4(<i>R</i>/<i>S</i>)-Guanidinylprolyl Collagen Peptides: On-Resin Synthesis, Complexation with Plasmid DNA, and the Role of Peptides in Enhancement of Transfection

Chimeric collagen peptides containing cationic 4­(<i>R</i>/<i>S</i>)-guanidinylproline are synthesized by in situ amidinylation of 4­(<i>R</i>/<i>S</i>)-aminoproline residues. These peptides uniquely enhance the transfection efficiency of GFP-encoded plasmid DNA (pRmHa3-GFP) into cells through efficient DNA condensation resulting from nonspecific electrostatic interactions of cationic guanidino groups and localize in subcytoplasmic organelles

Fluorous Peptide Nucleic Acids: PNA Analogues with Fluorine in Backbone (γ-CF₂-<i>apg</i>-PNA) Enhance Cellular Uptake

Fluorous PNA analogues possessing fluorine as inherent part of aminopropylglycine (<i>apg</i>) backbone (γ-CF₂-<i>apg</i> PNA) have been synthesized and evaluated for biophysical and cell penetrating properties. These form duplexes of higher thermal stability with cRNA than cDNA, although destabilized compared to duplexes of standard <i>aeg</i>-PNA. Cellular uptake of the fluorinated γ-CF₂-<i>apg</i> PNAs in NIH 3T3 and HeLa cells was 2–3-fold higher compared to that of nonfluorinated <i>apg</i> PNA, with NIH 3T3 cells showing better permeability compared to HeLa cells. The backbone fluorinated PNAs, which are first in this class, when combined with other chemical modifications may have potential for future PNA-based antisense agents

Aminomethylene Peptide Nucleic Acid (<i>am</i>-PNA): Synthesis, Regio-/Stereospecific DNA Binding, And Differential Cell Uptake of (α/γ,<i>R</i>/<i>S</i>)<i>am-</i>PNA Analogues

Inherently chiral, cationic <i>am-</i>PNAs having pendant aminomethylene groups at α­(<i>R</i>/<i>S</i>) or γ­(<i>S</i>) sites on PNA backbone have been synthesized. The modified PNAs are shown to stabilize duplexes with complementary cDNA in a regio- and stereo-preferred manner with γ­(<i>S</i>)-<i>am</i> PNA superior to α­(<i>R</i>/<i>S</i>)-<i>am</i> PNAs and α­(<i>R</i>)-<i>am</i> PNA better than the α­(<i>S</i>) isomer. The enhanced stabilization of <i>am</i>-PNA:DNA duplexes is accompanied by a greater discrimination of mismatched bases. This seems to be a combined result of both electrostatic interactions and conformational preorganization of backbone favoring the cDNA binding. The <i>am</i>-PNAs are demonstrated to effectively traverse the cell membrane, localize in the nucleus of HeLa cells, and exhibit low toxicity to cells

Clickable <i>C</i>^γ‑Azido(methylene/butylene) Peptide Nucleic Acids and Their Clicked Fluorescent Derivatives: Synthesis, DNA Hybridization Properties, and Cell Penetration Studies

Synthesis, characterization, and DNA complementation studies of clickable C^γ-substituted methylene (<i>azm</i>)/butylene (<i>azb</i>) azido PNAs show that these analogues enhance the stability of the derived PNA:DNA duplexes. The fluorescent PNA oligomers synthesized by their click reaction with propyne carboxyfluorescein are seen to accumulate around the nuclear membrane in 3T3 cells

Fluorinated Peptide Nucleic Acids with Fluoroacetyl Side Chain Bearing 5‑(F/CF₃)‑Uracil: Synthesis and Cell Uptake Studies

