Supramolecular Assembly in Live Cells Mapped by Real-Time Phasor-Fluorescence Lifetime Imaging.

The complex dynamics and transience of assembly pathways in living systems complicate the understanding of these molecular to nanoscale processes. Current technologies are unable to track the molecular events leading to the onset of assembly, where real-time information is imperative to correlate their rich biology. Using a chemically designed pro-assembling molecule, we map its transformation into nanofibers and their fusion with endosomes to form hollow fiber clusters. Tracked by phasor-fluorescence lifetime imaging (phasor-FLIM) in epithelial cells (L929, A549, MDA-MB 231) and correlative light-electron microscopy and tomography (CLEM), spatiotemporal splicing of the assembly events shows time-correlated metabolic dysfunction. The biological impact begins with assembly-induced endosomal disruption that reduces glucose transport into the cells, which, in turn, stymies mitochondrial respiration.

Spatiotemporally Controlled Photolabeling of Genetically Unmodified Proteins in Live Cells.

Selective labeling of the protein of interest (POI) in genetically unmodified live cells is crucial for understanding protein functions and kinetics in their natural habitat. In particular, spatiotemporally controlled installation of the labels on a POI under light control without affecting their original activity is in high demand but is a tremendous challenge. Here, we describe a novel ligand-directed photoclick strategy for spatiotemporally controlled labeling of endogenous proteins in live cells. It was realized with a designer labeling reagent skillfully integrating the photochemistries of 2-nitrophenylpropyloxycarbonyl and 3-hydroxymethyl-2-naphthol with an affinity ligand. Highly electrophilic ortho-naphthoquinone methide was photochemically released and underwent a proximity coupling reaction with nucleophilic amino acid residues on the POI in live cells. With fluorescein as a marker, this photoclick strategy enables time-resolved labeling of carbonic anhydrase subtypes localized either on the cell membrane or in the cytoplasm and a discriminable visualization of their metabolic kinetics. Given the versatility underlined by facilely tethering other functional entities (e.g., biotin, a peptide short chain) via acylation or (in cell) Huisgen cycloaddition, this affinity-driven photoclick chemistry opens up enormous opportunities for discovering dynamic functions and mechanistic interrogation of endogenous proteins in live cells.

The C-terminal 32-mer fragment of hemoglobin alpha is an amyloidogenic peptide with antimicrobial properties.

Antimicrobial peptides (AMPs) are major components of the innate immune defense. Accumulating evidence suggests that the antibacterial activity of many AMPs is dependent on the formation of amyloid-like fibrils. To identify novel fibril forming AMPs, we generated a spleen-derived peptide library and screened it for the presence of amyloidogenic peptides. This approach led to the identification of a C-terminal 32-mer fragment of alpha-hemoglobin, termed HBA(111-142). The non-fibrillar peptide has membranolytic activity against various bacterial species, while the HBA(111-142) fibrils aggregated bacteria to promote their phagocytotic clearance. Further, HBA(111-142) fibrils selectively inhibited measles and herpes viruses (HSV-1, HSV-2, HCMV), but not SARS-CoV-2, ZIKV and IAV. HBA(111-142) is released from its precursor by ubiquitous aspartic proteases under acidic conditions characteristic at sites of infection and inflammation. Thus, HBA(111-142) is an amyloidogenic AMP that may specifically be generated from a highly abundant precursor during bacterial or viral infection and may play an important role in innate antimicrobial immune responses.

Nopadiene: A Pinene-Derived Cyclic Diene as a Styrene Substitute for Fully Biobased Thermoplastic Elastomers.

