Judicious addition of photolabile caging groups (PCGs) to protect biologically im- portant molecules, has enabled the development of many powerful chemical tools for the study of biological processes. These tools have the potential to be activated in a cellular setting by irradiation with light of appropriate wavelengths, restoring functionality, with excellent spatial and temporal control. This D. Phil. dissertation highlights two biologically relevant examples where PCGs can be applied: (i) 4,5-dimethoxynitrobenzyl (DMNB) caged derivatives of GSH and ESG, were synthesised and uncaging of the DMNB group at 350 nm to reveal the free α-carboxylic acid of the glycine residue was demonstrated. These molecules have the potential to probe the binding mode within the KefC KTN binding domain, a ligand-gated K+ efflux system, critical for bacterial response to electrophilic assault. The molecules are currently with collaborators awaiting further evaluation. (ii) The wavelength-dependent application of PCGs toward the study of protein post- translational modifications (PTMs) was developed. PTMs modulate protein function and have a ubiquitous role in a diverse range of cellular functions. A wavelength selective sequential pair of PCGs was developed and demonstrated in a tripeptide, using the diethylamino coumarin (DEACM), which was cleaved at 420 nm, and the DMNB caging group, which was subsequently cleaved at 350 nm. A chromatically orthogonal pair of PCGs, with possible applications in both organic synthesis and biological systems, incorporating the BODIPY-based and DEACM caging groups, which could be cleaved at 530 nm and 420 nm, respectively, was also developed. Photolysis was conducted on a tripeptide and hexapeptide and an in vitro application was demonstrated where when caged, the peptide was not susceptible to peptidolysis when incubated with enzymes. Following irradiation at 420 nm, the DEACM group was uncaged and the peptide underwent peptidolysis in the presence of Endoproteinase AspN, whereas following irradiation at 530 nm to uncage the BODIPY group, the peptide underwent peptidolysis in the presence of Endoproteinase LysC. These results not only provide conclusive evidence of the ability of this pair of caging groups to act in a wavelength orthogonal sense but also demonstrates the potential applicability of the pair to biochemical studies. This pair of PCGs represents the first example of an orthogonal pair of caging groups where both PCGs could be cleaved, at wavelengths longer than 400 nm, in a sequence independent manner.

<p>The ability to manipulate the coordination chemistry of metal ions has significant ramifications for the study and treatment of metal-related health concerns, including iron overload, UV skin damage, and microbial infection among many other conditions. To address this concern, chelating agents that change their metal binding characteristics in response to external stimuli have been synthesized and characterized by several spectroscopic and chromatographic analytical methods. The primary stimuli of interest for this work are light and hydrogen peroxide.</p><p>Herein we report the previously unrecognized photochemistry of aroylhydrazone metal chelator ((E)-N&#8242;-[1-(2-hydroxyphenyl)ethyliden]isonicotinoylhydrazide) (HAPI) and its relation to HAPI metal binding properties. Based on promising initial results, a series of HAPI analogues was prepared to probe the structure-function relationships of aroylhydrazone photochemistry. These efforts elucidate the tunable nature of several aroylhydrazone photoswitching properties.</p><p>Ongoing efforts in this laboratory seek to develop compounds called prochelators that exhibit a switch from low to high metal binding affinity upon activation by a stimulus of interest. In this context, we present new strategies to install multiple desired functions into a single structure. The prochelator 2-((E)-1-(2-isonicotinoylhydrazono)ethyl)phenyl (E)-3-(2,4-dihydroxyphenyl)acrylate (PC-HAPI) is masked with a photolabile trans-cinnamic acid protecting group that releases umbelliferone, a UV-absorbing, antioxidant coumarin along with a chelating agent upon UV irradiation. In addition to the antioxidant effects of the coumarin, the released chelator (HAPI) inhibits metal-catalyzed production of damaging reactive oxygen species. Finally a peroxide-sensitive prochelator quinolin-8-yl (Z)-3-(4-hydroxy-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)acrylate (BCQ) has been prepared using a novel synthetic route for functionalized cis-cinnamate esters. BCQ uses a novel masking strategy to trigger a 90-fold increase in fluorescence emission, along with the release of a desired chelator, in the presence of hydrogen peroxide.</p> / Dissertation

