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Tribenzotriquinacene (TBTQ) is a polycyclic aromatic framework with a particularly rigid, C3v symmetrical, bowl-shaped core bearing three mutually fused indane wings. It has been discussed as a defect center for a nanographene by Kuck and colleagues. Therefore, extended TBTQ structures are promising models for saturated defect structures in graphene and graphene like molecules and could be used to investigate the role of defects for the electronic properties of graphene. With this motivation, three different pi-extended TBTQ derivatives have been synthesized in this work. Several different Scholl reaction conditions were tried to obtain fully annulated product of hexaphenyl substituted TBTQ. The desired benzannulated TBTQ derivative could not be obtained due to unfavourable electron density in the respective positions of the molecule and increased reactivity of the bay position of the precursor. As an another method for benzannulation is the on-surface synthesis of graphene flakes and can be carried out using electron beams e.g. in a tunneling microscope (STM). According to our previous research, the parent system TBTQ and centro-methyl TBTQ on silver and gold surfaces showed that the gas phase deposition of these molecules gives rise to the formation of highly ordered two-dimensional assemblies with unique structural features. This shows the feasibility for the formation of defective graphene networks starting from the parent structures. Therefore, the same deposition technique was used to deposit Me-TBTQ(OAc)3Ph6, and investigate the molecular self-assembly properties directly on the surface of Cu (111). In summary, the substrate temperature dependent self-assembly of Me-TBTQ(OAc)3Ph6 molecules on Cu(111), shows the following evolution of orientations. At room temperature, molecules form dimers, which construct a higher-coverage honeycomb lattice. Furthermore, one of the acetyl group located in the bay positions of the TBTQ core is cleaved and the remaining two induce the metal-molecule interaction. It was presumed that by increasing the temperature to 393 K, the remaining acetyl and methyl groups would beeliminated from the molecular structure.In addition, the smaller TBTQ-Ph6 molecules preferably lie flat on Cu(111) crystal and allowing the molecules to settle into a C3-symmetry and form a dense hexagonal structure.
The objective of this thesis was the synthesis and characterisation of two linear multifunctional PEG-alternatives for bioconjugation and hydrogel formation: i) Hydrophilic acrylate based copolymers containing peptide binding units and ii) hydrophilic polyether based copolymers containing different functional groups for a physical crosslinking.
In section 3.1 the successful synthesis of water soluble and linear acrylate based polymers containing oligo(ethylene glycol) methyl ether acrylate with either linear thioester functional 2-hydroxyethyl acrylate, thiolactone acrylamide, or vinyl azlactone via the living radical polymerisation technique Reversible Addition Fragmentation Chain Transfer (RAFT) and via free-radical polymerisation is described. The obtained polymers were characterized via GPC, 1H NMR, IR and RAMAN spectroscopy.
The RAFT end group was found to be difficult to remove from these short polymer chains and accordingly underwent the undesired side reaction aminolysis with the peptide during the conjugation studies. Besides that, polymers without RAFT end groups did not show any binding of the peptide at the thioester groups, which can be improved in future by using higher reactant concentrations and higher amount of binding units at the polymer. Polymers containing the highly reactive azlactone group showed a peptide binding of 19 %, but unfortunately this function also underwent spontaneous hydrolysis before the peptide could even be bound. In all cases, oligo(ethylene glycol) methyl ether acrylate was used with a relatively high molecular weight (Mn = 480 Da) was used, which eventually was efficiently shielding the introduced binding units from the added peptide. In future, a shorter monomer with Mn = 300 Da or less or hydrophilic N,N’-dialkyl acrylamide based polymers with less steric hindrance could be used to improve this bioconjugation system. Additionally, the amount of monomers containing peptide binding units in the polymer can be increased and have an additional spacer to achieve higher loading efficiency.
