Template effects of vesicles in dynamic covalent chemistry

Vesicle lipid bilayers have been employed as templates to modulate the product distribution in a dynamic covalent library of Michael adducts formed by mixing a Michael acceptor with thiols. In methanol solution, all possible Michael adducts were obtained in similar amounts. Addition of vesicles to the dynamic covalent library led to the formation of a single major product. The equilibrium constants for formation of the Michael adducts are similar for all of the thiols used in this experiment, and the effect of the vesicles on the composition of the library is attributed to the differential partitioning of the library members between the lipid bilayer and the aqueous solution. The results provide a quantitative approach for exploiting dynamic covalent chemistry within lipid bilayers.

Vesicle lipid bilayers have been employed as templates to modulate the product distribution in a dynamic covalent library of Michael adducts formed by mixing a Michael acceptor with thiols.     

Transmembrane signal transduction by cofactor transport

Information processing and cell signalling in biological systems relies on passing chemical signals across lipid bilayer membranes, but examples of synthetic systems that can achieve this process are rare. A synthetic transducer has been developed that triggers catalytic hydrolysis of an ester substrate inside lipid vesicles in response to addition of metal ions to the external vesicle solution. The output signal generated in the internal compartment of the vesicles is produced by binding of a metal ion cofactor to a head group on the transducer to form a catalytically competent complex. The mechanism of signal transduction is based on transport of the metal ion cofactor across the bilayer by the transducer, and the system can be reversibly switched between on and off states by adding cadmium(ii) and ethylene diamine tetracarboxylic acid input signals respectively. The transducer is also equipped with a hydrazide moiety, which allows modulation of activity through covalent conjugation with aldehydes. Conjugation with a sugar derivative abolished activity, because the resulting hydrazone is too polar to cross the bilayer, whereas conjugation with a pyridine derivative increased activity. Coupling transport with catalysis provides a straightforward mechanism for generating complex systems using simple components.

Synthetic transducers transport externally added metal ion cofactors across the lipid bilayer membrane of vesicles to trigger catalysis of ester hydrolysis in the inner compartment. Signal transduction activity is modulated by hydrazone formation.     

Controlled dimerization of artificial membrane receptors for transmembrane signal transduction

In biology, membrane-spanning proteins are responsible for the transmission of chemical signals across membranes, including the signal recognition-mediated conformational change of transmembrane receptors at the cell surface, and a trigger of an intracellular phosphorylation cascade. The ability to reproduce such biological processes in artificial systems has potential applications in smart sensing, drug delivery, and synthetic biology. Here, an artificial transmembrane receptors signaling system was designed and constructed based on modular DNA scaffolds. The artificial transmembrane receptors in this system are composed of three functional modules: signal recognition, lipophilic transmembrane linker, and signal output modules. Adenosine triphosphate (ATP) served as an external signal input to trigger the dimerization of two artificial receptors on membranes through a proximity effect. This effect induced the formation of a G-quadruplex, which served as a peroxidase-like enzyme to facilitate a signal output measured by either fluorescence or absorbance in the lipid bilayer vesicles. The broader utility of this modular method was further demonstrated using a lysozyme-binding aptamer instead of an ATP-binding aptamer. Therefore, this work provides a modular and generalizable method for the design of artificial transmembrane receptors. The flexibility of this synthetic methodology will allow researchers to incorporate different functional modules while retaining the same molecular framework for signal transduction.

An artificial transmbrane signal transducer was developed through the chemical input-mediated dimerization of artificial DNA transmembrane receptors and the subsequent activation of a cascade of events inside the vesicles.     

A system for artificial light signal transduction via molecular translocation in a lipid membrane

Light signal transduction pathways are the central components of mechanisms that regulate plant development, in which photoreceptors receive light and participate in light signal transduction. Chemical systems can be designed to mimic these biological processes that have potential applications in smart sensing, drug delivery and synthetic biology. Here, we synthesized a series of simple photoresponsive molecules for use as photoreceptors in artificial light signal transduction. The hydrophobic structures of these molecules facilitate their insertion into vesicular lipid bilayers, and reversible photoisomerization initiates the reciprocating translocation of molecules in the membrane, thus activating or deactivating the hydrolysis reaction of a precatalyst in the transducer for an encapsulated substrate, resulting in a light controllable output signal. This study represents the first example of using simplified synthetic molecules to simulate light signal transduction performed by complex biomolecules.

