Overcoming PNA Synthesis Challenges with the PurePep® Chorus Automated Peptide Synthesizer

Quickly optimize PNA synthesis conditions using the versatile, multi-channel PurePep Chorus.

Peptide nucleic acid (PNA for short) is a DNA analog with a peptide-like backbone and exhibits a variety of unique physicochemical properties. The main-chain backbone of PNA consists of N-(2-aminoethyl)glycine linked to nucleic acid bases via methylene carbonyl groups.

Figure 1: Schematic diagram of the structure of peptide nucleic acid and its binding to DNA.

Peptide nucleic acids, which are non-ionic nucleic acid analogs, do not carry a negative charge, and thus there is no electrostatic repulsion between PNA and DNA or RNA. As a result, the stability and specificity of their binding are greatly enhanced. Their hybridization capability with DNA or RNA molecules is significantly superior to that of DNA/DNA or DNA/RNA hybrids, as evidenced by their high hybridization stability, excellent specificity in sequence recognition, and resistance to degradation by nucleases and proteases.

PNAs can inhibit gene expression by binding to complementary RNAs. Moreover, under certain conditions, they can also bind to double-stranded DNA sequences via a strand invasion mechanism, thereby promoting gene expression. Based on these advantageous characteristics, PNAs have been widely used in various fields over the past decade. Their high affinity and specificity have made them not only promising therapeutic agents but also powerful tools in diagnostic applications.

As basic research on PNA continues to deepen and new technologies keep emerging, PNA will demonstrate even greater performance and broader application prospects.

Current Challenges

The synthesis of PNAs poses significant challenges due to the tendency for polymerization during the coupling process and the numerous side reactions that occur during deprotection. To address the issue of polymerization, a common strategy is to use an excess of reagents in the condensation reaction. However, the N-terminal protected PNA monomer reagents used in this synthesis are relatively expensive, which has limited the widespread adoption of this approach. Therefore, it is essential to develop a synthesis process that is both cost-effective and efficient in order to facilitate the successful commercialization of PNAs.

Optimizing synthesis conditions on PurePep Chorus

In this article, we will demonstrate the rapid optimization of synthesis conditions for PNAs using the PurePep Chorus automated synthesizer.

PNA 1 will be used as a reference sequence to investigate various synthetic parameters, such as the molar excess ratio of PNA monomers, reaction time, and temperature. The conditions yielding the highest purity of PNA 1 will be applied to synthesize the other five sequences, which differ in length and purine content (adenine and guanine) (see Table 1). Purity is a critical factor in evaluating the synthesis process; due to the significant steric hindrance of PNA monomers, incomplete condensation reactions are prone to occur, resulting in low purity.

Table 1: PNA Sequence Information Involved in This Case

PNA 1 was synthesized using the PurePep Chorus peptide synthesizer, with a synthesis yield of 25 μmol. The Rink Amide MBHA resin, with a substitution degree of 0.27 mmol/g, was employed, and the Fmoc/tBu orthogonal protection strategy was followed. Deprotection of the Fmoc groups on the resin was carried out using a 20% piperidine DMF solution, reacting at room temperature for 5 minutes × 2 times. The coupling reaction was performed using a system consisting of HCTU and NMM; specific reaction details are provided in Table 2. After completion of the coupling reaction, capping was performed using a 10% acetic anhydride DMF solution, reacting at room temperature for 5 minutes. Finally, after the last deprotection step, the resin was placed in a cleavage mixture (TFA:TIS:H2O = 95:2.5:2.5) and allowed to react at room temperature for 4 hours.

The above operations can be performed on Chorus by setting different protocols in the software according to the required conditions. The software automatically calculates the necessary dosage, and the instrument runs automatically, saving manual labor and ensuring strict adherence to the experimental protocol.

Table 2: Study of Condensation Conditions

From the data analysis in Table 2, we can see that when the stoichiometric ratio of PNA is increased to 10 times, the purity of the crude product reaches 81%. However, given the high cost of the synthesis raw material—Fmoc-PNA monomer—this design is not easily acceptable. When the stoichiometric coefficient of PNA is reduced to 4 times, the purity of the crude product drops to 72%. At this point, while keeping the stoichiometric coefficient of PNA at 4 times, raising the reaction temperature to 45°C and reducing the condensation time to 5 minutes further lowers the purity of the crude product to 61%. This clearly indicates that a reaction time of only 5 minutes is too short for the condensation reaction. Under these same conditions, but with the reaction time extended to 10 minutes (Experiment D), the crude peptide purity reached 82%, which is comparable to the result obtained in Experiment A. Experiment E was conducted to further investigate the reaction conditions: the stoichiometric coefficient was adjusted to 2, and two separate addition reactions were performed. Using the same total amount of reaction raw materials and maintaining the same condensation time, the purity of the crude product improved by an additional 5%.

We determined the synthesis conditions based on the highest purity of PNA 1 product. The remaining sequences will be synthesized using the same approach as PNA 1 (i.e., Method E). The results are shown in Table 3.

Table 3: Crude results of synthesizing the remaining five PNA strands using Method E.

The crude purity of the products obtained after synthesizing the aforementioned five PNAs ranged from 66% to 91%, a result that is satisfactory for peptide nucleic acids. Interestingly, PNA products with higher purine content exhibited even higher purity.

Figure 2: Chromatograms of the five PNA products, acquired using a UPLC-UV system at a wavelength of 210 nm. The UPLC system is a Waters H-class UPLC/ESI-MS, and the chromatographic column is C18 (1.7 μm, 2.1 × 50 mm). The mobile phase consists of a mixture of 0.1% TFA in water (A) and acetonitrile (B).

Summary

In this application note, we present a detailed discussion of a method for optimizing PNA synthesis using the PurePep Chorus system. Initially, we employed a condensation reaction at room temperature for 1 hour using 10-fold molar equivalents of PNA. This approach yielded crude products with a purity of 81%. We were committed to reducing the amount of reactants used and minimizing their impact on the purity of the crude product, thereby making the reaction more economical, sustainable, and aligned with the principles of green chemistry. Ultimately, we developed a method that not only reduced the amount of reactants but also achieved higher product purity. By adopting a two-step condensation protocol—where each addition of reactants was performed at twice the molar equivalent—and carrying out the reaction at 45°C for 5 minutes, we were able to increase the purity of crude PNA 1 from 81% to 87%. Finally, this optimized method was successfully applied to the synthesis of five additional PNA sequences.

For peptide nucleic acids of varying lengths and different purine contents, this method can yield excellent products. The PurePep Chorus peptide synthesizer is an excellent tool for this work, as the synthesis protocol can be flexibly adjusted according to need—for example, by modifying reaction stoichiometric coefficients, repetition times, temperatures, and durations. Meanwhile, the instrument’s automation and the software’s automatic calculation functions help minimize manual operations, reduce the risk of human error, and allow the optimized synthesis conditions to be easily applied to the synthesis of other PNAs.

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