Specific enzymatic cleavage and payload release from peptide-based hybrid nanocapsules

Within this thesis, new approaches for the concepts of peptide-polymer conjugates and peptide-based hybrid nanomaterials are investigated. In the first part, the synthesis of a triblock polymer-peptide-polymer is carried out following a typical peptide coupling reaction, both in solution and on solid-phase. The peptide sequence is chosen, so that it is cleaved by an enzyme preparation of trypsin. End-functionalized polystyrene is used as a model hydrophobic polymer and coupled to the peptide sequence. The results show successful coupling reactions in both methods, while the solid phase method produced a more defined product. Suspensions, consisting of peptide-polymer conjugates particles, are prepared in water by ultrasonication. In contact with the enzyme, the peptide constituting the conjugated particles is cleaved. This demonstrates the enzymatic cleavage in heterophase of enzymatic sequence bond to hydrophobic polymers, and is of great interest for the encapsulation and delivery of hydrophobic molecules.rnA second approach is the preparation of peptide-based hybrid nanocapsules. This is achieved by interfacial polyaddition in inverse miniemulsion with the peptide sequence functionalized with additional amino acids. A method suitable to the use of a peptide sequence for interfacial polyaddition was developed. It is shown that, the polarity of the dispersed phase influences the structures prepared, from particle-like to polymeric shell with a liquid core.rnThe peptide sequence is equipped with a FRET pair (more exactly, an internally-quenched fluorescent system) which allows the real-time monitoring of the enzymatic cleavage of the recognition site. This system shows the successful cleavage of the peptide-based nanocapsules when trypsin preparation is added to the suspensions. A water-soluble fluorescent polymer is efficiently entrapped and its possible use as marker for the capsules is highlighted. Furthermore, a small water-soluble fluorescent dye (SR-101) is successfully encapsulated and the encapsulation efficiency as a function of the functionality of the peptide and the amount of comonomer equivalent (toluene diisocyanate) is studied. The dye is encapsulated at such a high concentration, that self-quenching occurs. Thus, the release of the encapsulated dye triggered by the enzymatic cleavage of the peptide results in a fluorescence recovery of the dye. The fluorescence recovery of the FRET pair in the peptide and of the encapsulated dye correlate well.rnFinally, nanocapsules based on a hepsin-cleavable peptide sequence are prepared. Hepsin is an enzyme, which is highly upregulated in prostate cancer cells. The cleavage of the nanocapsules is investigated with healthy and “cancerous” (hepsin-expressing) cell cultures. The degradation, followed via fluorescence recovery of the FRET system, is faster for the suspensions introduced in the hepsin expressing cell cultures.rnIn summary, this work tackles the domain of responsive nanomaterials for drug delivery from a new perspective. It presents the adaptation of the miniemulsion process for hybrid peptide-based materials, and their successful use in preparing specific enzyme-responsive nanoparticles, with hydrophilic payload release properties.rn

Uptake mechanism, intracellular trafficking and endo-lysosomal pH monitoring of polystyrene nanoparticles

