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N\(^6\)-methyladenosine (m\(^6\)A) is an important modified nucleoside in cellular RNA associated with multiple cellular processes and is implicated in diseases. The enzymes associated with the dynamic installation and removal of m\(^6\)A are heavily investigated targets for drug research, which requires detailed knowledge of the recognition modes of m\(^6\)A by proteins. Here, we use atomic mutagenesis of m\(^6\)A to systematically investigate the mechanisms of the two human m\(^6\)A demethylase enzymes FTO and ALKBH5 and the binding modes of YTH reader proteins YTHDF2/DC1/DC2. Atomic mutagenesis refers to atom-specific changes that are introduced by chemical synthesis, such as the replacement of nitrogen by carbon atoms. Synthetic RNA oligonucleotides containing site-specifically incorporated 1-deaza-, 3-deaza-, and 7-deaza-m\(^6\)A nucleosides were prepared by solid-phase synthesis and their RNA binding and demethylation by recombinant proteins were evaluated. We found distinct differences in substrate recognition and transformation and revealed structural preferences for the enzymatic activity. The deaza m\(^6\)A analogues introduced in this work will be useful probes for other proteins in m\(^6\)A research.
In aqueous environment, hydrophobic interactions play an important role for DNA. The introduction of modifications based on hydrophobic aromatic moieties offers additional ways for controlling recognition and reactivity of functional groups in DNA. Modifications are introduced through an artificial backbone or in the form of an extension of the nucleobases, resulting in additional properties of the DNA.
This dissertation focuses on the use of hydrophobic units for the functionalization of DNA.
In the first part of the work, the tolane (i. e. diphenylacetylene) motif was used in combination with the acyclic backbone of GNA and BuNA to generate recognition units in the DNA context. Fluorination of the aromatic rings in the tolane moiety provided the basis for a supramolecular language based on arene-fluoroarene interactions. The specific recognition was investigated by thermodynamic, kinetic and NMR spectroscopic methods.
In the second part of the work, deoxyuridine derivatives with a hydrophobic aromatic modification were prepared and incorporated into DNA duplexes. The irradiation with UV light led to a [2+2] cycloaddition reaction between two modified nucleosides in the DNA. This reaction product was structurally characterized and the reaction was used in various biochemical and nanotechnological DNA applications.
Dipolar merocyanines are very attractive supramolecular building blocks, as they combine interesting functional properties with strong, directional intermolecular interactions. The pyridine dioxocyano-pyridine (PYOP) chromophore (Chapter 2.2), used in this thesis, stands out because of its exceptionally high ground state dipole moment (g ~ 17 D), in combination with the option to retain good solubility also in unpolar solvents, by decoration with solubilizing groups.
The reliable binding motif of anti-parallel -stacking due to dipole-dipole interactions has allowed the design of molecular building blocks that form assemblies of predictable geometry. The intense unstructured charge transfer UV/Vis absorption band (eg ~ 10.7 D) is a result of the dominant contribution of the zwitterionic resonance structure which brings the PYOP chromophore just beyond the cyanine limit in solvents of low polarity (c2 = 0.60, 1,4 dioxane). The high sensitivity of the S0 – S1 UV/Vis absorption band to the environment manifests itself in a pronounced negative solvatochromism and strong H-type exciton coupling within -stacked PYOP assemblies. In accordance with the classical molecular exciton theory, an increasing hypsochromic shift of the dominant absorption band of these H aggregates can be observed as the stack size increases up to about six chromophores, where it levels out at about max ~ 440 nm (CHCl3). This allows a uniquely simple estimation of the number of interacting chromophores within the self-assembled structure from a single UV/Vis absorption spectrum of an aggregate.
The defined and well investigated PYOP dimer formation was employed in this thesis to probe the applicability and limitations of concentration-, temperature-, and solvent-dependent self-assembly studies (Chapter 3). Straightforward theoretical models to evaluate datasets of concentration-, temperature-, and solvent-dependent UV/Vis absorption by nonlinear regression analysis were derived for the case of dimer formation (Chapter 2.1). Although the dimer model is well known and widely applied in literature, this detailed derivation is helpful to understand assumptions and potential problems of the different approaches for the determination of thermodynamic parameters. This helps to decide on the most appropriate method to analyse a system of interest. In this regard it should be noted that covering a large portion of the self-assembly process with the experimental data is a prerequisite for the accuracy of the analysis. Additionally, many of the insights can also be transferred to other self-assembly systems like supramolecular polymerization or host-guest interactions.
