Abstract not available

Abstract not available

Arsonoacetic acid, H₂O₃As¹CH₂COOH, and arsenoacetic acid, punitively [AsVCH₂COOH]₂ have been synthesised according to historical literature methods, and have been characterised using modern techniques. Arsonoacetic acid was shown by an X-ray crystal structure analysis to be a molecular species with an extensive hydrogen bonding network in the crystal. Arsenoacetic acid proved to be more enigmatic. Electrospray mass spectra suggested it consisted of a mixtures of rings (RAs)n, n = 3-11, with n = 6 dominating. This was partly confirmed by a crystal structure of (AsCH₂COOH)₆ (as the pyridine solvate). On the other hand, ¹H and ¹³C NMR gave spectra that indicated only a single component for arsenoacetic acid. The formation of crystals of the high temperature phase of elemental sulfur, β-S8, at ambient temperature is also discussed.

This thesis describes the investigation of the coordination and supramolecular chemistry of three different types of pyridine-containing ligand with a selection of Ag(I), Cu(I), Cu(II) and Cd(II) salts. The ligand types are flexible and four-armed, rigid and four-armed and bent with two rigid arms. All the ligands also display the ability to form additional supramolecular interactions. Chapter one introduces supramolecular chemistry and crystal engineering and covers background on several areas of current interest in these fields. Network structures, both coordination polymers and hydrogen-bonded systems, are discussed and topological analysis as a method of describing and comparing network structures is introduced. An outline of the ligand design, choice of transition metals and anions is given. Chapter two provides a review of flexible four-armed pyridine-containing ligands and their use in coordination chemistry. The synthesis and characterisation of three flexible four-armed ligands 1,2,4,5-tetrakis(2-pyridylmethyl-sulfanylmethyl)benzene (2tet), 1,2,4,5-tetrakis(3-pyridylmethyl-sulfanylmethyl)benzene (3tet) and 1,2,4,5-tetrakis(4-pyridylmethyl-sulfanylmethyl)benzene (4tet) are given. The synthesis and characterisation of the Ag(I), Cu(II) and Cd(II) complexes formed with these three ligands are also given. The complex of [Cd(2tet)(NO₃)₄] was structurally characterised by X-ray diffraction and was found to be a discrete species. The complexes {[Ag₂(3tet)](ClO₄)₂}n̲, {[Ag₂(3tet)](PF₆)₂}n̲, {[Ag₂(3tet)](CF₃CO₂)₂}n̲, {[Ag₂(4tet)]-(ClO₄)₂�2MeCN�2CHCl₃}n̲, {[Ag₂(4tet)](PF₆)₂�6MeCN}n̲ and {[Ag₂(4tet)](ClO₄)₂-�3H₂O}n̲ were likewise structurally characterised by X-ray diffraction. All these complexes were three-dimensional coordination polymers. A comparison of the seven structures is given at the end of the chapter. Chapter three reviews rigid four-armed pyridine-containing ligands and their use in coordination chemistry. The preparation of the rigid four-armed ligand 2,3,4,5-tetrakis(4-pyridyl)thiophene (pyth) is given. The synthesis and characterisation of the Ag(I), Cu(I) and Cd(II) complexes formed with this ligand are also given. The complexes [Ag(pyth)](BF₄)�3MeCN�CH₂Cl₂}n̲, [Ag(pyth)](PF₆)�MeCN�CH₂Cl₂}n̲, [Ag(pyth)]-(CF₃SO₃)�2MeCN�CH₂Cl₂}n̲, [Cu(pyth)](PF₆)�MeCN�CH₂Cl₂}n̲ and [(Cu₂I₂)(pyth)]-(BF₄)�1/2CH₂Cl₂�H₂O}n̲ were structurally characterised by X-ray diffraction. The complex with CuI was a two-dimensional coordination polymer, and the other four complexes were three-dimensional coordination polymers. A comparison of the five structures is given at the end of the chapter. Chapter four begins with a review of rigid angular bridging ligands and their use in coordination and supramolecular chemistry. The preparation of the ligand bis(4-pyridyl)amine (bpa) is given. The structural arrangement of bpa in the solid state was determined by X-ray diffraction. Complexes of Ag(I), Cu(I), Cu(II) and Cd(II) formed with this ligand were synthesised and characterised. The complexes {[Ag(bpa)(MeCN)](CF₃SO₃)}n̲, {[Ag(bpa)](PF₆)�MeCN}n̲, {[Ag(bpa)](ClO₄)-�2MeCN}n̲, {[Ag(bpa)](ClO₄)}n̲, {[Ag(bpa)](NO₃)}n̲, [(Cu₂I₂)(bpa)₂]n̲, {[Cu(bpa)₂Cl₂]-�3DMF�3/2H₂O}n̲, {[Cd(bpa)₂(NO₃)(H₂O)](NO₃)}n̲, {[Cd(bpa)₂(SO₄)(H₂O)]�3H₂O}n̲, [Cd(bpaH)₂(SO₄)₂(H₂O)₂]�2MeCN and {[Cd(bpa)(SCN)₂]�1/5iPrOH}n̲ were structurally characterised by X-ray diffraction. All complexes with Ag(I) were one-dimensional coordination polymers, with two of them helical, the other three zigzag. Both complexes with Cu(I) and (II) were two-dimensional coordination polymers. One complex with CdSO₄ was discrete, with the bpa ligands mono-protonated, but all other three other Cd(II) complexes were three-dimensional coordination polymers. Seven of these complexes showed hydrogen-bonding interactions linking them together to form supramolecular structures of higher dimensionalities. A comparison of the twelve structures is given at the end of the chapter. Chapter five is a brief summary of the outcomes of this thesis.

