Antimicrobial Potency of Nor-Pyochelin Analogues and Their Cation Complexes against Multidrug-Resistant Pathogens.

The opportunistic pathogen Pseudomonas aeruginosa develops increasing resistance toward even the most potent antibiotics. Like other bacteria, the pathogen produces a number of virulence factors including metallophores, which constitute an important group. Pseudomonads produce the iron-chelating metallophore (siderophore) pyochelin, which, in addition to its iron-scavenging ability, is an effector for the transcriptional regulator PchR in its FeIII-bound form (ferripyochelin). In the present study, docking studies predicted a major ferripyochelin binding site in PchR, which prompted the exploration of nor-pyochelin analogues to produce tight binding to PchR, and thereby upregulation of the pyochelin metabolism. In addition, we investigated the effects of using the analogues to bind the antimicrobial cations GaIII and InIII. Selected analogues of nor-pyochelin were synthesized, and their GaIII- and InIII-based complexes were assessed for antimicrobial activity. The results indicate that the GaIII complexes inhibit the pathogens under iron-limited conditions, while the InIII-based systems are more effective in iron-rich media. Several of the GaIII complexes were shown to be highly effective against a multidrug-resistant P. aeruginosa clinical isolate, with minimum inhibitory concentrations (MICs) of ≤1 µg/mL. Similarly, two of the InIII-based systems were particularly effective against the isolate, with an MIC of 8 µg/mL. These results show high promise in comparison with other, traditionally potent antibiotics, as the compounds generally indicated low cytotoxicity toward mammalian cells. Preliminary mechanistic investigations using pseudomonal transposon mutants suggested that the inhibitory effects of the InIII-based systems could be due to acute iron deficiency as a result of InIII-bound bacterioferritin.

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.

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.

The mechanism of action of auranofin analogs in B. cenocepacia revealed by chemogenomic profiling.

Drug repurposing efforts led to the discovery of bactericidal activity in auranofin, a gold-containing drug used to treat rheumatoid arthritis. Auranofin kills Gram-positive bacteria by inhibiting thioredoxin reductase, an enzyme that scavenges reactive oxygen species (ROS). Despite the presence of thioredoxin reductase in Gram-negative bacteria, auranofin is not always active against them. It is not clear whether the lack of activity in several Gram-negative bacteria is due to the cell envelope barrier or the presence of other ROS protective enzymes such as glutathione reductase (GOR). We previously demonstrated that chemical analogs of auranofin (MS-40 and MS-40S), but not auranofin, are bactericidal against the Gram-negative Burkholderia cepacia complex. Here, we explore the targets of auranofin, MS-40, and MS-40S in Burkholderia cenocepacia and elucidate the mechanism of action of the auranofin analogs by a genome-wide, randomly barcoded transposon screen (BarSeq). Auranofin and its analogs inhibited the B. cenocepacia thioredoxin reductase and induced ROS but did not inhibit the bacterial GOR. Genome-wide, BarSeq analysis of cells exposed to MS-40 and MS-40S compared to the ROS inducers arsenic trioxide, diamide, hydrogen peroxide, and paraquat revealed common and unique mediators of drug susceptibility. Furthermore, deletions of gshA and gshB that encode enzymes in the glutathione biosynthetic pathway led to increased susceptibility to MS-40 and MS-40S. Overall, our data suggest that the auranofin analogs kill B. cenocepacia by inducing ROS through inhibition of thioredoxin reductase and that the glutathione system has a role in protecting B. cenocepacia against these ROS-inducing compounds.IMPORTANCEThe Burkholderia cepacia complex is a group of multidrug-resistant bacteria that can cause infections in the lungs of people with the autosomal recessive disease, cystic fibrosis. Specifically, the bacterium Burkholderia cenocepacia can cause severe infections, reducing lung function and leading to a devastating type of sepsis, cepacia syndrome. This bacterium currently does not have an accepted antibiotic treatment plan because of the wide range of antibiotic resistance. Here, we further the research on auranofin analogs as antimicrobials by finding the mechanism of action of these potent bactericidal compounds, using a powerful technique called BarSeq, to find the global response of the cell when exposed to an antimicrobial.

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.

Maltose-Derivatized Fluorescence Turn-On Imaging Probe for Bacteria Detection.

