Solid-Phase Synthesis of Difficult Purine-Rich PNAs through Selective Hmb Incorporation: Application to the Total Synthesis of Cell Penetrating Peptide-PNAs

Abstract

Antisense oligonucleotide (ASO)-based drug development is gaining significant momentum following the recent FDA approval of Eteplirsen (an ASO based on phosphorodiamidate morpholino) and Spinraza (2′-O-methoxyethyl-phosphorothioate) in late 2016. Their attractiveness is mainly due to the backbone modifications which have improved the in vivo characteristics of oligonucleotide drugs. Another class of ASO, based on peptide nucleic acid (PNA) chemistry, is also gaining popularity as a platform for development of gene-specific therapy for various disorders. However, the chemical synthesis of long PNAs, which are more target-specific, remains an ongoing challenge. Most of the reported methodology for the solid-phase synthesis of PNA suffer from poor coupling efficiency which limits production to short PNA sequences of less than 15 residues. Here, we have studied the effect of backbone modifications with Hmb (2-hydroxy-4-methoxybenzyl) and Dmb (2,4-dimethoxybenzyl) to ameliorate difficult couplings and reduce “on-resin” aggregation. We firstly synthesized a library of PNA dimers incorporating either Hmb or Dmb and identified that Hmb is superior to Dmb in terms of its ease of removal. Subsequently, we used Hmb backbone modification to synthesize a 22-mer purine-rich PNA, targeting dystrophin RNA splicing, which could not be synthesized by standard coupling methodology. Hmb backbone modification allowed this difficult PNA to be synthesized as well as to be continued to include a cell-penetrating peptide on the same solid support. This approach provides a novel and straightforward strategy for facile solid-phase synthesis of difficult purine-rich PNA sequences.

Modular Synthesis of Trifunctional Peptide-oligonucleotide Conjugates via Native Chemical Ligation

“…The PNA was synthesized using previously established Fmoc/Bhoc solid-phase synthesis protocols ( Tailhades et al, 2017 ) and functionalized with cysteine at the N-terminus (5′-end). A polyethylene glycol spacer (miniPEG) was introduced between the PNA and the cysteine.…”

Conjugation Approaches for Peptide-Mediated Delivery of Oligonucleotides Therapeutics

“…3a). [34,35] Winkler and co-workers developed an on-resin phase transfer reaction to form an amide link between peptides and non-PNA oligonucleotides (Fig. 3b).…”

Automated Fast-Flow Synthesis of Peptide Nucleic Acid Conjugates

“…In-line UV-visible monitoring combined with liquid chromatography-mass spectrometry (LC-MS) and high-performance liquid chromatography (HPLC) product characterization allows for rapid optimization of PNA synthesis conditions. We began the optimization with a manually synthesized 4-mer PNA in ~4 h of in-lab effort 10,13 with 57% crude purity (Table 1, entry 1). During the manual synthesis, base can promote transamidation of the nucleobase to the N-terminus, resulting in an isomeric product (15%), similarly, transamidation on the main chain can lead to formation of a ketopiperazine that gets washed away, causing deletion of the N-terminal PNA unit (4%), and the benzhydryloxycarbonyl (Bhoc) protecting group can be cleaved by piperidine to yield a nucleobase adduct (2%) (supporting information S6).…”

“…PNA synthesis is limited by strong on-resin aggregation and side-reactions including deletion, rearrangement, isomerization, and nucleobase additions result in low isolated yields of the desired product. 10 Most of the reported synthetic PNA sequences are limited to fewer than 15 residues, especially for purine-rich sequences. 10 Efforts to improve synthetic efficiency are commonplace, such as extensive capping and frequent double-couplings, but with a great sacrifice of convenience.…”

“…10 Most of the reported synthetic PNA sequences are limited to fewer than 15 residues, especially for purine-rich sequences. 10 Efforts to improve synthetic efficiency are commonplace, such as extensive capping and frequent double-couplings, but with a great sacrifice of convenience. Therefore, automation technology 11 is needed to improve the PNA synthesis efficiency and in the meanwhile, reduce its complexity.…”