The concentration-dependent analysis is the most straightforward method to investigate self-assembly equilibria. No additional assumptions, besides mass balance and mass action law, are required. Since it includes the least number of parameters (only K, if M/D are known), it is the most, or even only, reliable method, to elucidate the self-assembly mechanism of an unknown system by model comparison. To cover a large concentration range, however, the compound must be soluble enough and generally sample amounts at least in the low mg scale must be available.
The temperature-dependent analysis has the advantage that all thermodynamic parameters G0, H0 and S0 can be obtained from a single sample in one automated measurement. However, the accessible temperature-range is experimentally often quite limited and dependent on the solvent. For systems which do not show the transition from monomer to aggregate in a narrow temperature range, as given for, e.g., cooperative aggregation or processes with a high entropy contribution, often not the entire self-assembly process can be monitored. Furthermore, the assumptions of temperature-independent extinction coefficients of the individual species as well as temperature-independent H0 and S0 must be met. Monte Carlo simulations of data sets demonstrated that even minor changes in experimental data can significantly impact the optimized values for H0 and S0. This is due to the redundancy of these two parameters within the model framework and even small thermochromic effects can significantly influence the results. The G0 value, calculated from H0 and S0, is, however, still rather reliable.
Solvent-dependent studies can often cover the entire self-assembly process from monomeric (agg = 0) to the fully aggregated state (agg = 1). However, for dyes with strong solvatochromic effects, such as the dipolar merocyanines investigated in this thesis, the results are affected. Also, the assumption of a linear relation of the binding energy G0 and the fraction of denaturating solvent f, which is based on linear free energy relationships between G0 and the solvent polarity, can lead to errors. Especially when specific solvent effects are involved.
For the evaluation of experimental data by nonlinear regression, general data analysis software can be used, where user-defined fit models and known parameters can be implemented as desired. Alternatively, multiple specialized programs for analysing self-assembly data are available online. While the latter programs are usually more user-friendly, they have the disadvantage of being a “black box” where only pre-implemented models can be used without the option for the user to adapt models or parameters for a specific system.
In Chapter 3 comprehensive UV/Vis absorption datasets are presented for the dimerization of merocyanine derivative 1 in 1,4-dioxane, which allowed for the first time a direct comparison of the results derived from concentration-, temperature-, and solvent-dependent self-assembly studies.
The results for the binding constant K and corresponding G0 from the concentration- and temperature-dependent analysis were in very good agreement, also in comparison to the results from ITC. For the temperature-dependent analysis, though, multiple datasets of samples with different concentration had to be evaluated simultaneously to cover a meaningful part of the self-assembly process. Furthermore, a significant dependence of the optimized parameters H0 and S0 on the wavelength chosen for the analysis was observed. This can be rationalized by the small thermochromic shifts of both the monomer and the dimer UV/Vis absorption band. The results from the solvent-dependent evaluation showed the largest deviation, as expected for the highly solvatochromic merocyanine dye.
However, even here by evaluation at 491 and 549 nm the deviation for G0 was only 2.5 kJ mol1 (9%) with respect to the results from the concentration-dependent analysis (G0 = 29.1 kJ mol1). Thus, despite the strong solvatochromism of the dipolar chromophore, it can still be considered a reliable method for estimating the binding strength. Furthermore, multiple repetitions of the concentration-, temperature-, and solvent-dependent studies provided insight into the reproducibility of the results and possible sources of experimental errors. In all cases, the deviations of the results were small (G0 < 0.4 kJ mol1) and within the same range as the fit error from the nonlinear regression analysis.
The insights from these studies were an important basis for the in-depth investigation of a more complex supramolecular system in Chapter 4, as a single method is often not enough to capture the full picture of a more complicated self-assembly process. To elucidate the anti-cooperative self-assembly of the chiral merocyanine 2, a combination of multiple techniques had to be applied.
Solvent-dependent UV/Vis absorption studies in CH2Cl2/MCH mixtures showed the step-wise assembly of the merocyanine monomer (max(M) = 549 nm, CH2Cl2) to first a dimer (max(D) = 498 nm, CH2Cl2/MCH 15:85) by dipole-dipole interactions, and then a -stacked higher aggregate (max(H) = 477 nm, MCH), with pronounced H-type coupling.