We report a maltose-derivatized fluorescence turn-on imaging probe, Mal-Cz, to detect E. coli and Staphylococci. The fluorescence turn-on is achieved through an intramolecular C-H insertion reaction of the perfluoroaryl azide-functionalized carbazole to give a fluorescent product. Confocal fluorescence microscopy confirmed the successful uptake of Mal-Cz by E. coli and Staphylococci upon photoactivation. The Mal-Cz probe could selectively detect E. coli and S. epidermidis in the presence of P. aeruginosa and M. smegmatis without interference from these bacteria. Both the photoactivation and bacteria detection can be accomplished using a hand-held UV lamp at 365 nm, with the limit of detection of 103 CFU/mL by the naked eye. Mal-Cz could also be used to detect E. coli and S. epidermidis spiked in milk by the naked eye under a hand-held UV lamp. The uptake of Mal-Cz requires metabolically active bacteria: the uptake was reduced in stationary phase bacteria and was diminished in bacteria that were killed by heating or treating with antibiotics or sodium azide. The uptake decreased with increasing concentration of added free maltose, indicating that Mal-Cz hijacked the maltose uptake pathways. In E. coli, the maltose transport systems, including maltoporin LamB, maltose binding protein MBP, and the maltose ATP binding cassette (ABC) transporter MalFGK2, are all critical for the transport of Mal-Cz. The uptake was diminished in the deletion mutants ΔLamB, ΔMalE, ΔMalF, and ΔMalK.

Synthesis of Glycopolymer Micelles for Antibiotic Delivery.

In this work, we designed biodegradable glycopolymers consisting of a carbohydrate conjugated to a biodegradable polymer, poly(lactic acid) (PLA), through a poly(ethylene glycol) (PEG) linker. The glycopolymers were synthesized by coupling alkyne end-functionalized PEG-PLA with azide-derivatized mannose, trehalose, or maltoheptaose via the click reaction. The coupling yield was in the range of 40-50% and was independent of the size of the carbohydrate. The resulting glycopolymers were able to form micelles with the hydrophobic PLA in the core and the carbohydrates on the surface, as confirmed by binding with the lectin Concanavalin A. The glycomicelles were ~30 nm in diameter with low size dispersity. The glycomicelles were able to encapsulate both non-polar (rifampicin) and polar (ciprofloxacin) antibiotics. Rifampicin-encapsulated micelles were much smaller (27-32 nm) compared to the ciprofloxacin-encapsulated micelles (~417 nm). Moreover, more rifampicin was loaded into the glycomicelles (66-80 µg/mg, 7-8%) than ciprofloxacin (1.2-2.5 µg/mg, 0.1-0.2%). Despite the low loading, the antibiotic-encapsulated glycomicelles were at least as active or 2-4 times more active than the free antibiotics. For glycopolymers without the PEG linker, the antibiotics encapsulated in micelles were 2-6 times worse than the free antibiotics.

Blood pH Analysis in Combination with Molecular Medical Tools in Relation to COVID-19 Symptoms.

The global outbreak of SARS-CoV-2/COVID-19 provided the stage to accumulate an enormous biomedical data set and an opportunity as well as a challenge to test new concepts and strategies to combat the pandemic. New research and molecular medical protocols may be deployed in different scientific fields, e.g., glycobiology, nanopharmacology, or nanomedicine. We correlated clinical biomedical data derived from patients in intensive care units with structural biology and biophysical data from NMR and/or CAMM (computer-aided molecular modeling). Consequently, new diagnostic and therapeutic approaches against SARS-CoV-2 were evaluated. Specifically, we tested the suitability of incretin mimetics with one or two pH-sensitive amino acid residues as potential drugs to prevent or cure long-COVID symptoms. Blood pH values in correlation with temperature alterations in patient bodies were of clinical importance. The effects of biophysical parameters such as temperature and pH value variation in relation to physical-chemical membrane properties (e.g., glycosylation state, affinity of certain amino acid sequences to sialic acids as well as other carbohydrate residues and lipid structures) provided helpful hints in identifying a potential Achilles heel against long COVID. In silico CAMM methods and in vitro NMR experiments (including 31P NMR measurements) were applied to analyze the structural behavior of incretin mimetics and SARS-CoV fusion peptides interacting with dodecylphosphocholine (DPC) micelles. These supramolecular complexes were analyzed under physiological conditions by 1H and 31P NMR techniques. We were able to observe characteristic interaction states of incretin mimetics, SARS-CoV fusion peptides and DPC membranes. Novel interaction profiles (indicated, e.g., by 31P NMR signal splitting) were detected. Furthermore, we evaluated GM1 gangliosides and sialic acid-coated silica nanoparticles in complex with DPC micelles in order to create a simple virus host cell membrane model. This is a first step in exploring the structure-function relationship between the SARS-CoV-2 spike protein and incretin mimetics with conserved pH-sensitive histidine residues in their carbohydrate recognition domains as found in galectins. The applied methods were effective in identifying peptide sequences as well as certain carbohydrate moieties with the potential to protect the blood-brain barrier (BBB). These clinically relevant observations on low blood pH values in fatal COVID-19 cases open routes for new therapeutic approaches, especially against long-COVID symptoms.

