This thesis reports the first synthesis and biophysical characterization of oligoribonucleotides (RNA) containing 3'-deoxy-3'-thiothymidine (3'-S-dT) units. Optimized conditions for incorporating 3'-S-dT into 21-nt RNA strands is also reported. The impact of 3'-S-dT substitutions on the structure and stability of RNA:RNA duplexes was investigated by circular dichroism (CD) and UV/thermal (Tm) studies. Generally, replacement of the 3'-oxygen by a sulphur atom resulted in a significant decrease in the thermal stability of the duplex and strong positional effects were observed. No significant disruption of the A-form helical structure of the duplexes was detected by CD, although in some cases 3'-S-dT inserts led to a reduction in base-base stacking and/or local distortion of the duplex. Assays revealed that short interfering RNA (siRNA) duplexes containing 3'-S-dT on the guide strand were not efficient at down-regulating gene products, but those containing 3'-S-dT on the passenger strand OR on both strands exhibited activities that often surpassed those of unmodified siRNA duplexes. Substitutions at the scissile phosphate position of the passenger strand suggest that the non-bridging 3'-oxygen is not involved in the coordination of a metal ion during the rate determining step in RISC-induced RNA cleavage. / Cette thèse présente la première synthèse et caractérisation biophysique d'oligoribonucleotides (ARN) contenant des unités de 3'-deoxy-3'-thiothymidine (3'-S-dT). Des conditions optimisées pour l'intégration de 3'-S-dT en 21-nt ARN sont aussi présentées. L'impact de la substitution de 3'-S-dT sur la structure et la stabilité des duplex ARN:ARN fut recherché systématiquement par l'entremise de dichroïsme circulaire (CD) et d'études UV/thermale (Tm). Généralement, le remplacement du 3'-oxygène par un atome de sulfure résulta en une baisse significative de la stabilité thermique du duplex, et des effets de positionnement important furent observés. Aucune disruption significative de la structure hélicoïdale A-form du duplex ne fut détectée par le CD, quoi que parfois des inserts de 3'-S-dT entraînèrent une réduction des interactions base-base et/ou une distorsion locale du duplex. Des essaies révélèrent que des duplex interférents d'ARN court (siRNA) contenant du 3'-S-dT sur le brin guide n'ont pas baissé la production de gènes, mais que celles contenant du 3'-S-dT sur le brin complémentaire OU sur les deux brins présentèrent des activités qui fréquemment surpassaient celles de duplex non-modifiés de siRNA. Les substitutions à la position phosphate de rupture du brin complémentaire suggèrent que l'étape déterminante du clivage par RISC n'implique pas le 3'-oxygène pour la coordination d'un ion de métal.

