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.

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.

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.

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.

Electrophilic Azides for Materials Synthesis and Chemical Biology.

Organic azides are involved in a variety of useful transformations, including nitrene chemistry, reactions with nucleophiles and electrophiles, and cycloadditions. The 1,3-dipolar cycloadditions of azides constitute a major class of highly reliable and versatile reactions, as shown by the development and rapid adoption of click chemistry and bioorthogonal chemistry. Metal-catalyzed azide-alkyne cycloaddition (Cu/RuAAC), the prototypical click reaction, has found wide utility in pharmaceutical, biomedical, and materials sciences. The strain-promoted, or distortion-accelerated, azide-alkyne cycloaddition eliminates the need for a metal catalyst.In the azide-mediated 1,3-dipolar cycloaddition reactions, azides are ambiphilic, i.e., HOMO-LUMO-controlled dipoles where both the HOMO and LUMO interact strongly with the dipolarophile. Azide-alkyne cycloaddition proceeds primarily through the HOMOazide-LUMOdipolarophile interaction, and electron-deficient dipolarophiles react more readily. The inverse-electron-demand reaction, involving the LUMOazide-HOMOdipolarophile interaction, is less common because of the low stability of electron-deficient azides such as acyl, sulfonyl, and phosphoryl azides. Nevertheless, there have been reports since the 1960s showing enhanced reaction kinetics between electron-poor azides and electron-rich dipolarophiles. Our laboratory has developed the use of perfluoroaryl azides (PFAAs), a class of stable electron-deficient azides, as nitrene precursors and for reactions with nucleophiles and electron-rich dipolarophiles. Perfluorination on the aryl ring also facilitates the synthesis of PFAAs and quantitative analysis of the products by 19F NMR spectroscopy.In this Account, we summarize key reactions involving electrophilic azides and applications of these reactions in materials synthesis and chemical biology. These electron-deficient azides exhibit unique reactivity toward nucleophiles and electron-rich or strained dipolarophiles, in some cases leading to new transformations that do not require any catalysts or products that are impossible to obtain from the nonelectrophilic azides. We highlight work from our laboratories on reactions of PFAAs with enamines, enolates, thioacids, and phosphines. In the reactions of PFAAs with enamines or enolates, the triazole or triazoline cycloaddition products undergo further rearrangement to give amidines or amides as the final products at rates of up to 105 times faster than their non-fluorinated anlogues. Computational investigations by the distortion/interaction activation strain model reveal that perfluorination lowers the LUMO of the aryl azide as well as the overall activation energy of the reaction by decreasing the distortion energies of the reactants to reach the transition states. The PFAA-enamine reaction can be carried out in a one-pot fashion using readily available starting materials of aldehyde and amine, making the reaction especially attractive, for example, in the functionalization of nanomaterials and derivatization of antibiotics for the preparation of theranostic nanodrugs. Similar fast kinetics was also observed for the PPAA-mediated Staudinger reaction, which proceeds at 104 times higher rate than the classic Staudinger ligation, giving stable phosphoimines in high yields. The reaction is biorthogonal, allowing cell-surface labeling with minimal background noise.

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.

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.

Tunable Liquid Chromatographic Separation of H/D Isotopologues Enabled by Aromatic π Interactions.

