Merging of a Supramolecular Ligand with a Switchable Luminophore - Light-Responsiveness, Photophysics and Bioimaging.

: In this contribution we report on a novel approach towards luminescent light-responsive ligands. To this end, cyanostilbene- guanidiniocarbonyl-pyrrole hybrids were designed and investigated. Merging of a luminophore with a supramolecular bioactive ligand bears numerous advantages by overcoming the typical drawbacks of drug-labelling, influencing the overall performance of the active species by attachment of a large luminophore. Here we were able to establish a simple and easily accessible synthesis route to different cyanostyryl-guanidininiocarbonyl-pyrrole (CGCP) derivatives. These compounds were investigated regarding their light-responsive double bond isomerisation, their molecular structures in single crystals by means of X-ray diffractometry, their emission properties by state of the art photophysical characterisation as well as bioimaging and assessment of cell toxicity.

Functional Linkers Support Targeting of Multivalent Tweezers to Taspase1.

Taspase 1 is a unique protease not only pivotal for embryonic development but also implicated in leukemias and solid tumors. As such, this enzyme is a promising while still challenging therapeutic target, and with its protein structure featuring a flexible loop preceding the active site a versatile model system for drug development. Supramolecular ligands provide a promising complementary approach to traditional small-molecule inhibitors. Recently, the multivalent arrangement of molecular tweezers allowed the successful targeting of Taspase 1's surface loop. With this study we now want to take the next logic step und utilize functional linker systems that not only allow the implementation of novel properties but also engage in protein surface binding. Consequently, we chose two different linker types differing from the original divalent assembly: a backbone with aggregation-induced emission (AIE) properties to enable monitoring of binding and a calix[4]arene scaffold initially pre-positioning the supramolecular binding units. With a series of four AIE-equipped ligands with stepwise increased valency we demonstrated that the functionalized AIE linkers approach ligand binding affinities in the nanomolar range and allow efficient proteolytic inhibition of Taspase 1. Moreover, implementation of the calix[4]arene backbone further enhanced the ligands' inhibitory potential, pointing to a specific linker contribution.

In situ Cyclization of Aromatic Thioethers in Emissive Materials to Generate Phosphorescent Dibenzothiophenes.

In this contribution, we explored the photocyclization of thioethers to highly substituted dibenzothiophenes (DBT) using solely UV-light without any need for additives. This cost-effective, robust and environmentally friendly approach yielded phosphorescent compounds, which were characterized through X-ray crystallography and state-of-the-art photophysical methods. The resulting DBTs feature ultralong photoluminescence lifetimes and quantum yields close to unity in frozen glassy matrices. The reaction mechanism was investigated in detail through a combination of quantum chemical calculations and experimental results, providing evidence that triplet states are involved in the cyclization process. Additionally, the photoreaction can also be induced within materials. For this purpose, the precursors can be integrated into polymer films or polymer resins suitable for 3D printing. Irradiation of these polymeric objects allows motifs with ultralong phosphorescence to be irreversibly inscribed through the proceeding photocyclization. The in situ photogeneration of DBTs from aromatic thioethers overcomes the observed incompatibilities regarding solubility in polymer resins for 3D printing.

Design Concepts for Solution and Solid-State Emitters - A Modern Viewpoint on Classical and Non-Classical Approaches.

For a long time, luminescence phenomena were strictly distinguished between the emission of isolated molecules in dilute solutions or close-packed structures such as in powders or aggregates. This changed with the breakthrough observation of dual-state efficient materials, which led to a rapid boost of publications examining the influence of structural features to achieve balanced emission with disregarded molecular surroundings. Some first general structural design concepts have already been proposed based on reoccurring patterns and pivotal motifs. However, we have found another way to classify these solution and solid-state emitters (SSSEs). Hence, this minireview aims to present an overview of published structural features of SSSEs while shining light on design concepts from a more generalized perspective. Since SSSEs are believed to bridge the gap of hitherto known aggregation-sensitive compound classes, we hope to give future scientists a versatile tool in hand to efficiently design novel luminescent materials.

Cationic Solution and Solid-State Emitters - Robust Imaging Agents for Cells, Bacteria, and Protists.

