Ultrasound-assisted Peptide Nucleic Acids synthesis (US-PNAS)

Abstract

Herein, we developed an innovative and easily accessible solid-phase synthetic protocol for Peptide Nucleic Acid (PNA) oligomers by systematically investigating the ultrasonication effects in all steps of the PNA synthesis (US-PNAS). When compared with standard protocols, the application of the so-obtained US-PNAS approach succeeded in improving the crude product purities and the isolated yields of different PNA, including small or medium-sized oligomers (5-mer and 9-mer), complex purine-rich sequences (like a 5-mer Guanine homoligomer and the telomeric sequence TEL-13) and longer oligomers (such as the 18-mer anti-IVS2-654 PNA and the 23-mer anti-mRNA 155 PNA). Noteworthy, our ultrasound-assisted strategy is compatible with the commercially available PNA monomers and well-established coupling reagents and only requires the use of an ultrasonic bath, which is a simple equipment generally available in most synthetic laboratories.

Keywords: Coupling Conditions; Peptide Nucleic Acid; Solid-Phase Synthesis; Sonochemistry; Ultrasonication.

Graphical abstract

Fig. 1

Representative comparison between the structures of PNA and DNA fragments. The different DNA and PNA backbones are highlighted in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Undesired side-reactions during manual PNA synthesis (A: N-Acyl transfer; B: cyclization).

Fig. 3

General synthetic scheme for the proposed ultrasound-assisted Solid Phase Peptide Nucleic Acids strategy (US-PNAS).

Fig. 4

Coupling agents screened in the US-PNAS oligomerization experiments.

Fig. 5

Optimization study for the US-PNAS of the model 5-mer PNA oligomer (Fmoc-TGACT-K-NH₂). PNA synthesis was attained in triplicate and the resulting crude purities are expressed as a percentage (mean values ± standard error of measurement (SEM), N = 3).

Fig. 6

Comparison of representative HPLC profiles of synthetic crudes of the model 5-mer PNA oligomer (Fmoc-TGACT-K-NH₂). Coupling reagents and synthetic methods are indicated. The crude purity was calculated by integrating the peaks at t_R = 13.15 min [analytical HPLC gradient 1 in HPLC and Mass spectrometry procedures section].

Fig. 7

Comparison of representative HPLC profiles of synthetic crudes of the model 5-mer PNA oligomer (Fmoc-TGACT-K-NH₂). Equivalents of reagents and reaction times are indicated. The crude purity was calculated by integrating the peaks at t_R = 13.15 min [analytical HPLC gradient 1 in HPLC and Mass spectrometry procedures section].

Fig. 8

Optimization study for the US-PNAS of the model 9-mer PNA oligomer (H-TACATGTC-K-NH₂). PNA synthesis was attained in triplicate and the resulting crude purities are expressed as a percentage (mean values ± standard error of measurement (SEM), N = 3).

Fig. 9

Comparison of representative HPLC profiles of synthetic crudes of the 9-mer PNA oligomer (H-TACACTGTC-K-NH₂) obtained employing the same reagent excess at a) 10 min and b) 5 min reaction times. Equivalents of reagents and reaction times are indicated in the figure legend. The crude purity was calculated by integrating the peaks at t_R = 9.40 min [analytical HPLC gradient 2 in HPLC and Mass spectrometry procedures section].

Fig. 10

Comparison of representative HPLC profiles of synthetic crudes of the TEL-13 PNA oligomer (H-CAGTTAGGGTTAG-K-NH₂). Equivalents of reagents and reaction times are indicated. The crude purity was calculated by integrating the peaks at t_R = 9.66 min, [analytical HPLC gradient 2 in HPLC and Mass spectrometry procedures section].

Fig. 11

HPLC profiles of a) 5- mer PNA (Fmoc-GGGGG-NH₂ t_R = 13.3 min) [analytical HPLC gradient 1 in HPLC and Mass spectrometry procedures section], b) 18-mer PNA (H-AEEA-TTGGTTGGTTTGTTACCT-Gly-NH₂ t_R = 11.1 min) [analytical HPLC gradient 2 in HPLC and Mass spectrometry procedures section], c) 23-mer PNA, H-AEEA-ACCCCTATCACAATTAGCATTAA-Gly-NH₂ t_R = 8.46 min and d) H-KKK-ACCCCTATCACAATTAGCATTAA-Gly-NH₂ t_R = 8.48 min and) [analytical HPLC Gradient 3 in HPLC and Mass spectrometry procedures section].