Design, Synthesis, and Analysis of Minor Groove Binder Pyrrolepolyamide-2′-Deoxyguanosine Hybrids

Pyrrolepolyamide-2′-deoxyguanosine hybrids (Hybrid 2 and Hybrid 3) incorporating the 3-aminopropionyl or 3-aminopropyl linker were designed and synthesized on the basis of previously reported results of a pyrrolepolyamide-adenosine hybrid (Hybrid 1). Evaluation of the DNA binding sequence selectivity of pyrrolepolyamide-2′-deoxyguanosine hybrids was performed by CD spectral and T m analyses. It was shown that Hybrid 3 possessed greater binding specificity than distamycin A, Hybrid 1 and Hybrid 2.


Introduction
Organic compounds capable of controlling gene expression may potentially be used as viable gene therapy agents. In this regard, one of the most important requirements the drug must possess is the ability to selectively distinguish target sequences from all other sequences within the genome. The development of antisense or antigene drugs comprising synthetic oligonucleotides has been investigated [1]. Furthermore, it was reported that pyrrolepolyamide molecules such as distamycin A (Dst) and netropsin (Net), which bind to the minor groove of DNA, can interfere with gene expression through sequence-specific recognition of DNA [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] (Figure 1). Baraldi et al. reported on the design, synthesis, and biological activity of hybrid compounds comprising a combination of Dst and uramustine (uracil mustard) [14]. Uramustine interacts with GC-rich regions and can alkylate guanine-N7. The hybrid compounds exhibited enhanced antitumor activity against the K562 Human Leukemia Cell Line compared to both distamycin A and uramustine derivatives. In this case, the interaction with DNA tends to be dominated by minor groove binding of the Dst moiety.
Given these findings, we expected that nucleosides bearing a minor groove binder (MGB) such as a pyrrolepolyamide, which possesses high affinity for the AAATT sequence, might be able to regulate gene expression. When the MGB polyamide-nucleoside hybrid acts on dsDNA, it is expected that complex formation would involve high affinity and sequence selectivity through minor groove-binding of the polyamide moiety and interactions between dsDNA and the nucleoside moiety of the hybrid such as those involving Hoogsteen-type hydrogen bonding. On the other hand, if assuming the hybrid is incorporated into DNA during DNA biosynthesis, it is expected that DNA replication and transcription would be obstructed through minor groovebinding of the hybrid polyamide moiety, incorporated near the target sequence of the DNA. Therefore, it is preferable that the pyrrolepolyamide be combined with the nucleoside site located on the minor groove side of a DNA duplex, for example, the 2 -position of the sugar moiety and the 2-exocyclic amino group of the guanine base. With this in mind, we previously designed and synthesized Hybrid 1, where the pyrrolepolyamide is linked at the 2 hydroxyl group of adenosine, as a lead compound for potential use as a new gene therapy agent [19,20]. Evaluation of the DNAbinding activity of Hybrid 1 by circular dichroism (CD) spectral and T m value analyses showed that although the ΔT m value (ca. 2 • C, Table 1) was small, Hybrid 1 displayed greater binding specificity than Dst and Net.
On the basis of these results, and for the purpose of developing a gene control agent through interaction with the DNA duplex and/or incorporation into DNA during DNA biosynthesis, we designed MGB polyamide-2 -deoxynucleoside hybrids, such as Hybrid 2 and Hybrid 3 combined with a pyrrolepolyamide using a linker at the 2-exocyclic amino group of the guanine base ( Figure 1). When considering a DNA strand with incorporated hybrid during DNA biosynthesis, it is expected that a short linker might result in high stabilization of the dsDNA. Therefore, we designed Hybrid 2 containing a 3-aminopropionyl linker used to the head-to-tail hairpin polyamide reported by Mrksich et al. [21]. Moreover, for investigations concerning the stabilization of dsDNA with the complementary strand, we designed Hybrid 3 containing an alkyl linker connected Journal of Nucleic Acids 3 directly at the 2-exocyclic amino group of the guanine base. Here we report on the synthesis and interaction of these hybrids on DNA duplexes.

UV Absorption Spectroscopy.
In an effort to assess the effect of the bound ligand (Hybrid 1, Hybrid 2, Hybrid 3, or Dst) on the thermal stability of the three duplexes (DNA 1-3), UV melting experiments were conducted in the absence and presence of each ligand. Absorbance versus temperature profiles were measured using a Shimazu TMSPC-8/UV-1600 spectrophotometer equipped with a thermoelectrically controlled cell holder at 260 nm and a heating rate of 1.0 • C/min. The concentration of each 11mer duplex was 4.3 μM, while the ligand concentrations varied from 0 to 12.9 μM.
We initially attempted the synthesis of the pyrrolepolyamide-2 -deoxyguanosine hybrid (Hybrid 2) by condensation of 3 ,5 -O-TIPDS-guanosine (12) and pyrrole amide trimer 11, prepared by coupling pyrrole amide trimer 9 with ethyl β-alanine, alkaline hydrolysis of the ester moiety, deprotection of the Boc group, and finally N-formylation. The desired compound was not obtained when using EDCI/DMAP or dicyclohexylcarbodiimide (DCC)/hydroxybenztriazole (HOBT) as condensation reagents due to the low reactivity of the exocyclic amino group of the guanine base. We then attempted the synthesis of Hybrid 2 using pyrrole amide trimer 10 and    sequence, were used ( Figure 2 and Table 1). Moreover, experiments employing Dst and compound 11 were conducted for comparison. Typical CD spectra of the uncomplexed DNA duplexes (DNA 1-3), indicative of DNA in the B-form conformation, are shown in Figure 2 (ligand = 0 μM). The addition of Hybrid 2, Hybrid 3, or Dst resulted in marked changes in the ellipticity θ, unlike the case with compound 11. In particular, an induced Cotton effect of bound ligand is indicated by the presence of an extra CD band centered at ∼325 nm for Hybrid 2 and Hybrid 3, and ∼333 nm for  Dst. Hybrid 3 showed selective binding affinity only with DNA 1, which contains the AAATT sequence (Figure 2), and stabilization of the DNA duplex (Table 1). On the other hand, Hybrid 2, combined with a pyrrolepolyamide at the 2exocyclic amino group of the guanine base through an amide bond using a 3-aminopropionyl linker, and Dst containing the cationic amidine edge which contributes to high DNA binding affinity for ligand binding [30][31][32][33], did not show any marked differences in sequence-specific binding affinity with the tested DNA.

Conclusions
We synthesized and evaluated pyrrolepolyamide-2deoxyguanosine hybrids (Hybrid 2 and Hybrid 3) incorporating 3-aminopropionyl or 3-aminopropyl linkers. Hybrid 3, combined with a pyrrolepolyamide at the 2exocyclic amino group of the guanine base through an N-alkyl bond using a 3-aminopropyl linker, showed high recognition of DNA containing the pyrrolepolyamide binding sequence. These results suggest that improvements in stability might be effected by varying the length of the linker. On the other hand, in a DNA strand with incorporated hybrid during DNA biosynthesis, the presence of a short linker, such as the 3-aminopropionyl moiety, might result in high stabilization of the dsDNA. These studies are currently underway and will be reported elsewhere. The minor groove binder pyrrolepolyamide-2 -deoxyguanosine hybrid presented in this report might potentially be of use as a sequence-specific gene therapy agent and could provide the basis for the development of a new series of antisense or antigene drugs.