Synthesis of 6-Alkynylated Purine-Containing DNA via On-Column Sonogashira Coupling and Investigation of Their Base-Pairing Properties

Synthetic unnatural base pairs have been proven to be attractive tools for the development of DNA-based biotechnology. Our group has very recently reported on alkynylated purine–pyridazine pairs, which exhibit selective and stable base-pairing via hydrogen bond formation between pseudo-nucleobases in the major groove of duplex DNA. In this study, we attempted to develop an on-column synthesis methodology of oligodeoxynucleotides (ODNs) containing alkynylated purine derivatives to systematically explore the relationship between the structure and the corresponding base-pairing ability. Through Sonogashira coupling of the ethynyl pseudo-nucleobases and CPG-bound ODNs containing 6-iodopurine, we have demonstrated the synthesis of the ODNs containing three NPu derivatives (NPu1, NPu2, NPu3) as well as three OPu derivatives (OPu1, OPu2, OPu3). The base-pairing properties of each alkynylated purine derivative revealed that the structures of pseudo-nucleobases influence the base pair stability and selectivity. Notably, we found that OPu1 bearing 2-pyrimidinone exhibits higher stability to the complementary NPz than the original OPu, thereby demonstrating the potential of the on-column strategy for convenient screening of the alkynylated purine derivatives with superior pairing ability.