The bicyclic 1,2-substituted, 1,3-diene monomer nopadiene (1R,5S)-2-ethenyl-6,6-dimethylbicyclo[3.1.1]hept-2-ene was successfully polymerized by anionic and catalytic polymerization. Nopadiene is produced either through a facile one-step synthesis from myrtenal via Wittig-olefination or via a scalable two-step reaction from nopol (10-hydroxymethylene-2-pinene). Both terpenoids originate from the renewable ß-pinene. The living anionic polymerization of nopadiene in apolar and polar solvents at 25 °C using organolithium initiators resulted in homopolymers with well-controlled molar masses in the range of 5.6-103.4 kg·mol-1 (SEC, PS calibration) and low dispersities (D) between 1.06 and 1.18. By means of catalytic polymerization with Me4CpSi(Me)2NtBuTiCl2 and (Flu)(Pyr)CH2Lu(CH2TMS)2(THF), the 1,4 and 3,4- microstructures of nopadiene are accessible in excellent selectivity. In pronounced contrast to other 1,3-dienes, the rigid polymers of the sterically demanding nopadiene showed an elevated glass temperature, Tg,∞ = 160 °C (in the limit of very high molar mass, Mn). ABA triblock copolymers with a central polymyrcene block and myrcene content of 60-75 mol %, with molar masses of 100-200 kg/mol were prepared by living anionic polymerization of the pinene-derivable monomers nopadiene and myrcene. This diene copolymerization resulted in thermoplastic elastomers displaying nanophase separation at different molar ratios (DSC, SAXS) and an upper service temperature about 30 K higher than that for traditional petroleum-derived styrenic thermoplastic elastomers due to the high glass temperature of polynopadiene. The materials showed good thermal stability at elevated temperatures under nitrogen (TGA), promising tensile strength and ultimate elongation of up to 1600%.

Chilling alcohol on the computer: isothermal compressibility and the formation of hydrogen-bond clusters in liquid propan-1-ol.

Molecular dynamics simulations have been performed to compute the isothermal compressibility [Formula: see text] of liquid propan-1-ol in the temperature range [Formula: see text] K. A change in behaviour, from normal (high T) to anomalous (low T), has been identified for [Formula: see text] at [Formula: see text] K. The average number of hydrogen bonds (H-bond) per molecule turns to saturation in the same temperature interval, suggesting the formation of a relatively rigid network. Indeed, simulation results show a strong tendency to form H-bond clusters with distinct boundaries, with the average largest size and width of the size distribution growing upon decreasing temperature, in agreement with previous theoretical and experimental studies. These results also emphasise a connection between the behaviour of [Formula: see text] and the formation of nanometric structures.

Confining the Sol-Gel Reaction at the Water/Oil Interface: Creating Compartmentalized Enzymatic Nano-Organelles for Artificial Cells.

Living organisms compartmentalize their catalytic reactions in membranes for increased efficiency and selectivity. To mimic the organelles of eukaryotic cells, we develop a mild approach for in situ encapsulating enzymes in aqueous-core silica nanocapsules. In order to confine the sol-gel reaction at the water/oil interface of miniemulsion, we introduce an aminosilane to the silica precursors, which serves as both catalyst and an amphiphilic anchor that electrostatically assembles with negatively charged hydrolyzed alkoxysilanes at the interface. The semi-permeable shell protects enzymes from proteolytic attack, and allows the transport of reactants and products. The enzyme-carrying nanocapsules, as synthetic nano-organelles, are able to perform cascade reactions when enveloped in a polymer vesicle, mimicking the hierarchically compartmentalized reactions in eukaryotic cells. This in situ encapsulation approach provides a versatile platform for the delivery of biomacromolecules.

Twisted Diindeno-Fused Dibenzo[a,h]anthracene Derivatives and their Dianions.

We report a facile synthesis of diindeno-fused dibenzo[a,h]anthracene derivatives (DIDBA-2Cl, DIDBA-2Ph, and DIDBA-2H) with different degrees of non-planarity using three substituents (chloro, phenyl, and hydrogen) of various sizes. The planarization of their cores, as evidenced by the decreased end-to-end torsional angles, was confirmed by X-ray crystallography. Their enhanced energy gaps with twisting were investigated by a combination of spectroscopic and electrochemical methods with density functional theory, which showed a transition from singlet open-shell to closed-shell configuration. Moreover, their doubly reduced states, DIDBA-2Ph2- and DIDBA-2H2- , were achieved by chemical reduction. The structures of dianions were identified by X-ray crystallographic analysis, which elucidated that the electron charging further distorted the backbones. The electronic structure of the dianions was demonstrated by experimental and theoretical approaches, suggesting decreased energy gaps with larger non-planarity, different from the neutral species.