A library of eight different cationic emitters with emission properties in solution and in solid-state (solution and solid-state emitters - SSSE) is presented. These compounds, bearing either ammonium or pyridinium groups, have been investigated regarding their photophysical properties as well as their potential application in biological imaging. Besides high quantum yields as well as a high degree of stability during the imaging process, it was additionally revealed that a broad range of biological targets can be addressed, such as different bacterial strains, human cells as well as protists. The reported SSSE approach employing the mentioned robust emitters for biological imaging, will contribute to a rapid and facile way to design and apply affordable emitters with outstanding properties. Additionally, these emitters will overcome the drawbacks of classical luminophores and agents featuring well-known aggregation-induced emission (AIE) or aggregation-caused quenching (ACQ) properties.

Deoxyestrone-based lipofection agents with solution- and solid-state emission properties.

In this contribution, three deoxyestrone-based emissive lipofection agents are reported. Because of a centrally incorporated terephthalonitrile motif, these ligands can be classified as solution and solid-state emitters (SSSEs). With the attachment of tobramycin, these amphiphilic structures are able to form lipoplexes, mediating gene transfection of HeLa and HEK 293T cells.

Pt(II) Complexes with Tetradentate C

A series of four regioisomeric Pt(II) complexes (PtLa-n and PtLb-n) bearing tetradentate luminophores as dianionic ligands were synthesized. Hence, both classes of cyclometallating chelators were decorated with three n-hexyl (n = 6) or n-dodecyl (n = 12) chains. The new compounds were unambiguously characterized by means of multiple NMR spectroscopies and mass spectrometry. Steady-state and time-resolved photoluminescence spectroscopy as well quantum chemical calculations show that the effect of the regioisomerism on the emission colour and on the deactivation rate constants can be correlated with the participation of the Pt atom on the excited state. The thermal properties of the complexes were studied by DSC, POM and temperature-dependent steady-state photoluminescence spectroscopy. Three of the four complexes (PtLa-12, PtLb-6 and PtLb-12) present an intriguing thermochromism resulting from the responsive metal-metal interactions involving adjacent monomeric units. Each material has different transition temperatures and memory capabilities, which can be tuned at the intermolecular level. Hence, dipole-dipole interactions between the luminophores and disruption of the crystalline packing by the alkyl groups are responsible for the final properties of the resulting materials.

Phosphorescence Induction by Host-Guest Complexation with Cyclodextrins - The Role of Regioisomerism and Affinity.

We present an in-depth investigation of cyclodextrin complexes with guest compounds featuring complexation-induced room temperature phosphorescence (RTP) in aqueous solution. Very interestingly, only the complexed regioisomers bearing lateral substituents on meta-position show RTP, whereas the stronger host-guest systems with para-substituted dyes show no RTP features. The reported systems were investigated regarding their complexation behavior in water using isothermal titration calorimetry and mass spectrometry. In the case of γ-CD very strong 1 : 1 inclusion complexes (Ka up to 5.13×105  M-1 ) were unexpectedly observed. It was found that not only a strong binding to the cyclodextrin cavity is needed to restrict motion, inducing the emission, but also the conformation inside the cavity plays a pivotal role - as supported by an extensive NMR study and MD simulations.

Covalent Attachment of Aggregation-Induced Emission Molecules to the Surface of Ultrasmall Gold Nanoparticles to Enhance Cell Penetration.

Three different alkyne-terminated aggregation-induced emission molecules based on a para-substituted di-thioether were attached to the surface of ultrasmall gold nanoparticles (2 nm) by copper-catalyzed azide-alkyne cycloaddition (click chemistry). They showed a strong fluorescence and were well water-dispersible, in contrast to the dissolved AIE molecules. The AIE-loaded nanoparticles were not cytotoxic and easily penetrated the membrane of HeLa cells, paving the way for an intracellular application of AIE molecules, e.g., for imaging.

Evolution of Artificial Arginine Analogues-Fluorescent Guanidiniocarbonyl-Indoles as Efficient Oxo-Anion Binders.

