Lewis Acid-Lewis Base Mediated Metal-Free Hydrogen Activation and Catalytic Hydrogenation

Organocatalysis, the use of organic molecules as catalysts, is attracting increasing attention as one of the most modern and rapidly growing areas of organic chemistry, with countless research groups in both academia and the pharmaceutical industry around the world working on this subject. The literature review of this thesis mainly focuses on metal-free systems for hydrogen activation and organocatalytic reduction. Since these research topics are relatively new, the literature review also highlights the basic principles of the use of Lewis acid-Lewis base pairs, which do not react irreversibly with each other, as a trap for small molecules. The experimental section progresses from the first observation of the facile heterolytical cleavage of hydrogen gas by amines and B(C6F5)3 to highly active non-metal catalysts for both enantioselective and racemic hydrogenation of unsaturated nitrogen-containing compounds. Moreover, detailed studies of structure-reactivity relationships of these systems by X-ray, neutron diffraction, NMR methods and quantum chemical calculations were performed to gain further insight into the mechanism of hydrogen activation and hydrogenation by boron-nitrogen compounds.

Effects of gender, and SLCO1B1 and CYP7A1 polymorphisms on plasma bile acids in humans and the pharmacokinetics of therapeutic ursodeoxycholic acid

Bile acids are important steroid-derived molecules essential for fat absorption in the small intestine. They are produced in the liver and secreted into the bile. Bile acids are transported by bile flow to the small intestine, where they aid the digestion of lipids. Most bile acids are reabsorbed in the small intestine and return to the liver through the portal vein. The whole recycling process is referred to as the enterohepatic circulation, during which only a small amount of bile acids are removed from the body via faeces. The enterohepatic circulation of bile acids involves the delicate coordination of a number of bile acid transporters expressed in the liver and the small intestine. Organic anion transporting polypeptide 1B1 (OATP1B1), encoded by the solute carrier organic anion transporter family, member 1B1 (SLCO1B1) gene, mediates the sodium independent hepatocellular uptake of bile acids. Two common SNPs in the SLCO1B1 gene are well known to affect the transport activity of OATP1B1. Moreover, bile acid synthesis is an important elimination route for cholesterol. Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme of bile acid production. The aim of this thesis was to investigate the effects of SLCO1B1 polymorphism on the fasting plasma levels of individual endogenous bile acids and a bile acid synthesis marker, and the pharmacokinetics of exogenously administered ursodeoxycholic acid (UDCA). Furthermore, the effects of CYP7A1 genetic polymorphism and gender on the fasting plasma concentrations of individual endogenous bile acids and the bile acid synthesis marker were evaluated. Firstly, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the determination of bile acids was developed (Study I). A retrospective study examined the effects of SLCO1B1 genetic polymorphism on the fasting plasma concentrations of individual bile acids and a bile acid synthesis marker in 65 healthy subjects (Study II). In another retrospective study with 143 healthy individuals, the effects of CYP7A1 genetic polymorphism and gender as well as SLCO1B1 polymorphism on the fasting plasma levels of individual bile acids and the bile acid synthesis marker were investigated (Study III). The effects of SLCO1B1 polymorphism on the pharmacokinetics of exogenously administered UDCA were evaluated in a prospective genotype panel study including 27 healthy volunteers (Study IV). A robust, sensitive and simple HPLC-MS/MS method was developed for the simultaneous determination of 16 individual bile acids in human plasma. The method validation parameters for all the analytes met the requirements of the FDA (Food and Drug Administration) bioanalytical guidelines. This HPLC-MS/MS method was applied in Studies II-IV. In Study II, the fasting plasma concentrations of several bile acids and the bile acid synthesis marker seemed to be affected by SLCO1B1 genetic polymorphism, but these findings were not replicated in Study III with a larger sample size. Moreover, SLCO1B1 polymorphism had no effect on the pharmacokinetic parameters of exogenously administered UDCA. Furthermore, no consistent association was observed between CYP7A1 genetic polymorphism and the fasting plasma concentrations of individual bile acids or the bile acid synthesis marker. In contrast, gender had a major effect on the fasting plasma concentrations of several bile acids and also total bile acids. In conclusion, gender, but not SLCO1B1 or CYP7A1 polymorphisms, has a major effect on the fasting plasma concentrations of individual bile acids. Moreover, the common genetic polymorphism of CYP7A1 is unlikely to influence the activity of CYP7A1 under normal physiological conditions. OATP1B1 does not play an important role in the in vivo disposition of exogenously administered UDCA.

