Vacuum Low-Temperature Microwave-Assisted Pyrolysis of Technical Lignins.

Cleavage by microwave-assisted pyrolysis is a way to obtain higher-value organic chemicals from technical lignins. In this report, pine kraft lignin (PKL), spruce and beech organosolv lignin (SOSL and BOSL), and calcium lignosulfonates from spruce wood (LS) were pyrolyzed at temperatures between 30 and 280 °C using vacuum low-temperature, microwave-assisted pyrolysis. The mass balance, energy consumption, condensation rate, and pressure changes of the products during the pyrolysis process were recorded. Phenolic condensates obtained at different temperatures during pyrolysis were collected, and their chemical composition was determined by GC-MS and GC-FID. The origin of the technical lignin had a significant influence on the pyrolysis products. Phenolic condensates were obtained in yields of approximately 15% (PKL and SOSL) as well as in lower yields of 4.5% (BOSL) or even 1.7% (LS). The main production of the phenolic condensates for the PKL and SOSL occurred at temperatures of approximately 140 and 180 °C, respectively. The main components of the phenolic fraction of the three softwood lignins were guaiacol, 4-methylguaiacol, 4-ethylguaiacol, and other guaiacol derivatives; however, the quantity varied significantly depending on the lignin source. Due to the low cleavage temperature vacuum, low-temperature, microwave-assisted pyrolysis could be an interesting approach to lignin conversion.

Monomer Release from Dental Resins: The Current Status on Study Setup, Detection and Quantification for In Vitro Testing.

Improvements in mechanical properties and a shift of focus towards esthetic dentistry led to the application of dental resins in various areas of dentistry. However, dental resins are not inert in the oral environment and may release monomers and other substances such as Bisphenol-A (BPA) due to incomplete polymerization and intraoral degradation. Current research shows that various monomers present cytotoxic, genotoxic, proinflammatory, and even mutagenic effects. Of these eluting substances, the elution of BPA in the oral environment is of particular interest due to its role as an endocrine disruptor. For this reason, the release of residual monomers and especially BPA from dental resins has been a cause for public concern. The assessment of patient exposure and potential health risks of dental monomers require a reliable experimental and analytical setup. However, the heterogeneous study design applied in current research hinders biocompatibility testing by impeding comparative analysis of different studies and transfer to the clinical situation. Therefore, this review aims to provide information on each step of a robust experimental and analytical in vitro setup that allows the collection of clinically relevant data and future meta-analytical evaluations.

Revisiting Reduction of CO2 to Oxalate with First-Row Transition Metals: Irreproducibility, Ambiguous Analysis, and Conflicting Reactivity.

Construction of higher C≥2 compounds from CO2 constitutes an attractive transformation inspired by nature's strategy to build carbohydrates. However, controlled C-C bond formation from carbon dioxide using environmentally benign reductants remains a major challenge. In this respect, reductive dimerization of CO2 to oxalate represents an important model reaction enabling investigations on the mechanism of this simplest CO2 coupling reaction. Herein, we present common pitfalls encountered in CO2 reduction, especially its reductive coupling, based on established protocols for the conversion of CO2 into oxalate. Moreover, we provide an example to systematically assess these reactions. Based on our work, we highlight the importance of utilizing suitable orthogonal analytical methods and raise awareness of oxidative reactions that can likewise result in the formation of oxalate without incorporation of CO2. These results allow for the determination of key parameters, which can be used for tailoring of prospective catalytic systems and will promote the advancement of the entire field.

A comparative in vitro study on monomer release from bisphenol A-free and conventional temporary crown and bridge materials.

This study aimed to investigate the release of common monomers from two conventional and two bisphenol A (BPA)-free temporary crown and bridge materials. Cylindrical samples of all materials were prepared (N = 90; five samples for each material and cycle of analysis). All samples were immersed in high-performance liquid chromatography (HPLC)-grade water and incubated for 1 h, 12 h, 24 h, and 7 days in an incubation shaker at 37°C and 112 rpm. Extraction was performed in accordance with ISO 10993-12. Eluted monomers were detected and quantified by HPLC coupled with ultraviolet-visible spectroscopy and mass spectrometry (HPLC-UV/Vis-MS). Analysis of BPA was performed by HPLC coupled with ultraviolet-visible spectroscopy (HPLC-UV/Vis) and positive results were verified by HPLC-tandem mass spectrometry (HPLC-MS/MS). Neither bisphenol A-glycidyl methacrylate (Bis-GMA) nor BPA was quantifiable in any of the crown and bridge samples investigated in the present study. However, all samples contained triethylene glycol dimethacrylate (TEGDMA) and/or urethane dimethacrylate (UDMA) after 24 h of incubation. Statistical analysis showed that significantly more UDMA was released from the BPA-free materials than from the conventional materials. All concentrations of UDMA measured were below the effective cytotoxic concentrations previously reported. However, for a few materials, especially BPA-free temporary crown and bridge materials, the levels of UDMA were above previously reported potentially harmful concentrations for local cells. As BPA-free materials were introduced as being more biocompatible than materials containing BPA, substitution of Bis-GMA with UDMA should be further investigated.