Photoisomerization chemistry was used to simulate light signal transduction, in which the light-controlled reciprocating translocation of molecules in lipids activates or deactivates the hydrolysis reaction for an encapsulated substrate.     

Supramolecular chemistry in lipid bilayer membranes

Lipid bilayer membranes form compartments requisite for life. Interfacing supramolecular systems, including receptors, catalysts, signal transducers and ion transporters, enables the function of the membrane to be controlled in artificial and living cellular compartments. In this perspective, we take stock of the current state of the art of this rapidly expanding field, and discuss prospects for the future in both fundamental science and applications in biology and medicine.

This perspective provides an overview of the current state of the art in supramolecular chemistry in lipid bilayer membranes, including receptors, signal transducers, catalysts and transporters, and highlights prospects for the future.     

Ratiometric detection of cysteine by a ferrocenyl Michael acceptor

An activated ferrocenyl enone (1) by an intramolecular hydrogen bond was utilized as a Michael acceptor type of chemodosimeter for cysteine. The probe has shown selective response toward cysteine over other natural amino acids through the Michael addition reaction of a cysteinyl thiol group to 1. When cysteine was added, the prominent color change of 1 was observed so that submillimolar concentrations of cysteine were detectable by the naked eye.     

Bioorthogonal regulation of DNA circuits for smart intracellular microRNA imaging

Catalytic DNA circuits represent a versatile toolbox for tracking intracellular biomarkers yet are constrained with low anti-interference capacity originating from their severe off-site activation. Herein, by introducing an unprecedented endogenous DNA repairing enzyme-powered pre-selection strategy, we develop a sequential and specific on-site activated catalytic DNA circuit for achieving the cancer cell-selective imaging of microRNA with high anti-interference capacity. Initially, the circuitry reactant is firmly caged by an elongated stabilizing duplex segment with a recognition/cleavage site of a cell-specific DNA repairing enzyme, which can prevent undesired signal leakage prior to its exposure to target cells. Then, the intrinsic DNA repairing enzyme of target cells can liberate the DNA probe for efficient intracellular microRNA imaging via the multiply guaranteed molecular recognition/activation procedures. This bioorthogonal regulated DNA circuit presents a modular and programmable amplification strategy for highly reliable assays of intracellular biomarkers, and provides a pivotal molecular toolbox for living systems.

An on-site bioorthogonal regulated DNA circuit was developed by introducing an endogenous DNA repairing enzyme-mediated sequential activation strategy to achieve cancer cell-selective microRNA imaging with high anti-interference ability.     

OvoAMtht from Methyloversatilis thermotolerans ovothiol biosynthesis is a bifunction enzyme: thiol oxygenase and sulfoxide synthase activities

Mononuclear non-heme iron enzymes are a large class of enzymes catalyzing a wide-range of reactions. In this work, we report that a non-heme iron enzyme in Methyloversatilis thermotolerans, OvoA_Mtht, has two different activities, as a thiol oxygenase and a sulfoxide synthase. When cysteine is presented as the only substrate, OvoA_Mtht is a thiol oxygenase. In the presence of both histidine and cysteine as substrates, OvoA_Mtht catalyzes the oxidative coupling between histidine and cysteine (a sulfoxide synthase). Additionally, we demonstrate that both substrates and the active site iron''s secondary coordination shell residues exert exquisite control over the dual activities of OvoA_Mtht (sulfoxide synthase vs. thiol oxygenase activities). OvoA_Mtht is an excellent system for future detailed mechanistic investigation on how metal ligands and secondary coordination shell residues fine-tune the iron-center electronic properties to achieve different reactivities.

Modulation of OvoA_Mtht''s dual activities: sulfoxide synthase and thiol oxygenase.     

