C35 Hopanoid Side Chain Biosynthesis: Reduction of Ribosylhopane into Bacteriohopanetetrol by a Cell‐Free System Derived from Methylobacterium organophilum

The major bacterial triterpenoids of the hopane series each consist of a C₃₀ triterpene hopane moiety and an additional nonterpene C₅ side chain derived from D ‐ribose and linked through its C‐5 carbon atom to the hopane side chain. Bacteriohopanetetrol and aminobacteriohopanetriol are the most common representatives of this natural product series, adenosylhopane and ribosylhopane being putative precursors. Deuterium‐labelled ribosylhopane was obtained by hemisynthesis and converted into deuterium‐labelled bacteriohopanetetrol in the presence of NADPH, thus giving evidence of this as yet unknown precursor‐to‐product relationship in the bacterial hopanoid metabolic pathway.     

Biosynthetic and Functional Color–Scent Associations in Flowers of Papaver nudicaule and Their Impact on Pollinators

Despite increasing evidence for biosynthetic connections between flower pigments and volatile compounds, examples of such relationships in polymorphic plant species remains limited. Herein, color–scent associations in flowers from Papaver nudicaule (Papaveraceae) have been investigated. The spectral reflectance and scent composition of flowers of four color cultivars was determined. We found that pigments and volatiles occur in specific combinations in flowers of P. nudicaule. The presence of indole in the bouquets is strongly associated with the occurrence of yellow pigments called nudicaulins, for which indole is one of the final biosynthetic precursors. Whereas yellow flowers emit an excess of indole, orange flowers consume it during nudicaulin production and lack the substance in their bouquet. By using the honeybee, Apis mellifera, evaluations were made on how color and scent affect the discrimination of these flowers by pollinators. Honeybees were able to discriminate artificial odor mixtures resembling those of the natural flower odors. Bees trained with stimuli combining colors and odors showed an improved discrimination performance. The results indicate that the indole moiety of nudicaulins and emitted indole might be products of the same biochemical pathway. We propose that conserved pathways account for the evolution of color–scent associations in P. nudicaule and that these associations positively affect flower constancy of pollinators.     

Expedient Introduction of β‐Methoxyacrylate Unit onto 3‐Substituted Indoles and Application of the Resulting Indole‐Dienes in Organocascade toward Indolino‐Polycyclics

An efficient methodology for introducing a β‐alkoxy acrylate unit onto the indolic C‐2 position of 3‐substituted indoles has been realized by oxidative radical alkylation of indoles with a xanthate bearing dialkyl acetal functionality and subsequent elimination of alcohol by treatment with 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU). The indole‐dienes thus prepared are quite stable and can be stored at room temperature for several months without obvious deterioration. Next, the indole‐dienes were successfully applied in an asymmetric cascade reaction with propargyl aldehyde catalyzed by MacMillan’s imidazolidinone and trichloroacetic acid (TCA), affording tetracyclic spiroindolines or tricyclic hydrocarbazoles in good to excellent yields with excellent ees. The practicality of this cascade was demonstrated by a gram scale preparation of a tetracyclic spiroindoline product with as low as 1 mol% catalyst loading and further elaboration of this spiroindoline compound into valuable intermediates for alkaloid synthesis.

     

Tubulin Photoaffinity Labeling with Biotin‐Tagged Derivatives of Potent Diketopiperazine Antimicrotubule Agents

NPI‐2358 ( 1 ) is a potent antimicrotubule agent that was developed from a natural diketopiperazine, phenylahistin, which is currently in Phase I clinical trials as an anticancer drug. To understand the precise recognition mechanism of tubulin by this agent, we focused on its potent derivative, KPU‐244 ( 2 ), which has been modified with a photoreactive benzophenone structure, and biotin‐tagged KPU‐244 derivatives ( 3 and 4 ), which were designed and synthesized for tubulin photoaffinity labeling. Introduction of the biotin structure at the p′‐position of the benzophenone ring in 2 exhibited reduced, but significant biological activities with tubulin binding, tubulin depolymerization and cytotoxicity in comparison to the parent KPU‐244. Therefore, tubulin photoaffinity labeling studies of biotin‐derivatives 3 and 4 were performed by using Western blotting analysis after photoirradiation with 365 nm UV light. The results indicated that tubulin was covalently labeled by these biotin‐tagged photoprobes. The labeling of compound 4 was competitively inhibited by the addition of diketopiperazine 1 or colchicine, and weakly inhibited by the addition of vinblastine. The results suggest that photoaffinity probe 4 specifically recognizes tubulin at the same binding site as anticancer drug candidate 1 , and this leads to the disruption of microtubules. Probe 4 serves well as a useful chemical probe for potent antimicrotubule diketopiperazines, much like phenylahistin, and it also competes for the colchicine‐binding site.     