Production of the Fragrance Geraniol in Peroxisomes of a Product-Tolerant Baker's Yeast.

Monoterpenoids, such as the plant metabolite geraniol, are of high industrial relevance since they are important fragrance materials for perfumes, cosmetics, and household products. Chemical synthesis or extraction from plant material for industry purposes are complex, environmentally harmful or expensive and depend on seasonal variations. Heterologous microbial production offers a cost-efficient and sustainable alternative but suffers from low metabolic flux of the precursors and toxicity of the monoterpenoid to the cells. In this study, we evaluated two approaches to counteract both issues by compartmentalizing the biosynthetic enzymes for geraniol to the peroxisomes of Saccharomyces cerevisiae as production sites and by improving the geraniol tolerance of the yeast cells. The combination of both approaches led to an 80% increase in the geraniol titers. In the future, the inclusion of product tolerance and peroxisomal compartmentalization into the general chassis engineering toolbox for monoterpenoids or other host-damaging, industrially relevant metabolites may lead to an efficient, low-cost, and eco-friendly microbial production for industrial purposes.

Supramolecular Self-Assembly of ß3 -Peptides Mediated by Janus-Type Recognition Units.

To gain mechanistic insights, natural systems with biochemical relevance are inspiring for the creation of new biomimetics with unique properties and functions. Despite progress in rational design and protein engineering, folding and intramolecular organization of individual components into supramolecular structures remains challenging and requires controlled methods. Foldamers, such as ß-peptides, are structurally well defined with rigid conformations and suitable for the specific arrangement of recognition units. Herein, we show the molecular arrangement and aggregation of ß3 -peptides into a hexameric helix bundle. For this purpose, ß-amino acid side chains were modified with cyanuric acid and triamino-s-triazine as complementary recognition units. The pre-organization of the ß3 -peptides leads these Janus molecule pairs into a hexameric arrangement and a defined rosette nanotube by stacking. The helical conformation of the subunits was indicated by circular dichroism spectroscopy, while the supramolecular arrangement was detected by dynamic light scattering and confirmed by high-resolution electrospray ionization mass spectrometry (ESI-HRMS).

Challenging the concept that eumelanin is the polymorphic brown banded pigment in Cepaea nemoralis.

The common grove snail Cepaea nemoralis displays a stable pigmentation polymorphism in its shell that has held the attention of scientists for decades. While the details of the molecular mechanisms that generate and maintain this diversity remain elusive, it has long been employed as a model system to address questions related to ecology, population genetics and evolution. In order to contribute to the ongoing efforts to identify the genes that generate this polymorphism we have tested the long-standing assumption that melanin is the pigment that comprises the dark-brown bands. Surprisingly, using a newly established analytical chemical method, we find no evidence that eumelanin is differentially distributed within the shells of C. nemoralis. Furthermore, genes known to be responsible for melanin deposition in other metazoans are not differentially expressed within the shell-forming mantle tissue of C. nemoralis. These results have implications for the continuing search for the supergene that generates the various pigmentation morphotypes.

Quantitation of eumelanin and pheomelanin markers in diverse biological samples by HPLC-UV-MS following solid-phase extraction.

Eumelanin and pheomelanin are well known and common pigments found in nature. However, their complex polymer structure and high thermostability complicate their direct chemical identification. A widely used analytical method is indirect determination using HPLC with UV detection of both types of melanin by their most abundant oxidation products: pyrrole-2,3-dicarboxylic acid (PDCA), pyrrole-2,3,5-tricarboxylic acid (PTCA), thiazole-4,5-dicarboxylic acid (TDCA), and thiazole-2,4,5-tricarboxylic acid (TTCA). An increasing interest in pigmentation in biological research led us to develop a highly sensitive and selective method to identify and quantify these melanin markers in diverse biological samples with complex matrices. By introducing solid-phase extraction (SPE, reversed-phase) following alkaline oxidation we could significantly decrease background signals while maintaining recoveries greater than 70%. Our HPLC-UV-MS method allows for confident peak identification via exact mass information in corresponding UV signals used for quantitation. In addition to synthetic melanin and Sepia officinalis ink as reference compounds eumelanin markers were detected in brown human hair and a brown bivalve shell (Mytilus edulis). Brown feathers from the common chicken (Gallus g. domesticus) yielded all four eumelanin and pheomelanin markers. The present method can be easily adapted for a wide range of future studies on biological samples with unknown melanin content.

