One‐Step Formation of “Chain‐Armor”‐Stabilized DNA Nanostructures

DNA‐based self‐assembled nanostructures are widely used to position organic and inorganic objects with nanoscale precision. A particular promising application of DNA structures is their usage as programmable carrier systems for targeted drug delivery. To provide DNA‐based templates that are robust against degradation at elevated temperatures, low ion concentrations, adverse pH conditions, and DNases, we built 6‐helix DNA tile tubes consisting of 24 oligonucleotides carrying alkyne groups on their 3′‐ends and azides on their 5′‐ends. By a mild click reaction, the two ends of selected oligonucleotides were covalently connected to form rings and interlocked DNA single strands, so‐called DNA catenanes. Strikingly, the structures stayed topologically intact in pure water and even after precipitation from EtOH. The structures even withstood a temperature of 95 °C when all of the 24 strands were chemically interlocked.     

The Molecular Basis of Conjugated Polyyne Biosynthesis in Phytopathogenic Bacteria

Polyynes (polyacetylenes), which are produced by a variety of organisms, play important roles in ecology. Whereas alkyne biosynthesis in plants, fungi, and insects has been studied, the biogenetic origin of highly unstable bacterial polyynes has remained a riddle. Transposon mutagenesis and genome sequencing unveiled the caryoynencin (cay) biosynthesis gene cluster in the plant pathogen B. caryophylli, and homologous gene clusters were found in various other bacteria by comparative genomics. Gene inactivation and phylogenetic analyses revealed that novel desaturase/acetylenase genes mediate bacterial polyyne assembly. A cytochrome P450 monooxygenase is involved in the formation of the allylic alcohol moiety, as evidenced by analysis of a fragile intermediate, which was stabilized by an in situ click reaction. This work not only grants first insight into bacterial polyyne biosynthesis but also demonstrates that the click reaction can be employed to trap fragile polyynes from crude mixtures.     

Chain‐Growth Click Polymerization of AB2 Monomers for the Formation of Hyperbranched Polymers with Low Polydispersities in a One‐Pot Process

Hyperbranched polymers are important soft nanomaterials but robust synthetic methods with which the polymer structures can be easily controlled have rarely been reported. For the first time, we present a one‐pot one‐batch synthesis of polytriazole‐based hyperbranched polymers with both low polydispersity and a high degree of branching (DB) using a copper‐catalyzed azide–alkyne cycloaddition (CuAAC) polymerization. The use of a trifunctional AB₂ monomer that contains one alkyne and two azide groups ensures that all Cu catalysts are bound to polytriazole polymers at low monomer conversion. Subsequent CuAAC polymerization displayed the features of a “living” chain‐growth mechanism with a linear increase in molecular weight with conversion and clean chain extension for repeated monomer additions. Furthermore, the triazole group in a linear (L) monomer unit complexed Cu^I, which catalyzed a faster reaction of the second azide group to quickly convert the L unit into a dendritic unit, producing hyperbranched polymers with DB=0.83.     

Iterative In Situ Click Chemistry Creates Antibody‐like Protein‐Capture Agents

Special agents for protein capture : Iterative in situ click chemistry (see scheme for the tertiary ligand screen) and the one‐bead–one‐compound method for the creation of a peptide library enable the fragment‐based assembly of selective high‐affinity protein‐capture agents. The resulting ligands are water‐soluble and stable chemically, biochemically, and thermally. They can be produced in gram quantities through copper(I)‐catalyzed cycloaddition.

     

Integrative Self‐Sorting: One‐Pot Synthesis of a Hetero[4]rotaxane from a Daisy‐Chain‐Containing Hetero[4]pseudorotaxane