Introduction
DNA is an essential biopolymer that plays a pivotal role in gene expression through replication and transferring genetic information. The functional basis of DNA lies in the formation of base pairs between A and T as well as G and C through the complementary hydrogen bonds, respectively. The selectivity of the base-pairing process, in combination with the facile programmability due to the countless permutations of base pair sequences, have made DNA an attractive platform for numerous applications in biotechnology [1,2] and nanotechnology [3][4][5], as well as drug discovery studies [6,7]. DNA-based technologies have immense potential, and efforts to harness them in a wide range of applications have resulted in the development of unnatural base pairs (UBPs) being extensively studied for the past several decades [8][9][10][11].
In our effort to expand the design repertoire of UBP, we have recently reported a new type of UBP, which include the base pairs N Pu-O Pz and O Pu-N Pz, consisting of alkynylated purine and pyridazine as base surrogates [31]. Our unnatural nucleobases are composed of three functional units: (1) purine and pyridazine core structures for maintaining the stacking interaction within the double-helix, (2) nucleobase-like heteroaromatics ("pseudo-nucleobases") as recognition units, and (3) alkyne spacers that dislocate the pseudo-nucleobases from the Watson-Crick interface (Figure 1a). In a case where 2-aminopyirimidine and 2-pyridone were adopted as the pseudo-nucleobases, these unnatural bases exhibit stable and selective base-pairing in duplex DNAs only when the combinations of the pseudo-nucleobases allow appropriate inter-base hydrogen bonding ( Figure  1b). While assessing the base-pairing properties of our alkynylated purine-pyridazine base pairs, we noticed that N Pu-O Pz and O Pu-N Pz exhibited somewhat different base-pairing stability. This suggested that the thermal stability of the alkynylated purine-pyridazine base pairs might depend on the structure of pseudo-nucleobases and that mechanistic studies may facilitate efforts to improve the base-pairing properties; however, oneby-one preparation of the corresponding phosphoramidites for solid-phase DNA synthesis imposes significant inefficiency in terms of investigating various alkynylated nucleosides. We therefore sought to explore the post-synthetic construction of the alkynylated nucleoside derivatives on DNA to facilitate the systematic and efficient preparation of the oligodeoxynucleotides (ODNs) containing the UBPs being studied. The post-synthesis modification of nucleic acids is a chemical approach in which the nucleoside analogs bearing reactive handles are first incorporated into ODNs and subsequently converted into the desired nucleoside analogs via conjugation chemistry. Various conversion chemistries, such as substitution reactions [32][33][34][35][36][37][38], click reactions [39,40], amide bond formation [41], oxime or hydrazone formation [42], as well as metal-catalyzed cross-coupling reactions [43][44][45][46][47][48][49][50][51][52][53][54][55], have been employed for the post-synthesis modification of canonical nucleosides. In addition, several unnatural nucleoside analogs have also been synthesized via the post-synthesis approach [56][57][58][59]. Hence, we circumvented the complicated and timeconsuming synthesis of the modified phosphoramidites by using a post-synthesis approach to investigate the relation between the structure and the base-pairing properties of While assessing the base-pairing properties of our alkynylated purine-pyridazine base pairs, we noticed that N Pu-O Pz and O Pu-N Pz exhibited somewhat different base-pairing stability. This suggested that the thermal stability of the alkynylated purine-pyridazine base pairs might depend on the structure of pseudo-nucleobases and that mechanistic studies may facilitate efforts to improve the base-pairing properties; however, one-by-one preparation of the corresponding phosphoramidites for solid-phase DNA synthesis imposes significant inefficiency in terms of investigating various alkynylated nucleosides. We therefore sought to explore the post-synthetic construction of the alkynylated nucleoside derivatives on DNA to facilitate the systematic and efficient preparation of the oligodeoxynucleotides (ODNs) containing the UBPs being studied. The post-synthesis modification of nucleic acids is a chemical approach in which the nucleoside analogs bearing reactive handles are first incorporated into ODNs and subsequently converted into the desired nucleoside analogs via conjugation chemistry. Various conversion chemistries, such as substitution reactions [32][33][34][35][36][37][38], click reactions [39,40], amide bond formation [41], oxime or hydrazone formation [42], as well as metal-catalyzed cross-coupling reactions [43][44][45][46][47][48][49][50][51][52][53][54][55], have been employed for the post-synthesis modification of canonical nucleosides. In addition, several unnatural nucleoside analogs have also been synthesized via the post-synthesis approach [56][57][58][59]. Hence, we circumvented the complicated and time-consuming synthesis of the modified phosphoramidites by using a post-synthesis approach to investigate the relation between the structure and the base-pairing properties of alkynylated nucleobases.
Herein, we describe the post-synthesis approach that allows the preparation of ODNs containing various alkynylated purine derivatives (Figure 2a). Specifically, we demon-strated the synthesis of ODNs containing three N Pu derivatives ( N Pu1, N Pu2, N Pu3) as well as three O Pu derivatives by subjecting the controlled pore glass (CPG)-bound ODNs containing 6-iodopurine ( I Pu) to Sonogashira coupling ( O Pu1, O Pu2, O Pu3) (Figure 2b). The base-pairing properties of each alkynylated purine derivative were investigated by UV melting temperature measurements, revealing that the structures of the pseudo-nucleobases influence the base pair stability and selectivity. Notably, we found that O Pu1 has superior base-pairing properties as compared to the original O Pu, which demonstrates the potential of the on-column strategy for the facile screening of alkynylated purine derivatives with optimal pairing ability.
Herein, we describe the post-synthesis approach that allows the preparation of ODNs containing various alkynylated purine derivatives (Figure 2a). Specifically, we demonstrated the synthesis of ODNs containing three N Pu derivatives ( N Pu1, N Pu2, N Pu3) as well as three O Pu derivatives by subjecting the controlled pore glass (CPG)-bound ODNs containing 6-iodopurine ( I Pu) to Sonogashira coupling ( O Pu1, O Pu2, O Pu3) (Figure 2b). The base-pairing properties of each alkynylated purine derivative were investigated by UV melting temperature measurements, revealing that the structures of the pseudo-nucleobases influence the base pair stability and selectivity. Notably, we found that O Pu1 has superior base-pairing properties as compared to the original O Pu, which demonstrates the potential of the on-column strategy for the facile screening of alkynylated purine derivatives with optimal pairing ability.