The thermodynamic evaluation of this data, however, suffered from the severe solvatochromism, especially of the monomeric species (max(M, CH2Cl2) = 549 nm, max(M, MCH) = 596 nm). Therefore, concentration-dependent studies were performed at three different temperatures (298, 323, 353 K) to elucidate the self-assembly mechanism and determine reliable thermodynamic parameters. The studies at elevated temperatures were hereby necessary, to obtain experimental data over a larger agg--range. Due to the pronounced difference in the thermodynamic driving force for dimerization and higher aggregate formation (KD/K5 = 6500) a concentration range exists in MCH where almost exclusively the dimer species of 2 is present, before further self-assembly by dispersion interactions occurs. Therefore, the data could be evaluated independently for the two self-assembly steps. The self-assembly of dimers into the higher aggregate could not be described by the isodesmic model but was fitted satisfactorily to a pentamer model. This rather small size of about ten -stacked PYOP chromophores was, furthermore, consistently indicated by AFM, VPO and DOSY NMR measurements. Based on 1D and 2D NMR data as well as the strong bisignate CD signal of the higher aggregate in combination with TD-DFT calculations, a P-helical stack is proposed as its structure. The small size can be rationalized by the anti-cooperative self-assembly mechanism and the sterical demand of the solubilizing trialkoxyphenyl and the chiral tetralin substituents. Additionally, the aliphatic shell formed by the solubilizing chains around the polar chromophore stack, can account for the exceptionally high solubility of 2 in MCH (> 15 mg mL1). These combined studies of the self-assembly process enabled the identification of suitable conditions for the investigation of fluorescence properties of the individual aggregate species. Aggregation-induced emission enhancement was observed for the almost non-emissive monomer (Fl(M) = 0.23%), which can be rationalized by the increasing rigidification within the dimer (Fl(D) = 2.3%) and the higher aggregate (Fl(H) = 4.5%). The helical chirality of the PYOP decamer stack, furthermore, gave rise to a strong CPL signal with a large glum value of 0.011.
The important conclusion of this thesis is that the temperature- and solvent-dependent analyses are valid alternatives to the classical concentration-dependent analysis to determine thermodynamic parameters of self-assembly equilibria. Although, for a specific supramolecular system, one approach might be favourable over the others for a variety of reasons. The experimental limitations often demand a combination of techniques to fully elucidate a self-assembly process and to gain insights in the aggregate structure. The anti-cooperative merocyanine self-assembly, which was described here for the first time for the PYOP merocyanine 2, is no exception. Besides the interest in the merocyanine assemblies from a structural and functional point of view, the insights gained from the presented studies can also be transferred to other self-assembly systems and be a guide to find the most appropriate analysis technique.
This work illustrates how the targeted tailoring of supramolecular cavities can not only accomplish high binding due to optimized stereoelectronic shape matches between host and guest but also how molecular engineering of the binding site by a refined substitution periphery of the cavity makes enantiospecific guest recognition and host mediated chirality transfer feasible. Moreover, an enzyme mimic, following the Pauling-Jencks model of enzyme catalysis was realized by the smart design of a PBI host composed of moderately twisted chromophores, which drives the substrate inversion according to the concepts of transition state stabilization and ground state destabilization. The results of this thesis contribute to a better understanding of structure-specific interactions in host-guest complexes as well as the corresponding thermodynamic and kinetic properties and represent an appealing blueprint for the design of new artificial complex structures of high stereoelectronic shape complementarity in order to achieve the goal of sophisticated supramolecular receptors and enzyme mimicry.
The present thesis introduce different synthetic strategies towards a variety of polycyclic aromatic dicarboximides (PADIs) with highly interesting and diverse properties. This included tetrachlorinated, tetraaryloxy- and tetraaryl-substituted dicarboximides, fused acceptor‒donor(‒acceptor) structures as well as sterically shielded rylene and nanographene dicarboximides. The properties and thus the disclosure of structure‒property relationships of the resulting dyes were investigated in detail among others with UV‒vis absorption spectroscopy, fluorescence spectroscopy, cyclic voltammetry and single crystal X-ray analysis. For instance, some of the fused and substituted PADIs offer strong absorption of visible and near infrared (NIR) light, NIR emission and low-lying LUMO levels. On the contrary, intriguing optical features in the solid-state characterize the rylene dicarboximides with their bulky N-substituents, while the devised sterically enwrapped nanographene host offered remarkable complexation capabilities in solution.
