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4,4'-Bis(2,6-difluoropyridin-4-yl)-1,1':4',1 ''-terphenyl (BDFPTP) molecules underwent dehydrocyclization and covalent coupling reactions on Au(111) according to scanning tunneling microscopy (STM) measurements and density functional theory (DFT) calculations. Self-assembly of the reactants in well-defined molecular domains prior to reaction could greatly enhance the regioselectivity of the dehydrocyclization reaction and suppress defluorinated coupling, demonstrating that self-assembly can efficiently steer on-surface reactions. Such a strategy could be of great importance in surface chemistry and widely applied to control on-surface reactions.

The aim of the present study was to design and synthesize a new class of compounds for the softening of hard water, i.e. for the removal of the divalent cations Ca2+ and Mg2+. To that end, a class of alternating aliphatic polyketones (copolymers of ethylene and propylene with carbon monoxide, PK30) was functionalized with a variety of amines employing the Paal-Knorr reaction, a relatively straightforward reaction-route to synthesize functional polyketones. The amino groups included aliphatic and aromatic structures with a molecular weight ranging from 74.1 to 129.2 g/mol. Elemental analysis was used to establish the degree of functionalization, whereas 1H-NMR spectroscopy was used to identify the molecular structure of the prepared polymers. Model compounds were used as reference for guiding structure determination and for studying the (relative) reaction kinetics.

The tetrodecamycins are a group of secondary metabolites that are characterized by the presence of a tetronate ring in their structure. Originally discovered for their antibiotic activity against Photobacterium damselae ssp. piscicida, the causative agent of pseudotuberculosis in fish, this family of molecules has also been shown to have potent antibiotic activity against methicillin-resistant Staphylococcus aureus. Due to their small size and highly cyclized nature, they represent an unusual member of the much larger group of bioactive molecules called the tetronates. Herein, we review what is known about the mechanism of action of these molecules and also present a hypothesis for their biosynthesis. A deeper understanding of the tetrodecamycins will provide a more holistic view of the tetronate-family, provide new chemical probes of bacterial biology, and may provide therapeutic lead molecules.

Covalent inhibition has undergone a resurgence and is an important modern-day drug design and chemical biology approach. To avoid off-target interactions and to fine-tune reactivity, the ability to accurately predict reactivity is vitally important for the design and development of safer and more effective covalent drugs. Several ligand-only metrics have been proposed that promise quick and simple ways of determining covalent reactivity. In particular, we examine proton affinity and reaction energies calculated with the density functional B3LYP-D3/6-311+G**//B3LYP-D3/6-31G* method to assess the reactivity of a series of α,β-unsaturated carbonyl compounds that form covalent adducts with cysteine. We demonstrate that while these metrics correlate well with experiment for a diverse range of small reactive molecules these approaches fail for predicting the reactivity of drug-like compounds. We conclude that ligand-only metrics such as proton affinity and reaction energies do not capture determinants of reactivity in situ and fail to account for important factors such as conformation, solvation, and intermolecular interactions.

Enteropeptidase, localized into the duodenum brush border, is a key enzyme catalyzing the conversion of pancreatic trypsinogen proenzyme to active trypsin, thereby regulating protein digestion and energy homeostasis. We report the discovery and pharmacological profiles of SCO‐792, a novel inhibitor of enteropeptidase. A screen employing fluorescence resonance energy transfer was performed to identify enteropeptidase inhibitors. Inhibitory profiles were determined by in vitro assays. To evaluate the in vivo inhibitory effect on protein digestion, an oral protein challenge test was performed in rats. Our screen identified a series of enteropeptidase inhibitors, and compound optimization resulted in identification of SCO‐792, which inhibited enteropeptidase activity in vitro, with IC 50 values of 4.6 and 5.4 nmol/L in rats and humans, respectively. In vitro inhibition of enteropeptidase by SCO‐792 was potentiated by increased incubation time, and the calculated K inact/KI was 82 000/mol/L s. An in vitro dissociation assay showed that SCO‐792 had a dissociation half‐life of almost 14 hour, with a calculated k off rate of 0.047/hour, which suggested that SCO‐792 is a reversible enteropeptidase inhibitor. In normal rats, a ≤4 hour prior oral dose of SCO‐792 effectively inhibited plasma elevation of branched‐chain amino acids in an oral protein challenge test, which indicated that SCO‐792 effectively inhibited protein digestion in vivo. In conclusion, our new screen system identified SCO‐792 as a potent and reversible inhibitor against enteropeptidase. SCO‐792 slowly dissociated from enteropeptidase in vitro and inhibited protein digestion in vivo. Further study using SCO‐792 could reveal the effects of inhibiting enteropeptidase on biological actions.