<p>The concept of light-activated "caged" metal ions was first introduced for Ca2+. These high affinity coordination complexes are activated by UV light to release calcium ions intracellularly and have found widespread use in understanding the many roles of calcium in biological processes. There is an unmet need for photocaging ligands for biologically relevant transition metal ions. Described here are the first examples of uncaging biologically important d-block metal ions using photoactive ligands. </p> <p>New nitrogen-donor ligands that contain a photoactive nitrophenyl group within the backbone have been prepared and evaluated for their metal binding affinity. Exposure of buffered aqueous solutions of apo-cage or metal-bound cage to UV light induces cleavage of the ligand backbone reducing the denticity of the ligands. Characterization of several caging compounds reveals that quantum efficiency and metal binding affinity can be tuned by modifications to the parent structure. The change in reactivity of caged vs. uncaged metal for promoting hydroxyl radical formation was demonstrated using the in vitro deoxyribose assay. The function of several of these compounds in vivo pre- and post-photolysis has been validated using MCF-7 cells. This strategy of caging transition metals ions is promising for applications where light can trigger the release of metal ions intracellularly to study metal trafficking and distribution, as well as, selectively impose oxidative stress and/or metal toxicity on malignant cells causing their demise.</p> / Dissertation

This work refers to the area of bio-nanotechnology and concerns the selective immobilization of DNA or other bio-template on microstructured gold contacts and which then permit a coordinated cooperation of several of these nanotemplate, e.g., within a microreactor. The immobilization of such nano-objects should be realized through functional thin polymer films which provide binding groups. Thus, the main aim of this work was the development of polymeric materials for thin functional films which permit to deposit on different substrates a wide variation of functional elements or metal structures and to achieve a pattern formation using optical grid methods. In order to realize this concept it was necessary to design and develop a polymer system based on suitable photolabile units and in addition having anchoring groups which attach on specific substrates like gold. In this terpolymer concept was aimed for which consists of three components with particular functions in suitable molar ratios, which allow the tune the properties of the materials, and provide: amino photolabile protected groups for the photolithographic creation of patterned areas with free amino groups, which are available for further modifications like attachment of colloids, metallization or attachment of DNA strands; disulfide derivative anchor groups providing anchoring capacity for gold surface and spacer groups for adjusting the film quality. These multifunctional terpolymers should be synthesised by free radical polymerisation of suitable monomers. Although these techniques are successful, they are limited by their complexity, rigorous synthetic demands, as well as incompatibility with many functional termolabile and highly reactive functionalities. To overcome these difficulties a polymerisation technique based on “living” free radical polymerisation has been used in this work. A highly efficient polymer-analogous modification allows to introduce the functionalities after the polymer construction reaction. The production of suitable prepolymers [poly(styrene-r-4-propargyl-oxystyrene)] was