<p>Carbon-carbon bond formation reactions are among the most important processes in chemistry, as they represent key steps in the synthesis of more complex molecules from simple precursors. This thesis describes mainly the development of novel regioselective applications of the mild and versatile palladium-catalyzed carbon-carbon coupling method, commonly known as the Heck reaction. In addition, this thesis will focus on environmentally friendly developments of the Heck reaction.</p><p>Novel ligand-controlled internal Heck vinylations of vinyl ethers and enamides to form branched electron-rich dienes were performed with high regioselectivity. The vinylation of 2-hydroxyethyl vinyl ether permits a chemoselective transformation of a vinylic triflate or bromide into a blocked α,β-unsaturated methyl ketone. Furthermore, a simple separation of the palladium catalyst was achieved with new fluorous-tagged bidentate ligands in combination with fluorous solid phase extraction. The reaction times could be reduced up to 1000 times with controlled microwave heating in the palladium-catalyzed reactions with, in the majority of cases, retained, high selectivity. </p><p>The development of a “green” regioselective arylation and vinylation method relying on an aqueous DMF-potassium carbonate system and excluding the toxic thallium salt has been accomplished. Ionic liquids as the versatile and environmentally friendly class of solvents have been used in rapid phosphine-free terminal Heck arylations with controlled microwave heating. Recycling of the catalytic medium was achieved after a simple product purification.</p>

Capillary electroseparation methods<b> </b>are exciting new techniques with very broad application areas and vast potential in pharmaceutical and biomedical analysis. To improve the limit of detection (LOD) capillary zone electrophoresis (CZE) has been combined with isotachophoretic (ITP) preconcentration in a single capillary. Using the ITP-CZE combination the LOD can be improved at least 100-fold. Laser-induced fluorescence (LIF) detection is more sensitive and more selective than the most common detection technique, UV, and the intensity and focusing capability of LIF fits well with the small dimensions in CZE. The total sensitivity enhancement attained for a new acetylcholinesterase inhibitor, NXX-066, by using ITP-CZE-LIF was more than 5500-fold compared to CZE-UV. Capillary electrochromatography (CEC) combines the high separation efficiency of CZE with the vast possibilities to improve selectivity of HPLC. We have examined different ways to solve the problem of extensively tailing peaks and studied the influence of the mobile phase composition on the electrochromatographic performance for a number of tricyclic antidepressants and related quaternary ammonium compounds. (1) Adding aliphatic amines to the mobile phase in reversed phase CEC. The effect on the chromatographic performance was coupled to the hydrophobicity of the additive and the amine of our choice was dimethyloctylamine. (2) Silica-based cation exchangers with different pore sizes. The large-pore materials promoted pore flow, but this had no positive influence on the performance. The small-pore (highest surface area) particles gave the best selectivity. (3) Designing special continuous beds. As the bed is covalently attached to the capillary wall, problems related to retaining frits are avoided. The stationary phase most suitable for our analytes had a molar ratio of 1:80 between the functional ligands, vinyl sulphonic acid and isopropyl groups, respectively. The LOD was lowered 26000-fold by dissolving the sample in a low-conducting medium.

Capillary electrophoresis-mass spectrometry has been used successfully for the analysis of a wide range of analytes such as chiral local anaesthetics, sulphonated reactive dyes and endogenous neurotransmitters and neuropeptides. The partial filling technique was used in CE-MS for chiral separation of bupivacaine and ropivacaine using the non-volatile selector β-cyclodextrin. By only partially filling the capillary with selector and using capillaries coated with polyacrylamide to suppress the electroosmotic flow, introduction of the selector into the mass spectrometer was avoided. An impurity of 0.25% of the R-enantiomer of ropivacaine in the S-form could be detected. The partial filling technique was developed further using CE employing two different selectors in separate plugs in the capillary. This enhanced the separation efficiency and offered greater flexibility in controlling the separation. By using transient-isotachophoresis (tITP)-CE-MS it was possible to concentrate and detect classical neurotransmitters and neuropeptides with masses ranging from 104 Da to 1642 Da. γ -Aminopropyltriethoxysilane coated capillaries were used to minimize adsorption of the peptides onto to capillary surface. Endogenous dopamine, glutamate, γ-aminobutyric acid (GABA), acetylcholine, methionine-enkephalin and substance P 1-7 were detected in the striatum of marmoset monkey. Sulphonated dyes obtained from single textile fibres were analysed using CE-MS. Capillary electrophoresis was found to be a good way of removing the excess amounts of glucose present in the sample that would otherwise interfere with the electrospray ionisation. Automatic function switching, originally developed for use together with liquid chromatography, was found to be a great method for acquiring MS/MS data when doing infusion experiments saving both time and sample without decreasing the quality of the MS/MS data. It was also found to be a more time efficient way than using the precursor ion scanning mode on the Q-TOF to obtain precursor ion data.

