Covalent Assembly Research Articles

In Situ Synthesis of Phenoxazine Dyes in Water: Application for “Turn‐On” Fluorogenic and Chromogenic Detection of Nitric Oxide**

AbstractThe synthesis of phenoxazine dyes was revisited in order to access these fluorescent N,O‐heterocycles under mild conditions. The combined sequential use of nitrosonium tetrafluoroborate (NOBF4) and triphenylphosphine enables the facile conversion of bis(3‐dimethylaminophenyl) ether into the methyl analogue of popular laser dye oxazine 1. The ability of nitrosonium cation (NO+) to initiate the domino reaction resulting in π‐conjugated phenoxazine molecules under neutral conditions, then led us to explore the feasibility of expanding it in aqueous media. Thus, we explored the use of reactive signaling molecule nitric oxide (NO) as a biological trigger of phenoxazine synthesis in water. The implementation of a robust analytical methodology based on fluorescence assays and HPLC‐fluorescence/‐MS analyses, have enabled us to demonstrate the viability of this novel fluorogenic reaction‐based process to selectively yield an intense “OFF‐ON” response in the near‐infrared (NIR‐I) spectral region. This study is an important step towards the popularization of the concept of “covalent assembly” in the fields of optical sensing, bioimaging and molecular theranostics.

Dissipative Organization of DNA Oligomers for Transient Catalytic Function

AbstractThe development of synthetic non‐equilibrium systems opens doors for man‐made life‐like materials. Yet, creating distinct transient functions from artificial fuel‐driven structures remains a challenge. Building on our ATP‐driven dynamic covalent DNA assembly in an enzymatic reaction network of concurrent ATP‐powered ligation and restriction, we introduce ATP‐fueled transient organization of functional subunits for various functions. The programmability of the ligation/restriction site allows to precisely organize multiple sticky‐end‐encoded oligo segments into double‐stranded (ds) DNA complexes. We demonstrate principles of ATP‐driven organization into sequence‐defined oligomers by sensing barcode‐embedded targets with different defects. Furthermore, ATP‐fueled DNAzymes for substrate cleavage are achieved by transiently ligating two DNAzyme subunits into a dsDNA complex, rendering ATP‐fueled transient catalytic function.

Dissipative Organization of DNA Oligomers for Transient Catalytic Function.

The development of synthetic non‐equilibrium systems opens doors for man‐made life‐like materials. Yet, creating distinct transient functions from artificial fuel‐driven structures remains a challenge. Building on our ATP‐driven dynamic covalent DNA assembly in an enzymatic reaction network of concurrent ATP‐powered ligation and restriction, we introduce ATP‐fueled transient organization of functional subunits for various functions. The programmability of the ligation/restriction site allows to precisely organize multiple sticky‐end‐encoded oligo segments into double‐stranded (ds) DNA complexes. We demonstrate principles of ATP‐driven organization into sequence‐defined oligomers by sensing barcode‐embedded targets with different defects. Furthermore, ATP‐fueled DNAzymes for substrate cleavage are achieved by transiently ligating two DNAzyme subunits into a dsDNA complex, rendering ATP‐fueled transient catalytic function.

Small-molecule fluorescent probes based on covalent assembly strategy for chemoselective bioimaging.

Fluorescent probes have been widely studied and applied in environment and health analysis, where among them small molecular “covalent assembly” probes are a novel type of reaction probes with many advantages, including no background interference, remarkable colorimetric change, rapid response, high sensitivity, and strong fluorescent signal. During the past decade, significant contributions have been made globally to both the application and mechanism of covalent assembly probes. In this review, we summarize the recent development of covalent assembly probes, classifying them based on different analytes, such as anions, metal ions, small biological molecules, reactive oxidative spices (ROS), reactive nitrogen species (RNS), nerve agent mimics, and enzymes, and introduce their detection mechanism in detail. Furthermore, the perspective on the next generation of covalent-assembly probes toward biomolecules imaging is presented.

A covalent cobalt diimine-dioxime - fullerene assembly for photoelectrochemical hydrogen production from near-neutral aqueous media.

The covalent assembly between a cobalt diimine-dioxime complex and a fullerenic moiety results in enhanced catalytic properties in terms of overpotential requirement for H2 evolution. The interaction between the fullerene moiety and PCBM heterojunction further allows for the easy integration of the cobalt diimine-dioxime – fullerene catalyst with a poly-3-hexylthiophene (P3HT):[6,6]-phenyl-C61-butyric acid methyl ester (PCBM) bulk heterojunction, yielding hybrid photoelectrodes for H2 evolution from near-neutral aqueous solutions.