In Situ Assembly of Platinum(II)-Metallopeptide Nanostructures Disrupts Energy Homeostasis and Cellular Metabolism.

Nanostructure-based functions are omnipresent in nature and essential for the diversity of life. Unlike small molecules, which are often inhibitors of enzymes or biomimetics with established methods of elucidation, we show that functions of nanoscale structures in cells are complex and can implicate system-level effects such as the regulation of energy and redox homeostasis. Herein, we design a platinum(II)-containing tripeptide that assembles into intracellular fibrillar nanostructures upon molecular rearrangement in the presence of endogenous H2O2. The formed nanostructures blocked metabolic functions, including aerobic glycolysis and oxidative phosphorylation, thereby shutting down ATP production. As a consequence, ATP-dependent actin formation and glucose metabolite-dependent histone deacetylase activity are downregulated. We demonstrate that assembly-driven nanomaterials offer a rich avenue to achieve broad-spectrum bioactivities that could provide new opportunities in drug discovery.

Panchromatic Absorption and Oxidation of an Iron(II) Spin Crossover Complex.

In order to expand and exploit the useful properties of d6-iron(II) and d5-iron(III) complexes in potential magnetic, photophysical, or magnetooptical applications, crucial ligand-controlled parameters are the ligand field strength in a given coordination mode and the availability of suitable metal and ligand frontier orbitals for charge-transfer processes. The push-pull ligand 2,6-diguanidylpyridine (dgpy) features low-energy π* orbitals at the pyridine site and strongly electron-donating guanidinyl donors combined with the ability to form six-membered chelate rings for optimal metal-ligand orbital overlap. The electronic ground states of the pseudo-octahedral d6- and d5-complexes mer-[Fe(dgpy)2]2+, cis-fac-[Fe(dgpy)2]2+, and mer-[Fe(dgpy)2]3+ as well as their charge-transfer (CT) and metal-centered (MC) excited states are probed by variable temperature UV/vis absorption, NMR, EPR, and Mössbauer spectroscopy, magnetic susceptibility measurements at variable temperature as well as quantum chemical calculations.

Nanodiamond Theranostic for Light-Controlled Intracellular Heating and Nanoscale Temperature Sensing.

Temperature is an essential parameter in all biological systems, but information about the actual temperature in living cells is limited. Especially, in photothermal therapy, local intracellular temperature changes induce cell death but the local temperature gradients are not known. Highly sensitive nanothermometers would be required to measure and report local temperature changes independent of the intracellular environment, including pH or ions. Fluorescent nanodiamonds (ND) enable temperature sensing at the nanoscale independent of external conditions. Herein, we prepare ND nanothermometers coated with a nanogel shell and the photothermal agent indocyanine green serves as a heat generator and sensor. Upon irradiation, programmed cell death was induced in cancer cells with high spatial control. In parallel, the increase in local temperature was recorded by the ND nanothermometers. This approach represents a great step forward to record local temperature changes in different cellular environments inside cells and correlate these with thermal biology.

Brush Conformation of Polyethylene Glycol Determines the Stealth Effect of Nanocarriers in the Low Protein Adsorption Regime.

For nanocarriers with low protein affinity, we show that the interaction of nanocarriers with cells is mainly affected by the density, the molecular weight, and the conformation of polyethylene glycol (PEG) chains bound to the nanocarrier surface. We achieve a reduction of nonspecific uptake of ovalbumin nanocarriers by dendritic cells using densely packed PEG chains with a "brush" conformation instead of the collapsed "mushroom" conformation. We also control to a minor extent the dysopsonin adsorption by tailoring the conformation of attached PEG on the nanocarriers. The brush conformation of PEG leads to a stealth behavior of the nanocarriers with inhibited uptake by phagocytic cells, which is a prerequisite for successful in vivo translation of nanomedicine to achieve long blood circulation and targeted delivery. We can clearly correlate the brush conformation of PEG with inhibited phagocytic uptake of the nanocarriers. This study shows that, in addition to the surface's chemistry, the conformation of polymers controls cellular interactions of the nanocarriers.

