| About ten years ago, PNA, a structural mimic of DNA in which the sugar-phosphate backbone is replaced by N-(2-aminoethyl)-glycine (aeg) linkage emerged as a potential antisense therapeutic agent. PNA has some advantages: (1) it is stable to cellular nucleases and proteases, (2) it hybridizes with complementary DNA or RNA (cDNA/RNA) sequences with high affinity, (3) it has low non-specific interaction with cellular contents and (4) easy synthesis by adoption of solid phase peptide synthesis chemistry. However, the major limitation of PNAs is their poor solubility in aqueous medium and achiral, and they bind to cDNA in both parallel (N-PNA/5'-DNA) and antiparallel (N-PNA/3'-DNA) modes. Based on the monomer synthesis from an amino acid, it seemed natural to substitute glycine with other amino acids in the preparation of the monomer. Various groups have tried to put chirality to PNA molecules by linking other amino acids, peptides, or oligonucleotides, at the terminus or by incorporation of chiral amino acids in place of glycine in the PNA backbone to attempt discriminating between parallel and antiparallel modes of binding.Tran.s-4-hydroxy-Z-proline is relatively easy to manipulate to access all four stereomers. The pyrrolidine ring in proline is a suitable unit for mimicking the ribose moiety in DNA. It has been proved to be a useful starting material to synthesize the conformationally constrained PNA monomers. In our laboratory, 4-hydroxyproline has also been utilized to synthesize PNA monomers in which nucleobase substitution is at the position of 4-C of the pyrrolidine ring. Among the homo-oligomers aepPNA T10-Lys-NH2, aepPNA Ai2-Lys-NH2, ampPNA T10-Lys-NH2 and ampPNA Aio-Lys-NH2 synthesized using these monomers only aepPNA Ai2-Lys-NH2 and ampPNA A10-Lys-NH2 showed hybridization with complementary DNA. We thought that the loss of affinity of these PNA analogues could be attributed to lack of flexibility of the backbone. In order to substantiate this assumption, we designed another PNA analogue: apmPNA. It has two differences from aepPNA and ampPNA we synthesized before: (1) nucleobase will not be directly but via a methlyene attached to the pyrrolidine ring and (2) the carbon attached to nucleobase in apmPNA is achiral. Those will make the backbone in the ampPNA have more flexibility. Tertiary amino structure in each unit could be expected to obtain a good solubility and a high affinity for negatively charged natural nucleic acids.In this paper, the detailed synthesis of the new chiral PNA monomers containing all four natural bases (A, C, T, G ) is described. The structures of these monomers and intermediates were determined by FAB-MS , 1H NMR, 1H-1H COSY, HMBC, IR and elements analysis.With the above Boc-protected monomers, the apmPNA oligomers: T10-Lys-NH2 , Lys-A8-Lys-NH2, A5GA2-Lys-NH2 and T*3tT*4-Lys-NH2 , (T*t)4-Lys-NH2 (T* aegPNA monomer; t apmPNA monomr) were assembled manully in a stepwise fashion by solid phase synthesis technique. The crude products were purified by sephadex gel filtration and RP-HPLC, and their structures were confirmed by FAB-MS.The hybridization properties of these ampPNA oligomers were evaluated by thermal-denaturation curve, TOF MS ES and NMR. However, the hybridization properties of these oligomers: T10-Lys-NH2, Lys-A8-Lys-NH2, A5GA2-Lys-NH2 and (T*t)4-Lys-NH2 with their DNA targets showed disappointing results. The hybrid between T*3tT*4-Lys-NH2 and dA8 showed a well defined single-phase melting profile, with a melting temperature (Tm) being 32 whereas the correspongding Tm of hybrid aegPNA T*8-Lys-NH2\ dAg was 42 . These data showed: (1) hybridization properties of the aepPNA and ampPNA could not be optimum by only increasing the flexibility of the backbones; (2) the effect of the apmPNA monomer on the structural perturbations in oligomeric aegPNA was additive.
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