Phosphono Bisbenzguanidines as Irreversible Dipeptidomimetic Inhibitors and Activity‐Based Probes of Matriptase‐2

Matriptase‐2, a type II transmembrane serine protease, plays a key role in human iron homeostasis. Inhibition of matriptase‐2 is considered as an attractive strategy for the treatment of iron‐overload diseases, such as hemochromatosis and β‐thalassemia. In the present study, synthetic routes to nine dipeptidomimetic inactivators were developed. Five active compounds ( 41 – 45 ) were identified and characterized kinetically as irreversible inhibitors of matriptase‐2. In addition to a phosphonate warhead, these dipeptides possess two benzguanidine moieties as arginine mimetics to provide affinity for matriptase‐2 by binding to the S1 and S3/S4 subpockets, respectively. This binding mode was strongly supported by covalent docking analysis. Compounds 41 – 45 were obtained as mixtures of two diastereomers and were therefore separated into the single epimers. Compound 45 A , with S configuration at the N‐terminal amino acid and R configuration at the phosphonate carbon atom, was the most potent matriptase‐2 inactivator with a rate constant of inactivation of 2790 m ^?1 s^?1 and abolished the activity of membrane‐bound matriptase‐2 on the surface of intact cells. Based on the chemotyp of phosphono bisbenzguanidines, the design and synthesis of a fluorescent probe ( 51 A ) by insertion of a coumarin label is described. The in‐gel fluorescence detection of matriptase‐2 was demonstrated by applying 51 A as the first activity‐based probe for this enzyme.     

Integration of the 1,2,3‐Triazole “Click” Motif as a Potent Signalling Element in Metal Ion Responsive Fluorescent Probes

In a systematic approach we synthesized a new series of fluorescent probes incorporating donor–acceptor (D‐A) substituted 1,2,3‐triazoles as conjugative π‐linkers between the alkali metal ion receptor N‐phenylaza‐[18]crown‐6 and different fluorophoric groups with different electron‐acceptor properties (4‐naphthalimide, meso‐phenyl‐BODIPY and 9‐anthracene) and investigated their performance in organic and aqueous environments (physiological conditions). In the charge‐transfer (CT) type probes 1 , 2 and 7 , the fluorescence is almost completely quenched by intramolecular CT (ICT) processes involving charge‐separated states. In the presence of Na⁺ and K⁺ ICT is interrupted, which resulted in a lighting‐up of the fluorescence in acetonitrile. Among the investigated fluoroionophores, compound 7 , which contains a 9‐anthracenyl moiety as the electron‐accepting fluorophore, is the only probe which retains light‐up features in water and works as a highly K⁺/Na⁺‐selective probe under simulated physiological conditions. Virtually decoupled BODIPY‐based 6 and photoinduced electron transfer (PET) type probes 3 – 5 , where the 10‐substituted anthracen‐9‐yl fluorophores are connected to the 1,2,3‐triazole through a methylene spacer, show strong ion‐induced fluorescence enhancement in acetonitrile, but not under physiological conditions. Electrochemical studies and theoretical calculations were used to assess and support the underlying mechanisms for the new ICT and PET 1,2,3‐triazole fluoroionophores.     

A Triazatruxene‐Based Glycocluster as a Fluorescent Sensor for Concanavalin A

A new triazatruxene‐based fluorescent glycocluster has been designed, synthesized, and fully characterized by NMR spectroscopy and mass spectrometry. Furthermore, its specific and selective binding properties with concanavalin A (Con A) have been investigated by fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and turbidity assay. The obtained results showed that the multivalent mannose‐modified triazatruxene exhibited specific binding with Con A, but no binding to peanut agglutinin (PNA) lectin or bovine serum albumin (BSA), corresponding to a two‐orders‐of‐magnitude higher affinity than that of monovalent mannose ligands. Most interestingly, a fluorescence enhancement of the triazatruxene‐based glycocluster was observed upon binding with Con A because of hydrophobic interactions involving sites close to the triazatruxene moiety. Furthermore, the inhibitory ability of the triazatruxene‐based glycocluster against ORN178‐ induced haemagglutination has been investigated by haemagglutination inhibition assay. The results indicated selective binding with ORN178.     

