Antimicrobial Delivery Using Metallophore-Responsive Dynamic Nanocarriers.

The increasing prevalence of multidrug-resistant (MDR) pathogens has promoted the development of innovative approaches, such as drug repurposing, synergy, and efficient delivery, in complement to traditional antibiotics. In this study, we present an approach based on biocompatible nanocarriers containing antimicrobial cations and known antibiotics. The matrices were prepared by coordinating GaIII or InIII to formulations of chitosan/tripolyphosphate or catechol-functionalized chitosan with or without encapsulated antibiotics, yielding particles of 100-200 nm in hydrodynamic diameter. MDR clinical isolates of Pseudomonas aeruginosa were found to be effectively inhibited by the nanocarriers under nutrient-limiting conditions. Fractional inhibitory concentration (FIC) indices revealed that cation- and antibiotic-encapsulated nanomatrices were effective against both Gram-negative and Gram-positive pathogens. Metallophores, such as deferoxamine (DFO), were probed to facilitate the sequestration and transport of the antimicrobial cations GaIII or InIII. Although the antimicrobial activities were less significant with DFO, the eradication of biofilm-associated bacteria showed promising trends against P. aeruginosa and Staphylococcus epidermidis. Interestingly, indium-containing compounds showed enhanced activity on biofilm formation and eradication, neutralizing P. aeruginosa under Fe-limiting conditions. In particular, InIII-cross-linked catechol-modified chitosan matrices were able to inhibit pathogenic growth together with DFO. The nanocarriers showed low cytotoxicity toward A549 cells and improvable CC50 values with NIH/3T3 cells.

Click-free imaging of carbohydrate trafficking in live cells using an azido photothermal probe.

Real-time tracking of intracellular carbohydrates remains challenging. While click chemistry allows bio-orthogonal tagging with fluorescent probes, the reaction permanently alters the target molecule and only allows a single snapshot. Here, we demonstrate click-free mid-infrared photothermal (MIP) imaging of azide-tagged carbohydrates in live cells. Leveraging the micromolar detection sensitivity for 6-azido-trehalose (TreAz) and the 300-nm spatial resolution of MIP imaging, the trehalose recycling pathway in single mycobacteria, from cytoplasmic uptake to membrane localization, is directly visualized. A peak shift of azide in MIP spectrum further uncovers interactions between TreAz and intracellular protein. MIP mapping of unreacted azide after click reaction reveals click chemistry heterogeneity within a bacterium. Broader applications of azido photothermal probes to visualize the initial steps of the Leloir pathway in yeasts and the newly synthesized glycans in mammalian cells are demonstrated.

Spontaneous and Selective Macrocyclization in Nitroaldol Reaction Systems.

Through a dynamic polymerization and self-sorting process, a range of lowellane macrocycles have been efficiently generated in nitroaldol systems composed of aromatic dialdehydes and aliphatic or aromatic dinitroalkanes. All identified macrocycles show a composition of two repeating units, resulting in tetra-ß-nitroalcohols of different structures. The effects of the building block structure on the macrocyclization process have been demonstrated, and the influence from the solvent has been explored. In general, the formation of the lowellanes was amplified in response to phase-change effects, although solution-phase structures were, in some cases, favored.

Small-molecule activators of a bacterial signaling pathway inhibit virulence.

The Burkholderia genus encompasses multiple human pathogens, including potential bioterrorism agents, that are often extensively antibiotic resistant. The FixLJ pathway in Burkholderia is a two-component system that regulates virulence. Previous work showed that fixLJ mutations arising during chronic infection confer increased virulence while decreasing the activity of the FixLJ pathway. We hypothesized that small-molecule activators of the FixLJ pathway could serve as anti-virulence therapies. Here, we developed a high-throughput assay that screened over 28,000 compounds and identified 11 that could specifically active the FixLJ pathway. Eight of these compounds, denoted Burkholderia Fix Activator (BFA) 1-8, inhibited the intracellular survival of Burkholderia in THP-1-dervived macrophages in a fixLJ-dependent manner without significant toxicity. One of the compounds, BFA1, inhibited the intracellular survival in macrophages of multiple Burkholderia species. Predictive modeling of the interaction of BFA1 with Burkholderia FixL suggests that BFA1 binds to the putative ATP/ADP binding pocket in the kinase domain, indicating a potential mechanism for pathway activation. These results indicate that small-molecule FixLJ pathway activators are promising anti-virulence agents for Burkholderia and define a new paradigm for antibacterial therapeutic discovery.

