No description available.

Thesis advisor: Mary F. Roberts / Myo-inositol and its phosphorylated derivatives are found across all domains of life, and these molecules play crucial roles in a wide variety of cellular processes. While the biosynthesis of inositol is an evolutionarily conserved pathway, there are a wide variety of enzymes that use inositol and its derivatives as substrates. This thesis explores two such enzymes; a phosphatidylinositol- specific phospholipase C (PI-PLC) produced by <i>Staphylococcus aureus</i>, and AF2372, a dual action inositol monophosphatase/ fructose bisphosphatase produced by the <i>Archaeoglobus fulgidus</i>. At the outset of this work, the structure of the <i>S. aureus</i> PI-PLC was unknown, but some interesting biochemical properties about the enzyme had been observed. The structure of AF2372 had been reported, but a structure had not yet been solved in the presence of osmolytes known to thermoprotect the enzyme. Both the <i>S. aureus</i> PI-PLC and AF2372 catalyze the cleavage of phosphorylated inositol compounds, but share no mechanistic, structural, or taxonomical similarities. Protein crystallography is a powerful tool, and with it I have been able to study these two enzymes at a molecular level, providing insight into complex biological questions about each enzyme. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The halogen bond has recently risen in prominence as a non-covalent interaction for use in supramolecular chemistry, allowing for the rational design of materials, pharmaceuticals, and functional molecules. The occurrence of the σ-hole opposite to the C-X covalent bond (X = F, Cl, Br, I) renders the halogen bond a highly directional and tuneable interaction, offering desirable features to crystal engineers. The halogen bond can be divided into its two components: the halogen bond donor bearing the halogen atom, and the electron-rich halogen bond acceptor. In this thesis, we investigate the nature of the halogen bond, its role in supramolecular assembly and impact on the local dynamics, along with developing synthetic methods to prepare this class of materials. We begin by fully characterizing the halogen bond donor by using 35Cl ultra-wideline solid-state nuclear magnetic resonance (NMR) spectroscopy on a series of single-component chloronitriles exhibiting the C-Cl···N halogen bond. We then perform the first modern nuclear quadrupole resonance (NQR) investigations of the halogen bond, observing the 79/81Br and 127I nuclei in a series of cocrystals exhibiting the C-Br···N and C-I···N halogen bond, respectively. Computational results attribute the observed increases in the quadrupolar coupling constants (CQ) to a reduction in the carbon-halogen σ-bonding contribution to V33 and an increase in the lone-pair and core orbital contributions, providing the first model of the electronic changes occurring on the halogen bond donor upon the formation of the halogen bond. Attention is then turned on characterizing the halogen bond acceptor and its surrounding environment, beginning by investigating a solid-state NMR approach relying on the 19F nucleus to characterize perfluorinated cocrystals. This strategy has reduced analysis times from hours to minutes while providing higher sensitivity and resolution, with the resulting chemical shifts permitting the unambiguous identification of the halogen bond and allowing for the refinement of X-ray crystal structures. The halogen bond acceptor is then investigated in a series of isomorphous dimers exhibiting both the halogen bond and hydrogen bond in the C≡C-I···X-···H-N+ motif, revealing the halogen bond’s relative contribution to the electric field gradient increasing in the order of Cl- > Br- > I-, contrasting the contributions of the hydrogen bond. We then explore the impact of the halogen bond on the surrounding environment, using the rotating methyl groups of 2,3,5,6-tetramethylpyrazine as a model. Upon the introduction of a halogen bond, we observe a reduction in the rotational energy barrier of 56% on average, overshadowing the 36% reduction observed in the hydrogen bonded cocrystals. This is the first instance of the halogen bond directly catalyzing the local dynamics, coining the term “dynamics catalyst”. These results provide an effective strategy of enhancing the dynamics in molecular systems, such as molecular machines, supramolecular catalyst, as well as correcting the faulty dynamics encountered in diseased proteins. The role of halogen bonding in crystal engineering is then explored, reporting the first supramolecular triangle, a series of discrete charged dimers, and supramolecular architectures built from 1,3,5-tri(iodoethynyl)-2,4,6-trifluorobenzene, with the potential of creating fully organic porous structures for gas absorption. Mechanochemistry is then investigated as a synthetic method, allowing for the preparation of cocrystals featuring 3-iodoethynylbenzoic acid as the donor, with the resulting structures exhibiting concurrent halogen and hydrogen bonding. Mechanochemical ball milling is shown to reduce preparation times of powdered cocrystals from days to a single hour, while using a fraction of the organic solvent. Lastly, we pioneer cosublimation as a solvent-free synthetic technique for rapidly preparing halogen bonded cocrystals, yielding quality single crystals within a few hours, and a microcrystalline product within 15 minutes. Among its advantages, cosublimation offers a significant acceleration of discovery, while eliminating the environmental footprint associated with conventional synthetic methods.