We report hydrogen/deuterium (H/D) isotope effects based on weak intermolecular interactions with polar functional groups and aromatic rings in liquid chromatography (LC). Various LC experiments with different aromatic analytes, separation media, and nonpolar mobile phases were conducted under normal phase LC conditions, where the hydrophobic interaction was completely suppressed. The separation media that had polar functional groups, such as silanol groups, allowed for higher separation efficiencies for the pairs of aromatic H/D isotopologues. In comparing the 13C NMR spectra of protiated and deuterated aromatic analytes, the electron density of the deuterated analyte was found to be slightly higher than that of the protiated analytes. In the case of silanol functional groups, aromatic rings of the analyte acted as donors through the OH-π interaction to hydrogen atoms in the silanol groups. Thus, the deuterated analytes were able to be greatly retained by the stronger OH-π interactions. Furthermore, a C70-fullerene bonded monolithic column (C70 column), which effectively provides CH-π interactions, allowed the opposite isotope effect. Briefly, an electrostatic attraction based on the dipole-(induced) dipole interaction dominated in the CH-π interactions, according to a van't Hoff analysis. Hence, the bonding lengths of the C-H or D bonds were sensitively affected, such that we were able to conclude that the CH-π interaction depended on the geometric effect. Applying these opposing H/D isotope effects, we were able to finally demonstrate effective H/D isotopologue separations by utilizing the complementary action of the OH-π and CH-π interactions.

Design and synthesis of theranostic antibiotic nanodrugs that display enhanced antibacterial activity and luminescence.

We report the modular formulation of ciprofloxacin-based pure theranostic nanodrugs that display enhanced antibacterial activities, as well as aggregation-induced emission (AIE) enhancement that was successfully used to image bacteria. The drug derivatives, consisting of ciprofloxacin, a perfluoroaryl ring, and a phenyl ring linked by an amidine bond, were efficiently synthesized by a straightforward protocol from a perfluoroaryl azide, ciprofloxacin, and an aldehyde in acetone at room temperature. These compounds are propeller-shaped, and upon precipitation into water, readily assembled into stable nanoaggregates that transformed ciprofloxacin derivatives into AIE-active luminogens. The nanoaggregates displayed increased luminescence and were successfully used to image bacteria. In addition, these nanodrugs showed enhanced antibacterial activities, lowering the minimum inhibitory concentration (MIC) by more than one order of magnitude against both sensitive and resistant Escherichia coli The study represents a strategy in the design and development of pure theranostic nanodrugs for combating drug-resistant bacterial infections.

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.

Differentiating π Interactions by Constructing Concave/Convex Surfaces Using a Bucky Bowl Molecule, Corannulene in Liquid Chromatography.

Convex-concave π conjugated surfaces in hemispherical bucky bowl such as corannulene (Crn) have shown increasing utility in constructing self-assembled new functional materials owing to its unique π electrons and strong dipole. Here, we investigate these specific molecular recognitions on Crn by developing new silica-monolithic capillary columns modified with Crn and evaluating their performance in the separation of different aromatic compounds by liquid chromatography (LC). We synthesized two Crn derivatives and conjugated them onto the surface of a silica monolith. The first Crn derivative was edge functionalized, which can undergo free inversion of a convex-concave surface. The second Crn derivative was synthesized by modifying the spoke of Crn, which suppresses the convex-concave inversion. Results of LC suggest that each surface showed different shape recognition based on π interaction. Furthermore, the concave surface of Crn showed strong CH-π interaction with a planar molecule, coronene, demonstrated by the shifts of the 1H NMR signals of both Crn and coronene resulting from the multiple interactions between Crn and π electrons in coronene. These results clearly demonstrated the presence of CH-π interactions at multiple points, and the role of shape recognition.

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.

The Sialic Acid-Dependent Nematocyst Discharge Process in Relation to Its Physical-Chemical Properties Is A Role Model for Nanomedical Diagnostic and Therapeutic Tools.

