Analyses of Arabidopsis ecotypes reveal metabolic diversity to convert D-amino acids.

For a long time D-enantiomers of proteinogenic L-amino acids were assumed to be physiologically irrelevant for plants. But there is growing evidence that D-amino acids (D-AAs) also fulfil important physiological functions in these organisms. However, the knowledge about the metabolic fate of D-AAs in plants is still scarce and more information about it is needed. To close this gap we established an optimized protocol for the processing and analysis of D- and L-AAs from large numbers of Arabidopsis lines. This included the application of 18 different D-AAs to seedlings, the extraction of free amino acids from the samples and the determination of 16 L-AAs and their corresponding D-enantiomers. To validate our approach we searched for genetic accessions with aberrant amino acid metabolism. Therefore we applied D-AAs on 17 ecotypes of Arabidopsis thaliana and analysed their free amino acid contents. These analyses confirmed the suitability of the system for the analysis of large sets of plant samples with enhanced velocity and improved accuracy. Furthermore, the resulting data led to the definition of standard amino acid profiles in response to D-AAs of Arabidopsis seedlings. Within these analyses the ecotype Landsberg erecta was found with aberrant metabolic patterns like drastically reduced capabilities to convert different D-AAs to D-alanine and D-glutamate. The presented experimental setup and results of this study offer starting points to dissect the metabolic pathway of D-AAs in plants.

A convenient and selective palladium-catalyzed aerobic oxidation of alcohols.

An efficient procedure for the oxidation of primary and secondary alcohols to aldehydes and ketones, respectively, with molecular oxygen under ambient conditions has been achieved. By applying catalytic amounts of Pd(OAc)2 in the presence of tertiary phosphine oxides (O=PR3) as ligands, a variety of substrates are selectively oxidized without formation of ester byproducts. Spectroscopic investigations and DFT calculations suggest stabilization of the active palladium(II) catalyst by phosphine oxide ligands.

Enantioselective zinc-catalyzed hydrosilylation of ketones using pybox or pybim ligands.

The combination of ZnEt(2) and chiral pyridinebisoxazoline (pybox) or pyridinebisimidazoline (pybim) ligands catalyzed the asymmetric hydrosilylation of aryl, alkyl, cyclic, heterocyclic, and aliphatic ketones. Under mild conditions, high yields and good enantioselectivities were achieved. ESI measurements allowed for the characterization of the active catalyst.

Uptake and conversion of D-amino acids in Arabidopsis thaliana.

The D-enantiomers of proteinogenic amino acids fulfill essential functions in bacteria, fungi and animals. Just in the plant kingdom, the metabolism and role of D-amino acids (D-AAs) still remains unclear, although plants have to cope with significant amounts of these compounds from microbial decay in the rhizosphere. To fill this gap of knowledge, we tested the inhibitory effects of D-AAs on plant growth and established a method to quantitate 16 out of 19 proteinogenic amino acids and their D-enantiomers in plant tissue extracts. Therefore, the amino acids in the extracts were derivatized with Marfey's reagent and separated by HPLC-MS. We used two ecotypes (Col-0 and C24) and a mutant (lht1) of the model plant Arabidopsis thaliana to determine the influence and fate of exogenously applied D-AAs. All of them were found in high concentrations in the plant extracts after application, even in lht1, which points to additional transporters facilitating the import of D-AAs. The addition of particular amino acids (D-Trp, D-Phe, D-Met and D-His) led to the accumulation of the corresponding L-amino acid. In almost all cases, the application of a D-AA resulted in the accumulation of D-Ala and D-Glu. The presented results indicate that soil borne D-AAs can actively be taken up and metabolized via central metabolic routes.

Laccase-catalyzed cross-linking of amino acids and peptides with dihydroxylated aromatic compounds.