What is the intracellular fate of nanoparticles (NPs) taken up by the cells? This question has been investigated for polystyrene NPs of different sizes with a set of molecular biological and biophysical techniques.rnTwo sets of fluorescent NPs, cationic and non-ionic, were synthesized with three different polymerization techniques. Non-ionic particles (132 – 846 nm) were synthesized with dispersion polymerization in an ethanol/water solution. Cationic NPs with 120 nm were synthesized by miniemulsion polymerization Particles with 208, 267 and 603 nm were produced by seeding the 120 nm particle obtained by miniemulsion polymerization with drop-wise added monomer and polymerization of such. The colloidal characterization of all particles showed a comparable amount of the surface groups. In addition, particles were characterized with regard to their size, morphology, solid content, amount of incorporated fluorescent dye and zeta potential. The fluorescent intensities of all particles were measured by fluorescence spectroscopy for calibration in further cellular experiments. rnThe uptake of the NPs to HeLa cells after 1 – 24 h revealed a much higher uptake of cationic NPs in comparison to non-ionic NPs. If the same amount of NPs with different sizes is introduced to the cell, a different amount of particles is present in the cell medium, which complicates a comparison of the uptake. The same conclusion is valid for the particles’ overall surface area. Therefore, HeLa cells were incubated with the same concentration, amount and surface area of NPs. It was found that with the same concentration always the same polymer amount is taking up by cells. However, the amount of particles taken up decreases for the biggest. A correlation to the surface area could not be found. We conclude that particles are endocytosed by an excavator-shovel like mechanism, which does not distinguish between different sizes, but is only dependent on the volume that is taken up. For the decreased amount of large particles, an overload of this mechanism was assumed, which leads to a decrease in the uptake. rnThe participation of specific endocytotic processes has been determined by the use of pharmacological inhibitors, immunocytological staining and immunofluorescence. The uptake of NPs into the endo-lysosomal machinery is dominated by a caveolin-mediated endocytosis. Other pathways, which include macropinocytosis and a dynamin-dependent mechanism but exclude clathrin mediated endocytosis, also occur as competing processes. All particles can be found to some extent in early endosomes, but only bigger particles were proven to localize in late endosomes. No particles were found in lysosomes; at least not in lysosomes that are labeled with Lamp1 and cathepsin D. However, based on the character of the performed experiment, a localization of particles in lysosomes cannot be excluded.rnDuring their ripening process, vesicles undergo a gradual acidification from early over late endosomes to lysosomes. It is hypothesized that NPs in endo-lysosomal compartments experience the same change in pH value. To probe the environmental pH of NPs after endocytosis, the pH-sensitive dye SNARF-4F was grafted onto amino functionalized polystyrene NPs. The pH value is a ratio function of the two emission wavelengths of the protonated and deprotonated form of the dye and is hence independent of concentration changes. The particles were synthesized by the aforementioned miniemulsion polymerization with the addition of the amino functionalized copolymer AEMH. The immobilization of SNARF-4F was performed by an EDC-coupling reaction. The amount of physically adsorbed dye in comparison to covalently bonded dye was 15% as determined by precipitation of the NPs in methanol, which is a very good solvent for SNARF-4F. To determine influences of cellular proteins on the fluorescence properties, a intracellular calibration fit was established with platereader measurements and cLSM imaging by the cell-penetrable SNARF-4F AM ester. Ionophores equilibrated the extracellular and intracellular pH.rnSNARF-4F NPs were taken up well by HeLa cells and showed no toxic effects. The pH environment of SNARF-4F NPs has been qualitatively imaged as a movie over a time period up to 1 h in pseudo-colors by a self-written automated batch program. Quantification revealed an acidification process until pH value of 4.5 over 24 h, which is much slower than the transport of nutrients to lysosomes. NPs are present in early endosomes after min. 1 h, in late endosomes at approx. 8 h and end up in vesicles with a pH value typical for lysosomes after > 24 h. We therefore assume that NPs bear a unique endocytotic mechanism, at least with regards to the kinetic involvedrn

