The Sutherland group recently demonstrated that from a mixture of oligoribonucleotide-2'- or 3'-phosphates the latter is chemoselectively acetylated. This is shown to mediate a template-directed ligation to give predominantly 3',5'-linked RNA that is acetylated at the ligation junction (acetyl-RNA). It was suggested that RNA emerged prebiotically via acetyl-RNA and also is proposed to have favourable genotypic properties due to greater propensity to form duplex structure. To study the properties of acetyl-RNA, their synthesis by solid-phase chemistry was required and described is the design of a 2'/3'-O-acetyl orthogonal protecting group strategy. Key to the orthogonal protecting group strategy is the use of (2-cyanoethoxy)carbonyl for the protection of the nucleobase exocyclic amines and a photolabile solid-phase linker group that allowed partial on-column deprotection. The synthesis of the 2'/3'-O-acetyl and 2'/3'-O-TBDMS phosphoramidites, in addition to preparation of a photolabile solid-phase support, are described. With the materials to hand the procedures for an automated synthesis of acetyl-RNA were optimised and several acetyl-RNA oligonucleotides were synthesised. The duplex stability of acetyl-RNA with up to four sites of 2'-O-acetylation were assessed by UV melting curve analysis. Remarkably, the acetyl groups caused a consistent decrease in Tm of between 3.0-3.2 °C. Thermodynamic parameters indicated a decrease in duplex stability that was consistent with a decrease in hydration of the minor groove resulting in a reduction of the stabilising hydrogen bonding network. The stability of a tetraloop was also found to decrease on acetylation. The acetylated- tetraloop it is able to form duplex at lower concentrations than the natural tetraloop. Additionally, it is more stable at high concentrations, indicating that acetyl-RNA favours duplex over other secondary structure. These properties are considered to give acetyl-RNA competitive advantage for their non-enzymatic replication. Aminoacylation of RNA is an important process in modern biology but the intermediacy of aminoacyl-adenylates is considered to be prebiotically implausible. A potentially prebiotic aminoacylation of nucleoside-3'-phosphates, selective for the 2'-hydroxyl, is presented. However, it was thought the aminoacylation yields could be improved and so a search for an alternative activator was conducted. Oligoribonucleotide-3'-phosphates were exposed to the aminoacylation conditions and selective aminoacylation at only the 2'-hydroxyl of the 3'-end was observed. In particular, the aminoacylation of a trimer lends support to Sutherland’s theory of a linked origin of RNA and coded peptide synthesis.

This work deals with the preparation of combinatorial libraries of peptides serving as platforms for proteolytic phenotyping. The primary objective was to prepare a solid phase fluorogenic peptide library and screen proteases by fluorescence. Further, the possibility of preparing solid phase DNA-encoded libraries was studied. Due to the non-reactivity of the specific proteases with the solid phase peptides, the solid phase was completely abandoned and DNA-encoded peptide library was prepared in the solution. Using this model of DNA-encoded dipeptide with terminal biotin, the new principle of testing proteolytic activities of proteases was verified. A combinatorial library of DNA-encoded hexapeptides was also prepared. Despite the low yield of the library, the possibility of DNA encoding, the amplifiability of the prepared molecules and the possibility of biotin-based separation were verified. The integrity of the hexapeptide sequence and the protease testing is the subject of further study.