In order to design potential biomaterials, we investigated the laccase-catalyzed cross-linking between L-lysine or lysine-containing peptides and dihydroxylated aromatics. L-Lysine is one of the major components of naturally occurring mussel adhesive proteins (MAPs). Dihydroxylated aromatics are structurally related to 3,4-dihydroxyphenyl-L-alanine, another main component of MAPs. Mass spectrometry and nuclear magnetic resonance analyses show that the epsilon-amino group of L-lysine is able to cross-link dihydroxylated aromatics. Additional oligomer and polymer cross-linked products were obtained from di- and oligopeptides containing L-lysine. Potential applications in medicine or industry for biomaterials synthesised via the three component system consisting of the oligopeptide [Tyr-Lys]10, dihydroxylated aromatics and laccase are discussed.

Synthesis of new N-analogous corollosporine derivatives with antibacterial activity by laccase-catalyzed amination.

Corollosporine isolated from the marine fungus Corollospora maritima and N-analogous corollosporines are antimicrobial substances. Owing to the basic structure of the N-analogous corollosporines, they have become an attractive target for laccase-catalyzed derivatisation. In this regard we report on the straightforward laccase-catalyzed amination of dihydroxylated arenes with N-analogous corollosporines. In biological assays the obtained amination products are more active than the parent compounds.

Preparation of novel unsymmetrical bisindoles under solvent-free conditions: synthesis, crystal structures, and mechanistic aspects.

Indole aziridines and their hydroxyl derivatives have been used for the preparation of a small library of novel functionalized bisindoles. Diversification of these building blocks by solvent-free C-C-bond formation on solid support yielded annulated Hymenialdisine analogues under mild reaction conditions. Indoles as C-nucleophiles form potentially pharmacologically active bisindoles through an electrophilic aromatic substitution pathway in good to excellent yields. Further transformations of the indole aziridines with H-, N-, and O-nucleophiles demonstrate their versatility as key intermediates in diversity oriented synthesis. The hydroxyl precursor leads also to unsymmetrical bisindoles under similar reaction conditions. Important intermediates and final library compounds were confirmed by X-ray analysis. Theoretical studies on these systems show the possible cationic intermediate in the substitution pathway.

Carbon-oxygen bond formation by fungal laccases: cross-coupling of 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide with the solvents water, methanol, and other alcohols.

Laccase-catalyzed reactions lead to oxidation of the substrate via a cation radical, which has been described to undergo proton addition to form a quinonoid derivative or nucleophilic attack by itself producing homomolecular dimers. In this study, for the substrate 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide, we show that, besides the quinonoid form of substrate, all products formed are nonhomomolecular ones. Indeed, without addition of a reaction partner, heteromolecular products are formed from the quinonoid form of the laccase-substrate and the solvents water or methanol present in the incubation assay. Consequently, in laccase catalyzed syntheses performed in aqueous solutions or in the presence of methanol or other alcohols, undesirable heteromolecular coupling reactions between the laccase substrate and solvents must be taken into account. Additionally, it could be shown at the example of methanol and other alcohols that C-O-bound cross-coupling of dihydroxylated aromatic substances with the hydroxyl group of aliphatic alcohols can be catalyzed by fungal laccases.

Antiviral Terpenoid Constituents of Ganoderma pfeifferi.

Four sterols and 10 triterpenes were isolated from the fruiting bodies of Ganoderma pfeifferi, including the three new triterpenes 3,7,11-trioxo-5alpha-lanosta-8,24-diene-26-al (lucialdehyde D, 1), 5alpha-lanosta-8,24-diene-26-hydroxy-3,7-dione (ganoderone A, 2), and 5alpha-lanosta-8-ene-24,25-epoxy-26-hydroxy-3,7-dione (ganoderone C, 3). The structures of 1-3 were determined on the basis of spectroscopic evidence. Antibacterial, antifungal, and antiviral activity were studied for some of the isolated compounds. Ganoderone A (2), lucialdehyde B (4), and ergosta-7,22-dien-3beta-ol (7) were found to exhibit potent inhibitory activity against herpes simplex virus.

Global expression profiling of Bacillus subtilis cells during industrial-close fed-batch fermentations with different nitrogen sources.