From carbazole-containing and pi-expanded triangular trinuclear porphyrinoids

Zusammenfassung:rnDie vorliegende Arbeit beschreibt das Design und die Synthese neuartiger Porphyrinoide anhand der Modifikation und der π-Systemausdehnung an der Peripherie des Porphyrin-Gerüsts. Die Darstellung künstlicher Porphyrine ist von Interesse, da neue physiko-chemischen Eigenschaften erhalten und untersucht werden können. Die in dieser Arbeit vorgestellten Porphyrinoide wurden mit Hilfe von modernen Synthesemethoden wie den metallkatalysierten Kreuzkupplungen und somit durch Aryl-Aryl Verknüpfungen aufgebaut.rnDer erste Teil dieser Arbeit befasst sich mit der Modifikation des Porphyrin-Gerüsts. Porphyrine bestehen aus jeweils zwei Pyrrol- und Pyrrolenin-Einheiten, welche systematisch ausgetauscht wurden. Die Pyrrol-Einheiten wurden durch Carbazol ersetzt, das sich formal vom Pyrrol durch Anfügen von zwei Benzogruppen ableitet und deshalb besonders gut geeignet ist. Die Pyrrolenin-Einheiten wurden aus folgenden Gründen durch andere Heterozyklen wie Pyridin, Pyrrol oder Triazol ersetzt: rn* Nachbildung des stabilen Porphyrin trans-NH-Tautomers (Carbazol und Pyridin)rn* Nachbildung von (NH)4-Liganden wie Calix[4]pyrrol (Carbazol und Pyrrol)rn* Vereinigung von N-H und C-H Wasserstoffbrücken-Donor-Einheiten in einem Makrozyklus (Carbazol und Triazol)rnDie Synthese eines drei-Zentren Porphyrinoids mit ausgedehntem π-System wird im zweiten Teil der vorliegenden Arbeit beschrieben. Dieses Thema basiert auf der aktuellen Entwicklung von nicht-Edelmetall basierten Katalysatoren für die Reduktion von Sauerstoff. Hier werden derzeit N4 makrozyklische Metallkomplexe, die mehrere katalytisch aktive Stellen aufweisen, untersucht. In diesem Zusammenhang, hat die Gruppe von Prof. Müllen einen neuartigen drei-Zentren-Komplex entwickelt. Ausgehend von diesen Erkenntnissen, dient diese Arbeit zur Verbesserung der katalytischen Aktivität des drei-Zentren-Komplex durch die Variation von verschieden Substituenten. Hierbei wurden zwei wesentliche Konzepte verfolgt:rn* Vernetzung durch die Bildung von Netzwerken oder durch Pyrolyse in der Mesophasern* Verbesserung des Katalysator-Trägermaterial-KontaktsrnNeben den Synthesen wurden die Eigenschaften und möglichen Anwendungen dieser neuartigen Materialen untersucht, wie z.B. als Liganden für Übergangsmetalle, als Anionenrezeptoren oder als Elektrokatalysatoren für die Reduktion von Sauerstoff. rn

Synthesis of novel ribose-based thioimidate n-oxides

This work is based on the study of new synthetic paths to obtain thioimidate N-oxides (TINOs) from D-ribose and to study their reactivity with the purpose to obtain ketonitrones. TINOs, aren’t well known molecules, but these enantiomerically pure backbones could be valuable intermediates in the synthesis of novel ketonitrones which are key intermediates in the synthesis of iminosugars. TINOs were discovered from the study of glucoraphanin, a particular glucosinolate, that unexpectedly cyclized into a TINO after desulfatation, by a spontaneous intramolecular Michael addition. The first part of this work was to synthetize the TINO 3 from D-ribose 1. The key step was the desilylative cyclisation of a suitably functionalized thiohydroximate 2. Based on precedent work developed in the laboratory, we could obtain the thiohydroximate from D-ribose. We then focused our studies on the cyclisation step trying to find the suitable substituents that could give the TINO in good yield by desilylative cyclisation. The second part of the project is to obtain ketonitrones 4 by palladiumcatalyzed coupling reaction.

The Synthesis of Macrocyclic Polystyrene by Sequential Atom Transfer Radical Reactions

A method for the production of macrocyclic polystyrene via ring closing of a linear !,"-dibrominated polystyrene by an Atom Transfer Radical Coupling (ATRC) reaction is described. The dibrominated polystyrene chain was produced from two simultaneous atom transfer radical polymerizations (ATRPs) originating from a dibrominated benzal bromide initiator. To ensure the retention of the halogen end groups polymerization was allowed to proceed to less than 50% conversion. Using this precursor in an intramolecular ATRC (ring closing) reaction was found to yield in excess of 90% cyclic product based on refractive index-gel permeation chromatography (GPC) analysis. The cyclic architecture of the polymer was verified by GPC, Nuclear Magnetic Resonance (NMR), and mass spectrometry analysis. The utility of this method has been expanded by the addition of 2-methyl-2-nitrosopropane to the coupling reaction, which allows for the coupling to proceed at a faster rate and to yield macrocycles with incorporated alkoxyamine functionality. The alkoxyamine functionality allows for degradation of the cycles at high temperatures (>125° C) and we hypothesize that it may allow the macrocycles to act as a macroinitiator for a ring expansion polymerization in future studies.