carried out with the help of a controlled synthesis methodology “nitroxide mediate radical polymerization&amp;quot; followed by polymer analogous reaction using one of the most efficient click reactions, the Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition between terminal acetylenes and azides to attach further functionalities through the formation of a stable 1,4-disubstituted 1,2,3-triazol ring . The combination of nitroxide mediated radical polymerization (NMRP) and click chemistry was used to produce well-defined random copolymer. It could already be shown that also block copolymers can be prepared which give the chance to combine nanostructure formation in block copolymers with special functionality. Thus, the special properties of these functional polymers like the capability for photopatterning and anchoring onto gold substrates make them very interesting for nanotechnology applications. / Diese Arbeit bezieht sich auf das Gebiet der Bionanotechnologie und betrifft ein neuartiges Verfahren zur selektiven Immobilisierung der DNA oder anderer Biomoleküle auf mikrostrukturierten Goldkontakten, welche dann ein koordiniertes Zusammenwirken von einzelnen Nanomolekülen ermöglichen, z.B. in einem Mikroreaktor. Die Immobilisierung solcher Nanoobjekte soll durch dünne Funktionsschichten realisiert werden, die die Anbindungsgruppen liefern. Folglich war das Hauptziel dieser Arbeit die Entwicklung von Polymermaterialien für dünne Funktionsschichten, die die Aufbringung einer großen Vielzahl von Funktionselementen oder metallischen Strukturen auf verschiedenen Substraten gestatten und die Strukturierung durch den Einsatz von lithographischen Methoden ermöglichen. Um dieses Konzept zu realisieren, war es notwendig, ein Polymersystem zu gestalten und zu entwickeln, welches auf geeignete photolabile Einheiten basiert und zusätzlich Ankergruppen hat, die mit spezifischen Substraten wie Gold verbunden ist. Dieses Terpolymerkonzept wurde gezielt aus drei Komponenten mit speziellen Funktionen in entsprechenden molaren Verhältnissen gebildet, die eine Abstimmung der Materialeigenschaften ermöglicht und folgendes bereitstellt: photolabile geschützte Aminogruppen für die photolitographische Strukturerzeugung mit freien Aminogruppen, welche für weitere Modifikationen verfügbar sind wie das Anhängen von Kolloiden, die Metallisierung oder Anfügung von DNA-Strängen; disulfide Derivate für die kovalente Anbindung auf der Goldoberfläche und Spacer-Gruppe für Verbesserung der Schichtenbildung. Diese multifunktionalen Terpolymere sollen durch eine freie radikalische Polymerisation von entsprechenden Monomeren synthetisiert werden. Obwohl diese Techniken erfolgreich sind, sind sie eingeschränkt durch ihre Komplexität, den strengen synthetischen Anforderungen, sowie der Inkompatibilität mit vielen funktionalen thermolabilen und hochreaktiven Funktionalitäten. Um diese Schwierigkeiten zu überwinden wurde eine Polymerisationstechnik für diese Arbeit genutzt, die auf der „lebenden“ freien radikalischen Polymerisation basiert. Eine hoch effiziente polymeranaloge Modifizierung erlaubt die Einführung von Funktionalitäten nach der Polymeraufbaureaktion. Die Herstellung von entsprechenden Präpolymeren Poly(Styrol-r-4-Propargyl-oxystyrol) wurde mittels einer kontrollierten Synthesemethodik „Nitroxid-mediated controled radical polymerisation“ (NMRP) durchgeführt, gefolgt von der Polymeranalogreaktion, die eine der effizientesten Click-Reaktion - die Cu(I) katalysierte 1,3-dipolar Cycloaddition von terminalen Alkinen an Aziden nach Huisgen nutzt, um weiter Funktionalitäten durch die Bildung eines stabilen 1,4-disubstituierten-[1,2,3]-Triazolringes anzufügen. Die Kombination von NMRP und Click-Chemie wurde zur Herstellung eines exakt definierten Random Copolymers genutzt. Es konnte bereits gezeigt werden, dass auch Blockcopolymere geschaffen werden können, die eine Möglichkeit zur Kombination von Nanostrukturformationen in Blockcopolymeren mit speziellen Funktionaltäten bieten. Folglich sind die speziellen Eigenschaften dieser Funktionalpolymere wie die Fähigkeit zur Photostrukturierung und Verankerung auf Goldsubstraten für nanotechnologische Anwendungen sehr interessant.

