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Large Stokes shift (LSS) fluorescent proteins (FPs) exploit excited state proton transfer pathways to enable fluorescence emission from the phenolate intermediate of their internal 4 hydroxybenzylidene imidazolone (HBI) chromophore. An RNA aptamer named Chili mimics LSS FPs by inducing highly Stokes-shifted emission from several new green and red HBI analogs that are non-fluorescent when free in solution. The ligands are bound by the RNA in their protonated phenol form and feature a cationic aromatic side chain for increased RNA affinity and reduced magnesium dependence. In combination with oxidative functional-ization at the C2 position of the imidazolone, this strategy yielded DMHBO\(^+\), which binds to the Chili aptamer with a low-nanomolar K\(_D\). Because of its highly red-shifted fluorescence emission at 592 nm, the Chili–DMHBO\(^+\) complex is an ideal fluorescence donor for Förster resonance energy transfer (FRET) to the rhodamine dye Atto 590 and will therefore find applications in FRET-based analytical RNA systems.
Efficient quadrupolar chromophores (A–pi–A) with triarylborane moieties as acceptors have been studied by the Marder group regarding their non‐linear optical properties and two‐photon absorption ability for many years. Within the present work, this class of dyes found applications in live‐cell imaging. Therefore, the dyes need to be water‐soluble and water‐stable in diluted aqueous solutions, which was examined in Chapter 2. Furthermore, the influence of the pi‐bridge on absorption and emission maxima, fluorescence quantum yields and especially the two-photon absorption properties of the chromophores was investigated in Chapter 3. In Chapter 4, a different strategy for the design of efficient two‐photon excited fluorescence imaging dyes was explored using dipoles (D–A) and octupoles (DA3). Finding the optimum balance between water‐stability and pi‐conjugation and, therefore, red‐shifted absorption and emission and high fluorescence quantum yields, was investigated in Chapter 5
The aim of this work was to synthesize and functionalize different bio-relevant nanomaterials like silica-coated superparamagnetic iron oxide nanoparticles (SPIONs) as contrast agents for T2 magnetic resonance imaging (MRI) and detonation nanodiamond (DND) with the neurohormone peptide allatostatin 1 (ALST1) and a fluorescent dye. Analytical techniques for the determination and quantification of surface functional groups like amines, azides, and peptides were also developed and established.
Thus, in the first part of the work, a TGF-1 binding peptide and allatostatin 1 (ALST1), both supposed to act as active tumour targeting vectors, were synthesized by solid-phase peptide synthesis (SPPS) and characterized by high pressure liquid chromatography (HPLC) and mass spectrometry. Then, azide-functionalized silica nanoparticles were synthesized by the Stöber process and characterized by transmission electron microscopy (TEM) and infrared spectroscopy (IR). The surface loading of amine and azide groups was determined by a new protocol. The azide groups were reduced with sodium boronhydride to amine and then functionalized with Fmoc-Rink Amide linker according to a standard SPPS protocol. Upon cleavage of Fmoc by piperidine, the resulting dibenzofulvene and its piperidine adduct were quantified by UV/Vis spectroscopy and used to determine the amount of amine groups on the nanoparticle surface. Then, ALST1 and related tyrosine- and phenylalanine substituted model peptides were conjugated to the azide-functionalized silica nanoparticles by copper(I)-catalyzed azide-alkyne dipolar cycloaddition (CuAAC). The successful peptide conjugation was demonstrated by the Pauly reaction, which however is only sensitive to histidine- and tyrosine-containing peptides. As a more general alternative, the acid hydrolysis of the peptides to their individual amino acid building blocks followed by derivatization with phenyl isothiocyanate (PITC) allowed the separation, determination, and quantification of the constituent amino acids by HPLC.
In the second part of the work, amine- and azide-functionalized silica-coated superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized by co-precipitation and subsequent silica-coated based on the Stöber process and characterized by TEM and IR. The amine surface loading was determined by the method already established for the pure silica systems. The azide surface loading could also be quantified by reduction with sodium boronhydride to amine groups and then conjugation to Fmoc-Rink amide linker. Upon cleavage of Fmoc with piperidine, the total amine surface loading was obtained. The amount of azide surface groups was then determined from the difference of the total amine surface loading and the amine surface loading. Thus, it was possible to quantify both amine and azide surface groups on a single nanoparticle system. Superparamagnetic iron oxide nanoparticles (SPIONs) are potent T2 contrast agents for magnetic resonance imaging (MRI). Due to their natural metabolism after injection into the blood stream, SPIONs mostly end up inside macrophages, liver, spleen or kidneys. To generate a potential target-specific SPION-based T2 contrast agent for MRI, the neurohormone peptide ALST1 was conjugated by CuAAC to the azide- and amine functionalized superparamagnetic iron oxide nanoparticles, since ALST1 is supposed to target difficult-to-treat neuroendocrinic tumours due to its analogy to galanin and somastatin receptor ligands. The organic fluorescent dye cyanine 5 (Cy5) was also conjugated to the silica-coated superparamagnetic iron oxide nanoparticles (SPIONs) via a NHS-ester to the amines to enable cell uptake studies by fluorescence microscopy. These constructs were characterized by TEM, dynamic light scattering (DLS), and IR. The amino acids of the conjugated ALST1 were determined by the HPLC method as described before for peptide-modified silica nanoparticle surfaces. Then, the relaxivity r2 was measured at 7 T. However, a r2 value of 27 L/mmolFe·s for the dual ALST1-/Cy5-functionalized silica-coated SPIONs was not comparable to T2 contrast agents in clinical use, since their relaxivity is commonly determined at 1.5 T, and no such instrument was available. However, it can be assumed that the synthesized dual
ALST1-/Cy5-functionalized silica-coated SPION would show a lower r2 at 1.5 T than at 7T. Commercial T2 MRI contrast agents like VSOP-C184 from Ferropharm show at r2 values of about 30 L/mmolFe·s at 1.5 T. Still, the relaxivity of the new material has some potential for application as a T2 contrast agent. Then, the material was used in cell uptake studies by fluorescence microscopy with the conjugated Cy5 dye as a probe. The dual
ALST1-/Cy5-functionalized silica-coated SPION showed a high degree of agglomeration with no cellular uptake unlike described for ALST1-functionalized nanoparticles in literature. It is assumed that upon agglomeration of the particles, constructs form which are unable to be internalized by the cellular endocytotic pathways anymore. As a future perspective, the tendency of the particle to agglomerate should be reduced by changing the coating material to polyethylene glycol (PEG) or chitosan, which are known to be bio-compatible, bio-degradable and prevent agglomeration.