On-Column Synthesis of Oligodeoxynucleotides Containing the Alkynylated Purine Derivatives
To demonstrate the feasibility of the on-column synthesis strategy, we planned to synthesize 15-mer ODNs containing O Pu and N Pu derivatives in the middle of the sequences by on-column Sonogashira coupling followed by a solid-phase elongation of the remaining part of the ODNs. In a previous study, we showed that the phosphoramidite building blocks of O Pu and N Pu can be synthesized by coupling I Pu deoxyriboside with ethynyl pseudo-nucleobases by Sonogashira coupling [31]. Thus, considering that CPGbound ODNs are compatible with the reaction conditions of Sonogashira coupling, we reasoned that the post-synthesis approaches using I Pu-containing ODN can be employed for the on-DNA synthesis of the alkynylated purine derivatives. To synthesize the ODNs containing I Pu at its 5′ terminal, the phosphoramidite of I Pu was synthesized as shown in Scheme 1. The 2′-deoxyadenosine 1 was acetylated and subsequently iodinated to afford protected 6-iodopurine 2′-deoxyriboside 3. The acetyl-protected I Pu 3 was then treated with methanolic ammonia to provide fully deprotected I Pu deoxyriboside 4. Finally, protection of the 5′-OH group with the 4,4′-dimethoxytrityl (DMTr) moiety, followed by phosphitylation at the 3′-OH group, afforded the phosphoramidite of I Pu 6. The phospho-

On-Column Synthesis of Oligodeoxynucleotides Containing the Alkynylated Purine Derivatives
To demonstrate the feasibility of the on-column synthesis strategy, we planned to synthesize 15-mer ODNs containing O Pu and N Pu derivatives in the middle of the sequences by on-column Sonogashira coupling followed by a solid-phase elongation of the remaining part of the ODNs. In a previous study, we showed that the phosphoramidite building blocks of O Pu and N Pu can be synthesized by coupling I Pu deoxyriboside with ethynyl pseudo-nucleobases by Sonogashira coupling [31]. Thus, considering that CPG-bound ODNs are compatible with the reaction conditions of Sonogashira coupling, we reasoned that the post-synthesis approaches using I Pu-containing ODN can be employed for the on-DNA synthesis of the alkynylated purine derivatives. To synthesize the ODNs containing I Pu at its 5 terminal, the phosphoramidite of I Pu was synthesized as shown in Scheme 1. The 2 -deoxyadenosine 1 was acetylated and subsequently iodinated to afford protected 6-iodopurine 2 -deoxyriboside 3. The acetyl-protected I Pu 3 was then treated with methanolic ammonia to provide fully deprotected I Pu deoxyriboside 4. Finally, protection of the 5 -OH group with the 4,4 -dimethoxytrityl (DMTr) moiety, followed by phosphitylation at the 3 -OH group, afforded the phosphoramidite of I Pu 6. The phosphoramidite 6 was then incorporated into ODN1 and ODN2 using an automated DNA synthesizer in a DMT-ON mode. At the same time, the corresponding ethynyl pseudo-nucleobases were prepared according to Schemes 2 and 3. The pseudo-nucleobase units for N Pu derivatives were synthesized by coupling halogenated aminoheteroaromatics with trimethylsilylacetylene (TMS-acetylene), followed by desilylation. Ethynyl pseudo-nucleobases for the O Pu derivatives were prepared similarly to TMS-ethyl-protected precursors.  At the same time, the corresponding ethynyl pseudo-nucleobases were prepared according to Schemes 2 and 3. The pseudo-nucleobase units for N Pu derivatives were synthesized by coupling halogenated aminoheteroaromatics with trimethylsilylacetylene (TMS-acetylene), followed by desilylation. Ethynyl pseudo-nucleobases for the O Pu derivatives were prepared similarly to TMS-ethyl-protected precursors. At the same time, the corresponding ethynyl pseudo-nucleobases were prepared cording to Schemes 2 and 3. The pseudo-nucleobase units for N Pu derivatives were s thesized by coupling halogenated aminoheteroaromatics with trimethylsilylacetyle (TMS-acetylene), followed by desilylation. Ethynyl pseudo-nucleobases for the O Pu der atives were prepared similarly to TMS-ethyl-protected precursors.  Having synthesized the CPG-bound ODNs containing I Pu at their 5′ terminal as well as ethynyl pseudo-nucleobases, we attempted the on-column synthesis of ODNs containing alkynylated purine derivatives. Initially, we tested whether on-column Sonogashira coupling could proceed with I Pu-containing ODN through the on-column construction of N Pu1 using CPG-bound ODN1 and the ethynyl compound 9 (Figure 3a). The coupling was performed by treating CPG-bound ODN1 with a DMF solution containing excess equivalents of alkyne 9, Pd(PPh3)4, copper (I) iodide, and triethylamine inside a column at ambient temperature. This reaction was conducted twice to confirm the coupling reaction. To check if the CPG-bound ODN had undergone coupling, an aliquot of the CPGs was subjected to deprotection by 28% NH4OH treatment at room temperature for 2 h, followed by treatment with 10% AcOH for 1 h, after which, the crude ODN was analyzed by RP-HPLC ( Figure 3b). The HPLC chart showed a major peak at 13 min, which was analyzed by MALDI-TOF MS (see Supplementary Materials) to confirm the formation of the desired ODN1 with N Pu1. After confirming the progress of the Sonogashira coupling reaction, the column was reattached to the DNA synthesizer for elongating the remaining part of the ODN1 in the Having synthesized the CPG-bound ODNs containing I Pu at their 5 terminal as well as ethynyl pseudo-nucleobases, we attempted the on-column synthesis of ODNs containing alkynylated purine derivatives. Initially, we tested whether on-column Sonogashira coupling could proceed with I Pu-containing ODN through the on-column construction of N Pu1 using CPG-bound ODN1 and the ethynyl compound 9 (Figure 3a). The coupling was performed by treating CPG-bound ODN1 with a DMF solution containing excess equivalents of alkyne 9, Pd(PPh 3 ) 4 , copper (I) iodide, and triethylamine inside a column at ambient temperature. This reaction was conducted twice to confirm the coupling reaction. To check if the CPG-bound ODN had undergone coupling, an aliquot of the CPGs was subjected to deprotection by 28% NH 4 OH treatment at room temperature for 2 h, followed by treatment with 10% AcOH for 1 h, after which, the crude ODN was analyzed by RP-HPLC ( Figure 3b). The HPLC chart showed a major peak at 13 min, which was analyzed by MALDI-TOF MS (see Supplementary Materials) to confirm the formation of the desired ODN1 with N Pu1.

