Development of a new PNA analogue as a potential antisense drug and tool for life-science studies

The work described in this thesis focuses on applying synthetic organic chemistry methods (supported by modem synthesizers and analytical techniques) for the preparation of peptides and modified nucleic acids in order to affect certain properties in these biomolecules, which can lead to their application as drugs and/or tools for life sciences studies.

The first part of the thesis reports the design and synthesis of a novel pyrrolidine-based peptide nucleic acid (PNA). Two enantiomers of the chiral pyrrolidine-containing unit were chemically synthesized and further incorporated into PNA fragments using modem automated solid-phase assembly methods. By comparison of their binding affinities to both DNA and RNA targets, it was found that such PNAs, due to their enhanced rigidity are able to recognize and bind to the complementary RNA strands with significantly larger affinity than to the complementary DNA. These findings may be utilized in the development of RNA-specific molecular probes, binding assays for cell extracts that contain mixtures of RNA and DNA, or targeting of viral RNAs, i.e. act as basis for the development of antisense drugs.

With the rapid advancement of biochemistry and related sciences, there is an increasing demand for synthetic peptides. The development and application of a new type of peptide-coupling agent is described. This coupling agent is superior in some ways to the most commonly used commercial agents, since peptides obtained by its action are generally of higher enantiopurity, i.e. a significant reduction of racemization of the amino acid during coupling is obtained. The potential use of this agent in linking peptide segments has been studied and discussed. So called 'segment coupling' becomes economically valuable when either large synthetic peptides are to be made, or when a number of peptides, consisting mainly of highly conserved parts with differences in only a few amino acid residues, are desired. Alternatively, this coupling agent could be used for solid phase synthesis of PNA sequences containing racemization-prone units.

As a spin-off from the above project, new sulfilimine derivatives of natural nucleosides have been successfully synthesized, isolated, characterized and studied. The synthesis of deoxycytidine and deoxyadenosine sulfilimine dimethyl and diphenyl sulfilimines is reported and the results of tests of their stability under a variety of conditions - mostly those relevant to oligonucleotide chemistry are discussed. The results of these stability studies lead to the conclusion that the sulfilimine group can serve as UV-detectable labels for short oligonucleotides. This label can be either transient or permanent (dimethyl or diphenyl sulfilimine, respectively) and can simplify the detection of tagged oligonucleotides during their purification and in assays, which can otherwise be a complicated task.

In the final part of this thesis it is demonstrated how a sequence-specific PNA clamp is used to suppress a dsDNA-digesting enzyme Exonuclease III. The rate of the enzymatic degradation has been studied in different environments and added-PNA concentrations. It was found that PNA indeed specifically suppresses the action of the enzyme, in addition to non-specific inhibition. This, previously non-documented finding, helps to better understand the mechanism of action of these enzymatic processes, as well as having potential applications within both DNA sequencing and gene therapy.

List of scientific papers

I. Slaitas A, Yeheskiely E (2001). Synthesis and hybridization of novel chiral pyrrolidine based PNA analogue. Nucleosides Nucleotides Nucleic Acids. 20(4-7): 1377-9.
https://pubmed.ncbi.nlm.nih.gov/11563026

II. Slaitas A, Yeheskiely E (2002). A novel N(pyrrolidinyl-2-methyl)glycine-based PNA with a strong preference for RNA over DNA. Eur J Org Chem. 2391-99.

III. Slaitas A, Yeheskiely E (2002). Fast and efficient peptide bond formation using bis-[alpha,alpha-bis(trifluoromethyl)-benzyloxy]diphenylsulfur. Part I. J Pept Res. 60(5): 283-91.
https://pubmed.ncbi.nlm.nih.gov/12383118

IV. Slaitas A (2004). Synthesis and reactivity of nucleoside sulfilimines. Phosphorus, Sulfur and Silicon. 179: 153-71.

V. Slaitas A, Ander C, Foldes-Papp Z, Rigler R, Yeheskiely E (2003). Suppression of exonucleolytic degradation of double-stranded DNA and inhibition of Exonuclease III by PNA. Nucleosides Nucleotides Nucleic Acids. 22(5-8): 1603-5.
https://pubmed.ncbi.nlm.nih.gov/14565476