Chemical Information Processing by a Responsive Chemical System.

Nature has an extraordinary capacity to precisely regulate the chemical reactivity in a highly complex mixture of molecules that is present in the cell. External stimuli lead to transient up- and downregulation of chemical reactions and provide a means for a cell to process information arriving from the environment. The development of synthetic chemical systems with life-like properties requires strategies that allow likewise control over chemical reactivity in a complex environment. Here, we show a synthetic system that mimics the initial steps that take place when a natural signal transduction pathway is activated. Monophosphate nucleosides act as chemical triggers for the self-assembly of nanoreactors that upregulate chemical reactions between reagents present at low micromolar concentrations. Different nucleotides template different assemblies and hence activate different pathways, thus establishing a distinct connection between input and output molecules. Trigger-induced upregulation of chemical reactivity occurs for only a limited amount of time because the chemical triggers are gradually removed from the system by enzymes. It is shown that the same system transiently produces different output molecules depending on the chemical input that is provided.

Formation of Catalytic Hotspots in ATP-Templated Assemblies.

The self-assembly of surfactant-based structures that rely for their formation on the combination of a thermodynamically controlled and a dissipative pathway is described. Adenosine triphosphate (ATP) acts as a high-affinity template and triggers assembly formation at low surfactant concentrations. The presence of these assemblies creates the conditions for the activation of a dissipative self-assembly process by a weak-affinity substrate. The substrate-induced recruitment of additional surfactants leads to the spontaneous formation of catalytic hotspots in the ATP-stabilized assemblies that cleave the substrate. As a result of the two self-assembly processes, catalysis can be observed at a surfactant concentration at which low catalytic activity is observed in the absence of ATP.

Persistent ATP-Concentration Gradients in a Hydrogel Sustained by Chemical Fuel Consumption.

We show the formation of macroscopic ATP-concentrations in an agarose gel and demonstrate that these gradients can be sustained in time at the expense of the consumption of a chemical fuel. The approach relies on the spatially controlled activation of ATP-producing and ATP-consuming reactions through the local injection of enzymes in the matrix. The reaction-diffusion system is maintained in a stationary non-equilibrium state as long as chemical fuel, phosphocreatine, is present. The reaction-diffusion system is coupled to a supramolecular system composed of monolayer protected gold nanoparticles and a fluorescent probe. As a result of this coupling, fluorescence signals emerge spontaneously in response to the ATP-concentration gradients. We show that the approach permits the rational formation of complex fluorescence patterns that change over time as a function of the evolution of the ATP-concentrations present in the system.

ATP Drives the Formation of a Catalytic Hydrazone through an Energy Ratchet Mechanism.

An energy ratchet mechanism is exploited for the synthesis of a molecule. In the presence of adenosine triphosphate (ATP), hydrazone-bond formation between an aldehyde and hydrazide is accelerated and the composition at thermodynamic equilibrium is shifted towards the hydrazone. Enzymatic hydrolysis of ATP installs a kinetically stable state, at which hydrazone is present at a higher concentration compared to the composition at thermodynamic equilibrium in the presence of the degradation products of ATP. It is shown that the kinetic state has an enhanced catalytic activity in the hydrolysis of an RNA-model compound.

Progressive Local Accumulation of Self-Assembled Nanoreactors in a Hydrogel Matrix through Repetitive Injections of ATP.

Cellular functions are regulated with high spatial control through the local activation of chemical processes in a complex inhomogeneous matrix. The development of synthetic macroscopic systems with a similar capacity allows fundamental studies aimed at understanding the relationship between local molecular events and the emergence of functional properties at the macroscopic level. Here, we show that a kinetically stable inhomogeneous hydrogel matrix is spontaneously formed upon the local injection of ATP. Locally, ATP templates the self-assembly of amphiphiles into large nanoreactors with a much lower diffusion rate compared to unassembled amphiphiles. The local depletion of unassembled amphiphiles near the injection point installs a concentration gradient along which unassembled amphiphiles diffuse from the surroundings to the center. This allows for a progressive local accumulation of self-assembled nanoreactors in the matrix upon repetitive cycles of ATP injection separated by time intervals during which diffusion of unassembled amphiphiles takes place. Contrary to the homogeneous matrix containing the same components, in the inhomogeneous matrix the local upregulation of a chemical reaction occurs. Depending on the way the same amount of injected ATP is administered to the hydrogel matrix different macroscopic distributions of nanoreactors are obtained, which affect the location in the matrix where the chemical reaction is upregulated.