This work describes the application of spiroligomers as serine hydrolases mimetics. Through collaboration with Kendall Houk's group, for the first time in the Schafmeister lab, we demonstrate that "theozymes" can be successfully used as models to design highly functionalized spiroligomer constructs for organocatalysis. We demonstrate a structure-function relationship between the structure of a series of bi-functional and tri-functional spiroligomer based transesterification catalysts and their catalytic activity. First, we designed and synthesized a series of stereochemically and regiochemically diverse bi-functional spiroligozymes to identify the best arrangement of a pyridine as a general base catalyst and an alcohol nucleophile to accelerate attack on vinyl trifluoroacetate as an electrophile. The best bifunctional spiroligozyme reacts with vinyl trifluoroacetate to form an acyl-spiroligozyme conjugate 2.7x103-fold faster than the background reaction with benzyl alcohol. We then incorporated an additional urea functional group to activate the acyl-spiroligozyme intermediate through hydrogen bonds and enable acyl transfer to methanol. The best trifunctional spiroligozyme carries out multiple turnovers and acts as a transesterification catalyst with k1/kuncat of 2.2x103 and k2/kuncat of 1.3x102. Quantum mechanical calculations identified four transition states in the catalytic cycle and provided a detailed view of every stage of the transesterification reaction. With the aim of accelerating the k2, we sought to design better oxyanion holes that hold multiple hydrogen bonding groups in close proximity of the catalytic groups. A macrocyclic motif would be a good candidate to force the oxyanion hole arm to arrange hydrogen-bonding groups in a precise three-dimensional constellation for transition state stabilization. In Chapter 4, we introduce an in silico designed macrocyclic spiroligomer, which overlays well with catalytic active site of an inhibitor bound-esterase. Finally, we detail our effort to develop new methodologies for rapidly synthesizing spiroligomers on solid-support. This would allow us to efficiently permute their structures for diverse applications such as organocatalysts, host molecules, and biologically related applications such as inhibiting protein-protein interactions. / Chemistry

This thesis research presents the synthesis and first application of bis-amino acid-based spiroligomers towards the development of organocatalysis, from small molecules to moderate size spiroligomers, and to macromolecules. By synthesizing a toolbox of cyclic monomers called "bis-amino acids", the Schafmeister group has developed an approach to construct both small and macromolecules named "Spiroligomers". These molecules arrange catalytic functional groups in a shape-persistent and programmable backbone. Unlike proteins and small peptides, spiroligomers do not fold; rather, their polycyclic backbone structures are controlled by the sequence and stereochemistry of the component monomers. Firstly, we demonstrated a structure/catalytic activity relationship together with computational modeling that suggests that a specific hydrophobic interaction between the modified pro4 catalyst and the aldehyde substrate is responsible for an observed rate enhancement in the aldol reaction. For the moderate size molecules, several spiroligomer libraries were prepared through solid phase or solution phase synthesis and screened for either the alcohol kinetic resolution reaction or the aldol reaction. The poor activity and selectivity suggest that the scaffolds involved cannot create the necessary chiral environment for asymmetric catalysis. Finally, a synthetic method of macromolecules using cross metathesis coupling was developed and a series of tetra-functionalized macrocyclic spiroligomers were synthesized. Three of these macromolecules were examined as asymmetric catalysts in the aldol reaction and gave moderate activity and selectivity. The NMR analysis of these macromolecules indicates their dynamic nature. As the first application of bis-amino acid based macromolecules in organocatalysis area, although these catalysts only generated moderate activity and selectivity, they provided evidence that changing the configuration of one stereocenter of the fourteen available within these macromolecules can alter the selectivity. This synthetic methodology also provides an effective way to create more complicated pocket like spiroligomer macromolecules for the future applications in catalysis and molecular recognition. / Chemistry

Yes / The duocarmycins are potent antitumour agents with potential in the development of antibody drug conjugates (ADCs) as well as being clinical candidates in their own right. In this paper, we describe the synthesis of a duocarmycin monomer (DSA) that is suitably protected for utilisation in solid phase synthesis. The synthesis was performed on a large scale and the resulting racemic protected Fmoc-DSA subunit was separated by supercritical fluid chromatography (SFC) into the single enantiomers. Application to solid phase synthesis methodology gave a series of monomeric and extended duocarmycin analogues with amino acid substituents. The DNA sequence selectivity was similar to previous reports for both the monomeric and extended compounds. The substitution at the C-terminus of the duocarmycin caused a decrease in antiproliferative activity for all of the compounds studied. An extended compound containing an alanine at the C-terminus was converted to the primary amide or to an extended structure containing a terminal tertiary amine but this had no beneficial effects on biological activity. / MJS was funded by Novartis and UEA. We thank the EPSRC Mass Spectrometry Service, Swansea. We thank Richard Robinson and Julia Hatto at Novartis for help in the large scale synthesis.