Bioorthogonal Probes for the Study of MDM2‐p53 Inhibitors in Cells and Development of High‐Content Screening Assays for Drug Discovery

To study the behavior of MDM2‐p53 inhibitors in a disease‐relevant cellular model, we have developed and validated a set of bioorthogonal probes that can be fluorescently labeled in cells and used in high‐content screening assays. By using automated image analysis with single‐cell resolution, we could visualize the intracellular target binding of compounds by co‐localization and quantify target upregulation upon MDM2‐p53 inhibition in an osteosarcoma model. Additionally, we developed a high‐throughput assay to quantify target occupancy of non‐tagged MDM2‐p53 inhibitors by competition and to identify novel chemical matter. This approach could be expanded to other targets for lead discovery applications.     

A Palette of Fluorescent Thiophene‐Based Ligands for the Identification of Protein Aggregates

By replacing the central thiophene unit of an anionic pentameric oligothiophene with other heterocyclic moities, a palette of pentameric thiophene‐based ligands with distinct fluorescent properties were synthesized. All ligands displayed superior selectivity towards recombinant amyloid fibrils as well as disease‐associated protein aggregates in tissue sections.     

A Covalent Reporter of β‐Lactamase Activity for Fluorescent Imaging and Rapid Screening of Antibiotic‐Resistant Bacteria

Bacterial resistance to antibiotics poses a great clinical challenge in fighting serious infectious diseases due to complicated resistant mechanisms and time‐consuming testing methods. Chemical reaction‐directed covalent labeling of resistance‐associated bacterial proteins in the context of a complicated environment offers great opportunity for the in‐depth understanding of the biological basis conferring drug resistance, and for the development of effective diagnostic approaches. In the present study, three fluorogenic reagents LRBL1–3 for resistant bacteria labeling have been designed and prepared on the basis of fluorescence resonance energy transfer (FRET). The hydrolyzed probes could act as reactive electrophiles to attach the enzyme, β‐lactamase, and thus facilitated the covalent labeling of drug resistant bacterial strains. SDS electrophoresis and MALDI‐TOF mass spectrometry characterization confirmed that these probes were sensitive and specific to β‐lactamase and could therefore serve for covalent and localized fluorescence labeling of the enzyme structure. Moreover, this β‐lactamase‐induced covalent labeling provides quantitative analysis of the resistant bacterial population (down to 5 %) by high resolution flow cytometry, and allows single‐cell detection and direct observation of bacterial enzyme activity in resistant pathogenic species. This approach offers great promise for clinical investigations and microbiological research.     

Total Synthesis and Activity of the Metallo‐β‐lactamase Inhibitor Aspergillomarasmine A

Resistance to β‐lactam antibiotics is mediated primarily by enzymes that hydrolytically inactivate the drugs by one of two mechanisms: serine nucleophilic attack or metal‐dependent activation of a water molecule. Serine β‐lactamases are countered in the clinic by several codrugs that inhibit these enzymes, thereby rescuing antibiotic action. There are no equivalent inhibitors of metallo‐β‐lactamases in clinical use, but the fungal secondary metabolite aspergillomarasmine A has recently been identified as a potential candidate for such a codrug. Herein we report the synthesis of aspergillomarasmine A. The synthesis enabled confirmation of the stereochemical configuration of the compound and offers a route for the synthesis of derivatives in the future.     

