Publications

doi:10.3390/metabo12070569

Abstract

Bacteria use two alternative pathways to synthesize nicotinamide adenine dinucleotide (NAD) from nicotinamide (Nam). A short, two-step route proceeds through nicotinamide mononucleotide (NMN) formation, whereas the other pathway, a four-step route, includes the deamidation of Nam and the reamidation of nicotinic acid adenine dinucleotide (NAAD) to NAD. In addition to having twice as many enzymatic steps, the four-step route appears energetically unfavourable, because the amidation of NAAD includes the cleavage of ATP to AMP. Therefore, it is surprising that this pathway is prevalent not only in bacteria but also in yeast and plants. Here, we demonstrate that the considerably higher chemical stability of the deamidated intermediates, compared with their amidated counterparts, might compensate for the additional energy expenditure, at least at elevated temperatures. Moreover, comprehensive bioinformatics analyses of the available >6000 bacterial genomes indicate that an early selection of one or the other pathway occurred. The mathematical modelling of the NAD pathway dynamics supports this hypothesis, as there appear to be no advantages in having both pathways.

doi:10.1021/acsomega.2c03163

Abstract

The geometry of a dye for dye-sensitized solar cells (DSSCs) has a major impact on its optical and electronic properties. The dye structure also dictates the packing properties and how well the dye insulates the metal–oxide surface from oxidants in the electrolyte. The aim of this work is to investigate the effect of planarizing the geometry of the common triarylamine donor, frequently used in dyes for DSSC. Five novel dyes were designed and prepared; two employ conventional triarylamine donors with thiophene and furan π-spacers, two dyes have had their donors planarized through one sulfur bridge (making two distinct phenothiazine motifs), and the final dye has been planarized by forming a double phenoxazine. The synthesis of these model dyes proved to be quite challenging, and each required specially designed total syntheses. We demonstrate that the planarization of the triarylamine donor can have different effects. When planarization was achieved by a 3,7-phenothiazine and double phenoxazine structures, improved absorption properties were noted, and a panchromatic absorption was achieved by the latter. However, an incorrect linking of donor and acceptor moieties has the opposite effect. Further, electrochemical impedance spectroscopy revealed clear differences in charge recombination depending on the structure of the dye. A drawback of planarized dyes in relation to DSSC is their low oxidation potentials. The best photovoltaic performance was achieved by 3,7-phenothazine with furan as a π-spacer, which produces a power conversion efficiency of 5.2% (Jsc = 8.8 mA cm–2, Voc = 838 mV, FF = 0.70).

doi:10.1021/acscatal.2c01864

Abstract

Gold catalysis has, over the past decades, provided innovative organic transformations under mild conditions with high chemoselectivities. It receives steadily growing attention thanks to its wide synthetic applicability. The catalytically active form, [Ln-Au]+, of ligated gold complexes, [Ln-Au-Cl], is formed via halide abstraction. This is typically achieved by anion exchange upon the addition of an appropriate silver salt to the reaction mixture. Herein, an alternative silver-free route for gold activation is presented, making use of halogen bonding to promote halide abstraction. We demonstrate that a catalytic amount of a strong halogen bond donor efficiently activates both gold(I) and gold(III) catalysts. Following the reaction, both the catalyst and the activator are easily recovered. Importantly, this not only reduces the metal waste in a gold-catalyzed process but also enables its upscaling, possibly opening new avenues for its use in industrial settings. Gold is an expensive and limited resource, and its recyclability is of supreme importance. Based on systematic reaction kinetics, NMR spectroscopic, and computational investigations, we describe the mechanism of halogen bond-activated gold(I/III) catalysis using cyclopropanation as a model reaction. Our discovery paves the way for the development of gold-mediated transformations that allow recycling of the precious gold catalyst and that may thereby become useful also for the large-scale generation of complex molecules.

doi:10.1016/j.carbpol.2022.119655

Abstract

Roots of Aconitum carmichaelii are used in Asian countries due to its content of bioactive alkaloids. In the production of root preparations, tons of leaves are usually discarded, leading to a huge waste of herbal material. The aim of this study is to investigate the polysaccharides in these unutilized leaves. A neutral polysaccharide (AL-N) appeared to be a mixture of heteromannans, and two purified acidic polysaccharides (AL-I-I and AL-I-II) were shown to be pectins containing a homogalacturonan backbone substituted with terminal β-Xylp-units. AL-I-I consisted of a type-I rhamnogalacturonan core, with arabinan and type-II arabinogalactan domains while AL-I-II was less branched. AL-N and AL-I-I were able to modulate the complement system, while AL-I-II was inactive. Interestingly, AL-N, AL-I-I and AL-I-II were shown to exert anti-inflammatory effects on porcine enterocyte IPEC-J2 cells. AL-I-I and AL-I-II were able to down-regulate the expression of toll-like receptor 4 (TLR4) and nucleotide-binding oligomerization domain 1 (NOD1).

doi:10.3390/ph15060668

Abstract

The identification and removal of all gross and microscopic tumor to render the patient disease free represents a huge challenge in ovarian cancer treatment. The presence of residual disease is an independent negative prognostic factor. Herein, we describe the synthesis and the “in vitro” evaluation of compounds as cyclooxygenase (COX)-1 inhibitors, the COX-1 isoform being an ovarian cancer biomarker, each bearing fluorochromes with different fluorescence features. Two of these compounds N-[4-(9-dimethylimino-9H-benzo[a]phenoxazin-5-ylamino) butyl]-2-(3,4-bis(4-methoxyphenyl)isoxazol-5-yl)acetamide chloride (RR11) and 3-(6-(4-(2-(3,4-bis(4-methoxyphenyl)isoxazole-5-yl)acetamido)butyl)amino-6-oxohexyl)-2-[7-(1,3-dihydro-1,1-dimethyl-3-ethyl 2H-benz[e]indolin-2-yl-idene)-1,3,5-heptatrienyl]-1,1-dimethyl-3-(6-carboxilato-hexyl)-1H-benz[e]indolium chloride, 23 (MSA14) were found to be potent and selective inhibitors of cyclooxygenase (COX)-1 “in vitro”, and thus were further investigated “in vivo”. The IC50 values were 0.032 and 0.087 µM for RR11 and 23 (MSA 14), respectively, whereas the COX-2 IC50 for RR11 is 2.4 µM while 23 (MSA14) did not inhibit COX-2 even at a 50 µM concentration. Together, this represented selectivity index = 75 and 874, respectively. Structure-based virtual screening (SBVS) performed with the Fingerprints for Ligands and Proteins (FLAP) software allowed both to differentiate highly active compounds from less active and inactive structures and to define their interactions inside the substrate-binding cavity of hCOX1. Fluorescent probes RR11 and 23 (MSA14), were used for preliminary near-infrared (NIR) fluorescent imaging (FLI) in human ovarian cancer (OVCAR-3 and SKOV-3) xenograft models. Surprisingly, a tumor-specific signal was observed for both tested fluorescent probes, even though this signal is not linked to the presence of COX-1.

doi:10.1038/s41598-022-11923-0

Abstract

Garden chervil, Anthriscus cerefolium (L.) Hoffm. is an important herb commonly applied in Norwegian large-scale commercial kitchens. This species is a highly enriched source of phenolics, containing 1260 mg gallic acid equivalents (GAE) 100–1 g DM, however, the individual phenolic compounds have been scarcely characterized. Here we report on the qualitative and quantitative content of phenolics in garden chervil. The structure of the main phenolic compound was elucidated to be the previously undescribed compound 1,3-dicaffeoyl-5-malonyl-δ-quinide (1) by means of 1D- and 2D NMR and high-resolution mass spectrometry. The known flavones apigenin 7-O-β-(2″-apiofuranosylglucopyranoside) (= apiin) (2), apigenin 7-(2″-apiosyl-6″-malonylglucoside) (3) and luteolin 7-glucoside (4) were also identified. Compound 3 is reported for the first time from this plant species. The main phenolic compound, 1,3-dicaffeoyl-5-malonyl-δ-quinide, exhibited moderate cytotoxicity towards acute monocytic leukaemia cells (MOLM-13) and rat kidney epithelial cells (NRK) with EC50 between 400 and 600 µM.

doi:10.1002/ejoc.202200437

Abstract

A selective and high-rate Ru-catalyzed cross-metathesis reaction of alkenes with vinylimidazole is disclosed. Cross-metathesis is known to operate less efficiently on N-heterocycles, but through optimization by means of statistical experimental design and multiple regression, optimal reaction conditions were identified that allowed for consistent high-yields without the need of overly complicated set-ups or additives. The method was tested on a series of terminal alkene reagents with a variety of different functional groups and it provides the corresponding target molecules in good to high yields.

doi:10.1016/j.rechem.2022.100360

Abstract

Au(III)-NHC-oxazolyl complexes are prepared by oxidation and anion exchange of the corresponding Au(I)Cl-NHC-oxazolyl precursors, which are synthesized from appropriate imidazolium pre-ligands. As the Au(III) complexes are too unstable for proper isolation and spectroscopic characterization, selective 15N NMR techniques provide valuable knowledge of N-coordination to gold by formation of N-ligated Au(III) complexes. The changes in 15N-shift values (Δδ15N), observed by 1H,15N-HMBC 2D NMR studies, going from Au(I)Cl, via Au(III)Cl3 to the C,N-Au(III) NHC-oxazolyl complexes, afford important information of the oxidation and the anion exchange processes. In particular, the huge up-field shift of the oxazoline-N by anion exchange (Δδ15NAE: −71.3 ppm), represents significant evidence that oxazoline-N-coordination to Au(III) takes place by anion exchange, and, hence, that the target six-membered bidentate C,N-Au(III)-NHC-oxazoline chelated complex is formed.

doi:10.1039/d2sc00855f

Abstract

Ruthenium–cyclic(alkyl)(amino)carbene (CAAC) catalysts, used at ppm levels, can enable dramatically higher productivities in olefin metathesis than their N-heterocyclic carbene (NHC) predecessors. A key reason is the reduced susceptibility of the metallacyclobutane (MCB) intermediate to decomposition via β-H elimination. The factors responsible for promoting or inhibiting β-H elimination are explored via density functional theory (DFT) calculations, in metathesis of ethylene or styrene (a representative 1-olefin) by Ru–CAAC and Ru–NHC catalysts. Natural bond orbital analysis of the frontier orbitals confirms the greater strength of the orbital interactions for the CAAC species, and the consequent increase in the carbene trans influence and trans effect. The higher trans effect of the CAAC ligands inhibits β-H elimination by destabilizing the transition state (TS) for decomposition, in which an agostic MCB Cβ–H bond is positioned trans to the carbene. Unproductive cycling with ethylene is also curbed, because ethylene is trans to the carbene ligand in the square pyramidal TS for ethylene metathesis. In contrast, metathesis of styrene proceeds via a ‘late’ TS with approximately trigonal bipyramidal geometry, in which carbene trans effects are reduced. Importantly, however, the positive impact of a strong trans-effect ligand in limiting β-H elimination is offset by its potent accelerating effect on bimolecular coupling, a major competing means of catalyst decomposition. These two decomposition pathways, known for decades to limit productivity in olefin metathesis, are revealed as distinct, antinomic, responses to a single underlying phenomenon. Reconciling these opposing effects emerges as a clear priority for design of robust, high-performing catalysts.

doi:10.1002/open.202200030

Abstract

Gold-catalyzed transformations of 1,3-diarylpropargyl alcohols and various aryl nucleophiles were studied. Selective tunable synthetic methods were developed for 1,1,3-triarylallenes, diaryl-indenes and tetraaryl-allyl target products by C3 nucleophilic substitution and subsequent intra- or intermolecular hydroarylation, respectively. The reactions were scoped with regards to gold(I)/(III) catalysts, solvent, temperature, and electronic and steric effects of both the diarylpropargyl alcohol and the aryl nucleophiles. High yields of triaryl-allenes and diaryl-indenes by gold(III) catalysis were observed. Depending on the choice of aryl nucleophile and control of reaction temperature, different product ratios have been obtained. Alternatively, tetraaryl-allyl target products were formed by a sequential one-pot tandem process from appropriate propargyl substrates and two different aryl nucleophiles. Corresponding halo-arylation products (I and Br; up to 95 % 2-halo-diaryl-indenes) were obtained in a one-pot manner in the presence of the respective N-halosuccinimides (NIS, NBS).

doi:10.1039/d2dt00371f

Abstract

Cyclometalated Au(III) complexes are of interest due to their catalytic, medicinal, and photophysical properties. Herein, we describe the synthesis of derivatives of the type (N,C)Au(OAcF)2 (OAcF = trifluoroacetate) and (N,C,C)AuOAcF by a cyclometalation route, where (N,C) and (N,C,C) are chelating 2-arylpyridine ligands. The scope of the synthesis is explored by substituting the 2-arylpyridine core with electron donor or acceptor substituents at one or both rings. Notably, a variety of functionalized Au(III) complexes can be obtained in one step from the corresponding ligand and Au(OAc)3, eliminating the need for organomercury intermediates, which is commonly reported for similar syntheses. The influence of substituents in the ligand backbone on the resulting complexes was assessed using DFT calculations, 15N NMR spectroscopy and single-crystal X-ray diffraction analysis. A correlation between the electronic properties of the (N,C) ligands and their ability to undergo cyclometalation was found from experimental studies combined with natural charge analysis, suggesting the cyclometalation at Au(III) to take place via an electrophilic aromatic substitution-type mechanism. The formation of Au(III) pincer complexes from tridentate (N,C,C) ligands was investigated by synthesis and DFT calculations, in order to assess the feasibility of C(sp3)–H bond activation as a synthetic pathway to (N,C,C) cyclometalated Au(III) complexes. It was found that C(sp3)–H bond activation is feasible for ligands containing different alkyl groups (isopropyl and ethyl), although the C–H activation is less energetically favored compared to a ligand containing tert-butyl groups.

doi:10.1055/s-0041-1737242

Abstract

Phenotype-guided transposon mutagenesis has emerged as a valuable tool to access cryptic metabolites encoded in bacterial genomes. Recently, the method was demonstrated by inducing silent biosynthetic gene clusters in Burkholderia thailandensis. Amongst the isolated metabolic products, thailandene A exhibited promising antibiotic activity. By assignment, the linear polyenic aldehyde contained a labile motif, where an ostensible chiral secondary alcohol was interlaced in an allylic and a homoallylic constellation. Our attention was drawn to the pseudo-symmetric relationship between the heterofunctionalities, indicating the transformation of a dodecapentaenedial scaffold. Centering on an iterative cross-coupling protocol, the assigned all-E-(12R)-hydroxydodecapentaenal moiety was assembled by combining Zincke chemistry with the MIDA-attenuated Suzuki reaction developed in the Burke laboratory. Thus, according to the devised strategy, the mixed 1,2-bisborylated vinyl linchpin was consecutively functionalized with 5-bromodienal derivatives in a doubly orthogonal fashion. However, when the synthetic material was matched against the bacterial isolate, inconsistencies were observed. A re-examination of the cryptic natural product was conducted by juxtaposing analytical data from experiment and density functional theory calculations, in which hydroperoxide was evaluated as a candidate metabolite present in the bacterial isolate.

doi:10.1007/s11307-021-01685-y

Abstract

Fluorescence imaging (FLI) using targeted near-infrared (NIR) conjugates aids the detection of tumour lesions pre- and intraoperatively. The optimisation of tumour visualisation and contrast is essential and can be achieved through high tumour-specificity and low background signal. However, the choice of fluorophore is recognised to alter biodistribution and clearance of conjugates and is therefore a determining factor in the specificity of target binding. Although ZW800-1, IRDye® 800CW and ICG are the most commonly employed NIR fluorophores in clinical settings, the fluorophore with optimal in vivo characteristics has yet to be determined. Therefore, we aimed to characterise the impact the choice of fluorophore has on the biodistribution, specificity and contrast, by comparing five different NIR fluorophores conjugated to folate, in an ovarian cancer model.

doi:10.1002/slct.202103796

Abstract

To front emergence of antibiotic resistance there is an urgent need for new therapeutics, and one seemingly relevant target is thymidylate monophosphate kinase (TMPK). Serendipitously, we discovered a naphthyl substituted pyrrolopyrimidine possessing activity towards E. coli TMPK. Based on this hit, synthesis, and screening of 61 fused pyrimidines were undertaken. The most potent derivatives were also counter assayed towards the human variant of the enzyme. Two of the inhibitors possessed promising drug-like properties and selectivity for E. coli TMPK. Although the initial pyrrolopyrimidine hit failed to have cellular activity, two alternative scaffolds were discovered providing starting points for further work.

doi:10.1016/j.carbpol.2021.118951

Abstract

Paenibacillus polymyxa is an avid producer of exopolysaccharides of industrial interest. However, due to the complexity of the polymer composition, structural elucidation of the polysaccharide remained unfeasible for a long time. By using a CRISPR-Cas9 mediated knock-out strategy, all single glycosyltransferases as well as the Wzy polymerases were individually deleted in the corresponding gene cluster for the first time. Thereby, it was observed that the main polymer fraction was completely suppressed (or deleted) and a pure minor fucose containing polysaccharide could be isolated, which was named paenan II. Applying this combinatorial approach, the monosaccharide composition, sequence and linkage pattern of this novel polymer was determined via HPLC-MS, GC–MS and NMR. Furthermore, we demonstrated that the knock-out of the glycosyltransferases PepQ, PepT, PepU and PepV as well as of the Wzy polymerase PepG led to the absence of paenan II, attributing those enzymes to the assembly of the repeating unit.

doi:10.1039/d1cc04356k

Abstract

A general Cu-catalyzed strategy for coupling cyclic imides and alkenylboronic acids by forming C(sp2)-N-bonds is reported. The method enables the practical and mild preparation of enimides. A large range of cyclic imides are allowed, and di- and tri-substituted alkenylboronic acids can be used. Full retention was observed in the configuration of the alkene double bond in the coupled products. The method is also applicable for preparing N-arylimides, using arylboronic acids as coupling partners. The usefulness of this strategy is exemplified by the convenient derivatization of the chemotherapy medication 5-flurouracil, the nucleoside uridine and the anti-epileptic drug phenytoin.

doi:10.1002/ejic.202100722

Abstract

A series of Cu(I) complexes of bidentate or tetradentate Schiff base ligands bearing either 1-H-imidazole or pyridine moieties were synthesized. The complexes were studied by a combination of NMR and X-ray spectroscopic techniques. The differences between the imidazole- and pyridine-based ligands were examined by 1H, 13C and 15N NMR spectroscopy. The magnitude of the 15Nimine coordination shifts was found to be strongly affected by the nature of the heterocycle in the complexes. These trends showed good correlation with the obtained Cu−Nimine bond lengths from single-crystal X-ray diffraction measurements. Variable-temperature NMR experiments, in combination with diffusion ordered spectroscopy (DOSY) revealed that one of the complexes underwent a temperature-dependent interconversion between a monomer, a dimer and a higher aggregate. The complexes bearing tetradentate imidazole ligands were further studied using Cu K-edge XAS and VtC XES, where DFT-assisted assignment of spectral features suggested that these complexes may form polynuclear oligomers in solid state. Additionally, the Cu(II) analogue of one of the complexes was incorporated into a metal-organic framework (MOF) as a way to obtain discrete, mononuclear complexes in the solid state.

doi:10.1039/d1fo02354c

Abstract

In this study, the Nelumbo nucifera leaf polysaccharide (NNLP) was isolated by hot water extraction and ethanol precipitation. The DEAE anion exchange chromatography and gel filtration were further performed to get the purified fraction NNLP-I-I, the molecular weight of which was 16.4 kDa. The monosaccharide composition analysis and linkage units determination showed that fraction NNLP-I-I was a pectic polysaccharide. In addition, the NMR spectra analysis revealed that NNLP-I-I mainly consisted of homogalacturonan backbone and rhamnogalacturonan I, with a long HG region, and short RG-I region with AG-II and 1-3 linked rhamnose as side chains. The biological studies demonstrated that NNLP-I-I displayed an antioxidant property through mediating the Nrf2-regulated intestinal cellular antioxidant defense, which could protect cultured intestinal cells from oxidative stress and improve the intestinal function of aged mice.

doi:10.1039/d1py00727k

Abstract

Renewable resources can provide a range of different polysaccharide blocks that can be used to prepare new types of stimuli-responsive polysaccharide-based block copolymers. Alginates are natural polysaccharides widely used as biomaterials. Functional properties depend on the content and distribution of the two 4-linked monomers (β-D-mannuronate (M) and α-L-guluronate (G)). Blocks of L-guluronate (Gn) are responsible for cooperative binding of calcium ions and hydrogel formation. Incorporation of such blocks in block polysaccharide copolymers would represent a new class of engineered, Ca-sensitive biomacromolecules. Dioxyamines and dihydrazides have recently been shown to be well suited for preparation of block polysaccharide structures. Here we first show that when applied to alginate blocks (Gn and Mn) the two types are both very reactive, but the detailed distribution of acyclic (E)- and (Z)-forms and cyclic N-pyranosides, reaction kinetics, conjugate stability, and the rate of Schiff base reduction with α-picoline borane differ considerably, also compared to other polysaccharides. Hence, alginate specific protocols were developed. The linkers introduce a highly flexible joint in otherwise semiflexible Gn-based diblocks. This was demonstrated by SEC-MALS using a symmetrical Gn-b-Gn diblock, which in solution can best be described according to a broken rod model. Ca-Induced self-assembly of Gn-b-dextran diblocks was studied by dynamic light scattering, demonstrating that well defined nanoparticles could be prepared for certain combinations of chain lengths. Taken together, this approach provides a new class of engineered, stimuli-responsive block polysaccharide copolymers solely based on natural resources.

