Antibody combining sites are constructed from limited set of germ-line gene segments, yet are capable of both recognizing a broad range of common epitopes and eliciting an adaptive response to newly encountered pathogens. Carbohydrate antigens generally do not draw T cell help and concomitant affinity maturation in the humoral response. Therefore, anti-carbohydrate responses must rely more heavily on the primary germ-line gene repertoire. Antibodies are usually thought of as highly specific. It has been suggested that polyspecificity and cross-reactivity in germ-line antibodies is necessary to provide the protective mechanisms required to broaden the potential number of antigens recognized; however, the molecular mechanism underlying polyspecificity is poorly understood. To investigate the phenomena of specificity, cross-reactivity and polyspecificity in germ-line antibodies my thesis focuses first on the unique LPS inner core oligosaccharide of Chlamydiaceae, which contains variations within the conserved inner core trisaccharide Kdo(2→8)Kdo(2→4)Kdo (3-deoxy-D-manno-oct-2-ulosonic acid). Antibodies raised against this family-specific trisaccharide showed strong V-region restriction with two sets of heavy and light chain V genes accounting for almost all clones isolated. These groups were named after their prototypic clones as the ‘S25-2 type’ and the ‘S25-23 type’. In contrast to the cross-reactive S25-2 and related antibodies, the S25-23 family of antibodies were shown to be specific for the Chlamydiaceae-specific trisaccharide antigen with no cross-reactivity to Kdo mono or disaccharides or to the Kdo(2→4)Kdo(2→4)Kdo trisaccharide antigen. Interest in S25-23 was sparked by its rare high μM affinity and strict specificity for the family-specific trisaccharide antigen. The structures of the antigen binding fragments of four S25-23-type mAbs have been determined to high resolution in complex with the Chlamydiaceae-specific epitope, revealing the molecular basis for their binding behaviour. The germ-line-encoded paratopes of these antibodies differ significantly from previously characterized S25-2-type mAbs. Unlike the terminal Kdo recognition pocket that promotes cross-reactivity in S25-2-type antibodies, S25-26 and the closely related S25-23 utilize a groove composed of germ-line residues to recognize the length of the trisaccharide antigen. Further S25-23-type antibodies are glycosylated on the variable heavy chain. Analysis of the glycan reveals a heterogeneous mixture with a common root structure that contains an unusually high number of terminal αGal-Gal moieties. One of the unliganded structures in S25-26 shows significant order in the glycan with appropriate electron density for nine residues. The elucidation of the three-dimensional structure of a Gal(α1→3)Gal containing N-linked glycan on a mAb variable heavy chain has potential clinical interest, as it has been implicated in allergic responses in patients receiving therapeutic antibodies. The second focus of my thesis research is the lipid A moiety of LPS, which is involved in septic shock. Though the lipid A epitope appears to be cryptic during infection with Gram-negative bacteria, there have been several reported instances of lipid A specific antibodies isolated from human sera. While these antibodies are strictly selective for lipid A, there are reports of polyspecificity of some anti-lipid A antibodies for single stranded DNA. In such cases, the breakdown of negative selection through polyspecificity has been reported to result in the unfortunate consequences of autoimmune disease. This thesis reports the first crystal structures of antibodies in complex with lipid A and single stranded nucleic acids, elucidating their mechanism for polyspecificity. Perhaps more importantly, the structures may yield clues to the genesis of autoimmune diseases such as systemic lupus erythematosus, thyroiditis, and rheumatic autoimmune diseases. / Graduate / 2020-04-18 / 0487 / 0982