Recently, DNA-PAINT single molecule localisation microscopy (SMLM) has shown great promise for quantitative imaging. However, labelling strategies so far have relied on approaches that are multivalent or affinity-based. Here, we demonstrate tagPAINT - the covalent labelling of expressed protein tags (SNAP tag and Halo tag) with single DNA docking strands for single molecule localisation microscopy via DNA-PAINT. We utilised tagPAINT for T-cell receptor signalling proteins at the immune synapse as a proof of principle.

A series of amphiphilic polymers bearing aliphatic secondary amines and hydroxyl groups have been synthesized showing different hydrophilic/hydrophobic balance. The synthesis is performed through the Paal-Knorr modification of a polyketone comprising both ethylene and propylene comonomers with N-(2-hydroxyethyl)ethylenediamine. The values of dicarbonyl conversion achieved were 19, 35, 51, and 63%, which allowed controlling the amphiphilia of the polymers: a lower carbonyl conversion degree implies a higher hydrophobia. On the other hand, photodegradation studies of a model nanosized pollutant pigment comprised of 1,3,5-triphenylformazan nanoparticles stabilized by poly(sodium 4-styrenesulfonate) have been performed in the absence and in the presence of the photocatalyst 5,10,15,20-tetrakis-(4-sulfonatophenyl)porphyrin, showing no catalytic action, since electrostatic repulsion minimize molecular contacts between the reactants. However, the synthesized polymers allow overcoming this problem. Due to their amphiphila, the polymers showing dicarbonyl conversion values of 35, 51, and 63% form complexes with the porphyrin and stabilize its non-self-aggregated tetraanionic form in water from basic pH up to pH 1.74, 1.82, and 2.76, respectively, the differences related with the polymeric relative hydrophilic/hydrophobic balance. Only the amphiphilic polymer showing a conversion degree of 35% acts as an adequate vehicle for the dye to photocatalyze the oxidation of 1,3,5-triphenylformazan confined in the nanoparticles, highlighting the potential of the Paal-Knorr modification of polyketones to achieve a fine tunning of polymeric properties to obtain a specific functionality: the positive charge of the complex and the high hydrophobia of the tuned polymer allow, respectively, attractive long-range electrostatic interactions with the nanoparticles and diffusion of the reactants into the nanoparticle hydrophobic environment.