doi:10.1016/j.jbc.2021.101084

Abstract

Among the extensive repertoire of carbohydrate-active enzymes, lytic polysaccharide monooxygenases (LPMOs) have a key role in recalcitrant biomass degradation. LPMOs are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides such as cellulose and chitin. Several LPMOs contain carbohydrate-binding modules (CBMs) that are known to promote LPMO efficiency. However, structural and functional properties of some CBMs remain unknown, and it is not clear why some LPMOs, like CjLPMO10A from the soil bacterium Cellvibrio japonicus, have multiple CBMs (CjCBM5 and CjCBM73). Here, we studied substrate binding by these two CBMs to shine light on their functional variation and determined the solution structures of both by NMR, which constitutes the first structure of a member of the CBM73 family. Chitin-binding experiments and molecular dynamics simulations showed that, while both CBMs bind crystalline chitin with Kd values in the micromolar range, CjCBM73 has higher affinity for chitin than CjCBM5. Furthermore, NMR titration experiments showed that CjCBM5 binds soluble chitohexaose, whereas no binding of CjCBM73 to this chitooligosaccharide was detected. These functional differences correlate with distinctly different arrangements of three conserved aromatic amino acids involved in substrate binding. In CjCBM5, these residues show a linear arrangement that seems compatible with the experimentally observed affinity for single chitin chains. On the other hand, the arrangement of these residues in CjCBM73 suggests a wider binding surface that may interact with several chitin chains. Taken together, these results provide insight into natural variation among related chitin-binding CBMs and the possible functional implications of such variation.

doi:10.1007/s11244-021-01468-3

Abstract

The selective transformation of 1-alkenes into E-olefins is a long-standing challenge in olefin metathesis. Density functional theory (DFT) calculations predict high E-selectivity for catalysts incorporating a bidentate, dianionic thio-indolate ligand within a RuXX'(NHC)(py)(= CHR) platform (NHC = N-heterocyclic carbene; py = pyridine). Such complexes are predicted to yield E-olefins by favoring anti-disposed substituents in the transition state expected to be rate-determining: specifically, that for cycloreversion of the metallacyclobutane intermediate. Three pyridine-stabilized catalysts Ru21a-c were synthesized, in which the thio-indolate ligand bears a H, Me, or Ph substituent at the C2 position, and the NHC ligand is the unsaturated imidazoline-2-ylidene Me(2)IMes (which bears N-mesityl groups and methyl groups on the C4,5 backbone). Single-crystal X-ray diffraction analysis of Ru21c confirms the ligand orientation required for E-selective metathesis, with the thio-indolate sulfur atom binding cis to the NHC, and the indolate nitrogen atom trans to the NHC. However, whereas the new complexes mediated metathetic exchange of their 2-thienylmethylidene ligand in the presence of the common metathesis substrates styrene and allylbenzene, no corresponding self-metathesis products were obtained. Only small amounts of 2-butene (73% (Z)-2-butene) were obtained in self-metathesis of propene using Ru21a. Detailed DFT analysis of this process revealed that product release is surprisingly slow, limiting the reaction rate and explaining the low metathesis activity. With the barrier to dissociation of (Z)-2-butene being lower than that of (E)-2-butene, the calculations also account for the observed Z-selectivity of Ru21a. These findings provide guidelines for catalyst redesign in pursuit of the ambitious goal of E-selective 1-alkene metathesis.

doi:10.1111/1750-3841.15855

Abstract

Enzymatic protein hydrolysates based on side stream materials from the fish-filleting industry are increasingly explored as food ingredients. However, intense sensory properties, and high salt contents, are often a limiting factor. Most of the sensory attributes, such as fish flavor and salty taste, can be ascribed to low-molecular-weight, water-soluble components, whereas bitterness is associated with small hydrophobic peptides. In this study, protein hydrolysates based on head and backbone residuals from Atlantic salmon (Salmo salar) and Atlantic cod (Gadus morhua) were produced using two different enzymes. The effects of micro- and nanofiltration on the chemical composition, protein recovery, and sensory properties of the final products were investigated. The choice of raw material and enzyme had negligible effects, whereas nanofiltration caused a considerable reduction in metabolites, ash, and the intensity of several sensory attributes. The intensity of bitterness increased after nanofiltration, indicating that small peptides associated with bitter taste were retained by the membrane. Total protein yield after microfiltration was 24%-29%, whereas 19%-24% were recovered in the nanofiltration retentate. Practical Application Enzymatic protein hydrolysates can be included in food products to increase the protein content, and as a nutritional supplement and/or functional ingredient; however, unpalatable and intense flavors limit applications. This study investigated the use of membrane filtration to improve flavor quality and reduce salt content in fish protein hydrolysates. Although some protein loss is unavoidable in micro- and nanofiltration, this study demonstrates the production of fish protein hydrolysates with >90% protein and peptide content, which is suitable for inclusion in foods.

doi:10.1002/cmdc.202100302

Abstract

FabF (3-oxoacyl-[acyl-carrier-protein] synthase 2), which catalyses the rate limiting condensation reaction in the fatty acid synthesis II pathway, is an attractive target for new antibiotics. Here, we focus on FabF from P. aeruginosa (PaFabF) as antibiotics against this pathogen are urgently needed. To facilitate exploration of this target we have set up an experimental toolbox consisting of binding assays using bio-layer interferometry (BLI) as well as saturation transfer difference (STD) and WaterLOGSY NMR in addition to robust conditions for structure determination. The suitability of the toolbox to support structure-based design of FabF inhibitors was demonstrated through the validation of hits obtained from virtual screening. Screening a library of almost 5 million compounds resulted in 6 compounds for which binding into the malonyl-binding site of FabF was shown. For one of the hits, the crystal structure in complex with PaFabF was determined. Based on the obtained binding mode, analogues were designed and synthesised, but affinity could not be improved. This work has laid the foundation for structure-based exploration of PaFabF.

doi:10.1093/glycob/cwab058

Abstract

Alginate is a major compound of brown macroalgae and as such an important carbon and energy source for heterotrophic marine bacteria. Despite the rather simple composition of alginate only comprising mannuronate and guluronate units, these bacteria feature complex alginolytic systems that can contain up to seven alginate lyases. This reflects the necessity of large enzyme systems for the complete degradation of the abundant substrate. Numerous alginate lyases have been characterized. They belong to different polysaccharide lyase (PL) families, but only one crystal structure of a family 17 (PL17) alginate lyase has been reported to date, namely Alg17c from the gammaproteobacterium Saccharophagus degradans. Biochemical and structural characterizations are helpful to link sequence profiles to function, evolution of functions and niche-specific characteristics. Here, we combined detailed biochemical and crystallographic analysis of AlyA3, a PL17 alginate lyase from the marine flavobacteria Zobellia galactanivorans DsijT, providing the first structure of a PL17 in the Bacteroidetes phylum. AlyA3 is exo-lytic and highly specific of mannuronate stretches. As part of an “alginate utilizing locus”, its activity is complementary to that of other characterized alginate lyases from the same bacterium. Structural comparison with Alg17c highlights a common mode of action for exo-lytic cleavage of the substrate, strengthening our understanding of the PL17 catalytic mechanism. We show that unlike Alg17c, AlyA3 contains an inserted flexible loop at the entrance to the catalytic groove, likely involved in substrate recognition, processivity and turn over.

doi:10.1021/jacs.1c04424

Abstract

Bimolecular catalyst decomposition is a fundamental, long-standing challenge in olefin metathesis. Emerging ruthenium–cyclic(alkyl)(amino)carbene (CAAC) catalysts, which enable breakthrough advances in productivity and general robustness, are now known to be extraordinarily susceptible to this pathway. The details of the process, however, have hitherto been obscure. The present study provides the first detailed mechanistic insights into the steric and electronic factors that govern bimolecular decomposition. Described is a combined experimental and theoretical study that probes decomposition of the key active species, RuCl2(L)(py)(═CH2) 1 (in which L is the N-heterocyclic carbene (NHC) H2IMes, or a CAAC ligand: the latter vary in the NAr group (NMes, N-2,6-Et2C6H3, or N-2-Me,6-iPrC6H3) and the substituents on the quaternary site flanking the carbene carbon (i.e., CMe2 or CMePh)). The transiently stabilized pyridine adducts 1 were isolated by cryogenic synthesis of the metallacyclobutanes, addition of pyridine, and precipitation. All are shown to decompose via second-order kinetics at −10 °C. The most vulnerable CAAC species, however, decompose more than 1000-fold faster than the H2IMes analogue. Computational studies reveal that the key factor underlying accelerated decomposition of the CAAC derivatives is their stronger trans influence, which weakens the Ru−py bond and increases the transient concentration of the 14-electron methylidene species, RuCl2(L)(═CH2) 2. Fast catalyst initiation, a major design goal in olefin metathesis, thus has the negative consequence of accelerating decomposition. Inhibiting bimolecular decomposition offers major opportunities to transform catalyst productivity and utility, and to realize the outstanding promise of olefin metathesis.

doi:10.1016/j.dyepig.2021.109553

Abstract

CytoCy5S™, a quenched, red-shifted fluorescent probe, has been used to exploit the imaging potential of the nitroreductase (NTR) reporter gene platform. Its use has been reported in a number of publications, however there are discrepancies in both the reported structure and its physicochemical properties. Herein, we aim to highlight these discrepancies and to define the best candidate of the four substrates under study for preclinical work in NTR reporting by optical applications. We report the synthesis, purification and characterisation of four NTR substrates, including alternately described structures currently referred by the name CytoCy5S. A comparative NTR enzymatic assay was performed to assess the spectroscopic characteristics of the different reductively activated probes. The NTR expressing triple-negative breast carcinoma cell line, MDA-MB-231 NTR+, was employed to compare, both in vitro and in vivo, the suitability of these fluorescent probes as reporters of NTR activity. Comparison of the reporting properties was achieved by flow cytometry, fluorescence microscopy and optical imaging, both in vivo and ex vivo. This study evaluated the different spectroscopic and biological characteristics of the four substrates and concluded that substrate 1 presents the best features for oncological in vivo preclinical optical imaging.

doi:10.1016/j.apcatb.2021.120349

Abstract

Mimicking natural photosynthesis by direct photoelectrochemical (PEC) reduction of CO2 to chemicals and fuels requires complex cell assemblies with limitations in selectivity, efficiency, cost, and stability. Here, we present a breakthrough cathode utilizing an oxygen tolerant formate dehydrogenase enzyme derived from clostridium carboxidivorans and coupled to a novel and efficient in situ nicotinamide adenine dinucleotide (NAD+/NADH) regeneration mechanism through interfacial electrochemistry on g-C3N4 films. We demonstrate stable (20 h) aerobic PEC CO2-to-formate reduction at close to 100 % faradaic efficiency and unit selectivity in a bio-hybrid PEC cell of minimal engineering with optimized Ta3N5 nanotube photoanode powered by simulated sunlight with a solar to fuel efficiency of 0.063 %, approaching that of natural photosynthesis.

doi:10.1016/j.fitote.2021.104920

Abstract

The resin of Canarium strictum Roxb. is used for rheumatism and asthma; the bark is used as a mosquito repellent. The major compounds in the resin are triterpenoids, but as no studies have been performed on the bark, this study investigated this economically important resource. Ten folk healers were interviewed about their medicinal uses of C. strictum. Resin and bark were extracted with dichloromethane followed by methanol using accelerated solvent extraction. The extracts were fractionated using different chromatographic methods, and isolated compounds were identified by NMR spectroscopy and GC–MS. Resin and bark extracts were investigated for DPPH radical scavenging, 15-lipoxygenase inhibition, effects on nitric oxide (NO) production in LPS-activated dendritic D2SC/I cells and toxicity against Artemia salina nauplii. Traditional healers used resin to treat colds, airway afflictions and rheumatoid arthritis. α-Amyrin and β-amyrin were identified as the major constituents in the dichloromethane resin extract. From the stem bark, procyanidins, gallic acid, methyl gallate, scopoletin, 3,3′-di-O-methylellagic acid 4-O-α-arabinofuranoside and elephantorrhizol (3,3′,4′,5,6,7,8-heptahydroxyflavan) were isolated and identified. By GC–MS, α-amyrin and β-amyrin and their acetates, lupeol, and taraxasterol were identified. Radical scavenging, 15-lipoxygenase inhibitory activity and inhibition of NO production was observed from resin and bark extracts, and no toxicity towards Artemia salina nauplii was found. Triterpenoids and procyanidins are the major compounds in C. strictum resin and stem bark, respectively. The high content of triterpenoids might contribute to anti-inflammatory effects and give a rationale for the widespread usage of the resin in India.

doi:10.1016/j.carbpol.2021.117991

Abstract

The mushroom cell wall contains polysaccharides that can activate cells of the innate immune system through receptors such as Toll-like receptors (TLR) and dectin-1. In the present study, Pleurotus eryngii polysaccharide fractions containing a 3-O methylated mannogalactan and (1→3)/(1→6)-β-d-glucans were isolated and extensively characterized by 2D NMR and methylation analysis. Traces of a (1→3)-α-d-glucan and a (1→2)-α-d-mannan were also observed. Affinity for TLR2, TLR2-TLR6 and dectin-1 using HEK-cells expressing the relevant receptor genes was tested. PeWN, containing the 3-O methylated mannogalactan, was inactive towards TLR2, whereas fraction PeWB, containing more β-glucan, activated the TLR2-TLR6 heterodimer. Activation of the human β-glucan receptor dectin-1 correlated with the amount of β-glucan in each fraction. Nitric oxide and cytokine supernatant levels of D2SC/1 dendritic cells stimulated with the P. eryngii fractions and interferon-γ were low to moderate. The results indicate that the immunomodulatory activity of water-soluble P. eryngii polysaccharide fractions is modest.

doi:10.1002/jsfa.11261

Abstract

Codonopsis pilosula and Codonopsis tangshen are plants widely used in traditional Chinese medicine. Two pectic polysaccharides from the roots of C. pilosula and C. tangshen named as CPP-1 and CTP-1 were obtained by boiling water extraction and column chromatography. The core structures of both CPP-1 and CTP-1 comprise the long homogalacturonan region (HG) as the backbone and the rhamnogalacturonan I (RG-I) region as the side chains. CPP-1 has methyl esterified galacturonic acid units and a slightly lower molecular weight than CTP-1. Biological testing suggested that CPP-1 and CTP-1 can protect IPEC-J2 cells against the H2O2-induced oxidative stress by up-regulating nuclear factor-erythroid 2-related factor 2 and related genes in IPEC-J2 cells. The different antioxidative activities of polysaccharides from different source of C. pilosula may be result of differences in their structures. All of the results indicated that pectic polysaccharides CPP-1 and CTP-1 from different species of C. pilosula roots could be used as a potential natural antioxidant source. These findings will be valuable for further studies and new applications of pectin-containing health products. © 2021 Society of Chemical Industry.

doi:10.1093/glycob/cwab025

Abstract

Mannuronan C-5 epimerases catalyze the epimerization of monomer residues in the polysaccharide alginate, changing the physical properties of the biopolymer. The enzymes are utilized to tailor alginate to numerous biological functions by alginate-producing organisms. The underlying molecular mechanism that control the processive movement of the epimerase along the substrate chain is still elusive. To study this, we have used an interdisciplinary approach combining molecular dynamics simulations with experimental methods from mutant studies of AlgE4, where initial epimerase activity and product formation were addressed with nuclear magnetic resonance spectroscopy, and characteristics of enzyme–substrate interactions were obtained with isothermal titration calorimetry and optical tweezers. Positive charges lining the substrate-binding groove of AlgE4 appear to control the initial binding of poly-mannuronate, and binding also seems to be mediated by both electrostatic and hydrophobic interactions. After the catalytic reaction, negatively charged enzyme residues might facilitate dissociation of alginate from the positive residues, working like electrostatic switches, allowing the substrate to translocate in the binding groove. Molecular simulations show translocation increments of two monosaccharide units before the next productive binding event resulting in mannuronate and guluronate (MG)-block formation, with the epimerase moving with its N-terminus towards the reducing end of the alginate chain. Our results indicate that the charge pair R343–D345 might be directly involved in conformational changes of a loop that can be important for binding and dissociation. The computational and experimental approaches used in this study complement each other, allowing for a better understanding of individual residues’ roles in binding and movement along the alginate chains.

doi:10.1093/nar/gkab195

Abstract

Uracil occurs at replication forks via misincorporation of deoxyuridine monophosphate (dUMP) or via deamination of existing cytosines, which occurs 2–3 orders of magnitude faster in ssDNA than in dsDNA and is 100% miscoding. Tethering of UNG2 to proliferating cell nuclear antigen (PCNA) allows rapid post-replicative removal of misincorporated uracil, but potential ‘pre-replicative’ removal of deaminated cytosines in ssDNA has been questioned since this could mediate mutagenic translesion synthesis and induction of double-strand breaks. Here, we demonstrate that uracil-DNA glycosylase (UNG), but not SMUG1 efficiently excises uracil from replication protein A (RPA)-coated ssDNA and that this depends on functional interaction between the flexible winged-helix (WH) domain of RPA2 and the N-terminal RPA-binding helix in UNG. This functional interaction is promoted by mono-ubiquitination and diminished by cell-cycle regulated phosphorylations on UNG. Six other human proteins bind the RPA2-WH domain, all of which are involved in DNA repair and replication fork remodelling. Based on this and the recent discovery of the AP site crosslinking protein HMCES, we propose an integrated model in which templated repair of uracil and potentially other mutagenic base lesions in ssDNA at the replication fork, is orchestrated by RPA. The UNG:RPA2-WH interaction may also play a role in adaptive immunity by promoting efficient excision of AID-induced uracils in transcribed immunoglobulin loci.

doi:10.1002/ejic.202100170

Abstract

Multidentate Schiff base ligands derived from a selection of biphenyl- and terphenyl polyamines were synthesized, and their reactivity towards divalent (Ni, Cu, Zn, Cd) and trivalent (Co, Y, Lu) metals was studied by single-crystal X-ray diffraction analysis, NMR spectroscopy, and UV/Vis spectroscopy for the Cu(II) complexes. Large variations in the resulting complexes were observed based on the relative position of the amine substituents in the parent triamines, as well as the electronic properties of the Schiff base ligand itself. Most notably, Schiff base ligands derived from a m-terphenyl-2,2’,2’’-triamine were found to coordinate in a tetradentate, pentadentate or hexadentate fashion, depending on the size and the valency of the corresponding metal center.

doi:10.3762/bjnano.12.21

Abstract

Seven gold(I) N-heterocyclic carbene (NHC) complexes were synthesized, characterized, and identified as suitable precursors for focused electron beam-induced deposition (FEBID). Several variations on the core Au(NHC)X moiety were introduced, that is, variations of the NHC ring (imidazole or triazole), of the alkyl N-substituents (Me, Et, or iPr), and of the ancillary ligand X (Cl, Br, I, or CF3). The seven complexes were tested as FEBID precursors in an on-substrate custom setup. The effect of the substitutions on deposit composition and growth rate indicates that the most suitable organic ligand for the gold precursor is triazole-based, with the best deposit composition of 15 atom % gold, while the most suitable anionic ligand is the trifluoromethyl group, leading to a growth rate of 1 × 10−2 nm3/e−.

doi:10.1038/s41467-021-21307-z

Abstract

Nicotinamide adenine dinucleotide (NAD) is a key molecule in cellular bioenergetics and signalling. Various bacterial pathogens release NADase enzymes into the host cell that deplete the host’s NAD+ pool, thereby causing rapid cell death. Here, we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa. The enzymes harbour a tuberculosis necrotizing toxin (TNT) domain and are predominately present in pathogenic species. The 1.6 Å X-ray structure of the homodimeric A. fumigatus protein reveals unique properties including N-linked glycosylation and a Ca2+-binding site whose occupancy regulates activity. The structure in complex with a substrate analogue suggests a catalytic mechanism that is distinct from those of known NADases, ADP-ribosyl cyclases and transferases. We propose that fungal NADases may convey advantages during interaction with the host or competing microorganisms.

doi:10.1021/acsomega.0c05642

Abstract

Methods for thermochemical conversion of biomass into renewable energy and materials rapidly increase in range and outreach. A focus on the target product streams for valorization is natural, yet several pretreatment steps and conversion methods also result in an aqueous byproduct, which has been given less attention. This paper aims to fill this knowledge gap in the existing literature on identification and quantification of organic components in such aqueous phases by reporting a fast and direct workup protocol combined with application of quantitative analytical nuclear magnetic resonance (NMR) spectroscopy. Laboratory workup procedures combined with subsequent proton NMR spectroscopy with water signal suppression using presaturation pulses during relaxation delay, noesygppr1d, have been established, evaluated, and approved by testing on three different Bruker BioSpin NMR spectrometers; an 850 MHz AVANCE III HD with a 5 mm TCI CryoProbe, a 600 MHz AVANCE NEO with a QCI CryoProbe, and a 500 MHz AVANCE with a 5 mm BBO room-temperature probe additionally confirmed the quantification method to be applicable. The analytical procedure identified furfural, methanol, acetic acid, and formic acid as the dominating compounds in the analyzed aqueous samples, which were process effluents generated by the patented Arbacore pellet production process using steam explosion of wood shavings. A selected range of quantitative results in the aqueous phase from large-scale steam explosion is included in the study. The described procedure provides excellent quantitative reproducibility with experimental series standard deviations of <1% (mM), is nondestructive, and can be automated on demand.

doi:10.1016/j.ijbiomac.2021.02.041

Abstract

Platycodonis Radix is widely used as homology of medicine and food in China; polysaccharides are thought to be one of its functional constituents. In this study, a pectic polysaccharide, PGP-I-I, was obtained from the root of the traditional medicine plant Platycodon grandiflorus through ion exchange chromatography and gel filtration. This was characterized being mainly composed of 1,5-α-L-arabinan and both arabinogalactan type I (AG-I) and II chains linked to rhamnogalacturonan I (RG-I) backbone linked to longer galacturonan chains. In vitro bioactivity study showed that PGP-I-I could restore the intestinal cellular antioxidant defense under the condition of hydrogen peroxide (H2O2) treatment through promoting the expressions of cellular antioxidant genes and protect against oxidative damages.

doi:10.1021/acs.joc.0c02913

Abstract

Herein, we report the stereoselective and convergent synthesis of resolvin E4, a newly identified specialized pro-resolving mediator. This synthesis proves the absolute configuration and exact olefin geometry. Key elements of the successful strategy include a highly stereoselective MacMillan organocatalytic oxyamination, a Midland Alpine borane reduction, and the use of a 1,4-pentadiyne unit as a linchpin building block. The application of reaction telescoping in several of the synthetic transformations enabled the preparation of the resolvin E4 methyl ester in 10\% yield over 10 steps (longest linear sequence). The physical property (UV–Vis and LC–MS/MS) data of synthetic resolvin E4 matched those obtained from biologically produced material.