<p>Globally of today, acquired immunodeficiency syndrome (AIDS) and malaria are two of the most threatening diseases known to mankind. The World Health Organization estimated that AIDS and malaria together claimed nearly 4 million lives in 2003 and many more were infected by the causative agent human immunodeficiency virus (HIV) and the <i>Plasmodium falciparum</i> (<i>P. falicparum</i>) parasite. Current treatment regims for HIV and <i>P. falicparum</i> infections are undermined by rapid emergence of drug-resistant strains and severe drug side-effects.</p><p>A resistance mechanism of the commonly selected K103N RT mutant towards three second generation non-nucleoside RT inhibitors (NNRTIs) is presented based on X-ray structures. Subtle changes in contacts between inhibitor and residue in position 103 aided the design of improved inhibitors. For the PR target, attempts have been made to structurally assist the development of diol-based protease inhibitors (PIs) with the aim of improving the anti-viral potency without reducing the inhibitory efficacy. It was shown that ortho- and meta-fluoro-substituted P1/P1’-benzyloxy side chains improved the anti-viral potency without affecting the accommodation to the S1/S1’ subsites. </p><p>The apparent increase in malaria resistance makes drug interventions of current targets increasingly complicated. A prominent new drug target is found in the parasite’s hemoglobin degradation pathway – the aspartic protease plasmepsin II (Plm II). The usefulness of Plm II as an anti-malarial target is presented supported by Plm II complexed with a novel inhibitor. Structurally it is shown that bulky P1- and P3-side-chains adopt a novel binding mode to the Plm II binding cleft with implications for further inhibitor development.</p>

Graduation date: 1995

Graduation date: 1994

Globally of today, acquired immunodeficiency syndrome (AIDS) and malaria are two of the most threatening diseases known to mankind. The World Health Organization estimated that AIDS and malaria together claimed nearly 4 million lives in 2003 and many more were infected by the causative agent human immunodeficiency virus (HIV) and the Plasmodium falciparum (P. falicparum) parasite. Current treatment regims for HIV and P. falicparum infections are undermined by rapid emergence of drug-resistant strains and severe drug side-effects. A resistance mechanism of the commonly selected K103N RT mutant towards three second generation non-nucleoside RT inhibitors (NNRTIs) is presented based on X-ray structures. Subtle changes in contacts between inhibitor and residue in position 103 aided the design of improved inhibitors. For the PR target, attempts have been made to structurally assist the development of diol-based protease inhibitors (PIs) with the aim of improving the anti-viral potency without reducing the inhibitory efficacy. It was shown that ortho- and meta-fluoro-substituted P1/P1’-benzyloxy side chains improved the anti-viral potency without affecting the accommodation to the S1/S1’ subsites. The apparent increase in malaria resistance makes drug interventions of current targets increasingly complicated. A prominent new drug target is found in the parasite’s hemoglobin degradation pathway – the aspartic protease plasmepsin II (Plm II). The usefulness of Plm II as an anti-malarial target is presented supported by Plm II complexed with a novel inhibitor. Structurally it is shown that bulky P1- and P3-side-chains adopt a novel binding mode to the Plm II binding cleft with implications for further inhibitor development.

