Adam, Volker: Synthetic evolution of azobenzene and arylazopyrazole photoswitches and translation into photoresponsive DNA nanosystems. - Bonn, 2019. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
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author = {{Volker Adam}},
title = {Synthetic evolution of azobenzene and arylazopyrazole photoswitches and translation into photoresponsive DNA nanosystems},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2019,
month = oct,

note = {Light as an external stimulus provides a powerful and versatile tool to control chemical reactions and biological systems. Light is easily available and permits a high spatial and temporal control while minimizing systemic contamination as often associated with invasive methods. Efficient photocontrol of a system of interest can be achieved by introduction of photochromic molecules which can undergo reversible interconversion from one conformation into another upon exposure to distinct wavelengths of light. Azobenzene thereby represents one of the best studied and most commonly used photochromic molecules in this context. Azobenzene molecules can be reversibly transformed from a planar trans conformation into a non-planar, sterically more challenging cis conformation by exposure to UV or visible light. Introduction of azobenzene into complex systems led to a series of applications ranging from light-controlled smart materials and nanodevices to light control over biological processes and cellular functions in vitro.
Introduction of azobenzene into desoxyribonucleic acid (DNA) afforded reversible photoinduced hybridization and dissociation of DNA double strands by means of UV or visible light irradiation. Coupling azobenzene to D-threoninol as a nucleobase surrogate led to the successful implementation into DNA backbones, thus paving the way for a series of photocontrolled applications. Photoresponsive DNA nanotechnology brought up structure-changing DNA nanoconstructs and devices as well as functional dynamic DNA nanotechnology, the in vitro control of biological and cellular processes up to the emerging field of photopharmacology. Despite the successful establishment of photocontrol in vitro, a translation towards in vivo systems in hampered by the mandatory use of UV light for cis isomerization, disrupting DNA and cellular tissue upon UV exposure. Furthermore, efficient azobenzene cis isomerization requires elevated irradiation temperatures and a high synthetic load inside DNA duplexes to achieve efficient duplex dissociation. Many efforts have been performed to circumvent these prerequisites by substituting D-threoninol for other diol linkers or adding substituents and functional groups to azobenzene moieties in different patterns to optimize photophysical properties. Yet, only few of these drawbacks could be overcome by the performed optimization studies.
Recent advances brought up a series of modified azobenzene molecules containing hetero atoms and functional moieties in all four ortho positions on both benzene rings. This substi-tution pattern results in a significant red-shift of isomerization wavelengths accompanied by an increased thermal stability of the cis state. Also replacement of benzene rings by heterocycles as in arylazopyrazoles led to near-quantitative conversion efficiencies accompanied by high thermal stabilities of the cis state up to three years. The recently reported molecules provide tools for a possible improvement of photocontrolled DNA duplex hybridization, bearing potential for improved response wavelengths, thermal stabilities of the cis state and conversion efficiencies between both isomers. Studies towards translation into DNA strands and DNA-based applications have not been reported to date.
The following dissertation catches up the potential improvement of photocontrolled DNA duplex hybridization. Based on the reported azobenzene and arylazopyrazole small molecules, a systematic feasibility study of azobenzene and arylazopyrazole photoswitches attached to D-threoninol was performed to optimize the photophysical properties and translate these into modified oligonucleotide strands. A pre-selection of synthetically accessible D-threoninol bound small molecule candidates was investigated empirically by UV/Vis spectroscopy in combination with an irradiation device providing high output LEDs to determine maximum response wavelengths. Reversed-phase HPLC and UV/Vis spectroscopy were utilized to quantify the maximum switching efficiencies and thermal stabilities of the cis states. Consecutive DMT-protection and phosphitylation of the D-threoninol bound photochromes enabled the implementation of selected photoswitches into DNA strands via solid phase DNA synthesis. A translation of optimized photophysical properties into DNA strands was investigated via reversed-phase HPLC quantification and UV/Vis spectroscopy involving systematically modified DNA strands and duplexes in direct comparison to the newly designed small molecules. Melting curve analysis of modified duplexes revealed improved duplex dissociation efficiencies even with a lower synthetic load as compared to the reported azobenzenes.
Translation of improved photophysical properties was further demonstrated by introduction of arylazopyrazoles into higher order DNA systems. A double-stranded DNA catenane was designed and assembled to further investigate coherent and independent switching properties using a combination of release oligomers each containing different types of photochromes. Not only the catenane system was designed to determine coherent photophysical properties, but also the catenane building blocks were used to qualitatively demonstrate individual switching properties.
Besides the development of new photochromes, further nanotechnological applications have been developed. Lipid-mediated self-assembling micellar nano-constructs were designed as carrier moieties for targeted transport of doxorubicin into cancer cells. The system consists of an aptamer-based cell receptor targeting unit combined with a photo-triggered doxorubicin release mechanism. Successful cell internalization and photo-triggered release of doxorubicin inside the cells was applied in vitro in H1838 non-small cell lung cancer cells.},

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