Formulas derived from theoretical physics provide important insights about the nematocyst discharge process of Cnidaria (Hydra, jellyfishes, box-jellyfishes and sea-anemones). Our model description of the fastest process in living nature raises and answers questions related to the material properties of the cell- and tubule-walls of nematocysts including their polysialic acid (polySia) dependent target function. Since a number of tumor-cells, especially brain-tumor cells such as neuroblastoma tissues carry the polysaccharide chain polySia in similar concentration as fish eggs or fish skin, it makes sense to use these findings for new diagnostic and therapeutic approaches in the field of nanomedicine. Therefore, the nematocyst discharge process can be considered as a bionic blue-print for future nanomedical devices in cancer diagnostics and therapies. This approach is promising because the physical background of this process can be described in a sufficient way with formulas presented here. Additionally, we discuss biophysical and biochemical experiments which will allow us to define proper boundary conditions in order to support our theoretical model approach. PolySia glycans occur in a similar density on malignant tumor cells than on the cell surfaces of Cnidarian predators and preys. The knowledge of the polySia-dependent initiation of the nematocyst discharge process in an intact nematocyte is an essential prerequisite regarding the further development of target-directed nanomedical devices for diagnostic and therapeutic purposes. The theoretical description as well as the computationally and experimentally derived results about the biophysical and biochemical parameters can contribute to a proper design of anti-tumor drug ejecting vessels which use a stylet-tubule system. Especially, the role of nematogalectins is of interest because these bridging proteins contribute as well as special collagen fibers to the elastic band properties. The basic concepts of the nematocyst discharge process inside the tubule cell walls of nematocysts were studied in jellyfishes and in Hydra which are ideal model organisms. Hydra has already been chosen by Alan Turing in order to figure out how the chemical basis of morphogenesis can be described in a fundamental way. This encouraged us to discuss the action of nematocysts in relation to morphological aspects and material requirements. Using these insights, it is now possible to discuss natural and artificial nematocyst-like vessels with optimized properties for a diagnostic and therapeutic use, e.g., in neurooncology. We show here that crucial physical parameters such as pressure thresholds and elasticity properties during the nematocyst discharge process can be described in a consistent and satisfactory way with an impact on the construction of new nanomedical devices.

Synthesis of Carbohydrate-Grafted Glycopolymers Using a Catalyst-Free, Perfluoroarylazide-Mediated Fast Staudinger Reaction.

Glycopolymers have gained increasing importance in investigating glycan-lectin interactions, as drug delivery vehicles and in modulating interactions with proteins. The synthesis of these glycopolymers is still a challenging and rigorous exercise. In this regard, the highly efficient click reaction, copper (I)-catalyzed alkyne-azide cycloaddition, has been widely applied not only for its efficiency but also for its tolerance of the appended carbohydrate groups. However, a significant drawback of this method is the use of the heavy metal catalyst which is difficult to remove completely, and ultimately toxic to biological systems. In this work, we present the synthesis of carbohydrate-grafted glycopolymers utilizing a mild and catalyst-free perfluorophenyl azide (PFPA)-mediated Staudinger reaction. Using this strategy, mannose (Man) and maltoheptaose (MH) were grafted onto the biodegradable poly(lactic acid) (PLA) by stirring a PFAA-functionalized PLA with a phosphine-derivatized Man or MH in DMSO at room temperature within an hour. The glycopolymers were characterized by ¹H-NMR, 19F-NMR, 31P-NMR and FTIR.

Dynamic Covalent Chemistry of Aldehyde Enamines: BiIII - and ScIII -Catalysis of Amine-Enamine Exchange.

The dynamic exchange of enamines from secondary amines and enolizable aldehydes has been demonstrated in organic solvents. The enamine exchange with amines was efficiently catalyzed by Bi(OTf)3 and Sc(OTf)3 (2 mol %) and the equilibria (60 mm) could be attained within hours at room temperature. The formed dynamic covalent systems displayed high stabilities in basic environment with <2 % by-product formation within one week after complete equilibration. This study expands the scope of dynamic C-N bonds from imine chemistry to enamines, enabling further dynamic methodologies in exploration of this important class of structures in systems chemistry.

Perfluoroaryl Azide Staudinger Reaction: A Fast and Bioorthogonal Reaction.