In this article, we present fluorescent guanidiniocarbonyl-indoles as versatile oxo-anion binders. Herein, the guanidiniocarbonyl-indole (GCI) and methoxy-guanidiniocarbonyl-indole (MGCI) were investigated as ethylamides and compared with the well-known guanidiniocarbonyl-pyrrole (GCP) concerning their photophysical properties as well as their binding behavior towards oxo-anions. Hence, a variety of anionic species, such as carboxylates, phosphonates and sulfonates, have been studied regarding their binding properties with GCP, GCI and MGCI using UV-Vis titrations, in combination with the determination of the complex stoichiometry using the Job method. The emission properties were studied in relation to the pH value using fluorescence spectroscopy as well as the determination of the photoluminescence quantum yields (PLQY). Density functional theory (DFT) calculations were undertaken to obtain a better understanding of the ground-lying electronic properties of the investigated oxo-anion binders. Additionally, X-ray diffraction of GCP and GCI was conducted. We found that GCI and MGCI efficiently bind carboxylates, phosphonates and sulfonates in buffered aqueous solution and in a similar range as GCP (Kass ≈ 1000-18,000 M-1, in bis-tris buffer, pH = 6); thus, they could be regarded as promising emissive oxo-anion binders. They also exhibit a visible fluorescence with a sufficient PLQY. Additionally, the excitation and emission wavelength of MGCI was successfully shifted closer to the visible region of the electromagnetic spectrum by introducing a methoxy-group into the core structure, which makes them interesting for biological applications.

Mapping the Regioisomeric Space and Visible Color Range of Purely Organic Dual Emitters with Ultralong Phosphorescence Components: From Violet to Red Towards Pure White Light.

We mapped the entire visible range of the electromagnetic spectrum and achieved white light emission (CIE: 0.31, 0.34) by combining the intrinsic ns-fluorescence with ultralong ms-phosphorescence from purely organic dual emitters. We realized small molecular materials showing high photoluminescence quantum yields (ΦL ) in the solid state at room temperature, achieved by active exploration of the regioisomeric substitution space. Chromophore stacking-supported stabilization of triplet excitons with assistance from enhanced intersystem crossing channels in the crystalline state played the primary role for the ultra-long phosphorescence. This strategy covers the entire visible spectrum, based on organic phosphorescent emitters with versatile regioisomeric substitution patterns, and provides a single molecular source of white light with long lifetime (up to 163.5 ms) for the phosphorescent component, and high overall photoluminescence quantum yields (up to ΦL =20 %).

Computational model predicts protein binding sites of a luminescent ligand equipped with guanidiniocarbonyl-pyrrole groups.

The 14-3-3 protein family, one of the first discovered phosphoserine/phosphothreonine binding proteins, has attracted interest not only because of its important role in the cell regulatory processes but also due to its enormous number of interactions with other proteins. Here, we use a computational approach to predict the binding sites of the designed hybrid compound featuring aggregation-induced emission luminophores as a potential supramolecular ligand for 14-3-3ζ in the presence and absence of C-Raf peptides. Our results suggest that the area above and below the central pore of the dimeric 14-3-3ζ protein is the most probable binding site for the ligand. Moreover, we predict that the position of the ligand is sensitive to the presence of phosphorylated C-Raf peptides. With a series of experiments, we confirmed the computational prediction of two C 2 related, dominating binding sites on 14-3-3ζ that may bind to two of the supramolecular ligand molecules.

Luminescent Amphiphilic Aminoglycoside Probes to Study Transfection.

We report the characterization of amphiphilic aminoglycoside conjugates containing luminophores with aggregation-induced emission properties as transfection reagents. These inherently luminescent transfection vectors are capable of binding plasmid DNA through electrostatic interactions; this binding results in an emission "on" signal due to restriction of intramolecular motion of the luminophore core. The luminescent cationic amphiphiles effectively transferred plasmid DNA into mammalian cells (HeLa, HEK 293T), as proven by expression of a red fluorescent protein marker. The morphologies of the aggregates were investigated by microscopy as well as ζ-potential and dynamic light-scattering measurements. The transfection efficiencies using luminescent cationic amphiphiles were similar to that of the gold-standard transfection reagent Lipofectamine® 2000.

Umbelliferone Decorated Water-soluble Zinc(II) Phthalocyanines - In Vitro Phototoxic Antimicrobial Anti-cancer Agents.