doi:10.3762/bjoc.17.18

Abstract

Chiral cyclam (1,4,8,11-tetraazacyclotetradecane) derivatives were synthesized stepwise from chiral mono-Boc-1,2-diamines and (dialkyl)malonyl dichloride via the open diamide-di-(N-Boc-amino) intermediates (65-91%). Deprotection and ring closure with a second malonyl unit afforded the cyclam tetraamide precursors (80-95%). The new protocol allowed preparation of the target cyclam derivatives (53-59%) by a final optimized hydride reduction. Both the open tetraamine intermediates and the cyclam derivatives successfully coordinated with AuCl3 to give moderate to excellent yields (50-96%) of the corresponding novel tetracoordinated N,N,N,N-Au(III) complexes with alternating five- and six-membered chelate rings. Testing of catalytic ability of the cyclam based N,N,N,N-Au(III) complexes demonstrated high catalytic activity of some complexes in selected test reactions (full conversion in 1-24h, 62-97% product yields).

doi:10.1038/s41598-020-79533-2

Abstract

The eye lens is a unique organ as no cells can be replaced throughout life. This makes it decisive that the lens is protected against damaging UV-radiation. An ultraviolet (UV)-absorbing compound of unknown identity is present in the aqueous humor of geese (wild and domestic) and other birds flying at high altitudes. A goose aqueous humor extract, that was believed to contain the UV protective compound which was designated as "compound X", was fractionated and examined using a variety of spectroscopic techniques including LC-MS and high field one- and two dimensional-NMR methods. A series of compounds were identified but none of them appeared to be the UV protective "compound X". It may be that the level of the UV protective compound in goose aqueous humor is much less than the compounds identified in our investigation, or it may have been degraded by the isolation and chromatographic purification protocols used in our investigations.

doi:10.3390/polym13020207

Abstract

Face masks have globally been accepted to be an effective protective tool to prevent bacterial and viral transmission, especially against indoor aerosol transmission. However, commercial face masks contain filters that are made of materials that are not capable of inactivating either SARS-CoV-2 or multidrug-resistant bacteria. Therefore, symptomatic and asymptomatic individuals can infect other people even if they wear them because some viable viral or bacterial loads can escape from the masks. Furthermore, viral or bacterial contact transmission can occur after touching the mask, which constitutes an increasing source of contaminated biological waste. Additionally, bacterial pathogens contribute to the SARS-CoV-2-mediated pneumonia disease complex, and their resistance to antibiotics in pneumonia treatment is increasing at an alarming rate. In this regard, herein, we report the development of a non-woven face mask filter fabricated with a biofunctional coating of benzalkonium chloride that is capable of inactivating more than 99% of SARS-CoV-2 particles in one minute of contact, and the life-threatening methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis (normalized antibacterial halos of 0.52 ± 0.04 and 0.72 ± 0.04, respectively). Nonetheless, despite the results obtained, further studies are needed to ensure the safety and correct use of this technology for the mass production and commercialization of this broad-spectrum antimicrobial face mask filter. Our novel protective non-woven face mask filter would be useful for many healthcare workers and researchers working in this urgent and challenging field.

doi:10.1128/aem.02399-20

Abstract

Enzymatic depolymerization of seaweed polysaccharides is gaining interest for the production of functional oligosaccharides and fermentable sugars. Herein, we describe a thermostable alginate lyase that belongs to Polysaccharide Lyase family 17 (PL17) and was derived from an Arctic Mid-Ocean Ridge (AMOR) metagenomics dataset. This enzyme, AMOR_PL17A, is a thermostable exolytic oligoalginate lyase (EC 4.2.2.26), which can degrade alginate, poly β-d-mannuronate and poly α-l-guluronate within a broad range of pH, temperature and salinity conditions. Site-directed mutagenesis showed that tyrosine Y251, previously suggested to act as catalytic acid, indeed is essential for catalysis; whereas mutation of tyrosine Y446, previously proposed to act as catalytic base, did not affect enzyme activity. The observed reaction products are protonated and deprotonated forms of the 4,5-unsaturated uronic acid monomer, Δ, two hydrates of DEH (4-deoxy-l-erythro-5-hexulosuronate), which are formed after ring opening and, finally, two epimers of a 5-membered hemiketal called 4-deoxy-d-manno-hexulofuranosidonate (DHF) formed through intramolecular cyclisation of hydrated DEH. The detection and NMR assignment of these hemiketals refine our current understanding of alginate degradation.Importance The potential markets for seaweed-derived products and seaweed processing technologies are growing, yet commercial enzyme-cocktails for complete conversion of seaweed to fermentable sugars are not available. Such an enzyme-cocktail would require the catalytic properties of a variety of different enzymes, where fucoidanases, laminarinases and cellulases together with endo- and exo-acting alginate lyases would be the key enzymes. Here we present an exo-acting alginate lyase that efficiently produces monomeric sugars from alginate. Since it is only the second characterized exo-acting alginate lyase capable of degrading alginate at industrially relevant higher temperatures of 60 {\textdegree}C, this enzyme may be of great biotechnological and industrial interest. In addition, in-depth NMR-based structural elucidation reveal previously undescribed rearrangement products of the unsaturated monomeric sugars generated from exo-acting lyases. The insight provided by the NMR assignment of these products facilitates future assessment of product formation by alginate lyases.

doi:10.24820/ark.5550190.p011.359

Abstract

The first gold(III)-oxazoline catalysed intramolecular tandem dihydroalkoxylations of alkynyl diols to give benzannulated 5,6-spiroketal products is reported. The results showed that Au(III)-bisoxazoline (BOX) and Au(III)-pyridine-oxazoline complexes are highly efficient catalysts for such spirocyclizations. The mono- and dibenzannulated 5,6-spiroketals were obtained in high yields (> 90 %) by rapid conversion of symmetrical and nonsymmetrical alkynyl diols, respectively. The Au(III)-BOX-BF4 catalyst generated minor spirocyclization enantioselectivity (up to 6 % ee). The choice of solvent was important for the outcome of the reactions. [GRAPHICS] .

doi:10.3390/foods10010038

Abstract

The focus on natural foods and ``clean'' labeled products is increasing and encourages development of new biobased ingredients. Fish solubles derived from downstream processing of side stream materials in the fish filleting industries have potential as emulsifiers based on their surface-active and emulsion stabilizing properties. The aim of this study was to evaluate and compare emulsion properties and critical micelle concentration (CMC) of direct protein extracts and protein hydrolysates based on fish backbones, and to identify associations between molecular weight distribution and process yield with the studied physicochemical properties. Protein extracts and enzymatic protein hydrolysates were produced based on two raw materials (cod and salmon backbones), two enzymes with different proteolytic specificity, and varying hydrolysis time. Emulsion activity index (EAI), emulsion stability index (ESI) and CMC were measured and compared with casein as a reference to protein-based emulsifiers. Protein hydrolysis was found to have negative impact on EAI and CMC, likely due to generation of small peptides disrupting the amphiphilic balance. The direct protein extracts had comparable EAI with casein, but the latter had superior ESI values. Protein hydrolysates with acceptable EAI could only be obtained at the expense of product yield. The study emphasizes the complexity of physicochemical properties of protein hydrolysates and discusses the challenges of achieving both good surface-active properties and high product yield.

doi:10.1002/ejoc.202001138

Abstract

Gold(III) coordination of new chiral polydentate (N,)N,O-pyridine based ligands is reported. Successful coordination afforded novel chiral N,N,O-tridentate Au(III) complexes with the 2-pyridyl-6-[(1S,2S,5R)-neomenthol-1-yl]pyridine ligand (1H, 13C, 15N NMR, HRMS, IR, XRD). The chiral 2-aryl-6-alkylpyridine alcohol ligands were prepared from 2,6-dibromopyridine by initial stereoselective addition to (–)-menthone and (+)-camphor, respectively, and subsequent Suzuki cross-coupling with a series arylboronic acids. Testing of catalytic activity in propargyl cyclopropanation demonstrated that the new N,N,O-ligated gold(III) complex was highly catalytic active and outperformed AuCl3.

doi:10.1007/s12104-020-09986-z

Abstract

The lytic polysaccharide monooxygenase JdLPMO10A is the N-terminal domain of the multimodular protein Jd1381. The isolated JdLPMO10A domain is one of the smallest chitin-active lytic polysaccharide monooxygenases known to date with a size of only 15.5 kDa. JdLPMO10A is a copper-dependent oxidative enzyme that depolymerizes chitin by hydroxylating the C1 carbon in the glycosidic bond. JdLPMO10A has been isotopically labeled and recombinantly expressed. Here, we report the 1H, 13C, 15N resonance assignment of JdLPMO10A. Secondary structural elements predicted based on the NMR assignment are in excellent agreement with the crystal structure of JdLPMO10A.

doi:10.1021/acs.macromol.0c01936

Abstract

Self-assembly of amphiphilic polymers into micelles is an archetypical example of a “self-confined” system due to the formation of micellar cores with dimensions of a few nanometers. In this work, we investigate the chain packing and resulting shape of Cn-PEOx micelles with semicrystalline cores using small/wide-angle X-ray scattering (SAXS/WAXS), contrast-variation small-angle neutron scattering (SANS), and nuclear magnetic resonance spectroscopy (NMR). Interestingly, the n-alkyl chains adopt a rotator-like conformation and pack into prolate ellipses (axial ratio ϵ ≈ 0.5) in the “crystalline” region and abruptly arrange into a more spheroidal shape (ϵ ≈ 0.7) above the melting point. We attribute the distorted spherical shape above the melting point to thermal fluctuations and intrinsic rigidity of the n-alkyl blocks. We also find evidence for a thin dehydrated PEO layer (≤1 nm) close to the micellar core. The results provide substantial insight into the interplay between crystallinity and molecular packing in confinement and the resulting overall micellar shape.

doi:10.3390/pharmaceutics12111080

Abstract

The recent emergence of resistance to colistin, an antibiotic of last resort with dose-limiting toxicity, has highlighted the need for alternative approaches to combat infection. This study aimed to generate and characterise alginate oligosaccharide (“OligoG”)–polymyxin (polymyxin B and E (colistin)) conjugates to improve the effectiveness of these antibiotics. OligoG–polymyxin conjugates (amide- or ester-linked), with molecular weights of 5200–12,800 g/mol and antibiotic loading of 6.1–12.9% w/w, were reproducibly synthesised. In vitro inflammatory cytokine production (tumour necrosis factor alpha (TNFα) ELISA) and cytotoxicity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) of colistin (2.2–9.3-fold) and polymyxin B (2.9–27.2-fold) were significantly decreased by OligoG conjugation. Antimicrobial susceptibility tests (minimum inhibitory concentration (MIC), growth curves) demonstrated similar antimicrobial efficacy of ester- and amide-linked conjugates to that of the parent antibiotic but with more sustained inhibition of bacterial growth. OligoG–polymyxin conjugates exhibited improved selectivity for Gram-negative bacteria in comparison to mammalian cells (approximately 2–4-fold). Both OligoG–colistin conjugates caused significant disruption of Pseudomonas aeruginosa biofilm formation and induced bacterial death (confocal laser scanning microscopy). When conjugates were tested in an in vitro “time-to-kill” (TTK) model using Acinetobacter baumannii, only ester-linked conjugates reduced viable bacterial counts (~2-fold) after 4 h. Bi-functional OligoG–polymyxin conjugates have potential therapeutic benefits in the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections, directly reducing toxicity whilst retaining antimicrobial and antibiofilm activities.

doi:10.1016/j.isci.2020.101785

Abstract

Heterologous expression of a biosynthesis gene cluster from Amycolatopsis sp. resulted in the discovery of two unique class IV lasso peptides, felipeptins A1 and A2. A mixture of felipeptins stimulated proliferation of cancer cells, while having no such effect on the normal cells. Detailed investigation revealed, that pre-treatment of cancer cells with a mixture of felipeptins resulted in downregulation of the tumor suppressor Rb, making the cancer cells to proliferate faster. Pre-treatment with felipeptins made cancer cells considerably more sensitive to the anticancer agent doxorubicin and re-sensitized doxorubicin-resistant cells to this drug. Structural characterization and binding experiments showed an interaction between felipeptins resulting in complex formation, which explains their synergistic effect. This discovery may open an alternative avenue in cancer treatment, helping to eliminate quiescent cells that often lead to cancer relapse.

doi:10.1021/acs.inorgchem.0c02720

Abstract

Gold(III) complexes are versatile catalysts offering a growing number of new synthetic transformations. Our current understanding of the mechanism of homogeneous gold(III) catalysis is, however, limited, with that of phosphorus-containing complexes being hitherto underexplored. The ease of phosphorus oxidation by gold(III) has so far hindered the use of phosphorus ligands in the context of gold(III) catalysis. We present a method for the generation of P,N-chelated gold(III) complexes that circumvents ligand oxidation and offers full counterion control, avoiding the unwanted formation of AuCl4-. On the basis of NMR spectroscopic, X-ray crystallographic, and density functional theory analyses, we assess the mechanism of formation of the active catalyst and of gold(III)-mediated styrene cyclopropanation with propargyl ester and intramolecular alkoxycyclization of 1,6-enyne. P,N-chelated gold(III) complexes are demonstrated to be straightforward to generate and be catalytically active in synthetically useful transformations of complex molecules.

doi:10.1016/j.dyepig.2020.108951

Abstract

To study the effect of pi-spacers on dye performance in DSSC, five novel phenothiazine dyes have been prepared where the number of successive thiophenes in the pi-spacer was increased from zero to four. Additionally, a dye bearing thieno[3,2-b]thiophene was synthesized, and compared to its unfused bithiophene analogue. When the number of thiophenes were two or more the light absorption properties was significantly better in the region 400-550 nm compared to the dye without a pi-spacer. DSSC device testing revealed that the reference dye without a pi-spacer gave the best overall performance producing a power conversion efficiency of 5.7% (J(SC) = 10.1 mA cm(-2), V-OC = 0.83 V, FF = 0.68), attributed to the superior V-OC of this dye. The incorporation of one and two thiophenes resulted in a similar performance as the reference, while incorporation of larger oligothiophenes proved to be deleterious to DSSC-performance. Electrochemical impedance spectroscopy indicates that the voltage drops and performance loss associated with these dyes are due to more facile recombination.

doi:10.3390/molecules25204848

Abstract

Phorbazoles are polychlorinated heterocyclic secondary metabolites isolated from a marine sponge and several of these natural products have shown inhibitory activity against cancer cells. In this work, a synthesis of the trichlorinated phorbazole B using late stage electrophilic chlorination was developed. The synthesis relied on the use of an oxazole precursor, which was protected with an iodine in the reactive 4-position, followed by complete chlorination of all pyrrole positions. Attempts to prepare phorbazole A and C, which contain a 3,4-dichlorinated pyrrole, were unsuccessful as the desired chlorination pattern on the pyrrole could not be obtained. The identities of the dichlorinated intermediates and products were determined using NMR techniques including NOESY/ROESY, 1,1-ADEQUATE and high-resolution CLIP-HSQMBC.

doi:10.1371/journal.pone.0232442

Abstract

Exosomes are vesicles involved in intercellular communication. Their membrane structure and core content is largely dependent on the cell of origin. Exosomes have been investigated both for their biological roles and their possible use as disease biomarkers and drug carriers. These potential technological applications require the rigorous characterization of exosomal blood brain barrier permeability and a description of their lipid bilayer composition. To achieve these goals, we have established a 3D static blood brain barrier system based on existing systems for liposomes and a complementary LC-MS/MS and 31P nuclear magnetic resonance methodology for the analysis of purified human plasma-derived exosome-like vesicles. Results show that the isolated vesicles pass the blood brain barrier and are taken up in endothelial cells. The compositional analysis revealed that the isolated vesicles are enriched in lyso phospholipids and do not contain phosphatidylserine. These findings deviate significantly from the composition of exosomes originating from cell culture, and may reflect active removal by macrophages that respond to exposed phosphahtidylserine.

doi:10.3390/molecules25194364

Abstract

Tridentate, bis-phenolate N-heterocyclic carbenes (NHCs) are among the ligands giving the most selective and active group 4-based catalysts for the copolymerization of cyclohexene oxide (CHO) with CO2. In particular, ligands based on imidazolidin-2-ylidene (saturated NHC) moieties have given catalysts which exclusively form polycarbonate in moderate-to-high yields even under low CO2 pressure and at low copolymerization temperatures. Here, to evaluate the influence of the NHC moiety on the molecular structure of the catalyst and its performance in copolymerization, we extend this chemistry by synthesizing and characterizing titanium complexes bearing tridentate bis-phenolate imidazol-2-ylidene (unsaturated NHC) and benzimidazol-2-ylidene (benzannulated NHC) ligands. The electronic properties of the ligands and the nature of their bonds to titanium are studied using density functional theory (DFT) and natural bond orbital (NBO) analysis. The metal–NHC bond distances and bond strengths are governed by ligand-to-metal σ- and π-donation, whereas back-donation directly from the metal to the NHC ligand seems to be less important. The NHC π-acceptor orbitals are still involved in bonding, as they interact with THF and isopropoxide oxygen lone-pair donor orbitals. The new complexes are, when combined with [PPN]Cl co-catalyst, selective in polycarbonate formation. The highest activity, albeit lower than that of the previously reported Ti catalysts based on saturated NHC, was obtained with the benzannulated NHC-Ti catalyst. Attempts to synthesize unsaturated and benzannulated NHC analogues based on Hf invariably led, as in earlier work with Zr, to a mixture of products that include zwitterionic and homoleptic complexes. However, the benzannulated NHC-Hf complexes were obtained as the major products, allowing for isolation. Although these complexes selectively form polycarbonate, their catalytic performance is inferior to that of analogues based on saturated NHC.

doi:10.1039/d0ob01745k

Abstract

A stereodivergent strategy has been devised to access the diene motif found in biologically active compounds from the Piperaceae family. Herein the first total syntheses of 2E,4E configured piperchabamide E (2) and its enantiomer (ent-2), as well as 2E,4Z configured scutifoliamide B (3), are narrated. The mainstay in the adopted approach is the gram-scale conversion of quaternized pyridine in a practical three-step sequence to access isomerically pure conjugated bromodiene esters 2E,4E8 and 2E,4Z9 by differential crystallization. Even though the developed oxidation protocol forms the basis of the entailed divergent strategy, the geometrical integrity of the involved bromodiene motive can be controlled by the choice of solvent. Thus, while oxidation of pure bromodienal 2E,4Z7 in methanol yields equal amounts of bromodiene esters 2E,4E8 and 2E,4Z9, only bromodiene ester 2E,4Z10 is formed in isopropanol. Subseqently, capitalizing on a stereoretentive Suzuki cross-coupling and direct amidation of the corresponding esters, the featured natural products can be accessed in five and six steps, respectively. The somewhat surprising (R)-configured amine portion, which has been assigned to piperchabamide E (2), is facilitated by a Curtius rearrangement. Following this, the actual amine portion is shown to be (S)-configured.

doi:10.4314/jasem.v24i8.26

Abstract

Malaria is a vector borne disease responsible for high morbidity, mortality and poverty in many tropical and subtropical countries. The disease is transmitted through a bite from an infected female Anopheles mosquito, amongst which Anopheles gambiae s.s serves as the most prevalent vector. The control of An. gambiae s.s population can therefore lead to a reduction in malaria spreading. Previous studies have reported the crude extracts of Kotschya thymodora to be active against the larvae of An. gambiae s.s and Culex quinquefasciatus. In this report the phytochemical study on the crude aqueous ethanolic extract of K. thymodora leaves led to isolation of vanillic acid (1) and protocatechuic acid (2). The structures of these compounds and mosquitoes larvicidal activity against An. gambiae s.s were established by using spectroscopic techniques and WHO protocol of 1996 respectively. The two phenolic acids exhibited a moderate mosquito larvicidal activity with LC50 of 77.35 μg/mL (vanillic acid, 1) and 62.4 μg/mL (protocatechuic acid, 2) after 48 hrs exposure time. This is the first report on the isolation of the two phenolic acids from plants belonging to the genus Kotschya and their larvicidal potential against An. gambiae s.s.

doi:10.1016/j.jaap.2020.104919

Abstract

Hydrothermal liquefaction (HTL) of biomass such as lignin could contribute to finding replacements for petroleum, both as a fuel and production of chemicals. The organic phase produced in formic acid assisted HTL of lignin has been extensively analyzed previously. The solid phase is routinely analyzed by elemental analysis, and the gas phase has also been studied. The aqueous phase, on the other hand, has received little attention so far and this paper aims to identify and quantify the organic compounds that remain in the aqueous phase after the workup of the organic phase. Using NMR with water suppression, this is achieved with simple sample preparation. The major components are identified using 2D NMR (HSQC spectra) together with proton spectra and 13C spectra as well as verification with standard samples. Their concentrations are determined based on 1H spectra with an added internal standard. An initial evaluation of the effect of temperature and catalyst in the formic acid assisted HTL is given to demonstrate the relevance of the approach. Methanol, formic acid, acetic acid, acetone, phenol, catechol, and dimethyl ether have been identified and quantified in aqueous samples from six different HTL-experiments. 76 %–86 % of the peak area of the proton spectra have been accounted for.

doi:10.1111/febs.15551

Abstract

Dysregulation of the biosynthesis of cholesterol and other lipids has been implicated in many neurological diseases, including Parkinson's disease. Misfolding of α‐Synuclein (α‐Syn), the main actor in Parkinson's, is associated with changes in a lipid environment. However, the exact molecular mechanisms underlying cholesterol effect on α‐Syn binding to lipids and as well as α‐Syn oligomerization and fibrillation remain elusive, as does the relative importance of cholesterol compared to other factors. We probed the interactions and fibrillation behaviour of α‐Syn using styrene‐maleic acid nanodiscs, containing zwitterionic and anionic lipid model systems with and without cholesterol. Surface plasmon resonance and thioflavin T fluorescence assays were employed to monitor α‐Syn binding, as well as fibrillation in the absence and presence of membrane models. 1H‐15N correlated NMR was used to monitor the fold of α‐Syn in response to nanodisc binding, determining individual residue apparent affinities for the nanodisc‐contained bilayers. The addition of cholesterol inhibited α‐Syn interaction with lipid bilayers, however significantly promoted α‐Syn fibrillation, with a more than a 20‐fold reduction of lag‐times before fibrillation onset. When α‐Syn‐bilayer interactions were analysed at an individual residue level by solution‐state NMR, we observed two different effects of cholesterol. In nanodiscs made of DOPC, the addition of cholesterol modulated the NAC part of α‐Syn, leading to stronger interaction of this region with the lipid bilayer. In contrast, in the nanodiscs comprising DOPC, DOPE and DOPG, the NAC part was mostly unaffected by the presence of cholesterol, while the binding of the N‐ and the C‐termini were both inhibited.