Lithium bis(amido)cuprates are an important class of bimetallic base, which can chemo- and regioselectively metalate aromatic compounds, via directed ortho cupration (DoCu). This thesis begins with an introduction to aspects of the chemistry of organolithium compounds, group 11 organometallic compounds and their lithium 'ate complexes. Examples of such synergic bases are presented and the introduction is concluded with a discussion of lithium bis(amido)cuprate bases, which along with their silver congeners, are the subject of this dissertation. In general, syntheses involve the addition of a lithium amide to a group 11 salt, resulting in the formation of a lithium bis(amido)cuprate or argentate. Structurally focussed work commences with the use of new amide ligands to develop heteroleptic bis(amido)cuprate systems. The reaction of mixtures of lithium amides with CuBr provides a series of novel Lipshutz-type and Gilman cuprates. Interesting structural features are uncovered, which are rationalised in terms of altered steric demands in the newly introduced amide ligands in these systems. CuSCN and CuOCN are investigated as inexpensive and safer alternatives to CuCN in cuprate formation. In the solid state, a series of Lipshutz-type cuprates (TMP)2Cu(SCN)Li2(L) (L = Et2O, THF, THP) are revealed, whose molecular conformations are infuenced by the identity of the Lewis base. However, in benzene solution, in situ conversion of Lipshutz-type to Gilman cuprate is found to occur. Moving to the synthetic setting, derivatisation of chloropyridines is attempted and gives functionalised halopyridines in 51-71 % yield. CuOCN is found to behave quite differently when reacted in the same way as CuSCN, whereby X-ray crystallography reveals structures in which Cu-Li substitution is apparent. The unique reactivity of CuOCN is interpreted with the aid of multinuclear NMR spectroscopy. A new route to Lipshutz-type cuprates is explored by the synthesis of (TMP)2Cu(OCN)Li2(THF) from Gilman cuprate and LiOCN. This avoids Cu-Li substitution. Meanwhile, reaction of lithium N,N-diisopropylamide with CuOCN also avoids metal disorder, to give a novel lithium cuprate-lithium amide adduct. Further advances in our understanding of group 11 'ate complexes are made by introducing silver as a spectroscopically active nucleus in the lithium argentates (TMP)2AgLi and (TMP)2Ag(CN)Li2(THF). In the solid state, these parallel the structures known for Gilman cuprate (TMP)2CuLi and Lipshutz cuprate (TMP)2Cu(CN)Li2(THF), respectively. In solution, NMR spectroscopy reveals features consistent with retention of these structures. Lastly, the formation of mixed Cu-Li aggregates from combining TMPLi and TMPCu in aromatic solvent are investigated. Surprising reactivity is uncovered, in which the aromatic solvent is metalated and incorporated into mixed-metal aggregates. This thesis concludes with a summary of the findings and suggestions for future work, including how the findings presented herein may be transformed into practical improvements to cuprate systems. In particular, the possibility that Gilman cuprate may be activated towards the metalation of aromatic substrates by the addition of sub-stoichiometric or catalytic amounts of a lithium salt additive is explored.

Glycosyltransferase enzymes synthesize glycosidic linkages, generating carbohydrates and carbohydrate-linked entities ranging from cellulose, starch, and chitin to glycolipids, glycopeptides, and natural product antibiotics. These syntheses involve stereo- and regio-specific sugar transfer from an activated donor molecule, often a UDP-sugar, to an acceptor molecule. Functionally, glycosyltransferases are classified as either “retaining” or “inverting” enzymes depending on whether the stereochemical linkage of the donor substrate is conserved in the product. While inverting glycosyltransfer is mechanistically straightforward, the retaining mechanism remains poorly understood. For retaining glycosyltransferases, the central question is whether transfer occurs via a front-face “SNi-like” mechanism or through a ‘double displacement’ mechanism that invokes a glycosyl-enzyme covalent intermediate. GTA and GTB are retaining enzymes that catalyze the final step in human ABO(H) blood group A and B antigen synthesis through UDP-GalNAc or UDP-Gal transfer, respectively, to the H-antigen disaccharide acceptor. Although they have been intensively characterized, the processes of substrate recognition, mobile loop organization, and product release in GTA and GTB has long resisted explanation. Further, the question of the retaining enzyme mechanism persists, though the covalent intermediate of the proposed double displacement mechanism has been detected via mass spectrometry experiments with GTA/GTB mutants. Building on previous investigations, we have aimed to characterize and have uncovered details of mechanism, substrate binding, loop organization, and product release using a combined kinetic and structural approach. These investigations are essential not only for understanding GTA, GTB, and retaining glycosyltransferases as a whole, but also for the rational design of inhibitors. Such inhibitors could selectively target, for example, bacterial glycosyltransferases and thus would represent a new class of antimicrobials. / Graduate

No description available.