Azide-Masked Fluorescence Turn-On Probe for Imaging Mycobacteria.

A fluorescence turn-on probe, an azide-masked and trehalose-derivatized carbazole (Tre-Cz), was developed to image mycobacteria. The fluorescence turn-on is achieved by photoactivation of the azide, which generates a fluorescent product through an efficient intramolecular C-H insertion reaction. The probe is highly specific for mycobacteria and could image mycobacteria in the presence of other Gram-positive and Gram-negative bacteria. Both the photoactivation and detection can be accomplished using a handheld UV lamp, giving a limit of detection of 103 CFU/mL, which can be visualized by the naked eye. The probe was also able to image mycobacteria spiked in sputum samples, although the detection sensitivity was lower. Studies using heat-killed, stationary-phase, and isoniazid-treated mycobacteria showed that metabolically active bacteria are required for the uptake of Tre-Cz. The uptake decreased in the presence of trehalose in a concentration-dependent manner, indicating that Tre-Cz hijacked the trehalose uptake pathway. Mechanistic studies demonstrated that the trehalose transporter LpqY-SugABC was the primary pathway for the uptake of Tre-Cz. The uptake decreased in the LpqY-SugABC deletion mutants ΔlpqY, ΔsugA, ΔsugB, and ΔsugC and fully recovered in the complemented strain of ΔsugC. For the mycolyl transferase antigen 85 complex (Ag85), however, only a slight reduction of uptake was observed in the Ag85 deletion mutant ΔAg85C, and no incorporation of Tre-Cz into the outer membrane was observed. The unique intracellular incorporation mechanism of Tre-Cz through the LpqY-SugABC transporter, which differs from other trehalose-based fluorescence probes, unlocks potential opportunities to bring molecular cargoes to mycobacteria for both fundamental studies and theranostic applications.

Self-Healable, Regenerable, and Degradable Dynamic Covalent Nitroalcohol Organogels.

Dynamic covalent gels are synthesized from an aromatic trialdehyde and α,ω-dinitroalkanes via the nitroaldol reaction in organic solvents. The gelation process can be fine-tuned by changing the starting nitroalkanes, solvents, feed concentration, catalyst loading, or reaction temperature. The resulting organogels demonstrate good structural integrity and excellent self-healing ability. Intact xerogels are produced upon drying, without damaging the network, and the solvent-free network can recover its gel form in the presence of an organic solvent. Furthermore, the crosslinked dynameric gel depolymerize to small molecules in response to excess nitromethane.

Turning on the Antimicrobial Activity of Gold Nanoclusters Against Multidrug-Resistant Bacteria.

In this work, we show that the addition of thiourea (TU) initiated broad-spectrum antimicrobial activity of otherwise inactive D-maltose-capped gold nanoclusters (AuNC-Mal). For example, AuNC-Mal/TU was effective against multidrug-resistant Pseudomonas aeruginosa with a minimum inhibitory concentration (MIC) of 1 µg mL-1 (2.5 µM [Au]) while having 30-60 times lower in vitro cytotoxicity against mammalian cells. The reaction of AuNC-Mal and TU generated the antimicrobial species of [Au(TU)2 ]+ and smaller AuNCs. TU increased the accumulation of Au in bacteria and helped maintain the oxidation state as AuI (vs. AuIII ). The modes of action included the inhibition of thioredoxin reductase, interference with the CuI regulation and depletion of ATP. Moreover, the antimicrobial activity did not change in the presence of colistin or carbonyl cyanide 3-chlorophenylhydrazone, suggesting that AuNC-Mal/TU was indifferent to the outer membrane barrier and to bacterial efflux pumps.

Polymerization, Stimuli-induced Depolymerization, and Precipitation-driven Macrocyclization in a Nitroaldol Reaction System.