Eumelanin and pheomelanin pigmentation in mollusc shells may be less common than expected: insights from mass spectrometry.

BACKGROUND: The geometric patterns that adorn the shells of many phylogenetically disparate molluscan species are comprised of pigments that span the visible spectrum. Although early chemical studies implicated melanin as a commonly employed pigment, surprisingly little evidence generated with more recent and sensitive techniques exists to support these observations. RESULTS: Here we present the first mass spectrometric investigations for the presence of eumelanin and pheomelanin in 13 different molluscan species from three conchiferan classes: Bivalvia, Cephalopoda and Gastropoda. In the bivalve Mytilus edulis we demonstrate that eumelanin mainly occurs in the outermost, non-mineralised and highly pigmented layer of the shell (often referred to as the periostracum). We also identified eumelanin in the shells of the cephalopod Nautilus pompilius and the marine gastropods Clanculus pharaonius and Steromphala adriatica. In the terrestrial gastropod Cepaea nemoralis we verify the presence of pheomelanin in a mollusc shell for the first time. Surprisingly, in a large number of brown/black coloured shells we did not find any evidence for either type of melanin. CONCLUSIONS: We recommend methods such as high-performance liquid chromatography with mass spectrometric detection for the analysis of complex biological samples to avoid potential false-positive identification of melanin. Our results imply that many molluscan species employ as yet unidentified pigments to pattern their shells. This has implications for our understanding of how molluscs evolved the ability to pigment and pattern their shells, and for the identification of the molecular mechanisms that regulate these processes.

Nanoscale Biodegradable Organic-Inorganic Hybrids for Efficient Cell Penetration and Drug Delivery.

We report a comprehensive study on novel, highly efficient, and biodegradable hybrid molecular transporters. To this end, we designed a series of cell-penetrating, cube-octameric silsesquioxanes (COSS), and investigated cellular uptake by confocal microscopy and flow cytometry. A COSS with dense spatial arrangement of guanidinium groups displayed fast uptake kinetics and cell permeation at nanomolar concentrations in living HeLa cells. Efficient uptake was also observed in bacteria, yeasts, and archaea. The COSS-based carrier was significantly more potent than cell-penetrating peptides (CPPs) and displayed low toxicity. It efficiently delivered a covalently attached cytotoxic drug, doxorubicin, to living tumor cells. As the uptake of fluorescently labeled carrier remained in the presence of serum, the system could be considered particularly attractive for the in vivo delivery of therapeutics.

"Reduced" Coumarin Dyes with an O-Phosphorylated 2,2-Dimethyl-4-(hydroxymethyl)-1,2,3,4-tetrahydroquinoline Fragment: Synthesis, Spectra, and STED Microscopy.

Large Stokes-shift coumarin dyes with an O-phosphorylated 4-(hydroxymethyl)-2,2-dimethyl-1,2,3,4-tetrahydroquinoline fragment emitting in the blue, green, and red regions of the visible spectrum were synthesized. For this purpose, N-substituted and O-protected 1,2-dihydro-7-hydroxy-2,2,4-trimethylquinoline was oxidized with SeO2 to the corresponding α,ß-unsaturated aldehyde and then reduced with NaBH4 in a "one-pot" fashion to yield N-substituted and 7-O-protected 4-(hydroxymethyl)-7-hydroxy-2,2-dimethyl-1,2,3,4-tetrahydroquinoline as a common precursor to all the coumarin dyes reported here. The photophysical properties of the new dyes ("reduced coumarins") and 1,2-dihydroquinoline analogues (formal precursors) with a trisubstituted C=C bond were compared. The "reduced coumarins" were found to be more photoresistant and brighter than their 1,2-dihydroquinoline counterparts. Free carboxylate analogues, as well as their antibody conjugates (obtained from N-hydroxysuccinimidyl esters) were also prepared. All studied conjugates with secondary antibodies afforded high specificity and were suitable for fluorescence microscopy. The red-emitting coumarin dye bearing a betaine fragment at the C-3-position showed excellent performance in stimulation emission depletion (STED) microscopy.

Transcription Factor SomA Is Required for Adhesion, Development and Virulence of the Human Pathogen Aspergillus fumigatus.