Gold Phosphonates as Transfer Agents in Palladium Catalyzed Hirao Reactions

Through a cross-coupling reaction, aryl phosphonates are produced in high yields when the corresponding aryl bromides are reacted with a gold phosphorylating agent in the presence of a palladium catalyst and an appropriate ligand. To the best of our knowledge, this transformation is the first example involving the transfer of a phosphonate functional group from a gold complex to palladium that has been reported. Throughout the investigation, three gold phosphorylating agents were screened for activity towards the phosphorylation of aryl bromides. Aryl bromides with electrondonating and electron-withdrawing groups were successfully employed in the crosscoupling reactions. All cross-coupling reactions were carried out in THF at room temperature (25ºC) or in a microwave reactor (CEM Discover) at 60ºC for 30 or 60 minutes. The effects of changing reaction parameters such as time, temperature, catalyst and free ligand loading have been investigated. All aryl bromide substrates tested in the cross-coupling reactions produced phosphorylated products.

Synthesis of Mid-Chain Functionalized Polymers

Polymers with mid-chain alkoxyamine functionality were synthesized by activating monohalogenated polymers in the presence of nitroso or nitrone radical traps. The resulting polymers were either polystyrene (PSt) homopolymers with a mid-chain alkoxyamine or PSt-poly(methyl acrylate) (PMA) diblock copolymers with an alkoxyamine unit at the junction between the segments. Monohalogenated polymers where synthesized by atom transfer radical polymerization (ATRP) and were then reacted to form polymer radicals in the presence of a radical trap, nitrone or nitroso. When only polystyrene radicals were reacted with the radical trap a dimer was formed with an alkoxyamine functionality in the center of the polymer chain. This functionality allowed the polymer chain to be cleaved in order to visualize the extent of the alkoxyamine functionality incorporation into the polymer chains. It was found that near quantitative alkoxyamine mid-chain functionality could be achieved by activating the PStBr in the presence of 10 equivalents of nitrone, 5 equivalents of copper bromide, and 2 equivalents of copper metal. Further reducing the amount of copper metal led to incomplete coupling, while increasing the equivalents beyond 2 generated polymer dimers with less than quantitative mid-chain functionality. Monochlorinated polystyrene (PStCl) precursors gave much poorer coupling results compared to reactions with PStBr, which is consistent with the stronger C-Cl bond resisting activation and the formation of the polystyryl radicals. When poly (methyl acrylate) (PMABr) is reacted with PStBr in the presence of a nitroso group at reduced temperatures (30 oC) block copolymers were selectively formed with an alkoxyamine functionality in the center. This was done by first activating the PSt-Br to form a polymer radical that would react with the radical trap to form a persistent radical on the oxygen. The PMA-Br, once activated, reacted with the radical on the oxygen to form the block copolymer. To test the amount of functionality incorporated, a coupling reaction was performed with no nitroso present, and found that no reaction occurred. This showed that the radical trap is essential for the coupling to occur, and cleavage of the diblock indicated that the alkoxyamine functionality was indeed incorporated into the diblock.

Novel unsymmetrically functionalized BEDT–TTF derivatives: synthesis, crystal structure and electrochemical characterization

Novel functionalized bis(ethylenedithio)tetrathiafulvalene (BEDT–TTF) derivatives 4 and 5 have been synthesized in good yields from cyano precursor via a cross-coupling reaction. Their redox potentials have been studied by cyclic voltammetry in a dichloromethane solution; this indicated that they are slightly weaker electron donors than BEDT–TTF. Compound 4 has been studied by X–ray crystallography; this revealed that, in the crystal, the molecules were held together by some unconventional C–H···N and C–H···S hydrogen bonds.

Heterocyclic carbene–metal-catalyzed Csp2–Csp2 and Csp–Csp2 couplings using nonmetallic substrates

The use of carbene ligands for transition-metal complexes has been developed in the last decades, being of special interest those carbenes derived from a nitrogen-containing heterocyclic system. An interesting variety of carbene-metal complexes has been tested in the Mizoroki-Heck reaction. In comparison, few examples can be found for the Matsuda-Heck version of this coupling reaction. Additionally, the Sonogashira coupling has been also catalyzed with different carbene-metal catalysts.

Biscarboxy-Functionalized Imidazole and Palladium as Highly Active Catalytic System in Protic Solvents: Methanol and Water

The coupling reaction between aryl bromides and boron reagents is efficiently catalyzed by an in situ generated palladium complex obtained from palladium(II) acetate (0.1 mol%) and 1,3-bis(carboxymethyl)imidazole (0.2 mol%). The catalytic system is very active in protic solvents, especially in methanol. Biaryl derivatives have been prepared in good isolated yields (up to >99%), and additionally styrene and stilbene derivatives have also been prepared by means of this protocol.

