Tuning the Polarity of Antibiotic-Cy5 Conjugates Enables Highly Selective Labeling of Binding Sites.

Multidrug-resistant bacteria pose a major threat to global health, even as newly introduced antibiotics continue to lose their therapeutic value. Against this background, deeper insights into bacterial interaction with antibiotic drugs are urgently required, whereas fluorescently labeled drug conjugates can serve as highly valuable tools. Herein, the preparation and biological evaluation of 13 new fluorescent antibiotic-Cy5 dye conjugates is described, in which the tuning of the polarity of the Cy5 dye proved to be a key element to achieve highly favorable properties for various fields of application.

Cyanine- and Rhodamine-Derived Alkynes for the Selective Targeting of Cancerous Mitochondria through Radical Thiol-Yne Coupling in Live Cells.

Despite their long history and their synthetic potential underlined by various recent advances, radical thiol-yne coupling reactions have so far only rarely been exploited for the functionalization of biomolecules, and no examples yet exist for their application in live cells - although natural thiols show widespread occurrence therein. By taking advantage of the particular cellular conditions of mitochondria in cancer cells, we have demonstrated that radical thiol-yne coupling represents a powerful reaction principle for the selective targeting of these organelles. Within our studies, fluorescently labeled reactive alkyne probes were investigated, for which the fluorescent moiety was chosen to enable both mitochondria accumulation as well as highly sensitive detection. After preliminary studies under cell-free conditions, the most promising alkyne-dye conjugates were evaluated in various cellular experiments comprising analysis by flow cytometry and microscopy. All in all, these results pave the way for improved future therapeutic strategies relying on live-cell compatibility and selectivity among cellular compartments.

Connecting Ruthenium Photocatalysis to 1,2-Dioxetane-Mediated Chemiluminescence: a Versatile Combination for Optical Detection and Read-Out.

Optical storage and photon quantification systems based on sensitive photoreactions have numerous applications. Herein, we report a highly efficient photocatalytic reaction, in which ruthenium photoredox catalysis is combined with a 1,2-dioxetane from which chemiluminescence can be triggered. In this system, blue light irradiation as optical input enables a defined inverse correlation with base-triggered, blue light emission as optical output. Comparison of readout by 1 H NMR and chemiluminescence, relative to previous optical input, underlines the reliability and usefulness of the ruthenium-dioxetane system for optical storage, sensing and ruthenium detection.

2-Fluoro-5-nitrophenyldiazonium: A Novel Sanger-Type Reagent for the Versatile Functionalization of Alcohols.

As a novel Sanger-type reagent, 2-fluoro-5-nitrophenyldiazonium tetrafluoroborate enabled the versatile functionalization of primary and secondary aliphatic alcohols. Based on a mild nucleophilic aromatic substitution of the fluorine atom under unprecedented, base-free conditions, the diazonium unit on the aromatic core of the resulting aryl-alkyl ether could be employed for such diverse transformations as radical C-H activation and cyclization, as well as palladium catalyzed cross-coupling reactions.

Visible Light Promoted, Catalyst-Free Radical Carbohydroxylation and Carboetherification under Mild Biomimetic Conditions.

Metal and catalyst-free carbohydroxylations and carboetherifications at room temperature have been achieved by a combination of beneficial factors including high aryl diazonium concentration and visible light irradiation. The acceleration of the reaction by visible light irradiation is particularly remarkable against the background that neither the aryldiazonium salt nor the alkene show absorptions in the respective range of wavelength. These observations point to weak charge transfer interactions between diazonium salt and alkene, which are nevertheless able to considerably influence the reaction course. As highly promising perspective, many more aryldiazonium-based radical arylations might benefit from simple light irradiation without requiring a photocatalyst or particular additive.

Synthetic Route to Phenyl Diazenes and Pyridazinium Salts from Phenylazosulfonates.