The transcription factor Flo8/Som1 controls filamentous growth in Saccharomyces cerevisiae and virulence in the plant pathogen Magnaporthe oryzae. Flo8/Som1 includes a characteristic N-terminal LUG/LUH-Flo8-single-stranded DNA binding (LUFS) domain and is activated by the cAMP dependent protein kinase A signaling pathway. Heterologous SomA from Aspergillus fumigatus rescued in yeast flo8 mutant strains several phenotypes including adhesion or flocculation in haploids and pseudohyphal growth in diploids, respectively. A. fumigatus SomA acts similarly to yeast Flo8 on the promoter of FLO11 fused with reporter gene (LacZ) in S. cerevisiae. FLO11 expression in yeast requires an activator complex including Flo8 and Mfg1. Furthermore, SomA physically interacts with PtaB, which is related to yeast Mfg1. Loss of the somA gene in A. fumigatus resulted in a slow growth phenotype and a block in asexual development. Only aerial hyphae without further differentiation could be formed. The deletion phenotype was verified by a conditional expression of somA using the inducible Tet-on system. A adherence assay with the conditional somA expression strain indicated that SomA is required for biofilm formation. A ptaB deletion strain showed a similar phenotype supporting that the SomA/PtaB complex controls A. fumigatus biofilm formation. Transcriptional analysis showed that SomA regulates expression of genes for several transcription factors which control conidiation or adhesion of A. fumigatus. Infection assays with fertilized chicken eggs as well as with mice revealed that SomA is required for pathogenicity. These data corroborate a complex control function of SomA acting as a central factor of the transcriptional network, which connects adhesion, spore formation and virulence in the opportunistic human pathogen A. fumigatus.

A Practical One-Pot Synthesis of Positron Emission Tomography (PET) Tracers via Nickel-Mediated Radiofluorination.

Invited for this months cover picture is the group of Professor Bernd Neumaier at the Institute of Radiochemistry and Experimental Molecular Imaging at the University Clinic of Cologne. The cover picture shows the differences in brain metabolism of a healthy young and a healthy old subject, as well as a patient suffering from Parkinsons disease (left to right) uncovered by 6-[(18)F]FDOPA-positron emission tomography (PET). Morbus Parkinson occurs when nerve cells that produce dopamine begin to die. The shortage of dopamine leads to movement problems in affected individuals. 6-[(18)F]FDOPA is extensively used to evaluate the progression of Parkinsons disease. Bold stick projections of this PET tracer, as well as a neuronal network, are seen in the background. Unfortunately, conventional procedures to produce 6-[(18)F]FDOPA are cumbersome. Thus, several recent developments aim at the simplification of this radiosynthesis. In our work, we studied the applicability of the recently reported Ni-mediated radiofluorination approach for daily routine production of 6-[(18)F]FDOPA. For more details, see the Full Paper on p. 457 ff.

A Practical One-Pot Synthesis of Positron Emission Tomography (PET) Tracers via Nickel-Mediated Radiofluorination.

Recently a novel method for the preparation of (18)F-labeled arenes via oxidative [(18)F]fluorination of easily accessible and sufficiently stable nickel complexes with [(18)F]fluoride under exceptionally mild reaction conditions was published. The suitability of this procedure for the routine preparation of clinically relevant positron emission tomography (PET) tracers, 6-[(18)F]fluorodopamine (6-[(18)F]FDA), 6-[(18)F]fluoro-l-DOPA (6-[(18)F]FDOPA) and 6-[(18)F]fluoro-m-tyrosine (6-[(18)F]FMT), was evaluated. The originally published base-free method was inoperative. However, a "low base" protocol afforded protected radiolabeled intermediates in radiochemical conversions (RCCs) of 5-18 %. The subsequent deprotection step proceeded almost quantitatively (>95 %). The simple one-pot two-step procedure allowed the preparation of clinical doses of 6-[(18)F]FDA and 6-[(18)F]FDOPA within 50 min (12 and 7 % radiochemical yield, respectively). In an unilateral rat model of Parkinsons disease, 6-[(18)F]FDOPA with high specific activity (175 GBq µmol(-1)) prepared using the described nickel-mediated radiofluorination was compared to 6-[(18)F]FDOPA with low specific activity (30 MBq µmol(-1)) produced via conventional electrophilic radiofluorination. Unexpectedly both tracer variants displayed very similar in vivo properties with respect to signal-to-noise ratio and brain distribution, and consequently, the quality of the obtained PET images was almost identical.

