The Design, Synthesis And Initial Discussion Of Tybridization Properties Of Chiral Peptide Nucleic Acids Containing Hydroxy And Imidazole Groups

PNA, a structural mimic of DNA in which the sugar-phosphate is replaced by N-(2-aminoethyl)-glycine linkage was first described by Nielsen et al. in 1991. PNA has some advantages over several other chemical modification of oligonucleotides: (1) PNA shows high bio-stability against nucleases and proteases; (2) it can readily pass though cell membrane; (3) it can hybridize specifically and tightly with complementary DNA or RNA sequences; (4) it can be well assembled by established solid phase peptide synthesis protocols.The introduction of hydroxy and imidazole, polar groups of side chains of natural chiral Z-serine and Z-histidine, to the backbone of classical PNA enhance its polarity and solubility in aqueous medium. In this study, we design to synthesize chiral PNA monomers containing Z-serine and Z-histidine residues. In order to enhance its solubility in aqueous medium we design to insert these PNA monomers into the backbone of classical PNA oligomers to assemble chiral PNA containing hydroxy and imidazole polar groups. The hybridization properties of the chiral PNA with complementary DNA and PNA will be evaluated by thermal-denaturation curve and effect of hydroxy and imidazole on hybridization stability will be discussed initially.In this paper, five chiral PNA monomers containing two natural bases (A, T) were prepared in modified methods to the literature. Two kinds of PNA monomers containing serine residue, N-(tert-butyloxycarbonylaminoethyl)-N-(thyininyl-1 -acetyl) -O-benzyl-serine (Ts) and N-(tert-butyloxycarbonylaminoethyl)-N-{[N~6-(benzoyl)ad enyl]-9-acetyl}-O-benzyl-serine (As), have been synthesized. Three kinds of PNA monomers containing histidine residue, N(a)-(tert-butyloxycarbonylaminoethyl)-N(a)-(thyminyl-1 -acetyl)-N((?))-(benzyloxymethyl)-histidine (Th), N(a)-(tert-butyloxy carbonylaminoethyl)-N(a)-{[N6-(benzoyl)adenyl]-9-acetyl}-N(t)-(benzyloxymethyl)-h istidine (Ah) and N(a)-(tert-butyloxycarbonylaminoethyl)-N(a)-(thyminyl-1-acetyl)-N(t)-(2,4-dinitrobenz)-histidine (18), have been prepared. PNA monomer 18 was designed and synthesized for the first time. The structures of PNA monomers and intermediates were determined by 1HNMR, ESI-MS and elements analysis.With the Boc-protected monomers above, two classical PNA (TPNA, APNA) and five chiral PNA oligomers (TPNA1, TPNA2, TPNA3, APNA1, APNA2)TPNA: Tg10-Lys-NH2APNA: Ag10-Lys-NH2TPNA1: Tg4-Th-Ts-Tg4-Lys-NH2TPNA2:Tg3-Th-Ts-Th-Ts-Tg3-Lys-NH2TPNA3:Tg3-Th-Th-Ts-Ts-Tg3-Lys-NH2APNA1: Ag4-Ah-As-Ag4-Lys-NH2APNA2:Ag3-Ah-As-Ah-As-Ag3-Lys-NH2were assembled manually in a stepwise fashion by solid phase synthesis technique. The crude products were purified by RP-HPLC, and their structures were confirmed by ESI-MS or MALDI-TOF-MS.The hybridization properties of these chiral PNA oligomers with complementary DNA and PNA were evaluated by thermal-denaturation curve. The referential results were listed below.PNA-DNA:T·A hybrid of PNA with complementary DNA: APNAi, APNA2 gave Tm values of 49℃ and 48.9℃ with dT10 and were 2.1℃ and 2.2℃ lower than that of APNA/dT10 (Tm=51.1℃); TPNA2 and dA10 showed a well defined single-phase melting profile, with a melting temperature (Tm) being 34.1℃. However, TPNA1, TPNA3 were found not to hybridize to dA10 at all.T·A·T hybrid of PNA with complementary DNA: APNA, APNAi gave Tm values of 51.9℃, 52.TC with dT10. APNA2 was found not to hybridize to dT10. Tm values of TPNA, and TPNA2 with dA10 were 57.3℃, 48.1℃. TPNA and TPNA1 were found not to hybridize to dA10 at all.PNA-PNA:T·A hybrid of PNA with complementary PNA: Tm values of APNA, and APNA2 with TPNA were 62℃, 58.3℃. TPNA1, TPNA2 and TPNA3 were found not to hybridize to APNA1 and APNA2.T·A·T hybrid of PNA with complementary PNA: TPNA gave Tm values of 63.4℃ and 61.2℃ with APNA,, APNA2 and were 1.5℃, 3.7℃ lower than that of (TPNA)2/APNA (Tm=64.9℃); TPNA,, TPNA and TPNA3 were found not to hybridize to APNA1 and APNA2 under these conditions.