Dynamic covalent polymers of different topology have been synthesized from an aromatic dialdehyde and α,ω-dinitroalkanes via the nitroaldol reaction. All dinitroalkanes yielded dynamers with the dialdehyde, where the length of the dinitroalkane chain played a vital role in determining the structure of the final products. For longer dinitroalkanes, linear dynamers were produced, where the degree of polymerization reached a plateau at higher feed concentrations. In the reactions involving 1,4-dinitrobutane and 1,5-dinitropentane, specific macrocycles were formed through depolymerization of the linear chains, further driven by precipitation. At lower temperature, the same systemic self-sorting effect was also observed for the 1,6-dinitrohexane-based dynamers. Moreover, the dynamers showed a clear adaptive behavior, displaying depolymerization and rearrangement of the dynamer chains in response to alternative building blocks as external stimuli.

Gold Nanoclusters as Nanoantibiotic Auranofin Analogues.

Auranofin, a gold(I)-complex with tetraacetylated thioglucose (Ac4 GlcSH) and triethylphosphine ligands, is an FDA-approved drug used as an anti-inflammatory aid in the treatment of rheumatoid arthritis. In repurposing auranofin for other diseases, it was found that the drug showed significant activity against Gram-positive but was inactive against Gram-negative bacteria. Herein, the design and synthesis of gold nanoclusters (AuNCs) based on the structural motif of auranofin are reported. Phosphine-capped AuNCs are synthesized and glycosylated, yielding auranofin analogues with mixed triphenylphosphine monosulfonate (TPPMS)/Ac4 GlcSH ligand shells. These AuNCs are active against both Gram-negative and Gram-positive bacteria, including multidrug-resistant pathogens. Notably, an auranofin analogue, a mixed-ligand 1.6 nm AuNC 4b, is more active than auranofin against Pseudomonas aeruginosa, while exhibiting lower toxicity against human A549 cells. The enhanced antibacterial activity of these AuNCs is characterized by a greater uptake of Au by the bacteria compared to AuI complexes. Additional factors include increased oxidative stress, moderate inhibition of thioredoxin reductase (TrxR), and DNA damage. Most intriguingly, the uptake of AuNCs are not affected by the bacterial outer membrane (OM) barrier or by binding with the extracellular proteins. This contrasts with AuI complexes like auranofin that are susceptible to protein binding and hindered by the OM barrier.

Activated Self-Resolution and Error-Correction in Catalytic Reaction Networks*.

Understanding the emergence of function in complex reaction networks is a primary goal of systems chemistry and origin-of-life studies. Especially challenging is to create systems that simultaneously exhibit several emergent functions that can be independently tuned. In this work, a multifunctional complex reaction network of nucleophilic small molecule catalysts for the Morita-Baylis-Hillman (MBH) reaction is demonstrated. The dynamic system exhibited triggered self-resolution, preferentially amplifying a specific catalyst/product set out of a many potential alternatives. By utilizing selective reversibility of the products of the reaction set, systemic thermodynamically driven error-correction could also be introduced. To achieve this, a dynamic covalent MBH reaction based on adducts with internal H-transfer capabilities was developed. By careful tuning of the substituents, rate accelerations of retro-MBH reactions of up to four orders of magnitude could be obtained. This study thus demonstrates how efficient self-sorting of catalytic systems can be achieved through an interplay of several complex emergent functionalities.

Configurational and Constitutional Dynamics of Enamine Molecular Switches.

Dual configurational and constitutional dynamics in systems based on enamine molecular switches has been systematically studied. pH-responsive moieties, such as 2-pyridyl and 2-quinolinyl units, were required on the "stator" part, also providing enamine stability through intramolecular hydrogen-bonding (IMHB) effects. Upon protonation or deprotonation, forward and backward switching could be rapidly achieved. Extension of the stator π-system in the 2-quinolinyl derivative provided a higher E-isomeric equilibrium ratio under neutral conditions, pointing to a means to achieve quantitative forward/backward isomerization processes. The "rotor" part of the enamine switches exhibited constitutional exchange ability with primary amines. Interestingly, considerably higher exchange rates were observed with amines containing ester groups, indicating potential stabilization of the transition state through IMHB. Acids, particularly BiIII , were found to efficiently catalyze the constitutional dynamic processes. In contrast, the enamine and the formed dynamic enamine system showed excellent stability under basic conditions. This coupled configurational and constitutional dynamics expands the scope of dynamic C-C and C-N bonds and potentiates further studies and applications in the fields of molecular machinery and systems chemistry.