In the third part of the work, the rhenium compound [ReBr(CO)3(L)] with L = 2-phenyl-1H-imidazo[4,5-f][1,10]phenanthroline and its manganese analogue were synthesized by heating the ligand and rhenium pentacarbonyl bromide or and manganese pentacarbonyl bromide respectively, in toluene. However, [MnBr(CO)3(L)] was unstable upon illumination by UV light at 365 nm. Thus, it was dismissed for further application. The photophysical properties of [ReBr(CO)3(L)] were explored, by determination of the excited-state life time by the time-correlated single-photon counting (TCSPC) method and the quantum yield by a fluorescence spectrometer equipped with an integration sphere. A value of = 455 ns, a Stokes shift of 197 nm and a rather low quantum yield =were found. Metal complexes are supposed to have superior properties compared to organic dyes due to their large Stokes shifts, long excited-state life times, and high quantum yields. Thus, amine- and azide-functionalized detonation nanodiamond (DND) as an alternative biological inert carrier system was functionalized with ALST1 to enhance its cell uptake properties. A luminescent probe for cell uptake studies using fluorescence microscopy was also attached, either based on the new rhenium complex or the commercially available organic dye Cy5, respectively. The aldehyde-functionalized rhenium complex was conjugated to the DND via oxime ligation, which is known to be a mild and catalyst-free conjugation method. The amount of peptide ALST1 on the DND was analyzed and quantified after acid hydrolysis and PITC derivatization by HPLC as described before. Then, the ALST1-/luminescent probe-functionalized DND was investigated for its photophysical properties by fluorescence spectroscopy. The Cy5-functionalized material showed a slightly lower fluorescence performance in aqueous solution than reported in literature and commercial suppliers with a life time < 0.4 ns and quantum yields not determinable by integration sphere due to the week signal intensity. The rhenium complex-functionalized material had a very low signal intensity in only aqueous medium, and thus determination of life times and quantum yield by fluorescence spectroscopy was not possible. After incubation with MDA-MB 231 cells, the Cy5-functionalized DND could easily be detected due to its red fluorescence. However, it was not possible to visualize the rhenium complex-functionalized DND with fluorescence microscopy due to the low fluorescence intensity of the complex in aqueous medium and the lack of proper filters for the fluorescence microscope. Cy5-functionalized DND did not show any cellular uptake in fluorescence microscopy after conjugation with ALST1. Since the nanodiamond surface is known to strongly adsorb peptides and proteins, it is assumed that the peptide chain is oriented perpendicular to the nanoparticle surface and thus not able to interact with cell membrane receptors to promote cell uptake of the particles. As a future perspective, the ALST1-promoted cellular uptake of the DND should be improved by using different linker systems for peptide conjugation to prevent adsorption of the peptide chain on the particle surface.
The new analytical methods for amino-, azide-, and peptide-functionalized nanoparticles have great potential to assist in the quantification of nanoparticle surface modifications by UV/Vis spectroscopy and HPLC. The determination of surface amine and azide groups based on the cleavage of conjugated Fmoc-Rink amide linker and detected by UV/Vis spectroscopy is applicable to all amine-/azide-functionalized nanomaterials. However, particles which form very stable suspension with the cleavage mixture can cause quantification problems due to scattering, making an accurate quantification of dibenzofulvene and its piperidine adduct impossible. The detection of tyrosine- and histidine-containing peptides based on the Pauly reaction is well-suited as a fast and easy-to-perform qualitative demonstration of successful peptide surface conjugation. However, its major drawback as a colourimetric approach is that coloured particles cannot be evaluated by this method. The amino acid analysis based on HPLC after acid hydrolysis of peptides conjugated to nanoparticle surfaces to its individual building blocks and subsequent derivatization with PITC, can be used on all nanomaterials with peptide or protein surface modification. It allows detection of amino acids down to picomolar concentrations and even enables analysis of very small peptide surface loadings. However, the resulting HPLC traces are difficult to analyze.
Three new analytical methods based on UV/Vis and HPLC techniques have been developed and established. They assisted in the characterization of the synthesized DND and SPIONs with dual functionalization by ALST1 and Cy5 or [ReBr(CO)3(L)], respectively. However, the nanomaterials showed no cellular uptake due to a high tendency to agglomerate. The cellular uptake should be improved and the tendency to agglomerate of the SPIONs should be reduced by changing the surface coating from silica to either PEG or chitosan. Furthermore, different linker systems for connecting peptides to DND surfaces should be synthesized and evaluated to reduce potential peptide chain adsorption.