The water soluble, linear and short polyether based polymers, so called polyglycidols, were successfully synthesized and modified as described in section 3.2. The obtained polymers were characterized using GPC, 1H NMR, 31P{1H} NMR, IR, and RAMAN spectroscopy. The allyl groups which were present up to 20 % were used for radical induced thiol-ene chemistry for the introduction of functional groups intended for the formation of the physically crosslinking hydrogels. For the positively charged polymers, first a chloride group had to be introduced for the subsequent nucleophilic substitution with the imidazolium compound. There, degrees of modifications were found in the range 40-97 % due to the repulsion forces of the charges, decreased concentration of active chloride groups, and limiting solution concentrations of the polymer for this reaction. For the negatively charged polymers, first a protected phosphonamide moiety was introduced with a deprotection step afterwards showing 100 % conversion for all reactions. Preliminary hydrogel tests did not show a formation of a three-dimensional network of the polymer chains which was attributed to the short backbone length of the used polymers, but the gained knowledge about the synthetic routes for the modification of the polymer was successfully transferred to longer linear polyglycidols. The same applies to the introduction of electron rich and electron poor compounds showing π-π stacking interactions by UV-vis spectroscopy.
Finally, long linear polyglycidyl ethers were synthesised successfully up to molecular weights of Mn ~ 30 kDa in section 3.3, which was also proven by GPC, 1H NMR, IR and RAMAN spectroscopy. This applies to the homopolymerisation of ethoxyethyl glycidyl ether, allyl glycidyl ether and their copolymerisation with an amount of the allyl compound ~ 10 %. Attempts for higher molecular weights up to 100 kDa showed an uncontrolled polymerisation behaviour and eventually can be improved in future by choosing a lower initiation temperature. Also, the allyl side groups were modified via radical induced thiol-ene chemistry to obtain positively charged functionalities via imidazolium moieties (85 %) and negatively charged functionalities via phosphonamide moieties (100 %) with quantitative degree of modifications. Hydrogel tests have still shown a remaining solution by using long linear polyglycidols carrying negative charges with long/short linear polyglycidols carrying positive charges. The addition of calcium chloride led to a precipitate of the polymer instead of a three-dimensional network formation representing a too high concentration of ions and therefore shielding water molecules with prevention from dissolving the polymer. These systems can be improved by tuning the polymers structure like longer polymer chains, longer spacer between polymer backbone and charge, and higher amount of functional groups.
The objective of the thesis was partly reached containing detailed investigated synthetic routes for the design and characterisation of functional polymers which could be used in future with improvements for bioconjugation and hydrogel formation tests.
A series of monomeric chirally substituted indolenine squaraine monomers were successfully synthesized and utilized for the construction of various oligo- and polymers, in order to study their chiroptical properties in terms of exciton chirality. The quaternary carbon atom at the 3-position of the indolenine subunit, as well as the alkyl side chain attached to the indolenine nitrogen were selected as the most suitable site for chiral functionalization.
For the C(3)-chiral derivatives, two synthetic routes depending on the desired substitution at the stereogenic center were established. The chiral side chains were prepared via Evans asymmetric alkylation where the resulting branching point at the 2 position constituted the chiral center. While the chiral substitution only had minor effects on the linear optical properties and geometric structure of the chromophore, all compounds exhibited a distinct and measurable CD signal that correlated with the distance of the chiral center to the central chromophore.
Polymers bearing chiral side chains exhibited a solvent- and temperature-dependent helix-coil equilibrium, which was influenced by the type of side chain used. CD spectroscopy revealed the helical conformation to possess a preferred twist sense, and temperature-dependent measurements showed the degree of homohelicity to be nearly complete in certain cases. Furthermore, a CPL signal was able to be obtained for the helical conformer of one polymer.
Various (co)oligo- and polymers comprising the C(3)-chiral monomers only displayed a solvent-independent J-type absorption behavior and thus did not form helical conformations in solution. CD spectroscopy revealed a solvent-dependent adoption of quasi-enantiomeric conformers, which was elucidated by quantum chemical TDDFT calculations.
In terms of the need of environmentally benign renewable and storable energy sources, splitting of water into hydrogen and oxygen by using sunlight is a promising approach. Hereby, water oxidation catalysts (WOCs) are required to perform the water oxidation comprising the transfer of four electrons to provide the reducing equivalents for producing hydrogen. The class of Ru(bda) (bda = 2,2'-bipyridine-6,6'-dicarboxylate) catalysts has proven to be efficient for this reaction.
