Excitonic Coupling in Fluorene-Based Bichromophoric Systems: Vibrational Quenching and the Transition from Weak to Intermediate Coupling.

Excimeric systems (i.e., excited dimers) have well served as model compounds for the study of the delocalization of electronic energy over weakly interacting chromophores. However, there remain relatively few isolated systems in which such interactions can be studied experimentally at a level to afford detailed comparisons with theory. In this Article, we examine a series of covalently and noncovalently linked dimers of fluorene, as a model aromatic chromophore, where the formation of excimers requires a π-stacked, cofacial orientation at van der Waals contact. Building upon a series of seminal prior studies that examined vibronic quenching of the excitation interaction in van der Waals dimers, the key question that we sought to address here is whether a single quenching factor could reproduce experimental excitonic splittings across a series of covalently and noncovalently linked bichromophoric systems built from the same chromophore. In comparing experimentally measured excitonic splittings with calculated static splittings using time-dependent density functional methods, we find that all systems save one fall on a line with a slope of 0.080(8), reflecting a vibrational quenching of roughly 1 order of magnitude. The outlier, which shows a significantly reduced quenching factor, represents a cyclophane-linked system where the fluorene moieties are constrained in a cofacial arrangement. We argue that this system evidences the transition from the weak to intermediate coupling regime.

Design and Synthesis of Cofacially-Arrayed Polyfluorene Wires for Electron and Energy Transfer Studies.

A study of cofacially arrayed π-systems is of particular importance for the design of functional materials for efficient long-range intra-chain charge transfer through the bulk semiconducting materials in the layers of photovoltaic devices. The effect of π-stacking between a pair of aromatic rings has been mainly studied in the form of cyclophanes, where aromatic rings are forced into a sandwich-like geometry, which extensively deforms the aromatic rings from planarity. The synthetic difficulties associated with the preparation of cyclophane-like structures has prevented the synthesis of many examples of their multi-layered analogues. Moreover, the few available multi-layered cyclophanes are not readily amenable to the structural modification required for the construction of D-spacer-A triads needed to explore mechanisms of electron and energy transfer. In this review, we recount how a detailed experimental and computational analysis of 1,3-diarylalkanes led to the design of a new class of cofacially arrayed polyfluorenes that retain their π-stacked structure. Thus, efficient synthetic strategies have been established for the ready preparation of monodisperse polyfluorenes with up to six π-stacked fluorenes, which afford ready access to D-spacer-A triads by linking donor and acceptor groups to the polyfluorene spacers via single methylenes. Detailed 1H NMR spectroscopy, X-ray crystallography, electrochemistry, and He(I) photoelectron spectroscopy of F2-F6 have confirmed the rigid cofacial stacking of multiple fluorenes in F2-F6, despite the presence of rotatable C-C bonds. These polyfluorenes (F2-F6) form stable cation radicals in which a single hole is delocalized amongst the stacked fluorenes, as judged by the presence of intense charge-resonance transition in their optical spectra. Interestingly, these studies also discern that delocalization of a single cationic charge could occur over multiple fluorene rings in F2-F6, while the exciton is likely localized only onto two fluorenes in F2-F6. Facile synthesis of the D-spacer-A triads allowed us to demonstrate that efficient triplet energy transfer can occur through π-stacked polyfluorenes; the mechanism of energy transfer crosses over from tunneling to hopping with increasing number of fluorenes in the polyfluorene spacer. We suggest that the development of rigidly held π-stacked polyfluorenes, described herein, with well-defined redox and optoelectronic properties provides an ideal scaffold for the study of electron and energy transfer in D-spacer-A triads, where the Fn spacers serve as models for cofacially stacked π-systems.

Design, Synthesis, Electronic Properties, and X-ray Structural Characterization of Various Modified Electron-Rich Calixarene Derivatives and Their Conversion to Stable Cation Radical Salts.