The synthesis of pyridazinium salts was achieved from readily available phenylazosulfonates in a single reaction step. The reaction proceeds via the formation of short-lived phenyldiazenes, which-owing to the strongly acidic conditions-are partially protonated. The phenyldiazenes then undergo a rapid cycloaddition to furans to give pyridazinium salts via elimination of water. The fact that the pyridazinium synthesis shows a low sensitivity toward oxygen, although phenyldiazenes occur as intermediates, can be explained by the very fast cycloaddition step and the partial protonation of the phenyldiazene.

Structure-guided development of selective M3 muscarinic acetylcholine receptor antagonists.

Drugs that treat chronic obstructive pulmonary disease by antagonizing the M3 muscarinic acetylcholine receptor (M3R) have had a significant effect on health, but can suffer from their lack of selectivity against the M2R subtype, which modulates heart rate. Beginning with the crystal structures of M2R and M3R, we exploited a single amino acid difference in their orthosteric binding pockets using molecular docking and structure-based design. The resulting M3R antagonists had up to 100-fold selectivity over M2R in affinity and over 1,000-fold selectivity in vivo. The crystal structure of the M3R-selective antagonist in complex with M3R corresponded closely to the docking-predicted geometry, providing a template for further optimization.

Shifted Selectivity in Protonation Enables the Mild Deuteration of Arenes Through Catalytic Amounts of Bronsted Acids in Deuterated Methanol.

Taking advantage of the "differentiating effect" of the solvent methanol, deuterations of electron-rich aromatic systems can be carried out under mild acid catalysis and thus under far milder conditions than known so far. The exceptional functional group tolerance observed under the optimized conditions, which even includes highly acid-labile groups, results from a hitherto unexploited shifted selectivity in protonation, and enabled simple and straightforward access to complex deuterium-labeled compounds.

Oxidative Formation of Disulfide Bonds by a Chemiluminescent 1,2-Dioxetane under Mild Conditions.

The oxidation of alkyl thiols to disulfides has been achieved under mild conditions using a chemiluminescent 1,2-dioxetane as a stoichiometric oxidant. Besides the mild and biocompatible reaction conditions, this approach offers the possibility to monitor the presence of thiols through oxidation and chemiluminescence of the remaining dioxetane.

N-Alkylaminoferrocene-Based Prodrugs Targeting Mitochondria of Cancer Cells.

Intracellular concentration of reactive oxygen species (e.g., H2O2) in cancer cells is elevated over 10-fold as compared to normal cells. This feature has been used by us and several other research groups to design cancer specific prodrugs, for example, N-alkylaminoferrocene (NAAF)-based prodrugs. Further improvement of the efficacy of these prodrugs can be achieved by their targeting to intracellular organelles containing elevated reactive oxygen species (ROS) amounts. For example, we have previously demonstrated that lysosome-targeted NAAF-prodrugs exhibit higher anticancer activity in cell cultures, in primary cells and in vivo (Angew. Chem. Int. Ed. 2017, 56, 15545). Mitochondrion is an organelle, where electrons can leak from the respiratory chain. These electrons can combine with O2, generating O2-• that is followed by dismutation with the formation of H2O2. Thus, ROS can be generated in excess in mitochondria and targeting of ROS-sensitive prodrugs to these organelles could be a sensible possibility for enhancing their efficacy. We have previously reported on NAAF-prodrugs, which after their activation in cells, are accumulated in mitochondria (Angew. Chem. Int. Ed. 2018, 57, 11943). Now we prepared two hybrid NAAF-prodrugs directly accumulated in mitochondria and activated in these organelles. We studied their anticancer activity and mode of action. Based on these data, we concluded that ROS produced by mitochondria is not available in sufficient quantities for activation of the ROS-responsive prodrugs. The reason for this can be efficient scavenging of ROS by antioxidants. Our data are important for the understanding of the mechanism of action of ROS-activatable prodrugs and will facilitate their further development.

