Synthesis Of Peptide Nucleic Acids Research Articles

Synthesis and properties of PNA containing a dicationic nucleobase based on N4-benzoylated cytosine

We report the synthesis of a peptide nucleic acid (PNA) monomer containing N4-bis(aminomethyl)benzoylated cytosine (BzC2+ base). The BzC2+ monomer was incorporated into PNA oligomers using Fmoc-based solid-phase synthesis. The BzC2+ base in PNA had two positive charges and exhibited greater affinity for DNA G base than the natural C base. The BzC2+ base stabilized PNA-DNA heteroduplexes through electrostatic attractions, even in high salt conditions. The two positive charges on the BzC2+ residue did not compromise the sequence specificity of PNA oligomers. These insights will aid the future design of cationic nucleobases.

Ultrasound-assisted Peptide Nucleic Acids synthesis (US-PNAS)

Herein, we developed an innovative and easily accessible solid-phase synthetic protocol for Peptide Nucleic Acid (PNA) oligomers by systematically investigating the ultrasonication effects in all steps of the PNA synthesis (US-PNAS). When compared with standard protocols, the application of the so-obtained US-PNAS approach succeeded in improving the crude product purities and the isolated yields of different PNA, including small or medium-sized oligomers (5-mer and 9-mer), complex purine-rich sequences (like a 5-mer Guanine homoligomer and the telomeric sequence TEL-13) and longer oligomers (such as the 18-mer anti-IVS2-654 PNA and the 23-mer anti-mRNA 155 PNA). Noteworthy, our ultrasound-assisted strategy is compatible with the commercially available PNA monomers and well-established coupling reagents and only requires the use of an ultrasonic bath, which is a simple equipment generally available in most synthetic laboratories.

The challenge of peptide nucleic acid synthesis.

Peptide nucleic acids (PNAs) are an important class of DNA/RNA mimics that can hybridize complementary chains of nucleic acids with high affinity and specificity. Because of this property and their metabolic stability, PNAs have broad potential applications in different fields. Consisting of a neutral polyamide backbone, PNAs are prepared following the method used for peptide synthesis. In this regard, they are prepared by the sequential coupling of the protected monomers on a solid support using a similar approach to solid-phase peptide synthesis (SPPS). However, PNA synthesis is a little more challenging due to issues of the difficulty on the preparation of monomers and their solubility. Furthermore, the PNA elongation is jeopardized by intra/inter chain aggregation and side reactions. These hurdles can be overcome using different protecting group strategies on the PNA monomer, which also dictate the approach followed to prepare the oligomers. Herein, the main synthetic strategies driven by the protecting group scheme are discussed. However, there is still ample scope for further enhancement of the overall process.

A safety-catch protecting group strategy compatible with Boc-chemistry for the synthesis of peptide nucleic acids (PNAs).

Peptide Nucleic Acids (PNAs) are an intriguing class of synthetic biomolecules with great potential in medicine. Although PNAs could be considered analogs of oligonucleotides, their synthesis is more like that of peptides. In both cases, a Solid-Phase Synthesis (SPS) approach is used. Herein, the advantage using Boc as a temporal protecting group has been demonstrated to be more favored than Fmoc. In this context, a new PNA SPS strategy has been developed based on a safety-catch protecting group scheme for the exocyclic nitrogen of the side-chain bases and the linker. Sulfinyl (sulfoxide)-containing moieties are fully stable to the trifluoroacetic acid (TFA) used to remove the Boc group, but they can be reduced to the corresponding sulfide derivatives, which are labile in the presence of TFA. The efficiency of this novel synthetic strategy has been demonstrated in the synthesis of the PNA pentamer H-PNA(TATCT)-βAla-OH.

Multicomponent Synthesis of Diaminopurine and Guanine PNA's Analogues Active against Influenza A Virus from Prebiotic Compounds.