Thesis (MTech (Chemistry))--Cape Peninsula University of Technology, 2016. / Inclusion compounds of two hydroxyl hosts with a variety of guests have been investigated. These host compounds are bulky molecules and have the ability to interact with smaller organic guests to form new compounds. The host 9-(1-naphthyl)-9H-xanthen-9-ol (H1), forms inclusion compounds with pyridine (PYR), N,N-dimethylacetamide (DMA), morpholine (MORP) and N-methyl-2-pyrrolidinone (NMP). The crystal structures of H1•NMP, H1•DMA and H1•MORP1 were successfully solved in the triclinic space group PĪ, whereas the inclusion compound H1•PYR crystallised in the monoclinic space group P21/c. A different inclusion compound involving morpholine, H1•MORP2 resulted from dissolution of H1 in a 1:1 molar ratio of MORP: DMA. H1•MORP2 crystallised in the space group PĪ. All of the abovementioned inclusion compounds demonstrated a host: guest ratio of 1:1 except for H1•MORP1 (host: guest ratio = 1: ). H1 interacts with pyridine and morpholine guests via (Host)O-H•••N(Guest) hydrogen bonds and via (Host)OH•••O(Guest) hydrogen bonds with N-methyl-2-pyrrolidinone and N,N-dimethylacetamide.

by Mok-Yin Chow. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 55-58). / Acknowledgement --- p.1 / Abstract --- p.2 / Contents --- p.3 / List of Figures --- p.4 / List of Tables --- p.5 / Chapter 1. --- Introduction --- p.6 / Chapter I. --- Chemistry of pseudohalides --- p.6 / Chapter II. --- Infrared spectroscopy of pseudohalides --- p.8 / Chapter III. --- Chemistry of metal carboxylates --- p.10 / Chapter IV. --- Infrared spectroscopy of carboxylates --- p.13 / Chapter V. --- Chemistry of betaine ligands --- p.14 / Chapter VI. --- Objectives of this research --- p.15 / Chapter 2. --- Experimental --- p.17 / Chapter I. --- Preparation --- p.17 / Chapter II. --- X-ray crystallography --- p.21 / Chapter 3. --- Results and discussion --- p.23 / Chapter I. --- "Isostructural complexes Co2(bet)2(N3)4 1,Zn2(bet)2(N3)42, Cd2(bet)2(N3)4 3, and Cd2(bet)2(NCO)4 4" --- p.23 / Chapter II. --- Copper(II) complex Cu2(bet)2(N3)2(N03)2 5 --- p.29 / Chapter III. --- Cadmium(II) complexes Cd3(bet)4(SCN)6(H20)2 6 and Cd(prbet)(NCS)2 7 --- p.34 / Chapter IV. --- Barium(II) complex Ba(pybet)2(NCS)2 8 --- p.43 / Chapter V. --- Cobalt(II) complex [Co(pybet)2(NCS)(H20)3]2[Co(NCS)4] 9 --- p.49 / Chapter VI. --- Conclusion --- p.53 / References --- p.55 / Publications based on work reported in this thesis --- p.59 / Appendix --- p.60

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 1981. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND SCIENCE. / Vita. / Includes bibliographical references. / by Thomas Clifford Alber. / Ph.D.

