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