We have designed and synthesized electron-rich calixarene derivatives, which undergo reversible electrochemical oxidation in a well-accessible potential range that allows the ready preparation and isolation of the corresponding cation radicals. Preparation of mono- or tetra-radical cation can be achieved by using stable aromatic cation-radical salts such as MA+•, MB+•, and NAP+• as selective organic oxidants. The cation radicals of calixarenes are stable indefinitely at ambient temperatures and can be readily characterized by UV-vis-NIR spectroscopy. These cation radicals bind a single molecule of nitric oxide within its cavity with remarkable efficiency.

Electron-Transfer-Induced Self-Assembly of a Molecular Tweezer Platform.

The design and synthesis of a new molecular tweezer (T-tmp) with electron-rich pincers are reported. The stable monocationic radicals and self-assembled dimeric radicals of this molecular tweezer platform were prepared by chemical oxidative titration. With the aid of DFT calculations, it was found that the dimeric radicals with syn-syn-syn conformer has the most stable structure, with the hole primarily delocalized between parallel stacked pyrenyl groups.

Role of Conserved Histidine and Serine in the HCXXXXXRS Motif of Human Dual-Specificity Phosphatase 5.

BACKGROUND: The mitogen-activated protein kinase (MAPK) pathway is functionally generic and critical in maintaining physiological homeostasis and normal tissue development. This pathway is under tight regulation, which is in part mediated by dual-specific phosphatases (DUSPs), which dephosphorylate serine, threonine, and tyrosine residues of the ERK family of proteins. DUSP5 is of high clinical interest because of mutations we identified in this protein in patients with vascular anomalies. Unlike other DUSPs, DUSP5 has unique specificity toward substrate pERK1/2. Using molecular docking and simulation strategies, we previously showed that DUSP5 has two pockets, which are utilized in a specific fashion to facilitate specificity toward catalysis of its substrate pERK1/2. Remarkably, most DUSPs share high similarity in their catalytic sites. Studying the catalytic domain of DUSP5 and identifying amino acid residues that are important for dephosphorylating pERK1/2 could be critical in developing small molecules for therapies targeting DUSP5. RESULTS: In this study, we utilized computational modeling to identify and predict the importance of two conserved amino acid residues, H262 and S270, in the DUSP5 catalytic site. Modeling studies predicted that catalytic activity of DUSP5 would be altered if these critical conserved residues were mutated. We next generated independent Glutathione-S-Transferase (GST)-tagged full-length DUSP5 mutant proteins carrying specific mutations H262F and S270A in the phosphatase domain. Biochemical analysis was performed on these purified proteins, and consistent with our computational prediction, we observed altered enzyme activity kinetic profiles for both mutants with a synthetic small molecule substrate (pNPP) and the physiological relevant substrate (pERK) when compared to wild type GST-DUSP5 protein. CONCLUSION: Our molecular modeling and biochemical studies combined demonstrate that enzymatic activity of phosphatases can be manipulated by mutating specific conserved amino acid residues in the catalytic site (phosphatase domain). This strategy could facilitate generation of small molecules that will serve as agonists/antagonists of DUSP5 activity.

Discovery and Characterization of Halogenated Xanthene Inhibitors of DUSP5 as Potential Photodynamic Therapeutics.