Synthesis of magnetic DDAB vesicles

The synthesis of magnetic vesicles is described. The vesicles are constituted by didodecyldimethylammonium bromide and have a diameter of about 1 m. An aqueous magnetic fluid, constituted by charged magnetic nanoparticles dispersed in water without surfactant, is encapsulated in the vesicle with a volume fraction in particles that may range up to 10%. The first step of the encapsulation is the synthesis of a multiple emulsion the intermediate oily phase being evaporated to obtain the DDAB bilayer. The magnetic vesicles thus synthesized align and change shape when a magnetic field is applied.     

Controlling transmembrane ion transport via photo-regulated carrier mobility

Stimuli-responsive transmembrane ion carriers allow for targeted and controllable transport activity, with potential applications as therapeutics for channelopathies and cancer, and in fundamental studies into ion transport phenomena. These applications require OFF–ON activation from a fully inactive state which does not exhibit background activity, but this remains challenging to achieve with synthetic transport systems. Here we introduce a novel mechanism for photo-gating mobile ion carriers, which involves modulating the mobility of the carriers within the lipid bilayer membrane. By appending a membrane-targeting anchor to the carrier using a photo-cleavable linker, the carrier''s ion transport activity is fully switched off by suppressing its ability to shuttle between the two aqueous-membrane interfaces of the bilayer. The system can be reactivated rapidly in situ within the membrane by photo-triggered cleavage of the anchor to release the mobile ion carrier. This approach does not involve direct functionalization of the ion binding site of the carrier, and so does not require the de novo design of novel ion binding motifs to implement the photo-caging of activity. This work demonstrates that controlling the mobility of artificial transport systems enables precise control over activity, opening up new avenues for spatio-temporally targeted ionophores.

Photo-gated anion transport is achieved by modulating the mobility of mobile carriers within a lipid bilayer membrane, using a photo-cleavable membrane anchor. This enables in situ, off–on activation of transport in vesicles.     

Simple Michael acceptor type coumarin derived turn-on fluorescence probes to detect cyanide in pure water

The present article describes design and synthesis of coumarin derived chemical reaction based chemodosimeter to recognize cyanide in aqueous medium. The Michael acceptor type coumarin derived probes 3 and 4 upon interaction with cyanide undergo Michael type nucleophilic addition reaction to form adducts in which fluorescence intensity was significantly enhanced, ‘turn-on’ by the change in intramolecular charge transfer (ICT) mechanism. The nonfluorescent color of solutions changed to a naked-eye sensitive fluorescent blue (switched-on). Job’s plot analysis revealed a 1:1 stoichiometry for the interaction between the probe and cyanide anion with a high detection limit.     

Ligand-free nickel catalyzed perfluoroalkylation of arenes and heteroarenes

We describe a “ligand-free” Ni-catalyzed perfluoroalkylation of heteroarenes to produce a diverse array of trfiluoromethyl, pentafluoroethyl and heptafluoropropyl adducts. Catalysis proceeds at room temperature via a radical pathway. The catalytic protocol is distinguished by its simplicity, and its wide scope demonstrates the potential in the late-stage functionalization of drug analogues and peptides.

A ligand-free, room temperature, Ni-catalyzed perfluoroalkylation of heteroarenes produced a diverse array of polyfluorinated adducts; potential in the late-stage functionalization of drugs and peptides is also demonstrated.     

Luminescent monomeric and dimeric Ru(ii) acyclic carbene complexes as selective sensors for NH3/amine vapor and humidity

A new class of luminescent bis(bipyridyl) Ru(ii) pyridyl acyclic carbene complexes with environmentally-sensitive dimerization equilibrium have been developed. Owing to the involvement of the orbitals of the diaminocarbene ligand in the emissive excited state, the phosphorescence properties of these complexes are strongly affected by H-bonding interactions with various H-bonding donor/acceptor molecules. With the remarkable differences in the emission properties of the monomer, dimer, and H-bonded amine adducts together with the change of the dimerization equilibrium, these complexes can be used as luminescent gas sensors for humidity, ammonia, and amine vapors. With the responses to amines and humidity and the corresponding change in the luminescence properties, a proof-of-principle for binary optical data storage with a reversible concealment process has been described.