Scheme 3. Synthesis of the ethynyl pseudo-nucleobases for O Pu derivatives.
Having synthesized the CPG-bound ODNs containing I Pu at their 5′ terminal as well as ethynyl pseudo-nucleobases, we attempted the on-column synthesis of ODNs containing alkynylated purine derivatives. Initially, we tested whether on-column Sonogashira coupling could proceed with I Pu-containing ODN through the on-column construction of N Pu1 using CPG-bound ODN1 and the ethynyl compound 9 (Figure 3a). The coupling was performed by treating CPG-bound ODN1 with a DMF solution containing excess equivalents of alkyne 9, Pd(PPh3)4, copper (I) iodide, and triethylamine inside a column at ambient temperature. This reaction was conducted twice to confirm the coupling reaction.
To check if the CPG-bound ODN had undergone coupling, an aliquot of the CPGs was subjected to deprotection by 28% NH4OH treatment at room temperature for 2 h, followed by treatment with 10% AcOH for 1 h, after which, the crude ODN was analyzed by RP-HPLC ( Figure 3b). The HPLC chart showed a major peak at 13 min, which was analyzed by MALDI-TOF MS (see Supplementary Materials) to confirm the formation of the desired ODN1 with N Pu1. After confirming the progress of the Sonogashira coupling reaction, the column was reattached to the DNA synthesizer for elongating the remaining part of the ODN1 in the After confirming the progress of the Sonogashira coupling reaction, the column was reattached to the DNA synthesizer for elongating the remaining part of the ODN1 in the DMT-Off mode ( Figure 4a). Before commencing the DNA elongation, the CPGs were treated with an excess amount of capping solution (tert-butylphenoxyacetyl acetic anhydride (Tac 2 O)/imidazole solution). We reasoned that this process would protect the NH 2 group on the pyridine moiety of N Pu1, which may otherwise cause branching of the ODN during DNA synthesis. Finally, the fully elongated ODN3 containing N Pu1 was deprotected with 28% NH 4 OH and analyzed by RP-HPLC ( Figure 4b). The major peak was isolated and analyzed by MALDI-TOF MS (see Supplementary Materials) to confirm the formation of the desired ODN3 containing N Pu1. It should be noted that we also attempted the on-column Sonogashira coupling reaction on the fully elongated 15-mer ODN3 containing I Pu in the middle. Although we could observe the formation of the ODNs containing the alkynylated purine derivatives, there was a non-negligible amount of the I Pu-containing strand remaining. Full conversion may be achieved by the optimization of the reaction conditions; however, we did not make any further attempt in this study.