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.

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.

Trehalose-Grafted Glycopolymer: Synthesis via the Staudinger Reaction and Capture of Mycobacteria.

A new trehalose-grafted poly(2-hydroxyethyl methacrylate) (HEMA) glycopolymer was synthesized via the perfluorophenyl azide (PFPA)-mediated Staudinger reaction between poly(HEMA-co-HEMA-PFPA) and a diphenylphosphine-derivatized trehalose. The reaction occurred rapidly at room temperature without the use of any catalyst, giving the trehalose glycopolymers over 68% yield after 1 h. The grafting density of trehalose can be controlled by the copolymer composition in poly(HEMA-co-HEMA-PFPA), resulting in 6.1% (TP1) or 37% (TP2) at 10:1 and 1:1 HEMA/HEMA-PFPA feed ratio, respectively. The trehalose glycopolymer was covalently attached on glass slides or silicon wafers using a thin film of poly(HEMA-co-HEMA-PFPA) as the adhesion layer, achieved through the C-H insertion reaction of the photogenerated singlet perfluorophenyl nitrene. To demonstrate the ability of the trehalose glycopolymer to capture mycobacteria, arrays of the trehalose glycopolymer were fabricated and treated with Mycobacterium smegmatis. Results from the optical, fluorescence, and scanning electron microscopy showed that mycobacteria were indeed captured on the trehalose glycopolymer. The amount of mycobacteria captured increased with the percent trehalose in the trehalose glycopolymer and also with the concentration of the trehalose glycopolymer. In addition, the captured bacteria could be visualized by the naked eye under the illumination of a hand-held UV lamp.

Trehalose-Modified Silver Nanoparticles as Antibacterial Agents with Reduced Cytotoxicity and Enhanced Uptake by Mycobacteria.

Silver nanoparticles (AgNPs) are potent antimicrobial agents, but their utility is limited due to their relatively high cytotoxicity. In this work, we used trehalose as the ligand to reduce the cytotoxicity of AgNPs without affecting their antimicrobial activities. Trehalose is a disaccharide that is unique to mycobacteria. We showed that trehalose-functionalized AgNPs, AgNP-Tre, drastically increased the viability of A549 cells, especially at high concentrations, for example, from 4% for AgNPs to 67% for AgNP-Tre at 64 µg/mL. The trehalose ligand slowed down the release of silver, and the amount of silver released from AgNP-Tre was less than half of that from AgNPs in the culture medium. Intriguingly, while the maltose (Mal) or tri(ethylene glycol) (TEG) ligand reduced the antibacterial activity of AgNPs against M. smegmatis (minimal inhibitory concentration (MIC) of AgNP-Mal and AgNP-TEG: 4 µg/mL for 7 nm AgNPs), the activity of AgNP-Tre was similar to that of AgNPs (MIC of AgNP-Tre: 1 µg/mL for 7 nm AgNPs). Uptake experiments revealed that the intracellular concentration of AgNP-Tre was 87 and 114% higher than those of AuNP-Mal and AgNP-TEG, respectively. The increased uptake was attributed to the enhanced interactions of AgNP-Tre with mycobacteria promoted by the trehalose ligand.

Using metal substrates to enhance the reactivity of graphene towards Diels-Alder reactions.

The Diels-Alder (DA) reaction, a classic cycloaddition reaction involving a diene and a dienophile to form a cyclohexene, is among the most versatile organic reactions. Theories have predicted thermodynamically unfavorable DA reactions on pristine graphene owing to its low chemical reactivity. We hypothesized that metals like Ni could enhance the reactivity of graphene towards DA reactions through charge transfer. The results indeed showed that metal substrates enhanced the reactivity of graphene in the DA reactions with a diene, 2,3-dimethoxy butadiene (DMBD), and a dienophile, maleic anhydride (MAH), with the activity enhancement in the order of Ni > Cu, and both are more reactive than graphene supported on silicon wafer. The rate constants were estimated to be two times higher for graphene supported on Ni than on silicon wafer. The computational results support the experimentally obtained rate trend of Ni > Cu, both predicted to be greater than unsupported graphene, which is explained by the enhanced graphene-substrate interaction reflected in charge transfer effects with the strongly interacting Ni. This study opens up a new avenue for enhancing the chemical reactivity of pristine graphene through substrate selection.

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.

