PNA Dimers Synthesis

In recent years, as tools for molecular biology and biotechnology, diagnostic agents and potential antisense drugs, PNAs have received significant attention. PNAs are mainly applied as oligonucleotide analogs, antisense/antigene experiments, and diagnostic tools. The cellular uptake pattern of PNA oligomers varies from clear endosomal localization to somewhat diffuse cytoplasmic and sometimes nuclear localization, depending on the specific combination of PNA sequence and cell type. This unique nucleic acid analogue can specifically recognize DNA or RNA fragments and forms duplexes through Watson-Crick and Hoogsteen base pairing. However, PNAs are chemically stable and resistant to enzymatic cleavage, making them difficult to degrade in cells. In order to improve the physico-chemical and biological properties of PNA, chemists have designed and prepared a wide variety of derivatives and analogues of PNA.

Our PNA Dimers Synthesis Technologies

• Boc/Z-based Methods

At BOC Sciences, the conventional Boc/Z-based synthesis strategy is used for the production of PNA dimer. This method has the advantage of high average coupling yields and easy scale-up to larger scale synthesis.

• Ugi Four-Component Reaction-based Methods

We have introduced a new approach to obtain PNA dimers by extending PNA chains. In this strategy, Ugi four-component condensation reaction is carefully designed and applied. Conformational rotamers are identified by using NMR and MD simulations. Our approach does not require prior preparation of PNA monomers since they are built up along with chain lengthening.

Application of Our PNA Dimers Synthesis

• Synthesis of Caffeic acid (CA)-PNA Dimers

Caffeic acid has high antioxidant activity. CA derivatives have gained much attention due to their antioxidant, antitumor and antimicrobial properties as well as their stability. The novel PNA-CA dimer has the potential to improve the biological properties of CA including antioxidant, cytotoxic, cytoprotective. At BOC Sciences, the PNA-CA dimer is prepared on resin by using our Fmoc solid-phase peptide synthesis method. The coupling activation is performed with reagents known for peptide synthesis, and each reaction step is carefully monitored by a UV-Fmoc test, measuring the UV absorbance of the sample after deprotection in DMF.

Synthesis Procedure

• Firstly, the PNA activator is mixed with the monomer to activate the carboxylic acid group of the protected monomer, which is then added to the resin.
• After the synthesis and deprotection of PNA dimer, excess CA is added, and the resin is washed with DMF at room temperature
• Then, the coupling reaction is carried out and CA-PNA dimer is separated from the resin
• The crude product is filtered and the solution is concentrated in high vacuum, followed by purification

Structure Confirmation and Characterization

In order to perform a complete physico-chemical characterization of the newly synthesized PNA-CA dimer, our experts carry out elemental analysis, NMR and HPLC analysis.

• Elemental analysis: Determination of the molecular formula of the PNA-CA dimer
• 2D spectroscopy: Further confirmation of identity
• NMR Characterization: 1D NMR spectra are recorded at 25°C. The temperature is recorded in a range of temperature between 25°C and 70°C. TOCSY and ROESY experiments are recorded with mixing times of 70 and 150 ms, respectively. The translational diffusion coefficient (Df) is also measured using the pulsed field gradient spin-echo (PFGSE) DOSY experiment
• HPLC: Separation of different rotamers of the PNA-CA dimers is achieved by reversed-phase HPLC.

Fig 1. Solid-phase synthesis of CA-PNA dimers. (Maria, G.; et al. 2013)

• Synthesis of PNA-RNA Dimers

We have designed a practical synthesis of peptide nucleic acid units with RNA nucleosides (PNA-RNA dimer), in which phosphoramidite chemistry is applied to place the PNA unit within RNA oligonucleotides.

Fig 2. One-step synthesis of chimeric PNA–RNA dimers. (Reuben, O.; et al. 2017)

• Synthesis of PNA-pPNA Dimers

PNA-pPNA dimer improves the physico-chemical and biological properties of PNAs, especially water solubility and cellular uptake. In our method, two types of pPNA monomers containing N-(2-hydroxyethyl)phosphonoglycine, or N-(2-aminoethyl)phosphonoglycine backbone are used in combination with PNA monomers representing N-(2-aminoethyl)glycine or N-(2-hydroxyethyl)glycine derivatives. The obtained PNA-pPNA dimer consists of PNA and pPNA extended or alternating PNA and pPNA monomers.

Synthesis Procedure

• Oligodeoxynucleotides are synthesized by the standard phosphoramidite method or by a phosphate ester method using O-type nucleophilic intramolecular catalysis
• Synthesis and purification of PNA monomers
• PNA-pPNA dimers are produced using solid-phase synthesis, and oligomers are purified by anion-exchange FPLC and/or gel electrophoresis
• The identity and purity of the obtained oligomers are confirmed by mass spectrometry (MALDI-TOF) and reversed-phase FPLC

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