Peptide nucleic acids (PNAs) play a key role in prebiotic chemistry as a chimera between RNA and proteins. We developed an alternative synthesis of bioactive PNA's diaminopurine and guanine analogues from prebiotic compounds, such as aminomalononitrile (AMN), urea, and guanidine, using a two-step multicomponent microwave-assisted and solvent-free approach in the presence of selected amino acids. The novel derivatives showed selective inhibitory activity against influenza virus A/Puerto Rico/8/34 H1N1 encompassing the range of nanomolar activity. Derivatives decorated with the tyrosine residue showed the highest inhibitory activity against the virus.

Machine Learning Guides Peptide Nucleic Acid Flow Synthesis and Sequence Design.

Peptide nucleic acids (PNAs) are potential antisense therapies for genetic, acquired, and viral diseases. Efficiently selecting candidate PNA sequences for synthesis and evaluation from a genome containing hundreds to thousands of options can be challenging. To facilitate this process, this work leverages machine learning (ML) algorithms and automated synthesis technology to predict PNA synthesis efficiency and guide rational PNA sequence design. The training data is collected from individual fluorenylmethyloxycarbonyl (Fmoc) deprotection reactions performed on a fully automated PNA synthesizer. The optimized ML model allows for 93% prediction accuracy and 0.97 Pearson's r. The predicted synthesis scores are validated to be correlated with the experimental high-performance liquid chromatography (HPLC) crude purities (correlation coefficient R2 = 0.95). Furthermore, a general applicability of ML is demonstrated through designing synthetically accessible antisense PNA sequences from 102315 predicted candidates targeting exon 44 of the human dystrophin gene, SARS-CoV-2, HIV, as well as selected genes associated with cardiovascular diseases, type II diabetes, and various cancers. Collectively, ML provides an accurate prediction of PNA synthesis quality and serves as a useful computational tool for informing PNA sequence design.

A Three‐Way Regioselective Synthesis of Amino Acid Decorated Imidazole, Purine and Pyrimidine Derivatives by Multicomponent Chemistry Starting from Prebiotic Diaminomaleonitrile

AbstractAmino acid decorated imidazole, pyrimidine and purine derivatives, resembling analogues of peptide nucleic acid's building blocks, have been synthesized by a three‐way multicomponent chemistry of diaminomaleonitrile, one of the most studied chemical precursors in prebiotic chemistry. The regioselectivity of the multicomponent reaction between diaminomaleonitrile, trimethyl orthoacetate and a selected panel of α‐amino acids, was controlled by the energy source used for triggering the condensation. Imidazole derivatives were obtained under thermal condition, while photochemical and combined photothermal conditions afforded pyrimidine and purine derivatives, respectively. The three synthetic pathways were independent of each other, the type of product being controlled by the chemical intermediates prevailing in the reaction mixture. In addition to the synthetic potential of these multicomponent reactions in the preparation of a large variety of heterocycles, the control of the synthesis of peptide nucleic acid's building blocks, simply by changing the nature of the energy source, further enlarged the chemical complexity available in prebiotic conditions for the emergence of pristine pre‐genetic codes.

Optimized Synthesis of Fmoc/Boc‐Protected PNA Monomers and their Assembly into PNA Oligomers.

AbstractContinuous advancement of application of peptide nucleic acid (PNA) oligomers encouraged exploration of rapid and efficient synthesis of PNA monomers and oligomers. Among the PNA monomers developed, only a few are commonly used in automated PNA synthesis. Herein, we report short and efficient protocols suitable for large‐scale synthesis of Fmoc/Boc‐protected PNA monomers with advantageous solubility properties; these also facilitate purification due to the traceless nature of the Boc protecting group. Initially, several coupling reagents were screened for assembly of a pentamer containing all four nucleobases, and then the most promising reagents were tested in the synthesis of a decamer. The Fmoc/Boc‐protected monomers proved compatible with both manual synthesis and assembly on an automated peptide synthesizer at room temperature or at 40 °C. As compared to the commonly used coupling agent, 1‐[bis(dimethylamino)methylene]‐1H‐1,2,3‐triazolo[4,5‐b]pyridinium 3‐oxide hexafluorophosphate (HATU), both 2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate (HBTU) and [ethyl cyano(hydroxyimino)acetato−O2]tri‐1‐pyrrolidinylphosphonium hexafluorophosphate (PyOxim) proved more favorable, with the latter being superior. A previously reported side reaction of guanine bases in the presence of benzotriazol‐1‐yl‐oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) was not observed with the phosphonium salts.