doi:10.1002/lipd.12277

Abstract

The uptake of specific fatty acids in humans is dependent on their position on the glycerol backbone. There is a great interest in methods that can access this information fast and accurately. By way of high‐resolution NMR, we have analyzed TAG extracted from pig and beef tissues and obtained quantitative data for the composition and regioisomeric distribution of all major unsaturated fatty acids usually found in these source materials, using a combination of manual integration and deconvolution of 13C NMR spectra. In addition, we have developed a method for determining composition and regioisomeric distribution of the two main saturated fatty acids found in pork (16:0, 18:0). The results are discussed in relation to species‐specific genetic characteristics of fatty acid and TAG biosynthesis. The developed method could support decisions related to breeding for desired fatty acid profiles, and stimulate further methodology developments using high field NMR.

doi:10.1002/adsc.202000909

Abstract

A general and selective Pd‐catalyzed cross‐coupling of aromatic boronic acids with vinyl‐imidazoles is disclosed. Unlike most cross‐coupling reactions, this method operates well in absence of bases avoiding the formation of by‐products. The reactivity is highly enhanced by the presence of nitrogen‐based ligands, in particular bathocuproine. The method involves MnO2 as oxidant for the oxidation Pd (0)→Pd (II), a much weaker oxidant than previously reported in the literature. This allows for the use of reactants that possess a multitude of functional groups. A scope and limitation study involving a series of 24 boronic acids, whereof 18 afforded TMs in yields in the range 41–95%. The disclosed method constitutes the first general method for the oxidative Heck cross‐coupling on the imidazole scaffold, which moreover operates with a selection of other heterocycles.

doi:10.3390/molecules25173782

Abstract

Nepeta curviflora Boiss. (Syrian catnip) is native to the Middle East. This medicinal plant is commonly used against nervous disorders, rheumatic pains, and high blood pressure. Herbal infusions prepared from various Nepeta spp. are extensively consumed as functional food. However, limited information has been known about the phenolic constituents of Syrian catnip. In this study, two acylated flavone 7-O-glucuronides, apigenin 7-O-(2″-O-(2‴-(E-caffeoyl)-β-glucuronopyranosyl)-β-glucuronopyranoside) (1) and luteolin 7-O-(2″-O-(2‴-(E-caffeoyl)-β-glucuronopyranosyl)-β-glucuronopyranoside) (2), along with the known phenolic compounds rosmarinic acid, caffeic acid, apigenin, and apigenin 7-O-β-glucopyranoside were isolated from the aerial parts of N. curviflora. The characterizations of these compounds were based on high-resolution mass spectrometry, UV, and extensive use of multidimensional NMR spectroscopy. The new compounds (1 and 2) were identified in the unmodified state and as dimethylesters.

doi:10.1038/s41598-020-69951-7

Abstract

Polysaccharides from plant biomass are the most abundant renewable chemicals on Earth and can potentially be converted to a wide variety of useful glycoconjugates. Potential applications of glycoconjugates include therapeutics and drug delivery, vaccine development and as fine chemicals. While anomeric hydroxyl groups of carbohydrates are amenable to a variety of useful chemical modifications, selective cross-coupling to non-reducing ends has remained challenging. Several lytic polysaccharide monooxygenases (LPMOs), powerful enzymes known for their application in cellulose degradation, specifically oxidize non-reducing ends, introducing carbonyl groups that can be utilized for chemical coupling. This study provides a simple and highly specific approach to produce oxime-based glycoconjugates from LPMO-functionalized oligosaccharides. The products are evaluated by HPLC, mass spectrometry and NMR. Furthermore, we demonstrate potential biodegradability of these glycoconjugates using selective enzymes.

doi:10.4155/fmc-2020-0127

Abstract

Tryptophan hydroxylase 1 (TPH1) catalyzes serotonin synthesis in peripheral tissues. Selective TPH1 inhibitors may be useful for treating disorders related to serotonin dysregulation. Results & methodology: Screening using a thermal shift assay for TPH1 binders yielded Compound 1 (2-(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one), which showed high potency (50% inhibition at 98 ± 30 nM) and selectivity for inhibiting TPH over related aromatic amino acid hydroxylases in enzyme activity assays. Structure-activity relationships studies revealed several analogs of 1 showing comparable potency. Kinetic studies suggested a noncompetitive mode of action of 1, with regards to tryptophan and tetrahydrobiopterin. Computational docking studies and live cell assays were also performed. Conclusion: This TPH1 inhibitor scaffold may be useful for developing new therapeutics for treating elevated peripheral serotonin.

doi:10.1002/ejic.202000589

Abstract

Zn complexes of Schiff base ligands derived from 2,2'‐diaminobiphenyls and salicylaldehyde derivatives were synthesized and characterized by NMR and single‐crystal X‐ray diffraction analysis. The detailed NMR studies suggest that the Zn complexes have a complicated behavior in solution, which is strongly dependent on the donating ability of the solvent, the steric properties of the ligand, as well as the concentration of the complex in the solvent. All these factors are decisive for the determination of the coordination number of the complex in solution. Furthermore, pentacoordinated Zn complexes of the aforementioned type, ligated by a series of nitrogen bases, were synthesized. NMR studies of the different complexes at different concentrations and temperatures, revealed information about their conformational stability. The differences were further examined by single‐crystal X‐ray diffraction analysis. In addition to the studies conducted on Zn complexes, comparative studies were conducted on a series of Cd complexes. Zn complexes of Schiff base ligands derived from 2,2'‐diaminobiphenyls were synthesized and studied by NMR. The complexes showed a complicated behavior in solution, strongly dependent on the nature of the solvent, the substitution pattern on the aromatic rings, and the concentration of the complexes. Similar behavior was observed in studies of the corresponding base‐ligated Zn complexes, obtained by reaction with different nitrogen bases.

doi:10.1073/pnas.2004277117

Abstract

Lytic polysaccharide monooxygenases (LPMOs) have unique catalytic centers, at which a single copper catalyzes the oxidative cleavage of a glycosidic bond. The mechanism by which LPMOs activate molecular oxygen is key to understanding copper (bio)catalysis but remains poorly understood, largely because the insoluble and heterogeneous nature of LPMO substrates precludes the use of usual laboratory techniques. Using an integrated NMR/EPR approach, we have unraveled structural and electronic details of the interactions of an LPMO from Bacillus licheniformis and β-chitin. EPR spectroscopy on uniformly isotope 15N-labeled 63Cu(II)-LPMO provided insight into substrate-driven rearrangement of the copper coordination sphere that predisposes the enzyme for O2 activation.

doi:10.1021/acs.organomet.0c00364

Abstract

A highly efficient mercury-free route, providing [C^N] cyclometalated symmetric dialkynyl gold(III) complexes with 2-(p-tolyl)pyridine (tpy) as bidentate ligands and using alkynyllithium reagents, was developed. With 1,9-decadiyne, an unprecedented representative of a di(bidentate) gold(III) complex, for the first time became accessible. By using Grignard instead of the alkynyllithium reagents, we succeeded in synthesizing a range of novel monoalkynyl gold(III) complexes in excellent yields (74–92%) and diastereoselectivity. Treating [(tpy)Au(C≡CTMS)2] with Bu4NF in THF gave a complete deprotection, and with K2CO3 in methanol, there was a selective deprotection of the TMS group trans to the pyridyl N atom, providing [(tpy)Au(C≡CH)(C≡CTMS)] as the major product. This new type of cyclometalated gold(III) complex bears two different alkynes as ancillary ligands. The further functionalization of the monoalkynyl (tpy)gold(III) complexes was investigated, offering a universal unprecedented and diastereoselective access to unsymmetrical dialkynyl (tpy)gold(III) complexes.

doi:10.1038/s41598-020-68789-3

Abstract

Bacterial alginate initially consists of 1–4-linked β-D-mannuronic acid residues (M) which can be later epimerized to α-L-guluronic acid (G). The family of AlgE mannuronan C-5-epimerases from Azotobacter vinelandii has been extensively studied, and three genes putatively encoding AlgE-type epimerases have recently been identified in the genome of Azotobacter chroococcum. The three A. chroococcum genes, here designated AcalgE1, AcalgE2 and AcalgE3, were recombinantly expressed in Escherichia coli and the gene products were partially purified. The catalytic activities of the enzymes were stimulated by the addition of calcium ions in vitro. AcAlgE1 displayed epimerase activity and was able to introduce long G-blocks in the alginate substrate, preferentially by attacking M residues next to pre-existing G residues. AcAlgE2 and AcAlgE3 were found to display lyase activities with a substrate preference toward M-alginate. AcAlgE2 solely accepted M residues in the positions − 1 and + 2 relative to the cleavage site, while AcAlgE3 could accept either M or G residues in these two positions. Both AcAlgE2 and AcAlgE3 were bifunctional and could also catalyze epimerization of M to G. Together, we demonstrate that A. chroococcum encodes three different AlgE-like alginate-modifying enzymes and the biotechnological and biological impact of these findings are discussed.

doi:10.1093/glycob/cwaa064

Abstract

Fucoidans are a diverse class of sulfated polysaccharides integral to the cell wall of brown algae, and due to their various bioactivities, they are potential drugs. Standardized work with fucoidans is required for structure–function studies, but remains challenging since available fucoidan preparations are often contaminated with other algal compounds. Additionally, fucoidans are structurally diverse depending on species and season, urging the need for standardized purification protocols. Here, we use ion-exchange chromatography to purify different fucoidans and found a high structural diversity between fucoidans. Ion-exchange chromatography efficiently removes the polysaccharides alginate and laminarin and other contaminants such as proteins and phlorotannins across a broad range of fucoidans from major brown algal orders including Ectocarpales, Laminariales and Fucales. By monomer composition, linkage analysis and NMR characterization, we identified galacturonic acid, glucuronic acid and O-acetylation as new structural features of certain fucoidans and provided a novel structure of fucoidan from Durvillaea potatorum with α-1,3-linked fucose backbone and β-1,6 and β-1,3 galactose branches. This study emphasizes the use of standardized ion-exchange chromatography to obtain defined fucoidans for subsequent molecular studies.

doi:10.1002/ejic.202000529

Abstract

A range of N,C‐chelated, cyclometalated gold(III) complexes Au(ppyR)X2 have been prepared and characterized by spectroscopic, crystallographic, and computational means. Here, ppyH is 2‐phenylpyridine dicarboxylic acid (series 1), ppyEt is diethyl 2‐phenylpyridine dicarboxylate (series 2), and X is trifluoroacetate OAcF (a), Cl (b), Br (c), or I (d) anion. The dihalo complexes 1b‐d and 2b‐d are obtained when Au(ppyR)(OAcF)2 (1a and 2a) are treated with HNO3/HX mixtures (aqua regiaX). Good to high yields are obtained with short reaction times (< 30 min) and simple work‐up. Notably, the strongly acidic medium does not cause protolytic cleavage of the Au–C or Au–N bonds in the chelate, nor is ester hydrolysis of complexes 2b‐d seen. Ethylene inserts into an Au–O bond of 1a and 1b, and the resulting trifluoroacetoxyethyl–Au complexes can be further elaborated in aqua regia without cleavage of the two Au–C bonds in the molecule. Facile, mutual halide exchange reactions between complexes with different halides (1b and 1d, 1c and 1d) were observed and led to formation of mixed‐halide complexes Au(ppyH)(X)(Y). These exchange reactions occurred with complete stereoselectivity. The stereoisomer produced was the one expected based on the relative trans influence of the halides (I > Br > Cl), i.e. the highest trans influence halide was located trans to N which is the lowest trans influence end of the chelate. These thermodynamic preferences were also investigated by DFT computations. Synthesis and characterization of N,C‐chelated, cyclometalated gold(III) complexes prepared by reaction of cyclometalated gold(III) bistrifluoroacetates with aqua regia solutions composed of mixtures of HNO3 and HCl, HBr, or HI. Mutual halide exchange reactions between complexes with different halides led to formation of mixed‐halide complexes Au(N^C)(X)(Y).

doi:10.1016/j.hal.2020.101853

Abstract

Ciguatera fish poisoning (CFP) is prevalent around the tropical and sub-tropical latitudes of the world and impacts many Pacific island communities intrinsically linked to the reef system for sustenance and trade. While the genus Gambierdiscus has been linked with CFP, it is commonly found on tropical reef systems in microalgal assemblages with other genera of toxin-producing, epiphytic and/or benthic dinoflagellates – Amphidinium, Coolia, Fukuyoa, Ostreopsis and Prorocentrum. Identifying a biomarker compound that can be used for the early detection of Gambierdiscus blooms, specifically in a mixed microalgal community, is paramount in enabling the development of management and mitigation strategies. Following on from the recent structural elucidation of 44-methylgambierone, its potential to contribute to CFP intoxication events and applicability as a biomarker compound for Gambierdiscus spp. was investigated. The acute toxicity of this secondary metabolite was determined by intraperitoneal injection using mice, which showed it to be of low toxicity, with an LD50 between 20 and 38 mg kg−1. The production of 44-methylgambierone by 252 marine microalgal isolates consisting of 90 species from 32 genera across seven classes, was assessed by liquid chromatography-tandem mass spectrometry. It was discovered that the production of this secondary metabolite was ubiquitous to the eight Gambierdiscus species tested, however not all isolates of G. carpenteri, and some species/isolates of Coolia and Fukuyoa.

doi:10.1021/acs.biomac.0c00620

Abstract

Diblock oligosaccharides based on renewable resources allow for a range of new but, so far, little explored biomaterials. Coupling of blocks through their reducing ends ensures retention of many of their intrinsic properties that otherwise are perturbed in classical lateral modifications. Chitin is an abundant, biodegradable, bioactive, and self-assembling polysaccharide. However, most coupling protocols relevant for chitin blocks have shortcomings. Here we exploit the highly reactive 2,5-anhydro-d-mannose residue at the reducing end of chitin oligomers obtained by nitrous acid depolymerization. Subsequent activation by dihydrazides or dioxyamines provides precursors for chitin-based diblock oligosaccharides. These reactions are much faster than for other carbohydrates, and only acyclic imines (hydrazones or oximes) are formed (no cyclic N-glycosides). α-Picoline borane and cyanoborohydride are effective reductants of imines, but in contrast to most other carbohydrates, they are not selective for the imines in the present case. This could be circumvented by a simple two-step procedure. Attachment of a second block to hydrazide- or aminooxy-functionalized chitin oligomers turned out to be even faster than the attachment of the first block. The study provides simple protocols for the preparation of chitin-b-chitin and chitin-b-dextran diblock oligosaccharides without involving protection/deprotection strategies.

doi:10.1016/j.mcat.2020.111009

Abstract

Cyclometalated gold(III) complexes Au(L)(OAcF)2 (L = phenylpyridine dicarboxylic diester (ppyde) or phenylpyridine dicarboxylic acid (ppydc)) have been prepared reacting Au(OAc)3 with corresponding phenyl pyridines (ppyde or ppydc) in trifluoroacetic acid (HOAcF) under microwave heating. Further treatment of Au(L)(OAcF)2 with aqua regia resulted in dichloro complexes Au(L)Cl2. Au-functionalized UiO-67 MOF has been synthesized by exchanging linkers of UiO-67 with Au(ppydc)Cl2, furnishing the MOF with (N^C)-cyclometalated Au(III) centers. The catalytic activities of the molecular cyclometalated complexes and the Au-incorporated MOF were studied in gold-catalyzed propargyl ester cyclopropanations. Almost all complexes and the MOF showed catalytic activity to the cyclopropanation product (up to 97% conversion), with a preference for the trans diastereoisomer (up to 14:86 d.r.). The recyclability of the most active molecular complex has also been investigated.

doi:10.1002/ejoc.202000599

Abstract

2‐Nitrophenylboronic acids serve as interesting starting materials for the construction of biphenyl‐ and terphenyl‐based amines if subjected to the Suzuki–Miyaura reaction. Unfortunately, these boronic acids suffer from low reactivity in Suzuki reactions, alongside their low stability in the presence of Pd. Herein, a general method for the construction of 2‐nitro‐substituted bi‐ and terphenyls is presented, with special emphasis on the synthesis of 2‐amino‐2'‐nitrobi‐ and terphenyls. Comparisons are made with other boronic acids that have some of the aforementioned issues. Finally, the application of the obtained 2‐amino‐2'‐nitrobi‐ and terphenyls as starting materials for the synthesis of bi‐ and terphenyl based di‐ and triamines is encountered for, with emphasis on the use of these amines as precursors for Schiff base ligands. In addition, the synthesis of some Zn complexes of these ligands is presented.

doi:10.1016/j.jff.2020.104025

Abstract

The fungus Inonotus obliquus has historically been used in traditional medicine in Europe and Asia. A melanin fraction and six triterpenoids were obtained from I. obliquus sclerotia, and evaluated in various bioassays including for immunomodulatory, cytotoxicity and enzyme-interacting properties. The water-soluble, nitrogen-free melanin fraction and the triterpenoids 3β-hydroxy-8,24-dien-21-al (1) and inotodiol (2) displayed potent activity in a human complement assay. The melanin fraction inhibited the complement cascade, whereas 1 and 2 activated the same cascade. Compound 2, as well as betulinic acid (3) and betulin (4) had anti-proliferative properties against the colon adenocarcinoma cell line HT29-MTX. Further, the melanin fraction and betulin-3-O-caffeate (6) reduced nitric oxide production in primary murine macrophages. Furthermore, the metabolites were nontoxic against the common gut bacteria E. coli and B. subtilis. The results demonstrate the anti-inflammatory and immunomodulatory effects of I. obliquus melanin and triterpenoids, which could potentially justify the consumption of this increasingly popular “edible” fungus.

doi:10.1021/acscatal.0c02206

Abstract

Ethylene is known to readily decompose ruthenium-based olefin metathesis catalysts, such as Grubbs second-generation catalyst (GII), by forming the unsubstituted ruthenacyclobutane (Ru-2) that may undergo a 1,2-H shift and liberate propene. The resulting alkylidene loss has been assumed to be irreversible. Yet, by reacting (SIMes)(η6-p-cymene)RuCl2 (1), the p-cymene-stabilized alkylidene-free fragment resulting from loss of propene from Ru-2, with ethylene, we show that the methylidene analogue of GII (GIIm) and other Ru alkylidenes are formed, along with catalytic amounts of propene and butenes, and can be stabilized by tricyclohexylphosphine (PCy3) at 50 °C in C6D6. An almost 20-fold increase in activity for ring-closing metathesis of diethyl diallylmalonate (DEDAM) on pretreatment of 1 with ethylene suggests that the reversibility of the alkylidene loss may be used to develop longer-lived metathesis catalysts and processes. Mechanistic density functional theory (DFT) calculations suggest that the connection between 1 and GIIm involves oxidative coupling of two ethylene molecules to form a key metallacyclopentane intermediate (M49). A 1,2-H shift in M49 gives the methyl-substituted ruthenacyclobutane M303, which, on cycloreversion, liberates propene and GIIm. Alternatively, successive H-shifts starting in M49 may give 1-butene (fast reaction) and 2-butene (slower) with a lower barrier than that of Ru alkylidene. The lower predicted barrier is consistent with butene, especially 1-butene, being the dominating product at the start of the experiments, in particular at lower temperature.

doi:10.3390/molecules25102349

Abstract

Direct C-H arylation coupling is potentially a more economical and sustainable process than conventional cross-coupling. However, this method has found limited application in the synthesis of organic dyes for dye-sensitized solar cells. Although direct C-H arylation is not an universal solution to any cross-coupling reactions, it efficiently complements conventional sp2−sp2 bond formation and can provide shorter and more efficient routes to diketopyrrolopyrrole dyes. Here, we have applied palladium catalyzed direct C-H arylation in the synthesis of five new 3,6-dithienyl diketopyrrolopyrrole dyes. All prepared sensitizers display broad absorption from 350 nm up to 800 nm with high molar extinction coefficients. The dye-sensitized solar cells based on these dyes exhibit a power conversion efficiency in the range of 2.9 to 3.4%.

doi:10.1016/j.ijbiomac.2020.05.083

Abstract

In this study, two pectic polysaccharides from stems of Codonopsis pilosula (CPSP-1) and C. tangshen (CTSP-1) were obtained by ion exchange chromatography and gel filtration. The molecular weight of CPSP-1 and CTSP-1 were 13.1 and 23.0 kDa, respectively. The results of structure elucidation indicated that both CPSP-1 and CTSP-1 are pectic polysaccharides with long homogalacturonan regions (HG) (some of galacturonic acid units were methyl esterified) and rhamnogalacturonan I (RG-I) regions. Side chains for CTSP-1 are both arabinogalactan type I (AG-I) and type II (AG-II), while CPSP-1 only has AG-II. The biological test demonstrated that CPSP-1 and CTSP-1 displayed an antioxidant property through mediating the intestinal cellular antioxidant defense system, which could protect cultured intestinal cells from oxidative stress induced oxidative damages and cell viability suppression. CPSP-1 and CTSP-I showed different bioactivities and mechanisms, which may be due to the difference in their structures.

doi:10.1016/j.seppur.2020.116959

Abstract

A series of 36 dual functionalized N-(poly)ethylen glycol N-allyl-/N-benzyl imidazolium and benzimidazolium ILs were synthesized for extractive desulfurization studies (EDS). The novel ILs were readily prepared from imidazole by a three-step dialkylation and anion exchange procedure and characterised (1H, 13C, 19F NMR; IR; HRMS positive/negative mode). The desulfurization studies were performed on a model oil with dibenzothiophene (DBT) and 4,6-dimethyldibenzothiophene (4,6-DMDBT) in n-dodecane. The N-allyl benzimidazolium based ILs exhibited higher extraction efficiency than corresponding imidazolium-based ILs (69 vs 59% DBT; 52 vs 29% 4,6-DMDBT). The EDS efficiency of the prepared ILs varied with the N-ethylen glycol chains length and by N-allyl or N-benzyl substitution on the imidazolium ILs. The results indicated that desulfurization efficiency is mainly affected by variation of the IL cation, as the IL anions (NTf2; N(CN)2-, SCN-, BF4-) only had minor impact on the extraction capacity of sulfur compounds. 1H NMR studies of IL-DBT solutions illustrated different EDS mechanisms, and specific interactions could explain the higher desulfurization capacity of dual functionalized benzimidazolium than the corresponding imidazolium ILs. 1H NMR confirmed that stronger π-π interactions between the planar aromatic electron system of benzimidazolium ILs and aromatic dibenzothiophenes play a major role for the high EDS extraction efficiency.