The structural complexity of mechanically interlocked molecules are very attractive to chemists owing to the challenges they present. In this article, novel mechanically interlocked molecules with a daisy‐chain‐containing hetero[4]rotaxane motif were efficiently synthesized. In addition, a novel integrative self‐sorting strategy is demonstrated, involving an ABB‐type (A for host, dibenzo‐24‐crown‐8 (DB24C8), and B for guest, ammonium salt sites) monomer and a macrocycle host, benzo‐21‐crown‐7 (B21C7), in which the assembled species in hydrogen‐bonding‐supported solvent only includes a novel daisy‐chain‐containing hetero[4]pseudorotaxane. The found self‐sorting process involves the integrative recognition between B21C7 macrocycles and carefully designed components simultaneously containing two types of secondary ammonium ions and a host molecule, DB24C8 crown ether. The self‐sorting strategy is integrative to undertake self‐recognition behavior to form one single species of pseudorotaxane compared with the previous report. This self‐sorting system can be used for the efficient one‐pot synthesis of a daisy‐chain‐containing hetero[4]rotaxane in a good yield. The structure of hetero[4]rotaxane was confirmed by ¹H NMR spectroscopy and high‐resolution electrospray ionization (HR‐ESI) mass spectrometry.     

Visualizing the Chain‐Flipping Mechanism in Fatty‐Acid Biosynthesis

The acyl carrier protein (ACP) from fatty acid synthases sequesters elongating products within its hydrophobic core, but this dynamic mechanism remains poorly understood. We exploited solvatochromic pantetheine probes attached to ACP that fluoresce when sequestered. The addition of a catalytic partner lures the cargo out of the ACP and into the active site of the enzyme, thus enhancing fluorescence to reveal the elusive chain‐flipping mechanism. This activity was confirmed by the use of a dual solvatochromic cross‐linking probe and solution‐phase NMR spectroscopy. The chain‐flipping mechanism was visualized by single‐molecule fluorescence techniques, thus demonstrating specificity between the Escherichia coli ACP and its ketoacyl synthase catalytic partner KASII.     

Fragment‐Based Discovery of a Dual pan‐RET/VEGFR2 Kinase Inhibitor Optimized for Single‐Agent Polypharmacology

Oncogenic conversion of the RET (rearranged during transfection) tyrosine kinase is associated with several cancers. A fragment‐based chemical screen led to the identification of a novel RET inhibitor, Pz‐1. Modeling and kinetic analysis identified Pz‐1 as a type II tyrosine kinase inhibitor that is able to bind the “DFG‐out” conformation of the kinase. Importantly, from a single‐agent polypharmacology standpoint, Pz‐1 was shown to be active on VEGFR2, which can block the blood supply required for RET‐stimulated growth. In cell‐based assays, 1.0 nM of Pz‐1 strongly inhibited phosphorylation of all tested RET oncoproteins. At 1.0 mg kg^?1 day^?1 per os, Pz‐1 abrogated the formation of tumors induced by RET‐mutant fibroblasts and blocked the phosphorylation of both RET and VEGFR2 in tumor tissue. Pz‐1 featured no detectable toxicity at concentrations of up to 100.0 mg kg^?1, which indicates a large therapeutic window. This study validates the effectiveness and usefulness of a medicinal chemistry/polypharmacology approach to obtain an inhibitor capable of targeting multiple oncogenic pathways.     

Identification of Living Legionella pneumophila Using Species‐Specific Metabolic Lipopolysaccharide Labeling

Legionella pneumophila is a pathogenic bacterium involved in regular outbreaks characterized by a relatively high fatality rate and an important societal impact. Frequent monitoring of the presence of this bacterium in environmental water samples is necessary to prevent these epidemic events, but the traditional culture‐based detection and identification method requires up to 10 days. Reported herein is a method allowing identification of Legionella pneumophila by metabolic lipopolysaccharide labeling which targets, for the first time, a precursor to monosaccharides that are specifically present within the O‐antigen of the bacterium. This new approach allows easy detection of living Legionella pneumophila, while other Legionella species are not labeled.     

Synthesis of Triazole‐Linked Amino Acid‐Aryl C‐Glycoside Hybrids via Click Chemistry as Novel PTP1B Inhibitors

Triazolyl phenylalanine and tyrosine‐aryl C‐glycoside hybrids were readily synthesized via microwave‐assisted Cu(I)‐catalyzed azide‐alkyne 1,3‐dipolar cycloaddition in high yields. Successive enzymatic assay identified the synthesized glycoconjugates as novel PTP1B inhibitors with low micromole‐ranged inhibitory activity and at least several‐fold selectivity over other homologous PTPs tested. In addition, the benzyl groups on glucosyl moiety were found crucial toward PTP1B inhibition.     