This report mainly discusses the development of the cysteine based PNA (CPNA), which is an analogue of PNAs. Peptide nucleic acids (PNA), a pseudopeptide DNA mimic, was discovered by Nielsen and his coworker in 1991. PNA is proved to sequence-specifically form a very stable duplex with complementary DNA and RNA strands through Watson-Crick base paring, and it is also capable of binding to duplex DNA by helix invasion. These intriguing properties of PNA implicated great potential for medical and biotechnical applications. Therefore, PNA has attracted many scientists in the fields of chemistry, biology, medicine including drug discovery and genetic diagnostics, molecular recognition. Due to its acyclic, achiral and neutral nature of the backbone, PNA has shown problems such as its poor aqueous solubility, poor cell permeability and instability of PNA-DNA duplexes and triplexes. Accordingly, many synthetic approaches have been directed toward developing modified backbones of PNA. Among those PNA analogs, only few examples including lysine-based monomers, guanidine-based peptide nucleic acids (GPNA) and the aminoethylprolyl PNA (aep-PNA) showed noticeable enhancements with regards to the daunting challenges mentioned above. Reported herein is the summary of our research endeavor to develop the CPNA oligomers with the great water-solubility and cell permeability. Chapter one briefly summarizs the background and history of the PNA as the front-runner of the antisense therapeutic agents. Chapter two discusses the novel protocols that enabled synthesis of the various versions of CPNA monomers for both Fmoc and Boc solid phase synthesis strategies. Chapter three includes the experimental procedures for solution phase preparation of the CPNA monomers. Chapter four starts with the introduction of solid phase synthesis strategy. After the brief review, our efforts on solid phase based synthesis of CPNA oligomers are discussed. Detailed procedures for the solid phase synthesis are summarized in Chapter five. Disclosed In the final chapter is a methodology which enables regioselective mono-acylation of hydrazines. Remarkably, this new protocol gives the mono-acylation on the less-reactive nitrogens of the hydrazines. Carbon disulfide takes the key role for this unique transformation. At the end of the dissertaion, selected NMR and Mass spectra are attached.

<p>Glycoconjugates are biologically important molecules with diverse functions. They consist of carbohydrates of varying size and complexity, attached to a non-sugar moiety as a lipid or a protein. Glycoconjugate structures are often very complex and their intricate biosynthetic pathways makes overexpression difficult. This renders the isolation of pure, structurally defined compounds from natural sources cumbersome. Therefore, to better address questions in glycobiology, synthetic glycoconjugates are an appealing alternative. In addition, synthetic methods allow for the preparation of non-natural glycoconjugates that can enhance the understanding of the influence of structural features on the biological responses.</p><p>In this thesis, synthetic methods for the preparation of glycoconjugates, especially glycolipid analogues, have been developed. These methods make use of solid-phase chemistry and are amenable to library synthesis of series of similar compounds. Solid-phase synthesis is a technique where the starting material of the reaction is attached to small plastic beads through a linker. This allows large excess of reagents to speed up the reactions and the sometimes difficult purifications of intermediate products are reduced to simple washings of the beads.</p><p>One problem with solid-phase synthesis is the difficulties to monitor the reactions and characterize the intermediate products. Gel-phase 19 F-NMR spectroscopy, using fluorinated linkers and protecting groups, is an excellent tool to overcome this problem and to monitor solid-phase synthesis of e.g. glycoconjugates. Two novel fluorinated linkers for the attachment of carboxylic acids have been developed and are presented in the thesis. These linkers can be cleaved with both acids of varying strengths and nucleophiles like hydroxide ions, and they are stable to glycosylation conditions. In addition, a novel filter reactor for solid-phase synthesis was designed. The reactor fits into an ordinary NMR spectrometer to facilitate the reaction monitoring with gel-phase 19 F-NMR spectroscopy.</p><p>The biological applications of the synthesized glycolipids were demonstrated in two different settings. The CD1d restricted binding of glycolipids carrying the monosaccharide α-GalNAc as carbohydrate could be detected on viable cells of mouse origin. CD1d is one of several antigen presenting molecules (the CD1 proteins) that presents lipids and glycolipids to circulating T-cells that in turn can initiate an immune response. The CD1 molecules are relatively sparsely investigated, and the method to measure glycolipid binding on viable cells, as described in the thesis, has the possibility to greatly enhance the knowledge of the structural requirements for CD1-binding.</p><p>Serine-based neoglycolipids with terminal carboxylic acids were used to prepare glycoconjugate arrays with covalent bonds to secondary amines on microtiter plates. Carbohydrate arrays have great possibilities to simplify the study of interactions between carbohydrates and e.g. proteins and microbes. The usefulness of the glycolipid arrays constructed in the thesis was illustrated with two lectins, RCA120 from Ricinus communis and BS-1 from Bandeiraea simplicifolia. Both lectins bound to the array of neoglycolipids in agreement with their respective specificity for galactosides.</p><p>Glycobiology is a large area of great interest and the methods described in this thesis can be used to answer a variety of glycoconjugaterelated biological questions.</p>

