Synthetic, Structural, and RNA Binding Studies on 2‐Aminopyridine‐Modified Triplex‐Forming Peptide Nucleic Acids

Abstract

The development of new RNA‐binding ligands is attracting increasing interest in fundamental science and the pharmaceutical industry. The goal of this study was to improve the RNA binding properties of triplex‐forming peptide nucleic acids (PNAs) by further increasing the pKa of 2‐aminopyridine (M). Protonation of M was the key for enabling triplex formation at physiological pH in earlier studies. Substitution on M by an electron‐donating 4‐methoxy substituent resulted in slight destabilization of the PNA–dsRNA triplex, contrary to the expected stabilization due to more favorable protonation. To explain this unexpected result, the first NMR structural studies were performed on an M‐modified PNA–dsRNA triplex which, combined with computational modeling identified unfavorable steric and electrostatic repulsion between the 4‐methoxy group of M and the oxygen of the carbonyl group connecting the adjacent nucleobase to PNA backbone. The structural studies also provided insights into hydrogen‐bonding interactions that might be responsible for the high affinity and unusual RNA‐binding preference of PNAs.

Triplex‐Forming Peptide Nucleic Acids with Extended Backbones

“…The most surprising discovery of our studies was the much higher stability of the M-modified PNA-dsRNA triple helix compared to the PNA-dsDNA complex. Follow-up NMR structural studies from our group [11] showed that the solution structure of the PNA-dsRNA triple helix was similar to the published crystal structure of PNA-DNA-PNA triplex. [12] Both adapted A-form-like helical conformations were the~5.7 Å spacing between the neighboring phosphate oxygens enabled PNA backbone amide NÀ H hydrogen-bonding to RNA or DNA phosphate oxygens ( Figure 2).…”

“…The methyl ester and Cbz protecting groups were cleaved using aqueous sodium hydroxide and hydrogenation, respectively, to give the M amino acid 11, which was treated with N-(9-fluorenylmethoxycarbonyloxy) succinimide (FmocÀ OSu) to give the target αextended M monomer 12. . PNA amide to RNA phosphate backbone hydrogen-bonding interactions (black dotted line) stabilize the PNA-dsRNA triplex; [11] the~5.7 Å spacing between neighboring phosphate oxygens matches well the distance between the backbone amide NÀ H in PNA. [11] Synthesis of PNA monomers having β-extended backbones started with the known Boc-protected 3-aminopropylglycine (Scheme 2).…”

“…PNA amide to RNA phosphate backbone hydrogen-bonding interactions (black dotted line) stabilize the PNA-dsRNA triplex; [11] the~5.7 Å spacing between neighboring phosphate oxygens matches well the distance between the backbone amide NÀ H in PNA. [11] Synthesis of PNA monomers having β-extended backbones started with the known Boc-protected 3-aminopropylglycine (Scheme 2). [16] Following the same synthetic strategy as in Scheme 1, HBTU-mediated coupling with thymine acetic acid 2 gave, after protecting group exchange, the target β-extended thymine monomer 17.…”

“…[12] Both adapted A-form-like helical conformations were the~5.7 Å spacing between the neighboring phosphate oxygens enabled PNA backbone amide NÀ H hydrogen-bonding to RNA or DNA phosphate oxygens ( Figure 2). [11] This hydrogen-bonding zipper would not be possible in the B-form dsDNA that has~7 Å spacing between the neighboring phosphate oxygens. Thus, favorable backbone hydrogen-bonding may be a major driving force for the PNA's preference to bind A-form dsRNA over Bform dsDNA.…”

“…Thus, favorable backbone hydrogen-bonding may be a major driving force for the PNA's preference to bind A-form dsRNA over Bform dsDNA. [11] Conversely, these studies suggested a hypothesis that extending the PNA's backbone by one À CH 2 À group may bring the distance between PNA amide NÀ H closer to 7 Å, favorable for hydrogen bonding to the B-form dsDNA phosphate oxygens. In other words, we hypothesized that PNA with CH 2 -extended backbone may bind tighter to B-form dsDNA than to the unmodified PNA.…”