The hereditary transthyretin (TTR) amyloidoses are rare, and in severe cases, fatal disorders caused by mutations in the TTR gene. The clinical picture is diverse, involving neuropathies and myopathies, and mainly depends on the causative mutation and the sites and rates of amyloid deposition. The ultimate aim of the field of research presented in this thesis is to prevent TTR amyloid disease. To reach this ambitious goal, a thorough understanding of the normal as well as the pathological properties of the protein is essential. Here, comparisons between TTR from humans and other species may provide valuable information. The three-dimensional structure of TTR from Gilthead sea bream (Sparus aurata) was determined at 1.75 Å resolution by X-ray crystallography, and was found to be structurally similar to human TTR. However, significant differences were observed in the area at and around β-strand D, an area believed to dissociate from the structure prior to amyloid formation, thereby allowing the β-strands A and B to participate in polymerization. During evolution, the preference of TTR for the thyroid hormones, 3,5,3’-triiodo-L-thyronine (T3) and 3,5,3’,5’-tetraiodo-L-thyronine (T4), has shifted. While human TTR has higher affinity for T4, the opposite is true in lower vertebrates, e.g. fish and reptiles, where T3 is the main ligand. We have determined two separate structures of sea bream TTR in complex with T3 and T4, both at 1.9 Å resolution, as well as the complex of human TTR with T3. A significantly wider entrance and narrower thyroid hormone binding channel suggest a structural explanation to the differences in thyroid hormone preference between human and piscine TTR. The Tyr114Cys substitution in TTR is associated with severe systemic amyloidosis. The mutation introduces a second cysteinyl group in the TTR monomer, and has been shown to inhibit the formation of fibril formation in vitro, promoting the formation of disulfide-bonded amorphous aggregates. To deduce the role of intermolecular disulfide bonds in fibril formation we characterized the TTR Cys10Ala/Tyr114Cys double mutant. Our results suggest that an intermolecular disulfide bond at position 114 enhances the exposure of Cys10, which promotes the formation of additional intermolecular disulfide-linked assemblies. Also, we were able to isolate a disulfide-linked dimeric form of this mutant that formed protofibrils in vitro, suggesting the architecture of TTR amyloid may be the result of different underlying structures rather than that of a highly stringent assembly. We have also been able to successfully adapt a method of protein pre-heating to enable crystallization, thereby succeeding in a particularly problematic protein crystallization experiment. By heating the protein solution, we succeeded in separating several forms of protein micro-heterogeneities from the properly folded protein species, thereby allowing the growth of well diffracting crystals. / Ärftlig transthyretinamyloidos är en ovanlig och i allvarliga fall dödlig proteininlagringssjukdom som orsakas av mutationer i genen för transthyretin. Den kliniska bilden är huvudsakligen beroende av den bakomliggande genförändringen samt amyloidlokaliseringen och -depositionshastigheten och omfattar vanligen neuropatier och myopatier av varierande grad. Det slutgiltiga målet med forskningsfältet som presenteras i denna avhandling är att förhindra eller bota transthyretinamyloidos. En förutsättning för att lyckas med detta ambitiösa mål är en ingående förståelse för proteinets grundläggande egenskaper, såväl i normalfallet som i de patologiska processerna, bland annat genom jämförande studier av humant och icke-humant transthyretin (TTR). Den tredimensionella röntgenkristallografiska strukturen av TTR från fisken guldsparid (Sparus aurata) bestämdes till en upplösning på 1,75Å och befanns vara strukturellt snarlik humant TTR. Signifikanta skillnader observerades emellertid i och kring β-sträng D, en region som tros dissociera från huvudstrukturen innan själva bildningen av amyloid. Enligt denna hypotes leder D-strängsdissociationen till exponering av β-strängarna A och B, vilka därmed kan delta i de reaktioner som bildar amyloid. Under evolutionen har bindningspreferenserna för thyroideahormonerna T3 (3,5,3’-trijod-L-thyronin) och T4 (3,5,3’,5’-tetrajod-L-thyronin) hos TTR ändrats. Humant TTR har högre affinitet för T4 än för T3, medan det motsatta förhållandet gäller för lägre vertebrater, t ex fisk, där T3 är den huvudsakliga liganden. Strukturerna bestämdes för guldsparid i komplex med T4 och med T3 till 1,9 Å upplösning, samt för humant TTR i komplex med T3 till 1,7 Å upplösning. Jämförande analyser visade på signifikanta skillnader i thyroideahormonbindningskanalen, vilken var vidare och grundare i fisk än i människa. Dessa strukturella skillnader kan delvis förklara olikheterna i hormonbindning mellan högre och lägre vertebrater. Substitutionen Tyr114Cys i TTR är kopplad till en allvarlig form av systemisk transthyretinamyloidos. Mutationen introducerar en andra cysteinylgrupp i TTR-monomererna, vilket förhindrar fibrillbildning in vitro, men gynnar bildningen av amorfa disulfidbundna aggregat. För att närmare studera betydelsen av disulfidbindningar vid fibrillbildning av detta protein så karakteriserades dubbelmutanten TTR Cys10Ala/Tyr114Cys. Baserat på våra resultat föreslår vi att intermolekylära disulfidbindningar i position 114 ökar exponeringen av Cys10, vilket förstärker tendensen att bilda ytterligare disulfidbundna aggregat. Vi isolerade även en disulfidbunden dimerisk form av dubbelmutanten som kan bilda protofibriller in vitro. Baserat på denna observation föreslår vi att transthyretinamyloids underliggande arkitektur är sammansatt och kan nås genom sammanfogning av olika substrukturer snarare än genom en strikt ordnad uppbyggnad. Vi har också modifierat och anpassat en metod för uppvärmning av proteiner för att möjliggöra kristallisation i ett synnerligen problematiskt proteinkristallisations-experiment. Genom uppvärmning av proteinlösningen lyckades vi separera olika former av mikroheterogeniteter från det rättveckade proteinet, som sedan bildade kristaller av god röntgendiffraktiv kvalitet.

