, resulting for I66F in the production of five more new compounds, including polytrichastreneâ B and polytrichastrolâ B, while A87T, A192V and the double exchange A87T, A192V gave a product shift towards wanju-2,5-diene.
Subject(s)
Chryseobacterium/enzymology , Hydro-Lyases/metabolism , Polyisoprenyl Phosphates/biosynthesis , Density Functional Theory , Molecular Conformation , Polyisoprenyl Phosphates/chemistryABSTRACT
A systematic computational study addressing the entire chemical space of guaianes in conjunction with an analysis of all known compounds shows that 1,3-hydride shifts are rare events in guaiane biosynthesis. As demonstrated here, 1,3-hydride shifts towards guaianes can only be realized for two stereochemically well defined out of numerous possible stereoisomeric skeletons. One example is given by the mechanism of guaia-4(15)-en-11-ol synthase from California poplar, an enzyme that yields guaianes with unusual stereochemical properties. The general results from DFT calculations were experimentally verified through isotopic-labeling experiments with guaia-4(15)-en-11-ol synthase.
Subject(s)
Sesquiterpenes, Guaiane , Isotope Labeling , StereoisomerismABSTRACT
The Ir-catalyzed conversion of prochiral tert-cyclobutanols to ß-methyl-substituted ketones proceeds under comparably mild conditions in toluene (45-110 °C) and is particularly suited for the enantioselective desymmetrization of ß-oxy-substituted substrates to give products with a quaternary chirality center with up to 95 % ee using DTBM-SegPhos as a chiral ligand. Deuteration experiments and kinetic isotope effect measurements revealed major mechanistic differences to related RhI -catalyzed transformations. Supported by DFT calculations we propose the initial formation of an IrIII hydride intermediate, which then undergoes a ß-C elimination (C-C bond activation) prior to reductive C-H elimination. The computational model also allows the prediction of the stereochemical outcome. The Ir-catalyzed cyclobutanol cleavage is broadly applicable but fails for substrates bearing strongly coordinating groups. The method is of particular value for the stereo-controlled synthesis of substituted chromanes related to the tocopherols and other natural products.
ABSTRACT
Two bacterial diterpene synthases (DTSs) from Chryseobacterium were characterised. The first enzyme yielded the new compound chryseodiene that closely resembles the known fusicoccane diterpenes from fungi, but its experimentally and computationally studied cyclisation mechanism is fundamentally different to the mechanism of fusicoccadiene synthase. The second enzyme produced wanjudiene, a diterpene hydrocarbon with a new skeleton, besides traces of the enantiomer of bonnadiene that was recently discovered from Allokutzneria albata.