doi:10.1021/acs.orglett.0c00642

Abstract

A general Cu-catalyzed, regioselective method for the N-3-arylation of hydantoins is described. The protocol utilizes aryl(trimethoxyphenyl)iodonium tosylate as the arylating agent in the presence of triethylamine and a catalytic amount of a simple Cu-salt. The method is compatible with structurally diverse hydantoins and operates well with neutral aryl groups or aryl groups bearing weakly donating/withdrawing elements. It is also applicable for the rapid diversification of pharmaceutically relevant hydantoins. A general Cu-catalyzed, regioselective method for the N-3-arylation of hydantoins is described. The protocol utilizes aryl(trimethoxyphenyl)iodonium tosylate as the arylating agent in the presence of triethylamine and a catalytic amount of a simple Cu-salt. The method is compatible with structurally diverse hydantoins and operates well with neutral aryl groups or aryl groups bearing weakly donating/withdrawing elements. It is also applicable for the rapid diversification of pharmaceutically relevant hydantoins.

doi:10.1073/pnas.1915376117

Abstract

Acetylation is an important feature of hemicellulose, altering the physical properties of the plant cell wall and limiting enzyme accessibility. Removal of acetyl groups from β-mannan is a key step toward efficient utilization of this glycan as a carbon source for gut microbiota and in biorefineries. We present detailed insight into mannan deacetylation by two highly substrate-specific acetyl-mannan esterases (AcMEs) from a prevalent gut commensal Firmicute, which cooperatively deacetylate complex galactoglucomannan. The three-dimensional structure of RiCE17 with mannopentaose in the active site has a unique two-domain architecture including a CBM35 and a SGNH superfamily hydrolytic domain. Discovery of β-mannan-specific esterases improves the understanding of an important step in dietary fiber utilization by gut commensal Firmicutes.β-mannans and xylans are important components of the plant cell wall and they are acetylated to be protected from degradation by glycoside hydrolases. β-mannans are widely present in human and animal diets as fiber from leguminous plants and as thickeners and stabilizers in processed foods. There are many fully characterized acetylxylan esterases (AcXEs); however, the enzymes deacetylating mannans are less understood. Here we present two carbohydrate esterases, RiCE2 and RiCE17, from the Firmicute Roseburia intestinalis, which together deacetylate complex galactoglucomannan (GGM). The three-dimensional (3D) structure of RiCE17 with a mannopentaose in the active site shows that the CBM35 domain of RiCE17 forms a confined complex, where the axially oriented C2-hydroxyl of a mannose residue points toward the Ser41 of the catalytic triad. Cavities on the RiCE17 surface may accept galactosylations at the C6 positions of mannose adjacent to the mannose residue being deacetylated (subsite -1 and +1). In-depth characterization of the two enzymes using time-resolved NMR, high-performance liquid chromatography (HPLC), and mass spectrometry demonstrates that they work in a complementary manner. RiCE17 exclusively removes the axially oriented 2-O-acetylations on any mannose residue in an oligosaccharide, including double acetylated mannoses, while the RiCE2 is active on 3-O-, 4-O-, and 6-O-acetylations. Activity of RiCE2 is dependent on RiCE17 removing 2-O-acetylations from double acetylated mannose. Furthermore, transacetylation of oligosaccharides with the 2-O-specific RiCE17 provided insight into how temperature and pH affects acetyl migration on manno-oligosaccharides.

doi:10.1002/ejoc.202000202

Abstract

(–)-Agelasine F (also known as ageline A) is a diterpene-adenine hybrid natural product isolated from marine sponges (Agelas species) and this compound is known to display cytotoxic activity against a variety of cancer cell lines as well as microorganisms. We herein report the first total synthesis of (–)-agelasine F. The commercially available and inexpensive monoterpenoid (S)-carvone was found to be a highly suitable starting material for the construction of the terpenoid part of the desired agelasine and controlling the stereochemistry of the target compound. Two alternative strategies from (S)-carvone were evaluated. Key-intermediates in the (–)-agelasine F synthesis are believed also to be valuable starting materials for total syntheses of other bioactive marine sponge metabolites. The synthetic route to (–)-agelasine F described herein is more efficient than previously published syntheses of racemic or ent-agelasine F.

doi:10.1021/acs.jafc.9b07828

Abstract

Nuclear magnetic resonance (NMR) metabolomics profiling was evaluated as a new tool in sensory assessment of protein hydrolysates. Hydrolysates were produced on the basis of different raw materials (cod, salmon, and chicken), enzymes (Food Pro PNL and Bromelain), and hydrolysis time (10 and 50 min). The influence of raw material and hydrolysis parameters on sensory attributes was determined by traditional descriptive sensory analysis and 1H NMR spectroscopy. The raw material had a major influence on the attribute intensity and metabolite variation, followed by enzyme and hydrolysis time. However, the formation of bitter taste was not affected by the raw material. Partial least-squares regression (PLSR) on 1H NMR and sensory data provided good models (Q2 = 0.55–0.89) for 11 of the 17 evaluated attributes, including bitterness. Significant metabolite–attribute associations were identified. The study confirms the potential prediction of the sensory properties of protein hydrolysates from cod, salmon, and chicken based on 1H NMR metabolomics profiling.

doi:10.1021/acsomega.9b04490

Abstract

Alginate is a linear copolymer composed of 1→4 linked β-d-mannuronic acid (M) and its epimer α-l-guluronic acid (G). The polysaccharide is first produced as homopolymeric mannuronan and subsequently, at the polymer level, C-5 epimerases convert M residues to G residues. The bacterium Azotobacter vinelandii encodes a family of seven secreted and calcium ion-dependent mannuronan C-5 epimerases (AlgE1–AlgE7). These epimerases consist of two types of structural modules: the A-modules, which contain the catalytic site, and the R-modules, which influence activity through substrate and calcium binding. In this study, we rationally designed new hybrid mannuronan C-5 epimerases constituting the A-module from AlgE6 and the R-module from AlgE4. This led to a better understanding of the molecular mechanism determining differences in MG- and GG-block-forming properties of the enzymes. A long loop with either tyrosine or phenylalanine extruding from the β-helix of the enzyme proved essential in defining the final alginate block structure, probably by affecting substrate binding. Normal mode analysis of the A-module from AlgE6 supports the results.

doi:10.1111/febs.15256

Abstract

Abstract Chromatin post-translational modifications are thought to be important for epigenetic effects on gene expression. Methylation of histone N-terminal tail lysine residues constitutes one of many such modifications, executed by families of Histone Lysine Methyltransferase (HKMTase). One such protein is ASHH2 from the flowering plant Arabidopsis thaliana, equipped with the interaction domain CW and the HKMTase domain, SET. The CW domain of ASHH2 is a selective binder of monomethylation at Lysine 4 on histone H3 (H3K4me1) and likely helps the enzyme dock correctly onto chromatin sites. The study of CW and related interaction domains have so far been emphasizing lock-key models, missing important aspects of histone-tail CW interactions. We here present an analysis of the ASHH2 CW-H3K4me1 complex using NMR and molecular dynamics, as well as mutation and affinity studies of flexible coils. β-augmentation and rearrangement of coils coincide with changes in the flexibility of the complex, in particular the η1, η3 and C-terminal coils, but also in the β1 and β2 strands and the C-terminal part of the ligand. Furthermore, we show that mutating residues with outlier dynamic behaviour affects the complex binding affinity despite these not being in direct contact with the ligand. Overall, the binding process is consistent with conformational selection. We propose that this binding mechanism presents an advantage when searching for the correct PTM state among the highly modified and flexible histone tails, and also that the binding shifts the catalytic SET domain towards the nucleosome.

doi:10.1002/ejoc.202000139

Abstract

Studies on gold(III) coordination of a series of prepared polydentate pyridine and quinoline based ligands are reported. Characterization (1H, 13C, 15N NMR, and XRD) of the novel gold(III) complexes, prepared in 31–98 % yield, revealed different coordination ability of the pyridine and quinoline nitrogen atoms. Testing of catalytic activity in cyclopropanation of propargyl ester and styrene demonstrated that all the new ligated gold(III) complexes were catalytically active and outperformed KAuCl4. The superior activity of the particular Au(III)‐pyridine‐oxazole complexes may indicate de‐coordination of the pyridine‐N ligand as a crucial step for efficient generation of catalytic activity.

doi:10.1039/c9dt04472h

Abstract

The synthesis and characterization of a series of N-heterocyclic carbene (NHC) complexes of Au(III), (NHC)AuCl3, is described. High yields are obtained when the corresponding Au(I) species (NHC)AuCl are oxidized with inexpensive aqua regia. The oxidation is in some cases accompanied by substitution and/or anti addition of Cl2 across the backbone C[double bond, length as m-dash]C bond of unsaturated NHC ligands.

doi:10.1002/solr.201900569

Abstract

Chenodeoxycholic acid (CDCA) is the most used antiaggregation additive in dye-sensitized solar cells since its introduction to the field in 1993. However, effective suppression of dye aggregation comes at the cost of reduced dye loading, a lower open-circuit voltage, and limited control of dye/additive distribution when cosensitizing with free CDCA. To combat this, herein, a novel dye design concept that uses the covalent attachment of a CDCA moiety to triarylamine sensitizers is reported. The CDCA substituents do not affect the photophysical or electrochemical properties of the sensitizers but have a positive effect on the photovoltaic performance with [Cu+/2+(tmby)2](TFSI)1/2 electrolyte (tmby = 4,4′,6,6′-tetramethyl-2,2′-bipyridine, TFSI = bis(trifluoromethanesulfonyl)imide). By ensuring a one-to-one ratio of dye and CDCA, paired with isotropic distributions of each component, this approach results in a higher-quality dye monolayer. Compared with the reference system, the novel approach reported herein gives a higher open-circuit voltage and power conversion efficiency (PCE). The best device is fabricated with the dye C6–CDCA, delivering a PCE of 6.84\% (8 μm TiO2, 1 mm CDCA, JSC = 8.64 mA cm−2, VOC = 1007 mV, and FF = 0.77).

doi:10.1021/acs.chemrestox.9b00385

Abstract

Deoxynivalenol (DON) is a trichothecene mycotoxin that is produced by several species of Fusarium, which may infect grain crops. DON, as well as other type-B trichothecenes, contain an alpha,beta-unsaturated carbonyl group that may react with sulfhydryl groups in, for example, amino acids and peptides. Such conjugates have been shown to occur in plants. Nucleophilic addition of thiols to the conjugated double bond in DON afforded several isomeric reaction products, and the thermodynamically favored isomers of DON-10-cysteine and DON-10-glutathione have been prepared and characterized previously. This study reports the preparation and characterization of the kinetically favored DON-10-cysteine isomer. We subsequently studied and compared the rate of the deconjugation reaction of the two DON-10-cysteine isomers and the thermodynamically favored DON-10-glutathione adduct. The deconjugation rate of the thermodynamically favored thiol conjugates was slow with half-lives of weeks even at pH 10.7, while the kinetically favored DON-10-cysteine isomer deconjugated within a few hours, affording free DON. We adapted a simple and rapid oxidation protocol in which the sulfide linkage was oxidized to a sulfoxide or sulfone that, when treated with the base, rapidly eliminated the adducted thiol as its sulfenate or sulfinate to afford free DON. The deconjugation reactions of the sulfoxides and sulfones of thermodynamically favored DON-10-thiols were complete within hours or minutes at pH 10.7, respectively. The increase in deconjugation rates for the kinetically favored DON-10-cysteine were less dramatic. Oxidation of sulfides to sulfoxides is known to occur in vivo, and thus, our data show that thiol-conjugated DON might become bioavailable via sulfide oxidation followed by elimination to regenerate DON. The oxidation-elimination approach could also be useful for the indirect quantification of DON-10-thiol conjugates in plant and animal tissues.

doi:10.1016/j.carbpol.2019.115748

Abstract

Highlights • Chitooligosaccharides of the type DnXA were obtained enzymatically. • Activation with a dioxyamine or a dihydrazide was studied in detail. • Major differences in kinetics, yields and distribution of reaction products. • One-pot reductive amination protocols with α-picoline borane were developed. • Comparison to dextran demonstrated major differences in conjugation kinetics. Reducing end activation of poly- and oligosaccharides by bifunctional dioxyamines and dihydrazides enables aniline-free and cyanoborohydride-free conjugation to aldehyde-containing molecules, particles and surfaces without compromising the chain structure. Chitosans are due to their polycationic character, biodegradability, and bioactivity important candidates for conjugation. Here, we present a kinetic and structural study of the conjugation of a dioxyamine and a dihydrazide to enzymatically produced chitooligosaccharides ranging from N,N’-diacetylchitobiose to a decamer, all having N-acetyl d-glucosamine at the reducing end. Conjugation of the dioxyamine resulted in mixtures of (E)- and (Z)-oximes and β-N-pyranoside, whereas the dihydrazide yielded cyclic N-glycosides. Reaction kinetics was essentially independent of DP. Stable secondary amines were in both cases obtained by reduction with α-picoline borane, but higher temperatures were needed to obtain acceptable reduction rate. Comparison to dextran oligomers shows that the nature of the reducing end strongly influences the kinetics of both the conjugation and reduction.

doi:10.1016/j.celrep.2019.11.023

Abstract

The translation initiation repressor 4E-BP2 is deamidated in the brain on asparagines N99/N102 during early postnatal brain development. This post-translational modification enhances 4E-BP2 association with Raptor, a central component of mTORC1 and alters the kinetics of excitatory synaptic transmission. We show that 4E-BP2 deamidation is neuron specific, occurs in the human brain, and changes 4E-BP2 subcellular localization, but not its disordered structure state. We demonstrate that deamidated 4E-BP2 is ubiquitinated more and degrades faster than the unmodified protein. We find that enhanced deamidated 4E-BP2 degradation is dependent on Raptor binding, concomitant with increased association with a Raptor-CUL4B E3 ubiquitin ligase complex. Deamidated 4E-BP2 stability is promoted by inhibiting mTORC1 or glutamate receptors. We further demonstrate that deamidated 4E-BP2 regulates the translation of a distinct pool of mRNAs linked to cerebral development, mitochondria, and NF-κB activity, and thus may be crucial for postnatal brain development in neurodevelopmental disorders, such as ASD.

doi:10.1021/acsomega.9b03043

Abstract

Carbohydrate-binding modules (CBM) play important roles in targeting and increasing the concentration of carbohydrate active enzymes on their substrates. Using NMR to get the solution structure of CBM14, we can gain insight into secondary structure elements and intramolecular interactions with our assigned nuclear overhauser effect peaks. This reveals that two conserved aromatic residues (Phe437 and Phe456) make up the hydrophobic core of the CBM. These residues are also responsible for connecting the two β-sheets together, by being part of β2 and β4, respectively, and together with disulfide bridges, they create CBM14’s characteristic “hevein-like” fold. Most CBMs rely on aromatic residues for substrate binding; however, CBM14 contains just a single tryptophan (Trp465) that together with Asn466 enables substrate binding. Interestingly, an alanine mutation of a single residue (Leu454) located behind Trp465 renders the CBM incapable of binding. Fluorescence spectroscopy performed on this mutant reveals a significant blue shift, as well as a minor blue shift for its neighbor Val455. The reduction in steric hindrance causes the tryptophan to be buried into the hydrophobic core of the structure and therefore suggests a preorganized binding site for this CBM. Our results show that both Trp465 and Asn466 are affected when CBM14 interacts with both (GlcNAc)3 and β-chitin, that the binding interactions are weak, and that CBM14 displays a slightly higher affinity toward β-chitin.

doi:10.3390/molecules24244485

Abstract

Phenothiazines are one of the more common dye scaffolds for dye-sensitized solar cells. However, these sensitizers are exclusively based on a 3,7-substitution pattern. Herein, we have synthesized and characterized novel 3,8-substituted phenothiazine dyes in order to evaluate the effect of auxiliary donor groups on the performance of this new dye class. The power conversion efficiency increased by 7%–10% upon insertion of an auxiliary donor in position 8 of the phenothiazine, but the structure of the auxiliary donor (phenyl, naphthyl, pyrene) had a low impact when electrodes were stained with chenodeoxycholic acid (CDCA) additive. In the absence of CDCA, the highest power conversion efficiency was seen for the phenyl-based sensitizer attributed to a higher quality dye-monolayer. By comparing the novel dyes to their previously reported 3,7- analogues, only subtle differences were seen in photophysical, electrochemical, and performance measurements. The most notable difference between the two geometries is a lowering of the oxidation potentials of the 3,8-dyes by 40–50 mV compared to the 3,7-analogues. The best auxiliary donor for the 3,8-phenothiazine dyes was found to be pyrenyl, with the best device delivering a power conversion efficiency of 6.23% (99 mW cm−2, 10 eq. CDCA, JSC = 10.20 mA cm−2, VOC = 791 mV, and FF = 0.765).

doi:10.1016/j.rechem.2019.100019

Abstract

The lignin to liquid (LtL) solvolysis is a type of hydrothermal liquefaction (HTL) that utilizes formic acid to facilitate the desired hydrodeoxygenation process to depolymerize lignin. Formic acid has been chosen due to its hydrogen donor ability, and it has proven to be a more efficient hydrogen donor than hydrogen gas and isopropanol. Based on previous experiments where the amount of carbon in the solid phase and organic phase together exceeded 100% relative to carbon in the lignin starting material, we set up experiments with 13C-labeled formic acid in order to investigate where the carbon form formic acid is incorporated. All the experiments were performed in duplicates, one with 13C-labeled formic acid and one with standard formic acid. The bio-oils from the experiments were analyzed by Nuclear Magnetic Resonance (NMR) and the spectra were compared. The 13C NMR spectra showed significant differences between oils made with standard formic acid and oils made with 13C-labeled formic acid, located in the section between δ 170 ppm and δ 180 ppm in the NMR spectra. The signal in this range arise from carbonyl bonds and shows that the carbon from formic acid ends up in the carbonyl structures of the bio-oil in compounds such as aromatic acids. This paper has also shown that the amount of formic acid plays a minor role in product composition compared to reaction temperature, which influences the product composition on a functional group level.

doi:10.1002/ejoc.201901623

Abstract

Synthesis, characterisation and catalytic activity of a series of novel σ,π‐dual gold(I) acetylide complexes are presented. σ,π‐Dual gold(I) complexes based on the JohnPhos ligand or the bridging chiral MeO‐BIPHEP ligand were generated from terminal alkynes in the presence of an organic base. The catalytic activity of the complexes was explored in a range of gold(I)‐catalysed reactions of propargylic alcohol derivatives and their catalytic and enantioselective potentials were compared to corresponding monogold(I) phosphane and chiral digold(I) diphosphane species.

doi:10.1016/j.ymthe.2019.11.010

Abstract

Mutations in hydroxymethylbilane synthase (HMBS) cause acute intermittent porphyria (AIP), an autosomal dominant disease where typically only one HMBS allele is mutated. In AIP, the accumulation of porphyrin precursors triggers life-threatening neurovisceral attacks and at long-term, entails an increased risk of hepatocellular carcinoma, kidney failure, and hypertension. Today, the only cure is liver transplantation, and a need for effective mechanism-based therapies, such as pharmacological chaperones, is prevailing. These are small molecules that specifically stabilize a target protein. They may be developed into an oral treatment, which could work curatively during acute attacks, but also prophylactically in asymptomatic HMBS mutant carriers. With the use of a 10,000 compound library, we identified four binders that further increased the initially very high thermal stability of wild-type HMBS and protected the enzyme from trypsin digestion. The best hit and a selected analog increased steady-state levels and total HMBS activity in human hepatoma cells overexpressing HMBS, and in an Hmbs-deficient mouse model with a low-expressed wild-type-like allele, compared to untreated controls. Moreover, the concentration of porphyrin precursors decreased in liver of mice treated with the best hit. Our findings demonstrate the great potential of these hits for the development of a pharmacological chaperone-based corrective treatment of AIP by enhancing wild-type HMBS function independently of the patients’ specific mutation.

doi:10.3390/antiox8120612

Abstract

In this case study, we explored quantitative 1H NMR (qNMR), HPLC-DAD, and the Folin-Ciocalteu assay (TPC) as methods of quantifying the total phenolic content of a green macroalga, Ulva intestinalis, after optimized accelerated solvent extraction. Tentative qualitative data was also acquired after multiple steps of purification. The observed polyphenolic profile was complex with low individual concentrations. The qNMR method yielded 5.5% (DW) polyphenols in the crude extract, whereas HPLC-DAD and TPC assay yielded 1.1% (DW) and 0.4% (DW) respectively, using gallic acid as the reference in all methods. Based on the LC-MS observations of extracts and fractions, an average molar mass of 330 g/mol and an average of 4 aromatic hydrogens in each spin system was chosen for optimized qNMR calculations. Compared to the parallel numbers using gallic acid as the standard (170 g/mol, 2 aromatic H), the optimized parameters resulted in a similar qNMR result (5.3%, DW). The different results for the different methods highlight the difficulties with total polyphenolic quantification. All of the methods contain assumptions and uncertainties, and for complex samples with lower concentrations, this will be of special importance. Thus, further optimization of the extraction, identification, and quantification of polyphenols in marine algae must be researched.

doi:10.1016/j.jmr.2019.106663

Abstract

The wettability of a reservoir rock is among the most important factors influencing oil recovery. This study presents how the application of a modified CPMG pulse sequence can determine the wettability of porous samples containing oil and water in the same pore network. This method can be applied as an average of the signal over the entire sample as well as with spatial resolution using slice selection. Correlations between dephasing in internal gradients () and the spectral linewidth () were used to calculate a fluid specific dimension of confinement (DOC) distribution for oil and water separately during forced displacement experiments performed inside the bore of an NMR spectrometer. Two samples, one water-wet and one oil-wet, were investigated. Data averaged over the entire sample show oil and water distribution at endpoint saturations ( and ) while spatially resolved data show which pores are initially occupied by the displacing fluid as well as the displacing efficiency. Both the data averaged over the entire sample and the spatially resolved data can, by itself, indicate wettability. As opposed to relaxation times as an indirect indication of wettability, the DOC distributions provide a direct fluid dimension, which more carefully describes the distribution of oil and water in the pore network.