Dual specific phosphatases (DUSPs) are an important class of mitogen-activated protein kinase (MAPK) regulators, and are drug targets for treating vascular diseases. Previously we had shown that DUSP5 plays a role in embryonic vertebrate vascular patterning. Herein, we screened a library of FDA-approved drugs and related compounds, using a para-nitrophenylphosphate substrate (pNPP)-based assay. This assay identified merbromin (also known as mercurochrome) as targeting DUSP5; and, we subsequently identified xanthene-ring based merbromin analogs eosin Y, erythrosin B, and rose bengal, all of which inhibit DUSP5 in vitro. Inhibition was time-dependent for merbromin, eosin Y, 2',7'-dibromofluorescein, and 2',7'-dichlorofluorescein, with enzyme inhibition increasing over time. Reaction progress curve data fit best to a slow-binding model of irreversible enzyme inactivation. Potency of the time-dependent compounds, except for 2',7'-dichlorofluorescein, was diminished when dithiothreitol (DTT) was present, suggesting thiol reactivity. Two additional merbromin analogs, erythrosin B and rose bengal also inhibit DUSP5, but have the therapeutic advantage of being less sensitive to DTT and exhibiting little time dependence for inhibition. Inhibition potency is correlated with the xanthene dye's LUMO energy, which affects ability to form light-activated radical anions, a likely active inhibitor form. Consistent with this hypothesis, rose bengal inhibition is light-dependent and demonstrates the expected red shifted spectrum upon binding to DUSP5, with a Kd of 690 nM. These studies provide a mechanistic foundation for further development of xanthene dyes for treating vascular diseases that respond to DUSP5 inhibition, with the following relative potencies: rose bengal > merbromin > erythrosin B > eosin Y.

Study of Förster Resonance Energy Transfer to Lipid Domain Markers Ascertains Partitioning of Semisynthetic Lipidated N-Ras in Lipid Raft Nanodomains.

Cellular membranes are heterogeneous planar lipid bilayers displaying lateral phase separation with the nanometer-scale liquid-ordered phase (also known as "lipid rafts") surrounded by the liquid-disordered phase. Many membrane-associated proteins were found to permanently integrate into the lipid rafts, which is critical for their biological function. Isoforms H and N of Ras GTPase possess a unique ability to switch their lipid domain preference depending on the type of bound guanine nucleotide (GDP or GTP). This behavior, however, has never been demonstrated in vitro in model bilayers with recombinant proteins and therefore has been attributed to the action of binding of Ras to other proteins at the membrane surface. In this paper, we report the observation of the nucleotide-dependent switch of lipid domain preferences of the semisynthetic lipidated N-Ras in lipid raft vesicles in the absence of additional proteins. To detect segregation of Ras molecules in raft and disordered lipid domains, we measured Förster resonance energy transfer between the donor fluorophore, mant, attached to the protein-bound guanine nucleotides, and the acceptor, rhodamine-conjugated lipid, localized into the liquid-disordered domains. Herein, we established that N-Ras preferentially populated raft domains when bound to mant-GDP, while losing its preference for rafts when it was associated with a GTP mimic, mant-GppNHp. At the same time, the isolated lipidated C-terminal peptide of N-Ras was found to be localized outside of the liquid-ordered rafts, most likely in the bulk-disordered lipid. Substitution of the N-terminal G domain of N-Ras with a homologous G domain of H-Ras disrupted the nucleotide-dependent lipid domain switch.

From Static to Dynamic: Electron Density of HOMO at Biaryl Linkage Controls the Mechanism of Hole Delocalization.

In order to extend the physical length of hole delocalization in a molecular wire, chromophores of increasing size are often desired. However, the effect of size on the efficacy and mechanism of hole delocalization remains elusive. Here, we employ a model set of biaryls to show that with increasing chromophore size, the mechanism of steady-state hole distribution switches from static delocalization in biaryls with smaller chromophores to dynamic hopping, as exemplified in the largest system, tBuHBC2 (i.e., "superbiphenyl"), which displays a vanishingly small electronic coupling. This important finding is analyzed with the aid of Hückel molecular orbital and Marcus-Hush theories. Our findings will enable the rational design of the novel molecular wires with length-invariant redox/optical properties suitable for long-range charge transfer.

Synthesis of Doubly Annulated m-Terphenyl-Based Molecular Tweezers and Their Charge-Transfer Complexes with DDQ as a Guest.

The synthesis of a doubly-annulated m-terphenyl-based tweezer platform has been developed, which affords ready incorporation of various pincer units from monobenzenoid to polybenzenoid electron donors. The complexation study with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) as guest has been carried out, and the crystal structure of T-Py∩DDQ reveals the sandwich-type binding mode in the solid state.

