Reductive Alkylation and Sequential Reductive Alkylation-Click Chemistry for On-Solid-Support Modification of Pyrrolidinyl Peptide Nucleic Acid

A methodology for the site-specific attachment of fluorophores to the backbone of pyrrolidinyl peptide nucleic acids (PNAs) with an α/β-backbone derived from d-prolyl-(1<i>S</i>,2<i>S</i>)-2-aminocyclopentanecarboxylic acid (acpcPNA) has been developed. The strategy involves a postsynthetic reductive alkylation of the aldehyde-containing labels onto the acpcPNA that was previously modified with (3<i>R</i>,4<i>S</i>)-3-aminopyrrolidine-4-carboxylic acid on the solid support. The reductive alkylation reaction is remarkably efficient and compatible with a range of reactive functional groups including Fmoc-protected amino, azide, and alkynes. This allows further attachment of readily accessible carboxyl-, alkyne-, or azide-containing labels via amide bond formation or Cu-catalyzed azide–alkyne cycloaddition (CuAAC, also known as click chemistry). The label attached in this way does not negatively affect the affinity and specificity of the pairing of the acpcPNA to its DNA target. Applications of this methodology in creating self-reporting pyrene- and thiazole orange-labeled acpcPNA probes that can yield a change in fluorescence in response to the presence of the correct DNA target have also been explored. A strong fluorescence enhancement was observed with thiazole orange-labeled acpcPNA in the presence of DNA. The specificity could be further improved by enzymatic digestion with S1 nuclease, providing a 9- to 60-fold fluorescence enhancement with fully complementary DNA and a less than 3.5-fold enhancement with mismatched DNA targets