This thesis reports work in three areas of X-ray crystallography. An initial chapter describes the structure of a protein, the methods based on the use of X-rays and computer analysis of diffraction patterns to determine crystal structure, and the subsequent derivation of the structure of part or all of a protein molecule. Work to determine the structure of the protein cytokine receptor-like factor 3 (CRLF3) leading to the successful generation of a structural model of a significant part of this molecule is then described in Chapter 2. A variety of techniques had to be deployed to complete this work, and the steps undertaken are described. Analysis was performed principally using phaser, using maximum likelihood methods. Areas for improvement in generating non-crystallographic symmetry (NCS) operators in existing programmes were identified and new and modified algorithms implemented and tested. Searches based on improved single sphere algorithms, and a new two-sphere approach, are reported. These methods showed improvements in many cases and are available for future use. In Chapter 4, work on determining the relative importance of low resolution and high intensity data in molecular replacement solutions is described. This work has shown that high intensity data are more important than the low resolution data, dispelling a common perception and helping in experimental design.

Imidazole was reacted with 3,1-tetrakis (N-phenlyacetamidato) dirhodium (II) to explore the chemistry of asymmetric dirhodium catalysts. The imidazolo 3,1-tetrakis (Nphenlyacetamidato) dirhodium (II) complex was synthesized and then characterized by Nuclear Magnetic Resonance and Ultraviolet-Visible spectroscopies as well as by single crystal X-ray Diffraction. Additionally, one copper and two molybdenum model cofactors were characterized by XRD to better understand their structure/function relationships. NMR results gave evidence of the formation of the 3,1-imidazole complex, and UV-Vis indicated that even in large excess imidazole was coordinated only to one axial site. The structure of the 3,1-imidazole complex was confirmed by XRD with the following refinement indicators: R1: 3.97%, wR2: 9.27%, GooF: 1.036. Model cofactors were also characterized by XRD and resulted in the following refinement indicators for Mo-1: R1: 4.27%, wR2: 9.15%, GooF: 1.074; for Cu-1: R1: 10.10%, wR2: 22.60%, GooF: 0.991, and for Mo-2: R1: 17.75%, wR2: 46.08%, GooF: 0.954.

Crystal engineering has recently emerged as a method of choice for the design and the construction of functional materials. Solid-state synthesis, of the most commonly studied aspects of crystal engineering, has been shown to provide access to molecular targets that are hardly obtainable using principles of conventional (i.e. solution-based) organic synthesis. Reactions in the solid state are, however, not routinely used in organic synthetic chemistry. The scarce use of solid-state reactions can be attributed to the difficulty of predicting molecular arrangements in the solid state, as well as to the lack of methodologies to control crystal packing. Template-directed solid-state synthesis is a recently developed modus operandi that enables control over reactivity within multi-component crystals. The thesis is focused on the application of template-directed solid-state approach to [2+2] photocycloaddition reactions in the solid state, as well as on the understanding of intermolecular interactions in crystals. Synthetic templates have been utilized to construct cocrystals that enable a class of hitherto underdeveloped organic solid-state reactions, namely [2+2] cross-photoaddition reactions. In addition, products derived form templated solid state reactions, namely tetrapyridylcyclobutanes, have been utilized to generate exceptional materials, such as thixothropic hydrogels based on nano-dimensional metal-organic particles. The utility of crystal engineering has also been expanded to the nanoscience and the development of nanomaterials. A crystallization method for the preparation of nano-dimensional cocrystals has been developed. The method has been shown to enable single-crystal-to-single-crystal [2+2] photodimerizations of olefins. In addition, nano-dimensional cocrystals have been shown to exhibit distinctive mechanical properties upon single-crystal-to-single-crystal transformations. In addition to solid-state reactions and materials derived therefrom, we systematically studied hierarchies of supramolecular synthons in pharmaceutical cocrystals comprised of multi-functional molecules. Pharmaceutical cocrystals have been recently shown to exhibit physical properties superior to those of parent drugs. Our studies involved xanthine alkaloids as pharmaceutical agents and a series of hydroxylated benzoic acids as cocrystal formers. Synthon hierarchies have been established for three xanthine alkaloids. We also discovered pharmaceutical salts that formed where cocrystallization was expected to occur. Reasons contributing to such unexpected salt formation were investigated using X-ray crystallography and computational methods. The established synthon hierarchies are expected to contribute to a better understanding of self-assembly processes in cocrystals that is crucial for the development of state-of-art drugs, and the design of organic reactions in the solid state.

