Macrocyclic Conformational Switch Coupled with Pyridinium-Induced PET for Fluorescence Detection of Adenosine Triphosphate.

The binding of nucleotides is crucial for signal transduction as it induces conformational protein changes, leading to downstream cellular responses. Synthetic receptors that bind nucleotides and transduce the binding event into global conformational rearrangements are highly challenging to design, especially those that operate in an aqueous solution. Much work is focused on evaluating functionalized dyes to detect nucleotides, whereas coupling of a nucleotide-induced conformational switching to a sensing event has not been reported to date. We disclose synthetic receptors that undergo a global conformational rearrangement upon nucleotide binding. Integrating naphthalimide and the pyridinium ion into the structure enables stabilization of the folded conformation and efficient fluorescence quenching. The binding of a nucleotide rearranges the receptor conformation and alters the strong fluorescence enhancement. The methylpyridinium-containing receptor demonstrated high sensing selectivity for adenosine 5'-triphosphate (ATP) and a record 160-fold fluorescence enhancement. It can detect fluctuations of ATP in HeLa cells and possesses low cytotoxicity. The developed systems present an attractive approach for designing ATP-responsive artificial molecular switches that operate in water and integrate a strong fluorescence response.

Effects of Ring Functionalization in Anthracene-Based Cyclophanes on the Binding Properties Toward Nucleotides and DNA.

Supramolecular recognition of nucleobases and short sequences is an emerging research field focusing on possible applications to treat many diseases. Controlling the affinity and selectivity of synthetic receptors to target desired nucleotides or short sequences is a highly challenging task. Herein, we elucidate the effect of substituents in the phenyl ring of the anthracene-benzene azacyclophane on the recognition of nucleoside triphosphates (NTPs) and double-stranded DNA. We show that introducing phenyl rings increases the affinity for NTPs 10-fold and implements groove and intercalation binding modes with double-stranded DNA. NMR studies and molecular modeling calculations support the ability of cyclophanes to encapsulate nucleobases as part of nucleotides.

Autofluorescent Artemisinin-Benzimidazole Hybrids via Organo-Click Reaction: Study of Antiviral Properties and Mode of Action in Living Cells.

Drug modification by a fluorescent label is a common tool for studying its mechanism of action with fluorescence microscopy techniques. However, the attachment of a fluorescent label can significantly alter the polarity, solubility, and biological activity of the investigated drug, and, as a result, the studied mechanism of action can be misrepresented. Therefore, developing efficient drugs, which are inherently fluorescent and can be tracked directly in the cell is highly favorable. Here an easy formation of fluorescent hybrid drugs is presented, generated by a combination of two readily available non-fluorescent pharmacophores via a non-cleavable linker using a Ramachary-Bressy-Wang organocatalyzed azide-carbonyl [3+2] cycloaddition (organo-click) reaction. All newly prepared fluorescent compounds showed strong anti-HCMV activity (EC50 down to 0.07±0.00 µM), thus presenting a very promising drug developmental basis compared to the approved drug ganciclovir (EC50 2.60±0.50 µM). Remarkably, in vitro fluorescent imaging investigation of new compounds revealed induced changes in mitochondrial structures, which is a phenotypical hallmark of antiviral activity. This approach opens up new vistas for the easy formation of potent fluorescent drugs from readily available non-fluorescent parent compounds and might facilitate insight into their mode of action in living cells, avoiding the requirement of linkage to external fluorescent markers.

Micromolar Affinity and Higher: Synthetic Host-Guest Complexes with High Stabilities.

The design of high-affinity synthetic host-guest complexes is of paramount importance because they are key elements in constructing unprecedented supramolecular assemblies, functional materials, molecular probes, artificial signal transduction events, and interfaces with the biological world. The present review article collects recent achievements in the design of 1 : 1 host-guest complexes with outstanding stabilities, i.e., exceeding 106  M-1 . The relationships between the measured thermodynamic constants and the structural parameters of the interacting species are analyzed. The design features of high-affinity hosts are discussed in light of their binding properties. Different solvents and different types of noncovalent interactions are considered for the stabilization of the complexes. Finally, some hints are provided for the design of future synthetic receptors displaying high affinity and selectivity.

Acenaphthenoannulation Induced by the Dual Lewis Acidity of Alumina.

We have discovered a dual (i. e., soft and hard) Lewis acidity of alumina that enables rapid one-pot π-extension through the activation of terminal alkynes followed by C-F activation. The tandem reaction introduces an acenaphthene fragment - an essential moiety of geodesic polyarenes. This reaction provides quick access to elusive non-alternant polyarenes such as π-extended buckybowls and helicenes through three-point annulation of the 1-(2-ethynyl-6-fluorophenyl)naphthalene moiety. The versatility of the developed method was demonstrated by the synthesis of unprecedented structural fragments of elusive geodesic graphene nanoribbons.