PEER REVIEW
6 of 17 treated with an excess amount of capping solution (tert-butylphenoxyacetyl acetic anhydride (Tac2O)/imidazole solution). We reasoned that this process would protect the NH2 group on the pyridine moiety of N Pu1, which may otherwise cause branching of the ODN during DNA synthesis. Finally, the fully elongated ODN3 containing N Pu1 was deprotected with 28% NH4OH and analyzed by RP-HPLC ( Figure 4b). The major peak was isolated and analyzed by MALDI-TOF MS (see Supplementary Materials) to confirm the formation of the desired ODN3 containing N Pu1. It should be noted that we also attempted the on-column Sonogashira coupling reaction on the fully elongated 15-mer ODN3 containing I Pu in the middle. Although we could observe the formation of the ODNs containing the alkynylated purine derivatives, there was a non-negligible amount of the I Pu-containing strand remaining. Full conversion may be achieved by the optimization of the reaction conditions; however, we did not make any further attempt in this study. Using the same protocol, we further prepared the ODN3 and ODN4 containing the other N Pu derivatives. The O Pu derivatives were prepared in a similar manner, apart from the deprotection step, in which the CPGs were treated with a ZnBr2 solution to remove the TMS-ethyl moiety on the pseudo-nucleobase prior to the 28% NH4OH treatment. The purity and structural integrity of the synthesized ODNs were confirmed by RP-HPLC ( Figure S1) and MALDI-TOF MS (Table 1) analyses. Taken together, these results demonstrated the utility of the post-synthesis method as a convenient approach for the preparation of ODNs containing alkynylated purine derivatives.  Using the same protocol, we further prepared the ODN3 and ODN4 containing the other N Pu derivatives. The O Pu derivatives were prepared in a similar manner, apart from the deprotection step, in which the CPGs were treated with a ZnBr 2 solution to remove the TMS-ethyl moiety on the pseudo-nucleobase prior to the 28% NH 4 OH treatment. The purity and structural integrity of the synthesized ODNs were confirmed by RP-HPLC ( Figure S1) and MALDI-TOF MS (Table 1) analyses. Taken together, these results demonstrated the utility of the post-synthesis method as a convenient approach for the preparation of ODNs containing alkynylated purine derivatives.

Base-Pairing Properties of the Alkynylated Purine Derivatives
Using the 15-mer ODNs incorporating the alkynylated purine derivatives, we investigated their base-pairing properties. To this end, the ODN3s and the complementary ODN4s incorporating different nucleosides at positions X and Y were annealed, and their thermal stabilities were determined by the UV melting temperature (T m ) measurement (Table 2, Figure S2). In the presence of 10 mM of sodium phosphate (pH 7.0) and 50 mM of NaCl, UV melting of DNA duplexes containing canonical G-C and A-T base pairs at the X-      (Table S1). Furthermore, all four N Pu derivatives exhibited significantly low  Subsequently, we investigated the base-pairing properties of the newly synthesized derivatives in comparison to the previously designed N Pu and O Pu ( Table 2) discriminate between N Pz and O Pz. These results indicated that the structure of the pseudonucleobases has a significant influence on pairing with the complementary bases of the N Pu derivatives. Similar trends were reproduced with the DNA duplexes containing inverted X-Y bases (Table S1). Furthermore, all four N Pu derivatives exhibited significantly low stability toward A, G, C, and T (Tables S2 and S3; T m = 43.5-48.2 • C), suggesting that the structural differences among the pseudo-nucleobases have little impact on the selectivity toward pairing with canonical nucleobases.

X-Y＝
Similarly, we also explored the base-pairing properties of the O Pu derivatives (Table 2). Of note, we found that O Pu1, bearing 2-pyrimidinone as a pseudo-nucleobase, exhibits higher affinity toward the complementary N Pz (T m = 53.2 • C). Its stability was higher than that of N (Table S1), and all three derivatives exhibited selectivity toward pairing with canonical nucleobases (Tables S2 and S3; T m = 43.4-47.9 • C). These results again confirmed that the pseudo-nucleobases play critical roles in the formation of the alkynylated purine-pyridazine pairs and that their structures have an influence on the selectivity and stability of the alkynylated purine-pyridazine base-pairing.