The focus of this dissertation is on the interactions of compounds containing group 12 and 16 elements. This work is presented in three major parts. First, the interaction of the synthetic dithiol N,N’-bis(2-mercaptoethyl)isophthalamide), abbreviated BDTH2, with selenite. Second, the interaction of cysteine with Cd(II) and the biologically relevant Cd-Cysteine crystal structure. Third, the green synthesis of CdSe quantum dots (QDs). The interaction of BDTH2 with selenite is different from the interactions with other metals and metalloids previously studied. Under ambient conditions, BDTH2 is oxidized to the disulfide, BDT(S-S), while selenite is reduced to elemental selenium. However, under carefully controlled conditions, the reaction of BDTH2 with selenite produces a mixture of BDT(S-S) and the covalently bound Se(II) species, BDT(S-Se-S). While the mixture could not be separated, experimental 77Se NMR combined with computational analysis confirmed the presence of BDT(S-Se-S). The interaction of the amino acid cysteine with Cd(II) was studied as a means to sequester, and potentially recycle, Cd(II) from bulk CdS waste. Single crystals of Cd(Cys)Cl·H2O were grown, and the crystal structure determined. Surprisingly, this is only the second structure to be determined by X-ray crystallography of a compound containing the Cd-Cysteine unit. Not only does this structure have biological relevance, but it also corrects a structure proposed in 1965. Using the knowledge gained from studying the interaction of BDTH2 with selenite, a green synthesis of water-soluble CdSe QDs by the reaction of selenite with Cd(Cys)Cl·H2O in water at room temperature was developed. This green method for the synthesis of CdSe QDs was extended to ZnSe and HgSe QDs. The mechanism of CdSe formation was investigated using Cd(II) combined with various thiols.