An Electron-Rich Calix[4]arene-Based Receptor with Unprecedented Binding Affinity for Nitric Oxide.

Calixarenes have found widespread application as building blocks for the design and synthesis of functional materials in host-guest chemistry. The ongoing desire to develop a detailed understanding of the nature of NO bonding to multichromophoric π-stacked assemblies led us to develop an electron-rich methoxy derivative of calix[4]arene (3), which we show exists as a single conformer in solution at ambient temperature. Here, we examine the redox properties of this derivative, generate its cation radical (3+. ) using robust chemical oxidants, and determine the relative efficacy of its NO binding in comparison with model calixarenes. We find that 3/3+. is a remarkable receptor for NO+ /NO, with unprecedented binding efficacy. The availability of precise experimental structures of this calixarene derivative and its NO complex, obtained by X-ray crystallography, is critically important both for developing novel functional NO biosensors, and understanding the role of stacked aromatic donors in efficient NO binding, which may have relevance to biological NO transport.

An electron-transfer induced conformational transformation: from non-cofacial "sofa" to cofacial "boat" in cyclotetraveratrylene (CTTV) and formation of charge transfer complexes.

Electro-active polychromophoric assemblies that undergo clam-like electromechanic actuation represent an important class of organic functional materials. Here, we show that the readily available cyclotetraveratrylene (CTTV) undergoes oxidation-induced folding, consistent with interconversion from a non-cofacial "sofa" conformation to a cofacial "boat" conformer. It is found that the non-cofacial "sofa" conformer of CTTV forms stable electron donor-acceptor complexes with chloranil and DDQ. Electron-transfer induced conformational transformation in CTTV provides a framework for the rational design of novel organic functional molecules.

Vertical vs. adiabatic ionization energies in solution and gas-phase: probing ionization-induced reorganization in conformationally-mobile bichromophoric actuators using photoelectron spectroscopy, electrochemistry and theory.

Ionization-induced structural and conformational reorganization in various π-stacked dimers and covalently linked bichromophores is relevant to many processes in biological systems and functional materials. In this work, we examine the role of structural, conformational, and solvent reorganization in a set of conformationally mobile bichromophoric donors, using a combination of gas-phase photoelectron spectroscopy, solution-phase electrochemistry, and density functional theory (DFT) calculations. Photoelectron spectral analysis yields both adiabatic and vertical ionization energies (AIE/VIE), which are compared with measured (adiabatic) solution-phase oxidation potentials (Eox). Importantly, we find a strong correlation of Eox with AIE, but not VIE, reflecting variations in the attendant structural/conformational reorganization upon ionization. A careful comparison of the experimental data with the DFT calculations allowed us to probe the extent of charge stabilization in the gas phase and solution and to parse the reorganizational energy into its various components. This study highlights the importance of a synergistic approach of experiment and theory to study ionization-induced structural and conformational reorganization.

π - π stacking vs. C-H/ π interaction: Excimer formation and charge resonance stabilization in van der Waals clusters of 9,9'-dimethylfluorene.

Studies of exciton and hole stabilization in multichromophoric systems underpin our understanding of electron transfer and transport in materials and biomolecules. The simplest model systems are dimeric, and recently we compared the gas-phase spectroscopy and dynamics of van der Waals dimers of fluorene, 9-methylfluorene (MF), and 9,9'-dimethylfluorene (F1) to assess how sterically controlled facial encumbrance modulates the dynamics of excimer formation and charge resonance stabilization (CRS). Dimers of fluorene and MF show only excimer emission upon electronic excitation, and significant CRS as evidenced in a reduced ionization potential for the dimer relative the monomer. By contrast, the dimer of F1 shows no excimeric emission, rather structured emission from the locally excited state of a tilted (non π-stacked) dimer, evidencing the importance of C-H/π interactions and increased steric constraints that restrict a cofacial approach. In this work, we report our full results on van der Waals clusters of F1, using a combination of theory and experiments that include laser-induced fluorescence, mass-selected two-color resonant two-photon ionization spectroscopy, and two-color appearance potential measurements. We use the latter to derive the binding energies of the F1 dimer in ground, excited, and cation radical states. Our results are compared with van der Waals and covalently linked clusters of fluorene to assess both the relative strength of π-stacking and C-H/π interactions in polyaromatic assemblies and the role of π-stacking in excimer formation and CRS.

