An introduction to nucleoside analogues containing heterocyclic sugar mimics and their synthesis is presented. This includes various ring sizes and different heteroatom combinations. concentrated on work between 1995-2000. The synthesis of novel nucleoside analogues from nitroimidazole precursors has been investigated. The regioisorners I-vinyl-4-nitroimidazole and I-vinyl-S-nitroimidazole have been synthesised from the readily available 4/S-nitroimidazole. Also synthesised from 4/S-nitroimidazoIe IS 3-vinyl-imidazo[ 4' ,S' :S,6]pyrido[2,3- d]pyrimidin-8-one, the pyridine stretched analogue of 3-vinylinosine. 1,3-Dipolar cycloaddition reactions of these three molecules are studied, with stabilised and unstabilised 1,3-dipolar compounds. to produce heterocyclic nucleoside analogues. Structures of these cycloadducts are investigated using nrnr studies to determine the regiochemistry of the reactions. This nrnr evidence is supported my MO calculations. Further studies have established synthetic routes to the pyridine stretched analogues of 2'-deoxyadenosine and 2'-deoxyinosine from deoxyribose and 4/5- nitroimidazole, involving directing the coupling of a chlorosugar to the sodium salt of 4-nitroimidazoIe to yield a maximum of the S-nitroimidazole isomer product

Thesis advisor: Larry W. McLaughlin / Nucleic acids are fundamental biomolecules responsible for all activities of a living cell. DNA serves as an instruction manual to the cell, containing blueprints and directions for all cellular processes, while RNA serves to carry out the messages held within DNA. Research into the structure, stability, and function of nucleic acids has revealed much about the origin and evolution of life. The ultimate goal of this work is to understand how molecules bind and associate within the minor groove of double stranded, helical DNA. A series of 2'-deoxyadenosine analogues are modified at the three position by replacing the N3-nitrogen with carbon. Substitution at this position is designed to emulate the effects of removing hydrogen bond acceptors, introducing steric bulk, and tethering functional groups of interest into the minor groove. These functional groups mimic small molecules that have been shown to bind within the minor groove of A-T rich sequences as well as serve as a platform for further substitution by fluorescent tags. The synthetic effort needed to obtain purine nucleosides containing each of these modifications was non-trivial. New methodologies unveiled directing and protecting strategies towards the desired isomer of these modified nucleosides in higher yields than those previously deemed acceptable. Application of these modified nucleosides into duplex DNA reveals thermodynamic parameters for how small molecules bind to the minor groove and the effects of introducing biomarkers into an unprecedented region of DNA. / Thesis (PhD) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

3'-Carboxymethyl-3'-deoxyadenosine derivatives were prepared from 2'-O-TBDMS-3'-deoxy-3'-[(ethoxycarbonyl)methyl]adenosine (1) via simple and efficient procedures. Conversion of 1 to 5'-azido-2'-O-TBDMS-3', 5'-dideoxy -3'-[(ethoxycarbonyl) methyl] adenosine (4) was accomplished via a novel one-pot method employing 5'-activation (TosCl) followed by efficient nucleophilic displacement with tetramethylguanidinium azide. Compound 4 was converted to a 5'-[(N-methylcarbamoyl)amino] derivative (5) via one-pot reduction/acylation employing H2/Pd-C followed by treatment with p-nitrophenyl N-methylcarbamate. The latter step of this two-step process required an efficient source of p-nitrophenyl N-methylcarbamate, thus a highly efficient new method for preparing p-nitrophenyl N-alkylcarbamate was developed. N6-phenylcarbamoyl groups were introduced by treatment with phenylisocyanate, and an efficient new method for lactonization of 2'-O-TBDMS-3'-deoxy-3'-[(ethoxycarbonyl)methyl]adenosines to give corresponding 2', 3'-lactones was also developed. Target compounds were evaluated for anti-HIV and anti-HIV integrase activities, but were not active at the concentrations tested.

No description available.