Efficient alkyne homocoupling catalysed by copper immobilized on functionalized silica

Copper immobilized on a functionalized silica support is a good catalyst for the homocoupling of terminal alkynes. The so-called Glaser-Hay coupling reaction can be run in air with catalytic amounts of base. The copper catalyst is active for multiple substituted alkynes, in both polar and non-polar solvents, with good to excellent yields (75-95%). Depending on the alkyne, full conversion can be achieved within 3-24 h. The catalyst was characterized by TGA, inductively coupled plasma and X-ray photoelectron spectroscopy. Leaching tests confirm that the catalyst is and remains heterogeneous. Importantly, the overall reaction requires only alkyne and oxygen (in this case, air) as reagents, making this a clean catalytic oxidative coupling reaction. © 2012 John Wiley & Sons, Ltd.

Novel approaches for the synthesis of C-5 modified pyrimidine nucleosides

The antiviral or anticancer activities of C-5 modified pyrimidine nucleoside analogues validate the need for the development of their syntheses. In the first half of this dissertation, I explore the Pd-catalyzed cross-coupling reaction of allylphenylgermanes with aryl halides in the presence of SbF 5/TBAF to give various biaryls by transferring multiple phenyl groups, which has also been applied to the 5-halo pyrimidine nucleosides for the synthesis of 5-aryl derivatives. To avoid the use of organometallic reagents, I developed Pd-catalyzed direct arylation of 5-halo pyrimidine nucleosides. It was discovered that 5-aryl pyrimidine nucleosides could be synthesized by Pd-catalyzed direct arylation of N3-free 5-halo uracil and uracil nucleosides with simple arenes or heteroaromatics in the presence of TBAF within 1 h. Both N3-protected and N3-free uracil and uracil nucleosides could undergo base-promoted Pd-catalyzed direct arylation, but only with electron rich heteroaromatics. ^ In the second half of this dissertation, 5-acetylenic uracil and uracil nucleosides have been employed to investigate the hydrogermylation, hydrosulfonylation as well as hydroazidation for the synthesis of various functionalized 5-vinyl pyrimidine nucleosides. Hydrogermylation of 5-alkynyl uracil analogues with trialkylgermane or tris(trimethylsilyl)germane hydride gave the corresponding vinyl trialkylgermane, or tris(trimethylsilyl)germane uracil derivatives. During the hydrogermylation with triphenylgermane, besides the vinyl triphenylgermane uracil derivatives, 5-[2-(triphenylgermyl)acetyl]uracil was also isolated and characterized and the origin of the acetyl oxygen was clarified. Tris(trimethylsilyl)germane uracil derivatives were coupled to aryl halides but with decent yield. Iron-mediated regio- and stereoselective hydrosulfonylation of the 5-ethynyl pyrimidine analogues with sulfonyl chloride or sulfonyl hydrazine to give 5-(1-halo-2-tosyl)vinyluracil nucleoside derivatives has been developed. Nucleophilic substitution of the 5-(β-halovinyl)sulfonyl nucleosides with various nucleophiles have been performed to give highly functionalized 5-vinyl pyrimidine nucleosides via the addition-elimination mechanism. The 5-(β-keto)sulfonyluracil derivative has also been synthesized via the aerobic difunctionalization of 5-ethynyluracil analogue with sulfinic acid in the presence of catalytic amount of pyridine. Silver catalyzed hydroazidation of protected 2'-deoxy-5-ethynyluridine with TMSN3 in the presence of catalytic amount of water to give 5-(α-azidovinyl)uracil nucleoside derivatives was developed. Strain promoted Click reaction of the 5-(α-azidovinyl)uracil with cyclooctyne provide the corresponding fully conjugated triazole product.^