The phenyl phosphate monoester in (Co$ sb2$(bpmp)($ mu$-PP)(OH)(OH$ sb2$)) (ClO$ sb4) sb2$ is hydrolyzed an unprecedented 10$ sp{11}$ times more rapidly than the corresponding unbound phosphate. Clean conversion of the bidentate monoester ((Co$ sb2$(bpmp)($ mu$-PP)(OH) (OH$ sb2$)) (ClO$ sb4) sb2$) to the tridentate inorganic phosphate ((Co$ sb2$(bpmp)($ mu, mu sp2$-Pi) (OH)) (ClO$ sb4$)), together with the crystal structure of an analog of phenylphosphonate, (Co$ sb2$(bpmp)($ mu$-PPO3)(OH)(OH$ sb2$)) (ClO$ sb4) sb2$ provide detailed mechanistic insight into the hydrolysis reaction. / The structure of (Co$ sb2$(bpmp)($ mu$-DPPO2)($ mu$-O$ sb2$)) (ClO$ sb4) sb2$ contains a diphenylphosphinate and a peroxide bridged between two cobalt(III) centers. Interestingly one of the oxygen atoms of the peroxide is only about 2.8 A away from the phosphorus of the coordinated phosphinate. When the bridging phosphinate is replaced with methyl phenylphosphonate (Co$ sb2$(bpmp)($ mu$-MPPO3)($ mu$-O$ sb2$)) (ClO$ sb4) sb2$, the ester bond is cleaved in aqueous DMSO (2:1 DMSO-d6/D$ sb2$O). The pseudo-first order rate constant for the cleavage of the methyl phenylphosphonate monoester bond in the complex is 5.3 x 10$ sp{-7}$ s$ sp{-1}$ at pH 7 (t$ sb{1/2}$ = 15 days) at 20$ sp circ$C. The estimated rate of base catalysed hydrolysis of the phosphonate is 1.4 $ times$ 10$ sp{-11}$M$ sp{-1}$s$ sp{-1}$ so that ca. 10$ sp{11}$ fold rate enhancement is observed. / The reactivity of dinuclear cobalt(III) complexes for cleaving phosphate diester in (Co$ sb2$(N3)$ sb2( mu$-OH)$ sb2( mu$-PO$ sb4$R$ sb2$)) can be significantly increased by decreasing the O-Co-O bond angle in the intermediate or transition state which forms a four membered ring. This can be achieved by increasing the N-Co-N ligands side bond angle opposite the O-Co-O angle. In (Co$ sb2$( (9) aneN3)$ sb2( mu$-OH)$ sb2( mu$-MPNPP)), the nitrogens are connected by two carbons whereas in (Co$ sb2$(tame)$ sb2( mu$-OH)$ sb2( mu$-MPNPP)), where tame is 1,1,1-tri(aminomethyl)ethane, the nitrogens are connected by three carbons. This results in a 10 fold increase in the reactivity for tame complex over (9) aneN3 complex. / There are three principal modes of activation that metal ions can provide for hydrolyzing phosphate esters. They are Lewis acid activation, nucleophiie activation and leaving group activation. All three modes of activation have been investigated in a three-metal system if the activation is additive. In the system that has a binding site on the leaving group for a third metal ion, double Lewis acid activation, double nucleophile activation and leaving group activation provides an unprecedented 10$ sp{17}$ fold rate enhancement for hydrolyzing a phosphate diester than in the corresponding third metal free system.

A dinuclear copper(II) complex has been investigated for promoting the transesterification of a phosphate diester, 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP), that is a model for RNA. Rate enhancements of over 10$ sp5$ have been obtained at neutral pH over the background hydroxide rate. For this study the dinucleating ligand 2,6-bis ((bis(benzimidazolymethyl)amino)methyl) -p-cresol was prepared and the reactivity of the copper(II) complex for cleaving 2-hydroxypropyl-4-nitrophenyl phosphate was determined. The two metals were shown to be acting cooperatively by comparing the reactivity of the dinuclear complex to that of a mononuclear complex, copper(II) 2-(bi(benzimidazolymethyl)amino)methyl-4,6-dimethylphenol. A mechanism involving double Lewis acid activation of HPNP is proposed to account for the 10$ sp5$ rate acceleration. / The reactivity of mononuclear Cu(II) and Zn(II) complexes of 2,9-diamino-1,10-phenanthroline (DAP) have been investigated for promoting the hydrolysis of phosphate diesters with good and poor leaving groups. The CuDAP and ZnDAP complexes were shown to be able to provide 10$ sp{10}$ and 10$ sp7$ orders of magnitude rate acceleration for hydrolyzing $2 sp prime,3 sp prime$-cAMP. A novel mechanism which combines metal ion catalysis with intramolecular hydrogen bonding has been proposed to account for the unprecedented rate acceleration. Crystallographic evidence and potentiometic titration data are provided, in addition to kinetic data, to support the proposed mechanism. The results with CuDAP are compared with the copper(II) complex of 2,9-dimethyl-1,10-phenanthroline (CuNeo) to evaluate the rate enhancement provided by intramolecular hydrogen bonding. It is found that intramolecular hydrogen bonding interactions increase the reactivity of CuDAP relative to CuNeo by a factor of 10$ sp3.$

