Dynamic combinatorial chemistry (DCC) is a novel approach to medicinal chemistry which integrates the synthesis and screening of small molecule libraries into a single step. The concept uses reversible chemical reactions to present a dynamic library of candidate structures to a template which selects and removes the best binder from equilibrium. Using this evolutionary process with a biopolymer template, such as a protein, leads to the protein directing the synthesis of its own best ligand. Biological DCC applications are extremely challenging since the thermodynamic criterion of reversibility has to be met under physiological conditions to ensure stability of the biomolecular template. The list of reversible reactions satisfying these stringent criteria is limited and is a major constraint on achieving both reaction and structural diversity in adaptive dynamic libraries. This thesis reports the development of a catalysed version of acylhydrazone dynamic libraries which are truly adaptive under protein-friendly conditions. In the presence of aniline as a trans-imination catalyst, acylhydrazone dynamic combinatorial libraries equilibrate rapidly at pH 6.2 and are switched off by an increase in pH. We designed acylhydrazone libraries targeting the enzyme superfamily Glutathione-S-Transferase (GST) using a scaffold aldehyde, 4-chloro-3-nitrobenzaldehyde, which is structurally related to a known GST substrate chlorodinitrobenzene. On interfacing these dynamic libraries with two different GST enzymes (SjGST from the helminth worm Schistosoma japonicum and hGSTP1-1, a human isoform and an important oncology drug target) we observed isoformselective amplification effects with two different acylhydrazones selected by the proteins. To explore the potential of anchoring in our DCC methodology we conjugated the endogenous GST ligand, glutathione (GSH) onto the scaffold chloronitrobenzaldehyde. The GSH recognition motif acts as an anchor and allows us to explore the hydrophobic binding site of the enzyme in a fragment-based approach. The presence of the glutathione moiety led to increased solubility of the library members and a DCC experiment with the enzymes led to the selection of conjugate hydrazones with significant binding ability. Multi-level dynamic libraries use multiple exchange processes in the same system to increase their accessible structural diversity. These exchange reactions may be orthogonal, where the different chemistries can be activated or deactivated independently of each other, or simultaneous, where all the processes are dynamic and crossover under the same conditions. Together, these interacting molecular networks provide an exciting experimental approach to the emerging field of systems chemistry. We demonstrate that two reversible reactions, conjugate addition of thiols to enones and hydrazone formation, are fully compatible and orthogonal to one another in a single dynamic library. Hydrazone exchange takes place at acidic pH, while conjugate addition operates at basic pH. Simple pH change can be used to switch between each process and establish two channels of reactivity.

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Ph.d.

Thesis (Ph. D.)--University of Wyoming, 2005. / Title from PDF title page (viewed on Oct. 16, 2007). Includes bibliographical references.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2007. / Title from title screen (site viewed Feb. 17, 2009). PDF text: viii, 383 p. : ill. (chiefly col.) ; 49.9 Mb. UMI publication number: AAT 3294904. Includes bibliographical references. Also available in microfilm and microfiche formats.

The development and identification of new biomaterials that can replace specific tissues and organs is desirable. In the presented PhD thesis polymer microarrays were applied for the screening of polyacrylates and polyurethanes and evaluation for material discovery for applications in the life sciences. In the first part of the thesis, the largest polymer microarray ever made with more than 7000 features was fabricated and subsequently used for the screening of polyacrylates that can control the fate of human embryonic stem cells. As stem cells have unique properties that offer the potential of replacing damaged or diseased tissue in future, the identification of cultivation substrates that can replace current biological and animal derived products was desirable. The water contact angle, roughness and cell doubling time of the cells on the identified polymers was determined and the stem cells characterised after 5 passages and compared to the currently most widely used animal derived substrate MatrigelTM. In the second part of the thesis, the development of a new polymer gradient microarray is presented. Initial studies involved the optimisation of printing parameters for the generation of linear polymer gradient lines and confirmed by XPS analysis. Cellular binding studies with the suspension cell line K562 and the adherent cell line HeLa were carried out and compared to previous binding studies to confirm the success of the concept. In further studies, the polymer gradients were functionalised with small molecules and proteins, allowing the generation of a protein gradient microarray with Semaphorin 3F. In binding studies with neuron cells it could be shown that the binding of the cells was concentration-dependent. The identification of polyacrylates for the effective and rapid activation and aggregation of platelets is described in the third part of the presented thesis. Here, polymer microarrays were applied for the binding of platelets in human blood samples. The amount of bound platelets as well as their activation state was compared to the natural agonist collagen by employing fluorescence intensity studies and scanning electron microscopy. In shear studies, the activation of the platelets by the polymers was evaluated under physiological conditions. The mechanism by which the polymer triggered the activation was further explored by protein binding studies. It was shown that the initial adsorption of fibrinogen and von Willebrand factor on the polymers lead to the adherence and aggregation of platelets. In the final part of the presented thesis, polymer microarrays were used to identify polymers that can sort and collect the precursor cells of platelets (megakaryocytes). For this purpose, the cell lines K562 and MEG-01 were used as cellular models. The identified polymers and the effect on the immobilised cells was further investigated by scanning electron microscopy, flow cytometry and miRNA studies. The adsorbed proteins on the different polymers were found to influence the cellular morphology on the different substrates.

Ph.D.

No description available.