Peptide nucleic acid (PNA) is a synthetic chemical similar to DNA, RNA. As the building block of PNA , the backbone of PNA consists of repeating units of N-2-(aminoethyl)-glycine assembled via peptide bonds. The unit structure of the PNA backbone is identical, with each monomer consisting of an aminoethyl group (-NH-CH2-CH2-NH2) linked to a glycine backbone (-CH2-CO-).
Fig.1 PNA consists of repeating units of N-(2-aminoethyl)-glycine linked by amide bonds. (Weiler et al., 1997)
There are three common methods for synthesizing backbones.
1. Alkylation reaction
With ethylenediamine or aminoacetonitrile as raw material, alkylation reaction is carried out with haloacetic acid derivatives. Applicable protection groups include 9-fluoromethoxycarbonyl (Fmoc), 4-methoxyphenyldiphenylmethyl (Mmt) and tert-butyloxycarbonyl (Boc).
2. Reduction reaction of schiff base
Although the Schiff base formed by reducing glycine ester and protected aminoacetaldehyde is only applicable to Boc protecting group, this method can be used to synthesize various PNA monomers with side chains with slight modification. For example, the Schiff base can be formed by reducing ethylenediamine and glyoxylic acid to obtain N - (2-aminoethyl) glycine, and then glycine can selectively connect appropriate protective groups, including Fmoc and Mmt. Or glycine is reduced to Boc aminoacetaldehyde, and then reacted with glycine ester.
3. Mitsunobu reaction
PNA backbones can be synthesized by Mitsunobu reaction between Boc protected aminoethanol and glycine ester protected by p-nitrophenyl methanesulfonyl (o-NBS).
In addition to the backbones section provided above, the following components are included.
The nucleotide bases of PNA are similar to those of DNA and RNA and include adenine (A), cytosine (C), guanine (G) and thymine (T) or uracil (U).
Linker groups are located between the backbone of the PNA and the nucleotide bases and provide the connection between them. These linker groups are usually short carbon chains.
Boc and Fmoc act as protecting groups that can protect the amine groups of amino acids in synthesis.
The introduction of protective groups prevents side reactions of amino acids, such as unspecific reactions with active esterifying agents. This helps to maintain the selectivity and purity of the synthesis.
By selecting appropriate conditions, Boc or Fmoc protecting groups can be selectively removed without affecting the presence of other protecting or functional groups.
The backbone of a PNA can be modified by introducing chemical groups on the aminoethyl or glycine monomers. These modifying groups can include aromatic groups, sugar groups, fluorescent dyes, and so on.
Choline groups can be introduced into the backbone of PNA to create choline-PNA (ch-PNA) molecules. The introduction of a choline moiety enhances the cell permeability and intracellular targeting of the PNA molecule, thereby increasing its efficacy as a drug delivery vehicle or antimicrobial agent.
Additional peptide chains can be introduced into the backbone of PNA to form peptide-PNA molecules. This modification can modulate the interaction of the PNA with the target molecule through the sequence and length of the peptide chain, extending the function and specificity of the PNA.
The unnatural backbone of PNA makes it highly resistant to enzymatic degradation, thus increasing its stability and durability in biological samples.
Due to the lack of oxygen atoms of sugars in the PNA backbone, PNA can form a tighter stack with the bases of DNA or RNA when the double-stranded structure of PNA is formed, resulting in a more compact structure.
The aminoethyl groups in the PNA backbone can form specific hydrogen bonds and van der Waals force interactions, resulting in high affinity and stability of the double-stranded structure formed by PNA and DNA or RNA.