Host-Guest Systems on the Surface of Functionalized Superparamagnetic Iron Oxide Nanoparticles (SPIONs) Utilizing Hamilton Receptors and Cyanurate Derivative Molecules.

The study of hydrogen bonding interactions at the level of functionalized nanoparticles remains highly challenging and poorly explored area. In this work, superparamagnetic iron oxide nanoparticles (SPIONs) were orthogonally functionalized using receptors bearing multiple hydrogen bonding motifs. Pristine SPIONs were modified by wet chemical processes with Hamilton receptors (hosts), or cyanurate-guest molecules linked to phosphonic acid moieties for monolayer functionalization. The modified surfaces were fully characterized and the number of attached ligands on the surface were determined. The host-guest interactions on the interface of modified SPIONs were investigated by using UV-Vis spectroscopic titrations. Functionalized SPIONs demonstrated two to three magnitudes stronger binding affinities as compared to the related molecular interactions in solution due to synergistic effects on complex surface environment. Higher supramolecular binding ratios of host-guest interactions on the modified surface were emerged. These studies provide fundamental insights into supramolecular complexations on the surface at solid-liquid interface systems with applications in engineered nanomaterials, nano-sensing devices, and drug delivery systems.

Binding and Sensing Properties of a Hybrid Naphthalimide-Pyrene Aza-Cyclophane towards Nucleotides in an Aqueous Solution.

Selective recognition of nucleotides with synthetic receptors is an emerging direction to solve a series of nucleic acid-related challenges in biochemistry. Towards this goal, a new aza-cyclophane with two different dyes, naphthalimide and pyrene, connected through a triamine linker has been synthesized and studied for the ability to bind and detect nucleoside triphosphates in an aqueous solution. The receptor shows Foerster resonance energy transfer (FRET) in fluorescence spectra upon excitation in DMSO, which is diminished dramatically in the presence of water. According to binding studies, the receptor has a preference to bind ATP (adenosine triphosphate) and CTP (cytidine triphosphate) with a "turn-on" fluorescence response. Two separate emission bands of dyes allow one to detect nucleotides in a ratiometric manner in a broad concentration range of 10-5-10-3 M. Spectroscopic measurements and quantum chemical calculations suggest the formation of receptor-nucleotide complexes, which are stabilized by dispersion interactions between a nucleobase and dyes, while hydrogen bonding interactions of nucleobases with the amine linkers are responsible for selectivity.

Ratiometric Detection of ATP by Fluorescent Cyclophanes with Bellows-Type Sensing Mechanism.

Pyrene-based cyclophanes have been synthesized with the aim to realize a bellows-type sensing mechanism for the ratiometric detection of nucleotide concentrations in a buffered aqueous solution. The sensing mechanism involves the encapsulation of a nucleobase between two pyrene rings, which affects the monomer-excimer equilibrium of the receptor in the excited state. The nature of the spacer and its connection pattern to pyrene rings have been varied to achieve high selectivity for ATP. The 1,8-substituted pyrene-based cyclophane with the 2,2'-diaminodiethylamine spacer demonstrates the best selectivity for ATP showing a 50-fold increase in the monomer-excimer emission ratio upon saturation with the nucleotide. The receptor can detect ATP within the biological concentrations range over a wide pH range. NMR and spectroscopic studies have revealed the importance of hydrogen bonding and stacking interactions for achieving a required receptor selectivity. The probe has been successfully applied for the real-time monitoring of creatine kinase activity.

Control of Photoisomerization of an Azoazacryptand by Anion Binding and Cucurbit[8]uril Encapsulation in an Aqueous Solution.

Control of isomerization of a receptor bearing multiple light-switchable subunits in a confined space is critical for the design of synthetic molecular machines. Toward this goal, a new azacryptand containing three azobenzene subunits has been developed, and its photoisomerization in an aqueous solution has been studied depending on anion coordination and recognition by a larger host-cucurbit[8]uril (CB[8]). The cryptand in its hexaprotonated form shows considerable affinity for fluoride and perchlorate, which in turn affects the isomer distribution of the receptor under UV-light irradiation, stabilizing the isomers of the cryptand with Z-configurations. CB[8] was found to be able to encapsulate the isomers of the cryptand by forming a Matryoshka-type complex. The irradiation of a 10:1 CB[8]-cryptand mixture has led to a selective conversion of the cryptand to the E,E,Z isomer inside CB[8]. It has been demonstrated that the addition of fluoride to the resulted complex induces the release of the cryptand as a major E,E,E isomer, while other studied anions were ineffective in this reaction. To our knowledge, this work presents a first example of a host-controlled photoisomerization of an anion receptor bearing multiple switching azobenzenes that model the function of naturally occurring chaperones.