Structural Impact of the Alkynylated Purine Derivatives on DNA Duplex
Finally, to assess the structural impact of the newly designed alkynylated purine derivatives' pairing with the pyridazines, we conducted circular dichroism (CD) spectroscopy measurements of the duplex DNAs. The CD spectra of the fully canonical duplex DNAs indicated the formation of a typical B-type structure, characterized by a positive band at about 270-290 nm and a negative band around 240 nm ( Figure 5). The duplex DNAs containing the N Pu derivatives, pairing with O Pz, as well as O Pu derivatives pairing with N Pz, exhibited similar CD spectra. Hence, these results confirmed that the duplex DNAs incorporating the alkynylated purine-pyridazine pairs do not cause significant structural disturbance of the double-helix structure, thereby demonstrating the robust base-pairing properties of the alkynylated purine and pyridazine derivatives.  (Table S1), and all three derivatives exhibited selectivity toward pairing with canonical nucleobases (Tables S2 and S3; Tm = 43.4-47.9 °C). These results again confirmed that the pseudo-nucleobases play critical roles in the formation of the alkynylated purine-pyridazine pairs and that their structures have an influence on the selectivity and stability of the alkynylated purine-pyridazine base-pairing.

Structural Impact of the Alkynylated Purine Derivatives on DNA Duplex
Finally, to assess the structural impact of the newly designed alkynylated purine derivatives' pairing with the pyridazines, we conducted circular dichroism (CD) spectroscopy measurements of the duplex DNAs. The CD spectra of the fully canonical duplex DNAs indicated the formation of a typical B-type structure, characterized by a positive band at about 270-290 nm and a negative band around 240 nm ( Figure 5). The duplex DNAs containing the N Pu derivatives, pairing with O Pz, as well as O Pu derivatives pairing with N Pz, exhibited similar CD spectra. Hence, these results confirmed that the duplex DNAs incorporating the alkynylated purine-pyridazine pairs do not cause significant structural disturbance of the double-helix structure, thereby demonstrating the robust base-pairing properties of the alkynylated purine and pyridazine derivatives.

Discussion
In this study, we aimed to develop a method to systematically synthesize ODNs incorporating different alkynylated purine derivatives. To this end, the on-column Sonogashira coupling reaction was investigated for the synthesis of the alkynylated purine derivatives from I Pu in ODNs and the corresponding ethynyl compounds. After confir-

Discussion
In this study, we aimed to develop a method to systematically synthesize ODNs incorporating different alkynylated purine derivatives. To this end, the on-column Sonogashira coupling reaction was investigated for the synthesis of the alkynylated purine derivatives from I Pu in ODNs and the corresponding ethynyl compounds. After confirmation of the reaction progress, subsequent elongation of the remaining part of the ODNs by solid-phase DNA synthesis successfully afforded the ODNs incorporating the N Pu and O Pu derivatives, bearing different pseudo-nucleobase moieties. To the best of our knowledge, this study demonstrated, for the first time, the utility of I Pu for on-column Sonogashira coupling with ODNs. Considering that the 6-iodopurine can be utilized as a substrate not only for Sonogashira coupling but also for other cross-coupling reactions, we believe that the present results would provide practical insights into the synthesis of various purine derivatives. We also confirmed that potential branching elongation at the amino group of the N Pu derivatives can be prevented by the on-column acylation using a capping reagent prior to the DNA synthesis. This procedure may find utility when it is necessary to continue DNA solid-phase synthesis after the on-column introduction of the chemical entity endowed with a nucleophilic character.
With the successful acquisition of ODNs incorporating N Pu1, N Pu2, and N Pu3, as well as O Pu1, O Pu2, and O Pu3, we further investigated their base-pairing properties. Interestingly, although their pseudo-nucleobases have similar hydrogen-bonding patterns, these derivatives exhibited different degrees of selectivity and stability as compared to the original N Pu and O Pu ( Figure S3). In particular, O Pu1 bearing 2-pyrimidinone as a pseudo-nucleobase exhibited increased stability toward N Pz, with the T m value being comparable to that of N Pu-O Pz. This may be attributed to the bifacial hydrogen-bonding ability of 2-pyrimidinone, which increased the frequency of hydrogen bonding between the tautomerizing pseudo-nucleobases (Figure 6a). In contrast, the introduction of pyridazinederived pseudo-nucleobases led to the loss of base-pairing selectivity and stability, as indicated by the decreased T m values obtained with O Pu2-N Pz and N Pu-O Pz pairs. This could be attributed to the presence of a nitrogen atom at the 2-position of the pyridazine ring whose lone pair may cause static repulsion with the pi-orbitals of the pyridazine core structure in the opposing N Pz or O Pz (Figure 6b). Such repulsion would prevent the formation of stable hydrogen bonds between the pseudo-nucleobases, thereby destabilizing the base-pairing of O Pu2 and N Pu2 with the complementary N Pz and O Pz, respectively. Overall, the present study demonstrated that the structure of the pseudo-nucleobase moiety is critical for the selectivity and stability of the alkynylated purine-pyridazine base-pairing and that the on-column synthesis approach would aid in the efficient selection of the appropriate pseudo-nucleobases for the alkynylated purine nucleoside. Further structural optimization of the alkynylated purine-pyridazine base pairs is currently in progress by our research group and will be reported in due course.

