Synthesis and Cellular Effect of a Novel Conjugate of Polyamide and Phospholipid

Abstract

Small molecules with the ability to target predetermined sequences of DNA would be valuable tools in molecular biology and potentially in human medicine.1-3) Synthetic polyamides consisting of N-methylpyrrole (Py) and Nmethylimidazole (Im) have received special attention due to their high DNA affinity, sequence specificity, and relatively small size. [4][5][6] These polyamides have been shown to interfere with protein-DNA interactions, and have the potential to modulate gene expression by designing to recognize specific DNA sequences. 5) For polyamides to be effective in gene regulation, they must reach their target DNA inside the cell. 5,7)However, there are few examples and only circumstantial evidence that suggest that polyamides bind to nuclear DNA targets in living eukaryotic cells. [7][8][9][10][11][12] In the present studies, we designed a conjugate in which a phosphatidylcholine (PC) was attached to the polyamide. The introduction of the phospholipid was expected to improve the permeability of the synthetic molecule through the cell membrane, guide the movement toward to nucleus in cells, and thereby reduce the cytotoxicity. The phospholipid is a primary component of biologicall membrane, and has proven to be noncytotoxic. In particular, the hydrophobic tail of the lipid is possible to greatly benefit the movement of the conjugate toward to nucleus in cells, and subsequently promote the interaction with nucleic DNA. 13) Results and DiscussionPolyamide 10 (O 2 N-PyPy-b-PyPy-b-OEt) and the targeted conjugate 13 (O 2 N-PyPy-b-PyPy-b-PC) were synthesized as shown in Chart 1. b-Alanine (b) functioned as a spacer both in polyamide 10 and conjugate 13. 10 is a regular structure among the synthetic polyamides.14,15) Based on the DNAbinding study results of natural product distamycin and netropsin 16,17) and the pairing rules of polyamides binding to DNA derived by Dervan 14) the polyamide 10 and the polyamide moiety in the designed conjugate 13 were presumed to have an ability of DNA-binding in A/T and T/A pairs rich region.Primarily, DCC coupling method was used in the synthesis.18) Direct coupling of b-alanine with pyrrole monomer Nmethyl-4-nitro-2-trichloroacetylpyrrole 1 without any coupling reagent generated monomer derivative 4 and 5, from which dimeric pyrrole amide 6 and 8 were prepared, respectively. These two dimers were among the key building blocks in the present synthesis. In the preliminary experiment of tetrameric amide synthesis, coupling of acid 7 of the dimer with the amino dimer generated from the hydrogenation of 6 was presumed to give the methyl ester of the tetrameric amide, which would be the desired polyamide and would also serve as another key building block for the conjugate. However, hydrogenation of the nitro group of 6 by the catalysis of palladium on carbon formed many side-products rather than the desired one detected by TLC method. While ethyl ester 8, instead of 6, prepared by the same method as that for the preparation of 6 was used in the reaction the desired reduction product...