Hydrogen-Bond Catalysis of Imine Exchange in Dynamic Covalent Systems.

The reversibility of imine bonds has been exploited to great effect in the field of dynamic covalent chemistry, with applications such as preparation of functional systems, dynamic materials, molecular machines, and covalent organic frameworks. However, acid catalysis is commonly needed for efficient equilibration of imine mixtures. Herein, it is demonstrated that hydrogen bond donors such as thioureas and squaramides can catalyze the equilibration of dynamic imine systems under unprecedentedly mild conditions. Catalysis occurs in a range of solvents and in the presence of many sensitive additives, showing moderate to good rate accelerations for both imine metathesis and transimination with amines, hydrazines, and hydroxylamines. Furthermore, the catalyst proved simple to immobilize, introducing both reusability and extended control of the equilibration process.

Design, Synthesis and Self-Assembly of Functional Amphiphilic Metallodendrimers.

A new family of alkynylated, amphiphilic dendrimers consisting of amidoamine linkers connected to 5,5'-functionalized 2,2'-bipyridine cores has been developed and evaluated in the formation of metallodendrimers of different generations and in self-assembly protocols. A convergent synthetic strategy was applied to provide dumbbell-shaped amphiphilic dendrimers, where the 2,2'-bipyridine cores could be coordinated to FeII centers to afford corresponding metallodendrimers. The ability of the metallic- and non-metallic dendritic structures to self-assemble into functional supramolecular aggregates were furthermore evaluated in aqueous solution. Spherical aggregates with sizes of a few hundred nanometers were generally produced, where controlled disassembly of the metallodendrimers through decomplexation could be achieved.

Dynamic Covalent Polymers for Biomedical Applications.

The rapid development of supramolecular polymer chemistry and constitutional dynamic chemistry over the last decades has made tremendous impact on the emergence of dynamic covalent polymers. These materials are formed through reversible covalent bonds, endowing them with adaptive and responsive features that have resulted in high interest throughout the community. Owing to their intriguing properties, such as self-healing, shape-memory effects, recyclability, degradability, stimuli-responsiveness, etc., the materials have found multiple uses in a wide range of areas. Of special interest is their increasing use for biomedical applications, and many examples have been demonstrated in recent years. These materials have thus been used for the recognition and sensing of biologically active compounds, for the modulation of enzyme activity, for gene delivery, and as materials for cell culture, delivery, and wound-dressing. In this review, some of these endeavors are discussed, highlighting the many advantages and unique properties of dynamic covalent polymers for use in biology and biomedicine.

Dynamic Covalent Kinetic Resolution.

Implemented with the highly efficient concept of Dynamic Kinetic Resolution (DKR), dynamic covalent chemistry can be a useful strategy for the synthesis of enantioenriched compounds. This gives rise to dynamic covalent kinetic resolution (DCKR), a subset of DKR that over the last decades has emerged as increasingly fruitful, with many applications in asymmetric synthesis and catalysis. All DKR protocols are composed of two important parts: substrate racemization and asymmetric transformation, which can lead to yields of >50% with good enantiomeric excesses (ee) of the products. In DCKR systems, by utilizing reversible covalent reactions as the racemization strategy, the substrate enantiomers can be easily interconverted without the presence of any racemase or transition metal catalyst. Enzymes or other chiral catalysts can then be adopted for the resolution step, leading to products with high enantiopurities. This tutorial review focuses on the development of DCKR systems, based on different reversible reactions, and their applications in asymmetric synthesis.

Formation and Out-of-Equilibrium, High/Low State Switching of a Nitroaldol Dynamer in Neutral Aqueous Media.

