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A series of metal-organic complexes which all contain two bridged dimolybdenum cores were synthesized and studied. Common building blocks involved in this series of syntheses include Mo2(DAniF)3(O2CCH3) (DAniF = N, N'-di-panisylformamidinate) and [Mo2(cis-DAniF)2(NCCH3)4](BF4)2. Bridges that were used to connect two different dimolybdenum cores in the synthesized structures include single metal complexes like ZnCl2 and Ni(acac)2 (acac = acetyl acetone), dimetal complex like Rh2(O2CCH3)4, as well as organic ligands like 1,2-dihydroxyl-4,5-dimethylaminbenzene and 1,3-dihydroxyl-2,5-dimethylaminbenzene. Several heterometallic supramolecules were obtained through self-assembly reactions. In these structures, the two dimolybdenum cores were bridged through different metal complexes; between these metal complexes and the molybdenum cores, isonicotinic acid anion acts as the key linkage. Depending on the geometry of the building blocks and their available binding site, these heterometallic supramolecules bear a variety of shapes, which include rod-like molecules with three metal centers, a squareshaped molecule with its four corners occupied by metal complexes, and also a zigzagshaped infinite metal complex chain. Although these molecules do show reversible redox peaks in electrochemistry studies, they demonstrated very poor electronic communication between the dimolybdenum centers. Possible explanations to this result may be that the dimolybdenum cores are far away from each other in these molecules (Mo2–Mo2 separation in compound 4, being 21 Å, is the longest among all dimolybdenum pairs synthesized to date) and that the calculated frontier orbital overlaps do not favor electron delocalization over the entire molecule. However, another type of molybdenum dimer of dimers where the dimolybdenum centers are united by conjugated organic ligands, namely 1,2-dihydroxyl- 4,5-dimethylaminbenzene and 1,3-dihydroxyl-2,5-dimethylaminbenzene, were also synthesized and found to bear significantly stronger electronic communication between the Mo2 centers. In fact, as electrochemistry reveals, these molecules demonstrated the greatest comproportionation constant values (Kc ~ 1014) than any other analogues synthesized so far. This interesting result is most likely due to the well conjugated linker ligands that would allow electrons on the metal centers to delocalize over the entire molecule. Computational studies of these compounds also show clear evidence of π overlapping in their molecular frontier orbitals.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19913号 / 理博第4213号 / 新制||理||1605(附属図書館) / 32999 / 京都大学大学院理学研究科物理学・宇宙物理学専攻 / (主査)教授 前野 悦輝, 教授 石田 憲二, 教授 川上 則雄 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

In this thesis we study non-relativistic, low-energy, s-wave scattering in a four-body spin-1/2 fermion system. The scattering is caused by an attractive twoermion contact interaction which is capable of producing a weakly bound state known as a dimer. This fourermion system is used to study the scattering of a two-dimer system. Using Hybrid Monte Carlo methods we compute the ground state energies of the system on a lattice. Luscher’s finite-volume formula and the Effective Range Expansion are used to calculate the dimer-dimer scattering length add and effective range rdd in terms of the fermionermion scattering length aff. Using these techniques we obtain the values add=aff = 0:60 +/- 0:04 and rdd=aff = 3:2 +/- 0:5. This scattering length shows excellent agreement with the numerous values in the literature. We also compare this effective range with the only currently known value in the literature.

Background: A D-dimer threshold <500ug/L has high negative predictive value (NPV) for venous thromboembolism (VTE), but is non-specific. Two strategies increase the specificity and utility (defined as the proportion of patients with a negative test) of D-dimer testing: 1) using a higher D-dimer threshold with increasing age (IAIT Strategy); and 2) using a high threshold in low clinical pretest probability (CPTP) patients and the standard threshold in moderate CPTP patients (CPTP Strategy). It is unknown whether the gain in specificity of the IAIT Strategy is simply due to using a higher threshold in some patients and whether the CPTP Strategy has better diagnostic accuracy than the IAIT Strategy. Methods: In a retrospective analysis of 1649 outpatients with suspected VTE, I compared the diagnostic accuracy of the IAIT Strategy to 1) its opposite: using a higher D-dimer threshold with decreasing age (DAIT strategy); 2) using a higher D-dimer threshold in all patients (Median Age Strategy); and 3) the CPTP Strategy. Results: The NPV of both the IAIT and DAIT Strategies was 99.6% and the NPV of the Median Age Strategy was 99.7%. The utility was almost identical in the IAIT and DAIT Strategies (50.9% vs. 50.6%) and greater in the Median Age Strategy (53.9%, p<0.001). The NPV of the CPTP and IAIT Strategies were 99.6% and 99.7%, respectively. The utility was higher in the CPTP Strategy than the IAIT Strategy (56.1% vs. 50.9%, p<0.001). Conclusions: The NPV and utility of using a higher D-dimer threshold in older patients (IAIT Strategy) is the same as using a higher D-dimer threshold in younger patients. The CPTP Strategy had the greatest utility while maintaining a high NPV and therefore appeared to be the optimal strategy of D-dimer interpretation. / Thesis / Master of Health Sciences (MSc)