Einflüsse der Photophysik und Photochemie von Cyaninfarbstoffen auf die Lokalisationsmikroskopie
(2023)
In den letzten Jahren haben sich hochauflösende Fluoreszenzmikroskopiemethoden, basierend auf der Lokalisation einzelner Fluorophore, zu einem leistungsstarken Werkzeug etabliert, um Fluoreszenzbilder weit unterhalb der Auflösungsgrenze zu generieren. Hiermit können räumliche Auflösungen von ~ 20 nm erzielt werden, was weit unterhalb der Beugungsgrenze liegt. Dabei haben zahlreiche Optimierungen und Entwicklungen neuer Methoden in der Einzelmolekül-Lokalisationsmikroskopie die Genauigkeit der orstspezifischen Bestimmung einzelner Fluorophore auf bis zu ~ 1 – 3 nm erhöht. Eine Auflösung im molekularen Bereich, weit unterhalb von ~ 10 nm bleibt allerdings herausfordernd, da die Lokalisationsgenauigkeit nur ein Kriterium hierfür ist. Allerdings wurde sich in den letzten Jahren überwiegend auf die Verbesserung dieses Parameters konzentriert. Weitere Kriterien für die fluoreszenzmikroskopische Auflösung sind dabei unter anderem die Markierungsdichte und die Kopplungseffizienz der Zielstruktur, sowie der Kopplungsfehler (Abstand zur Zielstruktur nach Farbstoffkopplung), die sich herausfordernd für eine molekulare Auflösung darstellen. Auch wenn die Kopplungseffizienz und -dichte hoch und der Kopplungsfehler gering ist, steigt bei Interfluorophordistanzen < 5nm, abhängig von den Farbstoffen, die Wahrscheinlichkeit von starken und schwachen Farbstoffwechselwirkungen und damit von Energieübertragungsprozessen zwischen den Farbstoffen, stark an. Daneben sollten Farbstoffe, abhänging von der Lokalisationsmikroskopiemethode, spezifische Kriterien, wie beispielsweise die Photoschaltbarkeit bei dSTORM, erfüllen, was dazu führt, dass diese Methoden häufig nur auf einzelne Farbstoffe beschränkt sind. In dieser Arbeit konnte mithilfe von definierten DNA-Origami Konstrukten gezeigt werden, dass das Blinkverhalten von Cyaninfarbstoffen unter dSTORM-Bedingungen einer Abstandsabhängigkeit aufgrund von spezifischen Energieübertragungsprozessen folgt, womit Farbstoffabstände im sub-10 nm Bereich charakterisiert werden konnten. Darüber hinaus konnte diese Abstandsabhängigkeit an biologischen Proben gezeigt werden. Hierbei konnten verschiedene zelluläre Rezeptoren effizient und mit geringem Abstandsfehler zur Zielstruktur mit Cyaninfarbstoffen gekoppelt werden. Diese abstandsabhänigen Prozesse und damit Charakterisierungen könnten dabei nicht nur spezifisch für die häufig unter dSTORM-Bedingungen verwendeten Cyaninfarbstoffen gültig sein, sondern auch auf andere Farbstoffklassen, die einen Auszustand zeigen, übertragbar sein. Darüber hinaus konnte gezeigt werden, dass hochauflösende dSTORM Aufnahmen unabhängig vom Farbstoffkopplungsgrad der Antikörpern sind, welche häufig für Standardfärbungen von zellulären Strukturen verwendet werden. Dabei konnte durch Photonenkoinzidenzmessungen dargelegt werden, dass aufgrund komplexer Farbstoffwechselwirkungen im Mittel nur ein Farbstoff aktiv ist, wobei höhere Kopplungsgrade ein komplexes Blinkverhalten zu Beginn der Messung zeigen. Durch die undefinierten Farbstoffabstände an Antikörpern konnte hier kein eindeutiger Energieübertragungsmechanismus entschlüsselt werden. Dennoch konnte gezeigt werden, dass Farbstoffaggregate bzw. H-Dimere unter dSTORM-Bedingungen destabilisiert werden. Durch die zuvor erwähnten DNA-Origami Konstrukte definierter Interfluorophordistanzen konnten Energieübertragungsmechanismen entschlüsselt werden, die auch für die Antikörper diverser Kopplungsgrade gültig sind. Des Weiteren konnten, ausgelöst durch komplexe Energieübertragungsprozesse höherer Kopplungsgrade am Antikörper, Mehrfarbenaufnahmen zellulärer Strukturen generiert werden, die über die spezifische Fluoreszenzlebenszeit separiert werden konnten. Dies stellt hier eine weitere Möglichkeit dar, unter einfachen Bedingungen, schnelle Mehrfarbenaufnahmen zellulärer Strukturen zu generieren. Durch die Verwendung des selben Farbstoffes unterschiedlicher Kopplungsgrade kann hier nur mit einer Anregungswellenlänge und frei von chromatischer Aberration gearbeitet werden. Neben den photophysikalischen Untersuchungen der Cyaninfarbstoffe Cy5 und Alexa Fluor 647 wurden diese ebenso photochemisch näher betrachtet. Dabei konnte ein neuartiger chemischer Mechanismus entschlüsselt werden. Dieser Mechanismus führt, ausgelöst durch Singulett-Sauerstoff (1O2), zu einer Photozerschneidung des konjugierten Doppelbindungssystems um zwei Kohlenstoffatome, was zu strukturellen und spektroskopischen Veränderungen dieser Farbstoffe führt. Auf Grundlage dieses Mechanismus konnte eine neue DNA-PAINT Methode entwickelt werden, die zu einer Beschleunigung der Aufnahmezeit führt.
