Development of an efficient and low-cost protocol for the manual PNA synthesis by Fmoc chemistry (original) (raw)
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Synthesis and Purification of Peptide Nucleic Acids
Current Protocols in Nucleic Acid Chemistry, 2001
Peptide nucleic acids (PNAs) are DNA analogs in which the normal phosphodiester backbone is replaced by 2-aminoethyl glycine linkages (Fig. 4.11.1). In spite of this significant chemical modification, the hybridization of PNAs with DNA or RNA follows normal rules for Watson-Crick pairing and occurs with high affinity. The fact that PNAs possess a dramatically different backbone and bind efficiently has created enormous interest in their application. This unit describes the synthesis and purification of PNAs. PNAs can be synthesized using either automated procedures (see Basic Protocol 1; Mayfield and Corey, 1999) or manual procedures (see Basic Protocol 2; Norton et al., 1995). The goal here is to provide the reader with the basic information necessary to initiate experiments with PNAs. NOTE: It is essential that the reagents used for PNA synthesis, whether automated or manual, be kept as anhydrous as possible, because contamination with water will result in incomplete coupling of monomers during synthesis. BASIC PROTOCOL 1 AUTOMATED SYNTHESIS OF PEPTIDE NUCLEIC ACIDS The automated synthesis of PNAs is illustrated in Figure 4.11.2. Automated synthesis is a convenient strategy for obtaining PNAs, and is performed using the Expedite synthesizer (Applied Biosystems). This instrument has been specially adapted for PNA synthesis, with proprietary software for FMOC chemistry instead of the phosphoramidite chemistry used for typical oligonucleotide synthesis. It is probably possible to adapt other synthesizers for PNA synthesis; however, the investigator will need to consider whether the number of PNAs needed justifies the time and expense required to adapt the instrumentation. The reagents used are available from Applied Biosystems separately or as an Expedite PNA kit. Two resins are available: a xanthen alkonic acid (XAL) resin and a peptide amide linker (PAL) resin. Although either is adequate for automated synthesis, the XAL resin is preferred because the cleavage step is considerably shorter (5 min according to Applied Biosystems, compared to 90 min for the PAL resin).
Solid-Phase synthesis of peptide nucleic acids
Journal of Peptide Science, 1995
Peptide nucleic acids (PNA) were synthesized by a modified Medield method using several improvements. Activation by O(benzotriazo1-1-yl)-1,1.3,3-tetramethyluronium hexafluorophosphate in combination with i n situ neutralization of the resin allowed efficient coupling of all four Boc-protected PNA monomers within 30 min. HPLC analysis of the crude product obtained from a fully automated synthesis of the model PNA oligomer H-CGGACTAAGTCCA?n;C-Gly-NH2, indicated an average yield per synthetic cycle of 97.1%. Ni-benzyloxycarbonyl-N3-methylimidazole triflate substantially outperformed acetic anhydride as a capping reagent. The resin-bound PNAs were successfully cleaved by the 'low-high' trifluoromethanesulphonic acid procedure.
Synthesis and analysis of peptide nucleic acid oligomers using Fmoc/acyl-protected monomers
Journal of the Chemical Society, Perkin Transactions 1, 2002
The optimization of PNA oligomer synthesis has been accomplished employing Fmoc/acyl-protected monomers on TentaGel and Wang resins. Among the tested activating agents (CMP, BET, HATU) the latter was of choice in solid phase syntheses. "Leakage" of TentaGel resin greatly hampers the solution and MS analyses. Synthesis and acyl group deprotection steps have been separately examined using Wang resin. Optimal conditions also worked well on the CPG support. HPLC and MS analyses of the PNA oligomers were carried out under various conditions. PERKIN 1266
Wiley Encyclopedia of Chemical Biology, 2007
Peptide nucleic acid (PNA) is a chimeric molecule that consists of hydrogen-bonding purine and pyrimidine heterocycles attached to a pseudopeptide backbone. The bases allow the recognition of specific DNA or RNA sequences, which results in hybrid multihelical structures. The lack of a negative charge on the PNA backbone eliminates Coulombic repulsion from target DNA and RNA strands, which results in high affinity hybridization. These properties have led to a diverse set of applications for PNA, which includes antisense/antigene inhibition of gene expression, various DNA/RNA detection assays, nucleic acid labeling and purification technologies, and programmed assembly of nanoscale materials. The modular design of PNA has led to the synthesis of several ''next-generation'' analogs that promise to improve PNA's performance in many existing applications as well as to open doors to new applications. Although most prior research on PNA has focused on its nucleic acid-like character, recent developments of the peptide-like aspects of PNA promise to stimulate additionally the work on this fascinating DNA mimic.
