The Diterpenoid Substrate Analogue 19-nor-GGPP Reveals Pronounced Methyl Group Effects in Diterpene Cyclisations.

The substrate analogue 19-nor-geranylgeranyl diphosphate (19-nor-GGPP) was synthesised and incubated with 20 diterpene synthases, resulting in the formation of diterpenoids in all cases. A total of 23 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.

Common Biosynthesis of Non-Canonical C16 Terpenes through a Fragmentation-Recombination Mechanism.

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.

Two Sesterterpene Synthases from Lentzea atacamensis Demonstrate the Role of Conformational Variability in Terpene Biosynthesis.

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.

Skeletal Rearrangements in the Enzyme-Catalysed Biosynthesis of Coral-type Diterpenes from Chitinophaga pinensis.

Two diterpene synthases from the bacterium Chitinophaga pinensis were characterised. The first enzyme mainly produced the rearranged diterpene palmatol, a compound known from octocorals, while the second enzyme made the new coral-type eunicellane chitinol. The mechanisms of both enzymes were deeply studied through isotopic labelling experiments, DFT calculations, and with a substrate analog containing a saturated double bond, resulting in the formation of derailment products that gave additional insights into the nature of the cyclisation cascade intermediates. The formation of coral-type diterpenes poses interesting questions on the functions of these compounds in organisms as different as bacteria and corals.

Biosynthesis of the Non-canonical C17 Sesquiterpenoids Chlororaphen A and B from Pseudomonas chlororaphis.

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.

Exploring the Glycosylation Reaction Inside the Resorcin[4]arene Capsule.

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.

Mechanistic Characterisation and Engineering of Sesterviolene Synthase from Streptomyces violens.

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.

Subrutilane-A Hexacyclic Sesterterpene from Streptomyces subrutilus.

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.

Mechanistic Characterisation of the Bacterial Sesterviridene Synthase from Kitasatospora viridis.

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 sesterviridene 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 sesterviridene A were obtained by chemical transformations, securing the NMR-based structural assignments. The absolute configuration of sesterviridene A was determined by chemical correlation using stereoselectively deuterated precursors and by anomalous dispersion X-ray crystallography. The cyclisation mechanism from GFPP to sesterviridene A was extensively studied through isotopic labelling experiments and DFT calculations.

Spiroluchuene A Synthase: A Cyclase from Aspergillus luchuensis Forming a Spirotetracyclic Diterpene.

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.

Mechanism of the Bifunctional Multiple Product Sesterterpene Synthase AcAS from Aspergillus calidoustus.

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 cytochrome P450 oxidation products of the sesterterpene hydrocarbons. Protein engineering of AcAS broadened the substrate scope and gave significantly improved enzyme yields.

The enzyme mechanism of patchoulol synthase.

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.

1,2- or 1,3-Hydride Shifts: What Controls Guaiane Biosynthesis?

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.

Enantioselective Cleavage of Cyclobutanols Through Ir-Catalyzed C-C Bond Activation: Mechanistic and Synthetic Aspects.

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.

Functional Switch and Ethyl Group Formation in the Bacterial Polytrichastrene Synthase from Chryseobacterium polytrichastri.

A reinvestigation of the linalool synthase from Chryseobacterium polytrichastri uncovered its diterpene synthase activity, yielding polytrichastrene A and polytrichastrol 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 polytrichastrene B and polytrichastrol B, while A87T, A192V and the double exchange A87T, A192V gave a product shift towards wanju-2,5-diene.

ω-Quinazolinonylalkyl aryl ureas as reversible inhibitors of monoacylglycerol lipase.

The serine hydrolase monoacylglycerol lipase (MAGL) is involved in a plethora of pathological conditions, in particular pain and inflammation, various types of cancer, metabolic, neurological and cardiovascular disorders, and is therefore a promising target for drug development. Although a large number of irreversible-acting MAGL inhibitors have been discovered over the past years, there are only few compounds known so far which inhibit the enzyme in a reversible manner. Therefore, much effort is put into the development of novel chemical entities showing reversible inhibitory behavior, which is thought to cause less undesired side effects. To explore a wide range of chemical structures as MAGL binders, we have applied a virtual screening approach by docking small molecules into the crystal structure of human MAGL (hMAGL) and envisaged a library of 45 selected compounds which were then synthesized. Biochemical investigations included the determination of the inhibitory potency on hMAGL and two related hydrolases, i.e. human fatty acid amide hydrolase (hFAAH) and murine cholesterol esterase (mCEase). The most promising candidates from theses analyses, i.e. three ω-quinazolinonylalkyl aryl ureas bearing alkyl spacers of three to five methylene groups, exhibited IC50 values of 20-41 µM and reversible, detergent-insensitive behavior towards hMAGL. Among these compounds, the inhibitor 1-(3,5-bis(trifluoromethyl)phenyl)-3-(4-(4-oxo-3,4-dihydroquinazolin-2-yl)butyl)urea (96) was selected for further kinetic characterization, yielding a dissociation constant Ki = 15.4 µM and a mixed-type inhibition with a pronounced competitive component (α = 8.94). This mode of inhibition was further supported by a docking experiment, which suggested that the inhibitor occupies the substrate binding pocket of hMAGL.