Aspartyl proteases can generally be inhibited by peptide mimics containing an uncleavable peptide bond isostere at the proposed cleavage site. One such peptide bond isostere is the hydroxyethylamine moiety, which in this thesis has successfully been incorporated in potential inhibitors of the HIV-1-protease as well as the malarial proteases plasmepsin I and II. The human immunodeficiency virus (HIV) has during the last 20 years given rise to a new fast-spread epidemic. The virus protease is one of the foremost targets for drug intervention. In an attempt to improve an earlier design, a P1'-anthranilic acid was exchanged for all four isomers of 2-aminocyclopentanecarboxylic acid, which were synthesized from racemic starting materials, the trans isomers via a novel synthetic route. None of the isomers enhanced potency as compared to the anthranilic acid. Because of increasing development of resistance, the pharmaceutical intervention with malaria is becoming rapidly more difficult. A prominent new target for drug research is the hemoglobin degradation pathway. Two of the many proteases involved in this pathway are plasmepsin I and II. Two series of peptide mimics with the hydroxyethylamine were prepared and tested against these enzymes as well as against the similar human protease cathepsin D. In the first series the central nitrogen of the target compounds is a secondary amine, derived from natural and unnatural amino acids, the side-chain of which was to bind in the S1'-site of the proteases. It was found that para-aryl substituted phenylalanines resulted in the most active inhibitors. While the P1- and P2-side-chains were kept constant at benzyl and isopropyl respectively, the P3 capping carboxylic acid was varied with a set of diverse carboxylic acids. It was found that many of the carboxylic acids were acceptable. A selection of compounds was tested for inhibition of parasite growth in infected human erythrocytes and found to be active. In the target compounds of the second series the P1'-side-chain was moved from the α-carbon of the initial amino acids to the adjacent nitrogen, thus rendering this a tertiary amine. The SAR of these compounds suggests that this side-chain cannot be larger than benzyl, which is in sharp contrast to the first series, where both isomers of phenylalanine (i.e. a benzyl group on the α-carbon) render inactive compounds. Most of the compounds show a good degree of selectivity for the plasmepsins over cathepsin D, even though a few good inhibitors of the human enzyme could be identified also.

Carbon-carbon bond formation reactions are among the most important processes in chemistry, as they represent key steps in the synthesis of more complex molecules from simple precursors. This thesis describes mainly the development of novel regioselective applications of the mild and versatile palladium-catalyzed carbon-carbon coupling method, commonly known as the Heck reaction. In addition, this thesis will focus on environmentally friendly developments of the Heck reaction. Novel ligand-controlled internal Heck vinylations of vinyl ethers and enamides to form branched electron-rich dienes were performed with high regioselectivity. The vinylation of 2-hydroxyethyl vinyl ether permits a chemoselective transformation of a vinylic triflate or bromide into a blocked α,β-unsaturated methyl ketone. Furthermore, a simple separation of the palladium catalyst was achieved with new fluorous-tagged bidentate ligands in combination with fluorous solid phase extraction. The reaction times could be reduced up to 1000 times with controlled microwave heating in the palladium-catalyzed reactions with, in the majority of cases, retained, high selectivity. The development of a “green” regioselective arylation and vinylation method relying on an aqueous DMF-potassium carbonate system and excluding the toxic thallium salt has been accomplished. Ionic liquids as the versatile and environmentally friendly class of solvents have been used in rapid phosphine-free terminal Heck arylations with controlled microwave heating. Recycling of the catalytic medium was achieved after a simple product purification.

The molecular-imprinting method has previously been used for the synthesis of artificial gel antibodies, highly selective for various proteins. In present study, we have synthesized artificial gel antibodies against haemoglobin, albumin and different forms of growth hormone with the aim to develop a simple and rapid procedure to measure the concentration of these protein biomarkers in samples of clinical interest.  A spectrophotometric method was developed to design a standard curve in the form of a straight line, whereby the true absorption (not the recorded “apparent” absorption) was plotted against a known protein concentration. The procedure, applied to quantitative analysis of albumin in human plasma and cerebrospinal fluid (CSF) from patients with ALS, indicated that  the concentration of this protein was significantly enhanced in CSF from patients with amyotrophic lateral sclerosis (ALS), compared to control samples. A low level of albumin was observed in plasma from ALS patients compared to controls. Additionally, free zone electrophoresis was employed to detect human growth hormone (GH) activity in hormone preparations purified from human pituitaries. We have successfully synthesized antibodies capable of discriminating between dimeric and monomeric GH in samples of clinical origin. To quantify these proteins a calibration curve has been designed, i.e. a plot of the electrophoretic mobility of the complex GH/gel antibody against the protein concentration in the sample, for instance serum or CSF. This method was also employed for qualitative and quantitative determinations of Somatropin, a non-glycosylated GH and glycosylated-GH in a body liquid. Our results indicate that by this technique one can “fish out” with high accuracy various proteins from both body fluids containing a great number of other proteins. It might well apply also to biomarker proteins for other diseases.