Duplex vs. folding: tuning the self-assembly of synthetic recognition-encoded aniline oligomers.

One of the challenges in the realization of synthetic oligomers capable of sequence-selective duplex formation is intramolecular folding interaction between complementary recognition units. To assess whether complementary hetero-oligomers can assemble into high fidelity duplex structures, the competing folding equilibria must be carefully considered. A family of recognition-encoded aniline oligomers were assembled via reductive amination of dianiline linkers and dialdehyde monomers, which were equipped with either a 2-trifluoromethylphenol or a phosphine oxide H-bond recognition unit. To test the possibility of 1,2-folding in mixed sequence oligomers, the self-assembly properties of the homo- and hetero-dimers were characterised by 19F and 1H NMR titration and dilution experiments in toluene and in chloroform. Three different systems were investigated with variations in the steric bulk around the H-bond acceptor unit and the length of the dianiline linker. For two systems, the hetero-dimers folded with intramolecular H-bonding in the monomeric state, reducing stability of the intermolecular duplex by two to three orders of magnitude compared with the corresponding homo-oligomers. However, the use of a long rigid linker as the backbone connecting two monomer units successfully prevents 1,2-folding and leads to the formation of a stable mixed sequence duplex in toluene.

Chemically Fueled Self-Assembly in Biology and Chemistry.

Life is a non-equilibrium state of matter maintained at the expense of energy. Nature uses predominantly chemical energy stored in thermodynamically activated, but kinetically stable, molecules. These high-energy molecules are exploited for the synthesis of other biomolecules, for the activation of biological machinery such as pumps and motors, and for the maintenance of structural order. Knowledge of how chemical energy is transferred to biochemical processes is essential for the development of artificial systems with life-like processes. Here, we discuss how chemical energy can be used to control the structural organization of organic molecules. Four different strategies have been identified according to a distinguishable physical-organic basis. For each class, one example from biology and one from chemistry are discussed in detail to illustrate the practical implementation of each concept and the distinct opportunities they offer. Specific attention is paid to the discussion of chemically fueled non-equilibrium self-assembly. We discuss the meaning of non-equilibrium self-assembly, its kinetic origin, and strategies to develop synthetic non-equilibrium systems.

Nucleotide-Selective Templated Self-Assembly of Nanoreactors under Dissipative Conditions.

Nature adopts complex chemical networks to finely tune biochemical processes. Indeed, small biomolecules play a key role in regulating the flux of metabolic pathways. Chemistry, which was traditionally focused on reactions in simple mixtures, is dedicating increasing attention to the network reactivity of highly complex synthetic systems, able to display new kinetic phenomena. Herein, we show that the addition of monophosphate nucleosides to a mixture of amphiphiles and reagents leads to the selective templated formation of self-assembled structures, which can accelerate a reaction between two hydrophobic reactants. The correct matching between nucleotide and the amphiphile head group is fundamental for the selective formation of the assemblies and for the consequent up-regulation of the chemical reaction. Transient stability of the nanoreactors is obtained under dissipative conditions, driven by enzymatic dephosphorylation of the templating nucleotides. These results show that small molecules can play a key role in modulating network reactivity, by selectively templating self-assembled structures that are able to up-regulate chemical reaction pathways.

Multimodal 19 F†NMR Dopamine Detection and Imaging with a Nanoparticle-Based Displacement Assay.