YÖK Tez ID: 617121 Bu çalışmada lakkaz enziminin karbodimit (EDC) ve hidroksisüksinimid (NHS) ile aktifleştirilmiş amin fonksiyonel silika nanotaneciklere kovalent bağlanma yöntemi ile immobilizasyonu gerçekleştirildi. İmmobilize enzimlerin özellikleri ve enzimatik renk giderimi araştırıldı ve serbest lakkaz enzim ile karşılaştırıldı. Michaelis-Menten sabiti (Km) ve maksimum reaksiyon hızı (Vmax) değerleri sırasıyla serbest enzim için 5,14 mM ve 1,217 mM.dakika-1 olarak bulundu. EDC ile aktifleştirilmiş immobilize enzim için Km ve Vmax değerleri de sırasıyla 21,28 mM ve 0,611 mM.dakika-1, NHS ile aktifleştirilmiş immobilize enzim için 21,12 mM ve 0,686 mM.dakika-1 olarak bulundu. Optimum pH değerleri serbest lakkaz için 5,0 ve immobilize enzimler için 6,0 olarak bulundu. Optimum sıcaklık serbest lakkaz ve immobilize enzimler için 40 oC olarak bulundu. + 4 °C' da 60 günlük depolama süresinin sonrasında serbest enzimin bağıl aktivitesindeki düşüş % 93 iken, EDC ve NHS ile immobilize enzimlerin aktivitelerindeki düşüş ise sırasıyla % 58 ve % 62,5 idi. EDC ve NHS kullanılarak hazırlanan immobilize enzimlerin 15 kez tekrar kullanım sonrasında orijinal aktivitelerini sırasıyla % 89 ve % 87 koruduğu gözlenmiştir. Metil oranjın renginin giderilmesinde kullanılan serbest ve EDC ve NHS ile immobilize enzimlerin renk giderme yüzdeleri sırasıyla % 59,4, % 69, % 67 olarak bulundu. In this study, the laccase enzyme was immobilized covalent bonding method to amin funtionalized silica nanoparticles preactivated with carbodimide (EDC) and hydroxysuccinimide (NHS). Properties of immobilized enzyme and enzymatic discoloration were investigated and compared with free laccase enzyme. The Michaelis-Mentenconstant (Km) and maximum reaction rate (Vmax) values were found to be 5.14 Mm and 1.217 mM.min-1 for the free enzyme, respectively. Km and Vmax values were of 21.28 mM and 0.611 mM.min-1, for immobilized enzyme with preactivated EDC, 21.12 mM and 0.686 mM.min-1, for immobilized enzyme with preactivated NHS, respectively. Optimum pH values were determined as 5,0 for free laccase and 6,0 for immobilized enzymes. Optimum temperature determined as 40 oC for free laccase and immobilized enzymes, respectively. After 60-days of storage period at + 4 oC, while the decreasing of the activity of free enzyme was of 93 %, the decreasing of the activities of immobilized enzyme with EDC and NHS were of were 58 % and 62,5 %, respectively. It was observed that after 15 reusing of the immobilized enzyme susing EDC and NHS, which retained their original activities of 89 % and 87 %, respectively. The decolorization percentages of the free and immobilized enzymes with EDC and NHS, which were used to decolorize the methyl orange, were determined as 59,4 %, 69 % and 67 %, respectively.

Single-chain polymer nanoparticles (SCNPs) obtained through chain collapse by intramolecular cross-linking are attracting increasing interest as components of all-polymer nanocomposites, among other applications. We present a dielectric relaxation study on the dynamics of mixtures of poly(vinyl methyl ether) (PVME) and polystyrene (PS)-based SCNPs with various compositions. Analogous dielectric measurements on a miscible blend of PVME with the linear precursor chains of the SCNPs are taken as reference for this study. Both systems present completely different behaviors: While the blend with the linear precursor presents dynamics very similar to that reported for PVME/PS miscible blends, in the PVME/SCNP mixtures there are an appreciable amount of PVME segments that are barely affected by the presence of SCNPs, which nearly vanishes only for mixtures with high SCNP content. Interestingly, in the frame of a simple two-phase system, our findings point towards the existence of a SCNP-rich phase with a constant PVME fraction, regardless of the overall concentration of the mixture. Moreover, the dynamics of the PVME segments in this SCNP-rich phase display an extreme dynamic heterogeneity, a signature of constraint effects., This research was funded by Eusko Jaurlaritza project code: IT-654-13 and the Ministerio de Economía y Competitividad project code: MAT2015-63704-P (MINECO/FEDER, UE).