Peptide and peptide nucleic acid syntheses using a DNA/RNA synthesizer
Biopolymers, 2014
The use of an ABI 394 DNA/RNA synthesizer for peptide and peptide nucleic acid (PNA) syntheses is described. No additional physical part or software is needed for the application. A commercially available large DNA synthesis column was used, and only about half of its volume was filled with resin when the resin was fully swollen. With additional space in the top portion of the column, agitation of reaction mixture was achieved by bubbling argon from the bottom without losing solution. Removing solutions from column was achieved by flushing argon from top to bottom. Two peptide and two PNA sequences were synthesized. Good yields were obtained in all the cases. The method is easy to follow by researchers who are familiar with DNA/RNA synthesizer. V
Bioconjugate chemistry, 2006
An efficient and highly versatile method for the synthesis of amino acid-modified peptide nucleic acid (PNA) monomers is described. By using solid-phase Fmoc techniques, such monomers can be assembled readily in a stepwise manner and obtained in high yield with minimal purification. Protected neutral hydrophilic, acidic, and basic amino acids were coupled to 2-chlorotrityl chloride resin. Following Fmoc removal, innovative conditions for the key step, reductive alkylation with N-Fmoc-aminoacetaldehyde, were developed to circumvent problems encountered with previously reported methods. Activation and coupling of pyrimidine and purine nucleobases to the resulting secondary amines afforded amino acid-modified PNA monomers. The mild reaction conditions utilized were compatible with sensitive and labile functional groups, such as tert-butyl ethers and tert-butyl esters. PNA monomers were obtained in 36-42% overall yield and very high purity, after cleavage and purification. Using standard solid-phase Fmoc chemistry, two of these monomers were incorporated with high coupling efficiency into a variety of modified PNA oligomers, including four tetradecamers designed to target bcl-2 mRNA. Such modified oligomers have the potential to enhance water solubility and cell portability, while maintaining hybridization affinity and promoting favorable biodistribution properties.
Peptide Nucleic Acids (PNAs), A Chemical Overview
Current Medicinal Chemistry, 2005
Peptide nucleic acid (PNA) is a nucleic acid analogue and a fully synthetic DNA/RNA-recognising ligand with a neutral peptide-like backbone. In spite of the large change on the backbone structure, PNA molecules bind strongly to complementary DNA and RNA sequences. Originally conceived as ligand for the recognition of double stranded DNA, the unique physico-chemical properties of PNAs have led to the development of a variety of research and diagnostic assays. The extraordinary properties of PNA may advance routine clinical tests and environmental analyses that will utilise the PNA technology. PNAs may also have an impact on in situ hybridisation, cytogenetics and industrial microbiology. This paper presents some recent achievements on peptide nucleic acids and discusses, from the viewpoint of literature, what the potential is and what the limitations of such compounds are. This review, which is not intended to be exhaustive, is mostly aimed at the current progress in PNA chemistry, structure, and hybridisation, highlighting some applications too.
