“There is plenty of room at the bottom”. These were the famous words of Richard P. Feynman in 1959 that led to the birth of nanotechnology and nanoscience. Electronic devices based on inorganic semiconductors have been part of our daily lives for the last 60 years. Their miniaturisation has occurred gradually over the years, however, according to Moore’s law the contemporary microelectronic industry’s “top-down” manufacturing technique will soon reach its limits. Therefore, the recent development and increased knowledge of organic semiconductors has led to a tendency to explore alternative avenues with a focus on the creation of electronic devices based on organic molecules. The invention of techniques such STM (1981) and AFM (1986) have facilitated this research, allowing the imaging and manipulation of surfaces and molecules at the nanometre scale (0.1-100 nm). The next step is therefore the development of methods for the controlled fabrication of molecular assemblies and their integration into usable macroscopic systems. In this respect, the “bottom-up” approach offers considerable advantages over any other methodology (i.e. “top-down”) for the construction of nanoscale functional materials and devices. This approach generally exploits the hierarchical self-assembly of functional molecules through multiple non-covalent interactions to prepare long range ordered and defect-free assemblies barely accessible through conventional covalent synthesis. However, an intrinsic drawback of investigating such systems in solution or in a crystal is that molecular components cannot be directly addressed on a nanometric scale. As a consequence, the best engineering methodology involves modifying the surfaces of bulk materials such as metals or semiconductors by deposition of functional organic materials. The modified surfaces are then characterised using scanning probe microscopies (e.g. STM, AFM). To this end, surface-confined, supramolecularly constructed, bi-dimensional (2D) networks, featuring regular porous domains (controllable both in shape and size) are of particular significance in this research domain because their cavities can be used as receptors for the confinement of other remotely controlled functional molecules (e.g. molecular switches, luminescent chromophores). Since these complex nanostructures could ultimately find applications as optoelectronic devices, research efforts in this domain have been gathering momentum in recent years. In Chapter 1, the reader is introduced to the methods employed to construct porous networks on surfaces via supramolecular interactions. The second part of the chapter deals with recent examples of recognition, selection and immobilisation of guest molecules within the cavities of the networks, which is followed in the third part with a discussion about surface assemblies that display structural features or functionality in the third dimension. The last section of the chapter is devoted to the construction of porous networks on surfaces via the interactions of biomimetic molecules (e.g. DNA), which leads to the objectives of the present doctoral project. Inspired by the self-assembly of DNA into nanoporous arrays, it was postulated that the Watson-Crick base pairing of oligonucleotide’s nucleobases would be ideal in preparing 2D porous networks with large receptor cavities. The idea was to covalently attach complementary single stranded oligonucleotides to rigid angular and linear unit core modules respectively, and then allow the two units to self-assemble on surfaces. However, instead of using DNA oligonucleotides, the use of peptide nucleic acid (PNA) oligonucleotides was proposed since more robust architectures would be obtained due to the higher duplex stability displayed by this class of biomimetic molecules. This doctoral dissertation describes the synthetic steps taken towards achieving this goal. The design of the angular and linear units bearing complementary PNA oligomers, required for the preparation of self-assembled nanoporous arrays is presented. However, prior to synthesizing these complex molecules, a simpler proof of principle was required to confirm that PNA duplexes could be formed on surfaces and also, whether the presence of chromophoric moieties (e.g. porphyrin) appended to the PNA strands had any effect on duplex formation and duplex stability. The molecule designed for this proof of principle was a self-complementary PNA dodecamer bearing a porphyrin adduct. The synthesis of the self-complementary PNA oligomer required for the preparation of the PNA-porphyrin adduct is described in the first part of Chapter 2. The main synthetic routes and protecting-group strategies used to prepare PNA monomers and oligomers are described first. This is followed by a discussion of the orthogonal protecting group strategies chosen for our project that would allow the isolation of PNA oligomers bearing protected nucleobases following resin-cleavage. This is contrary to the general norm in existing strategies wherein resin-cleavage and nucleobase deprotection is carried out in situ, however, it was required in our synthetic strategy since the terminal amino group of the PNA oligomers was required for further solution phase reactions. To this end, two protecting group strategies were proposed, a Fmoc/Mmt and Fmoc/Cbz-protecting group strategies. The solid support chosen for the Fmoc/Mmt strategy was Tentagel featuring a base-cleavable linker. Due to the failure to hydrolyse the linker during the resin-cleavage step, the Fmoc/Mmt strategy was abandoned. In the second strategy, an acid-cleavable Rink amide resin was chosen as the solid support, therefore a Fmoc/Cbz-protecting group strategy was chosen since it would allow the TFA-mediated cleavage of the oligomer from the resin, without the deprotection of the Cbz groups from the nucleobases. The preparation of the target PNA oligomer (sequence: TTAATTAATTAA) using the Fmoc/Cbz strategy is described in the next section. First, the required monomers for the oligomer synthesis were prepared using established procedures. Then, following reports of the advances in microwave assisted solid phase peptide synthesis claiming improved purity of oligomer products using short coupling times, the solid phase PNA oligomerisation was attempted using