The aim of this work was to explore the structural and functional diversity of polypeptides that are found in plants. Expanding knowledge of simililarities between plant use of these compound and animal use promises exceptional opportunities for finding, from plant research, new structures with biomedical and biotechnological potential. A fractionation protocol was developed and applied to many plant species, providing fractions enriched in polypeptides, amenable to chemical and biological evaluation. From one species, the common field pansy (Viola arvensis), a 29-amino-acid residue polypeptide was isolated, named varv A, which revealed a remarkable macrocyclic structure (i.e., N- and C-termini are joined) stabilised by three knotted disulfides. Varv A, together with an increasing number of homologous peptides, form the currently known peptide family of cyclotides. Their stable structure makes them an attractive scaffold for protein engineering. In addition, they display a wide range of biological activities (e.g., antimicrobial, cytotoxic, and insecticidal). As a part of this work, the cytotoxic effects of varv A and two other isolated cyclotides were evaluated in a human cell-line panel: all were active in the low µM range. Most likely, these effects involve pore formation through cell membranes. Cyclotides were found to be common in the plant family Violaceae; with eleven cyclotides isolated and sequenced from V. arvensis, V. cotyledon, and Hybanthus parviflorus. For six members of the genus Viola, cyclotide expression profiles were examined by liquid chromatography-mass spectrometry (LC-MS): all expressed notably complex mixtures, with single species containing more than 50 cyclotides. These profiles reflect the evolution of the genus. To assess these mixtures, a rational strategy for MS based amino acid sequencing of cyclotides was developed, circumventing inherent structural problems, such as low content of positively charged amino acids and the macrocyclic structure. This was achieved by aminoethylation of cysteines, which, following tryptic digestion, produced fragments of size and charge amenable to MS analysis. This method was also modified and used for mapping of disulfide bonds. Methods for isolation and characterisation developed in this work may prove useful not only for further studies on macrocyclic polypeptides from plants, but also for other plant peptides and disulfide-rich peptides from animals.

Hepatitis C Virus (HCV) is the leading cause of chronic liver disease worldwide as well as the primary indication for liver transplantation. More than 3% of the world’s population is chronically infected with HCV and there is an urgent need for effective therapy. NS3 protease, a viral enzyme required for propagation of HCV in humans, is a promising target for drug development in this area. This thesis addresses the design, synthesis and biochemical evaluation of new HCV NS3 protease inhibitors. The main objective of the thesis was the synthesis of peptide-based protease inhibitors of the bifunctional full-length NS3 enzyme (protease-helicase/NTPase). Three types of inhibitors were synthesized: i) classical serine protease inhibitors with electrophilic C-terminals, ii) product-based inhibitors with a C-terminal carboxylate group, and iii) product-based inhibitors with C-terminal carboxylic acid bioisosteres. The developmental work included the establishment of an improved procedure for solid-phase peptide synthesis (SPPS) in the N-to-C direction, in contrast to the C-to-N direction of classical SPPS methods. This inverse method facilitated synthesis of the peptides modified at the C-terminal. The potency of more than seventy newly synthesized inhibitors was assessed in an in vitro assay using the native form of the protease, i.e. the full-length NS3. The structure-activity relationship (SAR) data achieved was different from SAR data obtained from the more widely used truncated NS3 (protease domain) assay, indicating that the helicase domain of NS3 participates in the binding of the inhibitors. The most potent inhibitors identified in this study contained a C-terminal phenyl acyl sulfonamide moiety, i.e. a carboxylic acid bioisostere. It is concluded that the acyl sulfonamide moiety is a promising P1-P1´ spanning entity, which may have potential for use in the development of more drug-like HCV protease inhibitors.