The nitroaldol reaction is demonstrated as an efficient dynamic covalent reaction in phosphate buffers at neutral pH. Rapid equilibration was recorded with pyridine-based aldehydes, and dynamic oligomerization could be achieved, leading to nitroaldol dynamers of up to 17 repeating units. The dynamers were applied in a coherent stimuli-responsive molecular system in which larger dynamers transiently existed out-of-equilibrium in a neutral aqueous system rich in formaldehyde, controlled by nitromethane.

Photoactivatable Fluorogens by Intramolecular C-H Insertion of Perfluoroaryl Azide.

Molecules, capable of fluorescence turn-on by light, are highly sought-after in spatio-temporal labeling, surface patterning, monitoring cellular and molecular events, and high-resolution fluorescence imaging. In this work, we report a fluorescence turn-on system based on photoinitiated intramolecular C-H insertion of azide into the neighboring aromatic ring. The azide-masked fluorogens were efficiently synthesized via a cascade nucleophilic aromatic substitution of perfluoroaryl azides with carbazoles. The scaffold also allows for derivatization with biological ligands, as exemplified with d-mannose in this study. This photoinitiated intramolecular transformation led to high yields, high photo-conversion efficiency, and well-separated wavelengths for photoactivation and fluorescence excitation. The mannose-derivatized structure enabled spatio-temporal activation and showed high contrast and signal amplification. Live cell imaging suggested that the mannose-tagged fluorogen was transported to the lysosomes.

A Versatile Catalyst-Free Perfluoroaryl Azide-Aldehyde-Amine Conjugation Reaction.

A tri-component reaction, involving an electrophilically-activated perfluoroaryl azide, an enolizable aldehyde and an amine, reacts readily at room temperature without any catalysts in solvents including aqueous conditions to yield a stable amidine conjugate. The versatility of this reaction is demonstrated in the conjugation of an amino acid without prior protection of the carboxyl group, and in the synthesize antibiotic-nanoparticle conjugates.

Acid-Assisted Direct Olefin Metathesis of Unprotected Carbohydrates in Water.

The ability to use unprotected carbohydrates in olefin metathesis reactions in aqueous media is demonstrated. By using water-soluble, amine-functionalized Hoveyda-Grubbs catalysts under mildly acidic aqueous conditions, the self-metathesis of unprotected alkene-functionalized α-d-manno- and α-d-galactopyranosides could be achieved through minimization of nonproductive chelation and isomerization. Cross-metathesis with allyl alcohol could also be achieved with reasonable selectivity. The presence of small quantities (2.5 vol %) of acetic acid increased the formation of the self-metathesis product while significantly reducing the alkene isomerization process. The catalytic activity was furthermore retained in the presence of large amounts (0.01 mm) of protein, underlining the potential of this carbon-carbon bond-forming reaction under biological conditions. These results demonstrate the potential of directly using unprotected carbohydrate structures in olefin metathesis reactions under mild conditions compatible with biological systems, and thereby enabling their use in, for example, drug discovery and protein derivatization.

QCM sensing of multivalent interactions between lectins and well-defined glycosylated nanoplatforms.

Quartz crystal microbalance (QCM) methodology has been adopted to unravel important factors contributing to the "cluster glycoside effect" observed in carbohydrate-lectin interactions. Well-defined, glycosylated nanostructures of precise sizes, geometries and functionalization patterns were designed and synthesized, and applied to analysis of the interaction kinetics and thermodynamics with immobilized lectins. The nanostructures were based on Borromean rings, dodecaamine cages, and fullerenes, each of which carrying a defined number of carbohydrate ligands at precise locations. The synthesis of the Borromeates and dodecaamine cages was easily adjustable due to the modular assembly of the structures, resulting in variations in presentation mode. The binding properties of the glycosylated nanoplatforms were evaluated using flow-through QCM technology, as well as hemagglutination inhibition assays, and compared with dodecaglycosylated fullerenes and a monovalent reference. With the QCM setup, the association and dissociation rate constants and the associated equilibrium constants of the interactions could be estimated, and the results used to delineate the multivalency effects of the lectin-nanostructure interactions.