Ru-Se Coordination: A New Dynamic Bond for Visible-Light-Responsive Materials.

Photodynamic bonds are stable in the dark and can reversibly dissociate/form under light irradiation. Photodynamic bonds are promising building blocks for responsive or healable materials, photoactivated drugs, nanocarriers, extracellular matrices, etc. However, reactive intermediates from photodynamic bonds usually lead to side reactions, which limit the use of photodynamic bonds. Here, we report that the Ru-Se coordination bond is a new photodynamic bond that reversibly dissociates under mild visible-light-irradiation conditions. We observed that Ru-Se bonds form via the coordination of a selenoether ligand with [Ru(tpy)(biq)(H2O)]Cl2 (tpy = 2,2':6',2″-terpyridine, biq = 2,2'-biquinoline) in the dark, while the Ru-Se bond reversibly dissociates under visible-light irradiation. No side reaction is detected in the formation and dissociation of Ru-Se bonds. To demonstrate that the Ru-Se bond is applicable to different operating environments, we prepared photoresponsive amphiphiles, surfaces, and polymer gels using Ru-Se bonds. The amphiphiles with Ru-Se bonds showed reversible morphological transitions between spherical micelles and bowl-shaped assemblies for dark/light irradiation cycles. The surfaces modified with Ru-Se-bond-containing compounds showed photoswitchable wettability. Polymer gels with Ru-Se cross-links underwent photoinduced reversible sol-gel transitions, which can be used for reshaping and healing. Our work demonstrates that the Ru-Se bond is a new type of dynamic bond, which can be used for constructing responsive, reprocessable, switchable, and healable materials that work in a variety of environments.

Dual Stimuli-Responsive Dynamic Covalent Peptide Tags: Toward Sequence-Controlled Release in Tumor-like Microenvironments.

Dynamic covalent chemistry (DCvC) has emerged as a versatile synthetic tool for devising stable, stimuli-responsive linkers or conjugates. The interplay of binding affinity, association and dissociation constants exhibits a strong influence on the selectivity of the reaction, the conversion rate, as well as the stability in aqueous solutions. Nevertheless, dynamic covalent interactions often exhibit fast binding and fast dissociation events or vice versa, affecting their conversion rates or stabilities. To overcome the limitation in linker design, we reported herein dual responsive dynamic covalent peptide tags combining a pH responsive boronate ester with fast association and dissociation rates, and a redox-active disulfide with slow formation and dissociation rate. Precoordination by boronic acid-catechol interaction improves self-sorting and selectivity in disulfide formation into heterodimers. The resulting bis-peptide conjugate exhibited improved complex stability in aqueous solution and acidic tumor-like extracellular microenvironment. Furthermore, the conjugate responds to pH changes within the physiological range as well as to redox conditions found inside cancer cells. Such tags hold great promise, through cooperative effects, for controlling the stability of bioconjugates under dilution in aqueous media, as well as designing intelligent pharmaceutics that react to distinct biological stimuli in cells.

Association Equilibria of Organo-Phosphoric Acids with Imines from a Combined Dielectric and Nuclear Magnetic Resonance Spectroscopy Approach.

Aggregates formed between organo-phosphoric acids and imine bases in aprotic solvents are the reactive intermediates in Brønsted acid organo-catalysis. Due to the strong hydrogen-bonding interaction of the acids in solution, multiple homo- and heteroaggregates are formed with profound effects on catalytic activity. Yet, due to the similar binding motifs-hydrogen-bonds-it is challenging to experimentally quantify the abundance of these aggregates in solution. Here we demonstrate that a combination of nuclear magnetic resonance (NMR) and dielectric relaxation spectroscopy (DRS) allows for accurate speciation of these aggregates in solution. We show that only by using the observables of both experiments heteroaggregates can be discriminated with simultaneously taking homoaggregation into account. Comparison of the association of diphenyl phosphoric acid and quinaldine or phenylquinaline in chloroform, dichloromethane, or tetrahydrofuran suggests that the basicity of the base largely determines the association of one acid and one base molecule to form an ion-pair. We find the ion-pair formation constants to be highest in chloroform, slightly lower in dichloromethane and lowest in tetrahydrofuran, which indicates that the hydrogen-bonding ability of the solvent also alters ion-pairing equilibria. We find evidence for the formation of multimers, consisting of one imine base and multiple diphenyl phosphoric acid molecules for both bases in all three solvents. This subsequent association of an acid to an ion-pair is however little affected by the nature of the base or the solvent. As such our findings provide routes to enhance the overall fraction of these multimers in solution, which have been reported to open new catalytic pathways.