Controlled chemical assembly of enzymes in cell lysate enabled by genetic-encoded nonstandard amino acids

Genetically encoded nonstandard amino acids (NSAAs) facilitate the covalent assembly of enzymes and enable their self-purification, enhanced activity and stability.

Charge reversal hairpin peptide modified synergy therapeutic nanoplatforms for tumor specific drug shuttling.

Given the distinct pathological features of neoplasm tissues, multifunctional responsive nanocarriers have been recently considered as promising candidates to optimize the chemotherapy regime. As a result, we propose a graphene oxide-based pH-responsive drug delivery system via covalent assembly of "hairpin-like" cell penetrating peptides with acid sensitive and charge reversal properties to realize superior tumor specificity and lower toxicity. Graphene oxide here can serve as high doxorubicin-loading nanosheets and facilitate swift drug release in response to laser irradiation, which provides an efficient platform for the synergy of photo-chemotherapy. Structurally, polyglycol conjugation on the graphene oxide surface fulfils the function of nanocomposite stabilization. After administration, the elaborately acid sensitive cell penetrating peptides maintain the hairpin structure under physiological conditions, while after entering the tumor acidic microenvironment, they undergo charge reversal and structural conversion to promote the cellular uptake of nanoparticles. The evaluation of nanocomposites in vitro revealed their negligible systematic toxicity and remarkable antitumor effects. In vivo experiments also confirmed the impressive stability and tumor-specific targeting for alleviating breast cancer. In conclusion, hairpin peptide modified graphene oxide nanoparticles show multiple merits including high drug carrying capacity, selective tumor penetration, responsive drug release and effective combination oncotherapy.

Extending Native Top-Down Electron Capture Dissociation to MDa Immunoglobulin Complexes Provides Useful Sequence Tags Covering Their Critical Variable Complementarity-Determining Regions.

Native top-down mass spectrometry (MS) is gaining traction for the analysis and sequencing of intact proteins and protein assemblies, giving access to their mass and composition, as well as sequence information useful for identification. Herein, we extend and apply native top-down MS, using electron capture dissociation, to two submillion Da IgM- and IgG-based oligomeric immunoglobulins. Despite structural similarities, these two systems are quite different. The ∼895 kDa noncovalent IgG hexamer consists of six IgG subunits hexamerizing in solution due to three specifically engineered mutations in the Fc region, whereas the ∼935 kDa IgM oligomer results from the covalent assembly of one joining (J) chain and 5 IgM subunits into an asymmetric “pentamer” stabilized by interchain disulfide bridges. Notwithstanding their size, structural differences, and complexity, we observe that their top-down electron capture dissociation spectra are quite similar and straightforward to interpret, specifically providing informative sequence tags covering the highly variable CDR3s and FR4s of the Ig subunits they contain. Moreover, we show that the electron capture dissociation fragmentation spectra of immunoglobulin oligomers are essentially identical to those obtained for their respective monomers. Demonstrated for recombinantly produced systems, the approach described here opens up new prospects for the characterization and identification of IgMs circulating in plasma, which is important since IgMs play a critical role in the early immune response to pathogens such as viruses and bacteria.

Light-Induced Formation/Scission of C-N, C-O, and C-S Bonds Enables Switchable Stability/Degradability in Covalent Systems.

The manipulation of covalent bonds could be directed toward degradable, recyclable, and sustainable materials. However, there is an intrinsic conflict between properties of stability and degradability. Here we report light-controlled formation/scission of three types of covalent bonds (C-N, C-O, and C-S) through photoswitching between equilibrium and nonequilibrium states of dynamic covalent systems, achieving dual benefits of photoaddressable stability and cleavability. The photocyclization of dithienylethene fused aldehyde ring-chain tautomers turns on the reactivity, incorporating/releasing amines, alcohols, and thiols reversibly with high efficiency, respectively. Upon photocycloreversion the system is shifted to kinetically locked out-of-equilibrium form, enabling remarkable robustness of covalent assemblies. Reaction coupling allows remote and directional control of a diverse range of equilibria and further broadens the scope. Through locking and unlocking covalent linkages with light when needed, the utility is demonstrated with capture/release of bioactive molecules, modification of surfaces, and creation of polymers exhibiting tailored stability and degradability/recyclability. The versatile toolbox for photoswitchable dynamic covalent reactions to toggle matters on and off should be appealing to many endeavors.