New Auranofin Analogs with Antibacterial Properties against Burkholderia Clinical Isolates.

Bacteria of the genus Burkholderia include pathogenic Burkholderia mallei, Burkholderia pseudomallei and the Burkholderia cepacia complex (Bcc). These Gram-negative pathogens have intrinsic drug resistance, which makes treatment of infections difficult. Bcc affects individuals with cystic fibrosis (CF) and the species B. cenocepacia is associated with one of the worst clinical outcomes. Following the repurposing of auranofin as an antibacterial against Gram-positive bacteria, we previously synthetized auranofin analogs with activity against Gram-negatives. In this work, we show that two auranofin analogs, MS-40S and MS-40, have antibiotic activity against Burkholderia clinical isolates. The compounds are bactericidal against B. cenocepacia and kill stationary-phase cells and persisters without selecting for multistep resistance. Caenorhabditis elegans and Galleria mellonella tolerated high concentrations of MS-40S and MS-40, demonstrating that these compounds have low toxicity in these model organisms. In summary, we show that MS-40 and MS-40S have antimicrobial properties that warrant further investigations to determine their therapeutic potential against Burkholderia infections.

Maltoheptaose-Presenting Nanoscale Glycoliposomes for the Delivery of Rifampicin to E. coli.

Liposomes, a nanoscale drug delivery system, are well known for their ability to improve pharmacokinetics and reduce drug toxicity. In this work, maltoheptaose (G7)-presenting glycoliposomes were synthesized and evaluated in the delivery of the antibiotic rifampicin. Two types of liposomes were prepared: nonfluid liposomes from l-α-phosphatidylcholine (PC) and cholesterol, and fluid liposomes from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol). G7-derivatized glycolipid, G7-DPPE (DPPE: 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), was incorporated into the liposomes at 21 and 14 µmol/mg to form nanoparticles of 75 ± 12 and 146 ± 14 nm for the nonfluid and fluid G7-glycoliposomes, respectively. The multivalent G7-glycoliposomes were characterized by lectin binding with concanavalin A (Con A). The dissociation constant K d between Con A and the nonfluid or fluid G7-glycoliposomes was 0.93 or 0.51 µM, which represented ~900- or 1600-fold stronger affinity than the binding between Con A and G7. The G7-glycoliposomes were loaded with rifampicin at 6.6 and 16 wt % encapsulation for the nonfluid and fluid G7-glycoliposomes, respectively. Introducing a carbohydrate in the liposomes slowed down the release of rifampicin, with the G7-glycoliposomes having the slowest release rate and the lowest permeability coefficient among the liposome formulations. The fluid G7-glycoliposomes lowered the minimal inhibitory concentration (MIC) of rifampicin against E. coli ORN208 by about 3 times, whereas liposomes without G7 or Man (d-mannose)-glycoliposomes showed no improvement in MIC. The rifampicin-loaded fluid G7-glycoliposomes demonstrated the best sustained antibacterial activity against E. coli, with up to 2 log reduction in the colony forming units at 4 × MIC after 24 h. Fluorescence resonance energy transfer and confocal fluorescence microscopy revealed stronger interactions of the bacterium with the fluid G7-glycoliposomes than other liposome formulations.

Synthesis and solution isomerization of water-soluble Au9 nanoclusters prepared by nuclearity conversion of [Au11(PPh3)8Cl2]Cl.

Water-soluble gold nanoclusters (AuNCs) are popular in biomedical applications such as bioimaging, labelling, drug delivery, and biosensing. Despite their widespread applications, the synthesis of water-soluble phosphine-capped AuNCs is not as straightforward as their organic-soluble equivalents. Organic soluble phosphine-passivated [Au9(L)8]3+ are 6-electron closed-shell AuNCs that are generally prepared via the reduction of a phosphine-Au(I) complex by NaBH4. A similar approach attempted for the water-soluble ligand triphenylphosphine monosulfonate (TPPMS) using [AuTPPMS]Cl resulted in a mixture of cluster sizes that required gel electrophoresis or fractional precipitation to isolate the Au9 product. In this work, we report the synthesis of water-soluble [Au9(L)8]3+ nanoclusters in high yield through the biphasic ligand exchange of [Au11(PPh3)8Cl2]Cl with water-soluble phosphines such as TPPMS and 4-(diphenylphosphino)benzoic acid (DPPBA). The small molecule byproducts can be completely removed by size-based separation methods, like size exclusion chromatography or dialysis, as confirmed by 31P and 1H nuclear magnetic resonance (NMR) as well as diffusion ordered spectroscopy (DOSY). Furthermore, [Au9(DPPBA)8]Cl3 underwent a visible pH- and temperature-induced isomerization in ethanol between the 'crown' and 'butterfly' isomers of [Au9(L)8]3+ which has not been previously reported. Cytotoxicity evaluation of these water-soluble nanoclusters gave CC50 values of 36 µg mL-1 and 70 µg mL-1 against A549 human alveolar epithelial cells, and 30 µg mL-1 and 40 µg mL-1 against NIH/3T3 mouse fibroblast cells for [Au9(TPPMS)8]Cl3 and [Au9(DPPBA)8]Cl3, respectively. For comparison, auranofin, an FDA-approved gold drug, is more than an order of magnitude more toxic with a CC50 value of 7.7 µg mL-1 against A549 cells.