This work refers to the area of bio-nanotechnology and concerns the selective immobilization of DNA or other bio-template on microstructured gold contacts and which then permit a coordinated cooperation of several of these nanotemplate, e.g., within a microreactor. The immobilization of such nano-objects should be realized through functional thin polymer films which provide binding groups. Thus, the main aim of this work was the development of polymeric materials for thin functional films which permit to deposit on different substrates a wide variation of functional elements or metal structures and to achieve a pattern formation using optical grid methods. In order to realize this concept it was necessary to design and develop a polymer system based on suitable photolabile units and in addition having anchoring groups which attach on specific substrates like gold. In this terpolymer concept was aimed for which consists of three components with particular functions in suitable molar ratios, which allow the tune the properties of the materials, and provide: amino photolabile protected groups for the photolithographic creation of patterned areas with free amino groups, which are available for further modifications like attachment of colloids, metallization or attachment of DNA strands; disulfide derivative anchor groups providing anchoring capacity for gold surface and spacer groups for adjusting the film quality. These multifunctional terpolymers should be synthesised by free radical polymerisation of suitable monomers. Although these techniques are successful, they are limited by their complexity, rigorous synthetic demands, as well as incompatibility with many functional termolabile and highly reactive functionalities. To overcome these difficulties a polymerisation technique based on “living” free radical polymerisation has been used in this work. A highly efficient polymer-analogous modification allows to introduce the functionalities after the polymer construction reaction. The production of suitable prepolymers [poly(styrene-r-4-propargyl-oxystyrene)] was carried out with the help of a controlled synthesis methodology “nitroxide mediate radical polymerization&amp;quot; followed by polymer analogous reaction using one of the most efficient click reactions, the Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition between terminal acetylenes and azides to attach further functionalities through the formation of a stable 1,4-disubstituted 1,2,3-triazol ring . The combination of nitroxide mediated radical polymerization (NMRP) and click chemistry was used to produce well-defined random copolymer. It could already be shown that also block copolymers can be prepared which give the chance to combine nanostructure formation in block copolymers with special functionality. Thus, the special properties of these functional polymers like the capability for photopatterning and anchoring onto gold substrates make them very interesting for nanotechnology applications. / Diese Arbeit bezieht sich auf das Gebiet der Bionanotechnologie und betrifft ein neuartiges Verfahren zur selektiven Immobilisierung der DNA oder anderer Biomoleküle auf mikrostrukturierten Goldkontakten, welche dann ein koordiniertes Zusammenwirken von einzelnen Nanomolekülen ermöglichen, z.B. in einem Mikroreaktor. Die Immobilisierung solcher Nanoobjekte soll durch dünne Funktionsschichten realisiert werden, die die Anbindungsgruppen liefern. Folglich war das Hauptziel dieser Arbeit die Entwicklung von Polymermaterialien für dünne Funktionsschichten, die die Aufbringung einer großen Vielzahl von Funktionselementen oder metallischen Strukturen auf verschiedenen Substraten gestatten und die Strukturierung durch den Einsatz von lithographischen Methoden ermöglichen. Um dieses Konzept zu realisieren, war es notwendig, ein Polymersystem zu gestalten und zu entwickeln, welches auf geeignete photolabile Einheiten basiert und zusätzlich Ankergruppen hat, die mit spezifischen Substraten wie Gold verbunden ist. Dieses Terpolymerkonzept wurde gezielt aus drei Komponenten mit speziellen Funktionen in entsprechenden molaren Verhältnissen gebildet, die eine Abstimmung der Materialeigenschaften ermöglicht und folgendes bereitstellt: photolabile geschützte Aminogruppen für die photolitographische Strukturerzeugung mit freien Aminogruppen, welche für weitere Modifikationen verfügbar sind wie das Anhängen von Kolloiden, die Metallisierung oder Anfügung von DNA-Strängen; disulfide Derivate für die kovalente Anbindung auf der Goldoberfläche und Spacer-Gruppe für Verbesserung der Schichtenbildung. Diese multifunktionalen Terpolymere sollen durch eine freie radikalische Polymerisation von entsprechenden Monomeren synthetisiert werden. Obwohl diese Techniken erfolgreich sind, sind sie eingeschränkt durch ihre Komplexität, den strengen synthetischen Anforderungen, sowie der Inkompatibilität mit vielen funktionalen thermolabilen und hochreaktiven Funktionalitäten. Um diese Schwierigkeiten zu überwinden wurde eine Polymerisationstechnik für diese Arbeit genutzt, die auf der „lebenden“ freien radikalischen Polymerisation basiert. Eine hoch effiziente polymeranaloge Modifizierung erlaubt die Einführung von Funktionalitäten nach der Polymeraufbaureaktion. Die Herstellung von entsprechenden Präpolymeren Poly(Styrol-r-4-Propargyl-oxystyrol) wurde mittels einer kontrollierten Synthesemethodik „Nitroxid-mediated controled radical polymerisation“ (NMRP) durchgeführt, gefolgt von der Polymeranalogreaktion, die eine der effizientesten Click-Reaktion - die Cu(I) katalysierte 1,3-dipolar Cycloaddition von terminalen Alkinen an Aziden nach Huisgen nutzt, um weiter Funktionalitäten durch die Bildung eines stabilen 1,4-disubstituierten-[1,2,3]-Triazolringes anzufügen. Die Kombination von NMRP und Click-Chemie wurde zur Herstellung eines exakt definierten Random Copolymers genutzt. Es konnte bereits gezeigt werden, dass auch Blockcopolymere geschaffen werden können, die eine Möglichkeit zur Kombination von Nanostrukturformationen in Blockcopolymeren mit speziellen Funktionaltäten bieten. Folglich sind die speziellen Eigenschaften dieser Funktionalpolymere wie die Fähigkeit zur Photostrukturierung und Verankerung auf Goldsubstraten für nanotechnologische Anwendungen sehr interessant.