ER REVIEW 9 of 17
necessary to continue DNA solid-phase synthesis after the on-column introduction of the chemical entity endowed with a nucleophilic character.
With the successful acquisition of ODNs incorporating N Pu1, N Pu2, and N Pu3, as well as O Pu1, O Pu2, and O Pu3, we further investigated their base-pairing properties. Interestingly, although their pseudo-nucleobases have similar hydrogen-bonding patterns, these derivatives exhibited different degrees of selectivity and stability as compared to the original N Pu and O Pu ( Figure S3). In particular, O Pu1 bearing 2-pyrimidinone as a pseudonucleobase exhibited increased stability toward N Pz, with the Tm value being comparable to that of N Pu-O Pz. This may be attributed to the bifacial hydrogen-bonding ability of 2pyrimidinone, which increased the frequency of hydrogen bonding between the tautomerizing pseudo-nucleobases (Figure 6a). In contrast, the introduction of pyridazinederived pseudo-nucleobases led to the loss of base-pairing selectivity and stability, as indicated by the decreased Tm values obtained with O Pu2-N Pz and N Pu-O Pz pairs. This could be attributed to the presence of a nitrogen atom at the 2-position of the pyridazine ring whose lone pair may cause static repulsion with the pi-orbitals of the pyridazine core structure in the opposing N Pz or O Pz (Figure 6b). Such repulsion would prevent the formation of stable hydrogen bonds between the pseudo-nucleobases, thereby destabilizing the base-pairing of O Pu2 and N Pu2 with the complementary N Pz and O Pz, respectively. Overall, the present study demonstrated that the structure of the pseudo-nucleobase moiety is critical for the selectivity and stability of the alkynylated purine-pyridazine basepairing and that the on-column synthesis approach would aid in the efficient selection of the appropriate pseudo-nucleobases for the alkynylated purine nucleoside. Further structural optimization of the alkynylated purine-pyridazine base pairs is currently in progress by our research group and will be reported in due course.

General Information
Chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), the Tokyo Chemical industry (Tokyo, Japan), FUJIFILM Wako Pure Chemical (Osaka, Japan), Kanto Chemical (Tokyo, Japan), and BLDpharm (Shanghai, China), and used without further purification. Reactions were conducted under argon atmosphere in oven-dried glassware unless otherwise specified. The products were isolated by column chromatography

Solid-Phase DNA Synthesis
The oligonucleotides were synthesized on a 1 µmol scale using a DNA automated synthesizer (Applied Biosystems, Waltham, MA, USA, 392 DNA/RNA Synthesizer) with conventional phosphoramidite chemistry. The reagents were purchased from Glen Research (Sterling, VA, USA) and Sigma-Aldrich, and the synthesis was conducted using ultramild phosphoramidites (phenoxyacetyl-dA (Pac-dA), Tac-dG, Ac-dC, T), with 0.25 M 5-benzylthio-1H-tetrazole (BTT) in CH 3 CN as an activator, 3% dichloroacetic acid (DCA) in CH 2 Cl 2 as a deblocking solution, and 5% Tac 2 O and 16% N-methylimidazole in THF as a capping reagent. The ODNs incorporating the original N Pu and O Pu were synthesized as previously reported [31]. Fully natural ODNs were purchased from Japan Bio Service Co., Ltd. (Osaka Japan).

