Current Status and Research Projects
The Nucleic Acids Laboratory of the Department of Medicinal Chemistry (University of Szeged, Hungary) was set up in 1996 and there were several overhauls of its infrastructure over the years. The steadily improving conditions resulted in the recent purchase of some important new instruments essential in organic synthesis in general and in nucleic acids synthesis and characterization in particular. Currently 6 full-time employed people are working in one laboratory and in an adjacent office.
The activity of the Laboratory focuses on the following directions of contemporary nucleic acids research:
Our Laboratory has expertise, among others, in peptide nucleic acids (PNA), unnatural nucleic acid mimics composed of a poly[N-(2-aminoethyl)glycine] backbone and nucleobases attached through an acetyl linkage. PNAs, despite their achiral and uncharged backbone, retain the unsurpassed selectivity of base-pairing of native nucleic acids. We have prepared novel PNA monomers with Fmoc (backbone) and different acyl (nucleobases) protecting groups which have been successfully used in the synthesis of PNA oligomers. In a recent project, funded by The Wellcome Trust and the European Union, we are cooperating with Dr. Nicola M. Howarth (Heriot-Watt University, Edinburgh, UK) on the synthesis of chiral azetidine PNA analogues starting from carbohydrate precursors. Our results suggest that the intramolecular 1,3-dipolar cycloaddition of carbohydrate-derived omega-unsaturated nitrones offer a very promising entry into this field.
We have developed a nanoelectrospray mass spectrometric method using lithium salts to afford salient adduct ions of nucleotide phosphoramidites. This would have been impossible with other mass spectrometric methods and completes the existing NMR methods (1H, 13C, 31P) for the characterization of these very sensitive compounds.
The notoriously difficult problem of the regioselective substitution (N9) of guanine has been succesfully tackled and exclusively 9-substituted guanine derivatives have been obtained employing 4-nitrobenzyl-protected guaninium salts. Three different methods have been elaborated for the differentiation of 9- and 7-alkylated guanines: a statistical method based on 13C NMR chemical shift differences, a 15N NMR method and a novel electrospray mass spectroscopic method the quantitative version of which gives 2-5% accuracy in determining the ratio of isomeric guanines.
In a quest for alternative nucleobases we have investigated the hydrogen bonding pattern of 3-methylated 6-aminouracil (3sau) and 5-methyled 6-aminouracil (5sau) with native nucleobases. In cooperation with the Protein Chemistry Resarch Group of our Department, it has been found (ab intio calculations) that there are two potential duplexes of 5sau and three possible duplexes of 3sau. Altogether seven dimers containing one or two bifurcating H-bonds have been found. Later on, five triplexes from ten possible calculated dimers have also been identified. In two of them the amino group of 6-aminouracil moiety takes part in H-bonding and there are H-bonds, too, between the first and third base of the triplexes causing an extra stabilization. Attempts towards the experimental verification of the above assumption (synthesis of PNA analogues containing the above nucleobases) are in progress.
The synthesis of DNA bioconjugates often requires 5'-thiol modified oligonucleotides. We have prepared novel crystalline 5'-thiol modifier phosphoramidite linkers allowing the preparation of DNA-peptide and DNA-PNA conjugates. The most frequently applied protecting group on thiol-modifier linkers is the trityl group. However, removal of this group is usually problematic: heavy metal cations (Ag+, Hg2+) are used for removal and the residual heavy ion impurity may hamper the application of the desired DNA in biological systems. Moreover, metal ion precipitation may result in significant loss of the DNA product, too. According to our recent results, 4,4',4"-trimethoxytrityl protected derivatives can be efficiently deprotected without metal ions and their purification was accomplished using standard PolyPakTM column.
In another project we aimed at enhancing the sensitivity of MS analysis of crude PNAs and peptides, by introducing positive charge onto the oligomer by preparation of their pyridinium and thiazolium derivatives and at indentifying the molecular ion of the product easier, based on the isotope distribution of bromine-containing labels. Our preliminary results show that this is possible and the labeled derivatives of apolar peptides allow sequencing by virtue of the presence of an ions in the mass spectra.
© Lajos Kovács, 1999-