Nanoconjugates composed of gold nanoparticles (core diameter=1.9 nm) coated with thioundecyl-d-glucopyranosides and fluorinated phenylboronic acids can detect diol-containing derivatives by means of 19 F NMR spectroscopic analysis. The spectra of nanoconjugate solutions display broad signals due to the fast relaxation of the 19 F nuclei caused by nanoparticle grafting. When dopamine is added, the formation of a boronate ester between the analyte and the fluorinated boronic acid causes the release of the latter in solution and consequent sharpening of the NMR signals. Dopamine can be selectively detected through magnetic resonance imaging (MRI) and NMR spectroscopic analysis with respect to glucose and galactose with a detection limit of 20 µm. The chemical shift of the released ester is diagnostic of the recognized analyte. Consequently, the sensor also enables the simultaneous detection of different analytes.

Minimal Self-Immolative Probe for Multimodal Fluoride Detection.

Two single-molecule, self-immolative fluoride probes, namely tert-butyldimethylsilyl-protected 2- and 4-difluoromethylphenol, are described. Compared to similar systems previously described, the probes are characterized by a simpler structure and straightforward, two-step preparation. Nevertheless, they allow the detection of fluoride ions at micromolar concentration by the naked eye, UV-vis absorption, and fluorescence. A detailed investigation of the self-immolative reaction reveals that the rate-limiting step is the release of the first fluoride ion from the difluoromethylphenolate intermediate. Moreover, the mutual position of the difluoromethyl- and tert-butyldimethylsilyl-protected residues has a relevant effect on the reactivity. Likely, a CF2H-O hydrogen bond in the 2-isomer increases the reactivity of the silyl ether toward hydrolytic cleavage but also stabilizes the phenolate intermediate, slowing the release of fluoride ions.

Synthetic sulfoglycolipids targeting the serine-threonine protein kinase Akt.

The serine-threonine protein kinase Akt, also known as protein kinase B, is a key component of the phosphoinositide 3-kinase (PI3K)-Akt-mTOR axis. Deregulated activation of this pathway is frequent in human tumors and Akt-dependent signaling appears to be critical in cell survival. PI3K activation generates 3-phosphorylated phosphatidylinositols that bind Akt pleckstrin homology (PH) domain. The blockage of Akt PH domain/phosphoinositides interaction represents a promising approach to interfere with the oncogenic potential of over-activated Akt. In the present study, phosphatidyl inositol mimics based on a ß-glucoside scaffold have been synthesized as Akt inhibitors. The compounds possessed one or two lipophilic moieties of different length at the anomeric position of glucose, and an acidic or basic group at C-6. Docking studies, ELISA Akt inhibition assays, and cellular assays on different cell models highlighted 1-O-octadecanoyl-2-O-ß-d-sulfoquinovopyranosyl-sn-glycerol as the best Akt inhibitor among the synthesized compounds, which could be considered as a lead for further optimization in the design of Akt inhibitors.

Photoswitchable NIR-Emitting Gold Nanoparticles.

Photo-switching of the NIR emission of gold nanoparticles (GNP) upon photo-isomerization of azobenzene ligands, bound to the surface, is demonstrated. Photophysical results confirm the occurrence of an excitation energy transfer process from the ligands to the GNP that produces sensitized NIR emission. Because of this process, the excitation efficiency of the gold core, upon excitation of the ligands, is much higher for the trans form than for the cis one, and t→c photo-isomerization causes a relevant decrease of the GNP NIR emission. As a consequence, photo-isomerization can be monitored by ratiometric detection of the NIR emission upon dual excitation. The photo-isomerization process was followed in real-time through the simultaneous detection of absorbance and luminescence changes using a dedicated setup. Surprisingly, the photo-isomerization rate of the ligands, bound to the GNP surface, was the same as measured for the chromophores in solution. This outcome demonstrated that excitation energy transfer to gold assists photo-isomerization, rather than competing with it. These results pave the road to the development of new, NIR-emitting, stimuli-responsive nanomaterials for theranostics.

Thiol-ene mediated neoglycosylation of collagen patches: a preliminary study.