A new class of selective ammonia/amine vapor and humidity sensors have been developed from the luminescent bis(bipyridyl) Ru(ii) pyridyl acyclic carbene complexes with environmentally-sensitive dimerization equilibrium.     

Intracellular Thiols and Photo‐Illumination Sequentially Activate Doubly Locked Molecular Probes for Long‐Term Cell Highlighting and Tracking with Precise Spatial Accuracy

A novel photoconvertible fluorescent probe, which can be activated by intracellular thiols, has been synthesized. Such a molecular probe comprises three parts: a 7‐aminocoumarin phototrigger, a thiol‐removable energy acceptor, and a caged fluorescein scaffold with intracellular thiols reactivity as the fluorescent reporter. Extracellularly, the energy acceptor blocks the emission of the coumarin that regulates the photocleavage and photoactivation of the fluorescein. Intracelluarly, the high concentration of thiols releases the energy acceptor, thus activating the S1 state of the phototrigger, which emits coumarin blue fluorescence for pre‐visualization and liberates the caged green‐fluorescent fluorescein to highlight the specific cell upon illumination. Compared to traditional photoactivated organic dyes, the intracellular thiols activated probe requires double activations: one by intracellular thiols and the other by light activation. The dual activations restrict fluorescence precisely inside live cells and at the particular spatial region of light activation, thus a probe with precise spatial accuracy in live cells.     

Polariton-assisted manipulation of energy relaxation pathways: donor–acceptor role reversal in a tuneable microcavity

Resonant interaction between excitonic transitions of molecules and localized electromagnetic field allows the formation of hybrid light–matter polaritonic states. This hybridization of the light and the matter states has been shown to significantly alter the intrinsic properties of molecular ensembles placed inside the optical cavity. Here, we have observed strong coupling of excitonic transition in a pair of closely located organic dye molecules demonstrating an efficient donor-to-acceptor resonance energy transfer with the mode of a tuneable open-access cavity. Analysing the dependence of the relaxation pathways between energy states in this system on the cavity detuning, we have demonstrated that predominant strong coupling of the cavity photon to the exciton transition in the donor dye molecule can lead not only to an increase in the donor–acceptor energy transfer, but also to an energy shift large enough to cause inversion between the energy states of the acceptor and the mainly donor lower polariton energy state. Furthermore, we have shown that the polariton-assisted donor–acceptor chromophores'' role reversal or “carnival effect” not only changes the relative energy levels of the donor–acceptor pair, but also makes it possible to manipulate the energy flow in the systems with resonant dipole–dipole interaction and direct energy transfer from the acceptor to the mainly donor lower polariton state. Our experimental data are the first confirmation of the theoretically predicted possibility of polariton-assisted energy transfer reversal in FRET systems, thus paving the way to new avenues in FRET-imaging, remote-controlled chemistry, and all-optical switching.

Polariton-assisted donor–acceptor role reversal in resonant energy transfer between organic dyes tagged with the terminus of the closed oligonucleotide-based molecular beacon strongly coupled to electromagnetic modes of a tuneable microcavity.     

Incorporation of nanogels within calcite single crystals for the storage,protection and controlled release of active compounds

Nanocarriers have tremendous potential for the encapsulation, storage and delivery of active compounds. However, current formulations often employ open structures that achieve efficient loading of active agents, but that suffer undesired leakage and instability of the payloads over time. Here, a straightforward strategy that overcomes these issues is presented, in which protein nanogels are encapsulated within single crystals of calcite (CaCO₃). Demonstrating our approach with bovine serum albumin (BSA) nanogels loaded with (bio)active compounds, including doxorubicin (a chemotherapeutic drug) and lysozyme (an antibacterial enzyme), we show that these nanogels can be occluded within calcite host crystals at levels of up to 45 vol%. Encapsulated within the dense mineral, the active compounds are stable against harsh conditions such as high temperature and pH, and controlled release can be triggered by a simple reduction of the pH. Comparisons with analogous systems – amorphous calcium carbonate, mesoporous vaterite (CaCO₃) polycrystals, and calcite crystals containing polymer vesicles – demonstrate the superior encapsulation performance of the nanogel/calcite system. This opens the door to encapsulating a broad range of existing nanocarrier systems within single crystal hosts for the efficient storage, transport and controlled release of various active guest species.