Radical Carbofluorination of Alkenes with Arylhydrazines and Selectfluor: Additives, Mechanistic Pathways, and Polar Effects.

Radical carbofluorination reactions starting from arylhydrazines and nonactivated alkenes, in which the C-F bond is formed through the use of Selectfluor, can be improved through the addition of anisole. Because direct trapping products could be detected only in trace amounts, anisole does primarily act as a reversible scavenger for the highly reactive ammonium radical dication released from Selectfluor in the C-F bond-forming step. As shown for three diverse substitution patterns, the main role of anisole is to prevent, or at least reduce, the undesired addition of the ammonium radical dication to the alkene, which in turn leads to an unfavorable consumption of the arylhydrazine-derived precursors required for carbofluorination. Moreover, besides the remarkable polar effects in radical trapping, this study shows that the Selectfluor-derived nitrogen-centered radical dication may add directly to alkenes, which has not been described so far.

Selective Functionalization of Graphene at Defect-Activated Sites by Arylazocarboxylic tert-Butyl Esters.

The development of versatile functionalization concepts for graphene is currently in the focus of research. Upon oxo-functionalization of graphite, the full surface of graphene becomes accessible for C-C bond formation to introduce out-of-plane functionality. Herein, we present the arylation of graphene with arylazocarboxylic tert-butyl esters, which generates aryl radicals after activation with an acid. Surprisingly, the degree of functionalization is related to the concentration of lattice vacancy defects in the graphene material. Consequently, graphene materials that are free from lattice defects are not reactive. The reaction can be applied to graphene dispersed in solvents and leads to bitopic functionalization as well as monotopic functionalization when the graphene is deposited on surfaces. As the arylazocarboxylic tert-butyl ester moiety can be attached to various molecules, the presented method paves the way to functional graphene derivatives, with the density of defects determining the degree of functionalization.

Molecular Mechanisms of Biased and Probe-Dependent Signaling at CXC-Motif Chemokine Receptor CXCR3 Induced by Negative Allosteric Modulators.

Our recent explorations of allosteric modulators with improved properties resulted in the identification of two biased negative allosteric modulators, BD103 (N-1-{[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimi-din2yl]ethyl}-4-(4-fluorobutoxy)-N-[(1-methylpiperidin-4-yl)methyl}]butanamide) and BD064 (5-[(N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl-2-[4-fluoro-3-(trifluoromethyl)phenyl]acetamido)methyl]-2-fluorophenyl}boronic acid), that exhibited probe-dependent inhibition of CXC-motif chemokine receptor CXCR3 signaling. With the intention to elucidate the structural mechanisms underlying their selectivity and probe dependence, we used site-directed mutagenesis combined with homology modeling and docking to identify amino acids of CXCR3 that contribute to modulator binding, signaling, and transmission of cooperativity. With the use of allosteric radioligand RAMX3 ([3H]N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]-N-[(1-methylpiperidin-4-yl)methyl]acetamide), we identified that F1313.32 and Y3087.43 contribute specifically to the binding pocket of BD064, whereas D1864.60 solely participates in the stabilization of binding conformation of BD103. The influence of mutations on the ability of negative allosteric modulators to inhibit chemokine-mediated activation (CXCL11 and CXCL10) was assessed with the bioluminescence resonance energy transfer-based cAMP and ß-arrestin recruitment assay. Obtained data revealed complex molecular mechanisms governing biased and probe-dependent signaling at CXCR3. In particular, F1313.32, S3047.39, and Y3087.43 emerged as key residues for the compounds to modulate the chemokine response. Notably, D1864.60, W2686.48, and S3047.39 turned out to play a role in signal pathway selectivity of CXCL10, as mutations of these residues led to a G protein-active but ß-arrestin-inactive conformation. These diverse effects of mutations suggest the existence of ligand- and pathway-specific receptor conformations and give new insights in the sophisticated signaling machinery between allosteric ligands, chemokines, and their receptors, which can provide a powerful platform for the development of new allosteric drugs with improved pharmacological properties.

