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  • br Introduction In addition to

    2021-04-28


    Introduction In addition to the canonical double stranded structure, DNA can form various higher order structures such as bulges, and various kinds of mismatches, triplexes, to the G-quadruplex (G4). Over the past decades, accumulating evidence has begun to emerge that these non-canonical structures have important functional roles in genetic regulation and are correlated to many diseases.2, 3 For instance, the T-T mismatch formation from expandable repeat CTG sequences is implicated in human neurological diseases such as the myotonic dystrophy type 1 (DM1). Among the discovered structures, the G4 structure has gained significant attentions for its emerging role in key processes such as replication, transcription and telomere maintenance.5, 6, 7 Due to its potential as a therapeutic target, G4 has been the object of intense studies, and a significant number of various small molecules has been reported as G4 binding ligands with encouraging results both in vitro and in vivo5, 8, 9 While the search for more promising G4 targeting ligands continues to be an active field for drug discovery, the chemical reaction, in particular covalent modification, has been suggested as one of the effective strategies to augment the stabilizing effect of small molecules to G4. Several ligands containing chemically-reactive moieties, such as quinone methides10, 11, oxirane, chlorambucil, and platinum complexes, were developed as G4 alkylating agents. In addition, several G4 photo-cross-linking probes, such as benzophenone and the ruthenium complex, have also been reported. Our group has exploited the vinyl chemistry to achieve covalent modification to higher-order nucleic Kartogenin structures in a highly selective, mild, and readily initiated manner. Recently, we reported the vinyldiaminotriazine (VDAT)-acridine conjugate probe (1) as an efficient alkylating agent to the T-T mismatch DNA (Fig. 1(A)). The design of the probe (1) was based on the triaminotriazine (TAT)-acridine compound (2), which was developed by Zimmerman’s group, as a binding molecule to stabilize the T-T or U-U complex by the acridine intercalation effect and hydrogen bonds (Fig. 1(A)). We found that our conjugate probe (1) was efficiently and selectively alkylated with thymine bases at the mismatch site. Along with this previous finding, we have also reported that 2-amino-vinylpurine (AVP) conjugated with acridine (3) (Fig. 1(B)) exhibited a potent alkylation reactivity toward thymine bases of the human telomere G4. In this structure of 3, we expected that the acridine moiety interacts with the G4 plane through a π-π stacking interaction, allowing the reactive moiety AVP to reach the loop of the G4 to react with the thymine bases. This finding has ultimately fueled our interest to attempt the G4 alkylation using the VDAT-acridine conjugates. In addition to the VDAT-acridine conjugate probe (1) developed in a previous study, we designed two new VDAT-acridine conjugate probes (4 and 5) (Fig. 1(C)). These probes had the modified acridine with different cationic side chains in order to increase the water solubility as well as to investigate the effect of the side chain on the G4 alkylation selectivity and reactivity.
    Results and discussion
    Conclusion This study highlighted the unexpected finding of the VDAT-acridine conjugate as a new G4 alkylating agent. The lead compound, the VDAT-acridine conjugate (1), which was originally designed for targeting the T-T mismatch structure, exhibited a high reactivity with topology selectivity toward the human telomere G-4 DNA in K+ buffer in addition to the T-T mismatch. Two other probes (4 and 5), with their modified acridine with cationic side chains, showed rather lower reactivities but better selectivities for G-4 over the T-T mismatch than that of 1. The binding affinity with the VDAT-acridine conjugate (1) to G-4 in K+ was slightly stronger than with the T-T mismatch. We have also shown that alkylation with 1 effectively stabilized the G-4 in K+ without any major disturbance or alteration of the structure conformation. Additionally, we have determined that the alkylation site of 1 is thymine at either the top or side loop of the G-4 by the primer extension method. In addition, the HPLC analysis of the hydrolyzed products of the G-4 alkylated DNA suggested that the alkylation proceeded at the N3 position of the thymine. Based on the findings, we propose that the acridine moiety would interact with G4, followed by the nucleophilic attack of the thymine on the vinyl group by a proximity effect as the plausible reaction mechanism.