<p>Cytochrome P450 (P450 or CYP) is an enzyme system involved in the oxygenation of a wide range of endogenous compounds as well as foreign chemicals and drugs. This thesis describes investigations of P450-catalyzed oxygenation of prostaglandins, linoleic and arachidoniacids.</p><p>The formation of bisallylic hydroxy metabolites of linoleic and arachidonic acids was studied with human recombinant P450s and with human liver microsomes. Several P450 enzymes catalyzed the formation of bisallylic hydroxy metabolites. Inhibition studies and stereochemic analysis of metabolites suggest that the enzyme CYP1A2 may contribute to the biosynthesis of bisallylic hydroxy fatty acid metabolites in adult human liver microsomes.</p><p>19<i>R</i>-Hydroxy-PGE and 20-hydroxy-PGE are major components of human and ovine semen, respectively. They are formed in the seminal vesicles, but the mechanism of their biosynthesis is unknown. Reverse transcription-polymerase chain reaction using degenerate primers for mammalian <i>CYP4</i> family genes, revealed expression of two nov P450 genes in human and ovine seminal vesicles. The full coding regions of the genes were cloned and the enzymes were expressed in a yeast system. The human enzyme was designated CYP4F8 and the ovine enzyme was designated CYP4F21. Comparison of their deduced protein sequenceshowed that they had 74 % amino acid identity. Recombinant CYP4F8 oxygenated two prostaglandin endoperoxides (PGH₁ and PGH₂) and three stable PGH₂ analogues int19-hydroxy metabolites. Oxygenation of these substrates was mirrored when incubated with microsomes isolated from human seminal vesicles. These results suggest that CYP4F8 is present in human seminal vesicles and that 19<i>R</i>-hydroxy-PGE is formed by CYP4F8-catalyze 19<i>R</i>-hydroxylation of PGH₁ and PGH₂, followed by PGE synthase-catalyzed isomerization. Studies of catalytic properties of recombinant CYP4F21 suggest that 20-hydroxy-PGE may be formed by similar mechanisms in ovine seminal vesicles. CYP4F8 is the first enzyme shown to hydroxylate prostaglandin endoperoxides.</p>

<p>Cholesterol is metabolized to a variety of important biological products in the body including bile acids and vitamin D. The present investigation is focused on enzymes that catalyze 7α-hydroxylation or 27-hydroxylation in the metabolism of cholesterol, oxysterols (side chain-hydroxylated derivatives of cholesterol) and vitamin D₃. The enzymes studied belong to the cytochrome P450 enzyme families CYP7 and CYP27.</p><p>The study describes purification of a cytochrome P450 enzyme fraction active in 7α-hydroxylation of 25-hydroxycholesterol, 27-hydroxycholesterol, dehydroepiandrosterone and pregnenolone from pig liver microsomes. Peptide sequence analysis indicated that this enzyme fraction contains an enzyme belonging to the CYP7B subfamily. The purified enzyme was not active towards cholesterol or testosterone. Purification and inhibition experiments suggested that hepatic microsomal 7α -hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone involves at least two enzymes, probably closely related.</p><p>The study shows that recombinantly expressed human and rat cholesterol 7α -hydroxylase (CYP7A) and partially purified pig liver cholesterol 7α -hydroxylase are active towards 20(<i>S</i>)-, 24-, 25- and 27-hydroxycholesterol. CYP7A was previously considered specific for cholesterol and cholestanol. The 7α -hydroxylation of 20(<i>S</i>)-, 25-, and 27-hydroxycholesterol in rat liver was significantly increased by treatment with cholestyramine, an inducer of CYP7A. Cytochrome P450 of renal origin showed 7α -hydroxylase activity towards 25- and 27-hydroxycholesterol, dehydroepiaundrosterone and pregnenolone but not towards 20(<i>S</i>)-, 24-hydroxycholesterol or cholesterol. The results indicate a physiological role for CYP7A as an oxysterol 7α -hydroxylase, in addition to the previously known human oxysterol 7α -hydroxylase CYP7B.</p><p>The role of renal sterol 27-hydroxylase (CYP27A) in the bioactivation of vitamin D₃ was studied with cytochrome P450 fractions purified from pig kidney mitochondria. Purification and inhibition experiments and experiments with a monoclonal antibody against CYP27A indicated that CYP27A plays a role in renal 25-hydroxyvitamin D₃ l α -hydroxylation.</p><p>The expression of CYP7A, CYP7B and CYP27A during development was studied. The levels of CYP27A in livers of newborn and six months old pigs were similar whereas the levels of CYP7A increased. The expression of CYP7B varied depending on the tissue. The expression of CYP7B increased with age in the liver whereas the CYP7B levels in kidney showed a marked age-dependent decrease.</p>