Synthesis of a novel conformationally rigid dimer 106 containing a cyclopropyl-phosphonate linkage is described. A novel 3'-phosphonate building block 105 is synthesized and characterized. During the synthesis of the common precursor 86, unusual base-promoted desilylation of epoxide 80 and vinylselenides 85 and 87 are reported. The instability of the tert-butyldimethylsilyl (TBDMS) and the tert-butyldiphenylsilyl (TBDPS) protecting groups may be attributed to the participation of the uracil C2-oxygen anion. / The synthesis of a number of novel bicyclic phosphonate analogs 92b to 95b is described. / In search of alternate cyclopropanation methods, dimesylate 113 was synthesized and characterized. Dimethyl methyl phosphonate anion treatment produced 2'-keto nucleoside derivative 114. The synthesis of a novel reactive epoxide equivalent, the 2 ',3'-cyclic sulfate 119, is described. Dimethyl methylphosphonate anion treatment produced a furane derivative 120 in trace amounts. Reaction of azide anion with cyclic sulfate proceeded regioselectively and gave only 3'-azido nucleoside 122. The observed regioselectivity is novel and may be of great interest to nucleic acid chemists.* / A novel class of galactose and lactose-based glycoconjugates 23 and 24 has been synthesized and fully characterized. During the preparation of these glycoconjugates, novel N-fluorenylmethoxycarbonyl (Fmoc) and N-carboxybenzyl (Cbz)-protected galatosylated and lactosylated linker intermediates 7, 8, 11 and 12 have been synthesized and characterized. / The synthesis of a new active cholane intermediates 19 and 26 is also described.* / *Please refer to dissertation for diagrams.

Chiral auxiliary 1,2-O-cyclopentylidene-5-deoxy-5-isopropylamino-alpha- D-xylofuranose 51 and its enantiomer 52 were synthesized from D-xylose and L-xylose respectively and treated with PC13 to form cyclic phosphochloridites 58 and 67, and then reacted with 5'-O- t-butyldimethylsilylthymidine to give diastereornerically pure cyclic phosphoramidites 60 and 68. They were coupled with 3'-O-t-butyldimethylsilylthymidine in the presence of 2-bromo-4,5-dicyanoimidazole 46 as catalyst, and then sulfurized with Beaucage's reagent to form protected dithymidine phosphorothioates 62 and 72 stereoselectively. After deprotection, Sp and Rp dithymidine phosphorothioates 65 and 66 were obtained respectively in 98% diastereomeric excess. The mechanism of the coupling reaction which involves only one inversion in the transformation of the phosphoramidites (60, 68) to the dithymidine phosphorothioates (65, 66) is discussed. The effect of the acidity of the catalyst on the stereoselectivity and the rate of the coupling reaction was studied. / The synthesis and separation of diastereomers of protected dithymidine boranophosphates Sp-107 and Rp-108 gave the dimers Sp-109 and Rp-110 in a stereospecific manner. These dimers will be incorporated into oligonucleotides to synthesize mixed backbone oligonucleotides. To confirm the stereochemistry of the dimers Sp-109 and Rp-110, the free dithymidine boranophosphates Sp- 115 and Rp-116 were synthesized, the configurations at the phosphorus atom of which were assigned by comparison with spectral data from the literature. / A stereoselective synthesis of a dinucleotide boranophosphate Sp- 115 with a de of >98%, using (S)-3-hydroxyl-4-(2-indolyl)butyronitrile 28-(S) as chiral auxiliary, is described. The conversion of phosphite triester to boranophosphate by BH3-Me2S-proceeds with retention of configuration at the phosphorus atom. The procedure may be adaptable to the solid phase synthesis.