Electrostatics Favor PNA : DNA Stability over Stereochemistry in Pyrrolidine‐Based Cationic Dual‐Backbone PNA Analogues

AbstractModifications to the peptide nucleic acid (PNA) backbone has been well known to alter the thermodynamical parameters of PNA : DNA complexes to broaden their utility for different applications. Electrostatic interactions between a modified PNA having a positively charged backbone and the negatively charged DNA has been shown to enhance thermal stabilities of PNA : DNA complexes at various instances. On the other hand, chiral introduction in PNA backbone leads to stereochemical preference that affects binding properties. However, the interplay between electrostatics and stereochemistry has not been systematically studied so far. Herein, we report the synthesis and biophysical characterization of cationic PNA named dapPNA, first of its kind, having a dual PNA backbone constituting of a pyrrolidine ring having a β‐substitution. One of the aims of this study was to investigate the role of electrostatics over stereochemical preferences. The results show that electrostatic attraction between cationic dapPNA and negatively charged DNA overcomes the unfavorable stereochemical effects and enhances stability of PNA : DNA complexes. Moreover, two different PNA backbones derived from a single PNA monomer expands the repertoire of pyrrolidine based PNA analogues.

Evolution of peptide nucleic acid with modifications of its backbone and application in biotechnology.

Peptide nucleic acids (PNAs) are getting prodigious interest currently in the biomedical and diagnostic field as an extremely powerful tool because of their potentiality to hybridize with natural nucleic acids. Although PNA has strong affinity and sequence specificity to DNA/RNA, there is a considerable ongoing effort to further enhance their special chemical and biological properties for potential application in numerous fields, notably in the field of therapeutics. The toolbox for backbone modified PNAs synthesis has been extended substantially in recent decades, providing a more efficient synthesis of peptides with numerous scaffolds and modifications. This paper reviews the various strategies that have been developed so far for the modification of the PNA backbone, challenging the search for new PNA systems with improved chemical and physical properties lacking in the original aegPNA backbone. The various practical issues and limitations of different PNA systems are also summarized. The focus of this review is on the evolution of PNA by its backbone modification to improve the cellular uptake, sequence specificity, and compatibility of PNA to bind to DNA/RNA. Finally, an insight was also gained into major applications of backbone modified PNAs for the development of biosensors.

Potential applications of peptide nucleic acid in biomedical domain.

Peptide Nucleic Acid (PNA) are DNA/RNA synthetic analogs with 2‐([2‐aminoethyl] amino) acetic acid backbone. They partake unique antisense and antigene properties, just due to its inhibitory effect on transcription and translation; they also undergo complementary binding to RNA/DNA with high affinity and specificity. Hence, to date, many methods utilizing PNA for diagnosis and treatment of various diseases namely cancer, AIDS, human papillomavirus, and so on, have been designed and developed. They are being used widely in polymerase chain reaction modulation/mutation, fluorescent in‐situ hybridization, and in microarray as a probe; they are also utilized in many in‐vitro and in‐vivo assays and for developing micro and nano‐sized biosensor/chip/array technologies. Earlier reviews, focused only on PNA properties, structure, and modifications related to diagnostics and therapeutics; our review emphasizes on PNA properties and synthesis along with its potential applications in diagnosis and therapeutics. Furthermore, prospects in biomedical applications of PNAs are being discussed in depth.