About 2.4 billion years ago, nature has fundamentally revolutionized life on earth by inventing the multi-subunit protein complex photosystem II, the only molecular machine in nature that catalyzes the thermodynamically demanding photosynthetic splitting of water into oxygen and reducing equivalents. Nature chose a distorted Mn4CaO5 cluster as catalyst, better known as oxygen-evolving complex (OEC), thus recognizing the need for transition metals to achieve high-performance catalysts. The curiosity has always driven mankind to mimic nature’s achievements, but the performance of natural enzymes such as the oxygen-evolving complex in photosystem II remain commonly unmatched. An important role in fine-tuning and regulating the activity of natural enzymes is attributed to the surrounding protein domain, which facilitates substrate preorganization within well-defined nanoenvironments.
In light of growing energy demands and the depletion of fossil fuels, the unparalleled efficiency of natural photosynthesis inspires chemists to artificially mimic its natural counterpart to generate hydrogen as a ‘solar fuel’ through the light-driven splitting of water. As a result, significant efforts have been devoted in recent decades to develop molecular water oxidation catalysts based on earth-abundant transition metals and the discovery of the Ru(bda) (bda: 2,2’ bipyridine-6,6’-dicarboxylate) catalyst family enabled activities comparable to the natural OEC. Similar to the natural archetypes, the design of homogeneous catalysts that interplay judiciously with the second coordination sphere of the outer ligand framework proved to be a promising concept for catalyst design. In this present thesis, novel supramolecular design approaches for enzyme like activation of substrate water molecules for the challenging oxidative water splitting reaction were established via tailor-made engineering of the secondary ligand environment of macrocyclic Ru(bda) catalysts.
Covalent crosslinking of DNA strands provides a useful tool for medical, biochemical and DNA nanotechnology applications. Here we present a light-induced interstrand DNA crosslinking reaction using the modified nucleoside 5-phenylethynyl-2’-deoxyuridine (\(^{Phe}\)dU). The crosslinking ability of \(^{Phe}\)dU was programmed by base pairing and by metal ion interaction at the Watson-Crick base pairing site. Rotation to intrahelical positions was favored by hydrophobic stacking and enabled an unexpected photochemical alkene-alkyne [2+2] cycloaddition within the DNA duplex, resulting in efficient formation of a \(^{Phe}\)dU-dimer after short irradiation times of a few seconds. A \(^{Phe}\)dU dimer-containing DNA was shown to efficiently bind a helicase complex, but the covalent crosslink completely prevented DNA unwinding, suggesting possible applications in biochemistry or structural biology.
Beyond the four canonical nucleosides as primary building blocks of RNA, posttranscriptional modifications give rise to the epitranscriptome as a second layer of genetic information. In eukaryotic mRNA, the most abundant posttranscriptional modification is N6-methyladenosine (m6A), which is involved in the regulation of cellular processes. Throughout this thesis, the concept of atomic mutagenesis was employed to gain novel mechanistic insights into the substrate recognition by human m6A reader proteins as well as in the oxidative m6A demethylation by human demethylase enzymes. Non-natural m6A atomic mutants featuring distinct steric and electronic properties were synthesized and incorporated into RNA oligonucleotides. Fluorescence anisotropy measurements using these modified oligonucleotides revealed the impact of the atomic mutagenesis on the molecular recognition by the human m6A readers YTHDF2, YTHDC1 and YTHDC2 and allowed to draw conclusions about structural prerequisites for substrate recognition. Furthermore, substrate recognition and demethylation mechanism of the human m6A demethylase enzymes FTO and ALKBH5 were analyzed by HPLC-MS and PAGE-based assays using the modified oligonucleotides synthesized in this work.