In this contribution we report on the synthesis, characterization and application of water-soluble zinc(II) phthalocyanines, which are decorated with four or eight umbelliferone moieties for photodynamic therapy (PDT). These compounds are linked peripherally to zinc(II) phthalocyanine by a triethylene glycol linker attached to pyridines, leading to cationic pyridinium units, able to increase the water solubility of the system. Beside their photophysical properties they were analyzed concerning their cellular distribution in human hepatocyte carcinoma (HepG2) cells as well as their phototoxicity towards HepG2 cells, Gram-positive (S. aureus strain 3150/12 and B. subtilis strain DB104) and Gram-negative bacteria (E. coli strain UTI89 and E. coli strain Nissle 1917). At low light doses and concentrations, they exhibit superb antimicrobial activity against Gram-positive bacteria as well as anti-tumor activity against HepG2. They are even capable to inactivate Gram-negative bacteria, whereas the dark toxicity remains low. These unique water-soluble compounds can be regarded as all-in-one type photosensitizers with broad applications ranges in the future.

Take your Positions and Shine: Effects of Positioning Aggregation-Induced Emission Luminophores within Sequence-Defined Macromolecules.

A luminophore with aggregation-induced emission (AIE) is employed for the conjugation onto supramolecular ligands to allow for detection of ligand binding. Supramolecular ligands are based on the combination of sequence-defined oligo(amidoamine) scaffolds and guanidiniocarbonyl-pyrrole (GCP) as binding motif. We hypothesize that AIE properties are strongly affected by positioning of the luminophore within the ligand scaffold. Therefore, we systematically investigate the effects placing the AIE luminophore at different positions within the overall construct, for example, in the main or side chain of the olig(amidoamine). Indeed, we can show that the position within the ligand structure strongly affects AIE, both for the ligand itself as well as when applying the ligand for the detection of different biological and synthetic polyanions.

Reversible Self-Assembly of Gold Nanoparticles Based on Co-Functionalization with Zwitterionic and Cationic Binding Motifs*.

We report a pH- and temperature-controlled reversible self-assembly of Au-nanoparticles (AuNPs) in water, based on their surface modification with cationic guanidiniocarbonyl pyrrole (GCP) and zwitterionic guanidiniocarbonyl pyrrole carboxylate (GCPZ) binding motifs. When both binding motifs are installed in a carefully balanced ratio, the resulting functionalized AuNPs self-assemble at pH 1, pH 7 and pH 13, whereas they disassemble at pH 3 and pH 11. Further disassembly can be achieved at elevated temperatures at pH 1 and pH 13. Thus, we were able to prepare functionalized nanoparticles that can be assembled/disassembled in seven alternating regimes, simply controlled by pH and temperature.

Tuning the solid-state emission of liquid crystalline nitro-cyanostilbene by halogen bonding.

The first example of halogen-bonded fluorescent liquid crystals based on the interaction of iodofluorobenzene derivatives with nitro-cyanostilbenes is reported. The systematic variation of the fluorination degree and pattern indicates the relevance of the halogen bond strength for the induction of liquid crystalline properties. The modular self-assembly approach enables the efficient tuning of the fluorescence behaviour and mesomorphic properties of the assemblies.

Smart Glycopolymeric Nanoparticles for Multivalent Lectin Binding and Stimuli-Controlled Guest Release.

The synthesis and self-assembly of a polymer featuring a self-complementary supramolecular binding motif guanidiniocarbonyl pyrrole carboxylate zwitterion (GCP-zwitterion) bearing lactose moieties are reported. The GCP-zwitterion acts as a cross-linker to facilitate self-assembly of the polymeric chain into nanoparticles (NPs) at neutral pH in an aqueous medium. The formation of polymeric NPs can be controlled by addition of external stimuli (acid or base), which disfavors self-assembly of the GCP-zwitterion because of protonation or deprotonation of the GCP units in the polymer chain. The small-sized (<40 nm) NPs have a hydrophobic cavity and accessible lactose units on the outer shell for multivalent lectin binding. The multivalent interaction between NPs and the lectin peanut agglutinin was confirmed by agglutination experiments. In addition, the stimuli-responsive property of NPs was exploited for the uptake and release of a hydrophobic guest Nile red. Furthermore, the selectivity toward different cell lines (HEK 296T, HeLa, and Hep2G) was tested, and a cellular uptake of cargo-loaded NPs was found for Hep2G cells bearing the lactose-specific asialogylcoprotein receptor, whereas all other cells showed no NP interaction.