The three major biopolymers, proteins, nucleic acids and glycoconjugates are mainly responsible for the information transfer, which is a fundamental process of life. The biological importance of proteins and nucleic acids are well explored and oligosaccharides in the form of glycoconjugates have gained importance recently. The β-(1→4) linked N-acetylglucosamine (GlcNAc) moiety is a frequently occurring structural unit in various naturally and biologically important oligosaccharides and related conjugates. Chitin which is the most abundant polymer of GlcNAc is widely distributed in nature whereas the related polysaccharide chitosan (polymer of GlcN and GlcNAc) occurs in certain fungi. Chitooligosaccharides of mixed acetylation patterns are of interest for the determination of the substrate specificities and mechanism of chitinases. In this report, we describe the chemical synthesis of three chitotetraoses namely GlcNAc-GlcN-GlcNAc-GlcN, GlcN-GlcNAc-GlcNAc-GlcN and GlcN-GlcN-GlcNAc-GlcNAc. Benzyloxycarbonyl (Z) and p-nitrobenzyloxycarbonyl (PNZ) were used for the amino functionality due to their ability to form the β-linkage during the glycosylation reactions through neighboring group participation and the trichloroacetimidate approach was utilized for the donor. Monomeric, dimeric acceptors and donors have been prepared by utilizing the Z and PNZ groups and coupling between the appropriate donor and acceptors in the presence of Lewis acid yielded the protected tetrasaccharides. Finally cleavage of PNZ followed by reacetylation and the deblocking of other protecting groups afforded the N,N’-diacetyl chitotetraoses in good yield. Successful syntheses for the protected diacetyl chitotetraoses by solid phase synthesis have also been described. / Die drei wichtigsten Biopolymere sind Proteine, Nukleinsäuren und Glykokonjugate. Sie sind von fundamentaler Bedeutung für lebenswichtige Prozesse, wie z.B. den Informationstransfer. Die biologische Bedeutung von Proteinen und Nukleinsäuren ist eingehend erforscht, während Oligosaccharide in Form von Glykokonjugaten erst in neuerer Zeit an Bedeutung gewonnen haben. Die β-(1→4) verknüpfte N-Acetylglucosamin (GlcNAc) Einheit kommt häufig als in vielen natürlichen und biologisch wichtigen Oligosacchariden und ihren Konjugaten vor. Chitin, ein Polymer von GlcNAc, ist in der Natur weit verbreitet, während das verwandte Polysaccharid Chitosan (Polymer of GlcN und GlcNAc) in gewissen Pilzen auftritt. Chitooligosaccharide gemischter Acetylierungsmuster sind von Bedeutung für die Bestimmung von Substratwirkungen und für den Mechanismus von Chitinasen. In dieser Arbeit beschreiben wir die chemische Synthese von drei Chitotetraosen, nämlich GlcNAc-GlcN-GlcNAc-GlcN, GlcN-GlcNAc-GlcNAc-GlcN and GlcN-GlcN-GlcNAc-GlcNAc. Benzyloxycarbonyl (Z) und p-Nitrobenzyloxycarbonyl (PNZ) wurden aufgrund ihrer Fähigkeit, die β-Verknüpfung während der Glykosylierung durch die Nachbargruppenbeteiligung zu steuern, als Aminoschutzgruppen verwendet. Zur Aktivierung der Donoren wurde die Trichloracetamidat Methode angewendet. Monomere und dimere Akzeptoren und Donoren wurden unter Verwendung von Z und PNZ Gruppen hergestellt. Die Kupplung von geeigneten Donoren und Akzeptoren in Gegenwart einer Lewis Säure ergaben die Tetrasaccharide. Schließlich ergab die Entschützung von PNZ, gefolgt von der Reacetylierung der Aminogruppe und Abspalten der übrigen Schutzgruppen die N,N’-Diacetylchitotetraosen in guten Ausbeuten. Weiterhin wird die erfolgreiche Synthese der geschützten Diacetylchitotetraosen durch Festphasensynthese beschrieben.