On Ammonia Binding to the Oxygen‐Evolving Complex of Photosystem II: A Quantum Chemical Study

A recent EPR study (M. Perrez Navarro et al., Proc. Natl. Acad. Sci.­ 2013 , 110, 15561) provided evidence that ammonia binding to the oxygen‐evolving complex (OEC) of photosystem II in its S₂ state takes place at a terminal‐water binding position (W1) on the “dangler” manganese center Mn_A. This contradicted earlier interpretations of ¹⁴N electron‐spin‐echo envelope modulation (ESEEM) and extended X‐ray absorption fine‐structure (EXAFS) data, which were taken to indicate replacement of a bridging oxo ligand by an NH₂ unit. Here we have used systematic broken‐symmetry density functional theory calculations on large (ca. 200 atom) model clusters of an extensive variety of substitution patterns and core geometries to examine these contradictory pieces of evidence. Computed relative energies clearly favor the terminal substitution pattern over bridging‐ligand arrangements (by about 20–30 kcal mol^?1) and support W1 as the preferred binding site. Computed ¹⁴N EPR nuclear‐quadrupole coupling tensors confirm previous assumptions that the appreciable asymmetry may be accounted for by strong, asymmetric hydrogen bonding to the bound terminal NH₃ ligand (mainly by Asp61). Indeed, bridging NH₂ substitution would lead to exaggerated asymmetries. Although our computed structures confirm that the reported elongation of an Mn–Mn distance by about 0.15 Å inferred from EXAFS experiments may only be reproduced by bridging NH₂ substitution, it seems possible that the underlying EXAFS data were skewed by problems due to radiation damage. Overall, the present data clearly support the suggested terminal NH₃ coordination at the W1 site. The finding is significant for the proposed mechanistic scenarios of OEC catalysis, as this is not a water substrate site, and effects of this ammonia binding on catalysis thus must be due to more indirect influences on the likely substrate binding site at the O5 bridging‐oxygen position.     

Site‐Directed Spin‐Labeling of DNA by the Azide–Alkyne ‘Click’ Reaction: Nanometer Distance Measurements on 7‐Deaza‐2′‐deoxyadenosine and 2′‐Deoxyuridine Nitroxide Conjugates Spatially Separated or Linked to a ‘dA‐dT’ Base Pair

Nucleobase‐directed spin‐labeling by the azide‐alkyne ‘click’ (CuAAC) reaction has been performed for the first time with oligonucleotides. 7‐Deaza‐7‐ethynyl‐2′‐deoxyadenosine ( 1 ) and 5‐ethynyl‐2′‐deoxyuridine ( 2 ) were chosen to incorporate terminal triple bonds into DNA. Oligonucleotides containing 1 or 2 were synthesized on a solid phase and spin labeling with 4‐azido‐2,2,6,6‐tetramethylpiperidine 1‐oxyl (4‐azido‐TEMPO, 3 ) was performed by post‐modification in solution. Two spin labels ( 3 ) were incorporated with high efficiency into the DNA duplex at spatially separated positions or into a ‘dA‐dT’ base pair. Modification at the 5‐position of the pyrimidine base or at the 7‐position of the 7‐deazapurine residue gave steric freedom to the spin label in the major groove of duplex DNA. By applying cw and pulse EPR spectroscopy, very accurate distances between spin labels, within the range of 1–2 nm, were measured. The spin–spin distance was 1.8±0.2 nm for DNA duplex 17 ( dA*^7,10 ) ?11 containing two spin labels that are separated by two nucleotides within one individual strand. A distance of 1.4±0.2 nm was found for the spin‐labeled ‘dA‐dT’ base pair 15 ( dA*⁷ ) ?16 ( dT*⁶ ). The ‘click’ approach has the potential to be applied to all four constituents of DNA, which indicates the universal applicability of the method. New insights into the structural changes of canonical or modified DNA are expected to provide additional information on novel DNA structures, protein interaction, DNA architecture, and synthetic biology.     

1,2,3‐Triazole Bridge as Conformational Constrain in β‐Hairpin Peptides: Analysis of Hydrogen‐Bonded Positions

Conformational constrained β‐hairpin peptides are useful tool to modulate protein–protein interactions. A triazole bridge in hydrogen‐bonded positions between two antiparallel strands induces a conformational stabilization of the β‐hairpin peptide. The entity of the stability of the β‐hairpin peptide depends on the length of the bridge.     

