Pyrrolidinyl peptide nucleic acids bearing hydroxy-modified cyclobutane building blocks: Synthesis and binding properties.

Abstract

The conformationally constrained pyrrolidinyl PNA with a dipeptide consisting of an alternating nucleobase-modified D-proline and a cyclic β-amino acid "spacer" exhibited improved nucleic acid binding properties compared to the original PNA. The pyrrolidinyl PNA with the four-membered ring spacer (1S,2S)-2-aminocyclobutanecarboxylic acid (acbcPNA) are among the best performed members of the pyrrolidinyl PNA family. However, these PNA suffer some limitations such as aqueous solubility and non-specific interactions due to their extreme hydrophobicity. In the present work, a hydroxy group is introduced onto the cyclobutane ring spacer of the acbcPNA with the aim of decreasing its hydrophobicity. To this end, a Fmoc/tBu ether-protected 4-hydroxy-2-aminocyclobutanecarboxylic acid building block was synthesized and resolved by chiral HPLC. Each enantiomer was used to synthesize the hydroxy-modified acbcPNA employing Fmoc solid-phase peptide synthesis. DNA/RNA binding studies indicated that the introduction of the hydroxy group to the acbcPNA decreases the binding affinity toward complementary DNA and RNA while maintaining the sequence and directional specificity of unmodified acbcPNA. The hydrophobicity of the hydroxy-modified acbcPNA decreased with the number of hydroxy groups added as indicated by the decrease in the logP values. Only two modifications were sufficient to decrease the logP by an order of magnitude without excessively lowering the binding affinity nor the specificity. This work thus demonstrated that the specific structural modifications for this type of PNA model can be performed in a modular fashion, which paves the way toward the future realization of improving hydrophilicity and nucleic acid binding affinity as well as specificity.