A detailed gene expression analysis of industrial-close Bacillus subtilis fed-batch fermentation processes with casamino acids as the only nitrogen source and with a reduced casamino acid concentration but supplemented by ammonia was carried out. Although glutamine and arginine are supposed to be the preferred nitrogen sources of B. subtilis, we demonstrate that a combined feeding of ammonia and casamino acids supports cell growth under fed-batch fermentation conditions. The transcriptome and proteome analyses revealed that the additional feeding of ammonia in combination with a reduced amino acid concentration results in a significantly lower expression level of the glnAR or tnrA genes, coding for proteins, which are mainly involved in the nitrogen metabolism of B. subtilis. However, the mRNA levels of the genes of the ilvBHC-leuABD and hom-thrCB operons were significantly increased, indicating a valine, leucine, isoleucine, and threonine limitation under these fermentation conditions. In contrast, during the fermentation with casamino acids as the only nitrogen source, several genes, which play a crucial role in nitrogen metabolism of B. subtilis (e.g., glnAR, nasCDE, nrgAB, and ureABC), were up-regulated, indicating a nitrogen limitation under these conditions. Furthermore, increased expression of genes, which are involved in motility and chemotaxis (e.g., hag, fliT) and in acetoin metabolism (e.g., acoABCL), was determined during the fermentation with the mixed nitrogen source of casamino acids and ammonia, indicating a carbon limitation under these fermentation conditions. Under high cell density and slow growth rate conditions a weak up-regulation of autolysis genes could be observed as well as the induction of a number of genes involved in motility, chemotaxis and general stress response. Results of this study allowed the selection of marker genes, which could be used for the monitoring of B. subtilis fermentation processes. The data suggest for example acoA as a marker gene for glucose limitation or glnA as an indicator for nitrogen limitation.

Synthesis and structure-activity relationships of 2-alkylidenethiazolidine-4,5-diones as antibiotic agents.

2-Alkylidenethiazolidine-4,5-diones were prepared by novel one-pot cyclizations of arylacetonitriles with isothiocyanates and ethyl 2-chloro-2-oxoacetate. The products show antibiotic activity against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus.

From a spin-off to the advantageous use in Diels-Alder reactions: a combined synthetic, spectroscopic and computational approach to N-(dienyl)acylamines.

The acid-catalyzed condensation of aldehydes and acetamide has been shown to provide a pool of diverse equilibrating species. For the first time, the synthesis of substituted 1-N-acylamino-1,3-butadienes has been accomplished directly in moderate yields upon telomerization of two molecules of aldehyde with one molecule of carboxamide. Detailed spectroscopic and computational investigations establish the favourable formation of all-trans aminodienes under the reaction conditions. Employment thereof as diene building blocks in Diels-Alder reactions allows for the synthesis of carbocyclic molecules with high stereocontrol.

Multicomponent coupling reactions for organic synthesis: chemoselective reactions with amide-aldehyde mixtures.

The acid-catalyzed condensation chemistry of simple amides and aldehydes provides a highly prolific source of diverse reactants for irreversible follow-up reactions. Amide-aldehyde mixtures have been successfully employed in multicomponent syntheses of N-acyl alpha-amino acids (via palladium-catalyzed amidocarbonylation) and various cyclohexene, cyclohexadiene, and benzene derivatives (via the amide-aldehyde-dienophile (AAD) reaction).

Unusual coupling reactions of aldehydes and alkynes: a novel preparation of substituted phthalic acid derivatives by automated synthesis.

Based upon a highly versatile multicomponent methodology, a new one-pot synthesis of substituted phthalic acid derivatives from alpha,beta-unsaturated aldehydes was developed. The reaction involves the intermediacy of an acetamidodiene species which undergoes Diels-Alder addition to diethyl acetylenedicarboxylate. The resultant acetamidocyclohexadiene is subject to elimination of acetamide under the reaction conditions to give rise to substituted diethyl phthalates in good yields. This domino condensation-cycloaddition-elimination sequence has been applied to a variety of alpha,beta-unsaturated aldehydes. Furthermore, we demonstrated the exploitation of parallelized and automated synthesis technology for the rapid screening of reaction conditions and compositions. Detailed studies revealed the catalytic role of the employed acetamide and the occurrence of a stereoselective 1,4-syn elimination pathway under standard conditions.