In this work, we prepared electrically conductive self-healing nanocomposites. The material consists of multi-walled carbon nanotubes (MWCNT) that are dispersed into thermally reversible crosslinked polyketones. The reversible nature is based on both covalent (Diels-Alder) and non-covalent (hydrogen bonding) interactions. The design allowed for us to tune the thermomechanical properties of the system by changing the fractions of filler, and diene-dienophile and hydroxyl groups. The nanocomposites show up to 1 &times, 104 S/m electrical conductivity, reaching temperatures between 120 and 150 &deg, C under 20&ndash, 50 V. The self-healing effect, induced by electricity was qualitatively demonstrated as microcracks were repaired. As pointed out by electron microscopy, samples that were already healed by electricity showed a better dispersion of MWCNT within the polymer. These features point toward prolonging the service life of polymer nanocomposites, improving the product performance, making it effectively stronger and more reliable.

In this work we present a reversible and toughened thermoset system based on the covalent incorporation of a furane functionalized ethylene-propylene rubber (EPM-Fu) into a thermoset furane functionalized polyketone (PK-Fu) via Diels-Alder (DA) reversible cross-linking with bismaleimide (b-MA). FT-IR and DSC analyses proved the reversible interaction between PK-Fu and EPM-Fu with b-Ma via DA and r-DA sequence. Likewise, thermo-mechanical experiments (DMTA) indicated the re-workability of the material with no evident differences in elastic and loss modulus after several heating cycles and recycling procedures. Moreover, a considerable increase in the softening point (tangent 6) was also found for the higher toughened system containing 12 wt% of EPM-Fu (neat thermoset T = 137 degrees C whereas toughened thermoset T = 155 degrees C). A two-fold increase in IZOD impact strength compared to the neat thermoset (up to 27 J/m) was also recorded by the toughened system. Overall, this approach clearly indicates that fully thermally reversible and toughened thermosets can be realized starting from mixtures of furan functionalized polyketone and EPM rubber, cross-linked via reversible Diels-Alder chemistry. (C) 2015 Elsevier Ltd. All rights reserved.

In the present work, microfibrillated cellulose (MFC) suspensions were produced by high-pressure homogenization and subsequently used to fabricate MFC membranes (C-1) by vacuum filtration followed by hot-pressing. A polyketone (PK50) was chemically modified by Paal-Knorr reaction to graft imidazole (IM) functional groups along its backbone structure. The resulting polymer is referred to as PK50IM80. By solution impregnation, C-1 was immersed in an aqueous solution of PK50IM80 and subsequently hot pressed, resulting in the fabrication of MFC/PK50IM80 composite membranes (C-IMP). Another method, referred to as solution mixing, consisted in adding MFC into an aqueous solution of PK50IM80 followed by vacuum filtration and hot-pressing to obtain MFC/PK50IM80 composite membranes (C-MEZC). C-IMP and C-MEZC were characterized by a wide range of analytical techniques including, X-ray photoelectron spectroscopy, Fourier-transform infrared chemical imaging, scanning electron microscopy, atomic force microscopy, dynamical mechanical analysis, tensile testing as well as streaming zeta potential, and compared to C-1 (reference material). The results suggested that C-IMP possess a more homogeneous distribution of PK50IM80 at their surface when compared to C-MEZC. C-IMP was found to possess significantly enhanced Young's modulus compared to C-1 and C-MEZC. The tensile strength of C-IMP was found to improve significantly compared to C-1, whereas C-1 possessed significantly higher tensile index than C-IMP and C-MEZC. Furthermore, the presence of PK50IM80 at the surface of MFC was found to significantly shift the isoelectric point (IEP) of the membranes from pH 2.3 to a maximum value of 4.5 for C-IMP. Above the IEP, C-IMP and C-MEZC were found to possess significantly less negative electrical surface charges (plateau value of −25 mV at pH 10) when compared to C-1 (plateau value of −42 mV at pH 10). Our approach may have implication to broaden the range of filtration applications of MFC-based membranes.