FHBC, a Hexa-peri-hexabenzocoronene-Fluorene Hybrid: A Platform for Highly Soluble, Easily Functionalizable HBCs with an Expanded Graphitic Core.

Materials based upon hexa-peri-hexabenzocoronenes (HBCs) show significant promise in a variety of photovoltaic applications. There remains the need, however, for a soluble, versatile, HBC-based platform, which can be tailored by incorporation of electroactive groups or groups that can prompt self-assembly. The synthesis of a HBC-fluorene hybrid is presented that contains an expanded graphitic core that is highly soluble, resists aggregation, and can be readily functionalized at its vertices. This new HBC platform can be tailored to incorporate six electroactive groups at its vertices, as exemplified by a facile synthesis of a representative hexaaryl derivative of FHBC. Synthesis of new FHBC derivatives, containing electroactive functional groups that can allow controlled self-assembly, may serve as potential long-range charge-transfer materials for photovoltaic applications.

From Intramolecular (Circular) in an Isolated Molecule to Intermolecular Hole Delocalization in a Two-Dimensional Solid-State Assembly: The Case of Pillarene.

To achieve long-range charge transport/separation and, in turn, bolster the efficiency of modern photovoltaic devices, new molecular scaffolds are needed that can self-assemble in two-dimensional (2D) arrays while maintaining both intra- and intermolecular electronic coupling. In an isolated molecule of pillarene, a single hole delocalizes intramolecularly via hopping amongst the circularly arrayed hydroquinone ether rings. The crystallization of pillarene cation radical produces a 2D self-assembly with three intermolecular dimeric (sandwich-like) contacts. Surprisingly, each pillarene in the crystal lattice bears a fractional formal charge of +1.5. This unusual stoichiometry of oxidized pillarene in crystals arises from effective charge distribution within the 2D array via an interplay of intra- and intermolecular electronic couplings. This important finding is expected to help advance the rational design of efficient solid-state materials for long-range charge transfer.

The Role of Torsional Dynamics on Hole and Exciton Stabilization in π-Stacked Assemblies: Design of Rigid Torsionomers of a Cofacial Bifluorene.

Exciton and charge delocalization across π-stacked assemblies is of importance in biological systems and functional polymeric materials. To examine the requirements for exciton and hole stabilization, cofacial bifluorene (F2) torsionomers were designed, synthesized, and characterized: unhindered (model) Me F2, sterically hindered tBu F2, and cyclophane-like C F2, where fluorenes are locked in a perfect sandwich orientation via two methylene linkers. This set of bichromophores with varied torsional rigidity and orbital overlap shows that exciton stabilization requires a perfect sandwich-like arrangement, as seen by strong excimeric-like emission only in C F2 and Me F2. In contrast, hole delocalization is less geometrically restrictive and occurs even in sterically hindered tBu F2, as judged by 160 mV hole stabilization and a near-IR band in the spectrum of its cation radical. These findings underscore the diverse requirements for charge and energy delocalization across π-stacked assemblies.

Poly-p-hydroquinone Ethers: Isoenergetic Molecular Wires with Length-Invariant Oxidation Potentials and Cation Radical Excitation Energies.

Typical poly-p-phenylene wires are characterized by strong interchromophoric electronic coupling with redox and optical properties being highly length-dependent. Herein we show that an incorporation of a pair of para-methoxy groups at each p-phenylene unit in poly-p-phenylene wires (i.e., PHEn) changes the nodal structure of HOMO that leads to length-invariant oxidation potentials and cation radical excitation energies. As such, PHEn represents a unique class of isoenergetic wires where hole delocalization mainly occurs via dynamic hopping and thus may serve as an efficient medium for long-range charge transfer. Availability of these wires will allow demonstration of long-range electron transfer via incoherent hopping using donor-bridge-acceptor systems with isoenergetic PHEn-based wires as bridges.