We report the direct imidization of naphthalene and perylene dicarboxylic anhydrides/esters with bulky ortho,ortho‐diaryl‐ and ortho,ortho‐dialkynylaniline derivatives. This imidization method uses n‐butyllithium as a strong base to increase the reactivity of bulky amine derivatives, proceeds under mild reaction conditions, requires only stoichiometric amounts of reactants and gives straightforward access to new sterically crowded rylene dicarboximides. Mechanistic investigations suggest an isoimide as intermediary product, which was converted to the corresponding imide upon addition of an aqueous base. Single‐crystal X‐ray diffraction analyses reveal dimeric packing motifs for monoimides, while two‐side shielded bisimides crystallize in isolated molecules without close π–π‐interactions. Spectroscopic investigations disclose the influence of the bulky substituents on the optical properties in the solid state.
Blending different low molecular weight gelators (LMWGs) provides a convenient route to tune the properties of a gel and incorporate functionalities such as fluorescence. Blending a series of gelators having a common bis-urea motif, and functionalised with different amino acid-derived end-groups and differing length alkylene spacers is reported. Fluorescent gelators incorporating 1- and 2-pyrenyl moieties provide a probe of the mixed systems alongside structural and morphological data from powder diffraction and electron microscopy. Characterisation of the individual gelators reveals that although the expected α-urea tape motif is preserved, there is considerable variation in the gelation properties, molecular packing, fibre morphology and rheological behaviour. Mixing of the gelators revealed examples in which: 1) the gels formed separate, orthogonal networks maintaining their own packing and morphology, 2) the gels blended together into a single network, either adopting the packing and morphology of one gelator, or 3) a new structure not seen for either of the gelators individually was created. The strong binding of the urea functionalities to anions was exploited as a means of breaking down the gel structure, and the use of fluorescent gel blends provides new insights into anion-mediated gel dissolution.
Single crystals of three at bay area tetraphenoxy‐substituted perylene bisimide dyes are grown by vacuum sublimation. X‐ray analysis reveals the self‐assembly of these highly twisted perylene bisimides (PBIs) in the solid state via imide–imide hydrogen bonding into hydrogen‐bonded PBI chains. The crystallographic insights disclose that the conformation and sterical congestion imparted by the phenoxy substituents can be controlled by ortho‐substituents. Accordingly, whilst sterically less demanding methyl and isopropyl substituents afford double‐stranded PBI chains of complementary P and M atropo‐enantiomers, single hydrogen‐bonded chains of homochiral PBIs are observed for the sterically more demanding ortho‐phenyl substituents. Investigation of the absorption and fluorescence properties of microcrystals and thin films of these PBIs allow for an unambiguous interpretation of these exciton systems. Thus, the J‐aggregates of the double‐stranded crystals exhibit a much larger (negative) exciton coupling than the single‐stranded one, which in contrast has the higher solid‐state fluorescence quantum yield.
The cyclic nucleotides cAMP and cGMP are two ubiquitous important second messengers, which regulate diverse physiological responses from vision and memory to blood pressure and thrombus formation. They act in cells via cAMP- and cGMP-dependent protein kinases (PKA and GK), cyclic nucleotide-gated channels and Epac. Although the concept of cyclic nucleotide signalling is well developed based on classical biochemical studies, these techniques have not allowed to analyze cAMP and cGMP in live cells with high temporal and spatial resolution. In the present study fluorescence resonance energy transfer was used to develop a technique for visualization of cAMP and cGMP in live cells and in vitro by means of fluorescent biosensors. Ligand-induced conformational change in a single nucleotide-binding domain flanked with green fluorescent protein mutants was used for dynamic, highly sensitive measurements of cAMP and cGMP. Such biosensors retained binding properties and chemical specificity of unmodified domains, allowing to image cyclic nucleotides in a physiologically relevant range of concentrations. To develop cAMP-sensors, binding domains of PKA, Epac and cAMP-gated HCN-channel were used. cGMP-sensors were based on single domains of GK and phosphodiesterases (PDEs). Sensors based on Epac were used to analyze spatio-temporal dynamics of cAMP in neurons and macrophages, demonstrating that cAMP-gradients travel with a high speed (~ 40 μm/s) throughout the entire cytosol. To understand the mechanisms of cAMP-compartmentation, kinetics properties of phosphodi-esterase (PDE2) were, next, analyzed in aldosterone producing cells. PDE2 is able to rapidly hydrolyze extensive amounts of cAMP, so that the speed of cAMP-hydrolysis is much faster than that of its synthesis, which might serve as a basis of compartmentation. cAMP-sensors were also used to develop a clinically relevant diagnostic method for reliable detection of β1-adrenergic receptor autoantibodies in cardiac myopathy patients, which has allowed to significantly increase the sensitivity of previously developed diagnostic approaches. Conformational change in a single binding domain of GK and PDE was, next, used to create novel fluorescent biosensors for cGMP. These sensors demonstrated high spatio-temporal resolution and were applied to analyze rapid dynamics of cGMP production by soluble and particulate guanylyl cyclases as well as to image cGMP in mesangial cells. In summary, highly sensitive biosensors for cAMP and cGMP based on single cyclic nucleotide-binding domains have been developed and used in various biological and clinically relevant applications.
