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　　Rapid optimization of PNA synthesis conditions using the multifunctional and multi-channel PurePep Chorus

Peptide nucleic acid (PNA) is a DNA analogue with a peptide like skeleton and various physical and chemical properties. The main backbone of PNA is composed of N (2-aminoethyl) - glycine and nucleic acid bases connected by methylene carbonyl groups.

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

Peptide nucleic acid non-ionic nucleic acid analogues, due to the non negative charge of PNA and the absence of electrostatic repulsion between DNA and RNA, have greatly improved stability and specificity in binding. Their hybridization ability with DNA or RNA molecules is much better than that of DNA/DNA or DNA/RNA, manifested in high hybridization stability, excellent specific sequence recognition ability, and resistance to hydrolysis by nucleases and proteases.

PNAs can inhibit gene expression by binding to complementary RNAs. And under specific conditions, gene expression can also be promoted by binding to DNA double stranded sequences through strand invasion mechanisms. Based on the above advantages and characteristics, PNA has been widely used in multiple fields in the past decade. Its high affinity and specificity make it not only a therapeutic agent, but also a powerful tool in diagnostic applications.

With the continuous deepening of basic research on PNA and the emergence of new technologies, PNA will demonstrate better performance and broader application prospects.

　　Current challenges

Due to the easy occurrence of polymerization during the synthesis process and the many side reactions during deprotection, the synthesis of PNAs presents significant challenges. The usual strategy for addressing the issue of easy aggregation is to use an excess of reagents in the condensation reaction. However, the price of PNA monomer reagents for N-terminal protection used in synthesis is relatively high, and this method has not been widely applied. Therefore, it is necessary to develop relatively economical and efficient synthesis processes to help PNAs successfully achieve commercialization.

　　Optimization of synthesis conditions on PurePep Chorus

In this article, we will demonstrate the rapid optimization of PNA synthesis conditions achieved on the PurePep Chorus automatic synthesizer.

PNA 1 will be used as a reference sequence to investigate different synthesis parameters, such as the excess coefficient of PNA monomers, reaction time, and temperature. The conditions for synthesizing PNA 1 with the highest purity will be used to synthesize five other sequences with different lengths and purine (adenine and guanine) contents (see Table 1). Purity is a key factor in evaluating synthesis, as the high steric hindrance of PNA monomers can easily lead to incomplete condensation reactions, resulting in low purity.

Table 1: PNA sequence information involved in this case

PNA 1 was synthesized using a PurePep Chorus peptide synthesizer with a synthetic equivalent of 25 μ mol. Rink Amide MBHA resin with a degree of substitution of 0.27 mmol/g was used, and the Fmoc/tBu orthogonal protection scheme was followed. The deprotection of Fmoc groups on the resin was carried out using a 20% piperidine DMF solution and reacted twice at room temperature for 5 minutes; The condensation reaction uses a system of HCTU and NMM, and the specific reaction details are shown in Table 2. After the condensation reaction is completed, perform the end capping operation using a 10% acetic anhydride DMF solution and react at room temperature for 5 minutes. After the final step of deprotection, place the resin in cutting solution (TFA: TIS: H2O=95:2.5:2.5) and react at room temperature for 4 hours.

The above operations can be set with different protocols on Chorus software according to the required conditions. The software automatically calculates the required amount of reagents, and the instrument runs automatically, saving more manual operation and ensuring that the experimental plan is strictly executed.

Table 2 Study on Condensation Conditions

From the data analysis in Table 2, we can find that when the reaction equivalent of PNA is 10 times, the purity of the crude product reaches 81%; However, due to the high price of the synthetic raw material Fmoc PVA monomer, the above design is not easily accepted. Reduce the reaction equivalence coefficient of PNA to 4 times and decrease the purity of the crude product to 72%. At this time, maintaining the reaction equivalence coefficient of PNA at 4 times, raising the reaction temperature to 45 ℃ and reducing the condensation time to 5 minutes, the purity of the crude product further decreased to 61%. It can be seen that a 5-minute reaction time is too short for condensation reactions. At this time, other conditions remained unchanged, and the reaction time was increased to 10 minutes alone (Experiment D). The purity of the crude peptide reached 82%, which was similar to the results of Experiment A. Experiment E was used to further investigate the reaction conditions, with the reaction equivalence coefficient adjusted to 2, and two sample addition reactions were conducted. The purity of the crude product increased by 5% using the same total amount of reaction materials and the same condensation time.

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

Table 3: Crude product results of synthesizing the remaining 5 PNA using method E

The crude purity of the 5 synthesized PNA products mentioned above is between 66-91%, which is satisfactory for peptide nucleic acids. It is interesting that PNA products with higher purine content have higher purity.

Figure 2: Chromatograms of 5 PNAs products collected using UPLC-UV system at a wavelength of 210nm. The UPLC system is Waters H-class UPLC/ESI-MS, and the chromatographic column is C18 (1.7 μ m, 2.1 x 50 mm). The mobile phase is a mixed solution of 0.1% TFA in water (A) and acetonitrile (B).

　　summary

In this application literature, we discussed in detail the method of optimizing PNA synthesis using the PurePep Chorus system. Initially, we used 10 times the reaction equivalent of PNA for condensation reaction at room temperature for 1 hour, which resulted in a crude product with a purity of 81%. We are committed to reducing the amount of reactants used and minimizing their impact on the purity of the crude product, making the reaction more economical, sustainable, and in line with the requirements of green chemistry. In the end, we developed a method that reduces the amount of reactants while maintaining high purity. The crude purity of PNA 1 can be increased from 81% to 87% by using 2 times the reaction equivalent for each sample addition and reacting for 5 minutes at 45 ℃. Finally, this method was applied to the synthesis of five other PNAs.

This method can produce very good products for peptide nucleic acids of different lengths and purine contents. The PurePep Chorus peptide synthesizer is a great tool for this job, as the synthesis scheme can be flexibly adjusted as needed; For example, modifying the reaction equivalence coefficient, number of repetitions, temperature, and time. At the same time, the automatic calculation function of automated instruments and software helps reduce manual operations, lower the risk of errors, and the optimized synthesis conditions can be easily applied to the synthesis of other PNAs.