Human cells possess multiple nucleoside transporters (NTs) that belong to either the human equilibrative or concentrative NT (hENT: hENT1/2/3/4; hCNT: CNT1/2/3) families. In the kidney, coupling of apical hCNT3 activities to basolateral hENT1/2 activities is hypothesized to mediate renal nucleoside proximal tubular absorption while apical ENT1 may have a role in secretion. The overall aim of this research was to increase understanding of the roles of hENTs and hCNTs in renal handling of physiological nucleosides and anti-cancer nucleoside analog drugs. This was achieved by investigating the distribution of hENTs and hCNTs in human kidney tissue and the function of hENTs and hCNTs in cellular uptake and transepithelial fluxes of nucleosides in cultured human renal proximal tubule cells (hRPTCs). Immunolocalization of hCNT3 and hENT1 in human kidney tissue revealed that hENT and hCNT3 were present in apical membranes of proximal tubules. Production and characterization of adherent hRPTC cultures demonstrated endogenous hCNT3, hENT1, and hENT2 activities. These results provided evidence for the involvement of hCNT3, hENT1, and hENT2 in renal handling of nucleosides. Comparison of adherent hRPTC cultures derived from kidneys from different individuals demonstrated that hCNT3 activities varied between cultures. Also, the extent of cellular uptake of fludarabine, an anti-cancer nucleoside drug, and degree of cytotoxicity was reflected in the different hCNT3 activities observed between cultures. These results suggested that hCNT3 plays an important role in fludarabine renal handling and is a determinant of potential renal toxicities. Production of polarized monolayer cultures of hRPTCs on transwell permeable inserts enabled the functional localization of hCNT3 and hENT1 to apical membranes and hENT2 to basolateral membranes. Transepithelial flux studies demonstrated that (i) apical-to-basolateral fluxes of adenosine were mediated by apical hCNT3 and basolateral hENT2, (ii) basolateral-to-apical fluxes of 2′-deoxyadenosine were mediated, in part, by apical hENT1 and basolateral hOATs, and (iii) apical-to-basolateral fluxes of fludarabine, cladribine, and clofarabine were mediated by apical hCNT3. These studies showed that coupling of apical hCNT3 to basolateral hENT2 mediates proximal tubular nucleoside reabsorption, that coupling of basolateral human organic anion transporters (hOATs) to apical hENT1 mediates proximal tubular nucleoside secretion, and that hCNT3 is a key determinant of fludarabine proximal tubular reabsorption and cytoxicity.

Two groups of growth regulators were described in Drosophila imaginal disc cell culture Cl.8+. Imaginal disc growth factors (IDGFs) belonging to chitinase-like protein family of carbohydrate binding proteins and Adenosine deaminase-related growth factors (ADGFs), which are active adenosine deaminases influencing homeostasis of key cellular metabolite adenosine. The functions of two of the IDGFs, as well as the effects of extracellular adenosine and its receptor were studied primarily in in vitro cell culture. Our results supported their roles in the regulation of cell survival and energy homeostasis especially in imaginal disc cells. Both the IDGFs and adenosine also play important roles in organismal responses to stress and infection and may interact in vivo.

A new series of N6,5'-bis-ureido-5'-amino-5'-deoxyadenosine derivatives was prepared and evaluated for anticancer activities using the NCI 60 panel of human cancers. Certain of the derivatives showed promising activities (low micromolar GI50's) against several of the representative cancers. These included cell lines from the following general cell types in the NCI 60: Leukemia, Breast, Central Nervous System, Non-Small Cell Lung, Ovarian, Prostate, Renal, and Colon cancers. Select compounds were also screened for their affinities for protein kinases. The synthesis of the compounds was straightforward and involved N6 acylation with arylisocyanates, preceded by activation and nucleophilic substitution of the 5'-position to give the desired 5'-azido-5'-deoxyadenosine derivatives. Reduction of the 5'-azido function with either H2/Pd-C, or Ph3P/H2O, gave the desired 5'-amino-5' deoxyadenosine products. Acylation of the 5'-amino group with N-methyl 4-nitrophenylcarbamate gave the N6,5'-bis-ureido-5'-amino-5' deoxyadenosine products. Yields ranged from good (50–75%) to excellent (75–95%) for all synthetic transformations.