Modified nucleosides not only expand the genetic alphabet, but are also extensively researched as drug candidates. In this thesis, the antiviral mechanism of the anti-SARS-CoV-2 drug remdesivir was investigated, which causes delayed stalling of the viral RNA-dependent RNA polymerase (RdRp). Novel remdesivir phosphoramidite building blocks were synthesized and used to construct defined RNA-RdRp complexes for subsequent studies by cryogenic electron microscopy (cryo-EM). It was found that the 1'-cyano substituent causes Rem to act as a steric barrier of RdRp translocation. Since this translocation barrier can eventually be overcome by the polymerase, novel derivatives of Rem with potentially improved antiviral properties were designed.
As central components of life, DNA and RNA encode the genetic information. However, RNA performs several functions that exceed the competences stated in the ‘central dogma of life‘. RNAs undergo extensive post-transcriptional processing like chemical modifications. Among all classes of RNA, tRNAs are the most extensively modified. Their modifications are chemically diverse and vary from simple methylations (e.g. m3C, m6A) to more complex residues, like isopentenyl group (e.g. i6A, hypermodifications: e.g. ms2i6A) or even amino acids (e.g. t6A). Depending on their location within the overall structure, modifications can have an impact on tRNA stability and structure, as well as affinity for the ribosome and translation efficiency and fidelity. Given the importance of tRNA modifications new tools are needed for their detection and to study their recognition by proteins and enzymatic transformations.
The chemical synthesis of these naturally occurring tRNA modifications as phosphoramidite building blocks is a prerequisite to incorporate the desired modification via solid-phase synthesis into oligonucleotides. With the help of the m3C, (ms2)i6A, and t6A oligonucleotides, the importance and impact of tRNA modifications was investigated in this thesis. To this end, the role of METTL8 as the methyltransferase responsible for the installation of the methyl group at C32 for mt-tRNAThr and mt-tRNASer(UCN) was resolved. Thereby, the respective adenosine modification on position 37 is essential for the effectiveness of the enzyme. Besides, by means of NMR analysis, CD spectroscopy, thermal denaturation experiments, and native page separation, the impact of m3C32 on the structure of the tRNA ASLs was shown. The modification appeared to fine-tune the tRNA structure to optimize mitochondrial translation. To investigate the regulation of the dynamic modification pathway of m3C, demethylation assays were performed with the modified tRNA-ASLs and the (α-KG)- and Fe(II)-dependent dioxygenase ALKBH1 and ALKHB3. A demethylation activity of ALKBH3 on the mt-tRNAs was observed, even though it has so far only been described as a cytoplasmic enzyme. Whether this is physiologically relevant and ALKBH3 present a mitochondrial localization needs further validation. In addition, ALKBH1 was confirmed to not be able to demethylate m3C on mt-tRNAs, but indications for a deprenylation and exonuclease activity were found. Furthermore, the aforementioned naturally occurring modifications were utilized to find analytical tools that can determine the modification levels by DNAzymes, which cleave RNA in the presence of a specific modification. Selective DNA enzymes for i6A, as well as the three cytidine isomers m3C, m4C, and m5C have been identified and characterized.
Besides the naturally occurring tRNA modifications, the investigation on artificially modified nucleosides is also part of this thesis. Nucleosides with specific properties for desired applications can be created by modifying the scaffold of native nucleosides.
During the pandemic, the potential of antiviral nucleoside analogues was highlighted for the treatment of the SARS-CoV-2 infection. For examinations of the potential drug-candidate Molnupiravir, the N4-hydroxycytidine phosphoramidite building block was synthesized and incorporated into several RNA oligonucleotides. A two-step model for the NHC-induced mutagenesis of SARS-CoV-2 was proposed based on RNA elongation, thermal denaturation, and cryo-EM experiments using the modified RNA strands with the recombinant SARS-CoV-2 RNA-dependent RNA polymerase. Two tautomeric forms of NHC enable base pairing with guanosine in the amino and with adenosine in the imino form, leading to error catastrophe after the incorporation into viral RNA. These findings were further corroborated by thermal melting curve analysis and NMR spectroscopy of the NHC-containing Dickerson Drew sequence. In conclusion, the anti-amino form in the NHC-G base pair was assigned by NMR analysis using a 15N-labeld NHC building block incorporated into the Dickerson Drew sequence.
This thesis also addressed the synthesis of a 7-deazaguanosine crosslinker with a masked aldehyde as a diol linker for investigations of DNA-protein interactions. The diol functional group can be unmasked to release the reactive aldehyde, which can specifically form a covalent bond with amino acids Lys or Arg within the protein complex condensin. The incorporation of the synthesized phosphoramidite and triphosphate building blocks were shown and the functionality of the PCR product containing the crosslinker was demonstrated by oxidation and the formation of a covalent bond with a fluorescein label.