Ever since the discovery of dye self-assemblies in nature, there have been tremendous efforts to exploit biomimetic supramolecular assemblies for tailored artificial photon processing materials. This feature necessarily has resulted in an increasing demand for understanding exciton dynamics in the dye self-assemblies. In a sharp contrast with pi-type aggregates, however, the detailed observation of exciton dynamics in H-type aggregates has remained challenging. In this study, as we succeed in measuring transient fluorescence from Frenkel state of π-stacked perylene tetracarboxylic acid bisimide dimer and oligomer aggregates, we present an experimental demonstration on Frenkel exciton dynamics of archetypal columnar π-π stacks of dyes. The analysis of the vibronic peak ratio of the transient fluorescence spectra reveals that unlike the simple π-stacked dimer, the photoexcitation energy in the columnar π-stacked oligomer aggregates is initially delocalized over at least three molecular units and moves coherently along the chain in tens of femtoseconds, preceding excimer formation process.
Electroactive Conjugated Polymers as Charge-Transport Materials for Optoelectronic Thin-Film Devices
(2005)
In this work the electrochemical and spectroelectrochemical properties of a series of pi-conjugated organic polymers were studied. The polymers were deposited on platinum electrodes or ITO-coated glass substrates by potentiodynamic electro-polymerisation of the corresponding monomeric precursor molecules. The electro-chemical and photophysical properties of the triarylborane monomers were studied in detail in order to estimate possible influences on the behaviour of the corresponding polymer. The first part of this work aimed at the synthesis and investigation of conjugated donor–acceptor polymers which combine the prerequisites of an OLED within one material: the transport of positive and negative charges and the formation of emissive excited states. With the carbazole-substituted oxadiazoles 1–3 it was shown that on the one hand the carbazole functionality is suitable for enabling the electrochemical polymerisation of the monomers and on the other hand it facilitates reversible p-doping of the resultant polymers. Although n-doping of poly-1–poly-3 is possible due to the electron-deficient oxadiazole rings, it causes the continuous degradation of these electron-acceptor units. Interestingly, this process does not influence the capability of p-doping of the polymers. With respect to its electrochemical and spectroelectrochemical properties the behaviour of the borane polymer poly-4 is absolutely identical with that of the oxadiazole polymers. Moreover, the optical excitation of poly-4 in the solid state leads to the emission of blue-green light which suggests that this polymer might also possess electroluminescent properties. AFM-measurements of poly-4 films on ITO-coated glass substrates revealed, that the film thickness can be controlled to a certain extent by the number of polymerisation redox cycles. It was shown from the electrochemical and photophysical properties of the triarylboranes 4–6 that the pi–pi-interaction between boron and nitrogen atoms is comparably weak in these molecules. This leads to an unexpected ground-state polarisation with a partially positive boron atom and a partially negative nitrogen atom. Moreover, it was found that TAB 4 possesses a lower symmetry than D3 in solution and that excitation energy can be transferred amongst the three subchromophores of 4. By titration experiments it was also demonstrated that TAB 4 can reversibly bind fluoride ions and that the binding event significantly influences the optical absorption characteristics of the chromophore. It can be assumed, that the above mentioned properties, which have a profound influence on the photophysical behaviour of these triarylborane chromophores, also determine the behaviour of the corresponding polymer in a solid state environment. The aim of the second part of this work was the investigation of purely n-conducting materials based on electron-deficient borane and viologen polymers. The corresponding precursor molecules should be polymerised on platinum electrodes by reductive electropolymerisation. However, a reductive polymerisation was not possible for the borane monomer 19 which is thought to be due to a strong localisation of the unpaired electron on the central boron atom of the radical anion. An electropolymerisation of the cyano-substituted bispyridinio-compound 17 failed because of the poor quality of CN– as a leaving group. Thus, a synthesis of the analogous isomer 18 was developed, in which the cyano-substituents were exchanged by the better leaving group Cl–. The viologen polymer poly-18, which can be regarded as an electron-deficient iso-electronic analogue of poly(para-phenylene), was successfully deposited on a platinum electrode by reductive electropolymerisation of 18. Poly-18 can be reversibly n-doped at comparably low potentials; however, at higher potentials the polymer is overcharged and destroyed irreversibly. As the synthetic strategy for 18 allows the variation of both spacer unit and leaving group in the last two steps of the reaction sequence, a series of analogous compounds can be easily synthesised using this route.
The synthesis and characterization of laterally extended azabora[5]‐, ‐[6]‐ and ‐[7]helicenes, assembled from N‐heteroaromatic and dibenzo[g,p]chrysene building blocks is described. Formally, the π‐conjugated systems of the pristine azaborole helicenes were enlarged with a phenanthrene unit leading to compounds with large Stokes shifts, significantly enhanced luminescence quantum yields (Φ) and dissymmetry factors (g\(_{lum}\)). The beneficial effect on optical properties was also observed for helical elongation. The combined contributions of lateral and helical extensions resulted in a compound showing green emission with Φ of 0.31 and |g\(_{lum}\)| of 2.2×10\(^{−3}\), highest within the series of π‐extended azaborahelicenes and superior to emission intensity and chiroptical response of its non‐extended congener. This study shows that helical and lateral extensions of π‐conjugated systems are viable strategies to improve features of azaborole helicenes. In addition, single crystal X‐ray analysis of configurationally stable [6]‐ and ‐[7]helicenes was used to provide insight into their packing arrangements.