In this thesis, ligand exchange processes in Ru(bda) complexes have been analyzed and the formation of multinuclear macrocyclic WOCs was studied. Based on the knowledge acquired by these studies, new multinuclear cyclic Ru(bda) complexes have been synthesized and their catalytic efficiencies in homogeneous water oxidation have been investigated. Going one step further for setting up functional devices, molecular WOCs have been immobilized on conducting or semiconducting supporting materials. Direct anchoring on carbon nanotubes generated a promising materials for further applications.
Nanodiamond (ND) is a versatile and promising material for bio-applications. Despite many efforts, agglomeration of nanodiamond and the non-specific adsorption of proteins on the ND surface when exposed to bio-fluids remains a major obstacle for biomedical applications. An assortment of branched and linear molecules with superior ability to colloidally stabilize nanoparticles in salt and cell media environment, for up to 30 days, was attached to the ND’s surface.
The building box system with azide as external groups offers a huge variety of binding with many molecules, such as drugs, dyes or targeting molecules, is possible. Clicking, for instance, zwitterions moieties to the chain protects ND surface from protein corona forming when the particles get in contact with biofluids containing proteins.
Thermogravimetric analysis results of the ND surface loading show a significant prevention of up to 98 % of the protein adsorption compared with NDs without zwitterionic headgroups and long colloidal stability when tetraethylene glycol (TEG) are attached to the surface.
The versatility of the modular system to bind not only zwitterionic chains but also clickable functional molecules to fluorescent nanodiamonds (fNDs) demonstrates the potential of the system at the nanodiamond. Using defect structures, such as nitrogen-vacancy (NV) centers, diamond particles, due to their widely non-toxic behavior, can be used as fNDs for photostable labeling, bioimaging and nanoscale sensing in living cells and organisms. To functionalize the fND surface a novel milling technique with diazonium salts was established to perform grafting on poorly reactive HPHT fNDs yielding in high surface loading and high negative zeta potential.
Combining the benefits of TEG and zwitterion containing groups with antibody enabled nucleus targeting ability on fND confirms the enhanced colloidal stability in living cells experiments for the first time. Furthermore, the results indicate an improved corona repulsion compared with fND without zwitterion containing headgroups. As a result, the circulation times were enlarged from 4 (fND without zwitterion chain but with antibody) to 17 (with antibody and zwitterion chains) hours.
In non-biomedical applications, the modular system can be used as a probe for heavy metals by binding it to dyes. Detection of metals in different environments with high selectivity and specificity is one of the prerequisites of the fight against environmental pollution with these elements. Pyrenes are well suited and known for fluorescence sensing in different media.
The applied sensing principle typically relies on the formation of intra- and intermolecular excimers, which is however limiting the sensitivity range due to masking of e.g. quenching effects by the excimer emission. This study shows a highly selective, structurally rigid chemical sensor based on the monomer fluorescence of pyrene moieties bearing triazole groups.
This probe can quantitatively detect Cu2+, Pb2+ and Hg2+ in organic solvents over a broad concentration range, even in the presence of ubiquitous ions such as Na+, K+, Ca2+ and Mg2+. The strongly emissive sensor’s fluorescence with a long lifetime of 165 ns is quenched by a 1:1 complex formation upon addition of metal ions in acetonitrile. Upon addition of a tenfold excess of the metal ion to the sensor, agglomerates with a diameter of about 3 nm are formed. Due to complex interactions in the system, conventional linear correlations are not observed for all concentrations. Therefore, a critical comparison between the conventional Job plot interpretation, the method of Benesi-Hildebrand, and a non-linear fit is presented. The reported system enables the specific and robust sensing of medically and environmentally relevant ions in the health-relevant nM range and could be used e.g. for the monitoring of the respective ions in waste streams.
Nonetheless, often these waste streams end up in sensitive aquacultures, where such sensor technology only works if the probe is water-soluble to monitor the spread and formation of environmental damage from heavy metals. Many chemosensors only work quantitatively in specific solvents and under highly pure conditions. In this thesis a method to stabilize water-insoluble chemosensors on nanodiamonds in saline water while maintaining the sensor efficacy and specificityas as well as colloidal stability is presented. Additionally, the sensor capability is retained in organic solvents. This study provides insight into the absorptivity of pyrene derivatives to the nanodiamond surface and a way to reversibly desorb them.
