Some Aspects of Nucleic Acids Chemistry

This thesis is divided into two parts based on a total of 8 papers: Part 1: Synthesis, physicochemical and biochemical studies of chemically modified oligonucleotides and their duplexes and triplexes. Potency of the chromophore conjugated DNA oligonucleotides as antigene and antisense gene repressors was evaluated. The effect of geometry, bulk and ¥ð-electron density of a series of chromophores, tethered at the 5'-end of oligonucleotides, as well as the effect of the linker nature, length and the attachment site of the chromophore to the oligo were explored based on the stability of the duplexes and triplexes. A dramatic improvement in the triplex stability with ara-U linked phenazine oligo (potent antigene) was achieved (¥ÄTm = 16.5¢ª C). A number of selected phenazine and dipyridophenazine tethered antisense oligos (AONs) and their phosphorothioate analogues were shown to form the AON/RNA hybrid duplexes with enhanced thermal stability. CD experiments revealed that these duplexes have the global structure unaltered from that of the native counterpart. RNase H degradation studies on three RNA targets having different degrees of folded structures showed that tethering of phenazine and dipyridophenazine increases the hydrolysis rates (potent antisense) of the target RNA, and that chemical nature of the chromophore influences the RNase H cleavage pattern. Further investigation at the RNA saturated conditions revealed that 3'-tethered chromophores influence the substrate recognition, and the kinetics of the cleavage by RNase H. Conjugation of different chromophores, charged polyaromatic systems and metal complexes with polyaromatic ligands at different sites of the AON revealed that RNase H is very sensitive to any modifications in the middle region of the AON/RNA duplex. On the contrary, any modification at the 3'-end of the AON regardless of the bulk of the substituent or presence of positive charge can be easily tolerated by the enzyme. Sensitivity of the RNase H towards the local structural changes in the AON/RNA hybrid was probed with a number of AONs containing a single 1-(1',3'-O-anhydro-©¬-D-psicofuranosyl)thymine with locked 3'-endo sugar conformation at different sites of AON. RNase H degradation studies revealed that the local conformational changes brought by the constrained nucleoside, although invisible by CD, span in the hybrid as far as 5 nucleotides toward the 5'-end of the AONs (3'-end of RNA), showing the unique transmission of the structural distortion from a single modification site. The results also showed that the structural requirements for the substrate binding and substrate cleavage by RNase H appear to be different. Part 2: Preparation of biologically important isotope labelled oligo-RNAs for the NMR structure determination in solution. Synthesis of the non-uniformly 13C5 labelled 29mer HIV-1 TAR RNA was achieved by solid-phase synthesis using 13C5 labelled ribonucleosides from 13C6-D-glucose). Two hammerhead forming RNAs (16mer and 25mer) were synthesized according to the Uppsala NMR-window strategy, where the sugar residues of the nucleosides forming stem I, II and the loop of the stem III of the resulting hammerhead complex were deuterated. UV melting and high resolution NMR structural studies showed that the 16mer RNA under quasiphysiological condition folds to a very stable hairpin structure, which prevents formation of a hammerhead RNA with the 25mer, primarily owing to thermodynamic reasons

Thiomethylation, Nitro Reduction and Tandem Reduction/SMe Insertion of Nitrogen Heterocycles Using BF3⦁SMe2

Herein, a general, solvent-free and straightforward thiomethylation of electron deficient heterocycles using BF3⦁SMe2 as a dual thiomethyl source and Lewis acidic activator is presented. A range of heterocycles including pyrimidine, pyrazine, pyridazine, thiazole and purine derivatives were successfully substituted using this method. An unexpected reductive property of BF3⦁SMe2 towards nitropyridines was also discovered including an intriguing tandem reduction/SMe insertion process in certain substrates. Notable features of the present work include its convenience and use of a non-malodorous reagent while the discovery of novel chemical transformations using BF3⦁SMe2 provides fundamental new insights into the reactivity of this commonly employed reagent

Antibacterial sulfonimidamide-based oligopeptides as type I signal peptidase inhibitors : Synthesis and biological evaluation