New synthetic strategies for treatment of the renal disease: synthesis of peptide nucleic acids

New synthetic strategies for treatment of the renal disease: synthesis of peptide nucleic acids

Identification of Optimized Peptide Nucleic Acid (PNA) Designs for Nuclease-Free Gene Editing in Sickle Cell Disease Hematopoietic Stem and Progenitor Cells (HSPCs)

Homozygous E6V alleles in the beta globin gene cause disease in the majority of sickle cell patients and the use of gene editing technologies to correct this mutation in hematopoietic stem cells offers the potential to cure disease. Approaches utilizing viral transduction or exogenous nucleases to facilitate gene editing merit consideration, but also invoke a number of safety and manufacturing concerns that are avoided through use of peptide-nucleic acids (PNAs) for triplex gene editing. In our approach, nanoparticle delivery of a synthetic PNA that binds the beta globin gene near the E6V mutation triggers high-fidelity endogenous gene repair pathways. Inclusion of a synthetic DNA correction oligo subsequently directs correction of the mutation. This methodology has previously been shown to facilitate gene repair in vivo in several murine models including sickle disease, beta-thalassemia, and cystic fibrosis (Quijano et al., [ASGCT abstr.] 2019, Bahal et al., 2016, McNeer et al., 2015). To date, there has been limited exploration of the structure-activity relationship of PNA designs and the resultant gene editing. Here we describe the design, synthesis, and structure-activity relationship of a hit-to-lead series of more than 300 PNAs and show with certain design features we can improve gene editing efficiency in both murine and human hematopoietic stem and progenitor cells. Our first round of PNA design and synthesis consisted of 45 PNAs within 200 nucleotides of the E6V allele, of which 18/45 (40 %) showed >/= 10% allele correction to wild-type HBB allele when tested exvivo in total bone marrow cultures derived from humanized sickle disease "Townes mice". The best PNA in this series consistently demonstrated ~ 20% editing with an ~ED50 of ~1-3 ug/ml of donor DNA or PNA. Editing observed in total mouse bone marrow was also robust in separate cultures of both Townes mouse c-kit(+) HSPCs and healthy donor human CD34(+) HSPCs. In our second round of PNA designs, considerations encompassed numerous factors including whether the PNA binds to the transcribed versus non-transcribed chromosomal strand, distance and location of the PNA relative to the mutation site and relative to the donor template sequence, substitutions on gamma-carbons of the PNA polyamide backbone, structure of the linker joining PNA nucleobase segments, incorporation of novel bases to eliminate off-target base pairing or aggregation to due self-complementarity, and inclusion of lysine residues to facilitate kinetics and affinity of the association with the poly-anionic backbone of target DNA sequences. At the time of this submission we have synthesized and assessed >250 additional PNA designs and identified 6 modifications that increase editing 2-4 fold over the initial designs. We will share data on the design and activity of these molecules, data on impact of combining design features, and next generation designs in the presentation. This is the first extensive study on the structure-activity relationship of PNAs for use in gene editing. Our data demonstrate a number of PNA parameters significantly improve gene editing activity. The combination of these findings with additional technological improvements offers the prospect of high-fidelity gene editing without use of any biologics (e.g., viruses or nucleases) that have inherent clinical risk and manufacturing challenges which may limit or prevent their implementation as a cure for sickle patients. Figure Disclosures Ebens: Trucode Gene Repair, Inc.: Employment, Equity Ownership, Patents & Royalties. Curd:Trucode Gene Repair, Inc.: Employment, Equity Ownership, Patents & Royalties. Sim:Trucode Gene Repair, Inc.: Employment, Equity Ownership, Patents & Royalties. Quijano:Yale University: Patents & Royalties; Trucode Gene Repair, Inc.: Consultancy, Research Funding. Tam:Trucode Gene Repair, Inc.: Employment, Equity Ownership, Patents & Royalties. Coull:Trucode Gene Repair, Inc.: Consultancy, Equity Ownership, Patents & Royalties. Huang:Trucode Gene Repair, Inc.: Employment, Equity Ownership. Hayes:Trucode Gene Repair, Inc.: Employment, Equity Ownership, Patents & Royalties. Fordyce:Trucode Gene Repair, Inc.: Employment, Equity Ownership, Patents & Royalties. Pearson:Trucode Gene Repair, Inc.: Employment. Whoriskey:Trucode Gene Repair, Inc.: Consultancy, Equity Ownership, Patents & Royalties. Srivastava:Trucode Gene Repair, Inc.: Employment, Equity Ownership, Patents & Royalties. Li:Trucode Gene Repair, Inc.: Employment, Equity Ownership, Patents & Royalties. Zhang:Trucode Gene Repair, Inc.: Employment, Equity Ownership, Patents & Royalties.