Moreover, the system proves that in presence of 95 % oxygen atmosphere while the fluoresce measurement the results of the do not vary from the one in argon atmosphere. Furthermore, the presence of common ions in water do not disturb the colloidal stability of the NDs and also no influence the sensor functionality and thus is highly promising candidate for measurement without cumbersome preparation steps.
RNA molecules play diverse roles in biological systems. Post-transcriptional RNA modifications and dynamic structures enhance the functional diversity of RNA. A prerequisite for studying their biological significance is the availability of reliable methods for the detection of RNA modifications and structures. Several promising approaches have been developed in the last few decades; however, efficient, and versatile tools are still required to study the dynamic features of RNA. This thesis focuses on the development of nucleic acid catalysts as a tool to address the current needs in studying RNA. The major part of this thesis aimed at the development of deoxyribozymes as a tool for the detection of RNA modifications. Using in vitro selection from a random DNA library, we found deoxyribozymes that are sensitive to N 6 -isopentenyladenosine (i6A), a native tRNA modification and structural analogue of m6A. The in vitro evolution identified three classes of DNA enzymes: AA, AB08, and AC17 DNAzymes that showed distinct response to i6A modification and showed strong discrimination between structural analogues, i.e., m6A and i6A. In the continuation of the project, we attempted to develop RNA-cleaving deoxyribozymes that differentially respond to monomethylated cytidine isomers, 3-methylcytidine (m3C), N4 - methylcytidine (m4C), and 5-methylcytidine (m5C). Several deoxyribozymes were identified from in vitro selection, which are selective for a specific methylated cytidine isomer. The characterization of AL112, AM101, AN05, and AK104 catalysts confirmed the successful evolution of modification-specific and general deoxyribozymes that showed a broad substrate scope. In order to accelerate the DNAzymes discovery, a high throughput sequencing method (DZ-seq) was established that directly quantifies the RNA cleavage activity and cleavage site from deep sequencing data. The libraries contained information about cleavage status, cleavage site and sequence of deoxyribozymes and RNA substrate. The fraction cleaved (FC) data obtained from Dz-seq was validated for a subset of deoxyribozmes using conventional gel based kinetic assay and showed a good linear correlation (R2 = 0.91). Dz-seq possesses a great potential for the discovery of novel deoxyribozymes for the analysis of various RNA modifications in the future. The second objective of the current study was the development of structure-specific RNA labeling ribozymes. Here, we attempted to develop ribozymes that targets RNA of interest by structure-specific interaction rather than base-pairing and focused on a specific RNA G-quadruplex as the target. Two subsequent selection experiments led to the identification of the adenylyltransferase ribozymes AO10.2 and AR9. The partial characterization of these catalysts showed that A010.2 was unable to recognize intact BCL2 structure, but it turned out as the first reported trans-active ribozyme that efficiently labeled uridine in a defined substrate RNA hybridized to the ribozyme. The other ribozyme AR9 was shown to serve as a trans-active, self-labeling ribozyme that catalyzed adenylyl transferase reaction in the presence of the intact BCL2 sequence. Based on these preliminary findings, we envision that AR9 could potentially serve as a reporter RNA by self-labeling in the presence of an RNA G-quadruplex. However, both AO10.2 and AR9 still require more detailed characterization for their potential applications.
Paclitaxel (PTX) is one of the leading drugs against breast and ovarian cancer. Due to its low solubility, treatment of the patients with this drug requires a very well-suited combination with a soluble pharmaceutical excipient to increase the bioavailability and reduce the strong side ef-fects. One efficient way to achieve this in the future could be the incorporation of PTX into pol-ymeric micelles composed of poly(2-oxazoline) based triblock copolymers (POL) which ena-bles PTX loadings of up to 50 wt.%. However, structural information at an atomic level and thus the knowledge of interaction sites within these promising but complex PTX-POL formula-tions were not yet available. Such results could support the future development of improved excipients for PTX and suitable excipients for other pharmaceutical drugs. Therefore, a solid-state MAS NMR investigation of these amorphous formulations with different POL-PTX com-positions was performed in this thesis as this gives insights of the local structure at an atomic level in its solid state. NMR in solution showed very broad 13C signals of PTX for this system due to the reduced mobility of the incorporated drug which exclude this as an analytical meth-od.