Novel Approaches for the Synthesis of C-5 Modified Pyrimidine Nucleosides

The antiviral or anticancer activities of C-5 modified pyrimidine nucleoside analogues validate the need for the development of their syntheses. In the first half of this dissertation, I explore the Pd-catalyzed cross-coupling reaction of allylphenylgermanes with aryl halides in the presence of SbF5/TBAF to give various biaryls by transferring multiple phenyl groups, which has also been applied to the 5-halo pyrimidine nucleosides for the synthesis of 5-aryl derivatives. To avoid the use of organometallic reagents, I developed Pd-catalyzed direct arylation of 5-halo pyrimidine nucleosides. It was discovered that 5-aryl pyrimidine nucleosides could be synthesized by Pd-catalyzed direct arylation of N3-free 5-halo uracil and uracil nucleosides with simple arenes or heteroaromatics in the presence of TBAF within 1 h. Both N3-protected and N3-free uracil and uracil nucleosides could undergo base-promoted Pd-catalyzed direct arylation, but only with electron rich heteroaromatics. In the second half of this dissertation, 5-acetylenic uracil and uracil nucleosides have been employed to investigate the hydrogermylation, hydrosulfonylation as well as hydroazidation for the synthesis of various functionalized 5-vinyl pyrimidine nucleosides. Hydrogermylation of 5-alkynyl uracil analogues with trialkylgermane or tris(trimethylsilyl)germane hydride gave the corresponding vinyl trialkylgermane, or tris(trimethylsilyl)germane uracil derivatives. During the hydrogermylation with triphenylgermane, besides the vinyl triphenylgermane uracil derivatives, 5-[2-(triphenylgermyl)acetyl]uracil was also isolated and characterized and the origin of the acetyl oxygen was clarified. Tris(trimethylsilyl)germane uracil derivatives were coupled to aryl halides but with decent yield. Iron-mediated regio- and stereoselective hydrosulfonylation of the 5-ethynyl pyrimidine analogues with sulfonyl chloride or sulfonyl hydrazine to give 5-(1-halo-2-tosyl)vinyluracil nucleoside derivatives has been developed. Nucleophilic substitution of the 5-(β-halovinyl)sulfonyl nucleosides with various nucleophiles have been performed to give highly functionalized 5-vinyl pyrimidine nucleosides via the addition-elimination mechanism. The 5-(β-keto)sulfonyluracil derivative has also been synthesized via the aerobic difunctionalization of 5-ethynyluracil analogue with sulfinic acid in the presence of catalytic amount of pyridine. Silver catalyzed hydroazidation of protected 2'-deoxy-5-ethynyluridine with TMSN3 in the presence of catalytic amount of water to give 5-(α-azidovinyl)uracil nucleoside derivatives was developed. Strain promoted Click reaction of the 5-(α-azidovinyl)uracil with cyclooctyne provide the corresponding fully conjugated triazole product.

Sustainable Catalysis : Rational Pd Loading on MIL-101Cr-NH2 for More Efficient and Recyclable Suzuki-Miyaura Reactions

Palladium nanoparticles have been immobilized into an amino-functionalized metal-organic framework (MOF), MIL-101Cr-NH2, to form Pd@MIL-101Cr-NH2. Four materials with different loadings of palladium have been prepared (denoted as 4-, 8-, 12-, and 16wt%Pd@MIL-101Cr-NH2). The effects of catalyst loading and the size and distribution of the Pd nanoparticles on the catalytic performance have been studied. The catalysts were characterized by using scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier-transform infrared (FTIR) spectroscopy, powder X-ray diffraction (PXRD), N-2-sorption isotherms, elemental analysis, and thermogravimetric analysis (TGA). To better characterize the palladium nanoparticles and their distribution in MIL-101Cr-NH2, electron tomography was employed to reconstruct the 3D volume of 8wt%Pd@MIL-101Cr-NH2 particles. The pair distribution functions (PDFs) of the samples were extracted from total scattering experiments using high-energy X-rays (60keV). The catalytic activity of the four MOF materials with different loadings of palladium nanoparticles was studied in the Suzuki-Miyaura cross-coupling reaction. The best catalytic performance was obtained with the MOF that contained 8wt% palladium nanoparticles. The metallic palladium nanoparticles were homogeneously distributed, with an average size of 2.6nm. Excellent yields were obtained for a wide scope of substrates under remarkably mild conditions (water, aerobic conditions, room temperature, catalyst loading as low as 0.15mol%). The material can be recycled at least 10times without alteration of its catalytic properties.