2 research outputs found
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
Hydrophilic and Cell-Penetrable Pyrrolidinyl Peptide Nucleic Acid via Post-synthetic Modification with Hydrophilic Side Chains
Peptide nucleic acid (PNA) is a nucleic acid mimic in which the
deoxyribose–phosphate was replaced by a peptide-like backbone.
The absence of negative charge in the PNA backbone leads to several
unique behaviors including a stronger binding and salt independency
of the PNA–DNA duplex stability. However, PNA possesses poor
aqueous solubility and cannot directly penetrate cell membranes. These
are major obstacles that limit in vivo applications of PNA. In previous
strategies, the PNA can be conjugated to macromolecular carriers or
modified with positively charged side chains such as guanidinium groups
to improve the aqueous solubility and cell permeability. In general,
a preformed modified PNA monomer was required. In this study, a new
approach for post-synthetic modification of PNA backbone with one
or more hydrophilic groups was proposed. The PNA used in this study
was the conformationally constrained pyrrolidinyl PNA with prolyl-2-aminocyclopentanecarboxylic
acid dipeptide backbone (acpcPNA) that shows several advantages over
the conventional PNA. The aldehyde modifiers carrying different linkers
(alkylene and oligoÂ(ethylene glycol)) and end groups (−OH,
−NH<sub>2</sub>, and guanidinium) were synthesized and attached
to the backbone of modified acpcPNA by reductive alkylation. The hybrids
between the modified acpcPNAs and DNA exhibited comparable or superior
thermal stability with base-pairing specificity similar to those of
unmodified acpcPNA. Moreover, the modified apcPNAs also showed the
improvement of aqueous solubility (10–20 folds compared to
unmodified PNA) and readily penetrate cell membranes without requiring
any special delivery agents. This study not only demonstrates the
practicality of the proposed post-synthetic modification approach
for PNA modification, which could be readily applied to other systems,
but also opens up opportunities for using pyrrolidinyl PNA in various
applications such as intracellular RNA sensing, specific gene detection,
and antisense and antigene therapy