doi:10.1016/j.ijbiomac.2019.10.034

Abstract

Highlights • Both nanomaterials induced cell proliferation at short term in PHBV films. • PHB/GO exhibits higher proliferative activity along time than PHBV/CNFs. • PHBV/GO exhibits higher enhancement of cell adhesion than PHBV/CNFs. • GO provides higher antibacterial activity than CNFs to PHBV films. • Similar enhancement of physical properties is provided by both nanomaterials. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a microbial biodegradable polymer with a wide range of potential industrial applications. However, its biomedical uses could increase exponentially if certain physical and biological properties were enhanced without compromising on the non-cytotoxic property of this biocompatible polymer. Graphene oxide (GO) nanosheets and carbon nanofibers (CNFs) have proven to be very promising reinforcing agents for the development of new composite materials. Therefore, PHBV films were prepared with 1% w/w of GO nanosheets or CNFs with the aim of enhancing their compression performance, thermal behaviour, wettability and cell adhesion using canine adipose-derived mesenchymal stem cells, and antibacterial activity against the model bacterium Staphylococcus aureus. The results of this study showed that both nanomaterials produced similar enhancements of the physical properties. However, PHBV/GO exhibited higher proliferative activity against time, cell adhesion and antibacterial activity than that of PHBV/CNFs. Nonetheless, both PHBV/GO and PHBV/CNFs composite films have shown considerable promise for biomedical applications.

doi:10.1002/anie.201912315

Abstract

A highly asymmetric AuIII η3‐allyl complex has been generated by treating Au(η1‐allyl)Br(tpy) (tpy=2‐(p‐tolyl)pyridine) with AgNTf2. The resulting η3‐allyl complex has been characterized by NMR spectroscopy and X‐ray crystallography. DFT calculations and variable temperature 1H NMR suggest that the allyl ligand is highly fluxional.

doi:10.1016/j.carbpol.2019.115594

Abstract

Highlights • High concentration of cellulose was dissolved in alkaline ionic liquids. • Ionic liquids with unconventional anions were synthesized. • Addition of polar solvent increased cellulose dissolution capacity of Ionic Liquids. • No functionalization of cellulose after dissolution in ionic liquids. • Energy efficient method for cellulose dissolution was developed. Alkaline ionic liquids (ILs) with unconventional organic anions were prepared and used for cellulose dissolution studies. High concentrations of cellulose were dissolved at room temperature in the phenolate based imidazolium IL [C2mim][OPh], combined with organic solvent, and up to 45 wt-% cellulose dissolution (wt-% MCC of weight IL) was readily achieved at 100 ºC. No functionalization of the regenerated cellulose was observed during the dissolution process (FTIR). Characteristic cellulose II XRD diffraction pattern was observed after IL dissolution and regeneration of MCC. The crystallinity index (CI) of the pretreated MCC was reduced from 93.2 % to 31 %. Inert conditions were not required for the cellulose dissolution experiments. This study indicates that the IL H-bond basicity is not the only key parameter determining their cellulose dissolution ability. The alkaline ILs represent an energy efficient and sustainable approach for cellulose dissolution.

doi:10.1111/febs.15117

Abstract

Efficient capture of glycans, the prime metabolic resources in the human gut, confers a key competitive advantage for gut microbiota members equipped with extracellular glycoside hydrolases (GHs) to target these substrates. The association of glycans to the bacterial cell surface is typically mediated by carbohydrate binding modules (CBMs). Here, we report the structure of Ri CBM86 appended to a GH family 10 xylanase from Roseburia intestinalis . This CBM represents a new family of xylan binding CBMs present in xylanases from abundant and prevalent healthy human gut Clostridiales. Ri CBM86 adopts a canonical β‐sandwich fold, but shows structural divergence from known CBMs. The structure of Ri CBM86 has been determined with a bound xylohexaose, which revealed an open and shallow binding site. Ri CBM86 recognizes only a single xylosyl ring with direct hydrogen bonds. This mode of recognition is unprecedented amongst previously reported xylan binding type‐B CBMs that display more extensive hydrogen‐bonding patterns to their ligands or employ Ca2+ to mediate ligand‐binding. The architecture of Ri CBM86 is consistent with an atypically low binding affinity (K D about 0.5 mm for xylohexaose) compared to most xylan binding CBMs. Analyses using NMR spectroscopy corroborated the observations from the complex structure and the preference of Ri CBM86 to arabinoxylan over glucuronoxylan, consistent with the largely negatively charged surface flanking the binding site. Mutational analysis and affinity electrophoresis established the importance of key binding residues, which are conserved in the family. This study provides novel insight into the structural features that shape low‐affinity CBMs that mediate extended bacterial glycan capture in the human gut niche.

doi:10.1016/j.foodchem.2019.125678

Abstract

Significant quantities of several important herbs are processed and consumed from Norwegian commercial kitchens annually although surprisingly the contents of polyphenols have been scarcely characterized. We here report on the qualitative and quantitative content of polyphenolic compounds from ten of the most utilized herbs. From parsley (Petroselinum crispum) var. Darki, isorhamnetin 3-(6″-malonylglucoside)-7-glucoside (2) and diosmetin 7-(2″-apiosyl-6″-malonylglucoside) (8) are reported for the first time, in addition to seven known flavonoids, some of which are reported for the first time from this plant species. Oregano, rosemary and thyme contained the highest amounts of total phenolics with maximum levels of 23.8, 24.2 and 23.4 mg GAE g−1 dry matter, respectively. Fresh herbs contained significantly higher quantities of phenolics than processed, dried herbs. Parsley, coriander, dill and thyme were the richest sources of flavonoids among the investigated herbs.

doi:10.1021/jacs.9b09907

Abstract

Significant quantities of several important herbs are processed and consumed from Norwegian commercial kitchens annually although surprisingly the contents of polyphenols have been scarcely characterized. We here report on the qualitative and quantitative content of polyphenolic compounds from ten of the most utilized herbs. From parsley (Petroselinum crispum) var. Darki, isorhamnetin 3-(6″-malonylglucoside)-7-glucoside (2) and diosmetin 7-(2″-apiosyl-6″-malonylglucoside) (8) are reported for the first time, in addition to seven known flavonoids, some of which are reported for the first time from this plant species. Oregano, rosemary and thyme contained the highest amounts of total phenolics with maximum levels of 23.8, 24.2 and 23.4 mg GAE g−1 dry matter, respectively. Fresh herbs contained significantly higher quantities of phenolics than processed, dried herbs. Parsley, coriander, dill and thyme were the richest sources of flavonoids among the investigated herbs.

doi:10.3389/fchem.2019.00625

Abstract

Structurally modified hydroxyl functionalized pyridinium ionic liquids (ILs), liquid at room temperature, were synthesized and characterized. Alkylated N-(2-hydroxyethyl)-pyridinium ILs were prepared from alkylpyridines via corresponding bromide salts by N-alkylation (65-93%) and final anion exchange (75-96%). Pyridinium-alkylation strongly influenced the IL physicochemical and electrochemical properties. Experimental values for the ILs physicochemical properties (density, viscosity, conductivity, and thermal decomposition temperature), were in good agreement with corresponding predicted values obtained by theoretical calculations. The pyridinium ILs have electrochemical window of 3.0-5.4 V and were thermally stable up to 405°C. The IL viscosity and density were measured over a wide temperature range (25-80°C). Pyridine alkyl-substitution strongly affected the partial positive charge on the nitrogen atom of the pyridinium cations, as shown by charge distribution calculations. On-going studies on Mg complexes of the new ILs demonstrate promising properties for high current density electrodeposition of magnesium.

doi:10.1073/pnas.1912741116

Abstract

Anthocyanins are key pigments of plants, providing color to flowers, fruit, and foliage and helping to counter the harmful effects of environmental stresses. It is generally assumed that anthocyanin biosynthesis arose during the evolutionary transition of plants from aquatic to land environments. Liverworts, which may be the closest living relatives to the first land plants, have been reported to produce red cell wall-bound riccionidin pigments in response to stresses such as UV-B light, drought, and nutrient deprivation, and these have been proposed to correspond to the first anthocyanidins present in early land plant ancestors. Taking advantage of the liverwort model species Marchantia polymorpha, we show that the red pigments of Marchantia are formed by a phenylpropanoid biosynthetic branch distinct from that leading to anthocyanins. They constitute a previously unreported flavonoid class, for which we propose the name "auronidin," with similar colors as anthocyanin but different chemistry, including strong fluorescence. Auronidins might contribute to the remarkable ability of liverworts to survive in extreme environments on land, and their discovery calls into question the possible pigment status of the first land plants.

doi:10.1073/pnas.1902346116

Abstract

Nicotinamide adenine dinucleotide (NAD) provides an important link between metabolism and signal transduction and has emerged as central hub between bioenergetics and all major cellular events. NAD-dependent signaling (e.g., by sirtuins and poly–adenosine diphosphate [ADP] ribose polymerases [PARPs]) consumes considerable amounts of NAD. To maintain physiological functions, NAD consumption and biosynthesis need to be carefully balanced. Using extensive phylogenetic analyses, mathematical modeling of NAD metabolism, and experimental verification, we show that the diversification of NAD-dependent signaling in vertebrates depended on 3 critical evolutionary events: 1) the transition of NAD biosynthesis to exclusive usage of nicotinamide phosphoribosyltransferase (NamPT); 2) the occurrence of nicotinamide N-methyltransferase (NNMT), which diverts nicotinamide (Nam) from recycling into NAD, preventing Nam accumulation and inhibition of NAD-dependent signaling reactions; and 3) structural adaptation of NamPT, providing an unusually high affinity toward Nam, necessary to maintain NAD levels. Our results reveal an unexpected coevolution and kinetic interplay between NNMT and NamPT that enables extensive NAD signaling. This has implications for therapeutic strategies of NAD supplementation and the use of NNMT or NamPT inhibitors in disease treatment.

doi:10.1016/j.molliq.2019.111353

Abstract

New dual functionalized N-oxyethylene-N-sulfopropyl imidazolium based ionic liquids (ILs) with unconventional anions for cellulose dissolution were synthesized and characterized (NMR, IR, HRMS, TGA, Td). The ILs were applied in studies on cellulose (MCC) dissolution. Results showed that varying the IL cation had greater impact on cellulose dissolution ability than the minor effect seen by anion exchange. Up to 20 wt-% MCC was dissolved in DMF/ILs solutions at 100°C. The dual functionalized ionic liquids (ILs) were synthesized via the corresponding N-oxyethylene imidazolium sulfonate zwitterions (ZIs), which were prepared (74–99%) through a double N-imidazole alkylation procedure, including 1,3-propanesultone ring opening. The original MCC and the regenerated cellulose samples were characterized by XRD analysis.

doi:10.1039/c9cc02695a

Abstract

A series of hafnium complexes were structurally identified showing high activity (up to 500 h−1) in the selective alternated copolymerization of epoxides with CO2 under low pressure.

doi:10.3390/gels5020023

Abstract

In this work, the mechanical properties and stability of alginate hydrogels containing functionalized alginates (peptide and β-cyclodextrin) were studied. There is an increasing interest in the modification of alginates to add functions such as cell attachment and increased solubility of hydrophobic drugs, for better performance in tissue engineering and drug release, respectively. Functionalization was achieved in this study via periodate oxidation followed by reductive amination, previously shown to give a high and controllable degree of substitution. Young's modulus and the stress at rupture of the hydrogels were in general lowered when exchanging native alginate with the modified alginate. Still, the gel strength could be adjusted by the fraction of modified alginate in the mixed hydrogels as well as the degree of oxidation. No notable difference in deformation at rupture was observed while syneresis was influenced by the degree of oxidation and possibly by the nature and amount of the grafted molecules. The mixed hydrogels were less stable than hydrogels with only native alginate, and modified alginate was released from the hydrogels. Furthermore, the hydrogels in general rather disintegrated than swelled upon saline treatments.

doi:10.1021/acs.jproteome.8b00801

Abstract

Glioblastoma is the most common and malignant brain tumor, and current therapies confer only modest survival benefits. A major obstacle is our ability to monitor treatment effect on tumors. Current imaging modalities are ambiguous, and repeated biopsies are not encouraged. To scout for markers of treatment response, we used NMR spectroscopy to study the effects of a survivin inhibitor on the metabolome of primary glioblastoma cancer stem cells. Applying high resolution NMR spectroscopy (1H resonance frequency: 800.03 MHz) to just 3 million cells per sample, we achieved sensitive and high resolving determinations of, e.g., amino acids, nucleosides, and constituents of the citric acid cycle. For control samples that were cultured, prepared, and measured at varying dates, peak area relative standard deviations were 15–20%. Analyses of unfractionated lysates were performed for straightforward compound identification with COLMAR and HMDB databases. Principal component analysis revealed that citrate levels were clearly upregulated in nonresponsive cells, while lactate levels substantially decreased following treatment for both responsive and nonresponsive cells. Hence, lactate and citrate may be potential markers of successful drug uptake and poor response to survivin inhibitors, respectively. Our metabolomics approach provided alternative biomarker candidates compared to spectrometry-based proteomics, underlining benefits of complementary methodologies. These initial findings make a foundation for exploring in vivo MR spectroscopy (MRS) of brain tumors, as citrate and lactate are MRS-visible. In sum, NMR metabolomics is a tool for addressing glioblastoma.

doi:10.1016/j.sbi.2019.02.015

Abstract

The discovery of oxidative cleavage of glycosidic bonds by enzymes currently known as lytic polysaccharide monooxygenases (LPMOs) has had a major impact on our current understanding of the enzymatic conversion of recalcitrant polysaccharides such as chitin and cellulose. The number of LPMO sequence families keeps expanding and novel substrate specificities and biological functionalities are being discovered. The catalytic mechanism of these LPMOs remains somewhat enigmatic. Recently, novel insights have been obtained from studies of enzyme–substrate complexes by X-ray crystallography, EPR, NMR, and modeling. Furthermore, it has been shown that LPMOs may carry out peroxygenase reactions, at much higher rates than monooxygenase reactions, which affects our understanding and exploitation of these powerful enzymes.

doi:10.3390/molecules24071199

Abstract

Platycodon grandiflorus is a plant widely used in traditional Chinese medicine, of which polysaccharides are reported to be the main components responsible for its bio-functions. In this work, the inulin-type fructan (PGF) was obtained by DEAE anion exchange chromatography from the water extracted from P. grandifloras. Characterization was performed with methanolysis, methylation, and NMR and the results showed that PGF is a β-(2-1) linked fructan, with terminal glucose and with a degree of polymerization of 2–10. In order to study its biofunctions, the prebiotic and immunomodulation properties were assayed. We found that PGF exhibited good prebiotic activity, as shown by a promotion on six strains of lactobacillus proliferation. Additionally, the PGF also displayed direct immunomodulation on intestinal epithelial cells and stimulated the expressions of anti-inflammatory factors. These results indicated that the inulin from P. grandiflorus is a potential natural source of prebiotics as well as a potential intestinal immunomodulator, which will be valuable for further studies and new applications.

doi:10.1021/acs.biomac.8b01796

Abstract

With the present accessibility of algal raw material, microbial alginates as a source for strong gelling material are evaluated as an alternative for advanced applications. Recently, we have shown that alginate from algal sources all contain a fraction of very long G-blocks (VLG), that is, consecutive sequences of guluronic acid (G) residues of more than 100 residues. By comparing the gelling properties of these materials with in vitro epimerized polymannuronic acid (poly-M) with shorter G-blocks, but comparable with the G-content, we could demonstrate that VLG have a large influence on gelling properties. Hypothesized to function as reinforcement bars, VLG prevents the contraction of the gels during formation (syneresis) and increases the Young’s modulus (strength of the gel). Here we report that these VLG structures are also present in alginates from Azotobacter vinelandii and that these polymers consequently form stable, low syneretic gels with calcium, comparable in mechanical strength to algal alginates with the similar monomeric composition. The bacterium expresses seven different extracellular mannuronan epimerases (AlgE1-AlgE7), of which only the bifunctional epimerase AlgE1 seems to be able to generate the long G-blocks when acting on poly-M. The data implies evidence for a processive mode of action and the necessity of two catalytic sites to obtain the observed epimerization pattern. Furthermore, poly-M epimerized with AlgE1 in vitro form gels with comparable or higher rigidity and gel strength than gels made from brown seaweed alginate with matching G-content. These findings strengthen the viability of commercial alginate production from microbial sources. With the present accessibility of algal raw material, microbial alginates as a source for strong gelling material are evaluated as an alternative for advanced applications. Recently, we have shown that alginate from algal sources all contain a fraction of very long G-blocks (VLG), that is, consecutive sequences of guluronic acid (G) residues of more than 100 residues. By comparing the gelling properties of these materials with in vitro epimerized polymannuronic acid (poly-M) with shorter G-blocks, but comparable with the G-content, we could demonstrate that VLG have a large influence on gelling properties. Hypothesized to function as reinforcement bars, VLG prevents the contraction of the gels during formation (syneresis) and increases the Young’s modulus (strength of the gel). Here we report that these VLG structures are also present in alginates from Azotobacter vinelandii and that these polymers consequently form stable, low syneretic gels with calcium, comparable in mechanical strength to algal alginates with the similar monomeric composition. The bacterium expresses seven different extracellular mannuronan epimerases (AlgE1-AlgE7), of which only the bifunctional epimerase AlgE1 seems to be able to generate the long G-blocks when acting on poly-M. The data implies evidence for a processive mode of action and the necessity of two catalytic sites to obtain the observed epimerization pattern. Furthermore, poly-M epimerized with AlgE1 in vitro form gels with comparable or higher rigidity and gel strength than gels made from brown seaweed alginate with matching G-content. These findings strengthen the viability of commercial alginate production from microbial sources.

doi:10.1007/978-981-13-7318-3_6

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze the cleavage of 1,4-glycosidic bonds various plant cell wall polysaccharides and chitin. In contrast to glycoside hydrolases, LPMOs are active on the crystalline regions of polysaccharides and thus synergize with hydrolytic enzymes. This synergism leads to an overall increase in the biomass-degradation activity of enzyme mixtures. Chitin-active LPMOs were discovered in 2010 and are currently classified in families AA10, AA11, and AA15 of the Carbohydrate-Active enZYmes database, which include LPMOs from bacteria, fungi, insects, and viruses. LPMOs have become important enzymes both industrially and scientifically and, in this chapter, we provide a brief introduction to chitin-active LPMOs including a summary of the 20+{\thinspace}chitin-active LPMOs that have been characterized so far. Then, we describe their structural features, catalytic mechanism, and appended carbohydrate modules. Finally, we show how chitin-active LPMOs can be used to perform chemo-enzymatic modification of chitin substrates.

doi:10.1016/j.jmr.2019.02.008

Abstract

This study presents a two-dimensional NMR pulse sequence for obtaining spatially resolved correlations between magnetic susceptibility induced internal gradients (G0), and both the apparent difference in magnetic susceptibility (Δχapp) and spectral frequency (Δν). G0-Δχapp correlations were utilized to generate spatially resolved pore size distributions, while the G0-Δν correlations were used to additionally evaluate sample heterogeneity. The spatially resolved measurements were performed on a water saturated heterogeneous porous sample which contains one layer of 5–50 μm glass spheres (top layer) and one layer of 140–165 μm glass spheres (bottom layer). The slice selection was validated by applying the pulse sequence on a liquid mineral oil and water sample as well as on the porous sample. The resulting spatially resolved pore size distributions show very good agreement with results from our previously published non slice selective pulse sequence in the 140–165 μm glass spheres and good agreement in the 5–50 μm glass spheres. The G0-Δν correlations correctly indicate a slightly higher degree of heterogeneity in the 5–50 μm glass spheres compared to the 140–165 μm glass spheres.

doi:10.1039/C9TA00472F

Abstract

Auxiliary donors are common design motifs for phenothiazine sensitizers for dye-sensitized solar cells. Despite this{,} there are only a few reports on their overall contribution to the photon-to-electron conversion efficiency. Twelve sensitizers have been prepared and investigated{,} including ten different auxiliary donors in addition to a control with no auxiliary donor. The various auxiliary donors improved the PCE by a modest 4–11%{,} and pyrene (AFB-19) was determined to be the most efficient auxiliary donor{,} with the best cell delivering a PCE of 5.36%. Included in the dye series was also the champion dye within the phenothiazine class. With a reported PCE of 12.1%{,} it would be an excellent phenothiazine reference dye. The high VOC of 0.83 V of this dye is worth further investigation{,} but the absorption and photovoltaic performance in this work does not correlate with the previously reported PCE of 12.1%.

doi:10.1039/C8DT04592E

Abstract

Thiolate-coordinated ruthenium alkylidene complexes can give high Z-selectivity and stereoretentivity in olefin metathesis. To investigate their applicability as heterogeneous catalysts{,} we have successfully developed a methodology to easily immobilize prototype ruthenium alkylidenes onto hybrid mesostructured silica via a thiolate tether. In contrast{,} the preparation of the corresponding molecular complexes appeared very challenging in solution. These prototype supported complexes contain small thiolates but still{,} they are slightly more Z-selective than their molecular analogues. These results open the door to more active and selective heterogeneous catalysts by supporting more advanced thiolate Ru-complexes.

doi:10.1016/j.tetlet.2019.01.043

Abstract

A new analogue of gambierone, 44-methylgambierone, was isolated from the benthic dinoflagellate Gambierdiscus australes collected from Raoul Island (Rangitahua/Kermadec Islands). This molecule has been previously reported as maitotoxin-3. The structure of 44-methylgambierone was elucidated using 1D- and 2D-nuclear magnetic resonance spectroscopy and mass spectrometry techniques. The nine-ring polyether backbone (A–I) and functional groups (carbonyl, terminal diol, 1,3-diene and monosulphate) are the same for both compounds with the addition of an olefinic methyl group being the only modification in 44-methylgambierone.