microwave irradiation. Three attempts were performed. The first, using a standard laboratory microwave, resulted in a complex mixture of products at the dodecamer stage. An improvement was observed in the results using the CEM discover SPS microwave which was specifically designed for solid phase synthesis, however, the crude dodecamer obtained was still inseparable from the by-products. Similar results were obtained with the CEM liberty microwave, which was an automated solid phase synthesis setup. Finally, utilising manual solid phase synthesis, the target PNA dodecamer was obtained. The HPLC chromatogram of crude PNA dodecamer obtained following resin cleavage displayed a single major product, which was subsequently purified. The oligomer was then deprotected by treatment with TMSI, and was analysed by mass spectrometry, which confirmed that the target dodecamer had been isolated. Section 2.2 described our efforts to prepare PNA-chromophore adducts. Following the isolation of the PNA dodecamer, attempts to covalently attach a porphyrin moiety to the resin-bound oligomer via an amide linkage failed, possibly due to steric hindrance. Subsequently, an azide linker was appended to the oligomer, and attempts to attach an acetylene functionalised porphyrin using a Cu(I)-catalysed 1,3-dipolar cycloaddition were performed. Unfortunately, this approach also did not yield the target adduct. These unsuccessful results paved the way to the development of a Cu(I)-free 1,3-dipolar cycloaddition that enabled the attachment of chromophores to the PNA oligomer. Recently published reports of Cu(I)-free 1,3-dipolar cycloaddition reactions applied on DNA oligomers offered inspiration towards this goal. The reported strategies involved the generation of a nitrile oxide species, which then reacted with either an alkene or an alkyne to form an isoxazoline or an isoxazole. Two methods of generating the nitrile oxide species were evaluated using anthracene derivatives. The first method involved the base-mediated dehydrochlorination of anthracene hydroximoyl chloride to yield the nitrile oxide, which then reacted with a dipolarophile that was introduced into the reaction mixture. The second approach to generating a nitrile oxide species involved treating an O-silylated hydroxamic acid derivative of anthracene with trifluoromethanesulfonic anhydride in the presence of a base (Carreira’s method). Following successful trapping of the nitrile oxides generated by both methods using trimethylsilyl ethylene as the dipolarophile, the reactions were applied on a resin-bound, acetylene-functionalised PNA dodecamer. Both methods yielded the target PNA-anthracene adduct. Since the nitrile oxide-acetylene 1,3-dipolar cycloaddition reaction had never been applied on porphyrins, a method had to be developed. Attempts to prepare a hydroximoyl chloride derivative of a porphyrin resulted in the decomposition of the macrocycle upon treatment with chlorinating agents (NCS, tert-BuOCl, and 1-chlorobenzotriazole), therefore, the hydroximoyl chloride method was abandoned in favour of the Carreira method. An O-silylated hydroxamic acid derivative of porphyrin was synthesized, and upon exposure to trifluoromethanesulfonic anhydride and Et3N, the nitrile oxide was generated and was trapped with a large excess (200 eq.) of trimethylsilyl ethylene yielding the target tetra-isoxazole porphyrin derivative in 62% yield, which corresponded to a yield of 89% per 1,3-dipolar cycloaddition. Optimisation of the reaction conditions using phenyl acetylene as the dipolarophile allowed similar yields to be obtained with only a 10 eq. excess of the acetylene. Having developed a protocol that was compatible with both PNA and porphyrin, the utility of the method to prepare a variety of PNA-chromophore adducts was tested. Hydroxamate derivatives of pyrene, porphyrin, phenanthroline and fluorescein chromophores were prepared. Subsequently, the corresponding nitrile oxide species were generated and were reacted with the resin-bound, acetylene-functionalised PNA dodecamer. The PNA-pyrene adduct was successfully isolated, however, the other target PNA-chromophore products were not isolated. The porphyrin nitrile oxide derivative was insoluble in the reaction medium, thus preventing the cycloaddition reaction from proceeding. In the case of the fluorescein hydroxamate, the presence of nucleophilic functional groups in the starting material were probably reactive towards the trifluoromethanesulfonic anhydride reagent, therefore it was unlikely that the nitrile oxide species was formed, and thus the cycloaddition reaction could not proceed. Finally, the reaction with the phenanthroline derivative yielded a new product, however mass spectrometry analysis indicated that it did not correspond the target PNA-phenanthroline adduct. Further work is currently underway to re-evaluate these reactions. In parallel to the synthetic work, a preliminary study into the deposition of PNA onto mica surfaces was investigated using AFM imaging. Deposition of drops of an aqueous solution of deprotected self-complementary PNA dodecamer onto clean mica surfaces using spin coating resulted in aggregates of PNA on the surface. Following annealing of the solution, a repeated deposition of a single drop of the solution resulted in a completely different surface assembly. The surface was saturated by what was thought to be PNA duplexes. This was confirmed by the deposition of drop of a solution that was diluted ten-fold which resulted in an AFM image where bright spot were intermitted by clean mica surface. Topographical analysis of the surface indicated that the bright spots were an average in 1 nm in height, which closely corresponds to the expected height of PNA duplexes, thus confirming that PNA duplexes could be deposited onto surfaces.
| Date of Award | 28 Apr 2011 |
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| Original language | French |
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| Awarding Institution | |
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| Supervisor | Davide BONIFAZI (Supervisor), Carmela Aprile (Jury), Stephane Vincent (President), Marcella BONCHIO (Jury), Annemieke MADDER (Jury) & Maurizio Prato (Co-Supervisor) |
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