PNA bearing 5-azidomethyluracil: a novel approach for solid and solution phase modification

Abstract

Fmoc- and Boc-protected modified monomers bearing 5-azidomethyluracil nucleobase were synthesized. Four different solid-phase synthetic strategies were tested in order to evaluate the application of this series of monomers for the solid-phase synthesis of modified PNA. The azide was used as masked amine for the introduction of amide-linked functional groups, allowing the production of a library of compounds starting from a single modified monomer. The azide function was also exploited as reactive group for the modification of PNA in solution via azide-alkyne click cycloaddition.

Figure 1. Synthesis of the modified monomer. (A) CH₂O, Et₃N in H₂O, 81%; (B) HCl 37%, 80%; (C) NaN₃ in DMF, 90%; (D) (1) BrCH₂COOEt, K₂CO₃ in DMF, 41% (2) BrCH₂COO^tBu, K₂CO₃ in DMF, 49%; (E) (1) NaOH in H₂O/MeOH 1:1 then HCl, 96% (2) TFA in DCM, 70%; (F) (1) EDC∙HCl, DhBtOH, DIPEA, tert-butyl 2-[(2-Fmoc-aminoethyl)amino]acetate hydrochloride in DMF, 96% (2) DCC, DhBtOH, DIPEA, ethyl 2-[(2-Fmoc-aminoethyl)amino]acetate in DMF, 77%; (G) (1) TFA in DCM, 86% (2) NaOH in H₂O/MeOH 1:1, 96%.

Figure 2. UPLC-ESI extracted ion current (XIC) traces of the cleaved resin bearing: (A) the azide function, (B) amine function after reduction with PMe₃ (peak at 4.40 min corresponding to the iminophosphorane intermediate of the Staudinger reaction), (C) pyrene moiety after coupling. XIC traces were obtained by extrapolating the multicharge signal of the three different PNAs: 1342.5, 1329.5, 967.4, 895.2, 886.9, 725.7, 671.8, 665.3, 580.9 (for mass spectra see Figs. S1 and S2).

Figure 3. Synthesis of PNA 3. (A) Introduction of monomer 8b during solid-phase synthesis; (B) azide reduction by trimethyl phosphine; (C) coupling of naphthalene‐2‐carboxylic acid activated by DIC/DhBtOH; (D) elongation of the strand to final sequence.

Figure 4. (A) HPLC‐UV traces of crude PNA 3-napht after modification; (B) HPLC‐UV traces of PNA after purification; (C) ESI-MS spectra of modified PNA 3. Peaks marked (*) are multicharge peaks due to source-fragmentation at the C5-methylene position of uracil.

Figure 5. (A) UPLC‐MS traces of PNA 4 after coupling with napht-gly; (B) ESI-MS spectra of the peak at 8.16 min.

Figure 6. UPLC-ESI extracted ion current (XIC) traces of PNA 1 and PNA 2 bearing the unreacted azide (A and B respectively) and after 45 min in presence of 1 equivalent of propargyl alcohol and Cu(I) (C and D respectively). XIC traces were obtained by extrapolating the multicharge signal of the different PNAs: 1468.1, 762.6 and 734.6 for chromatogram (A and C); 1259.5, 1231.6, 840.1, 821.2, 630.3 and 616.2 for chromatogram (B and D) (for mass spectra see Figs. S1 and S2).