New acyclic dibenzyloxy disulfides substituted in the para position were prepared by a modified procedure in very high yield ( ca. 85%). Each of them were found to display an AB quartet in the 1H NMR spectrum, suggesting the possible existence of the isomeric thionosulfite structure (RO)2S=S in contrast to the more commonly proposed linear structure R-O-S-S-O-R. This structure question was addressed by preparing a series of closely related compounds including the analogous disulfide R2S2, tetrasulfide R2S4, "oxytrisulfide" R-O-S3-R, sulfoxylate R-O-S-O-R, sulfite (RO)2S=O, cyclic thionosulfite and corresponding cyclic sulfite, followed by comparative 1H and 13C NMR studies. The solid state structure was established to be linear according to X-ray analysis with somewhat short S-S bond length compared with conventional disulfide structures. The possible interconversion between the linear and the branched isomer was ruled out following an extensive spectroscopic study including 17O NMR, IR, Raman and UV. / The solid/solution structure was established to be linear with a rigid gauche conformation dependent of a barrier to rotation of ca. 18 kcal mol-1 (Tc = 75°C). This barrier was responsible for the diastereotopicity of the adjacent benzylic protons, giving rise to asymmetric induction, and permitted the detection of the enantiomers with the chiral shift reagent Eu(hfc)3 in chloroform at room temperature. The existence of rotational diastereomers was demonstrated by 1H and 13C NMR for chiral dialkoxy disulfides prepared from enantiomerically pure chiral and racemic alcohols. / A temperature dependent NMR study revealed that dibenzyloxy disulfides can deliver singlet diatomic sulfur S2. The thermolysis was studied in different solvents in the presence of different dienes. The disulfide adduct and the diene were comparably competitive for the S2 transfer. Tetrasulfide adducts were obtained in the presence of dimethyl and diphenyl butadiene, but converted to the disulfide adduct upon triphenylphosphine treatment. The related "oxytrisulfide" was demonstrated to also transfer the S2 unit to dienes. A brief analysis of a known thionosulfite has shown its potential to transfer S2 units to dienes. Mechanistic considerations were proposed to rationalize the olefinic compounds formed. / Triphenylphosphine desulfurization of dialkoxy disulfides yielded the corresponding sulfoxylates. The kinetics of the conversion of these to the corresponding sulfinates RO(S=O)R were examined and found to be first order; the Arrhenius parameters were determined.

The diastereoselective synthesis of phosphorothioates and methylphosphonates has been investigated. It has been found that indole is a good leaving group, and that it can form a stable indolephosphorine and can be stereospecifically substituted by a nucleoside in the presence of DBU. / Chiral indole-oxazaphosphorines 31, 39, 44, 56 were synthesized and their reactivities were investigated. The cyano derivative 56 turned out to be a good chiral precursor in the diastereoselective synthesis of a T-T phosphorothioate dimer 65, with a diastereomeric excess larger than 96%. The reaction of cyano, monomer 63 on solid support was investigated. It was found that alkylphosphonate 64 was obtained as the major product, rather than the expected phosphorothioate 65.* / A novel internucleoside coupling reagent 87 was developed for the synthesis of methylphosphonates, in which the indole group can be replaced by a nucleoside within several minutes in the presence of DBU. Several chiral auxiliaries were tested for the stereoselective synthesis of methylphosphonates. A diastereomerically enriched monoester 104 (66% de) was synthesized.* / *Please refer to dissertation for diagrams.

A new approach has been studied for solution-phase oligosaccharide syntheses using low molecular weight poly(ethylene glycol) monomethyl ethers (MPEG, average Mn = 550 and 750) as oligomer supports. A complex branched mannononaose (Man9) has been synthesized by this method in a very concise way to yield a large quantity of final product. Attempt has been made towards the synthesis of a heptasaccharide phytoalexin elicitor (HPE). / Model linear mannotetraose and branched mannopentaose were first tested using the novel HMB low molecular weight poly(ethylene glycol) linker. The use of this linker as the oligomer support retains the normal advantages of polymer-supported solution synthesis of oligosaccharides. Purification of the supported synthons by flash column chromatography on silica gel is greatly simplified. Another advantage of this approach is that the reaction can be monitored readily by the usual arsenal of spectroscopic techniques. Both compounds were obtained in over 10% overall yields. / In order to synthesize properly protected glycosyl donors for the construction of the mannononaose, the regioselective reductive ring opening of benzylidene acetals in carbohydrates with BH3/Bu2BOTf and regioselective acylation of hexopyranosides with pivaloyl chloride have been developed. BH 3/Bu2OTf is an effective reagent to reductively cleave 4,6- O-benzylidene acetals of various hexopyranosides to the corresponding 4-O-benzyl ethers. 4,6-O-isopropylidene acetals can be similarly cleaved. Common protecting groups are stable to the reaction conditions. On the other hand, the regioselectivity in the acylation of hexopyranosides with pivaloyl chloride in pyridine was studied. Manno- and galacto-pyranosides were regioselectively acylated to give the 3- O-pivaloylated compounds in good yields. Both methodologies have been used to provide properly protected mannoside donors and acceptors for polymannan syntheses. The regioselective reductive ring opening of 4,6- O-benzylidene acetals of hexopyranosides has also been demonstrated by concise syntheses of two building blocks of a heptasaccharide phytoalexin elicitor (HPE). / A concise synthesis of the mannan residue of a highly branched mannose type oligosaccharide present on the viral coat of HIV-1 from only two monosaccharide building blocks was achieved. The synthesis requires only five coupling steps instead of the seven or more steps required for the conventional block synthesis. In addition, even though the synthesis of HPE has not been completed to the last step, it showed that adjustment of the length of the poly(ethylene glycol) oligomer support could result in better overall yields. In both cases, 1H-1H COSY and 1H-13C HMQC NMR spectroscopies have proven to be extremely useful in monitoring the progress of oligosaccharide construction. These syntheses demonstrate that oligomer-supported solution synthesis using low molecular weight poly(ethylene glycol) is a powerful tool for the rapid and efficient preparation of complex oligosaccharides.