Attenuation of Human Lysozyme Amyloid Fibrillation by ACE Inhibitor Captopril: A Combined Spectroscopy, Microscopy, Cytotoxicity, and Docking Study.

Misfolding proteins could form oligomers or amyloid fibers, which can cause a variety of amyloid-associated diseases. Thus, the inhibition of protein misfolding and fibrillation is a promising way to prevent and treat these diseases. Captopril (CAP) is an angiotensin-converting enzyme inhibitor (ACEI) that is widely used to treat diseases such as hypertension and heart failure. In this study, we found that CAP inhibits human lysozyme (HL) fibrillation through the combination techniques of biophysics and biochemistry. The data obtained by thioflavin-T (ThT) and Congo red (CR) assays showed that CAP hindered the aggregation of HL amyloid fibrils by reducing the ß-sheet structure of HL amyloid, with an IC50 value of 34.75 ± 1.23 µM. Meanwhile, the particle size of HL amyloid decreased sharply in a concentration-dependent approach after CAP treatment. According to the visualization of atomic force microscopy (AFM) and transmission electron microscopy (TEM), we verified that in the presence of CAP, the needle-like fibers of HL amyloid were significantly reduced. In addition, CAP incubation dramatically improved the cell survival rate exposed to HL fibers. Our studies also revealed that CAP could form hydrogen bonds with amino acid residues of Glu 35 and Ala 108 in the binding pocket of HL, which help in maintaining the α-helical structure of HL and then prevent the formation of amyloid fibrillation. It can be concluded that CAP has antiamyloidogenic activity and a protective effect on HL amyloid cytotoxicity.

Imaging, Identification and Inhibition of Microorganisms Using AIEgens.

Microorganisms, including bacteria, viruses and fungi, are ubiquitous in nature. Some are extremely beneficial to life on Earth, whereas some cause diseases and disrupt normal human physiology. Pathogenic microorganisms can also undergo mutations and develop resistance to antimicrobial agents, which complicates diagnostic and therapeutic regimens. This calls for continuing efforts to develop new strategies and tools that can provide fast, sensitive and accurate diagnosis, as well as effective treatment of ever-evolving infectious diseases. Aggregation-induced emission luminogens (AIEgens) have shown promise in imaging, identification and inhibition of various microbial species. Compared to conventional organic fluorophores, AIEgens can offer improved photostability, and have found utilities in imaging microorganisms. AIEgens have been shown to detect microbial viability and differentiate among different microbial strains. Theranostic AIEgens that integrate imaging and killing of microbes have also been developed. This review highlights examples in the literature where AIEgens have been employed as molecular probes in the imaging, discrimination and killing of bacteria, viruses and fungi.

On the Reactivity Enhancement of Graphene by Metallic Substrates towards Aryl Nitrene Cycloadditions.

Pristine graphene is fairly inert chemically, and as such, most application-driven studies use graphene oxide, or reduced graphene oxide. Using substrates to modulate the reactivity of graphene represents a unique strategy in the covalent functionalization of this otherwise fairly inert material. It was found that the reactivity of pristine graphene towards perfluorophenyl azide (PFPA) can be enhanced by a metal substrate on which graphene is supported. Results on the extent of functionalization, defect density, and reaction kinetics all show that graphene supported on Ni (G/Ni) has the highest reactivity toward PFPA, followed by G/Cu and then G/silicon wafer. DFT calculations suggest that the metal substrate stabilizes the physisorbed nitrene through enhanced electron transfer to the singlet nitrene from the graphene surface assisted by the electron rich metal substrate. The G/Ni substantially stabilizes the singlet nitrene relative to G/Cu and the free-standing graphene. The product structure is also predicted to be substrate dependent. These findings open up opportunities to enhance the reactivity of pristine graphene simply through the selection of the substrate. This also represents a new and powerful approach to increasing the reactivity of singlet nitrenes through direct electronic communication with graphene.