Chemical Tuning of Exciton versus Charge-Transfer Excited States in Conformationally Restricted Arylene Cages.

Covalent assemblies of conjugated organic chromophores provide the opportunity to engineer new excited states with novel properties. In this work, a newly developed triple-stranded cage architecture, in which meta-substituted aromatic caps serve as covalent linking groups that attach to both top and bottom of the conjugated molecule walls, is used to tune the properties of thiophene oligomer assemblies. Benzene-capped and triazine-capped 5,5'-(2,2-bithiophene)-containing arylene cages are synthesized and characterized using steady-state and time-resolved spectroscopic methods. The conformational freedom and electronic states are analyzed using time-dependent density functional theory. The benzene cap acts as a passive spacer whose electronic states do not mix with those of the chromophore walls. The excited state properties are dominated by through-space interactions between the chromophore subunits, generating a neutral Frenkel H-type exciton state. This excitonic state undergoes intersystem crossing on a 200 ps time scale while the fluorescence output is suppressed by a factor of 2 due to a decreased radiative rate. Switching to a triazine cap enables electron transfer from the chromophore-linker after the initial excitation to the exciton state, leading to the formation of a charge-transfer state within 10 ps. This state can avoid intersystem crossing and exhibits red-shifted fluorescence with enhanced quantum yield. The ability to interchange structural modules with different electronic properties while retaining the overall cage morphology provides a new approach for tuning the properties of discrete chromophore assemblies.

Electrostatic and Covalent Assemblies of Anionic Hydrogel-Coated Gold Nanoshells for Detection of Dry Eye Biomarkers in Human Tears.

Although dry eye is highly prevalent, many challenges exist in diagnosing the symptom and related diseases. For this reason, anionic hydrogel-coated gold nanoshells (AuNSs) were used in the development of a label-free biosensor for detection of high isoelectric point tear biomarkers associated with dry eye. A custom, aldehyde-functionalized oligo(ethylene glycol)acrylate (Al-OEGA) was included in the hydrogel coating to enhance protein recognition through the formation of dynamic covalent (DC) imine bonds with solvent-accessible lysine residues present on the surface of select tear proteins. Our results demonstrated that hydrogel-coated AuNSs, composed of monomers that form ionic and DC bonds with select tear proteins, greatly enhance protein recognition due to changes in the maximum localized surface plasmon resonance wavelength exhibited by AuNSs in noncompetitive and competitive environments. Validation of the developed biosensor in commercially available pooled human tears revealed the potential for clinical translation to establish a method for dry eye diagnosis.

"Covalent-Assembly"-Triggered Striking Far-Red to near-Infrared Emitting Fluorescent Probe for Abrupt Detection of Nerve-Agent Mimic (DCP): Real Time Application in Monitoring the Presence of Trace Amounts in Soil and Live Cells.

Detection of chemical warfare agents (CWA) by simple and rapid methods with real-sample applications are quite inevitable in order to ease the threats to living systems caused by uncertain terror attacks and wars. Herein we have developed the first far-red to near infra-red (NIR) probe based on a covalent assembly approach for the detection of trace amounts of nerve agent mimic diethyl chloro phosphate (DCP) in soil and their fluorescent bio imaging in live cells. The probe features abrupt fluorescence turn on sensing of DCP with fluorescence quantum yield Φ = 0.622. It senses DCP selectively over other analytes in excellent sensitivity with a detection limit of 6.9 nM. In real time, the probe treated strips were employed to detect the DCP vapor effectively with eye catching fluorescence response. The presence of trace amounts of these acute warfare agents in the environment were monitored by soil analysis. Further fluorescent bio imaging was carried out to monitor trace level DCP in living cells using the HeLa cell line.

Constructing covalent organic nanoarchitectures molecule by molecule via scanning probe manipulation

Constructing low-dimensional covalent assemblies with tailored size and connectivity is challenging yet often key for applications in molecular electronics where optical and electronic properties of the quantum materials are highly structure dependent. We present a versatile approach for building such structures block by block on bilayer sodium chloride (NaCl) films on Cu(111) with the tip of an atomic force microscope, while tracking the structural changes with single-bond resolution. Covalent homo-dimers in cis and trans configurations and homo-/hetero-trimers were selectively synthesized by a sequence of dehalogenation, translational manipulation and intermolecular coupling of halogenated precursors. Further demonstrations of structural build-up include complex bonding motifs, like carbon–iodine–carbon bonds and fused carbon pentagons. This work paves the way for synthesizing elusive covalent nanoarchitectures, studying structural modifications and revealing pathways of intermolecular reactions.