This thesis examined the enzyme dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) from the pathogen Mycobacterium tuberculosis. DHDPS is a validated antibiotic target for which no potent inhibitor based on substrates, intermediates or product has been found. The importance of the homotetrameric quaternary structure in E. coli DHDPS has been demonstrated by the 100-fold decrease in activity observed in a dimeric variant, DHDPS-L197Y, created by site-directed mutagenesis. This suggested a new approach for inhibitor design: targeting the dimer-dimer interface and disrupting tetramer formation. DHDPS catalyzes the first committed step in the biosynthetic pathway of meso-diaminopimelic acid, a critical component of the mycobacterial cell wall. In this study, wild-type M. tuberculosis DHDPS was thoroughly characterized and compared with the E. coli enzyme. A coupled assay was used to obtain the kinetic parameters for M. tuberculosis DHDPS: KM(S) ASA = 0.43 (±0.02) mM, KMpyruvate = 0.17 (±0.01) mM, and kcat = 138 (±2) s 1. Biophysical techniques showed M. tuberculosis DHDPS to exist as a tetramer in solution. This is consistent with the crystal structure deposited as PDB entry 1XXX. The crystal structure of M. tuberculosis DHDPS showed active-site architecture analogous to E. coli DHDPS and a dimeric variant of M. tuberculosis DHDPS was predicted to have reduced enzyme activity. A dimeric variant of M. tuberculosis DHDPS was engineered through a rationally designed mutation to analyze the effect of disrupting quaternary structure on enzyme function. A single point mutation resulted in a variant, DHDPS-A204R, with disrupted quaternary structure, as determined by analytical ultracentrifugation and gel-filtration chromatography. DHDPS-A204R was found to exist in a concentration-dependent monomer-dimer equilibrium, shifted towards dimer by the presence of pyruvate, the first substrate that binds to the enzyme. The secondary and tertiary structure of DHDPS-A204R was analogous to wild-type M. tuberculosis DHDPS as judged by circular dichroism spectroscopy and X ray crystallography, respectively. Surprisingly, this disrupted interface mutant had similar activity to the wild type enzyme, with a kcat of 119 (±6) s-1; although, the affinity for its substrates were decreased: KM(S) ASA = 1.1 (±0.1) mM, KMpyruvate = 0.33 (±0.03) mM. These results indicated that disruption of tetramer formation does not provide an alternative direction for drug design for DHDPS from M. tuberculosis. Comparison with the recently discovered dimeric DHDPS from Staphylococcus aureus shed further light on the role of quaternary structure in DHDPS. In M. tuberculosis DHDPS-A204R and the naturally dimeric enzyme, the association of monomers into the dimer involves a greater buried surface area and number of residues than found in E. coli DHDPS-L197Y. This provides a framework to discriminate which DHDPS enzymes are likely to be inactive as dimers and will direct future work targeting the dimer-dimer interface of DHDPS as an approach for drug design.

Pulsed-beam Fourier transform microwave spectroscopy (PBFTMS) was used to determine the rotational structure of N-hydroxypyridine-2(1H)-thione. PBFTMS was also used to determine the rotational structure of a hydrogen dimer between propiolic acid and formic acid. Rotational constants and quadrupole coupling constants were determined. Calculations (MP2/DFT) were utilized in predicting the isotopic structures. Isotopic data (D, and ¹³C) and normal isotopomers collected were used in establishing of key structural parameters such as bond length and bond angles.

This thesis describes the design and synthesis of small molecule derivatives and their polypeptide conjugates as high affinity binders for proteins: the D-dimer protein (D-dimer), a biomarker for diagnosis of thromboembolic diseases; human myeloperoxidase (MPO), a biomarker for cardiovascular diseases; and chitinases, potential targets for asthma therapy. The interactions between the synthetic binder molecules and those proteins were evaluated by surface plasmon resonance (SPR) biosensor analysis and fluorescence spectroscopy. Competition SPR experiments or other methods proved that the small molecule components of the binder molecules were critical for binding and specifically bound to the original binding site of small molecules. The binder molecules consisted of a 42-residue helix-loop-helix polypeptide conjugated to a small molecule via aliphatic spacers of suitable length. The small molecules could be any type of moderately binding structure. In the binder development for the D-dimer, the tetrapeptide GPRP with a dissociation constant Kd of 25 μM was used and the affinity of 4C15L8GPRP obtained was estimated to be approximately 3 nM. In the binder development for MPO, salicylhydroxamic acid (SHA) with Kd of 2 μM was used and the affinity of 4C37L34C11SHA obtained was estimated to be approximately 0.4 nM. In the binder development for chitinases, a theobromine derivative (pentoxifylline) with a Kd of 43±10 μM was used and the affinity of 4C37L34-P obtained was estimated to be considerably higher than that of pentoxifylline. The binder molecules were identified from a 16-membered pool of candidates obtained by conjugating the small molecules to each member of a set of 16 designed polypeptides. The affinities were greatly enhanced by 2-3 orders of magnitude, compared to the small molecule. The polypeptides did not bind to the proteins with measurable affinities. The discovery of these new synthetic binders for protein targets can pave the way to diagnostic tests in vivo or in vitro, independent of antibodies.