Despite the relevance of carbohydrates as cues in eliciting specific biological responses, the covalent surface modification of collagen-based matrices with small carbohydrate epitopes has been scarcely investigated. We report thereby the development of an efficient procedure for the chemoselective neoglycosylation of collagen matrices (patches) via a thiol-ene approach, between alkene-derived monosaccharides and the thiol-functionalized material surface. Synchrotron radiation-induced X-ray photoelectron spectroscopy (SR-XPS), Fourier transform-infrared (FT-IR), and enzyme-linked lectin assay (ELLA) confirmed the effectiveness of the collagen neoglycosylation. Preliminary biological evaluation in osteoarthritic models is reported. The proposed methodology can be extended to any thiolated surface for the development of smart biomaterials for innovative approaches in regenerative medicine.

Arabinose 5-phosphate isomerase as a target for antibacterial design: studies with substrate analogues and inhibitors.

Structural requirements of D-arabinose 5-phosphate isomerase (KdsD, E.C. 5.3.1.13) from Pseudomonas aeruginosa were analysed in detail using advanced NMR techniques. We performed epitope mapping studies of the binding between the enzyme and the most potent KdsD inhibitors found to date, together with studies of a set of newly synthesised arabinose 5-phosphate (A5P) mimetics. We report here the first experimental evidence that KdsD may bind the furanose form of A5P, suggesting that catalysis of ring opening may be an important part of KdsD catalysis.

Epoxide opening versus silica condensation during sol-gel hybrid biomaterial synthesis.

Hybrid organic-inorganic solids represent an important class of engineering materials, usually prepared by sol-gel processes by cross-reaction between organic and inorganic precursors. The choice of the two components and control of the reaction conditions (especially pH value) allow the synthesis of hybrid materials with novel properties and functionalities. 3-Glycidoxypropyltrimethoxysilane (GPTMS) is one of the most commonly used organic silanes for hybrid-material fabrication. Herein, the reactivity of GPTMS in water at different pH values (pH 2-11) was deeply investigated for the first time by solution-state multinuclear NMR spectroscopic and mass spectrometric analysis. The extent of the different and competing reactions that take place as a function of the pH value was elucidated. The NMR spectroscopic and mass spectrometric data clearly indicate that the pH value determines the kinetics of epoxide hydrolysis versus silicon condensation. Under slighly acidic conditions, the epoxy-ring hydrolysis is kinetically more favourable than the formation of the silica network. In contrast, under basic conditions, silicon condensation is the main reaction that takes place. Full characterisation of the formed intermediates was carried out by using NMR spectroscopic and mass spectrometric analysis. These results indicate that strict control of the pH values allows tuning of the reactivity of the organic and inorganic moities, thus laying the foundations for the design and synthesis of sol-gel hybrid biomaterials with tuneable properties.

Sequence-selective duplex formation and template effect in recognition-encoded oligoanilines.

A new family of duplex-forming recognition encoded oligomers, capable of sequence selective duplex formation and template directed synthesis, was developed. Monomers equipped with both amine and aldehyde groups were functionalized with 2-trifluoromethylphenol or phosphine oxide as H-bond recognition units. Duplex formation and assembly properties of homo- and hetero-oligomers were studied by 19F and 1H NMR experiments in chloroform. The designed backbone prevents the undesired 1,2-folding allowing sequence-selective duplex formation, and the stability of the antiparallel duplex is 3-fold higher than the parallel arrangement. Dynamic combinatorial chemistry was exploited for the templated synthesis of complementary oligomers, showing that an aniline dimer can template the formation of the complementary imine. The key role of the H-bond recognition confers to the system the ability to discriminate a mutated donor monomer incapable of H-bonding. Sequence selective duplex formation combined with the template effect makes this system an attractive target for further studies.

Detergent-aided polymersome preparation.