In a first study, crystalline PTX was structurally characterized by solid-state NMR as no com-plete 13C spectrum assignment and no 1H NMR data existed for the solid state. In addition, the asymmetric unit of the PTX crystal structure consists of two molecules (Z'=2) that can only be investigated in its solid state. As crystalline PTX in total has about 100 different 13C and 1H chemical shifts with very small differences due to Z’=2, and furthermore, its unit cell consisting of more than 900 atoms, accompanying GIPAW (CASTEP) calculations were required for NMR signal assignments. These calculations were performed using the first three available purely hydrous and anhydrous PTX structures, which were determined by XRD and published by Vel-la-Zarb et al. in 2013. Within this thesis, is was discovered that two investigated batches of commercially available PTX from the same supplier both contained an identical and so far un-known PTX phase that was elucidated by PXRD as well as solid-state NMR data. One of the two batches consists of an additional phase that was shown to be very similar to a known hy-drated phase published in 2013.[1] By heating the batch with the mixture of the two phases un-der vacuum, it is transformed completely to the new dry phase occurring in both PTX batches. Since the drying conditions to obtain anhydrous PTX in-situ on the PXRD setup described by Vella-Zarb et. al.[1] were much softer than ours, we identify our dry phase as a relaxed version of their published anhydrate structure. The PXRD data of the new anhydrate phase was trans-ferred into a new structural model, which currently undergoes geometry optimization. Based on solid-state NMR data at MAS spinning frequencies up to 100 kHz, a 13C and a partial 1H signal assignment for the new anhydrous structure were achieved. These results provided sufficient structural information for further investigations of the micellar POL-PTX system.
In a second study, the applicability and benefit of two-dimensional solid-state 14N-1H HMQC MAS NMR spectra for the characterization of amorphous POL-PTX formulations was investi-gated. The mentioned technique has never been applied to a system of similar complexity be-fore and was chosen because around 84% of the small-molecule drugs contain at least one nitrogen atom. In addition, the number of nitrogen atoms in both POL and PTX is much smaller than the number of carbons or hydrogens, which significantly reduces the spectral complexity. 14N has a natural abundance of 99.6% but leads to quadrupolar broadening due to its nuclear spin quantum number I = 1. While this is usually undesirable due to broadening in the resulting 1D 14N NMR spectra, this effect is explicitly used in the 2D 14N-1H HMQC MAS experiment. The indirect 14N measurement can avoid the broadening while maintaining the advantage of the high natural abundance and making use of the much more dispersed signals due to the additional quadrupolar shifts as compared to 15N.
This measurement method could be successfully applied to the complex amorphous POL-PTX mixtures. With increasing PTX loading of the formulations, additional peaks arise as spatial proximities of the amide nitrogens of POL to NH or OH groups of PTX. In addition, the 14N quadrupolar shift of these amide nitrogens decreases with increasing PTX content indicating a more symmetric nitrogen environment. The latter can be explained by a transformation of the trigonal planar coordination of the tertiary amide nitrogen atoms in pure POL towards a more tetrahedral environment upon PTX loading induced by the formation of hydrogen bonds with NH/OH groups of PTX.
In the third and last project, the results of the two abovementioned studies were used and ex-tended by solid state 13C and two-dimensional 1H-13C as well as 1H-1H MAS NMR data with the aim to derive a structural model of the POL-PTX formulations at an atomic level. The knowledge of the NMR signal assignments for crystalline PTX was transferred to amorphous PTX (present in the micelles of the formulations). The 13C solid-state NMR signals were evalu-ated concerning changes in chemical shifts and full widths of half maximum (FWHM) for the different PTX loadings. In this way, the required information about possible interaction sites at an atomic level becomes available. Due to the complexity of these systems, such proximities often cannot be assigned to special atoms, but more to groups of atoms, as the individual de-velopments of line widths and line shifts are mutually dependent. An advantageous aspect for this analysis was that pure POL already forms unloaded micelles. The evaluation of the data showed that the terminal phenyl groups of PTX seem to be most involved in the interaction by the establishment of the micelle for lowest drug loading and that they are likely to react to the change in the amount of PTX molecules as well. For the incorporation of PTX in the micelles, the following model could be obtained: For lowest drug loading, PTX is mainly located in the inner part of the micelles. Upon further increasing of the loading, it progressively extends to-ward the micellar shell. This could be well shown by the increasing interactions of the hydro-phobic butyl chain of POL and PTX, proceeding in the direction of the polymer backbone with rising drug load. Furthermore, due to the size of PTX and the hydrodynamic radius of the mi-celles, even at the lowest loading, the PTX molecules partially reach the core-shell interface of the micelle. Upon increasing the drug loading, the surface coverage with PTX clusters increas-es based on the obtained model approach. The latter result is supported by DLS and SANS data of this system. The abovementioned results of the 14N-1H HMQC MAS investigation of the POL-PTX formulations support the outlined model.