On-Column Synthesis of ODNs Incorporating the Alkynylated Purine Derivatives
Under argon atmosphere, Solution A containing CuI (7.5 mM) in a mixture of TEA-DMF (3:7), and Solution B containing each alkynylated pseudo-nucleobase (22.5 mM) and Pd(PPh 3 ) 4 (7.5 mM) in a mixture of TEA-DMF (3:7), were prepared and degassed through argon bubbling for 30 min. Solution A (100 µL) and Solution B (400 µL) were transferred to individual disposable syringes and attached onto the column containing CPG-bound ODN1 or ODN2 (DMT-ON, 0.2 µmol). The reaction was initiated by mixing the solution inside the column. After 2 h at room temperature, the solution was discarded, and the CPGs were washed sequentially with DMF, CH 3 CN, H 2 O, EDTA solution (1.0 M in H 2 O, pH 8.0), H 2 O, and CH 3 CN. The same procedure was performed twice. After drying the CPGs under a vacuum overnight, the remaining part of the ODNs was elongated using the DNA synthesizer, as described in the previous section. In case of synthesizing ODNs containing the N Pu derivatives, the CPGs were treated with capping solution on the DNA synthesizer five times (60 s each) for protecting the NH 2 group prior to the elongation.

Deprotection and Purification of the Synthesized ODNs
The CPG-bound ODNs incorporating N Pu derivatives were treated with 28% NH 4 OH (1 mL) at room temperature for 2 h. The CPGs were filtered off, and the filtrate was evaporated using a centrifugal evaporator. The ODNs containing O Pu derivatives were deprotected in a similar manner, except that the CPGs were treated with zinc bromide solution (500 µL, prepared by dissolving 2.5 g of ZnBr 2 in a mixture of 1.5 mL of i-PrOH and 1.5 mL of CH 3 NO 2 ) at room temperature for 6 h prior to NH 4 OH treatment. The crude oligonucleotides were purified by reverse-phase HPLC using a JASCO HPLC system (PU-2089 plus, UV-2075 plus, CO-2067 plus) equipped with a Nacalai Tesque COSMOSIL 5C 18 -MS-II column (4.6 × 250 mm). The column oven was set to 50 • C and a peak was detected at 254 nm. The following buffer system was used: buffer A: 0.1 M triethylammonium acetate (TEAA), pH 7.0 in H 2 O, buffer B: acetonitrile. A flowrate of 1 mL/min with a gradient of 9% to 10% of buffer B in 30 min was applied for the purification. The structural integrity of the synthesized oligonucleotides was analyzed by MALDI-TOF mass measurement using a Bruker Daltonics Autoflex Speed instrument, with a mixture of 3-hydroxypicolinic acid and diammonium hydrogen citrate as a matrix.

UV Melting Temperature Measurement of the Duplex DNAs
A solution (100 µL) of equimolar amounts of ODN3 and complementary ODN4 (2 µM each) in the buffer solution containing 10 mM of sodium phosphate buffer (pH 7.0) and 50 mM of NaCl was heated at 85 • C and gradually cooled down to room temperature for annealing. UV melting curves were recorded with a quartz cell with a 1 cm path length at temperatures between 20 and 85 • C using the JASCO V-730 UV-visible spectrophotometer (Jasco, Oklahoma City, OK, USA), with a temperature controller at a ramping and scanning rate of 1.0 • C/min at 260 nm. Each T m value is presented as an average of three measurements.

CD Spectroscopy Measurement of the Duplex DNAs
The DNA solutions for CD measurement were prepared as described above. CD spectra were recorded on a JASCO J-720WI circular dichroism spectrometer equipped with a Peltier temperature controller using a micro quartz cell with a 1 cm path length. The ellipticity was recorded at 25 • C with wavelengths from 450 to 220 nm.