Surface proteins on microorganisms that build up the oral biofilm are key players in the formation of the biofilm. Antigen I/II proteins are surface adhesins found on virtually all oral streptococci and share a conserved multi-domain architecture. These adhesins bind surface components on other bacteria and on host cells. Thus, they are crucial for the development of the biofilm.     The objective of this thesis work is the structural characterization of the large multi-domain Antigen I/II protein SspB from the primary colonizing commensal bacterium Streptococcus gordonii. The crystal structure of the variable domain of SspB was determined to 2.3 Å resolution. The domain comprises a β-supersandwich and a putative binding cleft stabilized by a calcium ion. Despite high similarity in the overall structure, the cleft within SspB is significantly smaller than the cleft within the homologous protein from Streptococcus mutans, indicating that different substrates may bind in the clefts. A screen for carbohydrate binding resulted in no hits for interaction with the SspB variable domain suggesting that the cleft may not be suitable for binding sugars. This thesis also presents the high resolution 1.5 Å structure of a truncated C-terminal domain of SspB, the first of an Antigen I/II C-domain. The structure contains two structurally related domains, each containing one calcium ion and one intramolecular isopeptide bond. The SspB protein shares the feature of intramoleular isopeptide bonds with other surface proteins from Gram positive bacteria, such as pili from Streptococcus pyogenes and Corynebacterium diphtheriae. Intramolecular isopeptide bonds are suggested to be a common feature for retaining stability in a harsh environment. The SspB adherence region, shown to be the recognition motif for Porphyromonas gingivalis attachment to S. gordonii, protrudes from the core protein as a handle available for recognition. In conclusion, this thesis work has provided new knowledge about the SspB protein and increased the understanding of the common structure of AgI/II proteins.