Disaccharide Residues are Required for Native Antifreeze Glycoprotein Activity.

Antifreeze glycoproteins (AFGPs) are able to bind to ice, halt its growth, and are the most potent inhibitors of ice recrystallization known. The structural basis for AFGP's unique properties remains largely elusive. Here we determined the antifreeze activities of AFGP variants that we constructed by chemically modifying the hydroxyl groups of the disaccharide of natural AFGPs. Using nuclear magnetic resonance, two-dimensional infrared spectroscopy, and circular dichroism, the expected modifications were confirmed as well as their effect on AFGPs solution structure. We find that the presence of all the hydroxyls on the disaccharides is a requirement for the native AFGP hysteresis as well as the maximal inhibition of ice recrystallization. The saccharide hydroxyls are apparently as important as the acetyl group on the galactosamine, the α-linkage between the disaccharide and threonine, and the methyl groups on the threonine and alanine. We conclude that the use of hydrogen-bonding through the hydroxyl groups of the disaccharide and hydrophobic interactions through the polypeptide backbone are equally important in promoting the antifreeze activities observed in the native AFGPs. These important criteria should be considered when designing synthetic mimics.

The Unique Versatility of the Double Metal Cyanide (DMC) Catalyst: Introducing Siloxane Segments to Polypropylene Oxide by Ring-Opening Copolymerization.

The combination of hydrophobic polydimethylsiloxane (PDMS) blocks with hydrophilic polyether segments plays a key role for silicone surfactants. Capitalizing on the double metal cyanide (DMC) catalyst, the direct (i.e., statistical) copolymerization of cyclic siloxanes and epoxides is shown to be feasible. The solvent-free one-pot copolymerization of hexamethylcyclotrisiloxane and propylene oxide results in the formation of gradient propylene oxide (PPO)-PDMS copolymers. Copolymers with up to 46% siloxane content with low dispersities (Р< 1.2) are obtained in the molecular weight range of 2100-2900 g mol-1 . The polymerization kinetics are investigated by pressure monitoring and in situ 1 H and in situ 29 Si NMR spectroscopy. Contact angle measurements reveal the impact of siloxane incorporation manifest in strongly increased hydrophobicity of PPO-PDMS copolymers and a glass transition of -95 °C for 46% SiO content. This unusual copolymerization offers promise for the synthesis of silicone/polyether polyols.

Endobronchial Valve (Zephyr) Treatment in Homogeneous Emphysema: One-Year Results from the IMPACT Randomized Clinical Trial.

