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This thesis describes the design and synthesis of compounds that are intended to inhibit serine and aspartic proteases. The first part of the text deals with preparation of inhibitors of the hepatitis C virus (HCV) NS3 serine protease. Hepatitis C is predominantly a chronic disease that afflicts about 170 million people worldwide. The NS3 protease, encoded by HCV, is essential for replication of the virus and has become one of the main targets when developing drugs to fight HCV. The inhibitors discussed here constitute surrogates for the widely used N-acyl-hydroxyproline isostere designated 4-hydroxy-cyclopentene. The stereochemistry of the 4-hydroxy-cyclopentene scaffold was determined by nuclear overhauser effect spectroscopy (NOESY) and the regiochemistry by heteronuclear multiple bond correlation (HMBC). The scaffold was decorated with different substituents to obtain both linear and macrocyclic HCV NS3 protease inhibitors that display low nanomolar activity. The second part of the thesis describes the design and synthesis of potential aspartic protease inhibitors. The hydroxyethylene motif was used as a noncleavable transition state isostere. The synthetic route yielded a pivotal intermediate with excellent stereochemical control, which was corroborated by NOESY experiments. This intermediate can be diversified with different substituents to furnish novel aspartic protease inhibitors. / <p>Report code: LIU-TEK-LIC-2006:45</p>

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Plants are exposed to many adverse factors during their life cycles. Abiotic stresses are significantly limiting plant growth and development. Abiotic stress response mechanisms involve compatible solute synthesis (e.g. sugars, sugar alcohols and amino acids). The aim of this study was to characterise the responses of plants from Plantago genus to different abiotic stresses (drought, salinity, cold and stress combination). The complete plants grew under in vitro conditions. I compared morphological parameters, selected metabolic parameters (carbohydrates balance and proline accumulation) under optimal conditions and stress exposure. This study was focused on plants from genus Plantago, because they differ from each other in their tolerance to the salinity. Both, the glycophyte and the halophyte species are described within this genus. Plantains produce besides widespread soluble carbohydrates (sucrose, glucose, fructose) also sugar alcohol sorbitol, which has been reported as a significant component of the stress response. In addition, the reaction of plants to different carbon and energy sources was tested. More severe growth inhibition of the glycophyte Plantago lanceolata compared to the halophyte P. maritima was observed under salt treatment. Significant accumulation of sorbitol was observed...

Current agriculture is facing a serious challenge of decreasing precipitation and irregular occurrence of drought periods including their unfavorable distribution during the vegetation season. This leads to growing interest in planting highly drought-resistant crops like sorghum. In comparison with other crops, sorghum excels in low water demand, though exhibits high susceptibility to low temperatures, which hampers its spread to new regions. Surprisingly, there is not enough information about the nature of sorghum's reaction to cold exposure. The aim of this diploma thesis was to describe reactions of young sorghum plants exposed to cold stress, low water availability and their combination and to verify the possibility of plant hardening through previous low-stress load. The special focus was paid to changes in carbohydrate metabolism, which plays generally very important role in plant defense reactions. The other analyzed physiological traits were leaf tissue osmotic potential, proline content and basic morphometric characteristics. Experimental design consisted of pot experiments conducted in growth chambers and the experiments performed under controlled conditions in vitro, using two sorghum genotypes "Ruzrok" and "01Z1800012". Both genotypes exhibited similar response to stress treatment....

Pyrrolo [2,1-c] [1,4] benzodiazepine (PBD) is a class of natural products obtained from various actinomycetes which have both anti-tumor and antibiotic activities and can bind to specific sequences of DNA. PBD-dilactam was initially produced using isatoic anhydride and (L)-proline which was then converted to the PBD-thiolactam using Lawesson's reagent. Reaction of thiolactam with hydrazine in ethanol afforded PBD-11-hydrazinyl. Condensation of 11-hydrazinyl PBD with aldehydes possessing various substitutions was performed to obtain (S,E)-11-[2-(arylmethylene) hydrazono] pyrrolo [2,1-c] [1,4] benzodiazepine derivatives. 1HNMR, 13C-NMR, DEPT, IR, GC-MS and X-ray crystallography were used for the characterization. Inhibition activity of the products were carried out using TEM-1, AmpC and P99 β-lactamases. A minimal inhibition growth of 25% was observed for one of the selected PBDs on cancer cell line. A promising result was observed on preliminary cannabinoid binding activity test on one of the compounds.