The effect of ligand structure on the reactivity of a diverse group of aqua-hydroxytetraazacobalt(III) complexes towards the hydrolysis of phosphate diesters and carboxylic esters has been investigated. It has been shown that the magnitude of the N-Co-N bond angle that lies opposite to the aqua-hydroxy ligands is influential in determining which complexes are best able to stabilize a strained four-membered ring, and this has been related to the relative rates at which these complexes promote reactions involving four-membered ring formation. The great range of rates of acid catalyzed decarboxylation of the carbonato derivatives of these complexes has been explained in terms of the same ligand-effect, and a correlation between the rate of this reaction and Co(III) aqua-hydroxy promoted phosphate diester hydrolysis and carboxylic ester hydrolysis has been established. / The relationship between reactivity of a complex towards phosphate diester hydrolysis and mode of binding of inorganic phosphate has been elucidated. / The true catalysis of methyl acetate hydrolysis by the two complexes ((13aneN$ sb4)$Co(OH)(OH$ sb2)$) $ sp{2+}$ and ((cyclen)Co(OH)(OH$ sb2)$) $ sp{2+}$ has been demonstrated, it has been shown that complexes which are capable of this kind of catalysis can bind acetate as a chelate.

Hydrosilation of $ alpha, beta$-unsaturated carbonyl compounds catalyzed by hydridotetrakis(triphenylphosphine)rhodium(I) (2.20) was found to be highly regioselective, depending on the type of silicon hydrides used. Diphenylsilane was found to give 1,2-hydrosilation (100%), whereas dimethylphenylsilane and other monohydrosilanes gave 1,4-addition (100%). / The kinetic isotope effect of the hydrosilation reaction was examined. A kinetic isotope effect of $k sb{H}/k sb{D}=2$ was observed in the hydrosilation of acetophenone (2.78) by dihydrosilane, $ rm H sb2SiPh sb2$ (2.21) and $ rm D sb2SiPh sb2$ (2.83), catalyzed by the complex 2.20. While a mixture of 1:1 labeled and unlabeled sec-phenethyl alcohols was obtained for the same reaction with monohydrosilane, $ rm HSiMe sb2Ph$ (2.14) and DSiMe$ sb2$Ph (2.86). / A mechanism was proposed to account for the regioselection and the kinetic isotope effect. With the proposed intermediate O in Chart 2.5,$ sp*$ when R $ ne$ H, the hydrosilation proceeds at the rhodium center to give the 1,4-selection product; when R = H, the reaction occurs at the silicon center to generate the 1,2-reduction product. / The reaction catalyzed by 2.20 was also diastereoselective, in both 1,2-hydrosilation (up to 100% de) and 1,4-hydrosilation (84% de) of $ alpha, beta$-unsaturated ketones. / A moderate enantiomeric excess was achieved in the asymmetric hydrosilation of prochiral ketones with chiral complexes derived from complex 2.20 with chiral phosphine ligands (up to 78% ee), and with chiral oxazolinyl compound 2.139 (up to 46% ee). Two C$ sb3$ symmetric ligands have been designed and synthesized, which were found to be too unstable to form C$ sb3$-metal complexes. ftn$ sp*$Please refer to the dissertation for diagram.