N-(2-Boc-aminoethyl)-N-(thymin-1-ylacetyl)glycine

[139166-80-6]  · C16H24N4O7  · (MW 384.38)

(achiral DNA-mimic, peptide nucleic acid monomer)

Alternate Name: {(2-tert-Butoxycarbonylaminoethyl)-[(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)acetyl]amino} acetic acid.

Physical Data: mp 122-123 °C.

Solubility: soluble in aqueous bases, aprotic organic solvents like DMF, NMP, and DMSO. Insoluble in unpolar organic solvents.

Form Supplied in: commercially available neat.

Handling, Storage, and Precautions: stable on prolonged storage at 4 °C; should be dried prior to use.

Synthesis of N-(2-Boc-aminoethyl)-N-(thymin-1-ylacetyl) glycine

N-(2-Boc-aminoethyl)-N-(thymin-1-ylacetyl)glycine was first prepared by Nielsen and co-workers.1 The title compound can be easily prepared by a convergent five-step synthesis, starting from 1,2-diaminoethane. This compound is monoprotected in quantitative yield using di-tert-butyldicarbonate (1).2

Alkylation of the N-(tert-butyloxycarbonyl)ethylenediamine with bromoacetic acid ethyl ester gives (2-tert-butoxycarbonylaminoethylamino)acetic acid ethyl ester (2).3

In an alternative synthesis, (2-tert-butoxycarbonylaminoethylamino)acetic acid ethyl ester is prepared by a reductive amination, which employs glycine ethyl ester and N-Boc-protected aminoacetaldehyde. The latter is accessed by a two-step synthesis, in which 3-amino-1,2-propanediol is allowed to react with di-tert-butyldicarbonate followed by periodate oxidation.4 The thymin-1-ylacetate is prepared in a one-step synthesis from thymine and bromoacetic acid (3).5

Coupling of the thymin-1-ylacetate to the Boc-protected aminoethylglycine backbone using dicyclohexylcarbodiimide as coupling reagent and subsequent saponification of the ethyl ester yields N-(2-Boc-aminoethyl)-N-(thymin-1-ylacetyl)glycine (4).6

The use of the Boc-protecting group prevents acyl migration during deprotection since the primary amine liberated remains protonated. In addition, Boc-protection increases the solubility of the monomer compared to the Fmoc-protected building block.

Peptide Nucleic Acid Synthesis

Peptide nucleic acid oligomers can be prepared on solid support following standard solid-phase synthesis protocols for peptide synthesis.7,8 PNA oligomers are mainly synthesized as C-terminal amides using MBHA polystyrene resins as solid support. Couplings are performed with HBTU or HATU as coupling reagent and preactivation of the monomers for one minute. HATU has given better coupling yields compared to HBTU. Excess of HATU should be avoided since free amino groups can be converted into guanidinium moieties by HATU.7 To obtain optimal coupling results, the coupling solutions should be highly concentrated. N-Methylpyrrolidone is superior to DMF as solvent since the monomers are more soluble in NMP. Another advantage of NMP is its ability to reduce aggregation during PNA synthesis. Aggregation is a severe problem in PNA synthesis, limiting strand lengths to about 30 nucleobases. Sequences containing a high number of purine bases are especially difficult to synthesize. Couplings are usually complete after 10 min. If necessary, unreacted amino groups can be capped using acetic anhydride as capping reagent. Deprotection of the terminal Boc-group is achieved with pure TFA within a reaction time of 3 min. Prior to the next coupling reaction, all traces of TFA have to be removed from the resin. This can be achieved by using DCM for the washing step. Exposure of thymine residues to nucleophiles should be minimized since thymine is a Michael acceptor. The application of standard peptide chemistry allows incorporation of amino acids like cysteine or lysine that allow post-synthetic modifications of the oligomer like attachment of fluorophores. Lysine also enhances the solubility of the PNA oligomer.7

Structural Properties

PNA is an oligonucleotide analogue consisting of an uncharged, achiral peptide backbone and a nucleic acid attached via an acetyl linker to the backbone. PNA exhibits a higher binding affinity towards DNA and RNA than oligonucleotides. Furthermore, PNA tightly binds to complementary PNA oligomers. In all these cases PNA shows a high sequence selectivity. The thermal stabilities for identical sequences follow the order PNA-PNA > PNA-RNA > PNA-DNA.9,10 The binding properties are fairly independent of the salt concentration.1,9,11 Furthermore, PNA oligomers are capable of displacing one strand of a DNA double-helix. These properties make PNA a very potent DNA-mimic and promise applications as a tool in the diagnostics and pharmaceutical fields.

The structure of PNA-DNA and PNA-RNA duplexes has been investigated by NMR techniques revealing that PNA-DNA duplexes resemble a B-form whereas PNA-RNA duplexes adopt an A-form.12 PNA-PNA duplexes form both left- and right-handed helices that are wide (28 Å) and, large pitched (18 bp), with the base pairs perpendicular to the helix axis.13

Homopyrimidine PNAs form PNA-DNA-PNA triplexes with dsDNA by displacing one DNA strand. The nucleobase interactions follow Watson-Crick and Hoogsteen base pairing rules.14 This finding has led to the development of bis-PNAs consisting of a strand anti-parallel to the target DNA for Watson-Crick recognition and a second covalently linked strand which is designed to bind the target DNA by Hoogsteen base pairing. These bis-PNAs show a higher mismatch discrimination due to the two-fold recognition process.15