This study examines the preparation of electrically conductive polymer networks based on furan-functionalised polyketone (PK-Fu) doped with multi-walled carbon nanotubes (MWCNTs) and reversibly crosslinked with bis-maleimide (B-Ma) via Diels-Alder (DA) cycloaddition. Notably, the incorporation of 5 wt.% of MWCNTs results in an increased modulus of the material, and makes it thermally and electrically conductive. Analysis by X-ray photoelectron spectroscopy indicates that MWCNTs, due to their diene/dienophile character, covalently interact with the matrix via DA reaction, leading to effective interfacial adhesion between the components. Raman spectroscopy points to a more effective graphitic ordering of MWCNTs after reaction with PK-Fu and B-Ma. After crosslinking the obtained composite via the DA reaction, the softening point (tan(&delta, ) in dynamic mechanical analysis measurements) increases up to 155 &deg, C, as compared to the value of 130 &deg, C for the PK-Fu crosslinked with B-Ma and that of 140 &deg, C for the PK-Fu/B-Ma/MWCNT nanocomposite before resistive heating (responsible for crosslinking). After grinding the composite, compression moulding (150 &deg, C/40 bar) activates the retro-DA process that disrupts the network, allowing it to be reshaped as a thermoplastic. A subsequent process of annealing via resistive heating demonstrates the possibility of reconnecting the decoupled DA linkages, thus providing the PK networks with the same thermal, mechanical, and electrical properties as the crosslinked pristine systems.

P2X2 receptors (P2X2R) exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.