No description available.

The reaction between the lithium enolate of cyclohexanone and phenyl vinyl sulfoxide resulted in the formation of the novel bicyclooctanol sulfoxides 215-217 and the monoalkylated sulfoxide 218. The effects of variation in reaction time, temperature and concentration were studied. Under optimal conditions (10 minutes, -10°C and 0.085 M) the ratio of the bicyclooctanol sulfoxides 215-217 (75% yield) to the monoalkylated sulfoxide 218 was 95:5. The bicyclooctanol sulfoxides 215-217 were characterised as the sulfone derivatives, bicyclooctanol sulfones 219 and 220. X-ray crystal structures were used to determine the relative stereochemistry of the bicyclooctanol sulfoxides 215-217 and the bicyclooctanol sulfones 219 and 220. Bicyclo[4.2.0]octano-1-ol formation was determined to occur via an ionic mechanism. Mechanistic studies were carried out using variations in reaction lighting and reaction solvent, conducting the reaction in the presence of a radical trap and quenching the reaction with a deuterium label. The role of the counterion was determined to be important in the formation of the bicyclooctanol sulfoxides 215-217. Sequestering lithium ions with HMPA and substituting lithium with potassium favoured alkylation. Substituting the lithium enolate of cyclohexanone with the dimethylaluminium enolate of cyclohexanone resulted in a different distribution of the bicyclooctanol sulfoxides 215-217 and the formation of bicyclooctanol sulfoxide 243. Transition states to account for these differences have been proposed. The stability of the bicyclooctanol sulfoxides under conditions of acid, base and heating was studied. Thermal ring opening of the bicyclooctanol sulfoxides 215 and 216 to the monoalkylated sulfoxides 218A and 218B respectively occurred with retention of the configuration at sulfur. The relative stereochemistry of the individual bicyclooctanol sulfoxides 215-217 was considered to account for the observed stability in each case. The reaction between the lithium enolate of cyclohexanone and (R)-(+)-p-tolyl vinyl sulfoxide 193 gave the bicyclooctanol tolyl sulfoxides 246, 251 and 252 and the monoalkylated tolyl sulfoxide 247. This showed that both bond rotation in the side chain of the intermediate and epimerisation at sulfur occurred in the bicyclo[4.2.0]octan-1-ol forming process. The presence of the sulfoxide functionality in phenyl vinyl sulfoxide was determined to be crucial to the formation of bicyclo[4.2.0]octan-1-ols. In the reaction with the lithium enolate of cyclohexanone, phenyl vinyl sulfide gave no reaction, phenyl vinyl sulfone gave the bicyclic disulfones 260-265, ethyl acrylate gave the diesters 266-268 and diphenylvinylphosphine oxide gave the phosphine oxide 269. The cyclobutanol 270 and the ketone 271 were the products resulting from the reaction between the reaction between the lithium enolate of acetophenone and phenyl vinyl sulfoxide. This demonstrated potential scope for the cyclisation process using both cyclic and acyclic ketones.