doi:10.3389/fmicb.2018.03139

Abstract

Streptomyces bacteria are recognized as an important source for antibiotics with broad applications in human medicine and animal health. Here, we report the isolation of a new lichen-associating Streptomyces sp. YIM 130001 from the tropical rainforest in Xishuangbanna (Yunnan, China), which displayed antibacterial activity against Bacillus subtilis. The draft genome sequence of this isolate strain revealed 18 putative biosynthetic gene clusters (BGCs) for secondary metabolites, which is an unusually low number compared to a typical streptomycete. Inactivation of a lantibiotic dehydrogenase-encoding gene from the BGC presumed to govern biosynthesis of a thiopeptide resulted in the loss of bioactivity. Using comparative HPLC analysis, two peaks in the chromatogram were identified in the extract from the wild-type strain, which were missing in the extract from the mutant. The compounds corresponding to the identified peaks were purified, and structure of one compound was elucidated using NMR. The compound, designated geninthiocin B, showed high similarity to several 35-membered macrocyclic thiopeptides geninthiocin, Val-geninthiocin and berninamycin A. Bioinformatics analysis of the geninthiocin B BGC revealed its close homology to that of berninamycins.

doi:10.1039/C8DT04364G

Abstract

Magnesium(II) complexes, [Mg2+(hfac−)3][Cation+], were prepared as solids from hydrophobic hexafluoroacetylacetonate ionic liquids ([Cation+][hfac−] ILs) and Mg(Tf2N)2. 1-Butyl-3-methylimidazolium ([C4mim]), N-butylpyridinium ([C4Pyr]), N-butyl-N-methylpiperidinium ([C4Pip]), N-hexyl-N-methylmorpholinium ([C6Morp]) and N-butyl-N-methylpyrrolidinium ([C4pyrr]) were used as cationic cores. The [C4Pip][hfac], [C4Pyr][hfac] and [C6Morp][hfac] ILs were prepared for the first time. New Mg(II) complexes, [C4mim][Mg(hfac)3], [C4Pip][Mg(hfac)3], [C4Pyr][Mg(hfac)3], [C6Morp][Mg(hfac)3] and [C4Pyrr][Mg(hfac)3], were obtained from the [hfac] based ILs. The crystal structures of the novel Mg(II) complexes show the coordination of three [hfac] anions to the Mg2+ ion through the two oxygen atoms of each [hfac] anion.

doi:10.1016/j.bse.2018.11.001

Abstract

For more than six decades, bumblebee death, which mainly occurs in August, has been assumed to be associated with intake of presumed toxic nectar from linden trees (Tilia spp.), a hypothesis which has been sustained by observations of a significant number of dead bumblebees under these trees during their flowering season. Several theories exist in current literature to account for these observations. The nectar has been assumed to contain compound(s) toxic to the bumblebees including the monosaccharide mannose, which cannot be metabolized by bumblebees. The presence of toxic compounds such as the alkaloid nicotine or pesticides of anthropogenic origin has also been indicated. However, none of the above suggested compounds have hitherto been properly characterized from the nectar. In the current paper we report on characterization of the composition of nectar of linden trees, under which a significant number of dead bumblebees were observed. The structure determinations were performed by selective 1D TOCSY NMR and extensive applications of 2D NMR spectroscopy. The nectar of the linden flowers was readily available in significant amounts during the entire period when dead bumblebees were observed under these trees. The nectar only contained non-toxic sugars such as α-glucopyranose, β-glucopyranose, sucrose, fructose and minor amounts of xylose. The nectar did not contain mannose, nor alkaloids or pesticides. Initial toxicity studies using brine shrimp lethality assay showed that the nectar did not exhibit any toxic effects even at concentrations higher than 1 mg/ml dry weight, providing disproving evidence against the assumption of the nectar's toxic character.

doi:10.1021/acsabm.8b00436

Abstract

The structure of fucoidan isolated from Laminaria hyperborea was elucidated and chemically tailored in order to obtain a clear structure–function relationship on bioactive properties with a minimal amount of variations among the tested molecules. Analysis revealed a sugar composition of 97.8% fucose and 2.2% galactose. Analysis of the glycosidic linkages showed (1→3)-α-l-fuco-pyranose (31.9%) to be the dominant residue, followed by 1→2-linked (13.2%) and 1→4-linked (7.7%) fuco-pyranose as well as a high degree of branching (22.4%). Inductively coupled plasma mass spectrometry (ICP-MS) revealed a sulfate content of 53.8% (degree of sulfation (DS) = 1.7). Raman spectroscopy determined SO4 located axial at 4C and equatorial at 2C as well as an absence of acetylation. SEC-MALS analysis determined a high molecular weight (Mw = 469 kDa), suggesting a highly flexible main chain with short side chains. Both chemical shifts of the fucoidan, proton, and carbon were assigned by NMR and revealed a highly heterogeneous structure in terms of glycosidic linkages. Bioactivity was assessed using a lepirudin-based whole blood model. The immediate responses by coagulation and complement cascades were measured by prothrombine factor 1 and 2 (PTF1.2) and the terminal complement complex (TCC). Cytokines involved in inflammation were detected in a 27-plex cytokine assay. Fucoidan with a high Mw and DS inhibited coagulation, complement, and the cytokines PDGF-BB, RANTES, and IP-10, while activating MCP-1. These effects were obtained at the concentration of 1000 ug/mL and partly at 100 ug/mL. In low concentrations (10 ug/mL), a coagulation stimulating effect of highly sulfated fucoidans (DS = 1.7, Mw = 469 kDa or 20.3) was obtained. These data point to a multitude of effects linked to the sulfation degree that needs further mechanistic exploration.

doi:10.1016/j.carres.2018.10.012

Abstract

Albatrellus ovinus, the sheep polypore, is a large, dense mushroom being rich in cell wall material. Polysaccharides were isolated by sequential extraction, enzymatic treatment and analyzed with respect to monosaccharide composition, glycosidic linkages by methylation and GC-MS as well as NMR spectroscopy. A fucogalactan composed of an (1 → 6)-α-d-galactan backbone with single α-l-Fucp residues attached at O-2 was identified in the hot water extract obtained after treatment with a protease and size exclusion chromatography. Both the hot water extract and the hot alkali extract contained an (1 → 4)-α-d-glucan whereas β-d-glucans were mainly present in the latter. Structural analysis suggested the presence of two different β-d-glucan backbone structures; a (1 → 6)-linked β-d-glucan with single β-d-Glcp residues at O-3 and also a (1 → 3)-linked β-d-glucan with branches in O-6. In addition there were identified short (1 → 2)-linked β-d-xylan and (1 → 3)-α-d-mannan chains.

doi:10.1016/j.hal.2018.10.006

Abstract

In the summer of 2012, over 750 dead and dying birds were observed at the Paul S. Sarbanes Ecosystem Restoration Project at Poplar Island, Maryland, USA (Chesapeake Bay). Clinical signs suggested avian botulism, but an ongoing dense Microcystis bloom was present in an impoundment on the island. Enzyme-linked immunosorbent assay (ELISA) analysis of a water sample indicated 6000 ng mL−1 of microcystins (MCs). LC-UV/MS analysis confirmed the presence of MC-LR and a high concentration of an unknown MC congener (m/z 1037.5). The unknown MC was purified and confirmed to be [D-Leu1]MC-LR using NMR spectroscopy, LC-HRMS and LC–MS2, which slowly converted to [D-Leu1,Glu(OMe)6]MC-LR during storage in MeOH. Lyophilized algal material from the bloom was further characterized using LC-HRMS and LC–MS2 in combination with chemical derivatizations, and an additional 24 variants were detected, including MCs conjugated to Cys, GSH and γ-GluCys and their corresponding sulfoxides. Mallard (Anas platyrhynchos) livers were tested to confirm MC exposure. Two broad-specificity MC ELISAs and LC–MS2 were used to measure free MCs, while ‘total’ MCs were estimated by both MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) and thiol de-conjugation techniques. Free microcystins in the livers (63–112 ng g−1) accounted for 33–41% of total microcystins detected by de-conjugation and MMPB techniques. Free [D-Leu1]MC-LR was quantitated in tissues at 25–67 ng g−1 (LC–MS2). The levels of microcystin varied based on analytical method used, highlighting the need to develop a comprehensive analysis strategy to elucidate the etiology of bird mortality events when microcystin-producing HABs are present.

doi:10.1016/j.mri.2018.10.002

Abstract

We have applied diffusion and relaxation Nuclear Magnetic Resonance experiments to investigate the translational and rotational mobility of adsorbents on quartz and calcite mineral surfaces. On both surfaces it was found that water is the dominant molecule. On the quartz surface the majority of water molecules have a relatively high degree of both rotational and translational mobility, while a minor fraction of water molecules, and all hydrocarbon molecules, have a significantly lower mobility. On the calcite surface the translational mobility is very low for all the adsorbed molecules, while there is a large diversity in rotational mobility, indicating that the hydrocarbon molecules are strongly attached to the surface, but that some part of each molecule still have a large degree of rotational mobility. Diffusion and relaxation experiments give a detailed description of both the molecular mobility of adsorbed species on these mineral surfaces, which leads to new insight with respect to aging processes on a molecular level.

doi:10.1007/s00706-018-2277-9

Abstract

A potential intermediate in the synthesis of several trans-clerodane natural products has been constructed employing a Diels–Alder reaction as a key-step. Two epimeric exo-adducts were formed in a 4:3 ratio in an EtAlCl2-mediated cycloaddition of O-silylated 2-vinylcyclohex-2-enol and N-tigloylisoxazolidinone. Both isomers were converted to the trans-clerodane intermediate in four steps; reductive removal of the oxazolidinone, followed by O-benzylation, removal of the silyl protecting group, and, finally, a Dess–Martin oxidation of the deprotected alcohol to the corresponding ketone. It was possible to transform both isomers from the cycloaddition into the final target, but the major isomer was converted at greater yields. An interesting discovery made during the work was that the desilylation demanded significantly different conditions depending on which isomer was deprotected. The fact that the cycloaddition not only resulted in an excellent exo-selectivity, but also that the alkene to a large extent approached from the least hindered side, opens the possibility for enantioselective synthesis of the target compound from (R) or (S) diene starting materials in the future.

doi:10.1007/s12104-018-9850-3

Abstract

The N-terminal domain (residues 28-165) from the glycoside hydrolase family 10 from Roseburia intestinalis (RiCBMx), has been isotopically labeled and recombinantly expressed in Escherichia coli. Here we report H, C and N NMR chemical shift assignments for this carbohydrate binding module (CBM).

doi:10.1007/s12104-018-9839-y

Abstract

The apo-form of the 24.4 kDa AA9 family lytic polysaccharide monooxygenase TaLPMO9A from Thermoascus aurantiacus has been isotopically labeled and recombinantly expressed in Pichia pastoris. In this paper, we report the H, C, and N chemical shift assignments, as well as an analysis of the secondary structure of the protein based on the secondary chemical shifts.

doi:10.1007/s11103-018-0759-0

Abstract

Euglena gracilis is a unicellular microalga showing characteristics of both plants and animals, and extensively used as a model organism in the research works of biochemistry and molecular biology. Biotechnological applications of E. gracilis have been conducted for production of numerous important compounds. However, chitin-mediated defense system intensively studied in higher plants remains to be investigated in this microalga. Recently, Taira et al. (Biosci Biotechnol Biochem 82:1090-1100, 2018) isolated a unique chitinase gene, comprising two catalytic domains almost homologous to each other (Cat1 and Cat2) and two chitin-binding domains (CBD1 and CBD2), from E. gracilis. We herein examined the mode of action and the specificity of the recombinant Cat2 by size exclusion chromatography and NMR spectroscopy. Both Cat1 and Cat2 appeared to act toward chitin substrate with non-processive/endo-splitting mode, recognizing two contiguous N-acetylglucosamine units at subsites - 2 and - 1. This is the first report on a chitinase having two endo-splitting catalytic domains. A cooperative action of two different endo-splitting domains may be advantageous for defensive action of the E. gracilis chitinase. The unicellular alga, E. gracilis, produces a chitinase consisting of two GH18 catalytic domains (Cat1 and Cat2) and two CBM18 chitin-binding domains (CBD1 and CBD2). Here, we produced a recombinant protein of the Cat2 domain to examine its mode of action as well as specificity. Cat2 hydrolyzed N-acetylglucosamine (A) oligomers (A , n = 4, 5, and 6) and partially N-acetylated chitosans with a non-processive/endo-splitting mode of action. NMR analysis of the product mixture from the enzymatic digestion of chitosan revealed that the reducing ends were exclusively A-unit, and the nearest neighbors of the reducing ends were mostly A-unit but not exclusively. Both A-unit and D-unit were found at the non-reducing ends and the nearest neighbors. These results indicated strong and absolute specificities for subsites - 2 and - 1, respectively, and no preference for A-unit at subsites + 1 and + 2. The same results were obtained from sugar sequence analysis of the individual enzymatic products from the chitosans. The subsite specificities of Cat2 are similar to those of GH18 human chitotriosidase, but differ from those of plant GH18 chitinases. Since the structures of Cat1 and Cat2 resemble to each other (99% similarity in amino acid sequences), Cat1 may hydrolyze the substrate with the same mode of action. Thus, the E. gracilis chitinase appears to act toward chitin polysaccharide chain through a cooperative action of the two endo-splitting catalytic domains, recognizing two contiguous A-units at subsites - 2 and - 1.

doi:10.1039/c8cc05489d

Abstract

A (N,CAr,CAlk) Au(III) pincer complex has been synthesized from Au(OAc)3 (OAc = OCOCH3) and 2-(3,5-di-tert-butylphenyl)pyridine (L1) involving a Csp3–H bond activation by electrophilic substitution. In agreement with DFT calculations, the resulting complex significantly improves the performance of Au(tpy)(OAcF)2 (tpy = 2-(p-tolyl)pyridine, OAcF = OCOCF3) in the catalytic trifluoroacetylation of acetylene.

doi:10.1002/pro.3451

Abstract

The catalytically crucial N-terminal histidine (His1) of fungal lytic polysaccharide monooxygenases (LPMOs) is post-translationally modified to carry a methylation. The functional role of this methylation remains unknown. We have carried out an in-depth functional comparison of two variants of a family AA9 LPMO from Thermoascus aurantiacus (TaLPMO9A), one with, and one without the methylation on His1. Various activity assays showed that the two enzyme variants are identical in terms of substrate preferences, cleavage specificities and the ability to activate molecular oxygen. During the course of this work, new functional features of TaLPMO9A were discovered, in particular the ability to cleave xyloglucan, and these features were identical for both variants. Using a variety of techniques, we further found that methylation has minimal effects on the pK of His1, the affinity for copper and the redox potential of bound copper. The two LPMOs did, however, show clear differences in their resistance against oxidative damage. Studies with added hydrogen peroxide confirmed recent claims that low concentrations of H O boost LPMO activity, whereas excess H O leads to LPMO inactivation. The methylated variant of TaLPMO9A, produced in Aspergillus oryzae, was more resistant to excess H O and showed better process performance when using conditions that promote generation of reactive-oxygen species. LPMOs need to protect themselves from reactive oxygen species generated in their active sites and this study shows that methylation of the fully conserved N-terminal histidine provides such protection.

doi:10.1074/jbc.RA118.004269

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze the oxidative cleavage of polysaccharides such as cellulose and chitin, a feature that makes them key tools in industrial biomass conversion processes. The catalytic domains of a considerable fraction of LPMOs and other carbohydrate-active enzymes (CAZymes) are tethered to carbohydrate-binding modules (CBMs) by flexible linkers. These linkers preclude X-ray crystallographic studies, and the functional implications of these modular assemblies remain partly unknown. Here, we used NMR spectroscopy to characterize structural and dynamic features of full-length modular ScLPMO10C from Streptomyces coelicolor. We observed that the linker is disordered and extended, creating distance between the CBM and the catalytic domain and allowing these domains to move independently of each other. Functional studies with cellulose nanofibrils revealed that most of the substrate-binding affinity of full-length ScLPMO10C resides in the CBM. Comparison of the catalytic performance of full-length ScLPMO10C and its isolated catalytic domain revealed that the CBM is beneficial for LPMO activity at lower substrate concentrations and promotes localized and repeated oxidation of the substrate. Taken together, these results provide a mechanistic basis for understanding the interplay between catalytic domains linked to CBMs in LPMOs and CAZymes in general.

doi:10.1021/acs.organomet.8b00218

Abstract

The reactivity of Au(OAcF)2(tpy) (1, OAcF = OCOCF3; tpy = 2-(p-tolyl)pyridine) toward a wide variety of different alkenes with various substitution patterns and different oxygen-based nucleophiles has been investigated. These reactions are two-step processes where a ligand substitution is followed by a nucleophilic addition furnishing Au(III) complexes with C(sp3) ligands. In this work we have found that the reactions always occur trans to tpy-N while the OAcF ligand remains in place trans to tpy-C. The nucleophilic addition takes place exclusively at the most substituted side of the double bond, in a Markovnikov manner, and the nucleophilic addition occurs in an anti fashion as can be seen from the reaction with the 2,3-disubstituted alkene trans-2-hexene. This study has provided valuable insight into the scope and regiochemistry of Au(III) mediated nucleophilic additions, which is of great importance for further development of Au(III) catalysis and alkene functionalization.

doi:10.1039/c8ra03757d

Abstract

We herein report the development of a convenient, regioselective, aromatic fluorination method of hypervalent iodonium ylides for synthesising fluoro-arenes on a preparative scale. This transition metal free, nucleophilic methodology provides good yields for sterically hindered substrates, irrespective of activation. The methodology simplifies reference synthesis for PET imaging.

doi:10.1007/978-1-4939-7877-9_16

Abstract

Lytic polysaccharide monooxygenases are copper-dependent enzymes that perform oxidative cleavage of glycosidic bonds in cellulose and various other polysaccharides. LPMOs acting on cellulose use a reactive oxygen species to abstract a hydrogen from the C1 or C4, followed by hydroxylation of the resulting substrate radical. The resulting hydroxylated species is unstable, resulting in glycoside bond scission and formation of an oxidized new chain end. These oxidized chain ends are spontaneously hydrated at neutral pH, leading to formation of an aldonic acid or a gemdiol, respectively. LPMO activity may be characterized using a variety of analytic tools, the most common of which are high-performance anion exchange chromatography system with pulsed amperometric detection (HPAEC-PAD) and MALDI-TOF mass spectrometry (MALDI-MS). NMR may be used to increase the certainty of product identifications, in particular the site of oxidation. Kinetic studies of LPMOs have several pitfalls and to avoid these, it is important to secure copper saturation, avoid the presence of free transition metals in solution, and control the amount of reductant (i.e., electron supply to the LPMO). Further insight into LPMO properties may be obtained by determining the redox potential and by determining the affinity for copper. In some cases, substrate affinity can be assessed using isothermal titration calorimetry. These methods are described in this chapter.

doi:10.1002/ejoc.201800419

Abstract

The strong ability of Au catalysts with bisoxazoline (BOX) ligands for combined fast propargyl cyclopropanation and subsequent in situ cis‐to‐trans isomerization is reported. AuI or AuIII catalysts selectively provided pure cis or trans products, respectively, and isolated cis isomers were shown to rapidly isomerize into trans products in the presence of BOX‐AuIII catalysts.

doi:10.1038/s41564-018-0132-8

Abstract

Metabolism of dietary glycans is pivotal in shaping the human gut microbiota. However, the mechanisms that promote competition for glycans among gut commensals remain unclear. Roseburia intestinalis, an abundant butyrate-producing Firmicute, is a key degrader of the major dietary fibre xylan. Despite the association of this taxon to a healthy microbiota, insight is lacking into its glycan utilization machinery. Here, we investigate the apparatus that confers R. intestinalis growth on different xylans. R. intestinalis displays a large cell-attached modular xylanase that promotes multivalent and dynamic association to xylan via four xylan-binding modules. This xylanase operates in concert with an ATP-binding cassette transporter to mediate breakdown and selective internalization of xylan fragments. The transport protein of R. intestinalis prefers oligomers of 4–5 xylosyl units, whereas the counterpart from a model xylan-degrading Bacteroides commensal targets larger ligands. Although R. intestinalis and the Bacteroides competitor co-grew in a mixed culture on xylan, R. intestinalis dominated on the preferred transport substrate xylotetraose. These findings highlight the differentiation of capture and transport preferences as a possible strategy to facilitate co-growth on abundant dietary fibres and may offer a unique route to manipulate the microbiota based on glycan transport preferences in therapeutic interventions to boost distinct taxa.

doi:10.1073/pnas.1719251115

Abstract

N-terminal (Nt) acetylation is a major protein modification catalyzed by N-terminal acetyltransferases (NATs). Methionine acidic N termini, including actin, are cotranslationally Nt acetylated by NatB in all eukaryotes, but animal actins containing acidic N termini, are additionally posttranslationally Nt acetylated by NAA80. Actin Nt acetylation was found to regulate cytoskeletal dynamics and motility, thus making NAA80 a potential target for cell migration regulation. In this work, we developed potent and selective bisubstrate inhibitors for NAA80 and determined the crystal structure of NAA80 in complex with such an inhibitor, revealing that NAA80 adopts a fold similar to other NAT enzymes but with a more open substrate binding region. Furthermore, in contrast to most other NATs, the substrate specificity of NAA80 is mainly derived through interactions between the enzyme and the acidic amino acids at positions 2 and 3 of the actin substrate and not residues 1 and 2. A yeast model revealed that ectopic expression of NAA80 in a strain lacking NatB activity partially restored Nt acetylation of NatB substrates, including yeast actin. Thus, NAA80 holds intrinsic capacity to posttranslationally Nt acetylate NatB-type substrates in vivo. In sum, the presence of a dominant cotranslational NatB in all eukaryotes, the specific posttranslational actin methionine removal in animals, and finally, the unique structural features of NAA80 leave only the processed actins as in vivo substrates of NAA80. Together, this study reveals the molecular and cellular basis of NAA80 Nt acetylation and provides a scaffold for development of inhibitors for the regulation of cytoskeletal properties.