We employ transient absorption from the deep-UV to the visible region and fluorescence upconversion to investigate the photoinduced excited-state intramolecular proton-transfer dynamics in a biologically relevant drug molecule, 2-acetylindan-1,3-dione. The molecule is a ß-diketone which in the electronic ground state exists as exocyclic enol with an intramolecular H-bond. Upon electronic excitation at 300 nm, the first excited state of the exocyclic enol is initially populated, followed by ultrafast proton transfer (≈160 fs) to form the vibrationally hot endocyclic enol. Subsequently, solvent-induced vibrational relaxation takes place (≈10 ps) followed by decay (≈390 ps) to the corresponding ground state.
The potential of human-induced pluripotent stem cells (hiPSCs) to be differentiated into cardiomyocytes (CMs) mimicking adult CMs functional morphology, marker genes and signaling characteristics has been investigated since over a decade. The evolution of the membrane localization of CM-specific G protein-coupled receptors throughout differentiation has received, however, only limited attention to date. We employ here advanced fluorescent spectroscopy, namely linescan Fluorescence Correlation Spectroscopy (FCS), to observe how the plasma membrane abundance of the β\(_1\)- and β\(_2\)-adrenergic receptors (β\(_{1/2}\)-ARs), labelled using a bright and photostable fluorescent antagonist, evolves during the long-term monolayer culture of hiPSC-derived CMs. We compare it to the kinetics of observed mRNA levels in wildtype (WT) hiPSCs and in two CRISPR/Cas9 knock-in clones. We conduct these observations against the backdrop of our recent report of cell-to-cell expression variability, as well as of the subcellular localization heterogeneity of β-ARs in adult CMs.
DNA-stabilized silver clusters (Ag-DNA) show excellent promise as a multi-functional nanoagent for molecular investigations in living cells. The unique properties of these fluorescent nanomaterials allow for intracellular optical sensors with tunable cytotoxicity based on simple modifications of the DNA sequences. Three Ag-DNA nanoagent designs are investigated, exhibiting optical responses to the intracellular environments and sensing-capability of ions, functional inside living cells. Their sequence-dependent fluorescence responses inside living cells include (1) a strong splitting of the fluorescence peak for a DNA hairpin construct, (2) an excitation and emission shift of up to 120 nm for a single-stranded DNA construct, and (3) a sequence robust in fluorescence properties. Additionally, the cytotoxicity of these Ag-DNA constructs is tunable, ranging from highly cytotoxic to biocompatible Ag-DNA, independent of their optical sensing capability. Thus, Ag-DNA represents a versatile live-cell nanoagent addressable towards anti-cancer, patient-specific and anti-bacterial applications.
Many dyes suffer from fast non-radiative decay pathways, thereby showing only short-lived excited states and weak photoluminescence. Here we show a pronounced fluorescence enhancement for a weakly fluorescent merocyanine (MC) dye by being co-facially stacked to other dyes in hetero-folda-trimer architectures. By means of fluorescence spectroscopy (lifetime, quantum yield) the fluorescence enhancement was explained by the rigidification of the emitting chromophore in the defined foldamer architecture and the presence of a non-forbidden lowest exciton state in H-coupled hetero-aggregates. This folding-induced fluorescence enhancement (FIFE) for specific sequences of π-stacked dyes points at a viable strategy toward improved fluorophores that relates to the approach used by nature in the green fluorescent protein (GFP).
Three different perfluoroalkylated borafluorenes (\(^{F}\)Bf) were prepared and their electronic and photophysical properties were investigated. The systems have four trifluoromethyl moieties on the borafluorene moiety as well as two trifluoromethyl groups at the ortho positions of their exo‐aryl moieties. They differ with regard to the para substituents on their exo‐aryl moieties, being a proton \(^{F}\)Xyl\(^{F}\)Bf, \(^{F}\)Xyl: 2,6‐bis(trifluoromethyl)phenyl), a trifluoromethyl group (\(^{F}\)Mes\(^{F}\)Bf, \(^{F}\)Mes: 2,4,6‐tris(trifluoromethyl)phenyl) or a dimethylamino group (p‐NMe\(_{2}\)‐\(^{F}\)Xyl\(^{F}\)Bf, p‐NMe\(_{2}\)‐\(^{F}\)Xyl: 4‐(dimethylamino)‐2,6‐bis(trifluoromethyl)phenyl), respectively. All derivatives exhibit extraordinarily low reduction potentials, comparable to those of perylenediimides. The most electron‐deficient derivative \(^{F}\)Mes\(^{F}\)Bf was also chemically reduced and its radical anion isolated and characterized. Furthermore, all compounds exhibit very long fluorescent lifetimes of about 250 ns up to 1.6 μs; however, the underlying mechanisms responsible for this differ. The donor‐substituted derivative p‐NMe\(_{2}\)‐\(^{F}\)Xyl\(^{F}\)Bf exhibits thermally activated delayed fluorescence (TADF) from a charge‐transfer (CT) state, whereas the \(^{F}\)Mes\(^{F}\)Bf and FXylFBf borafluorenes exhibit only weakly allowed locally excited (LE) transitions due to their symmetry and low transition‐dipole moments.
In TFIIH the Arch domain of XPD is mechanistically essential for transcription and DNA repair
(2020)
The XPD helicase is a central component of the general transcription factor TFIIH which plays major roles in transcription and nucleotide excision repair (NER). Here we present the high-resolution crystal structure of the Arch domain of XPD with its interaction partner MAT1, a central component of the CDK activating kinase complex. The analysis of the interface led to the identification of amino acid residues that are crucial for the MAT1-XPD interaction. More importantly, mutagenesis of the Arch domain revealed that these residues are essential for the regulation of (i) NER activity by either impairing XPD helicase activity or the interaction of XPD with XPG; (ii) the phosphorylation of the RNA polymerase II and RNA synthesis. Our results reveal how MAT1 shields these functionally important residues thereby providing insights into how XPD is regulated by MAT1 and defining the Arch domain as a major mechanistic player within the XPD scaffold.
