Fluorescently Labeled Substrates for Monitoring α1,3‐Fucosyltransferase IX Activity

Fucosylation is often the final process in glycan biosynthesis. The resulting glycans are involved in a variety of biological processes, such as cell adhesion, inflammation, or tumor metastasis. Fucosyltransferases catalyze the transfer of fucose residues from the activated donor molecule GDP‐β‐L ‐fucose to various acceptor molecules. However, detailed information about the reaction processes is still lacking for most fucosyltransferases. In this work we have monitored α1,3‐fucosyltransferase activity. For both donor and acceptor substrates, the introduction of a fluorescent ATTO dye was the last step in the synthesis. The subsequent conversion of these substrates into fluorescently labeled products by α1,3‐fucosyltransferases was examined by high‐performance thin‐layer chromatography coupled with mass spectrometry as well as dual‐color fluorescence cross‐correlation spectroscopy, which revealed that both fluorescently labeled donor GDP‐β‐L ‐fucose‐ATTO 550 and acceptor N‐acetyllactosamine‐ATTO 647N were accepted by recombinant human fucosyltransferase IX and Helicobacter pylori α1,3‐fucosyltransferase, respectively. Analysis by fluorescence cross‐correlation spectroscopy allowed a quick and versatile estimation of the progress of the enzymatic reaction and therefore this method can be used as an alternative method for investigating fucosyltransferase reactions.     

Hyperspectral unmixing of Raman micro-images for assessment of morphological and chemical parameters in non-dried brain tumor specimens

Hyperspectral unmixing is an unsupervised algorithm to calculate a bilinear model of spectral endmembers and abundances of components from Raman images. Thirty-nine Raman images were collected from six glioma brain tumor specimens. The tumor grades ranged from astrocytoma WHO II to glioblastoma multiforme WHO IV. The abundance plots of the cell nuclei were processed by an image segmentation procedure to determine the average nuclei size, the number of nuclei, and the fraction of nuclei area. The latter two morphological parameters correlated with the malignancy. A combination of spectral unmixing and non-negativity constrained linear least squares fitting is introduced to assess chemical parameters. First, endmembers of the most abundant and most dissimilar components were defined that represent all data sets. Second, the content of the obtained components’ proteins, nucleic acids, lipids, and lipid to protein ratios were determined in all Raman images. Except for the protein content, all chemical parameters correlated with the malignancy. We conclude that the morphological and chemical information offer new ways to develop Raman-based classification approaches that can complement diagnosis of brain tumors. The role of non-linear Raman modalities to speed-up image acquisition is discussed.

Catalytic Methylation of C?H Bonds Using CO2 and H2

Formation of C C bonds from CO₂ is a much sought after reaction in organic synthesis. To date, other than C H carboxylations using stoichiometric amounts of metals, base, or organometallic reagents, little is known about C C bond formation. In fact, to the best of our knowledge no catalytic methylation of C H bonds using CO₂ and H₂ has been reported. Described herein is the combination of CO₂ and H₂ for efficient methylation of carbon nucleophiles such as indoles, pyrroles, and electron‐rich arenes. Comparison experiments which employ paraformaldehyde show similar reactivity for the CO₂/H₂ system.     

Identification and Affinity-Quantification of ß-Amyloid and α-Synuclein Polypeptides Using On-Line SAW-Biosensor-Mass Spectrometry

Bioaffinity analysis using a variety of biosensors has become an established tool for detection and quantification of biomolecular interactions. Biosensors, however, are generally limited by the lack of chemical structure information of affinity-bound ligands. On-line bioaffinity-mass spectrometry using a surface-acoustic wave biosensor (SAW-MS) is a new combination providing the simultaneous affinity detection, quantification, and mass spectrometric structural characterization of ligands. We describe here an on-line SAW-MS combination for direct identification and affinity determination, using a new interface for MS of the affinity-isolated ligand eluate. Key element of the SAW-MS combination is a microfluidic interface that integrates affinity-isolation on a gold chip, in-situ sample concentration, and desalting with a microcolumn for MS of the ligand eluate from the biosensor. Suitable MS- acquisition software has been developed that provides coupling of the SAW-MS interface to a Bruker Daltonics ion trap-MS, FTICR-MS, and Waters Synapt-QTOF- MS systems. Applications are presented for mass spectrometric identifications and affinity (K_D) determinations of the neurodegenerative polypeptides, ß-amyloid (Aß), and pathophysiological and physiological synucleins (α- and ß-synucleins), two key polypeptide systems for Alzheimer’s disease and Parkinson’s disease, respectively. Moreover, first in vivo applications of αSyn polypeptides from brain homogenate show the feasibility of on-line affinity-MS to the direct analysis of biological material. These results demonstrate on-line SAW-bioaffinity-MS as a powerful tool for structural and quantitative analysis of biopolymer interactions.