doi:10.1007/s12104-018-9811-x

Abstract

The ASHH2 CW domain is responsible for recognizing the methylation state at lysine 4 of histone 3 N-terminal tails and implicated in the recruitment of the ASHH2 methyltransferase enzyme correctly to the histones. The ASHH2 CW domain binds H3 lysine motifs that can be either mono-, di-, or tri-methylated [ARTK(meX)QTAR, where X denotes the number of methylations], but binds strongest to monomethylated instances (Kd values reported in the range of 1 µm to 500 nM). Hoppmann et al. published the uncomplexed NMR structure of an ASHH2 CW domain in 2011. Here we document the assignment of a shortened ASHH2 CW construct, CW42, with similar binding affinity and better expression yields than the one used to solve the uncomplexed structure. We also perform 1H–15N HSQC-monitored titrations that document at which protein–peptide ratios the complex is saturated. Backbone resonance assignments are presented for this shortened ASHH2 CW domain alone and bound to an H3 histone tail mimicking peptide monomethylated on lysine 4 (ARTK(me1)QTAR). Likewise, the assignment was also performed for the protein in complex with the dimethylated (ARTK(me2)QTAR) and trimethylated (ARTK(me3)QTAR) peptide. Overall, these two latter situations displayed a similar perturbation of shifts as the mono-methylated instance. In the case of the monomethylated histone tail mimic, side-chain assignment of CW42 in this complex was performed and reported in addition to backbone assignment, in preparation of a future solution structure determination and dynamics characterization of the CW42–ARTK(me1)QTAR complex.

doi:10.1016/j.carbpol.2017.12.041

Abstract

The aim of this paper was to perform a comprehensive characterization of polysaccharides isolated from the interior (IOI) and exterior (IOE) parts of the fungus Inonotus obliquus. Pre-extraction with DCM and MeOH, followed by water and alkali extraction and ethanol precipitation gave two water extracts and two alkali extracts. Neutral and acidic polysaccharide fractions were obtained after anion-exchange chromatography of the water extracts. The neutral polysaccharides (60-73 kDa) were heterogeneous and branched and consisted of a (1→3)-linked β-Glc backbone with (1→6)-linked kinks in the chain at approximately every fifth residue, with branches of (1→6)-linked β-Glc in addition to substantial amounts of (1→6)-linked α-Gal with 3-O-methylation at about every third Gal residue. The acidic polysaccharide fractions (10-31 kDa) showed similar structural motifs as the neutral fractions differing mainly by the presence of (1→4)-linked α-GalA and α-GlcA. β-Xyl, α-Man and α-Rha were also present in varying amounts in all fractions. No major structural differences between the IOI and IOE fractions were observed. An alkaline polysaccharide fraction (>450 kDa) was obtained from the IOI alkali extract, and consisted mainly of (1→3)- and (1→6)-linked β-Glc and (1→4)-linked β-Xyl. Several of the fractions showed in vitro immunomodulatory effect by increasing NO production in the murine macrophage and dendritic cell lines J774.A1 and D2SC/1. Most fractions managed to increase NO production only at the highest concentration tested (100 µg/ml), while the neutral fraction IOE-WN activated potent NO production at 10 µg/ml and was considered the most promising immunomodulating fraction in this study.

doi:10.1002/anie.201711006

Abstract

A convergent and stereoselective total synthesis of the previously assigned structure of azaspiracid-3 has been achieved via a late stage NHK coupling to form the C21‒C22 bond with the C20 configuration unambiguously established from L-(+)-tartaric acid. Post-coupling steps involved oxidation to an ynone, modified Stryker reduction of the alkyne, global deprotection, and oxidation of the primary alcohol to the carboxylic acid. The synthetic product matched naturally occurring azaspiracid-3 by mass spectrometry, but differed both chromatographically and spectroscopically.

doi:10.1002/anie.201711008

Abstract

The previously accepted structure of the marine toxin azaspiracid-3 is revised based upon an original convergent and stereoselective total synthesis of the natural product. The development of a structural revision hypothesis, its testing, and corroboration are reported. Synthetic (6R,10R,13R,14R,16R,17R, 19S,20S,21R,24S,25S,28S,30S,32R,33R,34R,36S,37S,39R)-azaspiracid-3 chromatographically and spectroscopically matched naturally occurring azaspiracid-3, whereas the previously assigned (20R)-epimer did not.

doi:10.1007/s12104-017-9772-5

Abstract

Human uracil N-glycosylase isoform 2—UNG2 consists of an N-terminal intrinsically disordered regulatory domain (UNG2 residues 1–92, 9.3 kDa) and a C-terminal structured catalytic domain (UNG2 residues 93–313, 25.1 kDa). Here, we report the backbone 1H, 13C, and 15N chemical shift assignment as well as secondary structure analysis of the N-and C-terminal domains of UNG2 representing the full-length UNG2 protein.

doi:10.1021/acs.organomet.7b00441

Abstract

Pyridine as a stabilizing donor ligand drastically improves the performance of ruthenium monothiolate catalysts for olefin metathesis in comparison with previous versions based on a stabilizing benzylidene ether ligand. The new pyridine-stabilized ruthenium alkylidenes undergo fast initiation and reach appreciable yields combined with moderate to high Z selectivity in self-metathesis of terminal olefins after only a few minutes at room temperature. Moreover, they can be used with a variety of substrates, including acids, and promote self-metathesis of ω-alkenoic acids. The pyridine-stabilized ruthenium monothiolate catalysts are also efficient at the high substrate dilutions of macrocylic ring-closing metathesis and resist temperatures above 100 °C during catalysis.

doi:10.1007/s12104-017-9759-2

Abstract

The apo-form of the 21.4 kDa catalytic domain and the 10.7 kDa carbohydrate binding domain of the AA10 family lytic polysaccharide monooxygenase ScLPMO10C from Streptomyces coelicolor have been isotopically labeled and recombinantly expressed in Escherichia coli. In this paper, we report the 1H, 13C, and 15N chemical shift assignments of each individual domain as well as an ensemble of the assignment for the full-length protein, including its approximately 30-amino acid long linker.

doi:10.1038/s41598-017-06855-z

Abstract

This paper reports that the abundances of endogenous cardiolipin and phosphatidylethanolamine halve during elongation of the Gram-positive bacterium Listeria innocua. The lyotropic phase behaviour of model lipid systems that describe these modulations in lipid composition indicate that the average stored curvature elastic stress of the membrane is reduced on elongation of the cell, while the fluidity appears to be maintained. These findings suggest that phospholipid metabolism is linked to the cell cycle and that changes in membrane composition can facilitate passage to the succeding stage of the cell cycle. This therefore suggests a means by which bacteria can manage the physical properties of their membranes through the cell cycle.

doi:10.1016/j.carbpol.2017.07.073

Abstract

Silver linden flowers contain different pectins (PSI-PSIII) with immunomodulating properties. PSI is a low-esterified pectic polysaccharide with predominant homogalacturonan region, followed by rhamnogalacturonan I (RGI) with arabinogalactan II and RGII (traces) domains. PSII and PSIII are unusual glucuronidated RGI polymers. PSIII is a unique high molecular weight RGI, having almost completely O-3 glucuronidated GalA units with >30% O-3 acetylation at the Rha units. Linden pectins induced reactive oxygen species (ROS) and NO generation from non-stimulated whole blood phagocytes and macrophages, resp., but suppressed OZP-(opsonized zymosan particles)-activated ROS generation, LPS-induced iNOS expression and NO production. This dual mode of action suggests their anti-inflammatory activity, which is known for silver linden extracts. PSI expressed the highest complement fixation and macrophage-stimulating activities and was active on intestinal Peyer{\textquoteright}s patch cells. PSIII was active on non-stimulated neutrophils, as it induced {\ss}2-integrin expression, revealing that acetylated and highly glucuronidated RGI exhibits immunomodulating properties via phagocytes.

doi:10.1016/j.carbpol.2017.07.011

Abstract

Two pectic (chPS-L1, chPS-L2) and one polyphenolic (chPP-L) fractions were obtained from lavender flowers after boiling water extraction, exhaustive removing of alcohol-soluble molecules and SEC. chPS-L1 (52.4kDa) contains mainly low-acetylated and high-methoxylated homogalacturonans (HG), and smaller rhamnogalacturonan (RG) I backbone fragments rich in 1,3,5-branched arabinan and arabinogalactan (AG) II side chains. chPS-L2 (21.8kDa) contains predominantly similarly esterified HG, followed by RGI with AGII structures and RGII. The prevalence of catechin and epicatechin in chPP-L indicates that they form weak interactions with pectins. chPS-L1 and chPS-L2 enhanced ß2-integrin expression on neutrophils, inducing ROS generation and macrophage NO production. Both the effects on ß2-integrin and high complement fixation activity of chPS-L1 were proposed for its inhibitory action against PMA- and OZP-activated ROS formation. This, together with suppression of NO generation after co-stimulation with chPS-L1 and LPS, suggested anti-inflammatory activity of studied pectins. Lavender polysaccharides expressed intestinal Peyer’s patch immunomodulating activity.

doi:10.1038/s41598-017-02382-z

Abstract

Chitin is an important structural component of numerous fungal pathogens and parasitic nematodes. The human macrophage chitotriosidase (HCHT) is a chitinase that hydrolyses glycosidic bonds between the N-acetyl-D-glucosamine units of this biopolymer. HCHT belongs to the Glycoside Hydrolase (GH) superfamily and contains a well-characterized catalytic domain appended to a chitin-binding domain (ChBDCHIT1). Although its precise biological function remains unclear, HCHT has been described to be involved in innate immunity. In this study, the molecular basis for interaction with insoluble chitin as well as with soluble chito-oligosaccharides has been determined. The results suggest a new mechanism as a common binding mode for many Carbohydrate Binding Modules (CBMs). Furthermore, using a phylogenetic approach, we have analysed the modularity of HCHT and investigated the evolutionary paths of its catalytic and chitin binding domains. The phylogenetic analyses indicate that the ChBDCHIT1 domain dictates the biological function of HCHT and not its appended catalytic domain. This observation may also be a general feature of GHs. Altogether, our data have led us to postulate and discuss that HCHT acts as an immune catalyser.

doi:10.1016/j.tet.2017.05.057

Abstract

We here report the preparation of a new 2,6,8-trisubstituted bicyclic tripeptidomimetic scaffold through TFA-mediated cyclization of a linear precursor containing three side chains. The introduction of a triphenylmethyl-protected thiol into carboxylic acid containing building blocks through sulfa Michael additions onto α,β-unsaturated hexafluoroisopropyl esters is described. The stereoselectivity of the bicycle formation was found to be somewhat lower than that previously reported for analogous 3,6,8-trisubstituted scaffolds. Moreover, the configuration of the linear precursor directs the stereochemical outcome of the cyclization differently when the R1 side chain is positioned on C2 in the bicycles (present work) instead of C3 (previous work). Tripeptidomimetic compounds based on the new scaffold were synthesized and evaluated for antagonistic potency toward CXCR4, and one compound (45a) displayed similar activity to earlier reported 3,6,8-tripeptidomimetic bicycles.

doi:10.1039/C6CP08506G

Abstract

Mixtures of surfactants and medium chained alcohols display an anomalous phase behaviour, with the formation of swollen micelles in mid-range surfactant concentrations, which transition into larger non-swollen aggregates when the surfactant concentration increases above a critical point. These alcohols also affect the adsorption behaviour of the surfactants. In this study, intermolecular proximities are measured for such systems by 1H–1H NMR dipolar correlation experiments, giving molecular localizations. The medium chained 1-heptanol and an anionic surfactant sodium dodecyl sulphate (SDS) are studied, both solubilized and adsorbed on alumina. Nuclear Overhauser Effect Spectroscopy (NOESY) shows that 1-heptanol localizes in both the palisade layer and in the core of SDS micelles when the 1-heptanol : SDS mole ratio increases beyond 2. The micelle diameter then increases with increasing 1-heptanol : SDS mole ratios due to more 1-heptanol partitioning in the micelle interior. When the micelle diameter increases beyond ∼6 nm, some SDS moves into the micelle interior, which may be a driving force for the structural transition at higher SDS concentrations. After being adsorbed on alumina, 1H–1H double-quantum magic angle spinning (DQ MAS) shows that SDS/1-heptanol bilayers are formed where 1-heptanol localizes in the palisade layer only, but with slightly different localizations compared to that in micelles. Three different 1-heptanol environments are identified on the surface by 2H NMR using 2H labelled 1-heptanol. However, in contrast to in solution, no 1-heptanol adsolubilizes in the bilayer interior.

doi:10.1016/j.carres.2017.02.003

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are key enzymatic players of lignocellulosic biomass degradation processes. As such, they have been introduced in cellulolytic cocktails for more efficient and less expensive lignocellulose saccharification. The recombinant production of LPMOs in bacteria for scientific investigations using vectors typically based on the T7 and lacUV5 promoters has been hampered by low yields. Reasons for this have been catabolite repression when producing the proteins in defined media with glucose as the sole carbon source, as well as the lack of an inducible expression system that allows controlled production of LPMOs that are correctly processed during translocation to the periplasmic space. A cassette vector design containing the XylS/Pm system was constructed and evaluated, showing that the expression cassette could easily be used for exchanging LPMO coding genes with or without signal sequences. The cassette was shown to reliably produce mature (translocated) LPMOs under controlled conditions that were achieved by using a low dosage (0.1 mM) of the Pm inducer m-toluic acid and a low (16 °C) cultivation temperature after induction. Furthermore, the signal sequences of five bacterial LPMOs were tested, and the signal sequence of LPMO10A from Serratia marcescens was found to give highest levels of recombinant protein production and translocation. The LPMO expression cassette was also evaluated in cultivations using defined media with glucose as the sole carbon source with a product yield of 7–22 mg per L of culture in shaking flasks. The integrity of the recombinant proteins were analyzed using NMR spectroscopy, showing that the system produced correctly processed and folded LPMOs. Finally, high cell-density cultivations of the recombinant strains were carried out, demonstrating stable protein production levels at similar relative yields (42–1298 mg per L of culture; 3.8–11.6 mg per OD600nm unit) as in shaking flasks, and showing the scale-up potential of the system.

doi:10.22203/ecm.v033a06

Abstract

Loss of articular cartilage from ageing, injury or degenerative disease is commonly associated with inflammation, causing pain and accelerating degradation of the cartilage matrix. Sulphated glycosaminoglycans (GAGs) are involved in the regulation of immune responses in vivo, and analogous polysaccharides are currently being evaluated for tissue engineering matrices to form a biomimetic environment promoting tissue growth while suppressing inflammatory and catabolic activities. Here, we characterise physical properties of sulphated alginate (S-Alg) gels for use in cartilage engineering scaffolds, and study their anti-inflammatory effects on encapsulated chondrocytes stimulated with IL-1β. Sulphation resulted in decreased storage modulus and increased swelling of alginate gels, whereas mixing highly sulphated alginate with unmodified alginate resulted in improved mechanical properties compared to gels from pure S-Alg. S-Alg gels showed extensive anti-inflammatory and anti-catabolic effects on encapsulated chondrocytes induced by IL-1β. Cytokine-stimulated gene expression of pro-inflammatory markers IL-6, IL-8, COX-2 and aggrecanase ADAMTS-5 were significantly lower in the sulphated gels compared to unmodified alginate gels. Moreover, sulphation of the microenvironment suppressed the protein expression of COX-2 and NF-κB as well as the activation of NF-κB and p38-MAPK. The sulphated alginate matrices were found to interact with IL-1β, and proposed to inhibit inflammatory induction by sequestering cytokines from their receptors. This study shows promising potential for sulphated alginates in biomimetic tissue engineering scaffolds, by reducing cytokine-mediated inflammation and providing a protective microenvironment for encapsulated cells.

doi:10.1016/j.orggeochem.2016.08.016

Abstract

Four hexacyclic oleananes having characteristic mass spectra with an intense fragment at m/z 325 were found in Late Cretaceous/Tertiary terrigenous oils and immature source rocks containing 18α-oleanane and other saturated pentacyclic compounds of higher plant origin. They are sometimes as abundant as 18α-oleanane itself. They were not found in oils without oleanane. The major hexacyclic oleanane, comprising 50-80% of the hexacyclic oleananes, was isolated using high performance liquid chromatography (HPLC) and characterized using nuclear magnetic resonance spectroscopy (NMR) as 18α-oleanane with the Me group at C-10 shifted to C-9 and with the Me at C-14 creating a new methylene bridge to C-10. Hexacyclic oleananes were not visible in m/z 191 chromatograms. They are most effectively detected by way of GC-MS-MS using the m/z 410 → 325 transition. Since few other compounds produce any m/z 410 → 325 signal, they may sometimes be easier to detect than regular and rearranged oleananes in samples with a low concentration of angiosperm markers. Natural precursor products have not been identified among plant triterpenoids, but might be related to (poly)functionalized oleanoids. Oleanoids hydroxylated at C-27 are obvious precursors. Oxic/clay-rich depositional environments seem to favour the formation of hexacyclic oleananes. A series of 2-alkylated oleananes had previously been characterized using NMR. Their hexacyclic counterparts were tentatively assigned in this study.

doi:10.1021/acs.jafc.6b02853

Abstract

Glutathione (GSH) conjugates of the mycotoxin 4-deoxynivalenol (DON), 1, have been detected in plants by LC-MS, but their identities were not confirmed due to a lack of standards. We have synthesized DON-GSH conjugates in alkaline solution. The major products 2 and 5 were isolated and their structures determined by mass spectrometry and NMR spectroscopy as GSH adducts at C-13 and C-10 (via epoxide and Michael addition, respectively) of 1. Other Michael addition products were also tentatively identified by LC-MS. Concentrations of 2 and 5 were determined by quantitative NMR and are suitable for use as quantitative standards for LC-MS studies of plant and animal metabolism of 1. LC-MS showed that in the presence of human glutathione S-transferases of the alpha and mu classes, the reaction of DON and GSH proceeded with a half-life of 17 h, identical with the rate of the uncatalyzed reaction rate, indicating an absence of catalysis.

doi:10.1021/acs.energyfuels.6b00799

Abstract

A series of novel C33–C35 hexacyclic benzohopanes (C33b-C35b) were identified in 39 samples of coal extracts and 39 crude oils of different ages from all over the world. C33b and C34b homologues were isolated, and their structures were determined by nuclear magnetic resonance. The structure of C35b benzohopane was proposed based on the mass spectrum and its similarity with the mass spectra of structurally defined C33b and C34b homologues. The structures of the C33b-C35b hexacyclic benzohopanes are closely related to isohopanes; both groups are typical for terrestrial organic matter and can be useful in the correlation analysis. A possible pathway of formation of these novel benzohopanes and their hopanoid precursors with an additional branch in the aliphatic side chain is proposed. C33b–C35b hexacyclic benzohopanes are stable up to the maturity level corresponding to random vitrinite reflectance (Rr) of ∼0.80%, which was demonstrated by analyzing the samples of different maturity and by the maturation simulation experiments: hydrous pyrolysis of two bituminous coals (Rr = 0.55 and 0.59%) and pyrolysis of an extracted bituminous coal (Rr = 0.56%) and its asphaltenes. This represents a confirmation that the formation of these novel benzohopanes is related to specific depositional conditions and microbial activity during diagenesis. Mature samples (Rr ≥ 0.8%) and hydrous pyrolysate of the bituminous coals (Rr < 0.60%) obtained at 330 °C show a distinct distribution of benzohopanes in comparison to immature and moderately mature samples, which is characterized by a low abundance of the "b" series benzohopanes and the presence of regular and numerous other benzohopane isomers. The latter most likely represent isomers of regular and novel benzohopanes with different substitution patterns on the aromatic ring. This isomerization of alkyl groups attached to the aromatic ring, leading to the formation of thermodynamically more stable isomers, is a well-known maturation scenario so far reported in the series of alkylated naphthalenes, phenanthrenes, and dibenzothiophenes. Therefore, in the same way, a distribution of benzohopanes can indicate thermal maturity. In addition to the novel benzohopanes, three series (2α, 2β, and 3β) of their methylated derivatives were identified in numerous samples. Finally, a novel C35 heptacyclic benzohopane with an additional cyclopentane ring was also observed in the studied samples, and its structure was tentatively identified based on the mass spectrum. Opposite to the hexacyclic C33b–C35b benzohopanes, the formation of the C35 heptacyclic benzohopane does not require a specific hopanoid precursor with two branches in the side chain. Therefore, this compound seems to have less geochemical significance than the new hexacyclic benzohopanes.

doi:10.1073/pnas.1602566113

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds using molecular oxygen and an external electron donor. We have used NMR and isothermal titration calorimetry (ITC) to study the interactions of a broad-specificity fungal LPMO, NcLPMO9C, with various substrates and with cellobiose dehydrogenase (CDH), a known natural supplier of electrons. The NMR studies revealed interactions with cellohexaose that center around the copper site. NMR studies with xyloglucans, i.e., branched β-glucans, showed an extended binding surface compared with cellohexaose, whereas ITC experiments showed slightly higher affinity and a different thermodynamic signature of binding. The ITC data also showed that although the copper ion alone hardly contributes to affinity, substrate binding is enhanced for metal-loaded enzymes that are supplied with cyanide, a mimic of O2 (-) Studies with CDH and its isolated heme b cytochrome domain unambiguously showed that the cytochrome domain of CDH interacts with the copper site of the LPMO and that substrate binding precludes interaction with CDH. Apart from providing insights into enzyme-substrate interactions in LPMOs, the present observations shed new light on possible mechanisms for electron supply during LPMO action.

doi:10.1007/s12104-016-9683-x

Abstract

The apo-form of the 23.3 kDa catalytic domain of the AA9 family lytic polysaccharide monooxygenase NcLPMO9C from Neurospora crassa has been isotopically labeled and recombinantly expressed in Pichia pastoris. In this paper, we report the 1H, 13C, and 15N chemical shift assignments of this LPMO.

doi:10.1016/j.carbpol.2016.03.052

Abstract

A comprehensive characterization of polysaccharides from the chanterelle was performed. Experiments included both linkage analysis by methylation and GC-MS, monosaccharide composition analysis by methanolysis, SEC-MALLS and several NMR experiments including COSY, HSQC, TOCSY, HSQC-TOCSY, NOESY, and HMBC. A 671kDa (1→6)-linked α-d-mannan with single and short (1→2)-linked side chains (WCcF1b) was isolated from the hot water extract, after protease treatment and fractionation by size exclusion chromatography. The hot 1M NaOH extract contained two types of β-glucan; a water soluble (1→6)-linked β-d-glucan with single and short (1→3)-linked β-glucan side chains (ACcSw) and a less branched (1→3)-linked β-d-glucan (ACcIw/ACcId), proposed to contain short side chains in O-4. Chemical shifts assignments of the α-mannan and β-glucan are presented as well as chemical shift values obtained from a (1→6)/(1→3)-β-d-glucan derived from Saccharomyces cerevisiae which was used as reference.

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