Highly pathogenic enteroviruses such as CVB3, EV D68 or EV71 have emerged as global threat for the human health with no treatment available to date. Polyprotein processing by the viral 3C protease (3Cpro) represents an essential step within the replication cycle of all enteroviruses, which spurred the development of anti-enterovirus agents by targeting the 3Cpro. Peptidomimetic analogues of the natural substrate are the most potent inhibitors developed so far. Yet, their inherent poor pharmacokinetic properties due to their peptidic nature limit clinical applicability. The primary aim of this thesis is the design of non-peptidic, covalent 3C protease inhibitors to cope with the impending threat of enterovirus infections. This was pursued through a computer-driven fragment-based approach in close collaboration with the group of Prof. Rademann. At first, the structural characteristics essential for covalent CVB3 3Cpro binding were derived from co-crystal structures and transferred into a 3D pharmacophore model, which served as basis for the subsequent fragment screening of an in-house small molecule collection. A virtual screening enriched selection of fragments was evaluated in a FRET-based enzyme kinetic assay, which led to the identification of a fragment hit with favorable 3Cpro binding characteristics. Due to its hydrolytic instability, scaffold hopping within commercial fragment space was pursued, which led the identification of additional 3Cpro binding fragments. Integration of protein mass spectrometry data into the established structural model enabled the rationalization of the fragment 3Cpro binding through a novel type of covalently binding substructure. For the fragment optimization, a rational de novo design workflow was developed, in which commercially available building blocks and synthesis oriented reaction rules are applied to grow the fragment within the active site of the 3Cpro in silico. Proposed fragment analogues were synthesized by the collaborating group according to the virtual design scheme, which led to the identification of 3Cpro inhibitors active in the micromolar range. In the second part of the thesis, the analysis of a noncompetitive 3Cpro inhibiting fragment, identified through the scaffold hopping, is described. Structure activity relationships derived from literature and through the biological evaluation of analogues were used for subsequent binding hypothesis generation. An allosteric pocket was identified through computational hot spot analysis and molecular dynamics simulations within the RNA binding site of the 3Cpro, which essential role in enterovirus genome replication provides biological relevance. A model for allosteric 3Cpro binding was developed through integration of the structure activity relationship data and statistical information of protein-ligand interaction patterns derived from molecular dynamics simulations. The reported findings provide a novel way to target the pivotal 3Cpro as our identified and rationally designed inhibitors represent a new structural basis for the development of non-peptidic anti-enteroviral drugs. In addition, the identified allosteric binding site enables a novel, complementing way to address this essential target and thus to combat enterovirus infections., Das Auftreten hochpathogener Enteroviren wie CVB3, EV D68 oder EV 71 stellt eine globale Gefahr für die menschliche Gesundheit dar. Die Spaltung des viralen Polyproteins durch die 3C Protease ist ein essentieller Schritt im viralen Replikationszyklus aller Enteroviren, weshalb sich die Entwicklung von Wirkstoffen auf die 3C Protease konzentriert hat. Die potentesten daraus hervorgegangenen Inhibitoren der 3C Protease sind Analoga des natürlichen Substrates, deren peptidische Struktur zu pharmakokinetischen Nachteilen führt und den klinischen Nutzen einschränkt. Das Ziel dieser Arbeit besteht in dem Design neuartiger, nicht-peptidischer Inhibitoren für die 3C Protease als Grundlage für Entwicklung anti-enteroviraler Wirkstoffe. Dies wurde über einen computergestützten, fragment-basierten Ansatz in Kooperation mit der Arbeitsgruppe von Prof. Rademann verfolgt. Zunächst wurden die für die kovalente CVB3 3C Protease Bindung essentiellen Strukturmerkmale aus Kristallstrukturen abgeleitet und in ein 3D-Pharmakophormodell überführt, das als Grundlage für ein sich anschließendes Fragment Screening einer in-house Molekülbibliothek diente. Die biologische Testung von durch das Bindungsmodell vorselektierten Fragmenten in einem FRET-basierten enzymkinetischen Assay lieferte einen Hit mit günstigen 3C Protease Bindungseigenschaften. Auf Grund dessen hydrolytischer Instabilität wurde eine Suche nach Strukturanaloga in Bibliotheken von kommerziell erhältlichen Fragmenten durchgeführt, die zur Identifizierung weiterer 3C Protease bindender Fragmente führte. Mit Hilfe massenspektrometrischer Daten konnte die Protein-Fragment Bindung über eine neuartige kovalent-bindende Substruktur aufgeklärt und eine rationale Bindungshypothese erstellt werden. Für die Optimierung der Fragmente wurde ein de novo-Design basierter Workflowentwickelt, in dem Moleküle in der aktiven Tasche der 3C Protease virtuell durch das Anknüpfen chemischer Bausteine optimiert werden. Vorgeschlagene Verbindungen wurden in der Kooperationsgruppe dem virtuellen Schema folgend synthetisiert und führten zur Identifizierung micro molarer 3C Protease Inhibitoren. Im zweiten Teil der Arbeit wird die Analyse eines nicht-kompetitiv bindenden Fragments beschrieben, welches ebenfalls im Rahmen der Suche nach Strukturanaloga identifiziert wurde. Strukturaktivitätsbeziehungen wurden durch die biologische Testung weiterer Analoga gewonnen oder der Literatur entnommen und für die anschließende Generierung von Bindungshypothesen verwendet. Eine allosterische Bindungsstelle wurde durch computer-basierte Hotspot Analysen und Molekulardynamik-Simulationen innerhalb der RNA-Bindungsdomäne der 3C Protease identifiziert, die aufgrund ihrer essentiellen Rolle für die Enterovirus Genomreplikation biologische Relevanz aufweist. Die Integration der erhaltenen Strukturaktivitätsbeziehungen und von Molekulardynamik-Simulationen abgeleiteten Protein-Ligand Interaktionsmustern ermöglichte die Generierung eines dynamischen Modells für die allosterische Bindung von Inhibitoren an die 3C Protease. Die im Rahmen dieser Arbeit identifizierten und rational entworfenen Inhibitoren ermöglichen einen neuen Ansatz zur Hemmung der 3C Protease für die Entwicklung nicht-peptidischer Wirkstoffe zur Therapie von Enterovirus Infektionen. Darüber hinaus ermöglicht die identifizierte allosterische Bindungsstelle eine ergänzende Möglichkeit, die Aktivität der 3C Protease zu modulieren.