Fragment‐Based Drug Design Facilitated by Protein‐Templated Click Chemistry: Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin

There is an urgent need for the development of efficient methodologies that accelerate drug discovery. We demonstrate that the strategic combination of fragment linking/optimization and protein‐templated click chemistry is an efficient and powerful method that accelerates the hit‐identification process for the aspartic protease endothiapepsin. The best binder, which inhibits endothiapepsin with an IC₅₀ value of 43 μm , represents the first example of triazole‐based inhibitors of endothiapepsin. Our strategy could find application on a whole range of drug targets.     

Synthesis and Extended Activity of Triazole‐Containing Macrocyclic Protease Inhibitors

Peptide‐derived protease inhibitors are an important class of compounds with the potential to treat a wide range of diseases. Herein, we describe the synthesis of a series of triazole‐containing macrocyclic protease inhibitors pre‐organized into a β‐strand conformation and an evaluation of their activity against a panel of proteases. Acyclic azido–alkyne‐based aldehydes are also evaluated for comparison. The macrocyclic peptidomimetics showed considerable activity towards calpain II, cathepsin L and S, and the 20S proteasome chymotrypsin‐like activity. Some of the first examples of highly potent macrocyclic inhibitors of cathepsin S were identified. These adopt a well‐defined β‐strand geometry as shown by NMR spectroscopy, X‐ray analysis, and molecular docking studies.     

Heptyl α‐D‐Mannosides Grafted on a β‐Cyclodextrin Core To Interfere with Escherichia coli Adhesion: An In Vivo Multivalent Effect

n‐Heptyl α‐D ‐mannoside (HM) has previously been identified as a nanomolar FimH antagonist able to prevent Escherichia coli adhesion. We have designed mono‐ and heptavalent glycoconjugates in which HM is tethered to β‐cyclodextrin (β‐CD) through short and long spacers. One‐pot click or co‐clicking procedures were developed to directly obtain the glycoconjugates from unprotected HM and β‐CD precursors. These FimH antagonists were examined biophysically and in vivo. Reverse titrations by isothermal calorimetry led to trapping of the short‐tethered heptavalent β‐CD in a complex with three FimH lectins. Combined dynamic light scattering and small‐angle X‐ray solution scattering data allowed the construction of a model of the FimH trimer. The heptavalent β‐CDs were shown to capture and aggregate living bacteria in solution and are therefore also able to aggregate FimH when attached to different bacteria pili. The first in vivo evaluation of multivalent FimH inhibitors has been performed. The heptavalent β‐CDs proved to be much more effective anti‐adhesive agents than monovalent references with doses of around 2 μg instilled in the mouse bladder leading to a significantly decreased E. coli load. Intravenously injected radiolabeled glycoconjugates can rapidly reach the mouse bladder and >2 μg concentrations can easily be retained over 24 h to prevent fluxing bacteria from rebinding.     

Synthesis of Stable Aminoacyl‐tRNA Analogues Containing Triazole as a Bioisoster of Esters

Stable analogues : An effective synthetic route involving cycloaddition between alkynes and azidonucleosides to afford a new class of stable aminoacyl‐tRNA analogues such as depicted is presented. Biological evaluation showed that theses new compounds act as potent inhibitors of FemX_Wv aminoacyl transferase, a novel drug target.

     

Efficient Access to Peptidyl‐RNA Conjugates for Picomolar Inhibition of Non‐ribosomal FemXWv Aminoacyl Transferase