Standard chemical DNA synthesis with isotope labels requires expensive reagents; moreover, a large excess of phosphoramadites (typically 50-100 fold) must be used. We developed a process where enzymatic cyclic solid phase synthesis of DNA allows for more economic reagent use. A DNA template was immobilized on an epoxy-activated solid support. This chemistry was chosen because the formed linkage is inert to high pH conditions. High efficiency of the covalent attachment was observed when the reaction was carried out in MgCl2/CAPS buffer. It was found that Mg2+ enables the reaction to be completed over a period of 14 h, compared to 72 h under standard conditions. DNA synthesis was carried in a cyclic fashion on a support bound DNA using Klenow fragment.

Glycoconjugates are biologically important molecules with diverse functions. They consist of carbohydrates of varying size and complexity, attached to a non-sugar moiety as a lipid or a protein. Glycoconjugate structures are often very complex and their intricate biosynthetic pathways makes overexpression difficult. This renders the isolation of pure, structurally defined compounds from natural sources cumbersome. Therefore, to better address questions in glycobiology, synthetic glycoconjugates are an appealing alternative. In addition, synthetic methods allow for the preparation of non-natural glycoconjugates that can enhance the understanding of the influence of structural features on the biological responses. In this thesis, synthetic methods for the preparation of glycoconjugates, especially glycolipid analogues, have been developed. These methods make use of solid-phase chemistry and are amenable to library synthesis of series of similar compounds. Solid-phase synthesis is a technique where the starting material of the reaction is attached to small plastic beads through a linker. This allows large excess of reagents to speed up the reactions and the sometimes difficult purifications of intermediate products are reduced to simple washings of the beads. One problem with solid-phase synthesis is the difficulties to monitor the reactions and characterize the intermediate products. Gel-phase 19 F-NMR spectroscopy, using fluorinated linkers and protecting groups, is an excellent tool to overcome this problem and to monitor solid-phase synthesis of e.g. glycoconjugates. Two novel fluorinated linkers for the attachment of carboxylic acids have been developed and are presented in the thesis. These linkers can be cleaved with both acids of varying strengths and nucleophiles like hydroxide ions, and they are stable to glycosylation conditions. In addition, a novel filter reactor for solid-phase synthesis was designed. The reactor fits into an ordinary NMR spectrometer to facilitate the reaction monitoring with gel-phase 19 F-NMR spectroscopy. The biological applications of the synthesized glycolipids were demonstrated in two different settings. The CD1d restricted binding of glycolipids carrying the monosaccharide α-GalNAc as carbohydrate could be detected on viable cells of mouse origin. CD1d is one of several antigen presenting molecules (the CD1 proteins) that presents lipids and glycolipids to circulating T-cells that in turn can initiate an immune response. The CD1 molecules are relatively sparsely investigated, and the method to measure glycolipid binding on viable cells, as described in the thesis, has the possibility to greatly enhance the knowledge of the structural requirements for CD1-binding. Serine-based neoglycolipids with terminal carboxylic acids were used to prepare glycoconjugate arrays with covalent bonds to secondary amines on microtiter plates. Carbohydrate arrays have great possibilities to simplify the study of interactions between carbohydrates and e.g. proteins and microbes. The usefulness of the glycolipid arrays constructed in the thesis was illustrated with two lectins, RCA120 from Ricinus communis and BS-1 from Bandeiraea simplicifolia. Both lectins bound to the array of neoglycolipids in agreement with their respective specificity for galactosides. Glycobiology is a large area of great interest and the methods described in this thesis can be used to answer a variety of glycoconjugaterelated biological questions.