SYNTHESIS AND DNA BINDING PROPERTIES OF PYRROLIDINYL PEPTIDE NUCLEIC ACID CONTAINING (2S,3S)-3-AMINOTETRAHYDROFURAN-2-CARBOXYLIC ACID SPACER

Abstract

Peptide nucleic acid (PNA) is an analogue of DNA with the negatively charged deoxyribose phosphate backbone is replaced with a peptide-like structure. PNA retains the ability to bind strongly and sequence specifically to DNA and RNA similar to natural DNA, but exhibit several remarkable differences such as the high chemical and biological stability, the electrostatic neutrality and the ability to invade into double stranded DNA. Recently, pyrrolidinyl PNA with a prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbone (acpcPNA) was introduced as a new conformationally constrained PNA analogue that exhibit several desirable properties including the high affinity and specificity in binding to DNA, the high preference for binding to DNA over RNA, the exclusive binding in antiparallel orientation and the inability to form self-hybrids. Nevertheless, the hydrophobic prolyl-ACPC backbone of acpcPNA showed non-specific interactions with many hydrophobic materials, including the commonly used polypropylene plastic tubes. This motivated a development of a new pyrrolidinyl PNA with a more hydrophilic backbone by replacement of the ACPC with a beta-amino acid containing a hydrophilic tetrahydrofuran ring. This requires a building block named N-Fmoc-(2S,3S)-3-aminotetrahydrofuran-2-carboxylic acid (SS-ATFC) pentafluorophenyl ester, which was successfully synthesized from 2-deoxy-d-ribose overall 12 steps in 3% yield. The ATFC building blocks were oligomerized with nucleobase-modified-D-proline monomers, represented as atfcPNA. The ATFC was incorporated in to the PNA structure to give a new atfcPNA, which could still bind to DNA and RNA, but with slightly to moderately lowered affinity and specificity (depending on the base sequence) when compared to the benchmark acpcPNA according to UV-Tm and CD spectroscopy. The higher polarity of the atfcPNA than acpcPNA was confirmed by reverse phase HPLC. Fluorescence experiments preformed on fluorescein labeled acpcPNA and atfcPNA clearly demonstrated the reduced non-specific binding of atfcPNA to plastic tubes. These results suggest that the replacement of the cyclopentane ring in acpcPNA with the tetrahydrofuran ring can reduce undesirable non-specific interactions without much affect on the DNA binding properties, which may have important implications in many practical applications of pyrrolidinyl PNA.