Peptide nucleic acids (PNAs) are DNA mimics where the sugar phosphate backbone of DNA is replaced by a peptide backbone consisting of N-(2-aminoethyl)glycine units. The purine nucleo- bases (Adenine A and Guanine G) and pyrimidine nucleobases (Cytosine C and Thymine T) are attached to the backbone through methylene carbonyl linkages.1
PNA oligomers have interesting properties such as their increased thermal and chemical stability and resistance to enzymatic degradation which drives their use in various
molecular biology, molecular diagnostic, microarray, biosensor and antisense applications.2 Here we synthesized two PNA oligomers (Figure 1) on a small scale using the Biotage® Initiator+ Alstra™ microwave peptide synthesizer.
Figure 1: PNA oligomers synthesized
PNA 1 incorporates a diaminoproprionic acid residue (Dap) (Figure 2) suitable for the post synthetic conjugation of different oligoethers3 as well as catalytic groups in PNA based artificial nucleases4 and here we present a bisimidazole derivative PNA 2. The incorporation of linkers into PNA oligomers is used to increase solubility, introduce functionality and for the conjugation of dyes such as Cy5.
Figure 2: Diaminoproprionic acid (Dap) linker.
All materials were obtained from commercial suppliers; Link technologies (peptide nucleic acid monomers, Fmoc-PNA-A(Bhoc)-OH, Fmoc-PNA-G(Bhoc)-OH, Fmoc-PNAC( Bhoc)-OH and Fmoc-PNA-T-OH), Iris Biotech GmbH (Fmoc-αN-Lys(εN-Boc)OH, Nα-Fmoc- Nβ-(p-methyltrityl)-L-2, 3-diaminopropionic acid (Fmoc-L-Dap(Mtt)-OH), Nα-Fmoc- Nβ Fmoc-L-2,3-diaminopropionic acid (Fmoc-L- Dap(Fmoc)-OH), trifluoroacetic acid (TFA), triisopropylsilane (TIS), and dichloromethane (DCM), N-methylpyrrolidone (NMP), piperidine)), Merck-Millipore (ethyl cyano(hydroxyimino) acetate (Oxyma) and N,N’ diisopropylcarbodiimide (DIC)), Life Technologies Europe (acetonitrile, lutidine, acetic anhydride ) and Biotage (Rink Amide ChemMatrix® resin). The benzhydry- loxycarbonyl (Bhoc) group was used for protecting the exocyclic amino groups of the nucleobases.
Mass spectrometry was performed on a Micromass LCT electro- spray ionization time-of-flight (ESI-TOF) mass spectrometer in acetonitrile–water 1:1 (v/v), 0.1% formic acid solutions. The molecular weights of the peptide nucleic acid conjugates were reconstructed from the m/z values using the mass deconvolu- tion program of the instrument (Mass Lynx software package).
The PNAs were prepared on a Biotage® Initiator+ Alstra™ microwave peptide synthesizer. PNA sequences were synthe- sized on Rink Amide ChemMatrix® resin (loading 0.47 mmol/g) on a 10 μmol scale in a 5 mL reactor vial. Fmoc deprotection was performed at room temperature (RT) in two stages by treating the resin with piperidine-NMP (1:4) for 3 min followed by piperidine-NMP (1:4) for 10 minutes. The resin was then washed with NMP (x5). PNA couplings were performed using 4 eq. of PNA monomer, 4 eq. oxyma and 4 eq. DIC in NMP. A coupling time of 6 min at 75 °C (microwave) was employed and then the resin was washed with NMP (x2). This was followed by an optional capping step using NMP-lutidine-acetic anhydride (89:6:5) for 1 min and then washing with NMP (x4). After the synthesis was completed, the resin was washed with NMP (x5), DCM (x5) and thoroughly dried. The PNAs were cleaved from the solid support by treatment with TFA-H2O-TIS (95:2.5:2.5) for 1.5 h at room temperature. The PNA products evaporated to dryness and then purified with an Ascentis Express Supelco Peptide ES-C18 column (2.7 µm, 150 × 4.6 mm) at 60 °C using a flow rate of 1 mL/min. The following solvent system was used: solvent A, water containing 0.1% TFA; solvent B, CH3CN: water containing 0.1% TFA (1:1; v/v). PNA 1 and 2 were purified using a linear gradient of 40% B for 30 min. Collected products were lyophilized. Water was added and the products were freeze dried again, which was then repeated once more.
The PNA sequence 1 was assembled using SPPS methods as described above with microwave heating during the coupling steps. The resin was washed and the PNA released from the solid support as described above to afford the desired PNA 1 with a crude purity of 87% and confirmed by ESI-TOF MS (Figure 3). MS (MALDI) m/z = 3290 [M+H]+, calcd for C130H169N69O37+H+: 3289.354.
Figure 3: Analytical HPLC trace and ESI-TOF MS of crude PNA 1.
For the synthesis of the conjugate PNA 2, the PNA sequence 1 was assembled using SPPS methods as described above with microwave heating during the coupling steps. This PNA
remained attached to the resin (Scheme 1). The Nβ-methyl trityl protection of diamino propionic acid was removed by treatment with 1% TFA in DCM for 1 min (x5), followed by washing with DCM and NMP. Nα-Fmoc- Nβ-Fmoc-L-2,3-diaminopropionic acid (5 eq.) was then coupled to the PNA using the standard coupling method described above followed by the standard removal of the two Dap Fmoc protecting groups.
Conjugation of a DMBz-imidazole moiety (2,6-dimethoxyben- zoyl-imidazole-4-acetic acid prepared separately. Prof. Roger Strömberg et al.) to the resin bound PNA was carried out using DMBz-imidazole (17 eq.), HATU (15 eq.), NMM (15 eq.) in 250 µl of NMP under microwave irradiation for 20 min at 55 °C. The resin was washed and the peptide released from the solid support as described above to afford the desired PNA 2 and confirmed by ESI-TOF MS (Figure 5). MS (MALDI) m/z = 3589 [M]+, calcd for C143H183N75O40: 3590.459.
Figure 4: Synthesis of PNA 2, a bisimidazole conjugate
Figure 5: Analytical HPLC trace and ESI-TOF MS of crude PNA 2
Here we have demonstrated the use of microwave heating for the small scale synthesis (10 μmol) of PNA oligomers where microwave heating was applied during coupling steps only. The Biotage® Initiator+ Alstra™ microwave peptide synthesizer is the ideal tool for the synthesis of PNAs and conjugates. With the ability to use low volumes and accurate dispensing using digital syringe pumps, makes the Initiator+ Alstra perfectly suited for small scale microwave peptide and peptidomimetic synthesis, especially when the use of expensive building blocks is required such as during PNA synthesis.
Prof. Roger Strömberg, Dr. Alice Ghidini, Dr. Merita Murtola and Mr. Lars Verdonck at the Karolinska Institutet, Huddinge, Sweden.
Literature number: AN110