Two Effective Methods to Synthesize High-Precision PNA Oligomers

Oligonucleotides are gaining significant attraction in drug development, diagnostic reagents, and molecular biology research tools. Despite the widespread application of oligonucleotides, many oligonucleotide analogs have been studied to modify and improve the physical, chemical, and biological properties of oligonucleotides due to their easy degradation by nucleases, poor thermal stability after hybridization, and other limitations in applications.

Peptide nucleic acid (PNA) is one of the more important oligonucleotide mimics. It is an artificially synthesized polymer similar to DNA or RNA. Notwithstanding the distinct change in PNA structure relative to oligonucleotides, the binding of PNA to complementary nucleic acids still follows the principle of base complementary pairing, and PNA presents higher affinity and stronger resistance to degradation by nucleases and proteolytic enzymes over nucleotides in the synthesis process.

The synthesis of PNA is first to synthesize the monomers, and then oligomerize the PNA monomers according to the standard peptide synthesis method. PNA oligomers are prepared from protected PNA monomers by solid-phase synthesis, and their protection strategies, condensation conditions, deprotection, and purification methods are similar to those of peptide synthesis. PNA oligomer synthesis can be achieved via Boc chemistry and Fmoc chemistry routes.

Chemical Synthesis of PNA Oligomers with Boc

Boc chemistry is a chemical technique using TFA removable Boc (tert-butyloxocarbonyl) as?-amino protective group and benzyl alcohol as side-chain protection. During synthesis, a Boc amino acid derivative is covalently cross-linked to the resin, the Boc is removed with TFA, the free amino-terminal is neutralized with triethylamine, then activated by DCC to couple with the next amino acid, and finally deprotected by HF or TFMSA methods. Many biological macromolecules have been successfully synthesized via the Boc method, such as active enzymes, artificial proteins, and so on.

At current, there are two modes, manual synthesis, and automatic synthesis, by Boc chemistry. Automation synthesis is the preferred method for the production of PNA oligomers because this method uses continuous flow instruments and batch synthesizers that can enable researchers to obtain high-yield and high-quality synthetic PNAs. Manual synthesis has the advantages of low cost and fast start-up.

Chemical Synthesis of PNA Oligomers with Fmoc

Fmoc-based PNA synthesis route consists of repeated cycles of deprotection, activation, coupling, and capping. Experts apply alkali-removable Fmoc as the protective group of?-amino group, TFA-removable tert-butoxy group to protect the side chain, 90% TFA-removable p-alkoxybenzyl alcohol resin. Consequently, the final deprotection can avoid strong acid treatment, and solid-phase synthesis also greatly reduces the difficulty of commercial purification.

Boc chemistry scheme has better synthetic quality while involving the use of hazardous chemicals. Therefore, the Fmoc method has become a commercially popular synthetic technology as it can provide high-quality synthetic products and avoid the involvement of strong corrosive chemicals at the same time. Besides, the synthesis of small-scale PNA oligomers can be quickly and readily performed on common DNA synthesis platforms. Furthermore, the Fmoc method makes it easy to prepare a wide range of PNA with fluorescent labels and other sensitive reporter groups.

Boc chemistry and Fmoc chemistry methods are highly relevant and appreciated to help us to explore and understand the multiple facets of PNA but, both technologies are also confronted with some problems such as expensive components and the purity of monomer. Accordingly, scientists strive to optimize the production technologies and platforms to improve the purity of raw materials and ensure the biological activity of the target PNA.

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