Construction Of Chirality-Modified Polyamides And Their Specific Recognition To B-DNA Base T

The pyrrole-imidazole (Py-Im) polyamides represent the only availab-le class of small synthetic molecules that can be designed to recognize vir-tually any predetermined B-DNA sequence in minor groove and permeate into nucleus to regulate gene expression in vitro & vivo cell due to affiniti-es and specificities that equal or exceed natural eukaryotic transcriptional regulatory proteins. They are mainly composed of N-methyl-pyrrole (Py), N-methylimidazole (Im), N-methyl-3-hydroxypyrrole (Hp) amino acids and aliphatic compounds including aliphatic amino acids. In these recogni-zing elements, stereochemically-modified aliphatic compounds as a part of constructing polyamides play a very important role in specifically recogniz-ing DNA sequencees.In this thesis,14 pyrrole-imidazole (Py-Im) polyamides including chi-ral amino acids:(R/S)-β-hydroxyl-y-aminobutyric acid (Rγβ-OH,Sγβ-OH), (R/S)-2,3-diaminopropionic acid(Rβα-NH2, Sβα-NH2), (1R,3S)-3-aminocyclo-pentanecarboxylic acid (RSCp), (1S,3R)-3-amino-cyclopentanecarboxylic acid (SRCp) as a T recognition element were designed,4 of which have been synthesized by Fmoc chemistry manual-solid-phase synthetic method. The binding properties of the stereochemically-modified Py-Im polyamides to predetermined DNA sequences including a core binding site were then systematically studied by surface Plasmon resonance (SPR) and computer-aided molecular simulation (MS) technologies. SPR and MS results revealed both that the pairing of chiral recognition elements:Rγβ-OH,Sγβ-OH, Rβα-NH2,Sβα-NH2, RSCp and SRCp side-by-side with β-alanine (β) in such polyamides sometimes significantly influenced the DNA binding affinity and recognition specificity of hairpin polyamides in the DNA minor groove compared with β/β and Py/Py pairings. More importantly, SPR study has shown that the polyamide Ac-Im-Sβα-NH2-ImPy-γ-ImPy-β-Py-β-Dp (β/Sβα-NH2) favorably binds to a DNA sequence including a core binding site 5’-TGCNCA-3’/5’-TGN:GCA-3’(N-N’=A-T) with dissociation equilibrium constant (KD) of 6.3×10-8M over N·N’=T·A with KD= 9.5×10-7M, with a 15-fold specificity. It has been suggested in MS data that a-NH2 of Sβα-NH2 residue is very close to 02 of the base T to form an effective hydrogen bond, leading to a stronger affinity in the recognition event with the polyamide including β/Sβα-NH2 pairing to 5’-TGCNCA-3’/5’-TGN’GCA-3’ (N·N’=A·T). Collectively, the pairing of Sβα-NH2 to β to form β/Sβα-NH2 in the polyamide may distinguish A·T from T·A, G·C and C·G Also, the binding characteristics with the polyamides Ac-ImIm-P-Imlm-γ-PyPy-Sγβ-OH-PyPy-β-Dp (Sγβ-OH/β) and Ac-ImIm-Sγβ-OH-ImIm-γ-PyPy-β-PyPy-β-Dp (β/Sγβ-OH) to DNA sequences:5’-TAATGGA/TGGATAA-375’-TTATCCT/ACCATTA-3’were investigated well using MS technology, suggesting that P-hydroxyl of Sγβ-OH is able to form a stronger hydrogen bond with O2 of the base T. It hints that (S)-β-hydroxyl-γ-aminobutyric acid (Sγβ-OH) possesses a potential for acting as a T recognition element. These results also indicate that the stereochemistry of the recognition elements, especially aliphatic amino acids may greatly alter binding affinity and recognition selectivity of hairpin polyamides to predetermined DNA sequences.