Anthraquinone (AQ) has been used in electron transfer studies in DNA. The focus of this dissertation is the synthesis of conjugates between AQ derivatives and 2’-deoxyadenosine (dA), which can be used to induce adenine oxidation in DNA. Different AQ derivatives were attached to dA via ethynyl or ethanyl linkers. If incorporated into DNA, these short linkers should enable regiocontrol for electron transfer from adenine within the DNA duplex structure. The challenge in working with anthraquinone-2’-deoxynucleosides conjugates is that they and their intermediates are insoluble in water and only sparingly soluble in most organic solvents. A strategy used to overcome this problem was the use of either tert-butyldiphenylsilyl (TBDPS) or 4’,4-dimethoxytrityl (DMTr) 5’-protected deoxynucleosides as starting materials. A water-soluble, ethynyl-linked AQ-dA conjugate with a 3’-benzyl hydrogen phosphate was synthesized using DMTr protection. The DMTr group was not stable to the hydrogenation required to make the ethanyl-linked AQ-dA conjugate with 3’-benzyl hydrogen phosphate. Hence the latter was synthesized starting with the TBDPS protecting group. Both of these syntheses were based on the Pd coupling between ethynylanthraquinone and 8-bromodeoxyadenosine derivatives. New conjugates between AQ and A, in which the AQ moieties have been modified with formyl, trifluoroacetyl and methyl ester groups as electron withdrawing substituents were also synthesized. The synthesis of these AQ-dA conjugates was based on Pd coupling between bromoanthraquinone and 8-ethynyldeoxyadenosine derivatives. This route avoided the use of ethynylanthraquinone derivatives that had extremely low solubility and photoinstability. Other anthraquinones with electron withdrawing groups (which should provide enhanced driving force to enable respective AQ derivative to oxidize adenine) were synthesized as models. Cyclic voltammetry showed that the conjugate with the two ester groups and ethynyl linker was the most easily reduced of the derivatives synthesized. Conjugates between AQ and dU were also synthesized. Those conjugates can potentially be used to oxidize guanine or adenine or they can be used as a deep trap for an electron in reduced DNA.

S-Adenosyl-L-methionine (SAM) is an essential metabolite for all domains of life. SAM- dependent reactions result in three major metabolites: S-adenosyl-L-homocysteine (SAH), methylthioadenosine (MTA), and 5'-deoxyadenosine (5'-dA). Each of these has been demonstrated to be feedback inhibitors of SAM dependent enzymes. Thus, each metabolite has a pathway to prevent inhibition through the salvage of nucleoside and ribose moieties. However, these salvage pathways are not universally conserved. In the anaerobic archaeal organism Methanocaldococcus jannaschii, the salvage of SAH, MTA, and 5'-dA, proceeds first via deamination to S-inosylhomocysteine (SIH), methylthioinosine (MTI), and 5'-deoxyinosine (5'-dI). The annotated SAH hydrolase from M. jannaschii is specific for SIH and the hydrolyzed product homocysteine is then methylated to methionine. The salvage of MTA is known to proceed through the methionine salvage pathway, however, an anaerobic route for the salvage of MTA is still mostly unknown. Only two enzymes from the methionine salvage pathway are annotated in M. jannaschii's proteome, a methylthioinosine phosphorylase (MTIP) and methylthioribose 1-phosphate isomerase (MTRI). These enzymes were shown to produce methylthioribulose 1-phosphate from MTI. Unfortunately, how MTI is converted to either 2-keto-(4-methylthio)butyrate or methionine remains unknown. The two enzymes involved in the salvage of MTI have also been demonstrated to be involved in the salvage of 5'-dI. Interestingly, there is little information on how 5'-dA or 5'-dI is recycled and it is proposed here to be the source of deoxysugars for the production methylglyoxal, a precursor for aromatic amino acids. MTIP and MTRI were demonstrated to produce 5-deoxyribulose 1-phosphate from 5'-dI. Additionally, two enzymes annotated as part of the pentose phosphate pathway, ribulose 5-phosphate 3-epimerase and transketolase, were able to convert 5-deoxyribulose 1-phosphate to lactaldehyde. Lactaldehyde was then reduced to methylglyoxal by an essential enzyme in methanogenesis, N5, N10-methylenetetahydromethanopterin reductase with NADPH. These results further demonstrate a novel route for the biosynthesis of methylglyoxal. Lastly, hypoxanthine produced from phosphorolysis of inosine, MTI, and 5'-dI was demonstrated to be reincorporated through the hypoxanthine/guanine phosphoribosyltransferase (Hpt) to IMP. Together these reactions represent novel pathways for the salvage of the SAM nucleoside and ribose moieties in M. jannaschii. / Ph. D.