A General Catalytic Hydroamidation of 1,3‐Dienes: Atom‐Efficient Synthesis of N‐Allyl Heterocycles,Amides, and Sulfonamides

Transition‐metal‐catalyzed hydroamination reactions are sustainable and atom‐economical C? N bond‐forming processes. Although remarkable progress has been made in the inter‐ and intramolecular amination of olefins and 1,3‐dienes, related intermolecular reactions of amides are still much less known. Control of the regioselectivity without analogous telomerization is the particular challenge in the catalytic hydroamidation of alkenes and 1,3‐dienes. Herein, we report a general protocol for the hydroamidation of electron‐deficient N‐heterocyclic amides and sulfonamides with 1,3‐dienes and vinyl pyridines in the presence of a catalyst derived from [{Pd(π‐cinnamyl)Cl}₂] and ligand L7 or L10 . The reactions proceeded in good to excellent yield with high regioselectivity. The practical utility of our method is demonstrated by the hydroamidation of functionalized biologically active substrates. The high regioselectivity for linear amide products makes the procedure useful for the synthesis of a variety of allylic amides.     

Convenient and Mild Epoxidation of Alkenes Using Heterogeneous Cobalt Oxide Catalysts

A general epoxidation of aromatic and aliphatic olefins has been developed under mild conditions using heterogeneous Co_xO_y–N/C (x=1,3; y=1,4) catalysts and tert‐butyl hydroperoxide as the terminal oxidant. Various stilbenes and aliphatic alkenes, including renewable olefins, and vitamin and cholesterol derivatives, were successfully transformed into the corresponding epoxides with high selectivity and often good yields. The cobalt oxide catalyst can be recycled up to five times without significant loss of activity or change in structure. Characterization of the catalyst by XRD, TEM, XPS, and EPR analysis revealed the formation of cobalt oxide nanoparticles with varying size (Co₃O₄ with some CoO) and very few large particles with a metallic Co core and an oxidic shell. During the pyrolysis process the nitrogen ligand forms graphene‐type layers, in which selected carbon atoms are substituted by nitrogen.     

De Novo 3D Structure Determination from Sub‐milligram Protein Samples by Solid‐State 100 kHz MAS NMR Spectroscopy

Solid‐state NMR spectroscopy is an emerging tool for structural studies of crystalline, membrane‐associated, sedimented, and fibrillar proteins. A major limitation for many studies is still the large amount of sample needed for the experiments, typically several isotopically labeled samples of 10–20 mg each. Here we show that a new NMR probe, pushing magic‐angle sample rotation to frequencies around 100 kHz, makes it possible to narrow the proton resonance lines sufficiently to provide the necessary sensitivity and spectral resolution for efficient and sensitive proton detection. Using restraints from such spectra, a well‐defined de novo structure of the model protein ubiquitin was obtained from two samples of roughly 500 μg protein each. This proof of principle opens new avenues for structural studies of proteins available in microgram, or tens of nanomoles, quantities that are, for example, typically achieved for eukaryotic membrane proteins by in‐cell or cell‐free expression.     

Catalytic Methylation of CH Bonds Using CO2 and H2

Formation of C? C bonds from CO₂ is a much sought after reaction in organic synthesis. To date, other than C? H carboxylations using stoichiometric amounts of metals, base, or organometallic reagents, little is known about C? C bond formation. In fact, to the best of our knowledge no catalytic methylation of C? H bonds using CO₂ and H₂ has been reported. Described herein is the combination of CO₂ and H₂ for efficient methylation of carbon nucleophiles such as indoles, pyrroles, and electron‐rich arenes. Comparison experiments which employ paraformaldehyde show similar reactivity for the CO₂/H₂ system.     

Disciformycins A and B: 12‐Membered Macrolide Glycoside Antibiotics from the Myxobacterium Pyxidicoccus fallax Active against Multiresistant Staphylococci

Two macrolide glycosides with a unique scaffold were isolated from cultures of the myxobacterium Pyxidicoccus fallax. Their structures, including absolute configurations, were elucidated by a combination of NMR, MS, degradation, and molecular modeling techniques. Analysis of the proposed biosynthetic gene cluster led to insights into the biosynthesis of the polyketide and confirmed the structure assignment. The more active compound, disciformycin B, potently inhibits methicillin‐ and vancomycin‐resistant Staphylococcus aureus.     

Cystobactamids: Myxobacterial Topoisomerase Inhibitors Exhibiting Potent Antibacterial Activity

The development of new antibiotics faces a severe crisis inter alia owing to a lack of innovative chemical scaffolds with activities against Gram‐negative and multiresistant pathogens. Herein, we report highly potent novel antibacterial compounds, the myxobacteria‐derived cystobactamids 1 – 3 , which were isolated from Cystobacter sp. and show minimum inhibitory concentrations in the low μg mL^?1 range. We describe the isolation and structure elucidation of three congeners as well as the identification and annotation of their biosynthetic gene cluster. By studying the self‐resistance mechanism in the natural producer organism, the molecular targets were identified as bacterial type IIa topoisomerases. As quinolones are largely exhausted as a template for new type II topoisomerase inhibitors, the cystobactamids offer exciting alternatives to generate novel antibiotics using medicinal chemistry and biosynthetic engineering.