Synthesis and DNA/RNA Binding Properties of Conformationally Constrained Pyrrolidinyl PNA with a Tetrahydrofuran Backbone Deriving from Deoxyribose

Sugar-derived cyclic β-amino acids are important building blocks for designing of foldamers and other biomimetic structures. We report herein the first synthesis of a C-activated <i>N</i>-Fmoc-protected <i>trans</i>-(2<i>S</i>,3<i>S</i>)-3-aminotetrahydrofuran-2-carboxylic acid as a building block for Fmoc solid phase peptide synthesis. Starting from 2-deoxy-d-ribose, the product is obtained in a 6.7% overall yield following an 11-step reaction sequence. The tetrahydrofuran amino acid is used as a building block for a new peptide nucleic acid (PNA), which exhibits excellent DNA binding affinity with high specificity. It also shows preference for binding to DNA over RNA and specifically in the antiparallel orientation. In addition, the presence of the hydrophilic tetrahydrofuran ring in the PNA structure reduces nonspecific interactions and self-aggregation, which is a common problem in PNA due to its hydrophobic nature

Pyrrolidinyl Peptide Nucleic Acid Homologues: Effect of Ring Size on Hybridization Properties

The effect of ring size of four- to six-membered cyclic β-amino acid on the hybridization properties of pyrrolidinyl peptide nucleic acid with an alternating α/β peptide backbone is reported. The cyclobutane derivatives (acbcPNA) show the highest <i>T</i>_m and excellent specificity with cDNA and RNA

Enantioselective Synthesis of 4‑Heterosubstituted Cyclopentenones

Racemic 4-hydroxycyclopentenone, readily derived from furfuryl alcohol, can be transformed via its <i>O</i>-Boc derivative to 4-acyloxy, 4-aryloxy-, 4-amino-, or 4-thio-substituted cyclopentenones with high enantioselectivity by palladium-catalyzed kinetic resolution via nucleophilic allylic substitutions. Applying this methodology, a short formal synthesis of <i>ent</i>-noraristeromycin was readily accomplished

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

Enantioselective Synthesis of 4‑Heterosubstituted Cyclopentenones

Racemic 4-hydroxycyclopentenone, readily derived from furfuryl alcohol, can be transformed via its <i>O</i>-Boc derivative to 4-acyloxy, 4-aryloxy-, 4-amino-, or 4-thio-substituted cyclopentenones with high enantioselectivity by palladium-catalyzed kinetic resolution via nucleophilic allylic substitutions. Applying this methodology, a short formal synthesis of <i>ent</i>-noraristeromycin was readily accomplished

Clickable and Antifouling Platform of Poly[(propargyl methacrylate)-<i>ran</i>-(2-methacryloyloxyethyl phosphorylcholine)] for Biosensing Applications

A functional copolymer platform, namely, poly­[(propargyl methacrylate)-<i>ran</i>-(2-methacryloyloxyethyl phosphorylcholine)] (PPgMAMPC), was synthesized by reversible addition–fragmentation chain-transfer polymerization. In principle, the alkyne moiety of propargyl methacrylate (PgMA) should serve as an active site for binding azide-containing molecules via a click reaction, i.e., Cu-catalyzed azide/alkyne cycloaddition (CuAAC), and 2-methacryloyloxyethyl phosphorylcholine (MPC), the hydrophilic monomeric unit, should enable the copolymer to suppress nonspecific adsorption. The copolymers were characterized using Fourier transform infrared (FTIR) and ¹H NMR spectroscopies. Thiol-terminated, PPgMAMPC-SH, obtained by aminolysis of PPgMAMPC, was immobilized on a gold-coated substrate using a “grafting to” approach via self-assembly. Azide-containing species, namely, biotin and peptide nucleic acid (PNA), were then immobilized on the alkyne-containing copolymeric platform via CuAAC. The potential use of surface-attached PPgMAMPC in biosensing applications was shown by detection of specific target molecules, i.e., streptavidin (SA) and DNA, by the developed sensing platform using a surface plasmon resonance technique. The copolymer composition strongly influenced the performance of the developed sensing platform in terms of signal-to-noise ratio in the case of the biotin–SA system and hybridization efficiency and mismatch discrimination for the PNA–DNA system

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 oligo­(ethylene glycol)) and end groups (−OH, −NH₂, 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

Inclusion Complexes between Amphiphilic Phenyleneethynylene Fluorophores and Cyclodextrins in Aqueous Media