Nitrogen Oxides and Nitric Acid Enable the Sustainable Hydroxylation and Nitrohydroxylation of Benzenes under Visible Light Irradiation.

A new type of waste recycling strategy is described in which nitrogen oxides or nitric acid are directly employed in photocatalyzed hydroxylations and nitrohydroxylations of benzenes. Through these transformations, otherwise costly denitrification can be combined with the synthesis of valuable compounds for various applications.

Microwave-Assisted Rapid One-Pot Synthesis of Fused and Non-Fused Indoles and 5-[18 F]Fluoroindoles from Phenylazocarboxylates.

Substituted indoles can be prepared from phenylazocarboxylates through a rapid one-pot sequence featuring a microwave-assisted Fischer indole synthesis as a key step. Considering that the phenylazocarboxylates may beforehand be modified by mild nucleophilic aromatic substitution, including the introduction of [18 F]fluoride, the overall strategy offers an attractive new access to 5-[18 F]fluoroindoles.

Radical Arylation of Anilines and Pyrroles via Aryldiazotates.

The radical arylation of anilines and pyrroles can be achieved under transition-metal- and catalyst-free conditions by using aryldiazotates in strongly alkaline aqueous solutions. The aryldiazotates act as protected diazonium ions, which do not undergo azo coupling with electron-rich aromatic substrates, but can still serve as an aryl radical source at slightly elevated temperatures. Based on an improved preparation of aryldiazotates in aqueous solution, homolytic aromatic substitutions of anilines and pyrroles were conducted with good overall yields and high regioselectivity. Moreover, DFT calculations provided further mechanistic insights.

Sustainable Synthesis of Balsalazide and Sulfasalazine Based on Diazotization with Low Concentrations of Nitrogen Dioxide in Air.

Low concentrations of nitrogen dioxide, which arises as a side product from a range of industrial processes, can effectively be recycled through the diazotization of anilines. The studies reported herein now demonstrate that the removal of nitrogen dioxide from gas streams is even more effective when hydrophilic anilines are used as starting materials. The diazonium salts, which are obtained in this way in up to quantitative yields, can directly be employed in azo coupling reactions, thus opening up an attractive route to the industrially important group of azo compounds.

Visible-Light-Induced, Catalyst-Free Radical Arylations of Arenes and Heteroarenes with Aryldiazonium Salts.

In the absence of a photocatalyst and other additives, the radical arylation of diverse arenes and heteroarenes has been achieved with aryldiazonium salts under visible-light irradiation from a blue light-emitting diode (LED). Although the course of some reactions can be rationalized by the formation of strongly light-absorbing charge-transfer (CT) complexes between the diazonium ion and the aromatic substrate, several further examples indicated that the simple presence of an aromatic substrate, showing only weak interactions to the diazonium ion, is fully sufficient to enable product formation.

Regioselective Radical Arylation of 3-Hydroxypyridines.

The titanium(III)-mediated radical arylation of 3-hydroxypyridines was found to proceed with high regioselectivity for the 2-position. Using aryldiazonium chlorides, which were prepared from the corresponding anilines, as aryl radical sources, a range of 3-hydroxy-2-phenylpyridines were obtained in moderate to good yields under simple reaction conditions. Reactions of ortho-carboxylic ester substituted phenyldiazonium salts directly provided tricyclic benzopyranopyridinones.

Strongly Directing Substituents in the Radical Arylation of Substituted Benzenes.

Although general interest in radical arylation reactions has grown rapidly in recent years, poor regioselectivities and the need to use a large excess of the radical-accepting arene have hindered their application to substituted benzenes. We now describe experimental and computational investigations into the substituent effects that lead to regioselective addition based on the recent discovery of anilines as outstanding substrates for radical arylations.