Up to three polychlorinated pyridyldiphenylmethyl radicals bridged by a triphenylamine carrying electron withdrawing (CN), neutral (Me), or donating (OMe) groups were synthesized and analogous radicals bridged by tris(2,6‐dimethylphenyl)borane were prepared for comparison. All compounds were as stable as common closed‐shell organic compounds and showed significant fluorescence upon excitation. Electronic, magnetic, absorption, and emission properties were examined in detail, and experimental results were interpreted using DFT calculations. Oxidation potentials, absorption and emission energies could be tuned depending on the electron density of the bridges. The triphenylamine bridges mediated intramolecular weak antiferromagnetic interactions between the radical spins, and the energy difference between the high spin and low spin states was determined by temperature dependent ESR spectroscopy and DFT calculations. The fluorescent properties of all radicals were examined in detail and revealed no difference for high and low spin states which facilitates application of these dyes in two‐photon absorption spectroscopy and OLED devices.
Two types of helically chiral compounds bearing one and two boron atoms were synthesized by a modular approach. Formation of the helical scaffolds was executed by the introduction of boron to flexible biaryl and triaryl derived from small achiral building blocks. All‐ortho‐fused azabora[7]helicenes feature exceptional configurational stability, blue or green fluorescence with quantum yields (Φ\(_{fl}\)) of 18–24 % in solution, green or yellow solid‐state emission (Φ\(_{fl}\) up to 23 %), and strong chiroptical response with large dissymmetry factors of up to 1.12×10\(^{-2}\). Azabora[9]helicenes consisting of angularly and linearly fused rings are blue emitters exhibiting Φ\(_{fl}\) of up to 47 % in CH\(_{2}\)Cl\(_{2}\) and 25 % in the solid state. As revealed by the DFT calculations, their P–M interconversion pathway is more complex than that of H1. Single‐crystal X‐ray analysis shows clear differences in the packing arrangement of methyl and phenyl derivatives. These molecules are proposed as primary structures of extended helices.
The 2- and 2,7- substituted para-N-methylpyridinium pyrene cations show high-affinity intercalation into ds-DNAs, whereas their non-methylated analogues interacted with ds-DNA/RNA only in the protonated form (at pH 5), but not at physiological conditions (pH 7). The fluorescence from non-methylated analogues was strongly dependent on the protonation of the pyridines; consequently, they act as fluorescence ratiometric probes for simultaneous detection of both ds-DNA and BSA at pH 5, relying on the ratio between intensities at 420 nm (BSA specific) and 520 nm (DNA specific), whereby exclusively ds-DNA sensing could be switched-off by adjustment to pH 7. Only methylated, permanently charged pyrenes show photoinduced cleavage of circular DNA, attributed to pyrene-mediated irradiation-induced production of singlet oxygen. Consequently, the moderate toxicity of these cations against human cell lines is strongly increased upon irradiation. Detailed studies revealed increased total ROS production in cells treated by the compounds studied, accompanied by cell swelling and augmentation of cellular complexity. The most photo-active 2-para-N-methylpyridinium pyrene showed significant localization at mitochondria, its photo-bioactivity likely due to mitochondrial DNA damage. Other derivatives were mostly non-selectively distributed between various cytoplasmic organelles, thus being less photoactive.
The subject of this thesis is the synthesis and characterization of PBI-based fluorescent metallosupramolecular polymers and cyclic arrays. Terpyridine receptor functionalized PBIs of predesigned geometry have been used as building blocks to construct desired macromolecular structures through metal-ion-directed self-assembly. These metallosupramolecular architectures have been investigated by NMR, UV/Vis and fluorescence spectroscopy, mass spectrometry, and atomic force microscopy.
Localization microscopy is a class of super-resolution fluorescence microscopy techniques. Localization microscopy methods are characterized by stochastic temporal isolation of fluorophore emission, i.e., making the fluorophores blink so rapidly that no two are
likely to be photoactive at the same time close to each other. Well-known localization microscopy methods include dSTORM}, STORM, PALM, FPALM, or GSDIM. The biological community has taken great interest in localization microscopy, since it can enhance the resolution of common fluorescence microscopy by an order of magnitude at little experimental cost.
However, localization microscopy has considerable computational cost since millions of individual stochastic emissions must be located with nanometer precision. The computational cost of this evaluation, and the organizational cost of implementing the complex algorithms, has impeded adoption of super-resolution microscopy for a long time.
In this work, I describe my algorithmic framework for evaluating localization microscopy data.
I demonstrate how my novel open-source software achieves real-time data evaluation, i.e., can evaluate data faster than the common experimental setups can capture them.
I show how this speed is attained on standard consumer-grade CPUs, removing the need for computing on expensive clusters or deploying graphics processing units.
The evaluation is performed with the widely accepted Gaussian PSF model and a Poissonian maximum-likelihood noise model.
I extend the computational model to show how robust, optimal two-color evaluation is realized, allowing correlative microscopy between multiple proteins or structures. By employing cubic B-splines, I show how the evaluation of three-dimensional samples can be made simple and robust, taking an important step towards precise imaging of micrometer-thick samples.
