Background

[PubMed]

Polyamides (PAM) constructed from N-methylpyrrole (Py), N-methylimidazole (Im), 3-chlorothiophene (Ct), and N-methylhydroxypyrrole (Hp) amino acids comprise a class of synthetic oligomeric ligands that bind to the minor groove of DNA (1, 2). The aromatic heterocycles in the PAM orientate antiparallel with respect to the Watson-Crick base pair (bp), which leads to a specific recognition of DNA sequences (3). The recognition process follows a series of pairing rules; i.e., an ImPy specifies for G·C, a PyPy binds both A·T and T·A, an HpPy discriminates T·A over A·T, and a CtPy prefers T·A over A·T at the N-terminus. These aromatic amino acids can be programmed to a strand with more than two residues to recognize longer DNA sequences; for example, an ImPyPy motif specifies for the five-bp sequence 5’-WGWCW-3’ (W=A, T) instead of 5’-WGWWW-3’ (4). More complicated PAM motifs can be designed by adding small molecules such as β-alanine or γ-aminobutyric acid to covalently link between two antiparallel PAM strands, yielding substantial increases in affinities and specificities. For instance, an eight-ring hairpin motif, which has a γ-aminobutyric acid (γ-turn) linker to connect the carboxylic terminus of one polyamide to the amino terminus of another, exhibits ~100-fold higher affinity for binding a six-bp DNA sequence compared to the unlinked homodimers (4). PAM are molecules that can permeate cell membranes and have been used in targeting a variety of DNA sequences in cell culture (5). The binding of PAM replaces the DNA-binding proteins and thus regulates the transcription of selected genes. The use of radiolabeled PAM aims at imaging gene regulations in vivo.

Fluorine-18 [¹⁸F], with a half-life of 109.7 min and low β⁺-energy (0.64 MeV), represents the ideal radionuclide for position emission tomography (PET). The ¹⁸F-produced positron is annihilated with an electron, leading to the emission of two 511-keV photons ~180º apart, which is detected coincidentally with PET. Various peptides have been successively fluorinated with multistep ¹⁸F-acylation, using ¹⁸F-labeled prosthetic groups such as amino-reactive ¹⁸F-labeling agent N-succinimidyl 4-[¹⁸F]fluorobenzoate (6). To increase labeling efficiency, the fluorination also can be conducted via a two-step synthetic approach in which an oxime is formed between an aminooxy group in the peptide and an ¹⁸F-labeled aldehyde such as 4-[¹⁸F]fluorobenzaldehyde (6). ImPyβImPyβImβ-C₃-¹⁸F ([¹⁸F]PIPAM5) is an ¹⁸F-labeled PAM used for PET that is obtained with the oxime ligation approach (5). [¹⁸F]PIPAM5 contains five aromatic amino acids connected with β-alanine, which is denoted as β and is also known as a five-ring β-linked motif. Its unlabeled form with an N-N-dimethylaminopropyl tail has been exhibit ability to upregulate the repressed gene frataxin in a cell culture mode of Friedreich’s ataxia (7).

Synthesis

[PubMed]

Harki et al. reported the synthesis of [¹⁸F]PIPAM5 (5). Initially, 4-[¹⁸F]-fluorobenzaldehyde was obtained by nucleophilic fluorination of a trimethylammonium benzaldehyde derivative with cyclotron-produced [¹⁸F]fluoride in the presence of 5,6-benzo-4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacos-5-ene (Kryptofix[2.2.2]). The β-linked PAM ImPyβImPyβImβ was synthesized on a Boc-β-alanine phenylacetamidomethyl resin according to standard protocols. Then the PAM was hydroxylamine-functionalized in DMF by reaction with tert-butyl-3-aminopropoxycarbamate in the presence of benzotriazolyloxy-tris-(pyrrolidino)-phosphonium hexafluorophosphate (PyBOP) and N,N,-diisopropylethylamine. Finally, the obtained ImPyβImPyβImβ-hydroxylamine was ligated with the 4-[¹⁸F]-fluorobenzaldehyde with aniline as a catalyst to produce [¹⁸F]PIPAM5 at radiochemical yield of 12%. The whole synthetic procedure was completed in 100 min after the end of bombardment.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Harki et al. used the cold PAM analog [¹⁹F]PIPAM5 to evaluate its affinity to DNA in vitro (5). Quantitative DNaseI footprint titrations were performed on the 5’-³²P-polymerase chain reaction fragment from plasmid pJWP-16. In this method, equilibrium mixtures of ³²P end-labeled DNA and a range of PAM concentrations were partially digested by DNase I followed by gel electrophoresis and autoradiography. The PAM bound DNA was protected from cleavage, which produced a band gap on the gel. Quantification of the binding fraction as a function of PAM concentration was used to the apparent association constant, 3.5 ± 2.1 × 10⁹ M^-1 for [¹⁹F]PIPAM5.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

References

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Nickols N.G. , Jacobs C.S. , Farkas M.E. , Dervan P.B. Improved nuclear localization of DNA-binding polyamides. Nucleic Acids Res. 2007; 35 (2):363–70. [PMC free article: PMC1802595] [PubMed: 17175539]

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Belitsky J.M. , Nguyen D.H. , Wurtz N.R. , Dervan P.B. Solid-phase synthesis of DNA binding polyamides on oxime resin. Bioorg Med Chem. 2002; 10 (8):2767–74. [PubMed: 12057666]

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Dervan P.B. , Edelson B.S. Recognition of the DNA minor groove by pyrrole-imidazole polyamides. Curr Opin Struct Biol. 2003; 13 (3):284–99. [PubMed: 12831879]

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Harki D.A. , Satyamurthy N. , Stout D.B. , Phelps M.E. , Dervan P.B. In vivo imaging of pyrrole-imidazole polyamides with positron emission tomography. Proc Natl Acad Sci U S A. 2008; 105 (35):13039–44. [PMC free article: PMC2529060] [PubMed: 18753620]

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Poethko T. , Schottelius M. , Thumshirn G. , Hersel U. , Herz M. , Henriksen G. , Kessler H. , Schwaiger M. , Wester H.J. Two-step methodology for high-yield routine radiohalogenation of peptides: (18)F-labeled RGD and octreotide analogs. J Nucl Med. 2004; 45 (5):892–902. [PubMed: 15136641]

7.

Burnett R. , Melander C. , Puckett J.W. , Son L.S. , Wells R.D. , Dervan P.B. , Gottesfeld J.M. DNA sequence-specific polyamides alleviate transcription inhibition associated with long GAA.TTC repeats in Friedreich's ataxia. Proc Natl Acad Sci U S A. 2006; 103 (31):11497–502. [PMC free article: PMC1544198] [PubMed: 16857735]