RATIONALE: The long-term safety and effectiveness of bronchoscopic lung volume reduction with Zephyr endobronchial valves in subjects with severe homogeneous emphysema with little to no collateral ventilation beyond 3 months have yet to be established. METHODS: Ninety-three subjects were randomized to either bronchoscopic lung volume reduction with Zephyr valves or standard of care (SoC) (1:1). Zephyr valve subjects were assessed at 3, 6, and 12 months. SoC subjects were assessed at 3 and 6 months; they were then offered crossover to Zephyr valve treatment. RESULTS: The mean group difference (Zephyr valve - SoC) for change in FEV1 from baseline to 6 months was 16.3 ± 22.1% (mean ± SD; p < 0.001). Secondary outcomes showed the mean between-group difference for the six-minute walk distance of +28.3 ± 55.3 m (p = 0.016); St. George's Respiratory Questionnaire, -7.51 ± 9.56 points (p < 0.001); modified Medical Research Council, -0.42 ± 0.81 points (p = 0.019); BODE index, -0.85 ± 1.39 points (p = 0.006); and residual volume of -430 ± 830 mL (p = 0.011) in favor of the Zephyr valve group. At 6 months, there were significantly more responders based on the minimal clinically important difference for these same measures in the Zephyr valve versus the SoC group. The clinical benefits were persistent at 12 months. The percentage of subjects with respiratory serious adverse events was higher in the Zephyr valve group compared to SoC during the first 30 days post-procedure but not statistically different for the Zephyr valve and SoC groups from 31 days to 6 months, and stable in the Zephyr valve group out to 12 months. There were 2 deaths in the SoC group in the 31-day to 6-month period and none in the Zephyr valve group out to 12 months. CONCLUSIONS: Bronchoscopic lung volume reduction with Zephyr valves in subjects with severe homogeneous emphysema and little to no collateral ventilation provides clinically meaningful change from baseline in lung function, quality of life, exercise capacity, dyspnea, and the BODE index at 6 months, with benefits maintained out to 12 months.

Dicyclopentaannelated Hexa-peri-hexabenzocoronenes with a Singlet Biradical Ground State.

Synthesis of two dicyclopentaannelated hexa-peri-hexabenzocoronene (PHBC) regioisomers was carried out, using nonplanar oligoaryl precursors with fluorenyl groups: mPHBC 8 with two pentagons in the "meta"-configuration was obtained as a stable molecule, while its structural isomer with the "para"-configuration, pPHBC 16, could be generated and characterized only in situ due to its high chemical reactivity. Both PHBCs exhibit low energy gaps, as reflected by UV-vis-NIR absorption and electrochemical measurements. They also show open-shell singlet ground states according to electron paramagnetic resonance (EPR) measurements and density functional theory (DFT) calculations. The use of fully benzenoid HBC as a bridging moiety leads to significant singlet biradical characters (y0 ) of 0.72 and 0.96 for mPHBC 8 and pPHBC 16, respectively, due to the strong rearomatization tendency of the HBC π-system; these values are among the highest for planar carbon-centered biradical molecules. The incorporation of fully unsaturated pentagons strongly perturbs the aromaticity of the parent HBC and makes the constituted benzene rings less aromatic or antiaromatic. These results illustrate the high impact of cyclopentaannelation on the electronic structures of fully benzenoid polycyclic aromatic hydrocarbons (PAHs) and open up a new avenue towards open-shell PAHs with prominent singlet biradical characters.

Three-Dimensional Pyrene-Fused N-Heteroacenes.

Four three-dimensional (3D) pyrene-fused N-heteroacenes (P1-P4) are designed and synthesized. From P1 to P4, their lengths are extended in an iterative way, where the thiadiazole unit can be reduced to diamine and the obtained diamines can be further condensed with the diketones with a thiadiazole unit. Compared to their two-dimensional counterparts, the solubility of these 3D pyrene-fused N-heteroacenes is improved by this 3D covalent linkage with two-dimensional units. The diameters of P1-P4 are 3.66, 6.06, 8.48 and 10.88 nm, respectively, and these 3D molecules are characterized by 1H, 13C and 2D NMR, MS, UV-vis, PL and CV spectra. Our strategy shows a promising way to large 3D pyrene-fused N-heteroacenes.

Sequence Programming with Dynamic Boronic Acid/Catechol Binary Codes.

The development of a synthetic code that enables a sequence programmable feature like DNA represents a key aspect toward intelligent molecular systems. We developed herein the well-known dynamic covalent interaction between boronic acids (BAs) and catechols (CAs) into synthetic nucleobase analogs. Along a defined peptide backbone, BA or CA residues are arranged to enable sequence recognition to their complementary strand. Dynamic strand displacement and errors were elucidated thermodynamically to show that sequences are able to specifically select their partners. Unlike DNA, the pH dependency of BA/CA binding enables the dehybridization of complementary strands at pH 5.0. In addition, we demonstrate the sequence recognition at the macromolecular level by conjugating the cytochrome c protein to a complementary polyethylene glycol chain in a site-directed fashion.