Hantavirus infection: Insights into entry, assembly and pathogenesis

Hantaviruses (family Bunyaviridae, genus Hantavirus) are enveloped viruses incorporating a segmented, negative-sense RNA genome. Each hantavirus is carried by its specific host, either a rodent or an insectivore (shrew), in which the infection is asymptomatic and persistent. In humans, hantaviruses cause Hemorrhagic fever with renal syndrome (HFRS) in Eurasia and Hantavirus cardiopulmonary syndrome (HCPS) in the Americas. In Finland, Puumala virus (genus Hantavirus) is the causative agent of NE, a mild form of HFRS. The HFRS-type diseases are often associated with renal failure and proteinuria that might be mechanistically explained by infected kidney tubular cell degeneration in patients. Previously, it has been shown that non-pathogenic hantavirus, Tula virus (TULV), could cause programmed cell death, apoptosis, in cell cultures. This suggested that the infected kidney tubular degeneration could be caused directly by virus replication. In the first paper of this thesis the molecular mechanisms involved in TULV-induced apoptosis was further elucidated. A virus replication-dependent down-regulation of ERK1/2, concomitantly with the induced apoptosis, was identified. In addition, this phenomenon was not restricted to TULV or to non-pathogenic hantaviruses in general since also a pathogenic hantavirus, Seoul virus, could inhibit ERK1/2 activity. Hantaviruses consist of membrane-spanning glycoproteins Gn and Gc, RNA-dependent RNA polymerase (L protein) and nucleocapsid protein N, which encapsidates the viral genome, and thus forms the ribonucleoprotein (RNP). Interaction between the cytoplasmic tails of viral glycoproteins and RNP is assumed to be the only means how viral genetic material is incorporated into infectious virions. In the second paper of this thesis, it was shown by immunoprecipitation that viral glycoproteins and RNP interact in the purified virions. It was further shown that peptides derived from the cytoplasmic tails (CTs) of both Gn and Gc could bind RNP and recombinant N protein. In the fourth paper the cytoplamic tail of Gn but not Gc was shown to interact with genomic RNA. This interaction was probably rather unspecific since binding of Gn-CT with unrelated RNA and even single-stranded DNA were also observed. However, since the RNP consists of both N protein and N protein-encapsidated genomic RNA, it is possible that the viral genome plays a role in packaging of RNPs into virions. On the other hand, the nucleic acid-binding activity of Gn may have importance in the synthesis of viral RNA. Binding sites of Gn-CT with N protein or nucleic acids were also determined by peptide arrays, and they were largely found to overlap. The Gn-CT of hantaviruses contain a conserved zinc finger (ZF) domain with an unknown function. Some viruses need ZFs in entry or post-entry steps of the viral life cycle. Cysteine residues are required for the folding of ZFs by coordinating zinc-ions, and alkylation of these residues can affect virus infectivity. In the third paper, it was shown that purified hantavirions could be inactivated by treatment with cysteine-alkylating reagents, especially N-ethyl maleimide. However, the effect could not be pin-pointed to the ZF of Gn-CT since also other viral proteins reacted with maleimides, and it was, therefore, impossible to exclude the possibility that other cysteines besides those that were essential in the formation of ZF are required for hantavirus infectivity.