Binding events of cyclodextrins (CyD's) in aqueous media are important for designing and explaining the host–guest chemistry applied in sensing and controlled release systems. A water-soluble tricationic compound (<b>3N</b>^<b>+</b>) with three branches of phenyleneethynylene fluorescent moieties and its related amphiphilic compounds (<b>3C</b>^<b>‑</b>, <b>N</b>^<b>0</b><b>N</b>^<b>+</b>, <b>N</b>^<b>+</b>, and <b>2N</b>^<b>+</b>) are employed as molecular probes in the systematic characterization of the supramolecular interactions with CyD's (α, β, and γ). The strong fluorescence enhancement, combined with induced circular dichroism (CD) signals and ¹H NMR data, is evidence of 1:1 static inclusion complexes of <b>3N</b>^<b>+</b>/γ-CyD and <b>2N</b>^<b>+</b>/γ-CyD. <b>3N</b>^<b>+</b> presents a structural design which can form inclusion complexation with γ-CyD with one of the highest binding constants of 3.0 × 10⁴. The relatively moderate fluorescence enhancement, shift of ¹H NMR signals, and weak induced CD signals indicate fast exchange complexation of β-CyD with the amphiphilic guest molecules. The interaction with α-CyD is perceived only for <b>N</b>^<b>0</b><b>N</b>^<b>+</b>, the only nonbranched fluorescent guest model, via its strong fluorescence enhancement. However, the lack of ¹H NMR signal splitting and the lack of induced CD signals suggest the noninclusion mode of binding between <b>N</b>^<b>0</b><b>N</b>^<b>+</b> and α-CyD

Sequential Flow Controllable Microfluidic Device for G‑Quadruplex DNAzyme-Based Electrochemical Detection of SARS-CoV‑2 Using a Pyrrolidinyl Peptide Nucleic Acid

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a significant health issue globally. Point-of-care (POC) testing that can offer a rapid and accurate diagnosis of SARS-CoV-2 at the early stage of infection is highly desirable to constrain this outbreak, especially in resource-limited settings. Herein, we present a G-quadruplex DNAzyme-based electrochemical assay that is integrated with a sequential flow controllable microfluidic device for the detection of SARS-CoV-2 cDNA. According to the detection principle, a pyrrolidinyl peptide nucleic acid probe is immobilized on a screen-printed graphene electrode for capturing SARS-CoV-2 DNA. The captured DNA subsequently hybridizes with another DNA probe that carries a G-quadruplex DNAzyme as the signaling unit. The G-quadruplex DNAzyme catalyzes the H2O2-mediated oxidation of hydroquinone to benzoquinone that can be detected using square-wave voltammetry to give a signal that corresponds to the target DNA concentration. The assay exhibited high selectivity for SARS-CoV-2 DNA and showed a good experimental detection limit at 30 pM. To enable automation, the DNAzyme-based assay was combined with a capillary-driven microfluidic device featuring a burst valve technology to allow sequential sample and reagent delivery as well as the DNA target hybridization and enzymatic reaction to be operated in a precisely controlled fashion. The developed microfluidic device was successfully applied for the detection of SARS-CoV-2 from nasopharyngeal swab samples. The results were in good agreement with the standard RT-PCR method and could be performed within 20 min. Thus, this platform offers desirable characteristics that make it an alternative POC tool for COVID-19 diagnosis

Sequential Flow Controllable Microfluidic Device for G‑Quadruplex DNAzyme-Based Electrochemical Detection of SARS-CoV‑2 Using a Pyrrolidinyl Peptide Nucleic Acid

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a significant health issue globally. Point-of-care (POC) testing that can offer a rapid and accurate diagnosis of SARS-CoV-2 at the early stage of infection is highly desirable to constrain this outbreak, especially in resource-limited settings. Herein, we present a G-quadruplex DNAzyme-based electrochemical assay that is integrated with a sequential flow controllable microfluidic device for the detection of SARS-CoV-2 cDNA. According to the detection principle, a pyrrolidinyl peptide nucleic acid probe is immobilized on a screen-printed graphene electrode for capturing SARS-CoV-2 DNA. The captured DNA subsequently hybridizes with another DNA probe that carries a G-quadruplex DNAzyme as the signaling unit. The G-quadruplex DNAzyme catalyzes the H2O2-mediated oxidation of hydroquinone to benzoquinone that can be detected using square-wave voltammetry to give a signal that corresponds to the target DNA concentration. The assay exhibited high selectivity for SARS-CoV-2 DNA and showed a good experimental detection limit at 30 pM. To enable automation, the DNAzyme-based assay was combined with a capillary-driven microfluidic device featuring a burst valve technology to allow sequential sample and reagent delivery as well as the DNA target hybridization and enzymatic reaction to be operated in a precisely controlled fashion. The developed microfluidic device was successfully applied for the detection of SARS-CoV-2 from nasopharyngeal swab samples. The results were in good agreement with the standard RT-PCR method and could be performed within 20 min. Thus, this platform offers desirable characteristics that make it an alternative POC tool for COVID-19 diagnosis