Fluorine incorporation into organic molecules imparts favorable physicochemical properties such as lipophilicity, solubility and metabolic stability necessary for drug action. Toward such applications using peptide nucleic acids (PNA), we herein report the chemical synthesis of fluorinated PNA monomers and biophysical studies of derived PNA oligomers containing fluorine in in the acetyl side chain (−CHF–CO−) bearing nucleobase uracil (5-F/5-CF3-U). The crystal structures of fluorinated racemic PNA monomers reveal interesting base pairing of enantiomers and packing arrangements directed by the chiral F substituent. Reverse phase HPLC show higher hydrophobicity of fluorinated PNA oligomers, dependent on the number and site of the fluorine substitution: fluorine on carbon adjacent to the carbonyl group induces higher lipophilicity than fluorine on nucleobase or in the backbone. The PNA oligomers containing fluorinated bases form hybrids with cDNA/RNA with slightly lower stability compared to that of unmodified aeg PNA, perhaps due to electronic effects. The uptake of fluorinated homooligomeric PNAs by HeLa cells was as facile as that of nonfluorinated PNA. In conjunction with our previous work on PNAs fluorinated in backbone and at N-terminus, it is evident that the fluorinated PNAs have potential to emerge as a new class of PNA analogues for applications in functional inhibition of RNA

Aza-PNA: Engineering E‑Rotamer Selectivity Directed by Intramolecular H‑bonding

The replacement of α(CH2) by NH in monomers of standard aeg PNA and its homologue β-ala PNA leads to respective aza-PNA monomers (1 and 2) in which the NαH can form either an 8-membered H-bonded ring with folding of the backbone (DMSO and water) or a 5-membered NαHαCO (water) to stabilize E-type rotamers. Such aza-PNA oligomers with exclusive E rotamers and intraresidue backbone H-bonding can modulate its DNA/RNA binding and assembling properties

5‑Amidodansyl‑U (U^D) Peptide Nucleic Acid (PNA) as a Fluorescent Sensor of the Local Dielectric Constant (ε) in PNA Duplexes: Major Grooves in PNA Duplexes Are More Hydrophobic Than Major Grooves in DNA–DNA Duplexes

Peptide nucleic acids (PNA) show great promise for the development of antisense drugs owing to their superior binding property with complementary DNA/RNA. They recognize complementary DNA/RNA/PNA via hydrogen bonding and electrostatic interaction whose strengths depend on their chemical environment. It is therefore important to understand the effects of local dielectrics in the major/minor grooves of PNA:DNA/RNA/PNA duplexes that influence its superior binding. By employing 5-amidodansyl U on PNA as a fluoroprobe of the local environment and measuring the polarity-sensitive Stokes shift, it is demonstrated that compared to the major groove of DNA–DNA duplexes, the analogous major groove of PNA:DNA/RNA/PNA duplexes is more hydrophobic (lower ε), and sequence-dependent polarity changes are seen in all PNA duplexes. The results highlight the effects of chemical modifications of backbone and base sequence in nucleic acids on the local environment of grooves, leading to a dielectric continuum that may have implications for the binding of ligands and macromolecules in grooves of nucleic acid duplexes

Fluorinated Peptide Nucleic Acids with Fluoroacetyl Side Chain Bearing 5‑(F/CF₃)‑Uracil: Synthesis and Cell Uptake Studies

Fluorine incorporation into organic molecules imparts favorable physicochemical properties such as lipophilicity, solubility and metabolic stability necessary for drug action. Toward such applications using peptide nucleic acids (PNA), we herein report the chemical synthesis of fluorinated PNA monomers and biophysical studies of derived PNA oligomers containing fluorine in in the acetyl side chain (−CHF–CO−) bearing nucleobase uracil (5-F/5-CF3-U). The crystal structures of fluorinated racemic PNA monomers reveal interesting base pairing of enantiomers and packing arrangements directed by the chiral F substituent. Reverse phase HPLC show higher hydrophobicity of fluorinated PNA oligomers, dependent on the number and site of the fluorine substitution: fluorine on carbon adjacent to the carbonyl group induces higher lipophilicity than fluorine on nucleobase or in the backbone. The PNA oligomers containing fluorinated bases form hybrids with cDNA/RNA with slightly lower stability compared to that of unmodified aeg PNA, perhaps due to electronic effects. The uptake of fluorinated homooligomeric PNAs by HeLa cells was as facile as that of nonfluorinated PNA. In conjunction with our previous work on PNAs fluorinated in backbone and at N-terminus, it is evident that the fluorinated PNAs have potential to emerge as a new class of PNA analogues for applications in functional inhibition of RNA

