## 1 Introduction  
Much attention has been paid to the design of fluorescent probes capable of selective binding to specific structures in DNAs and RNAs [1; 2]. This class of fluorescent probes has been designed to bind abasic (apyrimidinic or apurinic; AP) sites [3; 4; 5; 6; 7; 8], bulges [9; 10], mismatched sites [11; 12] and overhanging structures [13; 14; 15], by which the biological functions of these noncanonical base-pairs have been examined. Moreover, these fluorescent probes have great potential to be applied for the analysis of target DNAs and RNAs based on their binding-induced fluorescence signaling. These probes also serve as the affinity-labeling agents in the label-free assays for detecting various analytes [16; 17; 18]. We developed AP site-binding ligands (APLs) that are conjugated with thiazole orange (TO) for the fluorescence sensing of orphan nucleobases in DNA/DNA duplexes (Figure 1A) [19; 20]. APLs can form the pseudo-base pairing with target orphan nucleobases, which allows the selective binding to the target nucleobase opposite the AP site [3; 4; 5; 6; 7]. On the other hand, TO unit connected with the APL unit through an appropriate linker can function as a fluorescent intercalator [21; 22]. The TO unit alone shows negligible fluorescence, but its fluorescence is greatly enhanced upon intercalation into the base pairs near the AP site. APL-TO conjugates thus enable the light-up sensing of target orphan nucleobase in the AP site-containing DNA (AP-DNA) duplexes. Such light-up probes are useful for a more sensitive analysis compared to fluorescence quenching probes. We succeeded in the detection of single base mutation in target DNA sequences based on the binding and light-up functions of APL-TO conjugates in combination with an AP-DNA probe (cf. Figure 1A). In addition, these conjugates were applicable to the sequence-selective analysis of microRNAs, a class of small non-coding RNAs associated with various diseases and cancers [23], by the selective binding of conjugates to target orphan nucleobases in the AP site-containing DNA/RNA (AP-DNA/RNA) hybrids. The properties of nucleobase-selectivity and fluorescence emission wavelength are tunable by adopting suitable APLs and cyanine dyes, respectively, which enables the development of multiplex analysis of target sequences by the simultaneous use of these conjugates in a single solution [19]. In this context, 2-amino-5,6,7-trimethyl-1,8-naphthpyridine (ATMND, Figure 1B) was previously used as an APL unit in the conjugate with TO unit for the light-up sensing of target orphan C in the AP-DNA and AP-DNA/RNA hybrid duplexes [19]. The binding selectivity of ATMND toward target C can be rationalized by the complementary base pairing of the N1-protonated form of ATMND with target C through hydrogen bonding (Figure 1B) [3]. However, ATMND inherently has only moderate selectivity for C over T because of the possible protonation at N8 that allows the recognition of T (Figure 1B). This should limit the application of the conjugate for the sequence analysis of target DNAs and microRNAs such as the discrimination of C against T or U. This work describes the enhanced binding selectivity of a 2-amino-1,8-naphthpyridine derivative for orphan C by the incorporation of a chloro group. We previously found that the introduction of the electron-withdrawing trifluoromethyl group into 2-amino-7-methyl-1,8-naphthrydine (AMND) led to remarkably enhanced C/T selectivity due to more favorable protonation at N1 compared to N8 [24]. However, the obtained CF\({}_{3}\)-AMND (Figure 1B) showed much weaker affinity compared to ATMND. Herein, 2-amino-5,7-dimethyl-1,8-naphthryidine (ADMND), an AMND derivative with an additional methyl group, was explored as the scaffold for the introduction of an electron-withdrawing group considering the fact that ADMND can bind more strongly to target C than AMND [3].