Oligopeptide boronates with a lipophilic tail are known to inhibit the type I signal peptidase in E. coli, which is a promising drug target for developing novel antibiotics. Antibacterial activity depends on these oligopeptides having a cationic modification to increase their permeation. Unfortunately, this modification is associated with cytotoxicity, motivating the need for novel approaches. The sulfonimidamide functionality has recently gained much interest in drug design and discovery, as a means of introducing chirality and an imine-handle, thus allowing for the incorporation of additional substituents. This in turn can tune the chemical and biological properties, which are here explored. We show that introducing the sulfonimidamide between the lipophilic tail and the peptide in a series of signal peptidase inhibitors resulted in antibacterial activity, while the sulfonamide isostere and previously known non-cationic analogs were inactive. Additionally, we show that replacing the sulfonamide with a sulfonimidamide resulted in decreased cytotoxicity, and similar results were seen by adding a cationic sidechain to the sulfonimidamide motif. This is the first report of incorporation of the sulfonimidamide functional group into bioactive peptides, more specifically into antibacterial oligopeptides, and evaluation of its biological effects

Design, synthesis, and in vitro biological evaluation of meta-sulfonamidobenzamide-based antibacterial LpxH inhibitors

New antibacterial compounds are urgently needed, especially for infections caused by the top-priority Gram-negative bacteria that are increasingly difficult to treat. Lipid A is a key component of the Gram-negative outer membrane and the LpxH enzyme plays an important role in its biosynthesis, making it a promising antibacterial target. Inspired by previously reported ortho-N-methyl-sulfonamidobenzamide-based LpxH inhibitors, novel benzamide substitutions were explored in this work to assess their in vitro activity. Our findings reveal that maintaining wild-type antibacterial activity necessitates removal of the N-methyl group when shifting the ortho-N-methyl-sulfonamide to the meta-position. This discovery led to the synthesis of meta-sulfonamidobenzamide analogs with potent antibacterial activity and enzyme inhibition. Moreover, we demonstrate that modifying the benzamide scaffold can alter blocking of the cardiac voltage-gated potassium ion channel hERG. Furthermore, two LpxH-bound X-ray structures show how the enzyme-ligand interactions of the meta-sulfonamidobenzamide analogs differ from those of the previously reported ortho analogs. Overall, our study has identified meta-sulfonamidobenzamide derivatives as promising LpxH inhibitors with the potential for optimization in future antibacterial hit-to-lead programs.De två sista författarna delar sistaförfattarskapetAuthors in the list of papers of Andrea Benediktsdóttir's thesis: Benediktsdottir A., Sooriyaarachchi S., Cao S., Ottosson N. E., Lindström S., Daina L., Bobileva O., Loza E., Hughes D., Jones A., Mowbray L. S., Zamaratski E., Sandström A., Karlén A.</p

Design, synthesis and in vitro biological evaluation of oligopeptides targeting E. coli type I signal peptidase (LepB)

Type I signal peptidases are potential targets for the development of new antibacterial agents. Here we report finding potent inhibitors of E. coli type I signal peptidase (LepB), by optimizing a previously reported hit compound, decanoyl-PTANA-CHO, through modifications at the N- and C-termini. Good improvements of inhibitory potency were obtained, with IC50s in the low nanomolar range. The best inhibitors also showed good antimicrobial activity, with MICs in the low μg/mL range for several bacterial species. The selection of resistant mutants provided strong support for LepB as the target of these compounds. The cytotoxicity and hemolytic profiles of these compounds are not optimal but the finding that minor structural changes cause the large effects on these properties suggests that there is potential for optimization in future studies.Maria De Rosa and Lu Lu contributed equally to this work.</p

Antibiotic class with potent in vivo activity targeting lipopolysaccharide synthesis in Gram-negative bacteria

Here, we describe the identification of an antibiotic class acting via LpxH, a clinically unexploited target in lipopolysaccharide synthesis. The lipopolysaccharide synthesis pathway is essential in most Gram-negative bacteria and there is no analogous pathway in humans. Based on a series of phenotypic screens, we identified a hit targeting this pathway that had activity on efflux-defective strains of Escherichia coli. We recognized common structural elements between this hit and a previously published inhibitor, also with activity against efflux-deficient bacteria. With the help of X-ray structures, this information was used to design inhibitors with activity on efflux-proficient, wild-type strains. Optimization of properties such as solubility, metabolic stability and serum protein binding resulted in compounds having potent in vivo efficacy against bloodstream infections caused by the critical Gram-negative pathogens E. coli and Klebsiella pneumoniae. Other favorable properties of the series include a lack of pre-existing resistance in clinical isolates, and no loss of activity against strains expressing extended-spectrum-beta-lactamase, metallo-beta-lactamase, or carbapenemase-resistance genes. Further development of this class of antibiotics could make an important contribution to the ongoing struggle against antibiotic resistance