As an outlook, the currently running geometry optimization and subsequently scheduled calcu-lation of the chemical shieldings of the newly obtained anhydrous PTX crystal structure can further improve the solid-state NMR characterization through determination of further spatial proximities among protons using the existing 2D 1H(DQ)-1H(SQ) solid-state MAS NMR spec-trum at 100 kHz rotor spinning frequency. The 2D 14N-1H HMQC MAS NMR experiments were shown to have great potential as a technique for the analysis of other disordered and amor-phous drug delivery systems as well. The results of this thesis should be subsequently applied to other micellar systems with varying pharmaceutical excipients or active ingredients with the goal of systematically achieving higher drug loadings (e.g., for the investigated PTX, the similar drug docetaxel or even different natural products). Additionally, it is planned to transfer the knowledge to another complex polymer system containing poly(amino acids) which offers hy-drogen bonding donor sites for additional intermolecular interactions. Currently, the POL-PTX system is investigated by further SANS studies that may provide another puzzle piece to the model as complementary measurement method in the future. In addition, the use of MD simu-lations might be considered in the future. This would allow a computerized linking of the differ-ent pieces of information with the aim to determine the most likely model.
Nucleic acids are not only one of the most important classes of macromolecules in biochemistry but also a promising platform for the defined arrangement of chromophores. Thanks to their precise organization by directional polar and hydrophobic interactions, oligonucleotides can be exploited as suitable templates for multichromophore assemblies with predictable properties. To expand the toolbox of emissive, base pairing nucleobase analogs several barbituric acid merocyanine (BAM) chromophores with tunable spectroscopic properties were synthesized and incorporated into RNA, DNA and glycol nucleic acid (GNA) oligonucleotides. A multitude of duplexes containing up to ten BAM chromophores was obtained and analysis by spectroscopic methods revealed the presence of dipolarly coupled merocyanine aggregates with properties
strongly dependent on the chromophore orientation toward each other and the backbone conformation. These characteristics were exploited for various applications such as FRET pair formation and polymerase chain reaction (PCR) experiments. The observed formation of higher-order aggregates implies future applications of these new oligonucleotide-chromophore systems as light-harvesting DNA nanomaterials. Besides oligonucleotide templated covalent assembly of chromophores also non-covalent nucleic acid-chromophore complexes are a broad field of research. Among these, fluorogenic RNA aptamers are of special interest with the most versatile ones based on derivatives of the GFP chromophore hydroxybenzylidene imidazolone (HBI). Therefore, new HBI-derived chromophores with an expanded conjugated system and an additional exocyclic amino group for an enhanced binding affinity were synthesized and analyzed in complex with the Chili aptamer. Among these, structurally new fluorogenes with strong fluorescence activation upon binding to Chili were identified which are promising for further derivatization and application as color-switching sensor devices for example.
A series of donor-acceptor macrocyclic architectures comprising oligothiophene strands that connect the imide positions of a perylene bisimide have been synthesized via a platinum-mediated cross-coupling strategy. The target structures were characterized by steady-state UV/Vis absorption, fluorescence and transient absorption spectroscopy, as well as cyclic and differential pulse voltammetry. Crystal structure analysis of the macrocycles revealed insights into the bridge arrangements. The properties of the macrocyclic bridges were compared to linear oligothiophene reference compounds which itself exhibited an unusual electrochemical effect.