Until now, most preparative methods used to form polymeric vesicles involve either organic cosolvents or sonication. In this communication, we demonstrate for the first time a detergent-aided method to produce polymersomes. Peptidic polymersomes were formed from the rod-rod block copolymer PBLG(36)-E, where PBLG is hydrophobic poly(gamma-benzyl l-glutamate) and E is a hydrophilic designed peptide. The block copolymer was first solubilized by detergent micelles in aqueous buffer, after which the concentration of detergent was reduced by dilution, transforming the particle morphology in solution from mixed micelles to polymersomes. The polymersome formation was monitored with dynamic light scattering and confirmed with transmission electron microscopy. Polymersomes with average diameters of approximately 300 nm were obtained as well as discs with average diameters of approximately 100 nm. This detergent-based method can be used to create polymersomes with a range of properties, as verified by its application to another biocompatible block copolymer, the flexible polybutadiene(46)-b-poly(ethylene glycol)(30). The technique will be particularly useful when delicate biomacromolecules such as (membrane) proteins, peptides, or nucleic acids are to be encapsulated in the polymersomes because the detergents used are compatible with these compounds, and the possible denaturing effect of sonication or organic solvents on the biological activity of the molecule of interest is avoided.

Supramolecular catalysis by recognition-encoded oligomers: discovery of a synthetic imine polymerase.

All key chemical transformations in biology are catalysed by linear oligomers. Catalytic properties could be programmed into synthetic oligomers in the same way as they are programmed into proteins, and an example of the discovery of emergent catalytic properties in a synthetic oligomer is reported. Dynamic combinatorial chemistry experiments designed to study the templating of a recognition-encoded oligomer by the complementary sequence have uncovered an unexpected imine polymerase activity. Libraries of equilibrating imines were formed by coupling diamine linkers with monomer building blocks composed of dialdehydes functionalised with either a trifluoromethyl phenol (D) or phosphine oxide (A) H-bond recognition unit. However, addition of the AAA trimer to a mixture of the phenol dialdehyde and the diamine linker did not template the formation of the DDD oligo-imine. Instead, AAA was found to be a catalyst, leading to rapid formation of long oligomers of D. AAA catalysed a number of different imine formation reactions, but a complementary phenol recognition group on the aldehyde reaction partner is an essential requirement. Competitive inhibition by an unreactive phenol confirmed the role of H-bonding in substrate recognition. AAA accelerates the rate of imine formation in toluene by a factor of 20. The kinetic parameters for this enzyme-like catalysis are estimated as 1 × 10-3 s-1 for k cat and the dissociation constant for substrate binding is 300 µM. The corresponding DDD trimer was found to catalyse oligomerisation the phosphine oxide dialdehyde with the diamine linker, suggesting an important role for the backbone in catalysis. This unexpected imine polymerase activity in a duplex-forming synthetic oligomer suggests that there are many interesting processes to be discovered in the chemistry of synthetic recognition-encoded oligomers that will parallel those found in natural biopolymers.

Two-component assembly of recognition-encoded oligomers that form stable H-bonded duplexes.

A new family of recognition-encoded oligomers that form stable duplexes in chloroform have been prepared. Monomer building blocks composed of dialdehydes functionalised with either a trifluoromethylphenol or phosphine oxide H-bond recognition unit were prepared. The dialdehydes were coupled with diamines by imine formation and then reduction to give homo-oligomers between one and three recognition units in length. Duplex formation was characterised by 19F and 1H NMR titration experiments in toluene and in chloroform. For duplexes formed between length complementary H-bond donor and acceptor homo-oligomers, an order of magnitude increase in stability was observed for every base-pair added to the duplex in chloroform. The effective molarity for the intramolecular H-bonds responsible for zipping up the duplex is 30 mM, which results in the fully assembled duplex in all cases. The uniform increase in duplex stability with oligomer length suggests that the backbone structure and geometry is likely to be compatible with the formation of extended duplexes in longer oligomers.

Time-gated fluorescence signalling under dissipative conditions.

Precise control over specific functions in the time domain is ubiquitous in biological systems. Here, we demonstrate time-gated fluorescence signalling under dissipative conditions exploiting an ATP-fueled self-assembly process. A temporal ATP-concentration gradient allows the system to pass through three states, among which only the intermediate state generates a fluorescent signal from a hydrophobic dye entrapped in the assemblies. The system can be reactivated by adding a new batch of ATP. The results indicate a strategy to rationally programme the temporal emergence of functions in complex chemical systems.