Nanocarriers have tremendous potential for the encapsulation, storage and delivery of active compounds.     

Short,enantioselective, gram-scale synthesis of (−)-zephyranthine

A reasonable synthesis design by strategically integrating functional group manipulation into the ring system construction resulted in a short, enantioselective, gram-scale total synthesis of (−)-zephyranthine. The concise route includes a catalytic Michael/Michael cascade for the asymmetric synthesis of a penta-substituted cyclohexane with three contiguous stereogenic centers, a remarkable 8-step one-pot operation to easily assemble the zephyranthine tetracyclic skeleton, the regioselective construction of a double bond in the C ring and an asymmetric dihydroxylation. This synthesis is also flexible and paves a potential path to a variety of cyclohexylamine-fused tricyclic or polycyclic alkaloids.

A reasonable synthesis design by strategically integrating functional group manipulation into the ring system construction resulted in a short, enantioselective, gram-scale total synthesis of (−)-zephyranthine.     

Intramolecular thiomaleimide [2 + 2] photocycloadditions: stereoselective control for disulfide stapling and observation of excited state intermediates by transient absorption spectroscopy

Thiomaleimides undergo efficient intermolecular [2 + 2] photocycloaddition reactions and offer applications from photochemical peptide stapling to polymer crosslinking; however, the reactions are limited to the formation of the exo head-to-head isomers. Herein, we present an intramolecular variation which completely reverses the stereochemical outcome of this photoreaction, quantitatively generating endo adducts which minimise the structural disturbance of the disulfide staple and afford a 10-fold increase in quantum yield. We demonstrate the application of this reaction on a protein scaffold, using light to confer thiol stability to an antibody fragment conjugate. To understand more about this intriguing class of [2 + 2] photocycloadditions, we have used transient absorption spectroscopy (electronic and vibrational) to study the excited states involved. The initially formed S₂ (π₁π*) excited state is observed to decay to the S₁ (n₁π*) state before intersystem crossing to a triplet state. An accelerated intramolecular C–C bond formation provides evidence to explain the increased efficiency of the reaction, and the impact of the various excited states on the carbonyl vibrational modes is discussed.

Tethered bromomaleimides are described which undergo thiol conjugation and rapid regio- and stereoselective photocycloadditions to serve as disulfide staples; with the excited state intermediates observed by transient absorption spectroscopies.     

Controlled microfluidic encapsulation of cells, proteins, and microbeads in lipid vesicles

Cells have been encapsulated inside lipid vesicles by using a new microfluidic lipid vesicle formulation technique. Lipid vesicles are formulated within minutes without using toxic lipid solvents. The encapsulation efficiency inside the vesicles is controlled by the microfluidic flows. Green fluorescent proteins (GFP), carcinoma cells, and bead encapsulated vesicles have mean diameters of 27.2 mum, 62.4 mum, and 55.9 mum, respectively. The variations of vesicle sizes are approximately 20% for the GFP and cell encapsulated vesicles and approximately 10% for the bead encapsulated vesicles.     

Molecular planting of a single organothiol into a “gap-site” of a 2D patterned adlayer in an electrochemical environment

The self-assembled inclusion of molecules into two-dimensional (2D) porous networks on surfaces has been extensively studied because 2D functional materials consisting of organic molecules have become an important research topic. However, the isolation of a single molecular thiol remains a challenging goal. Here, we report a method of planting and isolating organothiols onto a 2D patterned organic adlayer at an electrochemical interface. In situ scanning tunneling microscopy revealed that the phase transition of an ovalene adlayer is electrochemically induced and that the gap site created by three ovalene molecules serves as a 2D molecular template to isolate thiol molecules and to standardize the distance between them via the formation of precise selective open spaces, suggesting that electrochemical “molecular planting” opens applications for 2D patterns of isolated single organothiol molecules.

Gap sites electrochemically created in the ovalene adlayer can accept a single thiol.