RNA metabolism includes all the processes required for RNA synthesis, maturation, and degradation in living cells. Ribonucleases (RNases) are involved in RNA maturation and degradation, two essential processes in gene expression and regulation in both prokaryotes and eukaryotes. Oligoribonuclease (Orn) has an important role in eliminating small oligonucleotides (nano-RNA), the last step in mRNA degradation. In E. coli, Orn is the only essential exoribonuclease. The enzyme has been shown to form a stable dimer, both in solution and in the crystalline form. Analysis of the three-dimensional structure of Orn allowed us to hypothesize that dimerization is essential for enzyme catalysis. In order to test the hypothesis, I analyzed a number of deletion and point mutants of Orn and determined that tryptophan 143 is essential for dimerization. A W143A mutant is unable to dimerize and has very little activity, similar to that of an active site mutant (D162A). The atomic structure of the W143A mutant, solved at a resolution of 1.9 Å, showed that although the overall three-dimensional fold is similar to that of the wild-type protein, minor differences exist that could account for the monomeric behavior in solution. A flexible Arg174 is repositioned into the cavity created by the missing Trp143. In this new orientation Arg174 protrudes into a hydrophobic pocket in the dimerization interface and is proposed to produce sufficient unfavorable interactions to keep the monomers apart in solution. All these data suggest that dimerization of Orn is essential for its activity. The human homolog of Orn, also known as small fragment nuclease (Sfn), has been shown to degrade short single-stranded RNA, the last step in mRNA decay. In order to determine the mechanism of action of Sfn and its role in the cell, we solved the crystal structure of a truncated form of Sfn at a resolution of 2.6 Å. This mutant form of Sfn lacks the C-terminal 21 amino acids (Sfn-∆C21) yet is as efficient as full length Sfn on model substrates. Interestingly, Sfn is not as active as E. coli Orn in in vitro assays. Analysis of the atomic structure revealed that the active site cleft in Sfn is narrower than the corresponding active site in E. coli. We propose a model for how this narrower cleft may explain the lower in vitro activity. Bacillus subtilis does not have an Orn homolog and until recently, the enzyme responsible for nano-RNA degradation in this organism was unknown. YtqI (also termed nrnA or nanoRNase), a protein unrelated to E. coli Orn, was recently shown to be responsible for the digestion of oligonucleotides in B. subtilis. In order to better understand the mechanism of action of YtqI, I solved its crystal structure at a resolution of 2.0 Å. The nuclease has a RecJ-like fold with two globular domains connected via a flexible linker that forms a central groove. On one side of the groove, the larger N-terminal domain harbors the putative active site, while on the opposite side, the C-terminal domain includes a putative RNA binding domain. The structure of YtqI provides insights into how this enzyme binds and digests oligoribonucleotides. The studies described here provide a better understanding of the mechanism of action for several exoribonucleases that act on nano-RNA oligonucleotides - Oligoribonuclease from E. coli, its close homolog in humans (Small fragment nuclease), as well as a functional homolog in Bacillus (YtqI). This work is relevant to understanding RNA metabolism, which is an essential process for survival of both eukaryotic and prokaryotic organisms.

Alginate is an exopolysaccharide secreted by Pseudomonas aeruginosa and is a major component of biofilms that infect the lungs of cystic fibrosis patients. Ten proteins have been implicated in alginate polymerization, modification and export, and are believed to assemble into a multi-protein complex that spans the cell envelope and coordinates the synthesis and secretion of alginate. AlgK is a protein encoded in the alginate biosynthetic operon, which is required for the secretion of high-molecular weight alginate. This study describes structural and functional studies of AlgK to improve our understanding of AlgK’s role in alginate biosynthesis. To shed light on the function of AlgK, C14-palmitic acid labeling and sucrose gradient fractionation studies confirmed that AlgK is an outer membrane lipoprotein. Cellular fractionation experiments also found that AlgK is involved in the proper localization of AlgE, the alginate secretion pore in the outer membrane. The structure of AlgK was determined to 2.5 Å resolution by X-ray crystallography and revealed that the protein folds into 22 alpha-helices that pack into a flexible right-handed solenoid. Closer examination of the amino acid sequence revealed that AlgK carries 9.5 tetratricopeptide repeat (TPR)-like elements. Given the role that TPR motifs generally play in protein-protein interaction and the assembly of multi-protein complexes, the presence of these motifs in AlgK suggests that it can bind to one or more proteins. Based on the results presented in this study, we propose that AlgK acts as a scaffold for the assembly of the alginate secretion complex. By mapping highly conserved residues onto the surface of our model, three putative sites of protein-protein interaction were identified. We hypothesize that the N-terminus of AlgK binds to AlgE in the outer membrane, and the C-terminus of AlgK binds to periplasmic and/or inner membrane Alg proteins, thereby acting as a linker between the inner and outer membrane components of the alginate biosynthetic complex. We further hypothesize that together AlgE and AlgK constitute a novel exopolysaccharide secretin. The alginate biosynthetic complex appears to be distinct from the canonical capsular polysaccharide systems currently described.