Unambiguous Assignment of Short‐ and Long‐Range Structural Restraints by Solid‐State NMR Spectroscopy with Segmental Isotope Labeling

We present an efficient method for the reduction of spectral complexity in the solid‐state NMR spectra of insoluble protein assemblies, without loss of signal intensity. The approach is based on segmental isotope labeling by using the split intein DnaE from Nostoc punctiforme. We show that the segmentally ¹³C,¹⁵N‐labeled prion domain of HET‐s exhibits significantly reduced spectral overlap while retaining the wild‐type structure and spectral quality. A large number of unambiguous distance restraints were thus collected from a single two‐dimensional ¹³C,¹³C cross‐correlation spectrum. The observed resonances could be unambiguously identified as intramolecular without the need for preparing a dilute, less sensitive sample.     

S‐Adenosyl‐Methionine‐Dependent Methyltransferases: Highly Versatile Enzymes in Biocatalysis,Biosynthesis and Other Biotechnological Applications

S‐adenosyl methionine (SAM) is a universal biological cofactor that is found in all branches of life where it plays a critical role in the transfer of methyl groups to various biomolecules, including DNA, proteins and small‐molecule secondary metabolites. The methylation process thus has important implications in various disease processes and applications in industrial chemical processing. This methyl transfer is catalysed by SAM‐dependent methyltransferases (MTases), which are by far the largest groups of SAM‐dependent enzymes. A significant amount is now known regarding the structural biology and enzymology of these enzymes, and, consequently, there is now significant scope for the development of new MTases and SAM analogues for applications from biomolecular imaging to biocatalytic industrial processes. This review will focus on current efforts in the manipulation of class I and V SAM‐dependent MTases and the use of synthetic SAM analogues, which together offer the best prospects for rational redesign towards biotechnological applications. Firstly, metabolic engineering of organisms incorporating small‐molecule MTases is discussed; this can be applied in a variety of areas from the industrial bioprocessing of flavourants and antibiotics to frontier research in biofuel production and bioremediation. Secondly, the application of MTases in combination with SAM analogues is reviewed; this allows the tagging of proteins and oligonucleotides with moieties other than the methyl group. Such tagging allows the isolation of the tagged biomolecule and aids its visualisation by a range of analytical methods. The review then summarises the potential advantages of MTase‐mediated chemistry and offers some future perspectives on downstream applications.     

The Biosynthesis of the Aroma Volatile 2‐Methyltetrahydrothiophen‐3‐one in the Bacterium Chitinophaga Fx7914

2‐Methyltetrahydrothiophen‐3‐one ( 3 ) is a volatile compound that plays an important role especially in food and flavour chemistry because it contributes to the aroma of several foodstuffs including wine. Although 3 can be formed by chemical reactions during food preparation, it is also produced by microorganisms. Recent studies with yeasts showed that methionine ( 1 ) is a potential precursor of 3 , but the mechanism of the transformation is unknown. The biosynthetic pathway leading to 3 in the bacterium Chitinophaga Fx7914 was probed. Extensive feeding experiments with differently labelled precursors by using liquid cultures of Chitinophaga Fx7914 were performed. The volatiles released by the bacterium were collected by using a closed loop stripping apparatus (CLSA) and analysed by GC–MS. The observed incorporation pattern of the precursors into 3 led to the elucidation of the biosynthetic pathway. One part of the compound 2 originates from homocysteine ( 15 ), which is transformed into 3‐mercaptopropanal ( 17 ). The second biosynthetic building block is pyruvate ( 14 ). An acyloin‐forming reaction furnishes the key intermediate 21 , which cyclises intramolecularly to a diol. Dehydration followed by tautomerisation lead to the cyclic ketone 3 , which is produced by the bacterium in racemic form.     

