Synthesis Of Novel Peptide Nucleic Acids And Studies Of Their Properties

Oligonucleotides are potentially useful for the regulation of genetic expression by sequence specific binding with DNA or mRNA. The principle, which is known as the antisense, provides a way to control protein synthesis at the nucleic acid level, which would even be more effective than inhibition of enzymes. Furthermore, a highly specific inhibition can be achieved by a relatively short antisense oligonucleotide, whose sequence can be derived directly from the sequence of the target nucleic acids. However, to put the antisense principle into practice, the antisense oligonucleotides must be sufficiently stable under physiological conditions, able to pass through the cell membrane, and bind specifically and tightly with the target nucleic acids. Because natural oligonucleotides are readily degraded by nucleases in vivo, there is considerable interest in synthetic oligonucleotide analogues, which are stable under physiological conditions. Recently, there has been interest in oligonucleotide analogues in which the sugar phosphate backbone is replaced by a peptide chain after the success of the so-called peptide nucleic acids.Peptide nucleic acids were first described by Nielsen at al. in 1991. Peptide nucleic acid has advantages over several other chemical modification of oligonucleotides. Peptide nucleic acids showed high bio-stability. Peptide nucleic acid oligomers are neither degraded by nucleases nor proteases and protected oligomers can be assembled by well established solid phase peptide synthesis protocols.In this study, we designed a novel peptide nucleic acid analogue of DNA. The designated PNA composed of N-(2-aminomethyl-4-nucleobase) tetrahydropyrrole acetic acid units.Boc-protection strategy was used to synthesize the new structural PNA backbone. Using trans-4-hydroxy-L-proline as starting material, after a series chemical reactions, the intermediate product of N-[2-(tert-butoxylcarbonylaminomethyl)-4-hydroxy]tetrahydropyrrole acetic acid methyl ester[I] was synthesized. That intermediate compound [I] with N3-benzoylthymine and N~2-isobutylryl-O~6-(4-nitrophenylethyl)guanine by the Mitsunobu reaction to give N-[2-(tert-Butoxylcarbonylaminomethyl)-4-(N~3-benzoylthymin-1-yl)] tetrahydropyrrole acetic acid methyl ester and N-[2-(tert-butoxylcarbonylaminomethyl)-4-(N~2-isotutyrylguanin-9-yl)]tetrahydropyrrole acetic acid methyl ester {after removal of the O~6-nitrophenylethyl group by treatment with DBU in pyridine}.The compound [I] was treated with toluene-p-sulfonyl chloride to give toluene-p-sulfonate. The toluene-p-sulfonate reacted with N~6-benzoyladenine and N~4-benzoylcytosine to give N-[2-(tert-butoxylcarbonylaminomethyl)-4-(N~6-benzoyladenin-9-yl)]tetrahydropyrrole acetic acid methyl ester and N-[2-(tert-butoxylcarbonylaminomethyl)-4-(N4-benzoylcytosin-1 -yl)]tetrahydrogen pyrrole acetic acid methyl ester. The methyl esters were hydrolysised to give corresponding new structural PNA monomers.Using the above Boc-protected monomers, the new PNA oligomers p(A10-Lys-NH2) and p(T10-Lys-NH2) were assembled manually in a stepwise fashion by solid phase synthesis technique. The crude products were purified by sephadex gel filtration and HPLC, and the structure were confirmed by MS.The hybridization properties of new structural PNA were evaluated by thermal-denaturation curve. Experiments showed there is hybrid between p(A,0-Lys-NH2) and dT10, could not hybridize between p(T10-Lys-NH2) and dA10.