Strikt anaerobe Bakterien wie Clostridium difficile und C. scatologenes verwenden GRE, um die chemisch ungünstige Decarboxylierung von 4-Hydroxyphenylacetat zu p-Cresol zu katalysieren. Das Enzymsystem besteht aus einer Decarboxylase und dem zugehörigen Aktivierungsenzym. Die 4-Hydroxyphenylacetat-Decarboxylase (4Hpad) besitzt zusätzlich zum Protein-basierten Glycinradikal eine weitere Untereinheit mit bis zu zwei [4Fe-4S] Clustern und repräsentiert hierdurch eine neue Klasse von Fe/S-Cluster-haltigen GREs, die aromatische Verbindungen umsetzen. Das Aktivierungsenzym (4Hpad-AE) weicht vom Standardtypus ab, indem es zusätzlich zum S-Adenosylmethionin(SAM)-bindenden [4Fe-4S]-Cluster (RS-Cluster) mindestens einen weiteren [4Fe-4S]-Cluster bindet. In dieser Studie wurden heterologe Expressions- und Reinigungsprotokolle für 4Hpad und 4Hpad-AE entwickelt. Kristallstrukturen von 4Hpad cokristallisiert mit den Substraten (4-Hydroxyphenylacetat, 3,4-Dihydroxyphenylacetat) und dem Inhibitor (4-Hydroxyphenylacetamid) zeigten geringe strukturelle Änderungen im aktiven Zentrum des Proteins. Die Radikalbildung am 4Hpad-AE wurde durch die Überprüfung einer klassischen reduktiven Spaltung von SAM zu den Reaktionsprodukten 5’-Deoxyadenosin und Methionin bestätigt. EPR- und Mössbauer-Spektroskopische Analysen zeigten, dass 4Hpad-AE mindestens einen zusätzlichen [4Fe-4S] Cluster neben dem einzelnen RS-Cluster enthält. Die katalytische Notwendigkeit eines zusätzlichen Clusters wurde durch eine Mutationsanalyse untersucht, wobei eine verkürzte Version des Enzyms ohne die zusätzliche Cystein-reiche Insertion konstruiert wurde. Das verkürzte Mutante ohne die Bindungsmotive für die zusätzlichen Cluster gekennzeichnet, die Konfiguration, Stöchiometrie und die Funktion der zusätzlichen Cluster diagnostizieren. / 4-hydroxyphenylacetate decarboxylase (4Hpad) is a two [4Fe-4S] cluster containing glycyl radical enzyme proposed to use a glycyl/thiyl radical dyad to catalyze the last step of tyrosine fermentation in Clostridium difficile and C. scatologenes by a Kolbe-type decarboxylation. The decarboxylation product p-cresol is a virulence factor of the human pathogen C. difficile. The small subunit of 4Hpad may have a regulatory function with the Fe/S clusters involved in complex formation and radical dissipation in the absence of substrate. The respective activating enzyme (4Hpad-AE) has one or two [4Fe-4S] cluster(s) in addition to the SAM-binding [4Fe-4S] cluster (RS cluster). The role of these auxiliary clusters is still under debate with proposed functions including structural integrity and conduit for electron transfer to the RS cluster. This study shows the optimized expression and purification protocols for the decarboxylase and the co-crystallization experiments and binding studies with 4-hydroxy-phenylacetate and 3,4-dihydroxyphenylacetate and with the inhibitor 4-hydroxy-phenylacetamide. The purification and characterization of active site mutants of decarboxylase are also done. Concerning 4-HPAD-AE, we report on the purification of code-optimized variants, and on spectroscopic and kinetic studies to characterize the respective i) SAM binding enthalpies, ii) rates for reductive cleavage of SAM and iii) putative functions of the additional Fe/S clusters. The truncated mutant lacking the binding motifs for the auxiliary clusters is characterized to diagnose the configuration, stoichiometry and function of the auxiliary clusters.