A photolabile terpolymer containing 6-nitroveratyloxycarbonyl (NVOC) protected amine, epoxy and trimethoxysilyl functionality in 1:3:2 monomer ratio was synthesized to template glass surfaces for specific site directed coupling of non-probe oligomers and probe oligonucleotides. Non-probe oligomers were introduced to the surface to control the environment of the probes by reducing probe-to-probe and probe-to-surface interactions. The trimethoxysilyl group served as the anchoring site for the terpolymer to be covalently bound to glass and silicon wafers. Amine terminated non-probe oligomers were coupled to the epoxy sites and thiolated 19-mer SMN1 probes were directed to the deprotected amine sites via the heterobifunctional linker, sulfosuccinimidyl-4-[maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC). Characterization of the terpolymer was done using 1H NMR, 13C NMR, MALDI-ToF and elemental analysis. NVOC deprotection was monitored by UV absorption, and surface characterization of the bound terpolymer on silicon wafers was investigated with XPS, ToF-SIMS, ellipsometry and static contact angle. Neutral polyethylene glycol (PEG), negatively charged methacrylic acid (MAA) oligomer and dC20 oligonucleotides were used as non-probe oligomers. The probe density on the surface was estimated to be 2.2 ± 0.3 x 10^12 molecules/cm2 and the presence of the oligomers on the surface did not significantly affect probe immobilization efficiency. The mixed films were functional for target hybridization and its selectivity towards partially-mismatched targets was investigated at different solution pH, ionic strength and temperature. It was demonstrated that pH can be tuned to ameliorate non-specific adsorption and ionic strength governed the selectivity of the surfaces. Improved selectivity was achieved at high salt concentration (1 M NaCl) on PEG and dC20 mixed films at room temperature. The MAA surface did not show significant improvements in selectivity. This indicated that charge of the oligomers does not dominate control of selectivity. The results suggested that the terpolymer construct played a role in depression of the melting temperature of the hybridized duplex to within 5 to 10 oC of room temperature. With the melting temperature shifted closer to room temperature, it is possible to improve selectivity for room temperature detections of single nucleotide polymorphism.

Les glucides sont omniprésents dans la nature et jouent un rôle clé dans divers processus de reconnaissance moléculaire. Nous avons réalisé la conception et la synthèse de quatre types de dérivés glucidiques qui se divisent en 4 chapitres. Le le` chapitre est consacré à la synthèse d'analogues C glycosidiques de l'arbutine, une hydroquinone glycosylée, inhibiteur de la tyrosinase utilisé pour le blanchissement de la peau. Le 2è' chapitre concerne la synthèse d'analogues C glycosidiques de la vitamine E avec comme objectif d'améliorer ses propriétés antioxydants. Le 3e' chapitre est consacré à la synthèse des glycosides photolabiles pour la réalisation de glycopuces. Via l'ouverture régiosélective du 4,6 0 (o nitro)benzylidene des methyl glycopyranosides, suivie d'une C 4 épimerization, plusieurs 6 0 (o nitro)benzyl glycopyranosides ont été préparés. Enfin, le chapitre 4 est consacré à la synthèse des C glycosyl amino acides comme inhibiteurs potentiels de PTP 1B, cible potentielle pour le traitement du diabète de type II. Les résultats biologiques montrent que ces molécules inhibent l'enzyme avec IC50 de l'ordre de micromolaire.