Influence of Pendant Chiral C^γ‑(Alkylideneamino/Guanidino) Cationic Side-chains of PNA Backbone on Hybridization with Complementary DNA/RNA and Cell Permeability

Intrinsically cationic and chiral C^γ-substituted peptide nucleic acid (PNA) analogues have been synthesized in the form of γ­(<i>S</i>)-ethyleneamino (<i>eam</i>)- and γ­(<i>S</i>)-ethyleneguanidino (<i>egd</i>)-PNA with two carbon spacers from the backbone. The relative stabilization (Δ<i>T</i>_m) of duplexes from modified cationic PNAs as compared to 2-aminoethylglycyl (<i>aeg</i>)-PNA is better with complementary DNA (PNA:DNA) than with complementary RNA (PNA:RNA). Inherently, PNA:RNA duplexes have higher stability than PNA:DNA duplexes, and the guanidino PNAs are superior to amino PNAs. The cationic PNAs were found to be specific toward their complementary DNA target as seen from their significantly lower binding with DNA having single base mismatch. The differential binding avidity of cationic PNAs was assessed by the displacement of DNA duplex intercalated ethidium bromide and gel electrophoresis. The live cell imaging of amino/guanidino PNAs demonstrated their ability to penetrate the cell membrane in 3T3 and MCF-7 cells, and cationic PNAs were found to be accumulated in the vicinity of the nuclear membrane in the cytoplasm. Fluorescence-activated cell sorter (FACS) analysis of cell permeability showed the efficiency to be dependent upon the nature of cationic functional group, with guanidino PNAs being better than the amino PNAs in both cell lines. The results are useful to design new biofunctional cationic PNA analogues that not only bind RNA better but also show improved cell permeability

Orchestration of Structural, Stereoelectronic, and Hydrogen-Bonding Effects in Stabilizing Triplexes from Engineered Chimeric Collagen Peptides (Pro^X‑Pro^Y‑Gly)₆ Incorporating 4(<i>R</i>/<i>S</i>)‑Aminoproline

Collagens are an important family of structural proteins found in the extracellular matrix with triple helix as the characteristic structural motif. The collagen triplex is made of three left-handed polyproline II (PPII) helices with each PPII strand consisting of repetitive units of the tripeptide motif X-Y-Gly, where the amino acids X and Y are most commonly proline (Pro) and 4<i>R</i>-hydroxyproline (Hyp), respectively. A C4-<i>endo</i> pucker at X-site and C4-<i>exo</i> pucker at Y-site have been proposed to be the key for formation of triplex, and the nature of pucker is dependent on both the electronegativity and stereochemistry of the substituent. The present manuscript describes a new class of collagen analogueschimeric cationic collagenswherein both X- and Y-sites in collagen triad are simultaneously substituted by a combination of 4­(<i>R</i>/<i>S</i>)-(OH/NH₂/NH₃⁺/NHCHO)-prolyl units and triplex stabilities measured at different pHs and in EG:H₂O. Based on the results a model has been proposed with the premise that any factors which specifically favor the ring puckers of C4-<i>endo</i> at X-site and C4-<i>exo</i> at Y-site stabilize the PPII conformation and hence the derived triplexes. The pH-dependent triplex stability uniquely observed with ionizable 4-amino substituent on proline enables one to define the critical combination of factors C4-(<i>exo</i>/<i>endo</i>), intraresidue H-bonding, stereoelectronic (<i>R</i>/<i>S</i>) and n → π* interactions in dictating the triplex strength. The ionizable NH₂ substituent at C4 in <i>R</i>/<i>S</i> configuration is thus a versatile probe for delineating the triplex stabilizing factors and the results have potential for designing of collagen analogues with customized properties for material and biological applications