Anthracene-Based Cyclophanes with Selective Fluorescent Responses for TTP and GTP: Insights into Recognition and Sensing Mechanisms.

Three anthracene-based cyclophanes were synthesized and their binding properties towards nucleoside triphosphates were studied. A new polycyclic amine derived from dearomatized anthracene was identified as a major side product in the cyclization reaction between 9,10-anthracenedicarboxaldehyde and diethylenetriamine. Its structure was determined by single-crystal X-ray analysis. The cyclophanes were found to form 1:1 complexes with all nucleoside triphosphates as well as with pyrophosphate in a buffered aqueous solution at pH 6.2. A turn-on fluorescence response was observed for all nucleotides except for GTP, which demonstrated strong fluorescence quenching. The strongest turn-on fluorescence was observed for the largest receptor 3 in the presence of thymidine triphosphate (TTP). Based on the NMR and fluorescence experiments, two major binding modes for nucleotide complexes were identified.

Molecular Receptors for Recognition and Sensing of Nucleotides.

Nucleotides are constituents of nucleic acids and they have a variety of functions in cellular metabolism. Synthetic receptors and sensors are required to reveal the role of nucleotides in living organisms and mechanisms of signal transduction events. In recent years, a large number of nucleotide-selective synthetic receptors have been devised, which utilize different molecular designs and sensing mechanisms. This Minireview presents recent progress in the design of synthetic molecular receptors for selective recognition of nucleotides in aqueous solution. The binding properties of receptors and the origins of their selectivity for a particular nucleotide are discussed.

Conformational Selection in Anion Recognition: cGMP-Selective Binding by a Naphthalimide-Functionalized Amido-Amine Macrocycle.

Amido-amine macrocycles with two and four naphthalimide dyes were designed to bind nucleoside monophosphates and oligonucleotides in an aqueous buffered solution. Anion-templated synthesis was used to direct the macrocyclization reaction to the [2+2] product, while high dilution conditions favored the formation of the [4+4] macrocycle with an unprecedented geometry, as revealed from the X-ray analysis. The [2+2] product was found to exhibit a remarkable binding strength and fluorescence response for cyclic guanosine monophosphate (cGMP) in an aqueous solution. To our knowledge, this is the first synthetic receptor for cGMP, which also demonstrates a high preference to bind guanine-rich sequences accomplished by a strong fluorescence quenching. The receptor conformation is very sensitive to the guest structure in an aqueous solution, thus modeling the adaptive behavior of proteins. The study of synthetic systems with a detectable conformational equilibrium represents a great potential for understanding highly specific and tightly regulated interactions in biological systems.

Straightforward Design of Fluorescent Receptors for Sulfate: Study of Non-Covalent Interactions Contributing to Host-Guest Formation.

A straightforward design of receptors for binding and sensing of sulfate in aqueous medium was developed. The design involves the connection of two naphthalimide-based pH probes through a hydrogen-bonding motif. The structure of the receptor-sulfate complex, predicted by DFT calculations, was unambiguously confirmed by NMR measurements. There are three major interactions stabilizing the host-guest complex: electrostatic interactions, hydrogen bonding, and stacking interactions of the dyes. Study of two control receptors containing either one dye or methyl amide groups instead of amides, revealed that electrostatic and hydrogen bonding interactions contribute the most to affinity and selectivity of receptors. The receptors can detect sulfate in a 1:1 THF-buffer mixture in pH window 3.6-4.5 demonstrating up to 7-fold fluorescence enhancement. To the best of our knowledge, the reported PET (photoinduced electron transfer) anion probes possess the largest response for sulfate in aqueous solution yet described.

Hybrid Macrocycles for Selective Binding and Sensing of Fluoride in Aqueous Solution.

Synthesis and anion binding properties of hybrid macrocycles containing ammonium and hydrogen bond donor groups are reported. Receptor properties were studied in a 10 mM MES buffer solution at pH 6.2, at which the receptors carry two positive charges at the secondary amine groups. Receptor 1 was found to bind fluoride with the highest affinity (105 M-1) and selectivity among the synthesized receptors. It was the only receptor that demonstrated fluorescence increase upon addition of fluoride. Other titration experiments with halides and oxyanions led to an anion-induced aggregation and fluorescence quenching. The mechanism of the particular turn-on fluorescence for fluoride was explained by the ability of receptor 1 to encapsulate several fluoride anions. Multiple anion coordination resulted in the protonation of the tertiary amine group and subsequent hindering of the PET process. 1H and 19F NMR titrations, single-crystal X-ray structure of chloride complex, and DFT calculation suggest that 1 can perfectly accommodate two fluoride anions in the inner cavity but only one chloride, keeping the second chloride in the outer coordination sphere. Thus, the importance of size selectivity, which is reflected in a collective behavior of molecules in an aqueous solution, represents a new strategy for the design of highly selective probes for fluoride functioning in an aqueous solution.