Peptidyl–RNA conjugates have various applications in studying the ribosome and enzymes participating in tRNA‐dependent pathways such as Fem transferases in peptidoglycan synthesis. Herein a convergent synthesis of peptidyl–RNAs based on Huisgen–Sharpless cycloaddition for the final ligation step is developed. Azides and alkynes are introduced into tRNA and UDP‐MurNAc‐pentapeptide, respectively. Synthesis of 2′‐azido RNA helix starts from 2′‐azido‐2′‐deoxyadenosine that is coupled to deoxycytidine by phosphoramidite chemistry. The resulting dinucleotide is deprotected and ligated to a 22‐nt RNA helix mimicking the acceptor arm of Ala‐tRNA^Ala by T4 RNA ligase. For alkyne UDP‐MurNAc‐pentapeptide, meso‐cystine is enzymatically incorporated into the peptidoglycan precursor and reduced, and L ‐Cys is converted to dehydroalanine with O‐(mesitylenesulfonyl)hydroxylamine. Reaction of but‐3‐yne‐1‐thiol with dehydroalanine affords the alkyne‐containing UDP‐MurNAc‐pentapeptide. The Cu^I‐catalyzed azide alkyne cycloaddition reaction in the presence of tris[(1‐hydroxypropyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]amine provided the peptidyl‐RNA conjugate, which was tested as an inhibitor of non‐ribosomal FemX_Wv aminoacyl transferase. The bi‐substrate analogue was found to inhibit FemX_Wv with an IC₅₀ of (89±9) pM , as both moieties of the peptidyl–RNA conjugate contribute to high‐affinity binding.     

Synthesis and Thermoreversible Gelation Properties of Main‐Chain Poly(pyridine‐2,6‐dicarboxamide‐triazole)s

A series of main‐chain poly(amide‐triazole)s were prepared by copper(I)‐catalyzed alkyne–azide AABB‐type copolymerizatons between five structurally similar diacetylenes 1 – 5 with the same diazide 6 . The acetylene units in monomers 1 – 5 possessed different degrees of conformational flexibility due to the different number of intramolecular hydrogen bonds built inside the monomer architecture. Our study showed that the conformational freedom of the monomer had a profound effect on the polymerization efficiency and the thermoreversible gelation properties of the resulting copolymers. Among all five diacetylene monomers, only the one, that is, 1 ‐Py(NH)₂ which possesses the pyridine‐2,6‐dicarboxamide unit with two built‐in intramolecular H bonds could produce the corresponding poly(amide‐triazole) Poly‐(PyNH)₂ with a significantly higher degree of polymerization (DP) than other monomers with a lesser number of intramolecular H bonds. In addition, it was found that only this polymer exhibited excellent thermoreversible gelation ability in aromatic solvents. A self‐assembling model of the organogelating polymer Poly‐(PyNH)₂ was proposed based on FTIR spectroscopy, XRD, and SEM analyses, in which H bonding, π–π aromatic stacking, hydrophobic interactions, and the structural rigidity of the polymer backbone were identified as the main driving forces for the polymer self‐assembly process.     

Modular Synthesis of Thermosensitive P(NIPAAm‐co‐HEMA)/β‐CD Based Hydrogels via Click Chemistry

Two kinds of representative polymers, poly(N‐isopropylacrylamide) (PNIPAAm) and β‐cyclodextrin (β‐CD) were selected and modified with azide and alkyne fucntional groups, respectively. When the solutions of these two modified polymers were mixed together, a cross‐linking reaction, a type of Huisgen's 1,3‐dipolar azide‐alkyne cycloaddition, occurred in the presence of Cu(I) catalyst. The strategy described here provides several advantages for the hydrogel formation including mild reaction conditions and controllable gelation rate. The resulted hydrogels were studied in terms of scanning electric microscopy (SEM), equilibrium swelling ratio and swelling/shrinking kinetics. The data obtained demonstrated the hydrogels had a porous structure as well as favorable thermosensitivity.

     

Chain‐Terminating and Clickable NAD+ Analogues for Labeling the Target Proteins of ADP‐Ribosyltransferases

ADP‐ribosyltransferases (ARTs) use NAD⁺ as a substrate and play important roles in numerous biological processes, such as the DNA damage response and cell cycle regulation, by transferring multiple ADP‐ribose units onto target proteins to form poly(ADP‐ribose) (PAR) chains of variable sizes. Efforts to identify direct targets of PARylation, as well as the specific ADP‐ribose acceptor sites, must all tackle the complexity of PAR. Herein, we report new NAD⁺ analogues that are efficiently processed by wild‐type ARTs and lead to chain termination owing to a lack of the required hydroxy group, thereby significantly reducing the complexity of the protein modification. Due to the presence of an alkyne group, these NAD⁺ analogues allow subsequent manipulations by click chemistry for labeling with dyes or affinity markers. This study provides insight into the substrate scope of ARTs and might pave the way for the further developments of chemical tools for investigating PAR metabolism.