The development of assays that detect changes in this methylation pattern of m6A could provide new insights into important biological processes. In the last project of this thesis, the influence of RNA methylation states on the structural properties of RNA was analyzed and a fluorescent nucleoside analog (8-vinyladenosine) as molecular tools for such assays was developed. Initial experiments with the fluorescent nucleoside analog N6-methyl-8-vinyladenosine (m6v8A) were performed and revealed a strong fluorescence enhancement of the free m6v8A nucleoside by the installation of the vinyl moiety at position 8.
Overall, this thesis contributes to various research topics regarding the application of naturally occurring and artificial nucleoside analogues. Starting with the chemical synthesis of RNA and DNA modifications, this thesis has unveiled several open questions regarding the dynamic (de-)methylation pathway of m3C and the mechanism of action of molnupiravir through in-depth analysis and provided the basis for further investigations of the protein complex condensin, and a new fluorescent nucleoside analog m6v8A.
In dieser Dissertation wird beschrieben, wie es durch systematische Anwendung unterschiedlicher Methoden zur Herstellung und Modifizierung von Diamant gezielt und verlässlich möglich ist, die Eigenschaften von Diamanten zu beeinflussen. Es wird gezeigt, wie durch Variation der Parameter bei dem Wachstum von Diamant Einfluss auf dessen Morphologie und Eigenschaften genommen werden kann. Des Weiteren wird ein Verfahren vorgestellt, mit dem die Oberfläche des Diamanten durch Ozon effizient oxidiert beziehungsweise reduziert werden kann. Um diese veränderte Oberflächenbelegung möglichst genau zu analysieren, wird im letzten Teil der Dissertation eine Methode zur qualitativen und quantitativen Analytik der Oberflächen von Kohlenstoffnanomaterialien beschrieben.
Proton‐coupled electron‐transfer (PCET) processes play a key role in biocatalytic energy conversion and storage, for example, photosynthesis or nitrogen fixation. Here, we report a series of bipyridine‐containing di‐ to tetranuclear Ru(bda) macrocycles 2 C–4 C (bda: 2,2′‐bipyridine‐6,6′‐dicarboxylate) to promote O−O bond formation. In photocatalytic water oxidation under neutral conditions, all complexes 2 C–4 C prevail in a folded conformation that support the water nucleophilic attack (WNA) pathway with remarkable turnover frequencies of up to 15.5 s\(^{−1}\) per Ru unit respectively. Single‐crystal X‐ray analysis revealed an increased tendency for intramolecular π‐π stacking and preorganization of the proximal bases close to the active centers for the larger macrocycles. H/D kinetic isotope effect studies and electrochemical data demonstrate the key role of the proximal bipyridines as proton acceptors in lowering the activation barrier for the crucial nucleophilic attack of H\(_{2}\)O in the WNA mechanism.
We introduce fluorescence-detected pump–probe microscopy by combining a wavelength-tunable ultrafast laser with a confocal scanning fluorescence microscope, enabling access to the femtosecond time scale on the micrometer spatial scale. In addition, we obtain spectral information from Fourier transformation over excitation pulse-pair time delays. We demonstrate this new approach on a model system of a terrylene bisimide (TBI) dye embedded in a PMMA matrix and acquire the linear excitation spectrum as well as time-dependent pump–probe spectra simultaneously. We then push the technique towards single TBI molecules and analyze the statistical distribution of their excitation spectra. Furthermore, we demonstrate the ultrafast transient evolution of several individual molecules, highlighting their different behavior in contrast to the ensemble due to their individual local environment. By correlating the linear and nonlinear spectra, we assess the effect of the molecular environment on the excited-state energy.
Mittels einer fünfstufigen Synthese wurde das 2,2´-Ditetracen als Modellsystem zur Erforschung von singlet fission-Prozessen hergestellt. Die Synthese wurde mit einer Gesamtausbeute von 21 % durchgeführt, wobei der Schlüsselschritt, die Kopplung der beiden Monomere, durch eine Suzuki-Kopplung erfolgte. Das gewünschte Produkt konnte nach gründlicher Reinigung mittels Gradientensublimation als leuchtend rote Einkristalle erhalten werden. Während die Emissionsspektren der Einzelmoleküle nahezu identisch sind, zeigen Untersuchungen mittels Photolumineszenzspektroskopie eine Rotverschiebung im Emissionsspektrum des Dimer-Einkristalls im Vergleich zum Einkristall des Tetracen-Monomers. Durch theoretische Berechnung konnte die Absenkung des S1-Zustands des Dimers im Kristall erklärt werden, wodurch die Energiebedingung für singlet fission (2 E(T1) ≤ E(S1)) nicht mehr erfüllt ist.