Naphthalimide-Based Polyammonium Chemosensors for Anions: Study of Binding Properties and Sensing Mechanisms.

New naphthalimide-based receptors for anions have been synthesized. Efficient synthetic routes have been discovered to functionalize the naphthalimide core with branched polyamines. Binding and sensing properties of the receptors were studied by potentiometric, NMR and fluorescence titrations. The receptors bind selectively to the pyrophosphate anion in buffered aqueous solutions. The receptors with more than six amine groups in the structure demonstrated the highest affinities for pyrophosphate. The fluorescence response towards anions was found to be dependent on the position of the amine groups relative to the naphthalimide core, and on the pH of the buffered solution. Three sensing mechanisms have been found that explain fluorescence responses of receptors towards anions in an aqueous solution.

Utilizing a pH-Sensitive Dye in the Selective Fluorescent Recognition of Sulfate.

A receptor containing amidopyrrole binding subunits and free amino groups, conjugated to a naphthalimide dye, has been designed and synthesized. The intrinsic selectivity of the binding motif for phosphate present in DMSO completely disappears in 10 % DMSO aqueous buffer at pH 3.6, at which the receptor is protonated. The electrostatic interactions between the receptor and an anion start to dominate, thus leading to selectivity for sulfate. The ability of the HSO4- anion to transfer the proton to the amino group during the recognition event suppresses the photoinduced electron transfer (PET) on the dye, resulting in a selective turn-on fluorescent response. The choice of pH of the solution for sensing is dictated by the pKa value of the dye.

Understanding Stacking Interactions between an Aromatic Ring and Nucleobases in Aqueous Solution: Experimental and Theoretical Study.

Stacking interactions between aromatic compounds and nucleobases are crucial in recognition of nucleotides and nucleic acids, but a comprehensive understanding of the strength and selectivity of these interactions in aqueous solution has been elusive. To this end, model complexes have been designed and analyzed by experiment and theory. For the first time, stacking free energies between five nucleobases and anthracene were determined experimentally from thermodynamic double mutant cycles. Three different experimental methods were proposed and evaluated. The dye prefers to bind nucleobases in the order (kcal/mol): G (1.3) > T (0.9) > U (0.8) > C (0.5) > A (0.3). The respective trend of interaction free energies extracted from DFT calculations correlates to that obtained experimentally. Analysis of the data suggests that stacking interactions dominate over hydrophobic effects in an aqueous solution and can be predicted with DFT calculations.

Investigation of structural mimetics of natural phosphate ion binding motifs.

Phosphates are ubiquitous in biology and nearly half of all proteins interact with their partners by means of recognition of phosphate residues. Therefore, a better understanding of the phosphate ion binding by peptidic structures is highly desirable. Two new receptors have been designed and synthesized and their anion binding properties in an acetonitrile solution have been determined. The structure of hosts mimics a part of the kinase active site that is responsible for the recognition of the phosphate residue. New hosts contain additional free amino groups with the aim to facilitate coordination of protonated anions, such as dihydrogen phosphate. According to spectrophotometric measurements, stepwise 1:1 and 1:2 binding modes have been observed for both receptors in the presence of acetate, hydrogen sulfate and dihydrogen phosphate. Compared with the acyclic receptor, the macrocyclic receptor has demonstrated a remarkably enhanced selectivity for dihydrogen phosphate over other anions. Fluorometric measurements have revealed different responses of the acyclic and macrocyclic receptors towards anions. However, in both cases, a 5-8 nm hypsochromic shift of fluorescence maximum has been observed upon interaction of acetate and dihydrogen phosphate with receptors.

Mesoporous supraparticles with a tailored solid-liquid-gas interface for visual indication of H2 gas and NH3 vapours.

Dual-gasochromic supraparticles that undergo rapid eye-readable and gas-specific colour changes upon reaction with hydrogen or ammonia are reported. This functionality is achieved by tailoring the solid-liquid-gas interface within the mesoporous framework of supraparticles via spray-drying.

Templating irreversible covalent macrocyclization by using anions.

Inorganic anions were used as templates in the reaction between a diamine and an activated diacid to form macrocyclic amides. The reaction conditions were found to perform the macrocyclization sufficiently slow to observe a template effect. A number of analytical methods were used to clarify the reaction mechanisms and to show that the structure of the intermediate plays a decisive role in determining the product distribution. For the macrocyclization under kinetic control, it was shown that the amount of a template, the conformational rigidity of building blocks, and the anion affinities of reaction components and intermediates are important parameters that one should take into consideration to achieve high yields.