Two Diterpene Synthases from Chryseobacterium: Chryseodiene Synthase and Wanjudiene Synthase.

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.

Hydrogen Peroxide Sensors Based on Fluorescence Quenching of the 2-AminobenzimidazoleFluorophore.

The fluorescence emission of the parent 2-aminobenzimidazole (ABZ, 1), the mono- and disubstituted derivatives (2, 3), 2-aminonaphthoimidazole (4), and 4-amino dinaphthodiazepine 5 (λem = 315-400 nm) is strongly quenched in the presence of aqueous hydrogen peroxide. The quenching process is dual: for diazepine 5, quenching is dynamic at lower H2O2 concentrations with linear reduction of the fluorescence lifetime from 4.3 to 2.6 ns. At higher H2O2 concentrations, a second species appears in the absorption and emission spectra with fluorescence lifetimes of 1.3 ns, indicating the formation of a new (ground-state) hydrogen-bonded ABZ-H2O2 complex (static quenching). Sensors 1 and 2 show also dual quenching that fits with a static 1:1 and 1:2 model with K1:1 = 8(11) M-1 and K1:2 = 21(147) M-1 for 1(2). The formation of a 1:2 complex (1:(H2O2)2) is also supported by density functional theory (DFT) calculations and spectra simulations.

Silanediol versus chlorosilanol: hydrolyses and hydrogen-bonding catalyses with fenchole-based silanes.

Biphenyl-2,2'-bisfenchyloxydichlorosilane (7, BIFOXSiCl2) is synthesized and employed as precursor for the new silanols biphenyl-2,2'-bisfenchyloxychlorosilanol (8, BIFOXSiCl(OH)) and biphenyl-2,2'-bisfenchyloxysilanediol (9, BIFOXSi(OH)2). BIFOXSiCl2 (7) shows a remarkable stability against hydrolysis, yielding silanediol 9 under enforced conditions. A kinetic study for the hydrolysis of dichlorosilane 7 shows a 263 times slower reaction compared to reference bis-(2,4,6-tri-tert-butylphenoxy)dichlorosilane (14), known for its low hydrolytic reactivity. Computational analyses explain the slow hydrolyses of BIFOXSiCl2 (7) to BIFOXSiCl(OH) (8, E a = 32.6 kcal mol-1) and BIFOXSiCl(OH) (8) to BIFOXSi(OH)2 (9, E a = 31.4 kcal mol-1) with high activation barriers, enforced by endo fenchone units. Crystal structure analyses of silanediol 9 with acetone show shorter hydrogen bonds between the Si-OH groups and the oxygen of the bound acetone (OH···O 1.88(3)-2.05(2) Å) than with chlorosilanol 8 (OH···2.16(0) Å). Due to its two hydroxy units, the silanediol 9 shows higher catalytic activity as hydrogen bond donor than chlorosilanol 8, e.g., C-C coupling N-acyl Mannich reaction of silyl ketene acetals 11 with N-acylisoquinolinium ions (up to 85% yield and 12% ee), reaction of 1-chloroisochroman (18) and silyl ketene acetals 11 (up to 85% yield and 5% ee), reaction of chromen-4-one (20) and silyl ketene acetals 11 (up to 98% yield and 4% ee).

Characterization of fatty acid amide hydrolase activity by a fluorescence-based assay.

Fatty acid amide hydrolase (FAAH) is involved in many human diseases, particularly cancer, pain and inflammation as well as neurological, metabolic and cardiovascular disorders. Therefore, FAAH is an attractive target for the development of low-molecular-weight inhibitors as therapeutics, which requires robust assays that can be used for high-throughput screening (HTS) of compound libraries. Here, we report the development of a fluorometric assay based on FAAH's ability to effectively hydrolyze medium-chain fatty acid amides, introducing N-decanoyl-substituted 5-amino-2-methoxypyridine (D-MAP) as new amide substrate. D-MAP is cleaved by FAAH with an 8-fold larger specificity constant than the previously reported octanoyl-analog Oc-MAP (Vmax/Km of 1.09 and 0.134 mL min-1 mg-1, respectively), with both MAP derivatives possessing superior substrate properties and much increased aqueous solubility compared to the respective p-nitroaniline compounds D-pNA and Oc-pNA. The new assay with D-MAP as substrate is highly sensitive using a lower enzyme concentration (1 µg mL-1) than literature-reported fluorimetric FAAH assays. In addition, D-MAP was validated in comparison to the substrate Oc-MAP for the characterization of FAAH inhibitors by means of the reference compounds URB597 and TC-F2 and was shown to be highly suitable for HTS in both kinetic and endpoint assays (Z' factors of 0.81 and 0.78, respectively).