PNA as an Antisense and Antigene Drug

Despite its name, PNA is neither a nucleic acid nor a peptide. As a result, PNA is stable against enzymatic degradation by both proteases and nucleases. In combination with the high DNA- and RNA-binding affinity, PNA's increased biostability has stimulated research into its potential as an antisense or antigene drug. It is able to inhibit transcription by blocking RNA polymerase as demonstrated for a T8-octamer PNA by Nielsen and co-workers.16 In contrast to other antisense agents like phosphorothioates, PNA does not induce RNase H-mediated cleavage of the RNA strand but acts via steric blocking of either RNA processing, transport into cytoplasm, or translation.17,18,19,20 Besides PNA, PNA-DNA-chimeras have been investigated as potential antisense agents. These chimeras can stimulate RNase H as well as RNase L activity and are therefore more effective antisense agents than pure PNA.21 PNA-DNA chimeras can also act as substrates for other oligonucleotide related enzymes like DNA-polymerases and reverse transcriptases.22 Both PNA and PNA-DNA-chimeras suffer from low cellular uptake. Several attempts to circumvent this problem have been made. Cationic liposomes have been used as carriers to deliver PNAs to the cytoplasm.23 Attaching antibodies to PNA also improves their ability to enter cells.24 Another way of enhancing cellular uptake is the coupling to a delivery peptide that accelerates cellular uptake significantly.25 Other peptide sequences, including a 35 residue peptide from HIV Tat, appear to have the same properties.26

PNA as a Tool in Molecular Biology

The chemical and biological stability as well as the sequence-specific binding properties of PNA has allowed for the development of promising molecular-biological tools. For example, fluorescently labelled PNA probes are valuable tools in FISH (fluorescence in situ hybridization) assays.27,28,29,30 PNAs have been used as capture probes for nucleic acid purification.31,32,33 The high-affinity DNA-binding has been advantageously employed as a means of tagging plasmid vectors with PNA-conjugates.34,35 PNA has been demonstrated to simplify Southern hybridization by enabling pre-gel hybridizations to be performed.36 Hybridization with PNA can protect DNA from enzymatic methylation thereby rendering the unmethylated DNA susceptible to restriction digestion (PNA-assisted rare cleavage).37 The binding of PNA to a specific nucleic acid sequence inhibits PCR-amplification (PCR clamping).38,39 This technique has been used to enhance the amplification of single base-pair gene variants by supressing the amplification of the wild-type allele.

PNA as a Tool in DNA Diagnostics

The favorable base-pairing properties of PNA-oligomers have fueled research aimed at the development of new diagnostic probes for the detection of specific nucleic acid sequences. The fabrication and usage of PNA-probe arrays enabled a hybridization-based DNA screening.40 For achieving a homogeneous DNA-detection, a linear PNA-oligomer was equipped with fluorescence donor and a fluorescence quencher yielding PNA-conjugates that begin to fluoresce upon hybridization.41 A real-time detection of DNA is also possible by appending intercalator dyes such as thiazol orange.42 It has been shown that the replacement of a nucleobase by thiazol orange can allow for the detection of single base mutations.43 PNA exhibits an unparalleled ability of MALDI-TOF/MS analysis in terms of molecular weight resolution and PNA-based MALDI-TOF genotyping has been used for detecting single nucleotide polymorphisms (SNP).44,45,46 Several point mutations were detected in a single assay (multiplex analysis) by using mass-spectrometrical monitoring of a PNA-based ligation reaction.47 Biosensors can provide an alternative means for the label-free detection of nucleic acid hybridizations.48 PNAs bound to a Biacore chip were shown to enable the differentiation of single base mutations in the DNA-analyte.49 A quartz crystal microbalance has been used for monitoring the weight increase that is observed when DNA-analytes bind to PNA-monolayer on a gold surface.50

Related Reagents.

{[2-(6-benzyloxycarbonylaminopurin-9-yl)acetyl]-(2-tert-butoxycarbonylaminoethyl)amino}acetic acid [Boc-PNA-A(Z) monomer]; {[2-(2-benzyloxycarbonylamino-6-oxo-1,6-dihydropurin-9-yl)acetyl]-(2-tert-butoxycarbonylaminoethyl)amino}acetic acid [Boc-PNA-G(Z) monomer]; {[2-(4-benzyloxycarbonylamino-2-oxo-2H-pyrimidin-1-yl)acetyl]-(2-tert-butoxycarbonylaminoethyl)amino}acetic acid [Boc-PNA-C(Z) monomer]; {[2-(9H-fluoren-9-ylmethoxycarbonylamino)ethyl]-[2-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)ethyl]amino}acetic acid [Fmoc-PNA-T monomer]; {[2-(6-benzhydryloxycarbonylamino-purin-9-yl)-ethyl]-[2-(9 H-fluoren-9-ylmethoxycarbonylamino)ethyl]amino}acetic acid [Fmoc-PNA-A(Bhoc) monomer]; {[2-(2-benzhydryloxycarbonylamino-6-oxo-1,6-dihydro-purin-9-yl)-ethyl]-[2-(9H-fluoren-9-yl-methoxycarbonylamino)ethyl]amino}acetic acid [Fmoc-PNA-G(Bhoc) monomer]; {[2-(4-benzhydryloxycarbonylamino-2-oxo-2H-pyrimidin-1-yl)ethyl]-[2-(9H-fluoren-9-yl-methoxycarbonylamino)ethyl]amino}acetic acid [Fmoc-PNA-C(Bhoc) monomer].


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Florian Münstermann & Oliver Seitz

Max-Planck-Institut für Molekulare Physiologie and Universität Dortmund, Germany



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