Pyrrolidinyl peptide nucleic acid with an oxetane-containing β-amino acid linker

Abstract

Peptide Nucleic Acid (PNA) is a nucleic acid mimic that exhibits excellent binding properties with complementary DNA and RNA. The PNA-DNA or PNA-RNA binding is stronger than the self-binding between natural nucleic acids, although the Watson-Crick base pairing is still strictly obeyed. The peptide-like structure of PNA contributed to its several distinct properties from natural nucleic acids including the higher chemical and biological stability. These aforementioned properties makes PNA useful in several applications such as diagnosis, therapeutic and other purposes such as tools in biological and materials sciences. Several variants of PNA is known to date. Among these, the conformationally constrained pyrrolidinyl peptide nucleic acid containing a dipeptide backbone derived from nucleobase-modified proline and a cyclic β-amino acid such as (1S,2S)-2-aminocyclopentanecarboxylic acid (acpc) is a notable PNA system since it show even higher binding affinity and sequence specificity to complementary DNA when compared to the original PNA. Changing the 2-aminocyclopentanecarboxylic acid spacer to (1S,2S)-2-aminocyclobutanecarboxylic acid (acbc) increased the DNA binding affinity of the resulting acbcPNA even further. Here in, a novel PNA was proposed based on the structure of acbcPNA by replacement of a methylene carbon atom in the cyclobutane ring with an oxygen atom to become an oxetane-containing pyrrolidinyl PNA in order to improve the solubility and reduce non-specific binding, without compromising the binding affinity and specificity. Towards this goal, there is a need to develop an efficient synthetic method for N-Fmoc-protected (2S,3S)-3-aminooxetane-2-carboxylic acid [Fmoc-(2S,3S)-epi-oxetin] required as a key building block. The compound was prepared via a 11 step-sequence reaction starting from the commercially available (Z)-but-2-ene-1,4-diol. A key reaction to introduce stereochemistry on target molecule is Jorgensen organocatalytic epoxidation to generate a chiral epoxide in 91-95% ee. The oxetane ring was formed following regioselective epoxide ring opening with azide, selective monotosylation and cyclization under basic conditions. The remaining steps involve functional group interconversion and protecting group manipulation, which afforded the desired target in 5.1% overall yield with 94% ee. The oxetane amino acid and pyrrolidinyl PNA monomers were used for synthesis of aocPNA carrying a GTAGATCACT sequence via Fmoc solid phase peptide synthesis (Fmoc-SPSS). The synthesis of aocPNA posed some unexpected challenges since it is susceptible to degradation under basic and acidic conditions, which is required for nucleobase deprotection and cleavage of aocPNA from the solid support. Based on mass spectrometry data and model NMR experiments, the decomposition under acidic conditions most likely began with hydrolysis of oxetane following by fragmentation of the amide bond connecting the oxetane and the proline moieties. Even if the nucleobase deprotection and cleavage conditions were carefully optimized, aocPNAs were obtain in low isolated yield (1.2-1.3%). In addition, aocPNA exhibits DNA and RNA binding capability in sequence specific fashion. However, the binding affinity was lower than acbcPNA and other pyrrolidinyl PNAs. Computational calculation suggested that the torsional angle between carbonyl and amide substituents on the oxetane amino acid is substantially different from the optimal value required for the PNA-DNA duplex.