Synthesis and optical properties of pyrrolidinyl peptide nucleic acid carrying fluorescence nucleobases

Abstract

Pyrrolidinyl peptide nucleic acid bearing D-prolyl-2-aminocyclopentanecarboxylic acid backbone (acpcPNA) is a nucleic acid mimic that shows superior binding affinity, stability and specificity to complementary DNA than the original PNA. This research focuses on the development of hybridization responsive fluorescence acpcPNA probes bearing fluorescent nucleobases that exhibit fluorescence increase upon hybridization to a specific nucleobase in the DNA target. The acpcPNA carrying the fluorescent nucleobases were synthesized via two different approaches, namely 1) pre-synthesized fluorescence PNA monomer as a building block that was subsequently incorporated into the PNA by solid phase synthesis and 2) post-synthetic modification of acpcPNA carrying a reactive precursor (5-iodouridine in this case) by palladium-catalyzed cross-coupling reactions. In the first approach, the 8-(pyrene-1-yl)ethynyl-adenine (APyE) monomer was synthesized by conventional methods and then incorporated into the acpcPNA probe via solid phase peptide synthesis. The APyE in acpcPNA can specifically recognize thymine base in the DNA target, and the base pairing is accompanied by a strong fluorescence enhancement. The behavior of APyE in acpcPNA is quite different from the case of DNA whereby the base pairing and fluorescence enhancement was not quite specific for dT. Moreover, fluorescence quenching was observed in mismatched APyE-acpcPNA and DNA hybrids, which was explained by the stacking of the pyrene moiety inside the duplex. Applications of the APyE-acpcPNA as a hybridization-responsive fluorescence probe for DNA detection was demonstrated. In the second approach, a versatile post-synthetic modification of 5-iodouracil-containing acpcPNA via a palladium catalyzed Suzuki-Miyaura and Sonogashira cross couplings to introduce 5-aryl or 5-arylalkynyl fluorophores into acpcPNA. The post-synthetic Sonogashira cross coupling in the solution phase allows efficient synthesis of 5-(pyren-1-yl)ethynyl-uracil (UPyE) acpcPNA probes that could not be obtained by solid phase synthesis due to the susceptibility of the 5-arylalkynyl uridine to cyclize to the corresponding furanouracil derivatives. The UPyE in acpcPNA specifically recognized base A in the DNA strand and showed a strong fluorescence increase similar to the DNA case, and thus could be useful as a hybridization-responsive fluorescence probe. The highly efficient post-synthetic modification of 5-iodouracil-modified acpcPNA in solution phase allows easy access to a wide range of fluorescence acpcPNA probe bearing heteroaryl (furan, thiophene, benzofuran and benzothiophene) substituted uracil via post-synthetic Suzuki-Miyaura cross coupling. Attachment of such small substituents did not significantly alter the base pairing strength and specificity as shown by thermal denaturation analyses. Unfortunately, none of these probes could discriminate between complementary and mismatched DNA targets since fluorescence increase was consistently observed due to the high stability of the mismatched hybrid. This study revealed that the behavior of the same modified nucleobase in acpcPNA may not necessarily be the same with DNA or other DNA analogues. The post-synthetic modification approach developed also opens up a new and efficient way to rapidly generate various modified acpcPNA probes which could lead to other new probes with superior performance in the future.