A convenient route to synthesize N2-(isobutyryl)-9-(carboxymethyl)guanine for aeg-PNA backbone

Synthesis of exclusive N2-(isobutyryl)-9-(carboxymethyl)guanine, an important moiety for peptide nucleic acid synthesis has been reported through a high-yielding reaction scheme starting from 6-chloro-2-amino purine. Crystal structures of two intermediates confirmed the formation of N9-regioisomer. This new synthetic route can potentially replace the conventional tedious method with moderate overall yield.

Synthesis of α-(R)-/γ-(S)-Dimethyl Substituted Peptide Nucleic Acid Submonomer Using Mitsunobu Reaction

One of the major challenges facing modern biochemical and biomedical technologies are finding molecular tools for diagnosis and detection of genetic diseases. In this connection, several classes of oligonucleotides have been developed that can recognize and bind to DNA and RNA with high affinity and sequence selectivity and withstand enzymatic degradation by proteases and nucleases; however, few can traverse the cell membrane on their own. One such promising class of nucleic acid mimics developed in the last two decades which showed good results in vitro, are the peptide nucleic acids (PNAs). New chiral α- and γ-peptide Nucleic Acid (PNA) submonomer with methyl substituents in pseudopeptide backbone were synthesized via Mitsunobu reaction. The α-(R)-/γ-(S)-configuration of the chiral centres will ensure the preorganization of the PNA oligomer into a right-handed helix. The results obtained showed that Boc/Fmoc-submonomer compatible with Boc-protocol PNAs solid-phase synthesis on an MBHA resin. We synthesized simple and efficient α-R-, γ-S-disubstituted PNA submonomer based on L-Ala and D-Ala with the construction of the intermediate pseudopeptide moiety by Mitsunobu reaction for subsequent use in the Boc-Protocol of solid phase PNA synthesis.

Synthesis and Application of LKγT Peptide Nucleic Acids.

Displaying ligands in a succinct and predictable manner is essential for elucidating multivalent molecular-level binding events. Organizing ligands with high precision and accuracy provides a distinct advantage over other ligand-display systems, such as polymers, because the number and position of the ligand(s) can be accurately and fully characterized. Here we describe the synthesis of peptide nucleic acids (PNAs), which are oligonucleotide mimics with a pseudopeptide backbone that can hybridize to oligonucleotides through Watson-Crick base pair to form highly predictable and organized scaffold for organizing a ligand. The ligand(s) are covalently attached to the PNA through a squarate coupling reaction that occurs between a free amine on the ligand and a free amine appended to the pseudopeptide backbone of the PNA. In this chapter we describe the synthesis of a LKγT monomer, which ultimately yields the free amine off the backbone of the PNA, incorporation of the monomer in a PNA oligomer, and the sequential squarate coupling to conjugate the ligand.

Metal Coordination to Ligand-Modified Peptide Nucleic Acid Triplexes.