The emergence and proliferation of Gram-negative bacteria expressing β-lactamases is a significant threat to human health. β-Lactamases are enzymes that degrade the β-lactam antibiotics (e.g., penicillins, cephalosporins, monobactams, and carbapenems) that we use to treat a diverse range of bacterial infections. Specifically, β-lactamases catalyze a hydrolysis reaction where the β-lactam ring common to all β-lactam antibiotics and responsible for their antibacterial activity, is opened, leaving an inactive drug. There are two groups of β-lactamases: serine enzymes that use an active site serine residue for β-lactam hydrolysis and metalloenzymes that use either one or two zinc ions for catalysis. Serine enzymes are divided into three classes (A, C, and D), while there is only one class of metalloenzymes, class B. Clavulanic acid, sulbactam, and tazobactam are β-lactam-based BLIs that demonstrate activity against class A and C β-lactamases; however, they have no activity against the class A KPC and MBLs, NDM and VIM. Avibactam and vaborbactam are novel BLIs approved in the last two years that have activity against serine carbapenemases (e.g., KPC), but do not inhibit MBLs. The overall goals of this project were to use X-ray crystallography to study the catalytic mechanism of serine β-lactamases with β-lactam antibiotics and to understand the mechanisms behind the broad-spectrum inhibition of class A β-lactamases by avibactam and vaborbactam. This project also set out to find novel inhibitors using molecular docking and FBDD that would simultaneously inactivate serine β-lactamases and MBLs commonly expressed in Gram-negative pathogenic bacteria. The first project involved examining the structural basis for the class A KPC-2 β-lactamase broad-spectrum of activity that includes cephalosporins and carbapenems. Three crystal structures were solved of KPC-2: (1) an apo-structure at 1.15 Å; (2) a complex structure with the hydrolyzed cephalosporin, cefotaxime at 1.45 Å; and (3) a complex structure with the hydrolyzed penem, faropenem at 1.40 Å. These complex structures show how alternative conformations of Ser70 and Lys73 play a role in the product release step. The large and shallow active site of KPC-2 can accommodate a wide variety of β-lactams, including the bulky oxyimino side chain of cefotaxime and also permits the rotation of faropenem’s 6-alpha-1R-hydroxyethyl group to promote carbapenem hydrolysis. Lastly, the complex structures highlight that the catalytic versatility of KPC-2 may expose a potential opportunity for drug discovery. The second project focused on understanding the stability of the BLI, avibactam, against hydrolysis by serine β-lactamases. A 0.83 Å crystal structure of CTX-M-14 bound by avibactam revealed that binding of the inhibitor impedes a critical proton transfer between Glu166 and Lys73. This results in a neutral Glu166 and neutral Lys73. A neutral Glu166 is unable to serve as a general base to activate the catalytic water for the hydrolysis reaction. Overall, this structure suggests that avibactam can influence the protonation state of catalytic residues. The third project centered on vaborbactam, a cyclic boronic acid inhibitor of class A and C β-lactamases, including the serine class A carbapenemase KPC-2. To characterize vaborbactam inhibition, binding kinetic experiments, MIC assays, and mutagenesis studies were performed. A crystal structure of the inhibitor bound to KPC-2 was solved to 1.25 Å. These data revealed that vaborbactam achieves nanomolar potency against KPC-2 due to its covalent and extensive non-covalent interactions with conserved active site residues. Also, a slow off-rate and long drug-target residence time of vaborbactam with KPC-2 strongly correlates with in vitro and in vivo activity. The final project focused on discovering dual action inhibitors targeting serine carbapenemases and MBLs. Performing molecular docking against KPC-2 led to the identification of a compound with a phosphonate-based scaffold. Testing this compound using a nitrocefin assay confirmed that it had micromolar potency against KPC-2. SAR studies were performed on this scaffold, which led to a nanomolar inhibitor against KPC-2. Crystal structures of the inhibitors complexed with KPC-2 revealed interactions with active site residues such as Trp105, Ser130, Thr235, and Thr237, which are all important in ligand binding and catalysis. Interestingly, the phosphonate inhibitors that displayed activity against KPC-2, also displayed activity against the MBLs NDM-1 and VIM-2. Crystal structures of the inhibitors complexed with NDM-1 and VIM-2 showed that the phosphonate group displaces a catalytic hydroxide ion located between the two zinc ions in the active site. Additionally, the compounds form extensive hydrophobic interactions that contribute to their activity against NDM-1 and VIM-2. MIC assays were performed on select inhibitors against clinical isolates of Gram-negative bacteria expressing KPC-2, NDM-1, and VIM-2. One phosphonate inhibitor was able to reduce the MIC of the carbapenem, imipenem 64-fold against a K. pneumoniae strain producing KPC-2. The same phosphonate inhibitor also reduced the MIC of imipenem 4-fold against an E. coli strain producing NDM-1. Unfortunately, no cell-based activity was observed for any of the phosphonate inhibitors when tested against a P. aeruginosa strain producing VIM-2. Ultimately, this project demonstrated the feasibility of developing cross-class BLIs using molecular docking, FBDD, and SAR studies.

Abstract not available