Thesis (Ph. D.)--Rutgers University, 2007. / "Graduate Program in Chemistry and Chemical Biology." Includes bibliographical references.

Nucleoside polyphosphates and their conjugates, such as nucleoside triphosphates, nucleoside tetraphosphates, sugar nucleotides, dinucleoside pyro- and higher order polyphosphates, 2’,3’-cyclic nucleoside monophosphates, and 2´-deoxynucleoside-5´-tetraphosphates in which a fluorescent label is attached to the terminal phosphate have many biological roles and have been developed into drugs. However, their synthesis remains a challenge. Several novel and efficient approaches to the synthesis of nucleoside polyphosphates and their conjugates were developed. In the first approach dinucleoside polyphosphates (NpnN’s where n = 2-4) are prepared via in situ trifluoroacetate protection and imidazolium activation of nucleoside 5’-monophosphates. This methodology was also used to prepare a substrate-intermediate analog of the reaction catalyzed by cytidine triphosphate synthase (CTPS) a recognized target for the development of antineoplastic, antiviral and antiprotozoal agents. The second approach uses sulfonylimidazolium salts as key reagents for generating highly reactive nucleotide donors. The procedure is rapid, produces a wide variety of nucleoside polyphosphates and their conjugates in high yield, does not require protection and subsequent deprotection of the nucleotide donors or acceptors and can be used to activate nucleoside mono-, di-, and triphosphates and a wide variety of acceptors. Finally an entirely new approach to the synthesis of nucleoside tetraphosphates (Np4’s), dinucleoside pentaphosphates (Np5N’s) and nucleoside tetraphosphates in which a fluorescent dye is attached to the terminal phosphate is described employing an activated form of cyclic trimetaphosphate as a novel phosphorylating agent. Attempts to prepare nucleoside triphosphates by subjecting unprotected ribonucleosides and 2’-deoxyribonucleosides to activated cyclic trimetaphosphate failed. Instead nucleoside 2’,3’-cyclic phosphates were obtained in good yield with the ribonucleoside substrates. This represents a new and convenient approach to the synthesis of this class of compounds.

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Mast cells are tissue resident members of the immune system. They have a wide range of functions and receptors including the FcεRI receptor, which gets activated by binding to IgE bound to an antigen. When the cells are activated in this manner, a process termed the LysRS- Ap4A-MITF signalling pathway occurs, resulting in the translocation of the Lys tRNA synthetase into the nucleus and an activation of its moonlighting activity - the production of diadenosine tetraphosphate (Ap4A). Ap4A is a dinucleoside polyphosphate, a type of ubiquitous molecule present in all domains of life. They are made up of two nucleosides joined together by a 5' to 5' phosphodiester bridge of variable lengths. Recently, these molecules have been shown to serve as non-canonical initiating nucleotides during bacterial transcription, where they function as 5' RNA caps, similar to the well-known 7- methylguanosine eukaryotic mRNA cap. In this thesis, I present proof of existence of Ap 4A capped RNA in mast cells, a previously unknown 5' RNA structure in eukaryotic cells, and I attempt to pinpoint its role in the activation of these cells and in the wider context of mast cell mediated immune response. Keywords: mast cells, RNA caps, Dinucleoside polyphosphates, Ap 4A, RNA modification, IgE, FcεRI receptor, Lysine tRNA synthetase

Für die Ätiopathogenese der Atherosklerose spielen eine Vielzahl von Mediatoren eine Rolle. Dabei werden durch das Endothel sowohl protektive als auch schädliche Mediatoren sezerniert. High Density Lipoproteine (HDL) stellen einen bedeutenden protektiven Marker für das kardiovaskuläre Risiko dar, u.a. durch die Aktivierung der endothelialen NO-Synthase (eNOS). HDL besteht zu 50 % aus Proteinen und zu 50 % aus Lipiden. Welche Komponenten des HDL für die eNOS Aktivierung verantwortlich sind, ist nicht bekannt gewesen. Im ersten Abschnitt dieser Promotionsarbeit konnte erfolgreich gezeigt werden, dass die Lysophospholipide, Sphingosin-1-Phosphat (S1P) und Sphinsosylphosphorylcholin (SPC), die strukturelle Bestandteile der Lipidfraktion von HDL darstellen, für einen Teil der HDL induzierten eNOS Aktivierung durch Stimulation des S1P3-Rezeptors verantwortlich sind. Diese eNOS Aktivierung wird durch den intrazellulären Einstrom von Calcium und durch die Aktivierung der Akt-Kinase induziert. Im zweiten Abschnitt dieser Promotionsarbeit konnte nachgewiesen werden, dass das oral verfügbare Lysophospholipid-basierte Medikament, FTY720, das ein strukturelles Analogon des S1P ist, den HDL induzierten Signaltransduktionsweg der eNOS Aktivierung in gleicher Weise induziert. Im dritten Abschnitt dieser Promotionsarbeit konnte ein neuartiges endothelabhängig sezerniertes gemischtes Dinukleosidpolyphosphat, Uridin-Adenosin-Tetraphosphat (Up4A), identifiziert werden. Up4A ist ein Agonist an den P2X- und P2Y-Purinrezeptoren. Up4A induziert bei Applikation in eine isoliert perfundierte Rattenniere hauptsächlich über die Aktivierung des P2X1-Rezeptors und des P2Y2/P2Y4-Rezeptors eine starke Vasokonstriktion im renalen Perfusionsgebiet mit einhergehender Erhöhung des mittleren renalen Perfusionsdrucks. Die direkte Infusion von Up4A in vivo in eine WKY-Ratte führt zu einer signifikanten Erhöhung des mittleren arteriellen Blutdrucks. / In the pathogenesis of atherosclerosis many mediators are included. Therefore the endothelium plays a crucial part by secreting protective but also deleterious factors. High density lipoproteins are an established protective factor in the risk profile of cardiovascular events especially by activating the endothelial NO synthase (eNOS). HDL is composed of 50 % proteins and 50 % lipids. Which component of HDL is responsible for the eNOS activation was not known. In the first part of this dissertation it could be shown, that the lysophospholipids, sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), which are structural compounds of the lipid fraction of HDL, are responsible for a significant part of the HDL induced eNOS activation by stimulating the specific S1P3 receptor. In the signal transduction mechanism the activation of Akt kinase and an influx of calcium is involved. In the second part of this dissertation it could be shown, that the orally active lysophospholipide based drug FTY720, which is a structural analogue of S1P, is able to induce the same signal transduction mechanism activated by HDL including the stimulation of the S1P3 receptor. In the last part of this dissertation a new endothelium dependent vasoconstrictor, the dinucleoside polyphosphate uridine-adenosine-tetraphosphate (Up4A), could be for the first time identified. Up4A is a potent agonist of the P2X- and P2Y-purinoceptors. Via activating the P2X1 receptor and the P2Y2/P2Y4 receptor Up4A induce a strong vasoconstriction in the renal perfusion system in the model of the isolated perfused rat kidney with an adjacent increase of the mean perfusion pressure. By injection of Up4A in vivo in a Wistar Kyoto rat the mean arterial pressure also increase significantly.