Oligomeric membranes for simultaneous chemical reaction and separation

Thin films of oligomers were synthesized on PTFE substrates and they were studied for separation of product from liquid chemical reaction mixture. Three different types of membranes were fabricated using a technique called ‘covalent molecular assembly’. Covalent molecular assembly is a synthesis technique in which a substrate is sequentially immersed in chemical solutions to grow molecular moieties with covalent bonding to the underlying layer of molecules on the substrate. The first membrane was oligoimide PMDA-ODA. The second membrane was crosslinked oligoamide TC-TAB. The third membrane was Nickel terminated oligoimide Ni-PMDA-ODA. The first two membranes were studied for Friedel-Crafts’ alkylation reaction between benzene and methyl chloride in presence of ferric chloride catalyst. With 30psia chloromethane pressure, the reaction proceeded to 100% conversion and the first two membranes provided a permeate of 99% pure toluene product. The third membrane was catalytically active to convert p-nitrophenol to p-aminophenol in presence of sodium borohydride in ethanol. The purity of the permeate stream reached 98% in 40 min and was stable for 2 h. Thus, novel membranes were synthesized for enhanced separations.

Hexaphyrin-cyclodextrin hybrids: Getting larger, getting confused

The diversity of hexaphyrin-cyclodextrin hybrids (HCD), previously formed from the covalent assembly of regular hexaphyrin and [Formula: see text]-cyclodextrin subunits, has been increased following two main directions: enlarging the confined space provided by the cyclodextrin and tuning the N-core of the hexaphyrin aiming at bimetallic complexes. The larger [Formula: see text]-cyclodextrin unit was selectively functionalized with aldehyde linkers on its primary rim which were further reacted with 2 eq. of pentafluorophenyl tripyrrane in acidic conditions, followed by DDQ oxidation. Doubly-linked HCD hybrids were obtained with structural variations in the hexaphyrin macrocycles depending on the MSA concentration (2 vs. 200 mM) producing either regular ([26] rectangular/[28] Möbius) or doubly N-confused dioxo ([26] rectangular) scaffolds. For the latter, two isomers were isolated featuring mirror transoid skeletons and long-side meso-linking patterns, giving rise to planar chirality at the origin of intense and opposite electronic circular dichroism (ECD) spectra. Zn(II) complexation was then investigated leading to two main findings. (i) The regular [28] Möbius HCD afforded a mixture of monometallic Möbius Zn(II) complexes in the presence of acac ligand. ECD spectroscopy indicated a chirality transfer from the [Formula: see text]-cyclodextrin favoring a [Formula: see text] Möbius twist, but opposite to that arising from the narrower [Formula: see text]-cyclodextrin congener ([Formula: see text] twist favored). (ii) Owing to N3O coordination boxes, dissymmetric bimetallic Zn(II) complexes were readily formed with the doubly N-confused dioxo HCDs through a positive cooperative process. Both metal centers bind a DMAP ligand in axial positions, which is of interest for the

Covalent Fixing of MoS2 Nanosheets with SnS Nanoparticles Anchored on g-C3N4/Graphene Boosting Fast Charge/Ion Transport for Sodium-Ion Hybrid Capacitors.

The sluggish layered structural sodium reaction kinetics and the easy restacking property are major obstacles hindering the practical application of MoS2-based electrodes for sodium storage. Herein, covalently assembled two-phase MoS2-SnS supported by a hierarchical graphitic carbon nitride/graphene (MoS2-SnS@g-C3N4/G) composite is constructed to improve cycling cyclability and rate performances for Na storage. The multiphase MoS2-SnS@g-C3N4/G is featured with a covalent assembly strategy and an interconnected network architecture. This unique structural design can not only enhance the conductivity and facilitate fast interfacial electron transport, which is confirmed by experiments and density functional theory, but also buffer the volumetric changes of MoS2-SnS. As a result, the as-obtained MoS2-SnS@g-C3N4/G anode delivers a high reversible capacity of 834 mA h g-1 at 0.1 A g-1, a high rate capability of 452 mA h g-1 at 5 A g-1, and a long-term cycling stability (320 mA h g-1 at 2 A g-1 with 54.7% retention after 500 cycles) for the Na half-cell. Coupling with activated carbon (AC), our MoS2-SnS@g-C3N4/G||AC sodium-ion hybrid capacitor delivers high energy/power densities (193.1 W h kg-1/90 W kg-1 and 41.5 W h kg-1/18,000 W kg-1) and a stable cycle life in the potential range of 0-4.0 V.