Weiterhin wurden mehrere mit Alkylgruppen und Vinylgruppen substituierte Tetracenderivate synthetisiert und diese mittels optischer und elektrochemischer Methoden auf ihre Eigenschaften hin untersucht. Es wurde bei allen synthetisierten Derivaten eine Rotverschiebung der Hauptbanden im Absorptionsspektrum beobachtet, was durch einen kleineren HOMO-LUMO-Abstand im Vergleich zum nicht substituierten Tetracen erklärt wird. Es wurde zudem eine erhöhte Stabilität dieser Derivate gegenüber Umwelteinflüssen wie Licht und Sauerstoff, die die Bildung von Endoperoxiden und Dimeren zur Folge haben, festgestellt. Dies kann auf sterische Effekte sowie die Stabilisierung des biradikalischen Zustands dieser Moleküle durch Hyperkonjugation und Resonanzeffekte zurückgeführt werden.
Perylene bisimides (PBIs) are among the best fluorophores but have to be enwrapped for optoelectronic applications by large and heavy substituents to prevent their ππ‐stacking, which is known to accelerate non‐radiative decay processes in the solid state. Here, light‐weight di‐tert‐butylsilyl groups are introduced to bridge 1,12‐dihydroxy and 1,6,7,12‐tetrahydroxy PBIs to afford sublimable dyes for vacuum‐processed optoelectronic devices. For both new compounds, this substitution provides a twisted and shielded perylene π‐core whose, via OSiObridges, rigid structure affords well‐resolved absorption and emission spectra with strong fluorescence in solution, as well as in the solid state. The usefulness of these dyes for vacuum‐processed optoelectronic devices is demonstrated in organic light‐emitting diodes (OLEDs) that show monomer‐like emission spectra and high maximum external quantum efficiency (EQEmax) values of up to 3.1% for the doubly silicon‐bridged PBI.
A series of novel imide‐functionalized C\(_{64}\) nanographenes is investigated as acceptor components in organic solar cells (OSCs) in combination with donor polymer PM6. These electron‐poor molecules either prevail as a monomer or self‐assemble into dimers in the OSC active layer depending on the chosen imide substituents. This allows for the controlled stacking of electron‐poor and electron‐rich π–scaffolds to establish a novel class of non‐fullerene acceptor materials to tailor the bulk‐heterojunction morphology of the OSCs. The best performance is observed for derivatives that are able to self‐assemble into dimers, reaching power conversion efficiencies of up to 7.1%.
A tolane-modified 5-ethynyluridine as a universal and fluorogenic photochemical DNA crosslinker
(2023)
We report the fluorescent nucleoside ToldU and its application as a photoresponsive crosslinker in three different DNA architectures with enhanced fluorescence emission of the crosslinked products. The fluorogenic ToldU crosslinking reaction enables the assembly of DNA polymers in a hybridization chain reaction for the concentration-dependent detectio of a specific DNA sequence.
We report the synthesis and spectroscopic analysis of RNA containing the barbituric acid merocyanine rBAM2 as a nucleobase surrogate. Incorporation into RNA strands by solid-phase synthesis leads to fluorescence enhancement compared to the free chromophore. In addition, linear absorption studies show the formation of an excitonically coupled H-type dimer in the hybridized duplex. Ultrafast third- and fifth-order transient absorption spectroscopy of this non-fluorescent dimer suggests immediate (sub-200 fs) exciton transfer and annihilation due to the proximity of the rBAM2 units.
Site-specific introduction of biorthogonal handles into RNAs is in high demand for decorating RNAs with fluorophores, affinity labels or other modifications. Aldehydes represent attractive functional groups for post-synthetic bioconjugation reactions. Here, we report a ribozyme-based method for the synthesis of aldehyde-functionalized RNA by directly converting a purine nucleobase. Using the methyltransferase ribozyme MTR1 as an alkyltransferase, the reaction is initiated by site-specific N1 benzylation of purine, followed by nucleophilic ring opening and spontaneous hydrolysis under mild conditions to yield a 5-amino-4-formylimidazole residue in good yields. The modified nucleotide is accessible to aldehyde-reactive probes, as demonstrated by the conjugation of biotin or fluorescent dyes to short synthetic RNAs and tRNA transcripts. Upon fluorogenic condensation with a 2,3,3-trimethylindole, a novel hemicyanine chromophore was generated directly on the RNA. This work expands the MTR1 ribozyme’s area of application from a methyltransferase to a tool for site-specific late-stage functionalization of RNA.