We report a fast Staudinger reaction between perfluoroaryl azides (PFAAs) and aryl phosphines, which occurs readily under ambient conditions. A rate constant as high as 18 m-1 s-1 was obtained between methyl 4-azido-2,3,5,6-tetrafluorobenzoate and methyl 2-(diphenylphosphanyl)benzoate in CD3 CN/D2 O. Furthermore, the iminophosphorane product was stable toward hydrolysis and aza-phosphonium ylide reactions. This PFAA Staudinger reaction proved to be an excellent bioothorgonal reaction. PFAA-derivatized mannosamine and galactosamine were successfully transformed into cell-surface glycans and efficiently labeled with phosphine-derivatized fluorophore-conjugated bovine serum albumin.

Fabrication of carbohydrate microarrays on a poly(2-hydroxyethyl methacrylate)-based photoactive substrate.

We report the fabrication of carbohydrate microarrays on a photoactive polymer, poly(HEMA-co-HEMA-PFPA), synthesized by RAFT copolymerization of 2-hydroxyethyl methacrylate (HEMA) and perfluorophenyl azide (PFPA)-derivatized HEMA (HEMA-PFPA). PFPA allows the covalent immobilization of carbohydrates whereas the HEMA polymer provides an antifouling surface, thus the microarrays can be used directly without pretreating the array with a blocking agent. The microarrays were prepared by spin-coating the polymer followed by printing the carbohydrates. Subsequent irradiation simultaneously immobilized the carbohydrates and crosslinked the polymer matrix. The obtained 3D carbohydrate microarrays showed enhanced fluorescence signals upon treating with a fluorescent lectin in comparison with a 2D microarray. The signals were acquired at a lower lectin concentration and a shorter incubation time. When treated with E. coli bacteria, the carbohydrate microarray showed results that were consistent with their binding patterns.

1,3-Dipolar cycloaddition reactivities of perfluorinated aryl azides with enamines and strained dipolarophiles.

The reactivities of enamines and predistorted (strained) dipolarophiles toward perfluoroaryl azides (PFAAs) were explored experimentally and computationally. Kinetic analyses indicate that PFAAs undergo (3 + 2) cycloadditions with enamines up to 4 orders of magnitude faster than phenyl azide reacts with these dipolarophiles. DFT calculations were used to identify the origin of this rate acceleration. Orbital interactions between the cycloaddends are larger due to the relatively low-lying LUMO of PFAAs. The triazolines resulting from PFAA-enamine cycloadditions rearrange to amidines at room temperature, while (3 + 2) cycloadditions of enamines and phenyl azide yield stable, isolable triazolines. The 1,3-dipolar cycloadditions of norbornene and DIBAC also show increased reactivity toward PFAAs over phenyl azide but are slower than enamine-azide cycloadditions.

Quantitative fluorine NMR to determine carbohydrate density on glyconanomaterials synthesized from perfluorophenyl azide-functionalized silica nanoparticles by click reaction.

A quantitative fluorine NMR ((19)F qNMR) method was developed to determine the carbohydrate density on glyconanomaterials. Mannose (Man)- and galactose (Gal)-conjugated silica nanoparticles (SNPs) were synthesized from perfluorophenyl azide (PFPA)-functionalized SNPs and propargylated Man or Gal by copper-catalyzed azide-alkyne cycloaddition (click reaction). After treating PFPA-SNPs or Man-SNPs with hydrofluoric acid followed by lyophilization, the remaining residues were directly subjected to (19)F NMR analysis. The density of PFPA on PFPA-SNP was determined to be 7.7 ± 0.2 × 10(-16) nmol/nm(2) and Man on Man-SNP to be 6.4 ± 0.2 × 10(-16) nmol/nm(2) giving a yield of ∼83% for the click coupling reaction. The apparent dissociation constant (Kd) of Man-SNPs with fluorescein isothiocyanate (FITC)-concanavalin A (Con A) was determined using a fluorescence competition assay to be 0.289 ± 0.003 µM, which represents more than 3 orders of magnitude affinity increase compared to free Man with Con A.