A challenging goal in nanotechnology is the precise and programmable arrangement of specific elements in nanosystems in the three-dimensional space. The use of ligand-modified nucleic acids represents an accurate and selective tool to achieve this goal when it comes to metal ion organization. The synthesis of peptide nucleic acid (PNA) monomers that contain ligands instead of nucleobases makes possible the creation of metal-mediated alternative base pairs and triplets at specific locations in PNA duplexes and triplexes, respectively. We report the formation of four- and six-coordinate metal complexes between PNA triplexes modified with 2,2'-bipyridine (Bpy) or 8-hydroxyquinoline (Q) ligands and 3d metal ions. These metal complexes function as alternative base triplets or pairs in that they increase the thermal stability of the triplexes if the stability constants of the metal complexes are relatively high. The increase in the triplex melting temperature correlates with the stability constants of the metal complexes with ligand-containing PNA determined by UV-vis titrations. The metal complexes coordinate two or three ligands although three bidentate ligands are in close proximity of each other within a triplex. Metal coordination to ligand-modified PNA triplexes was further studied by electron paramagnetic resonance (EPR) spectroscopy and circular dichrosim (CD) spectroscopy. EPR spectroscopy indicated the formation of a square planar [CuQ2] complex between Cu2+ and Q-containing PNA triplex. Taken together, the spectroscopic results indicate that in the presence of 1 equiv of Fe2+ or Ni2+ the majority, but not all, of the Bpy-containing PNA triplexes contain [MBpy3] complexes, with a minority of them being metal free. We attribute this behavior to a supramolecular chelate effect exerted by the triplex, which favors the formation of tris-ligand complexes, that is balanced by the steric interactions between the metal complex and the adjacent nucleobase triplets, which decrease the stability of the complex and triplex. In contrast, the very high stability of square planar [MQ2] complexes of Cu2+ and Ni2+ leads to formation of bis-ligand complexes instead of tris-ligand complexes with Q3-containing PNA triplexes. The metal-containing PNA triplexes have a terminal l-lysine and adopt a left-handed chiral structure in solution. The handedness of the PNA triplex determines that of the metal complexes formed with the Bpy-containing PNA triplexes.

A Pot-economical Liquid-phase Peptide Nucleic Acid Synthesis Enabled by a Soluble Tag-assisted Method

In a peptide nucleic acid (PNA), the base sequences are arranged onto a backbone of repeating units of N-(2-aminoethyl)glycine instead of on phosphodiester-linked riboses. PNA is a promising platform for the design of novel chemical probes because PNAs can form complementary base pairs with DNA and RNA that exhibit high enzymatic stability. Described herein is a pot-economical liquid-phase PNA synthesis achieved using a soluble tag-assisted method. Intramolecular trapping of the activated PNA monomer occurs much faster than the undesired intermolecular “double hit”. Consequently, excess PNA monomers do not have to be rinsed away after each coupling, enabling one-pot coupling and deprotection without the need for precipitation.

Preparation of Anti-miR PNAs for Drug Development and Nanomedicine.

Peptide Nucleic Acids (PNAs) are oligonucleotide mimics that can be used to block the biological action of microRNA, thus affecting gene expressionpost-transcriptionally. PNAs are obtained with solid-phase peptide synthesis, and can be easily conjugated to other peptides. Conjugation with R8-Peptide or modification of the PNA backbone (at C5 or C2 carbon) with arginine side chains allows efficient cellular uptake. The present protocol describes the synthesis of cationic PNAs that can be used alone as drugs or for efficient co-delivery in suitable inorganic nanocarriers.

A One-Pot Synthesis of Highly Functionalized Purines.

Highly substituted purines were synthesized in good to high yields through a one-pot straightforward metal-free scalable method, using the Traube synthesis adapted to Vilsmeier-type reagents. From 5-amino-4-chloropyrimidines, new 9-aryl-substituted chloropurines and intermediates for peptide nucleic acid synthesis were prepared. Variant procedures allowing a rapid synthesis of ribonucleosides and 7-benzylpurine from 5-amidino-6-aminopyrimidines are also reported to illustrate the high potential of this versatile toolbox. This route appears to be particularly interesting in the field of nucleic acids for a direct and rapid access to various new 8-alkylpurine nucleosides.