I uncover the behavior and limits of localization algorithms in the face of increasing emission densities.
Finally, I show up algorithms to extend localization microscopy to common biological problems.
I investigate cellular movement and motility by considering the in vitro movement of myosin-actin filaments. I show how SNAP-tag fusion proteins enable imaging with bright and stable organic fluorophores in live cells. By analyzing the internal structure of protein clusters, I show how localization microscopy can provide new quantitative approaches beyond pure imaging.
Background: The human receptor tyrosine kinase MET and its ligand hepatocyte growth factor/scatter factor are essential during embryonic development and play an important role during cancer metastasis and tissue regeneration. In addition, it was found that MET is also relevant for infectious diseases and is the target of different bacteria, amongst them Listeria monocytogenes that induces bacterial uptake through the surface protein internalin B. Binding of ligand to the MET receptor is proposed to lead to receptor dimerization. However, it is also discussed whether preformed MET dimers exist on the cell membrane.
Results: To address these issues we used single-molecule fluorescence microscopy techniques. Our photobleaching experiments show that MET exists in dimers on the membrane of cells in the absence of ligand and that the proportion of MET dimers increases significantly upon ligand binding.
Conclusions: Our results indicate that partially preformed MET dimers may play a role in ligand binding or MET signaling. The addition of the bacterial ligand internalin B leads to an increase of MET dimers which is in agreement with the model of ligand-induced dimerization of receptor tyrosine kinases.
The main objective of this thesis was the design and synthesis of perylene bisimide dyes with sufficient water-solubility for the construction of self-assembled architectures in aqueous solutions. Beside these tasks another goal of this project was the control over the self-assembly process in terms of aggregate size and helicity, respectively. Within this thesis an appropriate synthesis for spermine-functionalized perylene bisimide dyes was developed and conducted successfully. The characterization of these building blocks and their course of self-assembly were investigated by NMR, UV/Vis and fluorescence spectroscopy as well as by atomic force and transmission electron microscopy. For the better understanding of the experimental results theoretical calculations were performed.
The interaction of synaptic proteins orchestrate the function of one of the most complex organs, the brain. The multitude of molecular elements influencing neurological correlations makes imaging processes complicated since conventional fluorescence microscopy methods are unable to resolve structures beyond the diffraction-limit.
The implementation of super-resolution fluorescence microscopy into the field of neuroscience allows the visualisation of the fine details of neural connectivity. The key element of my thesis is the super-resolution technique dSTORM (direct Stochastic Optical Reconstruction Microscopy) and its optimisation as a multi-colour approach. Capturing more than one target, I aim to unravel the distribution of synaptic proteins with nanometer precision and set them into a structural and quantitative context with one another. Therefore dSTORM specific protocols are optimized to serve the peculiarities of particular neural samples.
In one project the brain derived neurotrophic factor (BDNF) is investigated in primary, hippocampal neurons. With a precision beyond 15 nm, preand post-synaptic sites can be identified by staining the active zone proteins bassoon and homer. As a result, hallmarks of mature synapses can be exhibited. The single molecule sensitivity of dSTORM enables the measurement of endogenous BDNF and locates BDNF granules aligned with glutamatergic pre-synapses. This data proofs that hippocampal neurons are capable of enriching BDNF within the mature glutamatergic pre-synapse, possibly influencing synaptic plasticity.
The distribution of the metabotropic glutamate receptor mGlu4 is investigated in physiological brain slices enabling the analysis of the receptor in its natural environment. With dual-colour dSTORM, the spatial arrangement of the mGlu4 receptor in the pre-synaptic sites of parallel fibres in the molecular layer of the mouse cerebellum is visualized, as well as a four to six-fold increase in the density of the receptor in the active zone compared to the nearby environment. Prior functional measurements show that metabotropic glutamate receptors influence voltage-gated calcium channels and proteins that are involved in synaptic vesicle priming. Corresponding dSTORM data indeed suggests that a subset of the mGlu4 receptor is correlated with the voltage-gated calcium channel Cav2.1 on distances around 60 nm.
These results are based on the improvement of the direct analysis of localisation data. Tools like coordinated based correlation analysis and nearest neighbour analysis of clusters centroids are used complementary to map protein connections of the synapse. Limits and possible improvements of these tools are discussed to foster the quantitative analysis of single molecule localisation microscopy data.
Performing super-resolution microscopy on complex samples like brain slices benefits from a maximised field of view in combination with the visualisation of more than two targets to set the protein of interest in a cellular context. This challenge served as a motivation to establish a workflow for correlated structured illumination microscopy (SIM) and dSTORM. The development of the visualisation software coSIdSTORM promotes the combination of these powerful super-resolution techniques even on separated setups. As an example, synapses in the cerebellum that are affiliated to the parallel fibres and the dendrites of the Purkinje cells are identified by SIM and the protein bassoon of those pre-synapses is visualised threedimensionally with nanoscopic precision by dSTORM.
In this work I placed emphasis on the improvement of multi-colour super-resolution imaging and its analysing tools to enable the investigation of synaptic proteins. The unravelling of the structural arrangement of investigated proteins supports the building of a synapse model and therefore helps to understand the relation between structure and function in neural transmission processes.