Synthesis, Analysis, Purification, and Intracellular Delivery of Peptide Nucleic Acids
Methods, 2001
in polymerase chain reactions (PCRs) (25) and for gene Peptide nucleic acids (PNAs) are nonionic DNA mimics. Their novel array analysis (26, 27). Full exploitation of the potential chemical properties may facilitate the development of selective and potent for recognition by PNAs requires knowledge of methods antisense and antigene strategies for regulating intracellular processes. for PNA synthesis, purification, characterization, and Described herein are procedures for the synthesis, purification, handling, delivery into cells. Here we describe methods that faciliand characterization of PNAs. A simple protocol for the lipid-mediated introduction of PNAs into in vitro cultures of mammalian cells is pro-tate the successful synthesis and use of PNAs. vided. ᭧ 2001 Academic Press DESCRIPTION OF METHODS Peptide nucleic acids (PNAs) (1-3) offer unique ad-A. Manual and Automated PNA Synthesis vantages for hybridization relative to RNA or DNA oli-PNAs can be synthesized using either manual or gonucleotides and their derivatives because PNAs posautomated protocols. Automated synthesis on an Expesess neutral N-(2-aminoethyl) linkages (Fig. 1). Despite dite 8909 (Applied Biosystems, Foster City, CA) (Fig. their dramatically altered geometry and charge, PNAs 2) uses Fmoc PNA monomers and offers a routine can bind to complementary sequences by Watson-Crick method for producing PNAs on a relatively small (2 base pairing and are able to discern single base mis-M) scale (28). Manual synthesis (9, 29-31) can be matches (4). Binding is characterized by high affinity accomplished using monomers protected by either t-(4) and high rates of association (5) and these advan-Boc or Fmoc protecting groups, although we find that tages combine to give PNAs a remarkable propensity yields are higher after t-Boc syntheses (http://hhmi.sfor invasion of duplex DNA (1, 6-9). The rules governwmed.edu/Labs/dc/DCHome.html). Manual syntheses ing the prediction of PNA hybridization to duplex DNA are relatively time consuming, but may be the most in vitro indicate that binding to inverted (7,9) or tricost effective way to obtain PNAs if only a few are nucleotide repeats (8) and polypurine-polypyrimidine needed. Manual synthesis also allows PNAs to be obsequences (10-13) are preferred. PNAs are also tained on a large scale (Ͼ20 M). Automated synthesis nuclease and protease resistant (14), suggesting that requires a substantial investment in instrumentation, they will be highly active in vivo. but is recommended if large numbers of PNAs are re-PNAs are being used in a variety of applications quired over the long term. PNAs can also be obtained (15,16) including purification of chromosomal DNA from PE Biosystems and other commercial sources. (8,17), induction of gene expression (18), inhibition of human telomerase (19-22), and inhibition of the expression of the pain receptors galanin (23) and neurotensin (24). Other studies have used PNAs as clamps B. Buffers RP-HPLC Buffer A: 0.1% (v/v) trifluoroacetic acid
Fmoc/Acyl protecting groups in the synthesis of polyamide (peptide) nucleic acid monomers
Journal of the Chemical Society, Perkin Transactions 1, 2000
The chemical synthesis of polyamide (peptide) nucleic acid (PNA) monomers 22-25 has been accomplished using Fmoc [N-(2-aminoethyl)glycine backbone], anisoyl (adenine), 4-tert-butylbenzoyl (cytosine) and isobutyryl/ diphenylcarbamoyl (guanine) protecting-group combinations, thus allowing oligomer synthesis on both peptide and oligonucleotide synthesizers. An alternative method for the preparation of (N 6-anisoyladenin-9-yl)acetic acid 7 is described using partial hydrolysis of a dianisoylated derivative. Different methods were studied for guanine alkylation including (a) Mitsunobu reaction; (b) low-temperature, sodium hydride-and (c) N,N-diisopropylethylaminemediated alkylation reactions to give preferentially N 9-substituted derivatives. Empirical rules are proposed for differentiating N 9 /N 7-substituted guanines based on their 13 C NMR chemical-shift differences.