| Peptide nucleic acids (PNA) were first described by Nielsen in 1991, in which the natural deoxyribose phosphate backbone of DNA was replaced by repeating N-(2-aminoethyl)glycineunits (Fig.1), the natural nucleobases attached through methylenecarbonyl linkers to the backbone. PNA is a good mimic of DNA or RNA, it can hybridize with complementary DNA or RNA strand through Watson-Crick base-pairing. In analogy to natural nucleic acids, homopyrimidine PNA oligomers form (PNA)2-DNA triplexes with complementary homo-purine nucleic acids and such triplexes show unprecedented high thermal stability. A consistent thermal stability was also observed in duplex formation, PNA-PNA > PNA-RNA > PNA-DNA > DNA-DNA, because the hybridization of PNA do not result in electronic repulsion, and therefore, it is not affected by ionic strength.PNA is much more stable chemically and biologically, compared to DNA and RNA. PNA can neither be recognized nor easily degraded by proteases or nucleases. Furthermore, its neutral structure avoids interactions with contents in blood plasma and non-specific interactions would be reduced. More importantly, the strategy for peptide solid-phase synthesis could be applied for PNA.But PNA has some drawbacks, such as poor water-solubility and bioavailability, and self-aggregation. Much progress have been made to improve these properties. Many novel PNAs were synthesized. The modifications are mainly focused on the backbone. It includes the changes of bond numbers in backbone of monomers, replacement of glycine with chiral amino acid for the introduction of functional groups atα-position orγ-position, as well as cyclic linkage between N-terminals and C-terminals.Nucleobase modification: This is focusing on some position of natural nucleobase or replacing natural nucleobase with groups containing nitrogen. Because the modified PNA sequence will be used to form higher structure, the modification on PNA monomers should not affect forming base-pairing. It includes introducing functional groups to 5-position of uracil, replacement of cytosine and adenine with isocytosine and 2,6-diamine-purine, heterocyclization of thymine and cytosine, and introducing fluorescent nucleobases.Simultaneous modifications on both backbone and nucleobases were seldom reported, because orthogonal protection suitable for peptide synthesis is difficult to be realized.Objective: PNA is a relatively chemically inert polymer to ensure its highly specific hybridization with complimentary sequences, but less active in biological or chemical process, and thus its potential applications in biotechnologies and new materials would be limited. Our research is on structure-activity relationship of peptide nucleic acids and new functions of these biopolymers, so functional groups were introduced to both backbone and nucleobase. The backbones with glycine replaced by serine or aspartate were used, thus, a hydroxyl or carboxyl, as well as a chiral center were introduced into the backbone. The 5-position of uracil was chosen as the position for the introduction of carboxyl group, because this position of the uracil are not involved in hydrogen-bonding. Two new PNA monomers were synthesized.Methods: According to requirement of the Boc/Bzl strategy for the solid-phase synthesis of PNA sequences, free carboxyl group and Boc-protected amino of the PNA monomers will be used. Semi- permanent protective group Bzl was used to protect hydroxyl and carboxyl groups in the side chain of amino acids in backbone, and the carboxyl group in the 5-position of uracil. Bzl could be removed by HF treatment after the sequence polymerization.Results: Two new PNA monomers were synthesized, they are N-(2-tbutyloxycarbonylethyl)-N-[5-(benzyloxy-carbonylethyl)uracil-l-acetyl]-O-benzyl-L-serine (1) and N-(2-tbutyloxycarbonylethyl)-N-[5-(benzyloxycarbonylethyl)ur acil-l-acetyl]-β-O-benzyester-L-aspartic acid (2). Other four known PNA monomers were also prepared for the building of random PNA sequences.(1) N-(2-tbutyloxycarbonylethyl)-N-[5-( benzyl oxy-carbonylethyl)uracil-1 -acetyl]-O-benzyl-L-serine1HNMR (DMSO-d6, 400 MHz): 12.84 (s, 1H, -COO(?)), 11.45 (s, 1H, -CO-N(?)-CO), 7.33-7.36 (m, 11H, 2-C6(?)5 and C=C(?)), 5.11 (s, 2H, -COO-C(?)2-Ar), 4.0-4.68 (m, 5H, -O-C(?)2-Ar, -C(?)-C(?)2-OBzl), 3.0-4.0 (m, 8H, -N-C(?)2-CO-N-, BocNH-C(?)2-C(?)2, -C-C(?)2CO-,), 1.35 [s, 9H, -C(C(?)3)3] ESI-MS (m/z): C32H38N4O10 638.26, found 637.6(2) N-(2-tbutyloxycarbonylethyl)-N-[5-(benzyloxy-carbonylethyl)uracil-1 -acetyl]-β-O-benzyester-L-aspartic acid 1HNMR(DMSO-d6, 400 MHz): 12.84(s, 1H, -COO(?)),11.45(s, 1H, -CO-N(?)-CO-), 6.93-7.37(m, 12H, 2-C6(?)5, C=C(?) and BocN(?)-), 5.10 (d, 4H, 2-C(?)2-Ar), 4.64(s,2H,-N-C(?)2-CO-), 4.39(t, 1H, -C(?)-CH2-), 2.76-3.35(m, 8H, -N-COC(?)2-N-, BocNH-C(?)2-C(?)2-, -CH-C(?)2-), 1.35[s, 9H, -C(C(?)3)3] ESI-MS (m/z): C33H38N4O11 666.68, found 665.5Conclusion:1 The orthogonality of protective strategy is suitable in the process of synthesis.2 On the process of preparation compound (3), lowering reaction temperature and dropwise rate can decreased the by-product.3 Compound (7) has lower solubility. Lower yeild was obtained by common esterification method. Condensing agent which can make high yield was used to esterize.4 Lowering the reaction temperature can decreased by-product when compound (8) react with tbutyl bromacetate .5 Pd(PPh3)4 can remove allyl group selectivity, but with lower yield.6 LiAlH4 was used to reduce compound (19). This reaction is very quick. About 7 min the reaction is done. Prolong time willlead to by-product.7 HBTU will lead to lower yield when the reagent has spaceobstacle.
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