MYC is a family of three regulator genes that codes for transcription factors. Expression of Myc proteins from MYC genes is found to be deregulated in 70 % of all cancer forms. The three human homologs C-Myc, N-Myc and L-Myc are mainly associated with cancer in the lymphatic system, nerve tissues and lung cancer, respectively. Even though N-Myc is associated with Neuroblastoma, the cancer variant that is most common among children, the field is focused towards C-Myc. The activation of C-Myc begins with phosphorylation of Serine 62, followed by trans-to-cis isomerisation of Proline 63. Then Threonine 58 becomes phosphorylated leading to that Serine 62 is dephosphorylated and subsequent cis-to-trans isomerisation of Proline 63, and C-Myc is marked for degradation. Cis-trans isomerisation is necessary for regulation of gene expression, and is therefore important to understand. Since N-Myc and C-Myc have identical sequences between residues 47 to residue 69, the hypothesis is that N-Myc is activated in the same manner, but this has not been confirmed. In this project the first 69 amino acids of N-Myc were analysed with NMR spectroscopy. This resulted in a near complete assignment of the major conformation, and of the alternative minor conformations as well. The traditional assignment experiments HNCACB, HN(CO)CACB, HNCO, HN(CA)CO in combination with CCH-TOCSY and HN(CCO)C revealed that the majority of the minor configurations can be explained by cis/trans isomerisation of prolines. In addition, the protein was analysed with direct carbon detected NMR spectroscopy to be able to detect the prolines.

Proline-rich antimicrobial peptides (PrAMPs) are promising candidates for the treatment of infections caused by highpriority human pathogens. Their mode of action consists of (I) passive diffusion across the outer membrane, (II) active transport through the inner membrane, and (III) inhibition of protein biosynthesis by blocking the exit tunnel of the 70S ribosome. We tested whether in vitro data on ribosomal binding and bacterial uptake could predict the antibacterial activity of PrAMPs against Gram-negative and Gram-positive bacteria. Ribosomal binding and bacterial uptake rates were measured for 47 derivatives of PrAMP Onc112 and compared to the minimal inhibitory concentrations (MIC) of each peptide. Ribosomal binding was evaluated for ribosome extracts from four Gram-negative bacteria. Bacterial uptake was assessed by quantifying each peptide in the supernatants of bacterial cultures. Oncocin analogues with a higher net positive charge appeared to be more active, although their ribosome binding and uptake rates were not necessarily better than for Onc112. The data suggest a complex mode of action influenced by further factors improving or reducing the antibacterial activity, including diffusion through membranes, transport mechanism, secondary targets, off-target binding, intracellular distribution, and membrane effects. Relying only on in vitro binding and uptake data may not be sufficient for the rational development of more active analogues.

In view of the global spread of multiresistant bacteria and the occurrence of panresistant bacteria, there is an urgent need for antimicrobials with novel modes of action. A promising class is antimicrobial peptides (AMPs), including them proline-rich AMPs (PrAMPs), which target the 70S ribosome to inhibit protein translation. Here, we present a new designer peptide, Api805, combining the N- and C-terminal sequences of PrAMPs Api137 and drosocin, respectively. Api805 was similarly active against two Escherichia coli B strains but was inactive against E. coli K12 strain BW25113. These different activities could not be explained by the dissociation constants measured for 70S ribosome preparations from E. coli K12 and B strains. Mutations in the SbmA transporter that PrAMPs use to pass the inner membrane or proteolytic degradation of Api805 by lysate proteases could not explain this either. Interestingly, Api805 seems not to bind to the known binding sites of PrAMPs at the 70S ribosome and inhibited in vitro protein translation, independent of release factors, most likely using a “multimodal effect”. Interestingly, Api805 entered the E. coli B strain Rosetta faster and at larger quantities than the E. coli K-12 strain BW25113, which may be related to the different LPS core structure. In conclusion, slight structural changes in PrAMPs significantly altered their binding sites and mechanisms of action, allowing for the design of different antibiotic classes.