Effect of pH variation and annealing on covalently assembled nanodiamond films

This work examines the effect of using low-ionic strength zwitterionic buffer and post-deposition thermal annealing on the microstructure and thermal conductivity of continuous nanocrystalline diamond films that are deposited via the directed covalent assembly of detonation nanodiamonds. Variation of the zwitterionic buffer pH was found to tune the surface coverage, film thickness, thermal conductivity, and film morphology as quantified by apparent porosity and pore size distribution. The sequential annealing of these films up to 400 °C, showed the anticipated severance of amide bonds holding the nanodiamonds together, followed by the detrimental aggregation of the nanodiamonds to segregated islands, losing film continuity and increasing apparent porosity. The change in thermal conductivity with annealing was subjective to buffer pH, suggesting that the morphology changes associated with annealing may have a complex relationship with thermal conductivity. The thermal conductivity data were further analyzed using a phonon hopping model to quantify the degree of phonon movement across boundaries. Overall, the results demonstrate a way to achieving porous, low-cost nanocrystalline diamond thin films with tunable film morphology and thermal conductivity.

Multiresponsive 2D Ti3C2Tx MXene via Implanting Molecular Properties.

The design and fabrication of active nanomaterials exhibiting multifunctional properties is a must in the so-called global "Fourth Industrial Revolution". In this sense, molecular engineering is a powerful tool to implant original capabilities on a macroscopic scale. Herein, different bioinspired 2D-MXenes have been developed via a versatile and straightforward synthetic approach. As a proof of concept, Ti3C2Tx MXene has been exploited as a highly sensitive transducing platform for the covalent assembly of active biomolecular architectures (i.e., amino acids). All pivotal properties originated from the anchored targets were proved to be successfully transferred to the resulting bioinspired 2D-MXenes. Appealing applications have been devised for these 2D-MXene prototypes showing (i) chiroptical activity, (ii) fluorescence capabilities, (iii) supramolecular π-π interactions, and (iv) stimuli-responsive molecular switchability. Overall, this work demonstrates the fabrication of programmable 2D-MXenes, taking advantage of the inherent characteristics of the implanted (bio)molecular components. Thus, the current bottleneck in the field of 2D-MXenes can be overcome after the significant findings reported here.

Supramolecular Nanodrugs Based on Covalent Assembly of Therapeutic Peptides toward In Vitro Synergistic Anticancer Therapy.

Therapeutic peptides have attracted significant attention in clinical applications due to their advantages in biological origination and good biocompatibility. However, the therapeutic performance of peptides is usually hindered by their short half-lives in blood and inferior activity. Herein, supramolecular nanodrugs of therapeutic peptides are constructed by covalent assembly of chemotherapeutic peptides through genipin cross-linking. The resulting nanodrugs have intense absorbance in the near-infrared region and high photothermal conversion efficiencies, leading to the possibility of photothermal therapy. The combination of photothermal therapy and chemotherapy using the nanodrugs shows synergistic therapeutic effects on cancer cells. Hence, covalent assembly not only maintains the chemotherapeutic activities of the peptides but also triggers supramolecular photothermal effects, demonstrating that the covalent assembly of therapeutic peptides through genipin cross-linking is an efficient approach in constructing supramolecular nanodrugs toward synergistic anticancer therapy.

Minimalist Design for a Hand-Held SARS-Cov-2 Sensor: Peptide-Induced Covalent Assembly of Hydrogel Enabling Facile Fiber-Optic Detection of a Virus Marker Protein.

The rampaging COVID-19 needs bioassaying methods of low cost and high robustness for those living in the poorly developed regions. Here, we propose such a method that does not need expensive and complicated equipment. Only a set of hand-held small devices is sufficient. A section along an optic fiber cable is stripped, so that laser light travelling through it will leak outside, while biosensing process taken place on this stripped section can form a new cladding layer of hydrogel, restoring the laser output of the fiber. A short peptide probe immobilized on the stripped section of the fiber can covalently capture a biomarker protein of SARS-Cov-2 from the serum sample. Through the cross-linking of the target protein with the interfering proteins in the serum sample, a hydrogel is covalently immobilized around the stripped section, highly resistant to detergent rinsing that is indispensable for removing nonspecific interference from the clinical sample. Using this "covalent biosensing" strategy, only one peptide probe is sufficient to simultaneously achieve ultrahigh affinity toward the biomarker protein of SARS-Cov-2 and effective signal amplification.