Synthetic Indolactam V Analogues as Inhibitors of PAR2‐Induced Calcium Mobilization in Triple‐Negative Breast Cancer Cells

Human proteinase‐activated receptor 2 (PAR2), a transmembrane G‐protein‐coupled receptor (GPCR), is an attractive target for a novel anticancer therapy, as it plays a critical role in cell migration and invasion. Selective PAR2 inhibitors therefore have potential as anti‐metastatic drugs. Knowing that the natural product teleocidin A2 is able to inhibit PAR2 in tumor cells, the goal of the present study was to elaborate structure–activity relationships and to identify potent PAR2 inhibitors with lower activity against the adverse target, protein kinase C (PKC). For this purpose, an efficient gram‐scale total synthesis of indolactam V (i.e., the parent structure of all teleocidins) was developed, and a library of derivatives was prepared. Some compounds were indeed found to exhibit high potency as PAR2 inhibitors at low nanomolar concentrations with improved selectivity (relative to teleocidin A2). The pseudopeptidic fragment bridging the C3 and C4 positions of the indole core proved to be essential for target binding, whereas activity and target selectivity depends on the substituents at N1 or C7. This study revealed novel derivatives that show high efficacy in PAR2 antagonism combined with increased selectivity.     

A Conserved Lysine in β‐Lactam Synthetase Assists Ring Cyclization: Implications for Clavam and Carbapenem Biosynthesis

β‐Lactam synthetase (β‐LS) is the paradigm of a growing class of enzymes that form the critical β‐lactam ring in the clavam and carbapenem antibiotics. β‐LS catalyzes a two‐stage reaction in which N²‐(2‐carboxyethyl)‐L ‐arginine is first adenylated, and then undergoes intramolecular ring closure. It was previously shown that the forward kinetic commitment to β‐lactam formation is high, and that the overall rate of reaction is partially limited to a protein conformational change rather than to the chemical step alone of closing the strained ring. β‐Lactam formation was evaluated on the basis of X‐ray crystal structures, site‐specific mutation, and kinetic and computational studies. The combined evidence clearly points to a reaction coordinate involving the formation of a tetrahedral transition state/intermediate stabilized by a conserved Lys. The combination of substrate preorganization, a well‐stabilized transition state and an excellent leaving group facilitates this acyl substitution to account for the strong forward commitment to catalysis and to lower the barrier of four‐membered ring formation to the magnitude of a protein conformational change.     

AstPT Catalyses both Reverse N1‐ and Regular C2 Prenylation of a Methylated Bisindolyl Benzoquinone

Prenylated bisindolyl benzoquinones exhibit interesting biological activities, such as antidiabetic or anti‐HIV activities. A number of these compounds, including asterriquinones, have been isolated from Aspergillus terreus. In this study, we identified two putative genes by genome mining, ATEG_09980 and ATEG_00702, which share high sequence similarity with the known bisindolyl benzoquinone prenyltransferase TdiB from Aspergillus nidulans. The coding sequences were cloned and overexpressed in E. coli. The overproduced recombinant proteins were purified to near homogeneity and used for enzyme assays with asterriquinone D in the presence of dimethylallyl diphosphate. HPLC analysis showed that product formation was only detected in enzyme assays with EAU29429 encoded by ATEG_09980, not in those with EAU39348 encoded by ATEG_00702. Product isolation and structure elucidation by NMR and MS analyses led to identification of N1‐reversely and C2‐regularly monoprenylated derivatives, as well as N1′,N1′′reversely, N1′‐reversely, C2′′‐regularly diprenylated derivatives. This proved that EAU29429 functions as an asterriquinone prenyltransferase (AstPT) and indicated the involvement of EAU29429 rather than EAU39348 in the biosynthesis of methylated asterriquinones. Furthermore, incubation of monoprenylated enzyme products with AstPT resulted in the formation of the diprenylated derivatives.