Locked by Design: A Conformationally Constrained Transglutaminase Tag Enables Efficient Site-Specific Conjugation.

Based on the crystal structure of a natural protein substrate for microbial transglutaminase, an enzyme that catalyzes protein crosslinking, a recognition motif for site-specific conjugation was rationally designed. Conformationally locked by an intramolecular disulfide bond, this structural mimic of a native conjugation site ensured efficient conjugation of a reporter cargo to the therapeutic monoclonal antibody cetuximab without erosion of its binding properties.

Genetically encoding lysine modifications on histone H4.

Post-translational modifications of proteins are important modulators of protein function. In order to identify the specific consequences of individual modifications, general methods are required for homogeneous production of modified proteins. The direct installation of modified amino acids by genetic code expansion facilitates the production of such proteins independent of the knowledge and availability of the enzymes naturally responsible for the modification. The production of recombinant histone H4 with genetically encoded modifications has proven notoriously difficult in the past. Here, we present a general strategy to produce histone H4 with acetylation, propionylation, butyrylation, and crotonylation on lysine residues. We produce homogeneous histone H4 containing up to four simultaneous acetylations to analyze the impact of the modifications on chromatin array compaction. Furthermore, we explore the ability of antibodies to discriminate between alternative lysine acylations by incorporating these modifications in recombinant histone H4.

Synthesis of ¹8F-labelled ß-lactams by using the Kinugasa reaction.

Owing to their broad spectrum of biological activities and low toxicity, ß-lactams are attractive lead structures for the design of novel molecular probes. However, the synthesis of positron emission tomography (PET)-isotope-labelled ß-lactams has not yet been reported. Herein, we describe the simple preparation of radiofluorinated ß-lactams by using the fast Kinugasa reaction between (18)F-labelled nitrone [(18)F]-1 and alkynes of different reactivity. Additionally, (18)F-labelled fused ß-lactams were obtained through the reaction of a cyclic nitrone 7 with radiofluorinated alkynes [(18)F]-6 a,b. Radiochemical yields of the Kinugasa reaction products could be significantly increased by the use of different Cu(I) ligands, which additionally allowed a reduction in the amount of precursor and/or reaction time. Model radiofluorinated ß-lactam-peptide and protein conjugates ([(18)F]-10 and (18)F-labelled BSA conjugate) were efficiently obtained in high yield under mild conditions (aq. MeCN, ambient temperature) within a short reaction time, demonstrating the suitability of the developed method for radiolabelling of sensitive molecules such as biopolymers.

Si-enterobactin from the endophytic Streptomyces sp. KT-S1-B5--a potential silicon transporter in Nature?

Si-enterobactin (2a), a hexacoordinated complex of the siderophore enterobactin (2b) with silicon as the central atom, was isolated from an endophytic Streptomyces sp. occurring in Piper guinensis roots. The structure and absolute configuration were determined from NMR and MS data, and by X-ray diffraction. The orientation of the molecule along the pseudo-3-fold axis shows that the coordination environment of the silicon atom complexed with three bidentate ligands is Δ. We assume that 2a or related complexes may be involved in the transport of silicon in plants, diatoms, or other silicon-dependent organisms.

Assembly and stepwise oxidation of interpenetrated coordination cages based on phenothiazine.

A breath of fresh air is sufficient for the eightfold S-monooxygenation of an interpenetrated double cage based on eight phenothiazine ligands and four square-planar-coordinated Pd(II) cations. Besides these two cages, which were both characterized by X-ray crystallography, an eightfold S-dioxygenated double-cage was obtained under harsher oxidation conditions.

Template control over dimerization and guest selectivity of interpenetrated coordination cages.

We have previously shown that the self-assembly of dibenzosuberone-based bis-monodentate pyridyl ligands L(1) with Pd(II) cations leads to the quantitative formation of interpenetrated coordination cages [BF4@Pd4L(1)8]. The BF4(-) anion inside the central cavity serves as a template, causing the outer two pockets to show a tremendous affinity for allosteric binding of two small chloride anions. Here we show that derivatization of the ligand backbone with a bulky aryl substituent allows us to control the dimerization and hence the guest-binding ability of the cage by the choice of the templating anion. Steric constraints imposed by L(2) prevent the large BF4(-) anion from serving as a template for the formation of interpenetrated double cages. Instead, a single isomer of the monomeric cage [Pd2L(2)4] is formed. Addition of the small anionic template Cl(-) permits dimerization, yielding the interpenetrated double cage [Cl@Pd4L(2)8], whose enlarged outer pockets show a preference for the binding of large anions such as ReO4(-).