Unraveling the Coulombic Forces in Electronically Decoupled Bichromophoric Systems during Two Successive Electron Transfers.

Coulombic forces are vital in modulating the electron transfer dynamics in both synthetic and biological polychromophoric assemblies, yet quantitative studies of the impact of such forces are rare, as it is difficult to disentangle electrostatic forces from simple electronic coupling. To address this problem, the impact of Coulombic interactions in the successive removal of two electrons from a model set of spirobifluorenes, where the interchromophoric electronic coupling is nonexistent, is quantitatively assessed. By systematically varying the separation of the bifluorene moieties using model compounds, ion pairing, and solvation, these interactions, with energies up to about 0.4 V, are absent at distances greater than about 9 Å. These findings can be (quantitatively) applied for the design of polychromophoric assemblies, whereby the redox properties of donors and/or acceptors can be tuned by judicious positioning of the charged groups to control the electron-transfer dynamics.

Serendipitous discovery of light-induced (In Situ) formation of an Azo-bridged dimeric sulfonated naphthol as a potent PTP1B inhibitor.

BACKGROUND: Protein tyrosine phosphatases (PTPs) like dual specificity phosphatase 5 (DUSP5) and protein tyrosine phosphatase 1B (PTP1B) are drug targets for diseases that include cancer, diabetes, and vascular disorders such as hemangiomas. The PTPs are also known to be notoriously difficult targets for designing inihibitors that become viable drug leads. Therefore, the pipeline for approved drugs in this class is minimal. Furthermore, drug screening for targets like PTPs often produce false positive and false negative results. RESULTS: Studies presented herein provide important insights into: (a) how to detect such artifacts, (b) the importance of compound re-synthesis and verification, and (c) how in situ chemical reactivity of compounds, when diagnosed and characterized, can actually lead to serendipitous discovery of valuable new lead molecules. Initial docking of compounds from the National Cancer Institute (NCI), followed by experimental testing in enzyme inhibition assays, identified an inhibitor of DUSP5. Subsequent control experiments revealed that this compound demonstrated time-dependent inhibition, and also a time-dependent change in color of the inhibitor that correlated with potency of inhibition. In addition, the compound activity varied depending on vendor source. We hypothesized, and then confirmed by synthesis of the compound, that the actual inhibitor of DUSP5 was a dimeric form of the original inhibitor compound, formed upon exposure to light and oxygen. This compound has an IC50 of 36 µM for DUSP5, and is a competitive inhibitor. Testing against PTP1B, for selectivity, demonstrated the dimeric compound was actually a more potent inhibitor of PTP1B, with an IC50 of 2.1 µM. The compound, an azo-bridged dimer of sulfonated naphthol rings, resembles previously reported PTP inhibitors, but with 18-fold selectivity for PTP1B versus DUSP5. CONCLUSION: We report the identification of a potent PTP1B inhibitor that was initially identified in a screen for DUSP5, implying common mechanism of inhibitory action for these scaffolds.

Dihedral-Angle-Controlled Crossover from Static Hole Delocalization to Dynamic Hopping in Biaryl Cation Radicals.

In cases of coherent charge-transfer mechanism in biaryl compounds the rates follow a squared cosine trend with varying dihedral angle. Herein we demonstrate using a series of biaryl cation radicals with varying dihedral angles that the hole stabilization shows two different regimes where the mechanism of the hole stabilization switches over from (static) delocalization over both aryl rings to (dynamic) hopping. The experimental data and DFT calculations of biaryls with different dihedral angles unequivocally support that a crossover from delocalization to hopping occurs at a unique dihedral angle where the electronic coupling (Hab ) is one half of reorganization (λ), that is, Hab =λ/2. The implication of this finding in non-coherent charge-transfer rates is being investigated.