Synthesis of Bifacial Peptide Nucleic Acids with Diketopiperazine Backbones

Abstract

We report a synthesis of bifacial peptide nucleic acids (bPNAs) with novel diketopiperazine (DKP) backbones that display unnatural melamine (M) bases, as well as native bases. To examine the structure-function scope of DKP bPNAs, we synthesized a set of bPNAs by using diaminopropionic acid, diaminobutyric acid, ornithine, and lysine derivatives to display the base-tripling motifs, which result in one, two, three, or four carbons linking the alpha carbon to the side-chain amine. Thermal denaturation of DNA hybrids with these bPNAs revealed that the optimal side-chain linkage was four carbons, corresponding to the lysine derivative. Accordingly, monomers displaying two bases per side-chain were prepared through double reductive alkylation of the ε-amine of Fmoc-lysine with acetaldehyde derivatives of adenine, cytidine, uridine, and melamine. With these building blocks in hand, DKP bPNAs were prepared to display a combination of native and synthetic (melamine) bases. Preliminary melting studies indicate binding signatures of cytidine- and melamine-displaying bPNAs to T-rich DNAs of noncanonical structure, though full characterization of this behavior is ongoing. The convenient and potentially scalable method described enables rapid access to DNA-binding scaffolds of low (<1 kD) molecular weight and previously established cell permeability. We expect that this straightforward and efficient approach to nucleic acid binders will enable studies on noncanonical nucleic acid hybridization.

Keywords: DNA binding; diketopiperazines; peptide nucleic acids; triplexes.

Figure 1

Overview of DKP bPNAs synthesized in this study. (a) Melamine-only DKP-bPNAs with side-chain alkyl lengths of 1, 2, 3, or 4 carbons, representing diaminopropionic acid (DAP), diaminobutyric acid (DAB), ornithine (O), and lysine (K), respectively. (b) DKP-bPNAs containing either a combination of melamine and native bases (1e–g) or only native bases (1h–j).

Figure 2

Acetaldehyde derivatives of melamine (4a), cytidine (4b), uridine (4c), and adenine (4d)

Figure 3

Thermal denaturation of bPNA-DNA hybrids with (A) dT₄C₄T₄ DNA, (B) dC₄T₄G₄ DNA and the bPNAs indicated. Reaction conditions: (A) 1:1 complexes and (B) 2:1 DNA:bPNA complexes in 1X PBS, pH 7.4. All experiments held a bPNA concentration of 2 μM.

Scheme 1

(a) Illustration of bPNA-DNA triplex hybridization through base triple formation with thymine and melamine (M) and (b) possible bPNA DKP backbone boat conformers that display melamine bases

Scheme 2

Preparation of bPNA monomers by double reductive alkylation of the Fmoc-amino acid derivative, following TFA cleavage of side-chain Boc protection. Derivatives 3a–d and 2a–d were monomers with side-chain length variation with R = M and a, b, c, and d representing n = 1–4 CH₂ units, respectively. Derivatives 3e–g and 2e–g were monomers with n = 4 and base variation, where e, f, and g have R = adenine, uridine, and cytidine, respectively. Reaction conditions: (a) NaBH₃CN, CH₃OH, 55 °C to RT (75–82%); (b) (i) TMSCl (3 equiv), CH₃OH, 55 °C, 1–6 h (85–90%), (ii) 16% NH₂NH₂ (74–80%).

Scheme 3

DKP synthesis. Reaction conditions c: HBTU (1 equiv), Et₃N (5 equiv), DMF (60–80 °C), 24 h, 65–72%.