A photolabile terpolymer containing 6-nitroveratyloxycarbonyl (NVOC) protected amine, epoxy and trimethoxysilyl functionality in 1:3:2 monomer ratio was synthesized to template glass surfaces for specific site directed coupling of non-probe oligomers and probe oligonucleotides. Non-probe oligomers were introduced to the surface to control the environment of the probes by reducing probe-to-probe and probe-to-surface interactions. The trimethoxysilyl group served as the anchoring site for the terpolymer to be covalently bound to glass and silicon wafers. Amine terminated non-probe oligomers were coupled to the epoxy sites and thiolated 19-mer SMN1 probes were directed to the deprotected amine sites via the heterobifunctional linker, sulfosuccinimidyl-4-[maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC). Characterization of the terpolymer was done using 1H NMR, 13C NMR, MALDI-ToF and elemental analysis. NVOC deprotection was monitored by UV absorption, and surface characterization of the bound terpolymer on silicon wafers was investigated with XPS, ToF-SIMS, ellipsometry and static contact angle. Neutral polyethylene glycol (PEG), negatively charged methacrylic acid (MAA) oligomer and dC20 oligonucleotides were used as non-probe oligomers. The probe density on the surface was estimated to be 2.2 ± 0.3 x 10^12 molecules/cm2 and the presence of the oligomers on the surface did not significantly affect probe immobilization efficiency. The mixed films were functional for target hybridization and its selectivity towards partially-mismatched targets was investigated at different solution pH, ionic strength and temperature. It was demonstrated that pH can be tuned to ameliorate non-specific adsorption and ionic strength governed the selectivity of the surfaces. Improved selectivity was achieved at high salt concentration (1 M NaCl) on PEG and dC20 mixed films at room temperature. The MAA surface did not show significant improvements in selectivity. This indicated that charge of the oligomers does not dominate control of selectivity. The results suggested that the terpolymer construct played a role in depression of the melting temperature of the hybridized duplex to within 5 to 10 oC of room temperature. With the melting temperature shifted closer to room temperature, it is possible to improve selectivity for room temperature detections of single nucleotide polymorphism.

The cytochrome P450 system plays an important role in metabolism of endogenous compounds and xenobiotics. This system consists of cytochrome P450, NADPH:cytochrome P450 oxidoreductase (CPR), cytochrome b5 and NADH:cytochrome b5 reductase (CYB5R3). Explanation of protein-protein interactions among these reaction partners is essential for understanding the function of the entire system. Covalent cross-linking is a favorable method for studying these interactions. In this work a photo-activatable analogue of amino acid L-methionine (L-photo-methionine) was used as a cross-linking agent. This work is focused on the organic synthesis of L-photo-methionine, expression and isolation of CPR and CYB5R3 as photoactivable proteins containing incorporated L-photo-methionine. Auxotrophic strain of E.coli B834 (DE3) and minimal media were used for the expression. CYB5R3 with incorporated L-photo-methionine was successfully expressed and isolated. The extent of L-photo-methionine incorporation was verified by mass spectrometry. Furthermore, the photo-initiated cross-linking of CYB5R3 with cytochrome b5 was tested. Key words: photolabile amino acid, protein expression, synthesis

The subject of this theses is a preparation of chemical UV dosimeters working on diverse principles and their printing. Three UV dosimeters were prepared. The first one is based on a photocalalytic reaction of titanium dioxide with redox dyes. Two other dosimeters are based on a reaction of a photolabile substance able to produce an intermediate reacting with a dye. Prepared dosimeter compositions were coated using a material roll-to-roll printing machine on a corona-treated PET foil. Prepared dosimeter compositions were calibrated for desired exposition to UV radiation doses. Validity of a reciprocity law was verified.