A Chemical Inhibitor of the Skp2/p300 Interaction that Promotes p53‐Mediated Apoptosis

Skp2 is thought to have two critical roles in tumorigenesis. As part of the SCF^Skp2 ubiquitin ligase, Skp2 drives the cell cycle by mediating the degradation of cell cycle proteins. Besides the proteolytic activity, Skp2 also blocks p53‐mediated apoptosis by outcompeting p53 for binding p300. Herein, we exploit the Skp2/p300 interaction as a new target for Skp2 inhibition. An affinity‐based high‐throughput screen of a combinatorial cyclic peptoid library identified an inhibitor that binds to Skp2 and interferes with the Skp2/p300 interaction. We show that antagonism of the Skp2/p300 interaction by the inhibitor leads to p300‐mediated p53 acetylation, resulting in p53‐mediated apoptosis in cancer cells, without affecting Skp2 proteolytic activity. Our results suggest that inhibition of the Skp2/p300 interaction has a great potential as a new anticancer strategy, and our Skp2 inhibitor can be developed as a chemical probe to delineate Skp2 non‐proteolytic function in tumorigenesis.     

Organic Spherical Nucleic Acids for the Transport of a NIR‐II‐Emitting Dye Across the Blood–Brain Barrier

DNA nanotechnology plays an increasingly important role in the biomedical field; however, its application in the design of organic nanomaterials is underexplored. Herein, we report the use of DNA nanotechnology to transport a NIR‐II‐emitting nanofluorophore across the blood–brain barrier (BBB), facilitating non‐invasive imaging of brain tumors. Specifically, the DNA block copolymer, PS‐b‐DNA, is synthesized through a solid‐phase click reaction. We demonstrate that its self‐assembled structure shows exceptional cluster effects, among which BBB‐crossing is the most notable. Therefore, PS‐b‐DNA is utilized as an amphiphilic matrix to fabricate a NIR‐II nanofluorephore, which is applied in in vivo bioimaging. Accordingly, the NIR‐II fluorescence signal of the DNA‐based nanofluorophore localized at a glioblastoma is 3.8‐fold higher than the NIR‐II fluorescence signal of the PEG‐based counterpart. The notably increased imaging resolution will significantly benefit the further diagnosis and therapy of brain tumors.     

Chemistry and Biology of Oligovalent β‐(1→2)‐Linked Oligomannosides: New Insights into Carbohydrate‐Based Adjuvants in Immunotherapy

A series of oligovalent carbohydrate assemblies (ranging from mono‐ to pentavalent), derived from three structurally different β‐linked or β‐(1→2)‐linked mannosides, has been chemically synthesized, and the respective compounds have been biologically evaluated in order to investigate their immunostimulatory properties. The Crich methodology for β‐mannosylation was successfully utilized to introduce the β‐linkages, and a click chemistry protocol was utilized to generate the oligovalent derivatives. A convenient protecting group strategy involving the simultaneous use of both p‐methoxybenzyl and benzylidene groups was employed, which allowed a simple and cost‐effective global deprotection step. The immunomodulatory properties of the synthesized multivalent mannosides were evaluated by assessing cytokine production in human white blood cell cultures. The Th2‐type cytokines interleukin‐4 and interleukin‐5 (IL‐4 and IL‐5), the Th1 cytokine interferon‐γ (IFN‐γ), the Treg cytokine IL‐10, and the pro‐inflammatory cytokine tumor necrosis factor (TNF) were included in the screening. A single trivalent acetylated mannobiose derivative was identified as a potent inducer of Treg and Th1 immune response, resulting in strong IL‐10 and moderate IFN‐γ productions dose‐dependently, while inducing no Th2 cytokine response. The immunomodulatory properties of this trivalent mannoside were further studied in vitro in allergen (Bet v)‐stimulated human peripheral blood mononuclear cell cultures of birch pollen allergic subjects. Stimulation with birch pollen induced strong IL‐4 and IL‐5 responses, which could be suppressed by the trivalent acetylated mannobiose derivative. The IL‐10 response was also suppressed, whereas the production of IFN‐γ was strongly enhanced. The results suggest that the identified lead compound has suppressive effects on the Th2‐type allergic inflammatory response and shows potential as a possible lead adjuvant for the specific immunotherapy of allergies.