The solvatochromic behavior of two donor-π bridge-acceptor (D-π-A) compounds based on the 2-(3-boryl-2-thienyl)thiazole π-linker and indandione acceptor moiety are investigated. DFT/TD-DFT calculations were performed in combination with steady-state absorption and emission measurements, along with electrochemical studies, to elucidate the effect of two different strongly electron-donating hydrazonyl units on the solvatochromic and fluorescence behavior of these compounds. The Lippert–Mataga equation was used to estimate the change in dipole moments (Δµ) between ground and excited states based on the measured spectroscopic properties in solvents of varying polarity with the data being supported by theoretical studies. The two asymmetrical D-π-A molecules feature strong solvatochromic shifts in fluorescence of up to ~4300 cm\(^{−1}\) and a concomitant change of the emission color from yellow to red. These changes were accompanied by an increase in Stokes shift to reach values as large as ~5700–5800 cm\(^{−1}\). Quantum yields of ca. 0.75 could be observed for the N,N-dimethylhydrazonyl derivative in nonpolar solvents, which gradually decreased along with increasing solvent polarity, as opposed to the consistently reduced values obtained for the N,N-diphenylhydrazonyl derivative of up to ca. 0.20 in nonpolar solvents. These two push–pull molecules are contrasted with a structurally similar acceptor-π bridge-acceptor (A-π-A) compound.
Bisdiynes undergo Pd(0)-catalyzed cyclization, forming azulene and naphthalene products. When dibenzylideneacetone is present in the reaction, it undergoes a [2+2+2] cyclization with the bisdiyne, forming cyclohexadiene derivatives. Ni(0) catalyzes the [2+2+2] cycloaddition of diynes with tolanes towards alkynylated o-terphenyl derivatives. The D-A substituted products are solvatochromic, fluorescent dyes with high quantum yields and short lifetimes. Bis-triarylborane tetrayne dyes were synthesized in both neutral and tetracationic forms, as potential DNA/RNA sensor. Both molecules are weakly fluorescent in solution and exhibit characteristic alkyne absorptions in the Raman spectra. Tributyl phosphine catalyzes the trans-hydroboration of 1,3-butadiynes with HBpin. We confirmed experimentally via NMR and HRMS experiments, that phosphine attack on the diyne is a key step in the catalytic cycle.
Temperature-responsive luminescent solar concentrators (LSCs) have been fabricated in which the Förster resonance energy transfer (FRET) between a donor–acceptor pair in a liquid crystalline solvent can be tuned. At room temperatures, the perylene bisimide (PBI) acceptor is aggregated and FRET is inactive; while after heating to a temperature above the isotropic phase of the liquid crystal solvent, the acceptor PBI completely dissolves and FRET is activated. This unusual temperature control over FRET was used to design a color-tunable LSC. The device has been shown to be highly stable towards consecutive heating and cooling cycles, making it an appealing device for harvesting otherwise unused solar energy.
Activating delayed fluorescence emission in a dilute solution via a non-covalent approach is a formidable challenge. In this report, we propose a strategy for efficient delayed fluorescence generation in dilute solution using a non-covalent approach via supramolecularly engineered cyclophane-based nanoenvironments that provide sufficient binding strength to π-conjugated guests and that can stabilize triplet excitons by reducing vibrational dissipation and lowering the singlet–triplet energy gap for efficient delayed fluorescence emission. Toward this goal, a novel biphenyl bisimide-derived cyclophane is introduced as an electron-deficient and efficient triplet-generating host. Upon encapsulation of various carbazole-derived guests inside the nanocavity of this cyclophane, emissive charge transfer (CT) states close to the triplet energy level of the biphenyl bisimide are generated. The experimental results of host–guest studies manifest high association constants up to 10\(^4\) M\(^{–1}\) as the prerequisite for inclusion complex formation, the generation of emissive CT states, and triplet-state stabilization in a diluted solution state. By means of different carbazole guest molecules, we could realize tunable delayed fluorescence emission in this carbazole-encapsulated biphenyl bisimide cyclophane in methylcyclohexane/carbon tetrachloride solutions with a quantum yield (QY) of up to 15.6%. Crystal structure analyses and solid-state photophysical studies validate the conclusions from our solution studies and provide insights into the delayed fluorescence emission mechanism.
Two different chromophores, namely a dipolar and an octupolar system, were prepared and their linear and nonlinear optical properties as well as their bioimaging capabilities were compared. Both contain triphenylamine as the donor and a triarylborane as the acceptor, the latter modified with cationic trimethylammonio groups to provide solubility in aqueous media. The octupolar system exhibits a much higher two‐photon brightness, and also better cell viability and enhanced selectivity for lysosomes compared with the dipolar chromophore. Furthermore, both dyes were applied in two‐photon excited fluorescence (TPEF) live‐cell imaging.
Novel dyes were prepared by simple “click CuAAC” attachment of a triarylborane–alkyne to the azide side chain of an amino acid yielding triarylborane dye 1 which was conjugated with pyrene (dye 2) forming a triarylborane–pyrene FRET pair. In contrast to previous cationic triarylboranes, the novel neutral dyes interact only with proteins, while their affinity to DNA/RNA is completely abolished. Both the reference triarylborane amino acid and triarylborane–pyrene conjugate bind to BSA and the hDPP III enzyme with high affinities, exhibiting a strong (up to 100-fold) fluorescence increase, whereby the triarylborane–pyrene conjugate additionally retained FRET upon binding to the protein. Furthermore, the triarylborane dyes, upon binding to the hDPP III enzyme, did not impair its enzymatic activity under a wide range of experimental conditions, thus being the first non-covalent fluorimetric markers for hDPP III, also applicable during enzymatic reactions with hDPP III substrates.