UV resonance Raman spectroscopy of the supramolecular ligand guanidiniocarbonyl indole (GCI) with 244 nm laser excitation.

Ultraviolet resonance Raman (UVRR) spectroscopy is a powerful vibrational spectroscopic technique for the label-free monitoring of molecular recognition of peptides or proteins with supramolecular ligands such as guanidiniocarbonyl pyrroles (GCPs). The use of UV laser excitation enables Raman binding studies of this class of supramolecular ligands at submillimolar concentrations in aqueous solution and provides a selective signal enhancement of the carboxylate binding site (CBS). A current limitation for the extension of this promising UVRR approach from peptides to proteins as binding partners for GCPs is the UV-excited autofluorescence from aromatic amino acids observed for laser excitation wavelengths >260 nm. These excitation wavelengths are in the electronic resonance with the GCP for achieving both a signal enhancement and the selectivity for monitoring the CBS, but the resulting UVRR spectrum overlaps with the UV-excited autofluorescence from the aromatic binding partners. This necessitates the use of a laser excitation <260 nm for spectrally separating the UVRR spectrum of the supramolecular ligand from the UV-excited autofluorescence of the peptide or protein. Here, we demonstrate the use of UVRR spectroscopy with 244 nm laser excitation for the characterization of GCP as well as guanidiniocarbonyl indole (GCI), a next generation supramolecular ligand for the recognition of carboxylates. For demonstrating the feasibility of the UVRR binding studies without an interference from the disturbing UV-excited autofluorescence, benzoic acid (BA) was chosen as an aromatic binding partner for GCI. We also present the UVRR results from the binding of GCI to the ubiquitous RGD sequence (arginylglycylaspartic acid) as a biologically relevant peptide. In the case of RGD, the more pronounced differences between the UVRR spectra of the free and complexed GCI (1:1 mixture) clearly indicate a stronger binding of GCI to RGD compared with BA. A tentative assignment of the experimentally observed changes upon molecular recognition is based on the results from density functional theory (DFT) calculations.

Naphthalonitriles featuring efficient emission in solution and in the solid state.

In this work, a series of γ-substituted diphenylnaphthalonitriles were synthesized and characterized. They show efficient emission in solution and in the aggregated state and their environment responsiveness is based on having variable substituents at the para-position of the two phenyl moieties. The excited state properties were fully investigated in tetrahydrofuran (THF) solutions and in THF/H2O mixtures. The size of the aggregates in aqueous media were measured by dynamic light scattering (DLS). The steady-state and time-resolved photoluminescence spectroscopy studies revealed that all the molecules show intense fluorescence both in solution and in the aggregated state. In THF solutions, a blue emission was observed for the unsubstituted (H), methyl- (Me) and tert-butyl- (t-Bu) substituted γ-diphenylnaphthalonitriles, which can be attributed to a weak π-donor capability of these groups. On the other hand, the methoxy- (OMe), methylsulfanyl- (SMe) and dimethylamino- (NMe2) substituted compounds exhibit a progressive red-shift in emission compared to H, Me and t-Bu due to a growing π-electron donating capability. Interestingly, upon aggregation in water-containing media, H, Me and t-Bu show a slight red-shift of the emission and a blue-shift is observed for OMe, SMe and NMe2. The crystal structure of Me allowed a detailed discussion of the structure-property relationship. Clearly, N-containing substituents such as NMe2 possess more electron-donating ability than the S-based moieties such as SMe. Moreover, it was found that NMe2 showed higher luminescence quantum yields (ΦF) in comparison to SMe, indicating that N-substituted groups could enhance the fluorescence intensity. Therefore, the π-donor nature of the substituents on the phenyl ring constitutes the main parameter that influences the photophysical properties, such as excited state lifetimes and photoluminescence quantum yields. Hence, a series of highly luminescent materials from deep blue to red emission depending on substitution and environment is reported with potential applications in sensing, bioimaging and optoelectronics.